SELF-ASSESSMENT REPORT 2007-2011

Transcription

“Victor Babes” National Institute of Pathology
SELF-ASSESSMENT REPORT
2007-2011
Table of Contents
1. Functional administrative structure diagram
2. General activity report
3. Activity report by team
4. Representative project
1. Functional administrative structure diagram
Management team:
- General director: Acad. Laurentiu M. POPESCU
- Scientific director: CSI Dr. Mihail Eugen HINESCU
- Director: CSI Dr. Bogdan Ovidiu POPESCU
- Economic director: Ec. Mariana GEORGESCU
Decision-making structures: Administrative Council (9 members), Scientific Council (13 members),
Board of directors (4 members).
Departments’ organization
PATHOLOGY
Dr. Carmen Ardeleanu
IMMUNOLOGY
Dr. Cornel Ursaciuc
BIOLOGY
Dr. Mihail Hinescu
Histopathology,
immunohistochemistry and
molecular diagnosis
Immunobiology
Medical genetics
Prof. Dr. Carmen Ardeleanu
Dr. Monica Neagu
Dr. Aurora Arghir
Ultrastructural pathology
Immunopathology
Cellular medicine
Dr. Mihaela Gherghiceanu
Dan Ciotaru
Dr. Mircea Leabu
Biochemistry
Animal husbandry
Molecular medicine
Dr. Cristiana Tanase
Bogdan Marinescu
Dr. Bogdan Popescu
Diagnosis center
Radiobiology
Georgeta Butur
Dr. Gina Manda
Since 2005 the Institute is certified SR EN ISO/CEI 9001:2008 for research activity and medical
services. Several laboratories received national accreditation: Histopathology and immunohistochemistry
laboratory (accreditation SR EN ISO/CEI 15189:2007), Biochemistry laboratory (accreditation SR EN
ISO/CEI 15189:2007), Animal Husbandry unit (accreditation SR EN ISO/CEI 17 025: 2005), Nuclear
unit (national authorization by CNCAN).
2
2. General activity report
Founded in 1887, “Victor Babes” National Institute of Pathology from Bucharest is the first institute of
biomedical research in Romania. Still at the forefront of fundamental research, the Institute is today a
center of reference for human disease diagnosis and monitoring.
Mission
Victor Babes Institute of Pathology mission is to conduct cutting edge research in the field of molecular
and cellular medicine for the knowledge-based scientific progress in the benefit of society.
We are using all organizational resources to address major societary needs in the area of health and to
provide scientifically sound instruments and solutions in the benefit of patients and health professionals.
The institute correlates the identified needs at national level with the scientific and health challenges at
European level, thus providing the most effective ways of access to state of the art knowledge / solutions
and acting as a scientific connection with health and research entities in Europe. The institute provides
support for strategic planning and decision at national level for policy makers in the field of biomedical
research and healthcare.
The institute’s mission is to expand the knowledge in biomedical and associated sciences by conducting
and supporting research, development, education / training and high-quality medical services. The
institute’s mission constructively influences the quality of life and healthcare services at national level.
The institute is committed to increase the international visibility of Romanian research in the field of
cellular and molecular medicine.
Sharing efforts with partner medical institutions (in an attempt to build a local translational research
community), the institute is responding with up-to-date solutions to major human health issues in cancer,
(neuro)degenerative diseases, immune disorders, nephropathology and cardiovascular diseases. The
institute, organized in three main research departments (pathology, immunology, biology) is using stateof the-art methodology to develop innovative diagnostic tools and personalized medicine strategies.
The advantage of having infrastructure and experts in different disciplines is that we can adjust our
projects along the way, depending on the priorities related to patient needs and in conjunction to the
European scientific trends. It is our commitment to be integrated in large scale multidisciplinary projects
in biomedical science, aiming at improving health and the quality of life.
Research focus: Multidisciplinary research in the field of cellular and molecular medicine, developed by
10 research teams
1. Telocytes: telocytes characterization; telocytes-stem cells tandem; telocytes in regenerative
medicine; in vitro and in vivo functional studies on telocytes
2. Surgical and molecular pathology: cell signaling pathways in malignant epithelial tumors;
molecular bases of therapy modulation in malignant tumors; genotypic profiles variability in cancer
3. Translational research in cancer: therapeutic targets in malignant tumors; prognostic and
predictive biomarkers in epithelial malignant tumors; molecular identification of etiologic factors in
infections and associated tumors
4. Ultrastructural pathology: basic research in fundamental mechanisms of cardiac regeneration from stem cell to heart tissue; 3D electron tomography of the caveolar microdomains in smooth
muscle cells; cellular and molecular mechanisms involved in glomerular pathology; cell
ultrastructure investigation
5. Proteomic biomarkers: proteomics technologies for biomarkers discovery in cancer; proteomics
biomarkers in pharmacological research; proteomics in the evaluation of environmental risks for
human health
6. Immunomodulation-immunodiagnosis: tumor immunology; biomarkers in autoimmune diseases;
cytokines and immunomodulation; innovative immunotherapies
7. Genomics and genetic diagnosis: genetics of neuropsychiatric disorders; genetic/epigenetic and
genomic biomarkers relevant for cancer onset and progression
8. Neurosciences: trophic factor receptors expression in central and peripheral nervous system; tight
junction proteins in brain and peripheral nerves; neurodegeneration models relevant to Alzheimer
and Parkinson diseases; neuromuscular pathology
3
9. Drug development and toxicology: pathologic mechanisms and drug targets in cardiovascular
diseases and rheumatoid arthritis; drug development - biological in vitro and in vivo screening;
immunotoxicology; radiobiology
10. Assay development and alternative testing: immune-based assay development for bacterial/viral
infections; cell-based assays development for drug assessment and nanomedicine.
Collaborations
National collaborations with partners having complementary expertise and infrastructure
International collaborations with Max Planck Institute, Graz University, University of Tuebingen,
Universite Catholique de Louvain, Ludwig Cancer Institute, University of Goteborg, University of
Turin, University of Medicine Florence, University of Athens, Cyprus Institute of Genetics and
Neurology, Center of Cardiovascular Research Aachen, Saint George’s University of London,
Descartes University of Paris, Hospital Cochin, Chinese Academy of Medical Sciences etc.
Project-based research
Research excellence in the field of life sciences is sustained by a broad array of research projects:
International projects: 4 bilateral projects with France, China and Cyprus, 1 NATO Science for Peace
project, 1 MNT-ERA NET project, 1 FP7 – People project, 1 project in the EU Education and CultureLifelong Learning Program.
Research structural funds: 2 POSCCE projects with foreign coordinator (Priority axes 2 – Competitivity
by research, technological development and innovation)
-
Proteomics technologies for cancer biomarkers discovery (coordinator Prof. S. Constantinescu)
Implementation of molecular tissue assays for cancer in Romania. State-of-the-art research
focused on personalized oncology (coordinator Prof. G. Bussolati)
Projects financed by the national research programs CEEX (Health, Biotech, Matnantech, Infosoc),
CNCSIS, PNII Partnerships and Capacities = 125; 21 projects were coordinated by INCD “Victor Babes”
and in 104 projects the institute participated as partner
o
o
5000000
35
30
number of projects
25
budget
4500000
4000000
3500000
3000000
20
2500000
15
2000000
budget (euros)
number of projects
EU Structural Funds
1500000
10
1000000
5
500000
0
0
2007
2008
2009
2010
2011
The significant cut-offs in number of granted projects and budgets are explained by lack of national
research competition calls in 2009 and 2010, and a dramatic decrease in research public funding as a
result of economic slow-down. The 2010-2011 up-trends reflect accessing of European structural funds.
Getting alternative financial support in the frame of Sectorial Operational Program for Increased
Competitiveness is an important advantage for research institutes. However, this opportunity was not a
long term option, since the current call topics do not fit the institute’s expertise/eligibility.
Projects financed by the European Social Fund: 3 projects focused on the training of personnel from the
national health system in the field of state-of-the-art biomedical techniques, aiming to implement new
diagnosis tools in clinical laboratories.
Infrastructure development projects: 1) Advanced infrastructure for molecular cytogenetic research; 2)
Upgrading of a biobank for tumor cells and nucleic acids by attaching an immunogenomics laboratory for
molecular screening in cancer; 3) Upgrade of research infrastructure for laboratory animals in INCD
"Victor Babes"; 4) Makeup of the most competitive laboratory in Romania for living cell direct study
under microscope in an incubator
Project for defining strategic priorities: “Cell therapy in regenerative medicine development. Strategic
priorities” – STRATEC, funded by the National Authority for Scientific Research. Through this project
4
the institute offered its expertise to the main research policy maker in Romania and our researchers
gained new insights in the field of cell therapies, paving the way to future research directions.
Major achievements
Publications
35
Publications in ISI journals
with non-zero rAIS = 99
number of papers
30
25
20
-
15
10
5
0
2007
2008
2009
2010
2011
total rAIS = 154,15918
rAIS/researcher = 2,03
total number of citations = 561
mean citations number/researcher = 7,38
year
60
200
50
150
rAIS
40
30
100
20
50
10
0
number of citations
The institute’s research activity
became significantly more visible at
international level during 2008-2011,
both as publications and citations
number in ISI ranked journals.
Considering the number of
contributing researchers (76), it is
obvious that the objective of the
institute’s strategy to enhance
international visibility was reached.
rAIS
citations
0
2007
2008
2009
2010
2011
year
Publications in ISI journals with zero rAIS = 48
Other publications
14
number of papers
12
77 papers published in non-ISI journals
10
9 books (1 published by Elsevier) and 20
book chapters (7 published by international
publishing houses)
8
6
4
2
0
2007
2008
2009
2010
2011
year
-
Patents
4 registered patents 1) Tetra-sulphonated porphyrin application for producing a dermatologic
therapy – photosensitizer; 2) Tetrapirolic compound asymmetrically substituted – synthesis and
biological evaluation; 3) Equipment and procedure for microwave irradiation in in vitro models with
concomitant registration of biological behavior in a fluorescence microscope; 4) Method of obtaining
yeast bioproduct enriched with chrome; 2 submitted patents.
The patent “Tetra-sulphonated porphyrin application for producing a dermatologic therapy –
photosensitizer” received Gold medal at Brussels Innova 2008, Special Prize of Rudy Demotte, Minister
President of the Walloon Government, Gold medal at The 37th International Exhibition of Inventions of
Geneva 2009 and Special Prize of the Ministry of Education of Russia, 2009; Gold medal at The
International Fair for Innovation, Moscow, 2009.
The project proposals of the Institute at the 2011 Call “Partnerships – Collaborative projects of applied
research” reflect our commitment to develop applied research in consortia with other public and private
R&D institutions, resulting in patents and publications.
Research results translated to the Diagnosis Center: New immunohistochemical algorithms for the
diagnosis of cancer; In situ hybridization for Epstein Barr virus in undifferentiated carcinoma of
nasopharynx and Hodgkin lymphoma; Fluorescent (FISH) and chromogen (CISH) in situ hybridization
for Her-2/neu gene amplification in breast and gastric carcinoma; FISH for bcl 2 translocation in
follicular malignant lymphoma; FISH for gene fusion in Ewing sarcoma; FISH for ALK gene
mutation in anaplastic malignant lymphoma and in bronchopulmonary adenocarcinoma; Molecular
5
detection of K-ras gene mutation in colon carcinoma; c-kit gene mutation in GISTs; EGFR gene
mutation in lung adenocarcinoma (in preparation).
Staff: 76 researchers, out of which 21 senior researchers and 19 PhD students, along with 39 technicians
and 34 NRDS personnel (situation at 15 December 2011), with a mean age of 44 years, represent a
critical mass for self-sustaining and further growth in biomedical research. We are committed to recruit
young scientists and to offer them adequate support to develop competitive research. The stability of
R&D personnel indicates that the institute assured appropriate conditions for research and career
development.
Major achievements of the institute’s human resources policy
-
Recruitment as project coordinators of foreign scientists with outstanding scientific visibility:
Prof. Stefan Constantinescu and Prof. Gianni Bussolati
-
Recruitment of young researchers with foreign experience in prestigious research institutions
and universities: Valeriu Cismasiu – specialization in stem cells: post-doctoral fellow at Lund
University, Stem Cell Center, Sweden (2007-2009) and EMBL Mouse Biology Unit, Monterotondo,
Italy; visiting scientist at Weatherall Institute of Molecular Medicine, Oxford, UK (2008-2009).
Andreea Tutulan-Cunita – specialization in molecular biology and genetics; master degree (20002002) and PhD (2002-2005) at Hiroshima University, Japan; postdoctoral fellowship (2005-2006) at
Manchester University, UK. Sevinci Pop – specialization in transcription activation, DNA-protein
interaction, chromatin structure and function, gene expression; doctoral fellowship (2000-2002) and
postdoctoral fellowship (2003-2008), University of Illinois at Urbana-Champaign, USA,. Georgeta
Cardos – doctoral fellowship (2004-2008) at Hamburg University, Germany
-
Training of our researchers in prestigious research laboratories
mean number of employees evolution of employees number by category
175
70
165
number of employees
number of employees
170
160
155
150
145
140
135
130
2008
50
2009
40
2010
30
2011
20
10
0
2007
2008
2009
2010
2011
4,5
y = 0,427x + 2,2871
R2 = 0,9917
4
3,5
CSI
CSII
CSIII
CS
AS
TS
AUX ADM
There is an obvious increase trend in the R&D/ADM
personnel ratio during the last 4 years, proving an
appropriate human resources policy according to the
main activity of the institute (research).
R&D/ADM staff ratio
number of emplyees
60
3
The personnel structure reflects the needs of the
institute for project development and for reaching
excellence in life sciences research.
2,5
2
1,5
1
0,5
1.200
2008
2009
2010
2011
equipment acquisition budget
(thousands of euros)
0
1.000
Infrastructure upgrading was one of the main
800
priorities of our strategic plan for 2007 - 2011.
600
Funds were obtained from 4 specific projects
400
financed by the national Capacities Program
200
and from research projects. Investments were
2007
focused on developing cytometry, imagistic,
genomics and proteomics research units, a
biobank for tumor cells and nucleic acids and the animal husbandry.
2008
2009
2010
2011
year
*
Taking together our achievements in the last 4 years, we conclude that “Victor Babes” Institute of
Pathology has a leading position in life science and biomedical research in Romania.
6
3. Activity reports by team
7
TEAM 1 - TELOCYTE - A NEW TYPE OF INTERSTITIAL CELL
Team leader: Laurentiu M. Popescu
Senior Researchers: Mihail Eugen Hinescu; Eugen Mandache
PostDoc researchers: Mihaela Gherghiceanu; Bogdan Ovidiu Popescu; Sanda Cretoiu
Laura Cristina Suciu; Valeriu Bogdan Cismasiu; Bogdan Gabriel Marinescu
PhD students: Catalin Gabriel Manole; Dragos Cretoiu; Mihnea Ioan Nicolescu
Technicians: Marin Teodor Regalia; Maria Dumitrescu; Rodica Stanca; Petrica Musat
L.M. Popescu, MD, PhD, Dr. h.c.mult., is currently Head of the National Institute of Pathology, Bucharest,
Romania. He is member of the National Academy of Sciences and of the Academy of Medical Sciences.
Recently, he became President Elect of the Federation of European Academies of Medicine, and of the
International Society for Adaptive Medicine. He published over 100 scientific articles in international peerreview journals and is cited more than 1500 times. He has a Hirsch Index of about 30. Professor Popescu
is Editor-in-Chief (and founder) of the Journal of Cellular and Molecular Medicine (Wiley/Blackwell), with a
5-year IF of 5. He is credited with the discovery of Telocytes.
Recently, our team discovered a new cell type in humans and mammals. We called recently (2010) these
cells Telocytes, replacing the term previously used by us – Interstitial Cajal-like Cells (acronym: ICLC).
This discovery is cited in more than 100 scientific papers, and very recently we are trying to impose the
concept of “Telocytes / Stem-Cells Tandem” existing and working in the so-called Stem Cell Niches. The
Telocyte concept is already adopted by many scientists: e.g. Eyden et al. – UK; Faussone-Pellegrini &
Bani – Italy; Gittenberger-de Groot et al. – The Netherlands; Klumpp et al – Germany; Kostin – Germany;
Polykandriotis – Greece; Zhou et al – China; Cantarero et al - Spain; Gard & Asirvatham – USA; Gevaert
et al. – Belgium; Marban et al – USA; Limana et al – Italy; Radenkovic – Serbia; Rupp et al. – Germany;
Russell – USA; Tommila – Finland, etc.
Major research topic is the connective tissue (CT) cellular and molecular biology with emphasis on
telocytes involvement in tissue physiology and pathology. CT represents the essential microenvironment
to coordinate body structure and function hosting and joining together three major systems (the
circulatory, nervous and immune systems) and integrating all others tissues and organs. More than
200,000 publications showed that CT is involved in organ development, renewing, repair, regeneration,
and tumour development. Recently, new types of cells (e.g. resident mesenchymal stem cells, resident
stem cells and telocytes) have been described but specific markers for the majority of the CT cells are still
missing. Their detection and discrimination in situ is highly biased by their almost exclusive
characterisation in vitro.
Major questions concerning CT biology must be addressed:

How many distinct cell types reside in the adult CT?
8

Are these cells tissue specific?

Do stem cells need a specific CT microenvironment to survive and differentiate?

How does the CT network change in specific pathological conditions?
We plan to perform a systematic inventory of the old and the new cell types of the CT, their molecular
profiling, tissue distribution and interactions with emphasis on fibroblasts, fibroblast-like cells, telocytes,
and mesenchymal stem cells.
Future research plans are to:

define specific phenotypic, genetic and functional markers for particular CT cells and to assess
their tissue specificity;

identify novel cellular, molecular and signalling networks involved in CT physiology and
remodelling;

re-evaluate homo/heterocellular and cell-to-matrix communication;

investigate cellular aspects of tumour-stroma interaction.
Our approach involves structural/ genomic/ transcriptomic/ proteomic/ functional assays using in situ/ in
vitro/ in vivo models to define the CT ‘connectomics’. CONNECT research could offer new ways for
regenerative medicine (e.g. poly cellular treatment instead of stem cell approach only) as well as antitumour therapies for stromal tumours.
Main expected outcomes of our research:
1) Better characterized resident CT cells (e.g. fibroblasts, mesenchymal stem cells, telocytes) and their
microenvironment
2) Endorsing markers for CT cells recognition in situ
3) Increasing knowledge about cellular and molecular mechanisms of tissue renewal
4) Better characterization of telocyte - the cell described in preliminary studies by our group
Existing equipment and facilities:
- electron microscopy unit (ultrastructural analysis, cellular tomography, array tomography):
Transmission electron microscope Morgagni 268 FEI, 100 kV; Olympus
MegaView CCD; Electron
microscope Tecnai G2 BioTwin Spirit FEI, 120 kV, single tilt holder; Olympus
MegaViewG2 CCD;
UV/Cryo-chamber EMS; LKB and RMC XL ultra-microtomes; diamond knifes, ovens, etc.
- identification and localization of molecules by immunofluorescence: laser scanning microscope
Nikon; microscopes Nikon E 600 with UV, CCD; 2 Nikon 200; Leica cryotom; immunostainer, paraffin
embedding station; biobank for tissues, cells and nucleic acids; deep freezing unit; Leica microtome.
- light microscopy facility: motorized AxioZ1 Zeiss microscope with light bright field and
epifluorescence, equipped with high resolution monochrome cooled CCD camera and image processing
software.
- cell culture facilities: Laminar flow hoods; CO2 Incubators; Centrifuges; Phase contrast microscope,
Sterilization unit, Ultra pure water system.
-
cytometry
unit:
fluorescence
and
confocal
microscopes,
flow-cytometer
Becton-Dickinson
FACSCalibur
9
- videomicroscopy: Biostation IM (Nikon Corp., Japan).
- cell layer impedance measurement: xCELLigence (Roche Diagnostics, Germany).
- microarray facility: Agilent DNA Microarray Scanner (G2565CA) with SureScan High-Resolution
Technology with Agilent Scan Control Software and Genomic Workbench; Agilent BioAnalyzer and
hybridization oven.
- molecular genetics facility: Corbett Palm Cycler PCR, Corbett Real Time PCR;
- nucleic acid isolation and manipulation facilities: chemical hoods, water baths, centrifuges,
spectrophotometer, transilluminator, freezers, ultrafreezers.
- microRNA qPCR: NanoDrop ND-1000 Spectrophotometer; QuantiMir RT -System Biosciences; iCycler
system and software- Bio-Rad.
- proteomics: SELDI-TOF mass spectrometry system, Luminex-xMAP; 2D and 2D-DIGE; software –
PDQuest; protein microarray platform.
- animal Models Core Unit (SR EN ISO 17025:2005)
- image processing: photographic laboratory; 4 Dell computers, 1 Siemens; Wacom Intuos 3 pen tablet;
software for image analysis: Axiovision Zeiss; ImageJ -NIH, iTEM FEICo, Explore3D FEICo, Amira.
International collaboration:

Prof. Maria-Simonetta Faussone-Pellegrini and Prof. Daniele Bani from Department of
Anatomy, Histology and Forensic Medicine, University of Florence, Italy

Prof. Ofer Binah from the Department of Physiology; Ruth and Bruce Rappaport Faculty of
Medicine, Technion Israel Institute of Technology, Haifa, Israel

Prof. Sawa Kostin from Max-Planck Institute for Heart and Lung Research, Franz Groedel
Institute, Bad Nauheim, Germany

Prof. Shengshou Hu from the Center for Cardiovascular Regenerative Medicine, Peking Union
Medical College, Chinese Academy of Medical Sciences, Beijing, China

Prof. Changyong Wang from the Tissue Engineering Research Center, Academy of Military
Medical Sciences, Beijing, China
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TEAM 2 - SURGICAL AND MOLECULAR PATHOLOGY
Team coordinator: CS1. Prof. Dr. Carmen Ardeleanu, Head of Pathology Department
Mission: Obtaining new tissue biomarkers regarding the structure and molecular profiles of
pathological processes (tumors, inflammations etc.)
Research focus:
 Molecular biology of malignant tumors focusing on inter- and intracellular signaling.:
exploring signaling pathways molecular factors
o development and implementation of high throughput methodologies using in vitro assays
with particular end-points, for identifying and characterization of new biomarkers of
cell signaling in tumors
o partnership with other research teams from clinical oncology, surgical clinics, for testing
and promoting new diagnostic and therapeutic tools potentially efficient in cancer
 Identifying new genes implication in tumor progression by means of genotypic and
phenotypic profiles
o implementation of advanced molecular methods for characterizing the genetic variability
of malignant tumors
 Developing and extending standardized methods for processing and stock tissues aiming to
obtain available results for the telepathological interpretation.
Research topics:
1. Cellular signaling pathways in malignant epithelial tumors
No
1.1
1.2
1.3
Research area / coordinator
Tumor-microenvironment interactions
CS1 Prof. Dr. Carmen Ardeleanu
CS3 Biol. Sp. Georgeta Butur
CS2.Dr. Dorel Arsene
CS3 Dr. Florina Vasilescu
CS Dr. Alina Grigore
Intercellular signaling in malignant lymphoma
CS3 Ass Prof. Dr. Camelia Dobrea
Dr. Florina Cionca
Signal transduction anomalies in epithelial malignant
tumors
CS3 Dr. Cristina Iosif
CS3 Dr. Florin Andrei
2. Molecular bases of therapy modulation in malignant tumors
No Research area / coordinator
Breast cancer molecular features
2.1
Ass.Prof. Dr. Maria Comanescu
Microsatellite instability of colon carcinoma
2.2 CS2 Ass.Prof. Camelia Vrabie
CS2 Prof. Dr. Maria Sajin
Proteinkinase receptors in lung carcinoma
2.3
Research contracts / budget
(Euro)
F20/140.968,04
F70/154586,78
F21/ 90.211,59
F40 / 127.272,73
Research contracts / budget (Euro)
CF10 / 68.181,82
F19 / 159.090,91
F69 / 45.271,59
11
3. Genotypic profiles variability in neoplasia
No. Research area / coordinator
Molecular genetics in epithelial malignant tumors
3.1 Senior Res Prof. Dr. Gianni Bussolati
Prof. Dr. Carmen Ardeleanu
CF8 / 1.231.125,00
F24/150.000,00
4. Phenotypic and genotypic profiles in inflammatory and degenerative diseases
No
(Euro)
Proapoptotic and antiapoptotic factors
F22/102.272,73
1.1
CS Dr. Alina Grigore
Cellular interactions in autoimmune diseases
F26/ 93.429,55
1.2
CS3 Ass Prof. Dr. Dana Terzea
Dr. Florina Cionca
Inflammatory disease of bowel
F72/ 45.454, 56
Ass.Prof. dr. Gasbriel Becheanu
1.3
F73/29.021,82
CS3.Dr. Cristina Iosif
F74/40.590,73
Research team
Name
CS1 Carmen Ardeleanu
CS 3 Biol sp. Georgeta
Butur
CSI Prof. Gianni Bussolati
C3 Florina Vasilescu
CS 3 Camelia Dobrea
C3 Cristina Iosif
CS Alina Grigore
Dr. Gabriel Becheanu
Dr. Maria Comanescu
Res Assist Adina Balan
Cs. Dr. Georgeta Cardos
CSII. Maria Sajin
Dr. Florina Cionca
ASC Staicu Viorela
Laboratory
Head of pathology
Department
Head of Diagnostic Center
Team coordinator
Name
CSIII. Dana Cristina
Terzea
Histopathology Department
CSII Dorel Eugen Arsene
CSII Camelia Vrabie
TS. Florina Alexandru
TS. Alina Anghel
TS. Valentina Muntean
TS. Elena Ion
TS. Tatiana Nora Petre
Ts. Florina Manda
TS. Daniel Anghel
TS. Monica Haghighat
Biol. Gaina Gisela
Laboratory

PhD students: molecular biology of nonsmallcell lung carcinoma; breast carcinoma molecular
features in young women; genic anomalies in colon carcinomas
 Post doc: Advanced studies in molecular biology and gene profiling by gene microarray in breast
triple negative breast carcinoma.
Training: 1. Systemic pathology , European School of pathology, Craiova: pancreas and liver– 2007 ( 3
researchers), gastro-intestinal pathology – 2008 (4 researchers), gynaecological pathology – 2009 (3
researchers), male genito-urinary tract pathology – 2010 (3 researchers), thyroid pathology – 2011 (3
researchers); 2. Agilent Gene microarray training for theory and practical applications (3 researchers). 3.
Stem cell implication in regenerative medicine, London, 06.10.2011 (1 researcher).
Methodological approach
o To apply in surgical and molecular medicine the know-how of our research team in human pathology,
cellular and molecular biology
12
- Histopathology , cytopathology, histochemistry, immunohistochemistry, in situ hybridization
(fluorescent and chromogenic), PCR, Real-time PCR, reverse -transcription PCR, PCR array, gene
profile microarray (morphological analysis of tumors, immune phenotyping on archived tissue, for
cell differentiation, typing secreting cells, identification of intercellular and intracellular signaling
factors, identification of diagnostic, prognostic and predictive factors in tumors, cellular activation
and proliferation biomarkers, apoptosis, amplification of genome sequences in infections and
tumors, genes mutations, gene profiles of tumors), etc.
Infrastructure
Histopathology unit: accreditation according to SR EN ISO 15189 for histopathology and
immunohistochemistry (microtome, automatic tissue processor, vacuum automatic tissue processor,
scientific microscopes, professional microscopes, routine staining machine); Laboratory of
Immunohistochemistry unit: (immunostainers, water baths, microwave oven, automatic cover splipping
machine); Hibridization compartment (hybridization plate, fluorescence microscope – Nikon 800, water
bath); Molecular diagnosis compartment (termocyclers, real-time PCR automatic system, GEL-Doc,
Nanodrop, nucleic acids extractor).
International project proposals
Proposal full title: Molecular workflow for the effective detection of multiplex HPV markers in cervical
cancer patients Proposal acronym: CERVIFLEX, Type of funding scheme: Collaborastive Project Small
and medium-scale focused research Work programme topics addressed: HEALTH.2010.1.2-1 Name of
the coordinating person: Prof. Dr. Giorgio Stanta
Bilateral Italian-Romanian project: Impact of immuno- and geno-typing for improving
diagnoses and planning treatment of human tumors. SUPPORT ACTION FOR BILATERAL
COOPERATTION, 2005-2008
TASTE: Telepathological assessment of histopathological and cytological techniques (financed)
Education and Culture-Lifelong Learning Programme, 2011-2014, Pr. Nr. 519108-LLP-2011-1-ITKA3-KA3MP
Partner institutions
1. University of Medicine and Pharmacy “Carol Davila”, Bucharest; 2. University of Bucharest; 3.
University of Medicine and Pharmacy Craiova, 4. “Gr.T.Popa” University of Medicine and Pharmacy,
Iassy, 5 “Victor Babes” University of Medicine and Pharmacy, Timisoara, 6 “Ion Cantacuzino” National
Institute of Immunology and Microbiology, 7 Universita degli Studi di Torino, Italy, 8 Roche Pharma
Publications
6 ISI publications A1,A2,A14,A28, A26,A71
We were involved in national project regarding cellular signaling pathways in malignant epithelial
tumors; molecular basis of therapy modulation in malignant tumors; genotypic profiles variability in
neoplasia; phenotypic and genotypic profiles in inflammatory and degenerative diseases
Other relevant publications: O2,O6, O10, O11, O16, O31, O40, O53, O61, O65, O95, O33,
O43, O54, O62, O66, O109.
Development plan
I. New research areas:
1. Improvement of molecular signature detection in cancer diagnosis, prognosis and therapy.
2. Applied oncology: Development of molecular approach of archived tumor tissue aiming to offer new
potential biomarkers of predictive value in malignant tumors.
3.Development and adapting molecular biology technologies for early detection of malignant tumors and
improvement of the therapy.
II. Patent submission for new molecular tools in cancer.
III. Validation of new experimental models for evaluation of tumor aggressiveness.
IV. Introduction of new validated investigation methods in medical practice.
13
TEAM 3 - TRANSLATIONAL MEDICAL RESEARCH IN CANCER
Team coordinator: CS3. Biol. Sp. Georgeta Butur, Head of Diagnostic Center
Mission: translation of high level technology in medical practice to improve survival and life quality of
cancer patients
Research focus:
 Development of molecular diagnosis
 Implementation of new prognostic and predictive biomarkers in malignant tumors
 Aplication of advanced methods for identification of molecular targets in cancer therapy.
Research topics:
1. Molecular identification of etiologic factors in infections and associated tumors
No
(Euro)
Molecular diagnosis of mycobacterial infections
F23/22.727,27
1.1
Res. Ass. Biol. Sp. Diana Teletin
Hepatitis viruses implications in chronic
lymphoproliferations
F18/ 39.897,73
1.2
CS3 Ass Prof. Dr. Camelia Dobrea
CS3 Biol sp. Georgeta Butur
Gene E4 expression of HPV in cervical lesions
F68 / 81.433,86
1.3
2. Prognostic and predictive biomarkers in epithelial malignant tumors
No Research area / coordinator
Biomarkers in malignant tumors
F67 / 14.09,91.
2.1
F24/150.000
2.2
2.3
Therapeutic targets in nonsmall cell lung carcinoma, liver
disease, melanoma
CS3 Dr. Camelia Dobrea
Prognostic markers in hepatocellular carcinoma, melanoma
Asso. Prof. Mariana Costache
Asso. Prof Dr. Gabriel Becheanu
Biomarkers of renal carcinoma involved in therapy
Dr. Mihaela Mihai
3. Therapeutic targets in malignant tumors
No. Research area / coordinator
High-throughput metodologies in personalized oncology)
3.1
Senior Res Prof. Dr. Gianni Bussolati
Molecular targets in the therapy of malignant tumors
3.2 CS1 Prof. dr. Carmen Ardeleanu
CS3 Biol.sp. Georgeta Butur
Immunohistochemical biomarkers in malignant tumors therapy
CS3 Dr. Florina Vasilescu, CS3 Dr. Camelia Dobrea, CS3
3.3
Dr. Florin Andrei, CS3 Dr. Cristina Iosif , CS Dr. Simona
Enache, CS Dr. Alina Grigore, Ass. Prof. Dr. Maria
Comanescu
2.4
F70 / 154.586, 78
F17/113.640
F25/45.450
F88/121.990
F90/16.540
F71 / 56.818,18
F95/ 68.181,82
CF8 / 1.231.125,00
Medical services contracts (ROCHE)
Medical services contract with
Pharma St. Invest., Medical Insurance
Agencies (Bucuresti,Dambovita,
Pitesti, Prahova, Vrancea, Buzau,
Ilfov, Bacau, Giurgiu, Calarasi)
14
Research team
Name
CS1 Carmen Ardeleanu
CS 3 Biol sp. Georgeta
Butur
C3 Florina Vasilescu
CS 3 Florin Andrei
CS 3 Camelia Dobrea
C3 Cristina Iosif
CS Simon Enache
CS Alina Grigore
CS Maria Neagu
Dr. Mihaela Mihai
Dr. Mariana Costache
Res Assist Adina Balan
Res Ass. Diana Teletin
Dr.Valentin Enache
Laboratory
Head of pathology
Department
Head of Diagnostic Center
Team coordinator
Name
Biol. Gaina Gisela
TS. Catalina Culda
TS. Florina Alexandru
TS. Alina Anghel
TS. Valentina Muntean
TS. Elena Ion
TS. Tatiana Nora Petre
TS. Daniel Anghel
TS. Monica Haghighat
TS. Elena Naita
Dr. Simona Chirlomez
TS. Georgiana Preda
TS. Georgeta Melinte
TS. Ene Eugenia
Laboratory
Res Ass. Staicu Viorela
PhD students: breast cancer stem cells, molecular features in GISTs, Parasitic infections in humans,
phenotypic and genotypic aspects in renal cell carcinoma, Endometrial carcinoma immunohistochemical and molecular characteristics.
Training: ESOT 2009, Paris (2 researchers), International course of pathology of digestive tract,
Bucharest, 2008 (5 researchers), 2009 (4 researchers), 2010 (6 researchers), 2011 (3 researchers),
Diagnostic histopathology of soft tissue tumors, Treviso Italy, 2008 (4 researchers), Breast pathology,
Harvard Medical School, Boston, 2007 (2 researchers).
- To improve the diagnostic in pathology of tumors
Histopathology , cytopathology, histochemistry, immunohistochemistry, in situ hybridization (fluorescent
and chromogenic), PCR, Real-time PCR, (morphological analysis of tumors, immune phenotyping on
archived tissue, for cell differentiation, typing secreting cells, identification of intercellular and
intracellular signaling factors, identification of diagnostic, prognostic and predictive factors in tumors,
cellular activation and proliferation biomarkers, amplification of genome sequences in infections and
tumors).
- To collaborate with medical institutes and clinical hospitals and ambulatories at national and
international level, with complementary expertise using high throughput metodologies.
Infrastructure
Diagnostic center for translation of know-how and the results of research as services to regional and
local health units, aiming to a higher quality of life and to improve the policy of healthcare by
personalized therapy .
Histopathology unit: accreditation according to SR EN ISO 15189 for histopathology and
immunohistochemistry (microtome, automatic tissue processor, vacuum automatic tissue processor,
scientific microscopes, professional microscopes, routine staining machine); Laboratory of
Immunohistochemistry unit (immunostainers, water baths, microwave oven, automatic cover splipping
machine); Hibridization compartment (hybridization plate, fluorescence microscope – Nikon 800, water
bath); Molecular diagnosis compartment (termocyclers, real-time PCR automatic system, GEL-Doc,
Nanodrop, nucleic acids extractor).
15
Characterization of early disseminating tumourigenic breast cancer cells with CD44+CD24-/low phenotype
and their microenvironment in the bone marrow and bone of patients with Breast cancer
Proposal acronym: METASTEM; Type of funding scheme: Collaborative Project: Small or medium scale
focused research project Work programme topics addressed: HEALTH-2007-2.4.1-6: Understanding and
fighting metastasis, Name of the coordinating person: Prof. Dr. T. Bauernhofer,
You have submitted a proposal to the Electronic Proposal Submission System. Your proposal is now
stored on the EPSS system with number 201269 for subsequent evaluation by the Commission.
(nonfinanced).
Partner institutions
1. University of Medicine and Pharmacy “Carol Davila”, Bucharest; 2. Medical services contract with
Pharma St. Invest.,3 Medical Insurance Agencies (Bucuresti,Dambovita, Pitesti, Prahova, Vrancea,
Buzau, Ilfov, Bacau, Giurgiu, Calarasi, 4. “Gr.T.Popa” University of Medicine and Pharmacy, Iassy, 5
“Victor Babes”University of Medicine and Pharmacy, Timisoara, 6“Ion Cantacuzino” National Institute
of Immunology and Microbiology, 7. Universita degli Studi di Torino, Italia, 8 Roche Pharma
Publications
1. Diagnosis of difficult cases: A1,A2,A26
- we were involved in national project regarding biomarkers in malignant tumors (lung, breast,
malignant lymphomas) and degenerative disease.
- Other relevant publication: O34, O44, O57, O63, O81, O35, O36, O48, O59, O82, O37, O39, O135,
O136.
Development plan
I. New research areas:
1. Development and introduction in the Diagnosis Center of new molecular tools (RT-PCR, microRNA,
PCR-array) applied on archived tumor tissue for a more accurate identification of new molecular
targets in personalised therapy.
2. Development of multi-skill teams (pathologists, genetists, biochemists, biologists, oncologists,
biophisicists, biostatisticians), for molecular diagnosis of malignant tumors by means of high-throughput
technologies.
II. Validation and patent submission for newly developed molecular tools in the diagnosis and
prognosis of cancer.
III. Theorical and practical training of pathologists aiming to improve the diagnosis in cancer for
applying novel personalized therapies.
16
TEAM 4 ‐ ULTRASTRUCTURAL PATHOLOGY LABORATORY The Ultrastructural Pathology laboratory (UPL) is fully equipped laboratory for transmission electron
microscopy with modern ancillary equipment and a new FEI Morgagni 100 kV Transmission Electron Microscope
(acquired in 2008) for ultrastructural investigation in diagnosis and research. Since 2009, UPL hosts also a Tecnai
G2 Spirit BioTWIN Transmission Electron Microscope with single tilt holder which undoubtedly will attract great
scientific projects focused on data collection for electron tomography. This technique allows 3D reconstruction of
large molecules, organelles and small cells.
The UPL is headed by Mihaela Gherghiceanu MD, PhD who has extensive experience in both diagnostic and
research applications of electron microscopy and electron tomography. Additional expertise is provided by Eugen
Mandache MD, PhD who has over 40 years of experience in the ultrastructural evaluation and diagnosis. He was
head of the laboratory until 2010. Technical support is provided by three highly trained electron microscopy
technicians and more than 400 samples are processed for ultrastructural investigation for diagnosis or research.
UPL TEAM AND ASSOCIATE RESEARCHERS
Mihaela Gherghiceanu, MD, PhD (48 ISI papers; 557 ISI citations, h-index 15)
Eugen Mandache, MD, PhD (25 ISI papers; 169 ISI citations, h-index 6)
Mihail Hinescu MD, PhD (30 ISI papers; 421 ISI citations, h-index 11)
Elena Moldoveanu, PhD (28 ISI papers; 82 ISI citations, h-index 5)
PostDoc: Laura Suciu MD, PhD; Marta Daciana PhD
PhD students: Gabriela Catalin Biol; Catalin Manole MD
EQUIPMENT
- Ultrastructural analysis _ electron microscopy and tomography.
Transmission electron microscopes: Morgagni 268 FEI, 100 kV with CCD; Tecnai G2 BioTwin Spirit FEI, 120 kV,
single tilt holder with CCD.
Ancillary electron microscopy equipment: RMC XL ultra-microtomes; UV/Cry-chamber EMS; Leica cryosubstitution system, diamond knifes, ovens, etc.
- Identification and localization of molecules by immunofluorescence: microscopes Nikon E 600 with UV, ZEISS
CCD 11Mp; Nikon 200; Leica cryotom.
- Image processing: photographic laboratory; 4 Dell and 1 Siemens computers; scanners; software for image
analysis (iTEM Olympus, FEI Explore3D, Amira Visage Imaging).
DIAGNOSIS
The UPL is a nationally recognized, reference center for diagnostic renal pathology and electron microscopy.
Diagnostic services are available and include diagnostic evaluation of kidney, muscle, nerve, skin, liver and other
biopsy material (200 specimens/year). In addition to routine ultrastructural techniques, we offer specialized
immunofluorescence analysis of biopsy specimens (e.g., kidney, skin).
RESEARCH
Research projects carried out in the last 5 years by senior researches of UPL were financed with more than
1.000.000 euro. Based on the results obtained from the ultrastructural studies performed in UPL, 36 articles (245 ISI
citations) have been published in international journals in the last 5 years. Ultrastructural analysis for “Telocytes”
project has been performed in UPL.
17
COLLABORATIVE PROJECTS (preliminary studies) ‐ Ultrastructure of hESC and iPSC derived cardiomyocytes ‐ collaborative project with Prof. Ofer Binah from the Department of Physiology; Ruth and Bruce Rappaport Faculty of Medicine, Technion Israel Institute of Technology, Haifa, Israel ‐ Zebra fish heart regeneration ‐collaborative project with Christopher Antos, Ph.D. laboratory Center for Regenerative Therapies; Technische Universitaet Dresden; Germany ‐ Engineered heart tissue ‐ collaborative project with Prof. Changyong Wang from the Tissue Engineering Research Center, Academy of Military Medical Sciences, Beijing, China ‐ Telocytes in human heart pathology ‐ collaborative project with Prof. Shengshou Hu from the Center for Cardiovascular Regenerative Medicine, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing, China FUTURE DIRECTIONS
Infrastructure investment plan and strategy Correlative Microscopy at cryo‐temperatures Integrated or correlative microscopy attempts to combine multidimensional information from complementary techniques to bridge the various resolution gaps and thus to be able to integrate structural information gathered from multiple levels of the biological hierarchy into one common framework. Cryo‐fluorescence microscopy for example can be exploited to navigate the cellular landscapes for features of interest before zooming in on these areas by cryo‐electron tomography. It offers an independent and unambiguous confirmation of the identity of the investigated features. For correlative studies we aim to acquire a cryo‐holder and cryo‐ultramicrotome. We also envisage the acquisition of a state‐of‐art confocal laser scanning microscope with 3‐5 channels for simultaneous detection of multiple markers in multi‐labelling protocols. Research focus
Cellular and molecular determinants of heart regeneration
Stem cell therapy for cardiac diseases has been started before an intrinsic regenerative capacity of heart to be proved
and accepted. The dogma that mammalian heart is terminally differentiated organ has been challenged by the reports
of few types of resident cardiac stem or progenitor cells. Moreover, a new type of interstitial cell – telocyte - has
been described in the adult heart and one important role seems to be nursing stem cells and progenitors in the
cardiac stem cell niches. We plan to study the cellular and developmental biology of cardiac stem niches and their
involvement in cardiac renewal considering that basic mechanisms governing its physiology are still unknown. By
extensive ultrastructural investigation (electron tomography included), confocal microscopy and miRNAs detection
we plan a basic research of telocytes and cardiac stem cell niches in normal, ageing and diseased mammalian heart.
We also will run a comparative study of regeneration in mammalian and zebrafish (known to have high regenerative
capacity) injured hearts. We will try to answer major question: there are one or more types of cardiac stem cell;
which cells are mandatory for cardiac renewal; which factors are most important in stem cell differentiation; how
newly formed cardiomyocytes are integrated in contractile myocardium; how all these are challenged in diseased
heart? All these questions must be answered before an effective cell therapy could be envisaged.
18
ULTRASTRUCTURAL PATHOLOGY LABORATORY DEVELOPMENT PLAN I. RESEARCH AREAS
1. Basic research in fundamental mechanisms of cardiac regeneration - from stem cell to heart tissue
 The identification and classification of somatic stem and progenitor cells that reside in the adult heart.
 The definition of heterocellular networks that direct stem cells toward a cardiac fate.
 Comparison of specific cardiac and hematopoietic stem/ progenitor cells by correlative microscopy
(fluorescent and ultrastructural analyses).
 The establishment of fate-mapping strategies to define the contribution of telocytes and selected
stem/progenitor cell populations to the cardiac lineage during development and after myocardial
injury.
2. Structural and molecular characterization of intercellular communication in the interstitial space
 Spatial structure in intercellular interactions - electron tomography of classical junctions
 Molecular and structural characterization of ‘atypical’ hetero-cellular junctions in the interstitial
space by correlative microscopy
2. Collaborative projects
 Specialized assistance for the research projects involving electron microscopy / tomography (research
design and technical expertise): virus ultrastructure and virus -cell interaction; nanoparticles
interaction with human cells; induced pluripotent stem cells characterization and tissue integration, etc.
 development of ongoing international collaborative projects
II. HUMAN RESOURCES DEVELOPMENT
 Researcher (junior or senior post-doc) for data collection, computer vision & image processing for
electron tomography of cells and macromolecules
 Electron Microscopy Research Technician to oversee the laboratory's facilities; to assist and train
visitor researchers
III. INFRASTRUCTURE DEVELOPMENT FOR CORRELATIVE MICROSCOPY
 confocal laser scanning microscope
 cryo-holder for TECNAI BioTwin
 cryo-ultramicrotome
19
TEAM 5 PROTEOMIC-BIOMARKERS
Team coordinator: CS2 Dr. Cristiana Tanase,
I. The quality of the results of the research activity
Research focus: Advanced proteomics: in discovery and application of proteomics biomarkers.
“Core concept”: application of multiple proteomics technologies/platforms in “biomarker driven”
research in diagnostics and pharmacology. Research areas:
- biomarker discovery and application as powerful diagnostics instruments in cancer,
- proteomics biomarkers in pharmacological research
- proteomics in the evaluation of environmental risks for human health,
- integration in European Research Area and medical services for biochemistry analyses.
Approach:
Multidisciplinary approach, based on integrating proteomics research with other key fields, generating
specific investigation platforms, addressing biomarker research in diagnostics, pharmacology and
toxicology. To apply in clinical diagnostics, pharmacotoxicology the know-how of our research team in
proteomics, pathology, immunology, cellular and molecular biology, bioinformatics. Key technologies
integrated: SELDI-TOF-MS, 2D-DIGE electrophoresis, multiplex-xMAP analysis, protein microarrays,
ELISA, cell-cultures.
Goals:
o To provide and translate research results in the field of biomarkers discovery (mainly in cancer)
into clinical applications; transferring towards hospitals, Ministry of Health and Health
Insurance House of technologies/protocols and/or services for the determination of biomarkers.
o To expand and integrate biomarkers research to other disease areas that may benefit from the use
of biomarkers in diagnostics, patient stratification, monitoring, etc.
o To collaborate within consortia joining institutions with complementary expertise and
multidisciplinary teams to apply individual and/or panels of biomarkers in the fields of molecular
diagnostics, pharmacology and risk assessment.
o Preparedness for the integration in European Research Area
Expertise: proteomics techniques (mass spectrometry, electrophoresis, multiplex-analysis,
immunoassays, cell cultres, (immuno)-toxicology, implementation of techniques for biomarker detection
(cytokines, chemokines, growth factors, signal transduction molecules, protein profiling, nucleic acids),
bioinformatics. The state-of-the-art technology available in these laboratories and the expertise of the
team allow complex, interdisciplinary studies
Research projects:
The Proteomics team is involved in 3 international ongoing projects in cancer research and drug
testing: CF14 - POS CCE 2.1.2, 152 “Proteomics technologies for cancer biomarkers discovery”; CF11 bilateral cooperation Romania- China;, “Biomarker discovery in digestive tract cancer and skin melanoma
using proteomic approaches” and CF16 - FP7- PIRSES-GA-2008-230816 “Natural Antidiabetic and
Antihypertensive Drugs. The team was involved in 19 national projects, as follows: cancer 7,
pharmacotoxicology 6, risk assessment 6, networking – 3.
Scientifc outcomes:
The results materialized in 15 articles, out of which 10 are published in journals with non-zero
relative Article Influence Score (cumulative score: 17,70719), 6 books/book-chapters at international
publishers, over 30 presentations at prestigious international conferences with ISI indexed abstract books,
of which 7 as invited speakers, 1 certified patent and 2 submitted patents.
Relevant results:
(1) The role of Caveolin-1 in cancer progression: we contributed to the annotation of this gene in
cancer pathology
(2) Evaluation of specific markers for proliferation, apoptosis and angiogenesis in cancers
(3) Key signalling molecules and the main microRNAs in pituitary and, digestive tumours
respectively.
(4) Importance of immune markers in the diagnosis, prognosis and therapy monitoring of cutaneous
melanoma.
(5) Integration of proteomic biomarkers with in vitro models for the assessment of safety and
efficacy of new potential drugs
20
Strategic scientific objectives and directions
The major target of the group is to enhance its performance in biomarkers studies, by continuing to
develop its output in proteomics biomarkers research. As established priorities for the medium and long
term, the team identifies:
a) Diagnostic “omics” – development of complex, high performance biomarker based tools with
application in the fields of diagnostics, monitoring, therapy optimization and personalization for
cancer, to foster the transfer the approach in other major diseases that will benefit of similar
instruments.
- Application of “Omics” technologies for high-throughput biomarker studies
- Signal transduction and other regulatory mechanisms in cancer and cancer stem cells
- Systems medicine approaches in biomarker discovery (integration of proteomics, miRNomics,
interactomics)
- Biomarker panels for early diagnostics, optimized and personalized therapies; integration of
proteomic, miRNA and other biomarkers.
- Development of “customized” detection instruments
- Translational research: transfer of protocols in clinical units
b) Pharmaco”omics” – profiling induced modulation of expression and activation key regulatory
molecules (signal transducers, miRNAs, cytokines) addressing oriented and personalized therapies.
Creating a “biomarker driven platform” and validated experimental models for the safety and efficacy
assessment in pharmacology and toxicology
II. Quality of human resources
The group comprizes 8 senior scientists, 5 young scientists (including 3 Ph.D. students), and 3 lab
technicians, with and involvement of 6,05 (scientists) and 2,8 (technicians) full time equivalents. Due to
the complementarities of basic and advanced trainings, the group is inter-disciplinary, covering expertizes
in medicine, molecular biology, proteomics, bioinformatics, toxicology, immunology. The group has an
average age of 37.7 years.
Name
Title
Involvment* Name
Role
Involvment*
Cristiana Tanase
MD, PhD, CS2 0.60
Mihail Hinescu
MD,
PHD, 0.1
CS1
Radu Albulescu
Biochemist,
0.25
Monica Neagu
Biochem,
0.25
PhD, CS1
PhD, CS2
Stefan
MD, PhD,
0.50
Mircea Leabu
Chem., Ph.D., 0.30
Constantinescu
Prof.
CS2
Ionela Daniela
Chemist, PhD 0.70
Carolina
Biochem.,
0.25
Popescu
student, CS
Constantin
PhD, CS3
Elena Codrici
Biologist, PhD 0.80
Irina Radu
Technician
0.80
student, CS3
Lucian Albulescu
Biochemist,
0.70
Nicoleta
Technician
1.00
PhD student
Constantin
Simona Mihai
MD, RA
1.00
Nicuta Lopazan
Technician
1.00
Alina-Ionela Nita
Economist,
0.80
RA
*see the structure of Research Teams
Continuous education and training: An estimated 6 persons/month spent in the last years for training,
covering advanced laboratory techniques in proteomics, bioinformatics etc. Examples of training
courses: International course Advanced Proteomics, St.George’s Medical Biomics Centre, University of
London, Protein Protein Interaction, European Institute of Bioinformatics, Cambridge, England (2011),
SELDI-TOF-Advanced Training Course, Biorad-Paris (2010), ProteinChip SELDI-Basic Training
Course, USA (2008), Luminex IS 2.3 Basic Training and Luminex Fundamental Assay Techniques,
Oosterhout, The Netherlands, 2D electrophoresis training, Prague, Protein Arrays for Biomarker
Discovery and Protein Expression Profiling and Profiling Kinases for Disease Biomarker and Drug
Target Discovery, Amsterdam (2007). Bursaries: FEBS 2010 Gothenburg, Sweden (2 persons), FEBS
2011, Turin, Italy (2 persons)
21
Exchanges of Personnel: PIRSES-GA-2008-230816 (CF16 Ongoing
Mobilities senior scientists: Invited speakers & Chairmen – Molecular Diagnostics Europe, 2011,
London; Cancer Proteomics, 2010, Berlin; Molecular Diagnostics Europe, 2010, Hannover; European
biomarkers Congress, 2010, Florence; Proteomics Europe Congress, 2009, Barcelona; BIT Life Sciences’
and Molecular Diagnostics, 2009, Beijing
III. Quality of Infrastructure and degree of exploitation
Based on a coherent policy, the group established and maintained a relevant rate of development of
research infrastructure, that parallels the progression of its scientific outcome. Thus, the group established
capabilities of research in proteomics that allows multiple, complementary investigations, such as de novo
discovery of novel biomarkers (supported by Mass spectrometric, bidimensional electrophoresis and
DIGE platforms), multiplex quantitative assays by fluorescence (Luminex xMAP and Luminex xTAG)
for cytokines, growth factors, hormones, signal transductors, miRNA, etc). Also, other techniques such as
ELISA assays, Western blot, on chip electrophoresis and cell culture assays are available and applied by
the group.
Domain
Equipment
Level of exploitation Year of purchase
2D electrophoresis
25%
2007
2D-DIGE (Typhoon 9000)
<25%
2011
SELDI-ToF-MS
75%
2008
Western blotting
25-50%
2007
Proteomics
Multiplex xMAP® technology
100%
2007
Protein microarray
< 25%
2010
Complete ELISA lines
100%
2007
MiniVIDAS
100%
2007
HITACHI 912
100%
2005
xCELLigence
25%
2010
In vitro
Complete unit for cell culture
75%
2007-2009
assays
Microplate Multimode Detector, Anthos 75%
2009
Zenyth 3100
Most of the existing equipment is purchased and installed after 2007, and based on functionally proven
“state of the art” technologies for both hardware and software components. Validation of the results
obtained using the key equipments (thus, of whole “operating chains” for each specific application) is a
standard procedure of the group. Interlaboratory comparisons (where such schemes are available) or staff
exchange/visits in other laboratories (such as at Biorad labs in Malverne, US, or in “Biomics Center” at
St. George’s University, London, UK
While mostly used (60% of the functioning time) for the projects coordinated by the senior members of
the group, the research infrastructure was also used in projects conducted by other groups, in which
individual members of the groups were involved (ca. 30%), while a quota was reserved also for activities
involving visiting scientists (e.g. in the NAAN project, during 2010-2011, when 5 scientists visited and
worked for 1 month each in the laboratory.
International collaboration – Biomics Medical Centre, Saint George’s University of London; Beijing
Institute of Genomics, Chinese Academy of Science and BioRad Proteomic Research Center, Malvern,
USA and in the FP7 project NAAN – Graz University, University of Lecce, Sekem Egypt..
Recognition at national and international level:
The group members are presently involved in international and national activities, as follows:
- National Expert in The member State Group of Innovative Medicines Initiative – Joint Undertaking
(IMI-JU), Expert in the Program Committee FP7 Health of the EC, Expert evaluators for the FP7 (themes
Health - 2010, 2011 calls, NMP - 2011 call), Expert evaluators for the programme “EuroNanoMed”,
(2010, 2011 calls), Expert-evaluators for National Plan 2 and for CNCSIS competitions.
- Board member of journals: Recent Patents on Biomarkers; Bentham Science Publisher Ltd.), Journal of
Immunoassay and Immunochemistry
- Peer reviewers for ISI indexed journals: International Journal of Cancer; Future Oncology; Acta
Endocrinologica; Recent Patents on Biomarkers; Journal of Immunoassay and Immunochemistry;
Medical Principles and Practice Toxicology in Vitro, Materials Science & Engineering B, JMPR
Medicinal Plants Research, Roumanian Biotechnology Letters.
22
TEAM 6 - IMMUNOMODULATION-IMMUNODIAGNOSIS
The “Immunomodulation-Immunodiagnosis” team is composed by 11 graduated
members with different specialties, and 4 auxiliaries, as follows:
1. Ursaciuc Cornel - MD, PhD, senior researcher 2, chief of Immunology Department, head of
the team – 80%
2. Neagu Monica - biochemist, PhD, senior researcher 2, chief of Immunobiology Laboratory –
30%
3. Manda Gina - biophysicist, PhD, senior researcher 2, chief of Radiobiology Laboratory –
30%
4. Ciotaru Dan - biologist, senior researcher 3, chief of Immunopathology Laboratory – 50%
5. Constantin Carolina - biochemist, PhD, senior researcher 3 – 30%
6. Surcel Mihaela - chemist, senior researcher 3 – 70%
7. Huica Radu - MD, Drd, researcher – 50%
8. Dobre Maria - biologist, researcher – 60%
9. Munteanu Adriana - biochemist, researcher – 100%
10. Neagoe Ionela - biologist, researcher – 30%
11. Pirvu Ioana - chemist, assistant researcher – 100%
12. Sorca Silvia - nurse – 100%
13. Caralicea Mariana - nurse – 30%
14. Pisica Mariana - technician – 30%
15. Dumitrascu Georgiana - medicine student, technician – 30%
Previous activity:
The main directions of research activity during last 5 years implied diverse pathology,
addressing directly, or participating in the development of topics such as:
 Immunomodulatory factors of tumor development
 Immune markers in melanoma
 Tumor immunogenomics
 Predictive biomarkers in inflammatory rheumatic diseases
 Therapeutic potential of environmental factors from salines and caves
 Innovative immunotherapies in tumors and autoimmune diseases
 Markers for diagnosis and prognosis of non-viral hepatitis
 Pannels of markers applicable in personalized medicine
 Cytokines and immunomodulation
This activity consisted in 5 original projects (developed as project coordinator) and 9
partner projects (developed as partner project responsive) with the above themes, funded by
CEEX and PN2 national research programs, as follows: F29-F37, F45-F47, F80, F114, CF5 (see
Annex 1). Besides, the team co-operated with other teams in “Victor Babeş” institute (Cellular
pathology, Molecular diagnosis technology transfer, Genetics, Drug development & toxicology,
Assay development) and other research groups in “Carol Davila” University of Medicine and
Pharmacy, as supplier of testing services.
The results of scientific activity can be measured, besides the projects applications, in a
list of 22 publications, like this: 5 ISI articles, 4 books or book chapters and 13 other relevant
publications (see Annex 2).
The team is also the contact associated partner in an infrastructure FP7 project:
- FP7 Capacities/2007 – “Biobanking and Biomolecular Resources Research
Infrastructure” (BBMRI) – European Commission Grant Agreement Number 212111 Associate Partner Project responsive Cornel Ursaciuc – 2008-2012
23
The team also was/is involved in several scientific service contracts:
- Immunofluorescence evaluation of test slides prepared for diagnosis of different serum
autoantibodies cathegories. “Tody Laboratories” SRL Bucharest, 2006-2009
- Experimental model and serum determinations in order to quantify the Simvastatin
effects on systemic inflammation, subclinical heart modifications, and early atherosclerosis in
rheumatoid arthritis. “Cantacuzino” Clinical Hospital Bucharest, 2007-2008
- Evaluation of cellular immune status by lymphocyte immunophenotyping and fagoburst
test in children with humoral immunodeficiency. IOMC Bucharest, 2007-2011, in progress
- Flow cytometry evaluation of irradiation and flavonoid effects on cell cycle and
apoptosis – data aquisition. „Carol Davila” University of medicine and Pharmacy Bucharest,
2010-2011, in progress
A constant activity and tradition of this group was performing of humoral and cellular
immunodiagnostic tests and hematology tests as currently medical services practised in the
“Victor Babeş” Institute's Diagnosis Center.
Members of the group also participate in sustaining the Cytometry Unit,
Immunogenomics Unit, Biobank and Microbiology Laboratory.
At a present, the team is involved as part of the work group in 5 “Victor Babeş”
institute’s project proposals and as partner project responsive in other 5 applications by other
institutions at the 2011 partner projects call of PN2 national research progamme.
Personnel:
The group includes various specialists covering the whole area of the biomedical
domains (2 medical doctors, 3 biologists, 3 biochemists, 2 chemists, 1 biophysicist). The mean
age of the graduated personnel is ~45 years, therefore a partial team rejuvenation is necessary for
the next period. We have in view physicians and biochemists, but this is dependent on the
financial support of the group during the future time. All the actual staff are good professionals
both in laboratory methods and theoretical knowledge and in the same time they are able to
perform teaching activity for learning students in immunology and immuno-detection. Part of the
staff are involved in formation activities developed through immunology courses organised by
Romanian Society of Immunology or human resources projects.
The team personnel policy is focused on stimulation of research creativity in order to
obtain competitive scientific results. Otherwise the members of the team have attended training
courses with the aim of updating their level of scientific information:
- Workshop of Fundamental Immunology , Bucharest, Romania, April 2007
- Immune system: genes, receptors and regulation, Hvar, Croatia, 2007
- Molecular biology in diagnosis and epidemiology of infectious diseases. INCDMI
”Cantacuzino” Bucharest, Romania, 2007
- The Course Molecular Diagnostics. The Erasmus Postgraduate School Molecular Medicine,
Rotterdam, 2007.
- Training in real time PCR at TATAA Biocenter, Prague, 2008
- The Advanced BD FACSCanto™ II and BD FACSDiva™ 6 Training, “Victor Babeş”
Institute, Bucharest, Romania, June 15-18 2009
- Epigenetics and new therapies in cancer. ESO-CNIO, Madrid, Spain, May 2007
- Cell culture Seminar, Bucharest, Romania, 2009
- Operator Training on BD FACSCanto II and BD FACSDiva 6.1.2, Heidelberg, Germany, June
2009
- 6th European Course on Clinical Cytometry, Valencia, Spain, September 2010
- 1st EFIS-EJI Intensive Educational Course in Clinical Immunology, Centre de Recherche des
Cordelieres, Paris, France, December 1-4 2010.
- Autumn Days of Cytometry, Bucharest, Romania, October 2011
24
Equipments:
The team has performance equipment, and is able to perform a lot of top methodologies
in view of accomplish the diverse scientific directions mentioned above. These equipments are
located in several units which the team are responsible with: Immunobiology, Immunopathology,
Immunoproteomics, Citometry, Immunogenomics, Immunomodulators. A list of purchased
equipments are presented in Annex 3.
One of the main intension is to upgrade the Immunogenomics unit endowment with a
hybridization unit for the gene microarray platform, a new bioanalyser for DNA/RNA evaluation
and a supplementary laminar flow hood for nucleic acid extraction. Besides, a refrigerated
centrifuge and a small autoclave unit are necessary for Immunopathology.
Perspectives:
Short- and medium term projects will most likely include acknowledged investigative
techniques applied in research or clinical immunological studies: flow cytometry,
immunofluorescence, serology, cell culture. Besides, additional investigative techniques such as
PCR-array and next-generation sequencing are considered to be developed as an enlargement of
immunogenomics branch of laboratory activity.
The team will continue several tools of the actual research and also will activate in the
diagnosis activity, formation area and biobanking, as:
- new scientific projects funded by national programs
- co-participants as team work in institute’s proposals
- partner institution in consortium projects
- service contracts in research or clinical immunology – every kind of immunological
investigation performed for beneficiaries from scientific or clinical area.
- continued diagnostic work pay activity in institute’s Diagnostic Center
- endowment possibilities reached by structural European projects (POS)
As constant preoccupations in the research activity will remain:
- autoimmune diseases and their diagnosis
- tumor immunogenomics
- cytokine modulation of cultured cells
- serum biomarkers in melanoma and other malignant tumors
Other directions will be approached in connection to the results that will be obtained at
the partner projects applied to the PN2 national program, whose outcome will appear in spring
2012.
Besides, our team will encourage internal and international collaborations (FP projects,
bilateral co-operations, service contracts, clinical trials) which implyes the laboratory as a data
supplier and scientific consulting collaborator. These will represent a supplementary funds
sources for scientific work and researchers mobilities.
The above mentioned options will serve also as data sources used for meeting
presentations, scientific articles or books.
25
TEAM 7 - GENOMICS AND GENETIC DIAGNOSIS
Research interests
genetics of neuropsychiatric disorders associated with intellectual disabilities (ID). Multiple genetic
anomalies are associated with ID phenotypes, and new data are reported at a high rate. However, the
underlying genetic mechanisms are seldom clear. Currently, we focus on the identification of genetic
aberrations associated with ID at genomic and cytogenetic level with the aim to further the knowledge
regarding the etiopathogeny of complex neuropsychiatric disorders.
characterization of genetic and epigenetic alterations underlying the initiation and progression of cancer
(hematologic neoplasms and solid tumors); cancer immunogenomics. The genetic abnormalities
associated with cancer bear a well-known clinical significance and are of outmost interest in the
diagnosis, risk assessment, and management of the disease; therefore, their characterization and
understanding brings multiple benefits for the medical science, and ultimately for the patients.
Epigenetic studies, due to the reversible nature of epigenetic modifications, have a great potential not
only for biomarkers discovery relevant for cancer detection and prognosis, but most importantly for
identifying new targets for epigenetic drugs. Presently, we focus on the detection and characterization
of these abnormalities at gene, chromosome and genome level, aiming to identify new aberrations and
their contribution to the malignant phenotype. We also started to investigate the epigenetic changes in
breast cancer cell lines, aiming to extend these studies to breast cancer diagnosis and prognosis.
Our experimental approaches cover molecular cytogenetics, molecular genetics, cell and molecular
biology, and genomics (immunogenomics included). The team uses state-of–the art infrastructure for
several applications, including: optical microscopy (transmitted light and epifluorescence), molecular
cytogenetic techniques (e.g. FISH, mBAND), „in house” preparation of FISH probes using bacterial
artificial chromosomes, PCR based techniques, and high-resolution genome wide approaches (DNA
microarray). The research strategy is based on using unique patient material obtained through close
collaboration with Clinical Departments from major tertiary hospitals. Additionally, our team makes
constant efforts to preserve the valuable biological material that it is not used in ongoing studies, for
future in depth or large scale approaches.
Research grants
Since 2007, the team participated in 6 national projects and 2 bilateral cooperation projects (RomaniaFrance). The projects were granted within national peer-reviewed competitions.
No.
Research area
Research grant ID
no/ funding(Euro)
1
Genetics research of neuropsychiatric disorders
F1, F10/
386,237
2
Hematologic neoplasms genetic characterization
F6, F96/ 213,635
3
Human foetal hepatic stem cells characterization
F3/68,181
4
Infrastructure project for genetic research
F4/425,452
5
Bilateral Cooperation projects – knowledge and expertise sharing in the F2, CF1/ Funding
field of genetic/genomic defects of complex pediatric neuropsychiatric for bilateral visits
disorders
Infrastructure
Up-grading the research infrastructure with state-of-the-art technologies has been the mainstay of our team
strategy. A major step forward was made with a grant dedicated to enhancing research capabilities (20072009). Consequently, microarray (Agilent platform) and molecular genetics facilities were developed; the
light microscopy facility was greatly improved by the acquisition of a motorized optical microscope.
Presently, the team uses the following research equipments/facilities:
Microarray facility: Agilent DNA Microarray C Scanner with Surescan, Agilent Scan Control, Feature
extraction and Genomic Workbench Software; Agilent BioAnalyzer 2100;
Molecular genetics facility: Corbett Real Time PCR, Corbett Palm Cycler PCR;
Nucleic acid isolation and manipulation facility: chemical hoods, water baths, cooling
centrifuges,spectrophotometer, BioDoc transilluminator, freezers, ultrafreezers;
Cell culture facility: safety cabinets, incubators;
Light microscopy facility: motorized Axio Z1 Zeiss microscope with examination in transmitted light bright
field and epifluorescence, equipped with high resolution monochrome cooled CCD camera; Metafer and
26
Ikaros software for scanning, automatic detection of metaphases, capture, karyotyping, FISH (standard, MFISH, m-BAND, Q- FISH, CGH).
Research team
Name
Laboratory
Aurora Arghir, CS3
Medical Genetics Laboratory
Andreea Tutulan-Cunita, CS 1)**
*
Sorina Papuc, CS
Magdalena Budisteanu, MD
Cornel Ursaciuc, CS2
Immunopathology Laboratory
Monica Dobre, CS
Radu Huica, CS3 *
Sevinci Pop, CS3 2)**
Cell Biology Laboratory
Valeriu Cismasiu, CS 3)**
Georgeta Cardos, CS 4)**
Pathology Department
Gisela Gaina CS3
Maria Neagu*, CS
Angela Petrescu, CS 3
IT Department
Agripina Lungeanu, CS1***
Ioana Borcan, Technician
Marioara Cristea, Technician
* PhD students
**1) Specialization in molecular biology and genetics (Master degree 2000-2002/ PhD 2002-2005 - Hiroshima
University, Japan; Postoctoral fellowship 2005-2006 - Manchester University, UK); 2) Doctoral fellowship: 20002002, Postoctoral fellowship: 2003-2008 University of Illinois at Urbana-Champaign, USA, Department of Cell and
Developmental Biology; 3) Specialization in molecular biology, protein biochemistry and cell biology within several
postdoctoral fellowships (Albany Medical Center, NY, USA; Stem Cell Center, Lund, Sweden; Wetherall Institute
of Molecular Medicine, Oxford, UK); 4) Doctoral fellowship: 2004-2008, Hamburg University, Germany.
*** Retired - 2010.04
Research / clinical collaboration
The research activities, focused on detecting clinically relevant genetic lesions in neuropsychiatric
disorders and cancer, add data to the body of knowledge and ultimately contribute to the improvement of
clinical management of the patients. This is achieved through close collaboration with groups from
clinical hospitals such as: “Prof. Dr Alexandru Obregia” Clinical Hospital of Psychiatry, Coltea Clinical
Hospital (Hematology Department), Emergency University Hospital Bucharest (Hematology
Department), other oncological departments.
In order to address the technological challenge of analyzing large amount of data generated by genomewide high through-put technologies (microarrays) our team recently established collaboration with a
bioinformatics/biostatistics group from “Al. I Cuza” University, Faculty of Computer Sciences.
Several international collaboration were established by our team with research groups from France
(Professor Jean-Michel Dupont team, Cochin Hospital, Paris), Germany (Professor Evelin Schrock
team, Carl Gustav Carus Faculty of Medicine, Dresden) and United States (Professor Kenneth Kosik
team, University of California, Santa Barbara). A long term collaboration has been developed with
Cochin Hospital through two bilateral cooperation (Romania-France) projects focused on genetic defects
underlying intellectual disabilities and autistic spectrum disorders, respectively. This collaboration
materialized in mutual visits and short-term staff trainings, exchange of technologies, and joint
publications. The cooperation with professor Kosik team focus on understanding the pathogenetic
mechanisms of neuropsychiatric disorders, such as microdeletion syndromes; it involves the generation of
induced pluripotent stem cells (obtained in professor Kosik lab from fibroblasts) with subsequent
differentiation into neuronal tissue in order to obtain a more accurate disease model. A recent
collaboration was established with Yolanda de Diego team from Carlos Haya Hospital, Malaga, Spain
on the topic of fragile X etiopathogeny and the perspective of antioxidative therapeutic strategy.
Publications: A49, A72, A93, A94, A95.
Training: 1. Advanced training in aCGH (Institute of Clinical Genetics, Carl Gustav Carus Faculty of
Medicine, Dresden, Germany) – 1 researcher; 2. Oligonucleotide Microarray training (Institute G.
Roussy, Paris, France) – 3 researchers; 3. Clinical Cytogenetics Course (European Cytogeneticists
Association, South Tyrol, Italy) - 1 researcher; 4. Courses of Dysmorphology 2006, 2008, 2009, 2011
27
(European Society of Human Genetics, Rome, Italy) – 1 researcher; 5. European Course on Genetics of
Mental Retardation (European Society of Human Genetics, Braga, Portugal) - 1 researcher; 6. Workshops
on laboratory accreditation, internal quality control and external quality assessment, (EuroGentest, Nice,
France and Berlin, Germany) – 2 researchers; 7. Project Management Course (AFPA, Bucharest,
Romania) – 2 researchers.
International project proposals: FP7 – ERC Starting grants, 2010 Call “Integrative analysis of DNA
methylation, miRNA and gene expression profiles in idiopathic autism” (not financed); FP7 – ERC
Starting grants, 2011 Call “Genome-wide analysis of drug responsiveness in attention deficit
hyperactive disorder” (under evaluation); FP7 - HEALTH-2012- INNOVATION-1 „A phase III
randomized double blind multicenter clinical trial to investigate the efficacy of the combination of
ascorbic acid and tocopherol versus placebo for the treatment of cognitive deficit and behavioral
problems in the fragile X syndrome. Functional relevance of the RAC1-GTPase as molecular target”
(under evaluation).
Perspectives - Since 2007, our team advanced from molecular cytogenetic and molecular genetic studies
of individual genes/ chromosomal regions towards genome-wide molecular strategies.
The envisaged strategy of our team consist of identification and characterization of constitutional and
acquired genomic abnormalities, by genome-wide molecular approaches and use of the resources
generated by the Human Genome Project.
Understanding the biological bases of complex neuropsychiatric disorders is one of the most
important medical challenges of the present time and most probably of the next years. Expanding
usage of whole-genome molecular strategies has given a new quality to the analysis of these disorders.
Thus, array-CGH can detect discrete copy-number changes and allows the definition of new clinical
syndromes. Additionally, the possibility to combine CNVs data with genotyping information (SNP
arrays) provides the advantage of adding high resolution and investigation of copy-neutral events. Our
team is involved in searching for clinically relevant genes/genomic regions through array-CGH studies,
with further interest in extending our investigations at transcriptomic, epigenetic/epigenomic as
well as model organism level. The collaboration with professor Kosik lab will allow us to participate
in the development of more accurate disease models for complex neuropsychiatric disorders such as
microdeletion syndromes.
Cancer is caused by genetic and epigenetic anomalies that alter the balance among cell proliferation,
survival, and differentiation. However, only a fraction of the cancer-associated genetic aberrations
have been identified. In this context, our team aims at identifying novel oncogenic lesions
relevant for pathogenesis as well as studying the cooperation between genetic events. On short and
medium term, our team intends to expand the area of interest toward the investigation of acquired
uniparental disomies, alterations of gene expression and epigenetic changes in relation with
disease onset and progression in cancer (hematologic malignancies, breast cancer, other solid tumors).
Identifying and characterizing immunogenomic markers and assessing their diagnostic and prognostic
impact is yet another objective of our team. A long term goal is to bring new evidence on the role of
microenvironment in tumorigenesis, with special emphasis on mesenchimal stem cells. By
analyzing the genetic/genomic abnormalities of tumor-associated cell populations, new factors at
interplay in the oncogenic process can be identified and new potential treatment targets be found.
While array-CGH is already in place and functioning in our institute, the expansion towards the
genome-wide investigation of copy-neutral alterations (e.g. uniparental disomy, loss of heterozygosity)
demands an upgrade of the existing technology (Agilent High Resolution Scanner Upgrade License License for C Scanner upgrade – allows CGH+SNP array as well as scanning of high definition
platforms).
For adding higher resolution – up to nucleotide level - to the genome-wide investigations, our team
foresees developing partnerships with neighbouring institutions (e.g. Carol Davila University of
Medicine and Pharmacy and Institute of Cellular Biology and Pathology “N. Simionescu”), as well as
foreign institution. By joining expertise and sharing resources (next-generation sequencing
technologies available in the above mentioned institutions) our team intend to expand its whole
genome molecular strategies towards hypothesis independent mutation screening in cancer and
complex neuropsychiatric disorders.
In order to exploit both the research and diagnostic potential of array-CGH, its introduction in
clinical laboratory as a diagnostic tool is one of our goals. In close collaboration with a bioinformatics
team as well as with clinical groups, we intend to device a reliable and sensitive method for the
investigation of genomic structural variation in several human pathologies.
28
TEAM 8 - NEUROSCIENCES
Evolution of the human resources
Neuroscience Team composition between 2007 and 2011
Period
Name
2007-2011 Bogdan O. Popescu, MD, PhD, senior researcher, neurologist
2008-2011 Emilia Manole, PhD, senior researcher, biologist
2007-2011 Paula Gratiela Chelu, MD
2007-2008 Raluca Colesniuc, researcher, biologist
2007-2011 Catalin Manole, MD, research assistant, PhD student
2007-2011 Dragos Cretoiu, MD, research assistant, PhD student
2009-2011 Mihnea Nicolescu, MD, research assistant, PhD student
2008-2010 Oana Romanitan, MD, PhD
2011
Laura Suciu, MD, PhD
2007-2011 Mariana Nicolae, technician
Team position
Head of laboratory
Member
Member
Member
Member
Member
Member
Member
Member
Member
In 2007-2009, two students were carried out their research work for the undergraduate thesis:
Radu Stoica and Maria Tuineag (on effects of antiepileptic drugs on neuronal apoptosis and
neuroplasticity).
In 2009-2011, Ana Maria Enciu, MD, PhD student, worked in the laboratory for her PhD thesis
(Molecular mechanisms in neurodegeneration).
Our team we have a balanced distribution of age (mean age of 38, 6 years) and sex (6 women, 4
men). Members of the team have different complementary expertise (both medicine and
biology). Dr. Bogdan O. Popescu has graduated a PhD in Neuroscience in Karolinska Institute,
Stockholm and works as well as a senior neurologist.
The dynamic of the research subjects and directions
Models and techniques currently used in laboratory are: cell cultures, animal models of
neurodegeneration, light microscopy (phase contrast, fluorescence, confocal), histology,
histochemistry, immunohistochemistry, Western blot, ELISA, PCR.
The main research directions of the laboratory during this period were: studies of the trophic
factor receptors expression in the central and peripheral nervous system, the distribution and
expression of the tight junction proteins in the brain, new neurodegeneration models relevant to
Alzheimer disease and Parkinson disease. Since 2008, we performed as well studies of
distribution and expression of different proteins involved in skeletal muscle and peripheral nerve
pathology.
The most important achievements
Grants
For the period 2007-2010 we obtained two research grants (PN II - Partnerships, 2007):
1. PN II 41-013 /2007: Expression and function of tight junction proteins – a study in
experimental models and in patients with dementia. Project Director (National Institute of
Pathology “Victor Babes”): Bogdan O. Popescu, MD, PhD; Partners: International Center
of Biodynamics, Bucharest and Bucharest Emergency University Hospital.
2. PN II 61-019/2007: Implementation and optimization of the technological process of
obtaining active therapeutic serum F (ab ') 2 against highly bacterial and viral pathogenic
agents. Project Director (National Institute for Microbiology and Immunology “Dr. I.
Cantacuzino”): Nadia Bucurenci, PhD; Project Director of the Partner 1 (National
Institute of Pathology “Victor Babes”): Bogdan O. Popescu, MD, PhD; Partner 2:
University of Medicine and Pharmacy “Carol Davila”, Bucharest.
For the period 2008-2011 we obtained two research grants (PN II - Partnerships, 2008):
1. PN II 42-124/2008: Molecular analysis of the proteins implicated in the main types of
peripheral neuropathies with a demyelinating component. Project Director (National
29
Institute of Pathology “Victor Babes”): Bogdan O. Popescu, MD, PhD; Partners:
University of Bucharest and Bucharest Emergency University Hospital.
2. PN II 42-133/2008: Cellular and molecular bases of muscle ageing. Project Director
(University of Bucharest): Emilia Manole, PhD; Project Director of the Partner 1
(National Institute of Pathology “Victor Babes”): Bogdan O. Popescu, MD, PhD; Partner
2: Colentina University Hospital.
Education
Starting with 2011, we contribute to the TDM project (Education of medical personnel - new
technologies for health system/molecular diagnosis-POSDRU/81/3.2/S/58819, coordinated by
our Institute. The Western blot expert from the Proteomics Section is Dr. Emilia Manole and the
Direct Immunofluorescence expert from the Molecular Imaging Section is Dr. Laura Suciu, both
from our laboratory.
Dr. Bogdan O. Popescu authored one textbook for medical students and two book chapters for
clinical neuroscience specialists. He is the Executive Editor of Romanian Journal of Neurology
(CNCSIS B+) and Secretary General of the Romanian Society of Neurology as well. Dr. Bogdan
O. Popescu served during this period as reviewer for several ISI journals and international grant
evaluations (European Science Foundation and American Alzheimer Association).
Publications
Since 2007, the members of our laboratory published 20 ISI full-text articles and 8 full-text
articles indexed in other international data bases and contributed with more than 50 lectures and
posters in international and national scientific meetings.
National scientific awards
 Dr. Bogdan O. Popescu - ‘Victor Babeş’ award of the Romanian Academy for medical
research activity – 2007.
 Dr. Bogdan O. Popescu - ‘Science and Art National Foundation Award of Excellence for
research in the field of Neuroscience and Neuropathology – 2010.
List of ISI publications (2007-2011)
1. Negreanu L, Popescu BO, Babiuc RD, Ene A, Bajenaru OA, Smarandache GC., Duodopa
infusion treatment: a point of view from the gastroenterologist., J Gastrointestin Liver Dis. 2011
Sep; 20 (3): 325-7.
2. Enciu AM, Constantinescu SN, Popescu LM, Mureşanu DF, Popescu BO., Neurobiology of
vascular dementia., J Aging Res. 2011; 2011: 401604. Epub 2011 Aug 17.
3. Enciu AM, Nicolescu MI, Manole CG, Mureşanu DF, Popescu LM, Popescu BO,
Neuroregeneration in neurodegenerative disorders, BMC Neurol. 2011 Jun 23; 11:75.
4. Enciu AM, Popescu BO, Gheorghisan-Galateanu A., MicroRNAs in brain development and
degeneration, Mol Biol Rep. 2011 Jun 5. [Epub ahead of print]
5. Popescu LM, Manole E, Serboiu CS, Manole CG, Suciu LC, Gherghiceanu M, Popescu BO,
Identification of telocytes in skeletal muscle interstitium: implication for muscle regeneration., J
Cell Mol Med. 2011 Jun; 15(6): 1379-92.
6. Popescu LM, Gherghiceanu M, Suciu LC, Manole CG, Hinescu ME., Telocytes and putative
stem cells in the lungs: electron microscopy, electron tomography and laser scanning microscopy,
Cell Tissue Res. 2011 Sep; 345(3): 391-403.
7. Hinescu ME, Gherghiceanu M, Suciu L, Popescu LM., Telocytes in pleura: two- and threedimensional imaging by transmission electron microscopy, Cell Tissue Res. 2011 Feb; 343(2):
389-97.
8. Nicolae L, Iacob G, Poparda M, Popescu BO, Case report. Gelastic seizures in a patient with
right gyrus cinguli astrocytoma, J Med Life. 2010 Oct-Dec; 3(4): 433-6.
9. Negreanu LM, Popescu BO, Babiuc RD, Ene A, Andronescu D, Băjenaru OA., Cutting the
Gordian knot: the blockage of the jejunal tube, a rare complication of Duodopa infusion
treatment, J Med Life. 2010 Apr-Jun; 3(2): 191-2.Erratum in: J Med Life. 2011 Jan-Mar; 4(1): 7
p following 123.
30
10. Bajenaru O, Tiu C, Moessler H, Antochi F, Muresanu D, Popescu BO, Novak P., Efficacy and
safety of Cerebrolysin in patients with hemorrhagic stroke, J Med Life. 2010 Apr-Jun; 3(2): 13743.
11. Dumitriu A, Popescu BO., Placebo effects in neurological diseases, J Med Life. 2010 Apr-Jun;
3(2): 114-21. Review.
12. Jianu DC, Muresanu DF, Bajenaru O, Popescu BO, Deme SM, Moessler H, Meinzingen SZ,
Petrica L., Cerebrolysin adjuvant treatment in Broca's aphasics following first acute ischemic
stroke of the left middle cerebral artery., J Med Life. 2010 Jul-Sep; 3(3): 297-307. Erratum in: J
Med Life. 2011 Jan-Mar; 4(1): 7 p following 123.
13. Muresanu DF, Alvarez XA, Moessler H, Novak PH, Stan A, Buzoianu A, Bajenaru O, Popescu
BO., Persistence of the effects of Cerebrolysin on cognition and qEEG slowing in vascular
dementia patients: results of a 3-month extension study, J Neurol Sci. 2010 Dec 15; 299(1-2):
179-83.
14. Hort J, O'Brien JT, Gainotti G, Pirttila T, Popescu BO, Rektorova I, Sorbi S, Scheltens P; EFNS
Scientist Panel on Dementia, EFNS guidelines for the diagnosis and management of Alzheimer's
disease, Eur J Neurol. 2010 Oct; 17(10): 1236-48.
15. Negreanu L, Popescu BO., Evaluation of successful treatment in achalasia with timed barium
esophagogram: revisiting an old friend, J Med Life. 2010 Jan-Mar; 3(1): 64-6.
16. Ghitoiu A, Rusu EC, Slăvoacă D, Aigyul E, Popescu BO., A hypertensive patient with multiple
intracerebral hemorrhages due to brain metastases., J Med Life. 2009 Oct-Dec; 2(4): 437-9.
17. Romanitan MO, Popescu BO, Spulber S, Băjenaru O, Popescu LM, Winblad B, Bogdanovic N.,
Altered expression of claudin family proteins in Alzheimer's disease and vascular dementia
brains, J Cell Mol Med. 2010 May;14(5):1088-100.
18. Suciu L, Nicolescu MI, Popescu LM., Cardiac telocytes: serial dynamic images in cell culture, J
Cell Mol Med. 2010 Nov; 14(11): 2687-92
19. Suciu L, Popescu LM, Gherghiceanu M, Regalia T, Nicolescu MI, Hinescu ME, FaussonePellegrini MS., Telocytes in human term placenta: morphology and phenotype, Cells Tissues
Organs. 2010; 192(5): 325-39.
20. Popescu BO, Toescu EC, Popescu LM, Bajenaru O, Muresanu DF, Schultzberg M, Bogdanovic
N., Blood-brain barrier alterations in ageing and dementia, J Neurol Sci. 2009 Aug 15; 283(1-2):
99-106. Review.
21. Cretoiu SM, Cretoiu D, Suciu L, Popescu LM., Interstitial Cajal-like cells of human Fallopian
tube express estrogen and progesterone receptors., J Mol Histol. 2009 Oct; 40(5-6): 387-94.
22. Suciu L, Popescu LM, Regalia T, Ardelean A, Manole CG., Epicardium: interstitial Cajal-like
cells (ICLC) highlighted by immunofluorescence, J Cell Mol Med. 2009 Apr; 13(4): 771-7.
23. Muresanu DF, Alvarez XA, Moessler H, Buia M, Stan A, Pintea D, Moldovan F, Popescu BO.,
A pilot study to evaluate the effects of Cerebrolysin on cognition and qEEG in vascular dementia:
cognitive improvement correlates with qEEG acceleration, J Neurol Sci. 2008 Apr 15; 267(1-2):
112-9
24. Popescu BO., Still debating a cause and diagnostic criteria for Alzheimer's disease, J Cell Mol
Med. 2007 Nov-Dec; 11(6): 1225-6
25. Oprica M, Hjorth E, Spulber S, Popescu BO, Ankarcrona M, Winblad B, Schultzberg M.,
Studies on brain volume, Alzheimer-related proteins and cytokines in mice with chronic
overexpression of IL-1 receptor antagonist., J Cell Mol Med. 2007 Jul-Aug; 11(4): 810-25.
26. Romanitan MO, Popescu BO, Winblad B, Bajenaru OA, Bogdanovic N., Occludin is
overexpressed in Alzheimer's disease and vascular dementia., J Cell Mol Med. 2007 May-Jun;
11(3): 569-79.
27. Cowburn RF, Popescu BO, Ankarcrona M, Dehvari N, Cedazo-Minguez A., Presenilin-mediated
signal transduction., Physiol Behav. 2007 Sep 10; 92(1-2): 93-7 Review.
28. Suciu L, Popescu LM, Gherghiceanu M., Human placenta: de visu demonstration of interstitial
Cajal-like cells, J Cell Mol Med. 2007 May-Jun; 11(3): 590-7.
31
TEAM 9 - DRUG DEVELOPMENT AND TOXICOLOGY
Team coordinator: CS2 Dr. Gina Manda, Head of Radiobiology Laboratory
Mission: applied biomedical research in drug development, medicinal chemistry and (immuno)toxicology
Research focus:
 Identification of pathology-relevant drug targets and action mechanism
 medicinal chemistry: screening & hits to leads (biological activity assessment)
o development and implementation of in vitro assays with particular end-points, for selecting
candidate drugs, for defining their action mechanism and safety profile
o assistance of partner research teams (organic and analytic chemists, pharmacists) in designing
and producing new compounds (libraries of synthetic or natural compounds), potentially
efficient in cancer, autoimmune, cardiovascular diseases etc.
o preclinical studies for these new compounds, both in vitro and in vivo in animal models
(selection of candidate compounds by in vitro studies, in vivo proof-of-action studies using
relevant animal models, in vitro and in vivo mechanistic studies and toxicological screening)

immunotoxicology
o implementation of advanced screening methods for characterizing the biological impact of
xenobiotics on human health (medicines, drugs of abuse, heavy metals, mycotoxins, ionizing
radiation etc)
Research topics:
1. Pathologic mechanisms and drug targets
No
Biomarkers and molecular targets in cardiovascular diseases
Prof. Dr. Elena MOLDOVEANU and CS3 Dr. Daciana MARTA
Immune targets in the treatment of rheumatoid arthritis
1.2
CS2 Dr. Gina MANDA
Integrin – extracellular matrix interactions in cell motility and metastasis
1.3
CS2 Dr. Mircea LEABU
2. Drug development
1.1
No
2.1
Novel nucleoside analogs for cancer therapy - CS2 Dr. Gina MANDA
Complexes of physiological, divalent transitional metals for cancer
therapy - CS2 Dr. Mircea LEABU
2.3 Functionalized nutrients - CS2 Cristiana TANASE
Alternative medicines: bioactive phytochemicals
2.4
CS2 Dr. Cristiana TANASE, CS2 Dr. Mircea LEABU
3. (Immuno)Toxicology
2.2
No
3.1
3.2
3.3
3.4
Immunotoxicology of mycotoxins: impact on the food chain and
development of counteracting agents (study in porcine model)
CS2 Dr. Gina MANDA
Heavy metals profiling in biological samples and food
CS3 Vasile PREOTEASA
Radiation-induced genotoxicity
CS3 Vasile PREOTEASA
Geno- and hepatotoxicity of xenobiotics
CS3 Bogdan Marinescu
Research contracts /
budget (Euro)
F49, F50, F51 / 336.381
F77 / 73.070
F108 / 100.676
budget (Euro)
F81, F82 / 188.653
F106 / 92.053
F41 /125.000
F38, F52, F53, F105 /
347.780
budget (Euro)
F76, F79 / 48.646
Nucleu Programme
Contract with Cernavoda
nuclear power plant
Contract with
S.C. ALCEDO S.R.L.
32
Publications
2. Pathologic mechanisms and drug targets: 7 ISI publications A13, A17, A20, A24, A36, A83,
A96, O15, 096, 098, 099 (cumulated rAIS 12,32 citations 18).
- biomarkers in vascular inflammatory pathology (pulmonary hypertension, heart failure and
antiphospholipid syndrome). In heart failure we add contribution to the recognition of LpPLA2 as a
marker of oxidative stress and vascular inflammation.
- in rheumatoid arthritis we highlighted particular networks of adaptive and innate immunity (B
lymphocytes-NK cells) and a key role of peripheral monocytes in mirroring disease outcome; we
showed that low doses of immunosuppressive agents (leflunomide) may exert a pro-inflammatory
action on pathologic monocytes.
3. Drug development: 12 ISI publications A12, A31, A41, A43, A57, A65, A67, A84, A88, A89,
A90, O28 (cumulated rAIS 5.69, citatons 12), 1 book chapter (O28).
22 new nucleoside analogs from 3 structural classes were investigated in vitro and 3 lead compounds with
anti-cancer activity were identified. A preliminary in vivo study on laboratory animals proved their
activity and showed a convenient toxicological profile for 2 of the lead compounds. Compounds will be
further developed, including by preliminary results-guided structural changes. We will investigate their
therapeutic application as radiosensitizers in cancer therapy and we will submit at least 1 national patent.
4. Toxicology: 3 ISI publications A71, A82, A92 (cumulated rAIS 2.43), 1 book chapter (O19), 071.
Accreditation of immunotoxicology assays according to SR EN ISO 15189 (Biochemistry Laboratory) F56/181.512 Euro
Research team
Name
Laboratory
Name
Laboratory
CS2 Gina Manda 1)**
CS Ionela Victoria Neagoe
CS3 Vasile Preoteasa
CS2 Mircea Leabu 2)**
3)
CS3 Sevinci Pop **
CS Andreea Oana Urs*
Cristina Mariana Niculite*
Mihaela Andreea Mocanu
Catalin Filipescu
CS3 Daciana Marta
Team coordinator, Head of
Radiobiology Laboratory.
Scientific secretary
Radiobiology Laboratory
Head of Nuclear Unit
Head of Cell Biology
Laboratory
Ultrastructural Pathology
Laboratory
CS3 Ciotaru Dan
CS3 Mihaela Surcel
CS3 Radu Huica*
CS2 Cristiana Tanase
CS1 Radu Albulescu
CS Daniela Popescu*
AS Lucian Albulescu*
CS Alina Grigore
CS Bogdan Marinescu
Ultrastructural Pathology
CS Gheorghita Isvoranu*
Laboratory
Technicians: Daniela Geogia, Sanda Sima, Cristina Vlad, Maria Paraschiv
CS3 Gabriela Catalin*
Head of Immunopathology
Laboratory
Head of Biochemistry
Laboratory
Biochemistry Laboratory
Head of Animal Care Unit,
Laboratory of experimental
models
Animal Care Unit, Laboratory
of experimental models
* 7 PhD students
** 1) Specialization in rheumatoid arthritis (idiotype/anti-idiotype network), INSERM, Paris, France; 2)
Post-doctoral fellow and visiting researcher, 2000-2005, University of Western Ontario, London, ON,
Canada; 3)Doctoral fellowship: 2000-2002, Postoctoral fellowship: 2003-2008 University of Illinois at
Urbana-Champaign, USA, Department of Cell and Developmental Biology.
o To apply in medicinal chemistry and toxicology the know-how of our research team in human
pathology, immunology, cellular and molecular biology
- cell cultures, flow cytometry (immune phenotyping, cellular activation and proliferation, generation
of reactive oxygen species, apoptosis), protein multiplexing (soluble factors profile, signal
transduction pathways), fluorescence and confocal microscopy, transmission electron microscopy,
impedance measurements for cell adhesion, radio-assays (biodistribution, cell functions), heavy
metals profiling in biological samples by ICP-MS etc.
o To collaborate within consortia joining institutions with complementary expertise and
multidisciplinary teams (biologists, biochemists, medical doctors, chemists, pharmacists, biophysicists
etc) for developing innovative compounds designed and produced by Romanian R&D teams and for
implementing new investigation methodologies for Drug development and Immunotoxicology.
33
Available infrastructure
Cell culture facilities - class II flow hoods, CO2 incubators, deep freezing unit; Cytometry unit - 2 flow
cytometers, fluorescence microscopes, confocal microscope; VIDEOCELL unit - xCELLigence system,
BioStation IM, fluorescence microscope, electrophoresis and western blot equipment, gas chromatograph;
Proteomics unit (Luminex platform); Histology unit; Animal Care Unit: 2008-2011 accreditation
according to SR EN ISO 17025 for B12 and B39 assays; Nuclear unit (certified by CNCAN): Canberra
Packard beta-counter, Sorcerer imager; ICP-MS unit: ICP-MS ULTRAMASS 700.
Infrastructure upgrading in the Animal Care Unit and in the Cellular Biology Laboratory (877.466
Euro from INFRAS and Capacities Programmes)
Collaborations
National collaborations: 1. University of Medicine and Pharmacy “Carol Davila”, Bucharest; 2.
University of Bucharest; 3. R&D Institute for Chemical-Pharmaceutical Research, Bucharest; 3. Institute
for Nuclear Physics and Engineering “Horia Hulubei”, Magurele; 4. Institute of Biology and Animal
Nutrition, Balotesti (IBNA); Private companies: Mecro Systems and SC Biotehnos SA; International
collaborations: 1. Universite Libre de Bruxelles, Laboratory of Pharmaceutical Chemistry, Brussels,
Belgium; 2. University of Thessaly, School of Health Sciences, Biochemistry Department, Greece.
1. FP7-NMP-2009-SMALL-3: Nanotechnologies for medical implants (not financed); 2.
IMI_Call_2009_5: Aberrant Immunity in Chronic Immune-mediated Diseases” (not financed); 3. FP7Low Dose Research towards Multidisciplinary Integration (DoReMi): Epidemiologic study - systemic
effects of low dose exposure to uranium on the immune status (not financed); 4. FP7 call Health-2013
Phosphodiesterases, inflammation endothelium dysfunction: a route to cardiovascular disease.
Coordinator: Laboratory of Pharmaceutical Chemistry, Faculty of Pharmacy, Universite Libre de
Bruxelles, , Brussels, Belgium (in preparation)
Development plan
I. New research areas: 1. Theranostics – Development of an integrated platform with fully 3D X-ray
tomographic and microbeam fluorescence / luminescence guidance capabilities and of particular
nanoparticles for simultaneous imaging and radiation therapy of tumors. Collaboration with the R&D
Institute of for Laser, Plasma and Radiation Physics
2. Implementation of „omics” and high-throughput technologies for drug development and
immunotoxicology (in collaboration with other teams from the institute)
3. Systemic effects of low dose exposure to ionizing radiation on the immune response; integration of the
Radiobiology Laboratory in the European research network DoReMi (Integrating Low Dose Research)
II. Patent submission for newly developed therapeutic compounds and design/screening of new ones
III. Accreditation of new experimental methods relevant for drug development and toxicology
IV. Introduction of new investigations in the Diagnosis Center (trace microelement profiling)
V. Enlargement of national and international collaborations within IMI, FP7 etc - Focus on private
companies
VI. Increased exploitation of existent infrastructure, to be translated in an increased number of
publications in ISI ranked journals. Development of Radiobiology laboratory’s specific infrastructure.
11 project proposals at the national 2011 Call “Partnerships – Collaborative projects of applied research” (under
evaluation)
Pathologic mechanisms and drug targets: 1. Fibro‐inflammatory biomarkers of early myocardial
remodeling in heart failure patients with preserved ventricular ejection fraction–Elena MOLDOVEANU;
2. Optimization of autologous bone marrow stem cell therapy in patients with ischemic heart failure–
Elena MOLDOVEANU 3. The importance of phosphodiesterase and thrombin in pulmonary fibrotic
diseases‐ possible future therapeutic targets – Daciana MARTA. Drug development: 1. New chemicals
and natural compounds for improving radiotherapy in cancer – radiosensitization, internal irradiation and
radioprotection-Gina MANDA; 2. Translation of coordinative chemistry in biology and therapy. Complex
compounds of physiological cations with thiosemicarbazones and tiocarbohyrdazones as antitumoral
and/or antibacterial drugs–Mircea LEABU; 3. New epigenetic drug – DHA – in breast cancer therapy and
biomarkers discovery–Sevinci POP; 4. Magnetic Nanostructures for Targeted Therapy – Sevinci POP; 5.
Tool design for pharmacovigilance. Prediction of life-threatening angioedema for patients treated with
renin-angiotensin system inhibitors and/or glyptins–Mircea LEABU. Toxicology: 1. Impact of feed
co‐contamination and mitigating solutions to increase feed safety, animal health and food quality – Gina
MANDA.
34
TEAM 10 - Assay Development and Alternative Testing
Team members
First and Last Name
Monica Neagu
Carolina Constantin
Dan Ciotaru
Mihaela Surcel
Cristiana Tanase
Radu Albulescu
Elena Codrici
Daniela Popescu
Lucian Albulescu
Emilia Manole
Gheorghita Izvoranu
Alina Nita
Angela Petrescu
TOTAL
Role
Team Leader
Member
Member
Member
Member
Member
Member
Member
Member
Member
Member
Member
Member
Involvement
0,50
0,50
0,3
0,3
0,30
0,15
0,2
0,25
0,2
0,3
0,3
0,2
0,5
4,00
Technical personnel
First and Last Name
Mariana Caralicea
Georgiana Dumitrascu
Mariana Pisica
Irina Radu
Laurentiu Anghelache
TOTAL
Role
Technician
Technician
Technician
Technician
Technician
Involvement
0.5
1
0.5
0,2
0.5
2.7
In the last 4 years our team was involved in the following research directions: Immune-based
assay development and Cell-based assays.
In the domain Immune-based assay development we are currently involved in innovative
immune-detection in infectious diseases through the cooperation with University of Tubingen
and University of Athens (NATO SfP 982838/2007). We have established a complete new
testing approach for early bacterial infection testing. The innovative assay development consists
in designing both new antibodies specific for C-terminal prothymosine peptide and
fluorescence-immunoassay detection methods. In the last 4 years we have published the
relation between the in vitro and in vivo bacterial infection and the release of a C-terminal
prothymosine peptide. In biological fluids the developed immune-assay can detect in
experimental mouse models the mentioned peptide as early as 2 hours post-infection. The
possibility to detect as early as 2 hours post-infection a marker by means of chemiluminometric
testing is a major breakthrough in this domain. The future of these results will be the
development of an original international patent and the technological transfer for developing an
easy-to-perform, sensitive and quick test for bio-terrorist attack. The research funds obtained in
the framework of this NATO SfP 982838/2007 project are 300,000 Euros for Romania.
In the domain of Cell-based assays development we are developing efficient
technology/workflow for drug potency identification with emphasis on nano-drugs for controlled
delivery. In the last 4 years through a long lasting collaboration with the National Institute for
35
Chemistry Bucharest we have developed several classes of photosensitizing compounds with
anti-tumoral effect. Target potency, cytotoxicity, and metabolic liabilities were evaluated and the
selected compounds entered the animal models testing. In animal models vivo efficacy was
tested when reliable models were available. The hit-to-lead process induced a close
collaboration between Assay Development Team and other teams, such as Proteomic and
Biomarkers and Drug Development. When a promising lead series has been identified, besides
publications, several composition-of-matter patents were accomplished and the internationally
recognized value was recognized by awards. The patent OSIM Mr. 00489/25.06.2008 entitled
“Tetra-sulphonated porphyrin application for producing a dermatologic therapy –
photosensitizer” received Gold medal at Brussels Innova 2008, Special Prize of Rudy Demotte,
Minister President of the Walloon Government, Gold medal at The 37th International Exhibition
of Inventions of Geneva 2009 and Special Prize of the Ministry of Education of Rusia, 2009;
Gold medal at The International Fair for Innovation, Moscow, 2009. Recognition of the last 10
years long-standing work performed by us in this domain, resides in our affiliation to the
international networks: COST D39 Metallo-Drug Design & Action (2006-2010); COST TD1002
European network on applications …in NanoMedicine and Life Sciences (2011-2015). In the
cell-based assay development we have developed specific equipments for cell imaging that
were subject for patent OSIM A/00351/2019 / 21.04.2010 entitled “Equipment and procedure for
microwave irradiation in in vitro models with concomitant registration of biological behaviour in a
fluorescence microscope”. Developing several nationally granted projects the funds obtained by
the research team in this domain heaves up to 850,000 Euros. Through cooperation with the
University of Lisbon and Technical Institute of Portalegre, we have developed several classes of
nano-compounds intended to be intracellular trackers in tumor cells and indicators for minimum
residual disease in blood circulation. The project has already a patent OSIM Nr. ROBOPI2/2009 entitled “Tetrapirolic compound asymmetrically substituted – synthesis and
biological evaluation” and until 2012 it will develop several others in the cell-based assay for
efficient drug delivery. The future of this research direction lays in increasing the nano-drug
specificity with emphasis in targeting tumour receptors and tissue markers for not only a
controlled delivery in time but as well in space. Through this MNT-ERA –NET 7050/2010 project
the team was financed with 115,000 Euros. The involved laboratories are affiliated to the
National Platform for Nanomedicine.
Related to the domain of cell-based assay the team leader is an active member of the
Commission for Advanced Therapies – European Medicine Agency and one team member is
evaluator for EuroNanoMed Projects.
Members of the Assay Development team are actively pear-reviewing for the following
scientific journals: Pigments and Dyes, Photochemical and Photobiological Sciences, Patents in
Biomarkers, Archives of Gerontology and Geriatrics, International Journal of Photoenergy,
Romanian Biotechnological Letters, Romanian Archives of Microbiology and Immunology,
Materials Science and Engineering B, Journal of Clinical Laboratory Analysis, International
Journal of Nanomedicine. Two team members are also members of the editorial boards and
invited editors for hot scientific topics in scientific journals such as Journal of
Immunoassay&Immunochemistry and Recent Patents on Biomarkers.
The personnel dynamics in our team is remarkable, namely we have a mean age of 40.2
years and equilibrated between young researchers and more experienced senior researchers
and have hosted through the international collaborations several PhD students that fulfilled their
36
thesis in subject developed by the research team. The knowledge up-grading of our team is
continuous such as, each of the team members attends international courses on up-to-date
technologies. In 2007 the following courses and training stages were attended - Profiling
Kinases and Phospho-Sites with Antibody-Based Methods for Disease Biomarker; Drug Target
Discovery and Protein Arrays for Biomarker Discovery and Protein Expression Profiling,
Amsterdam, Holland; In 2008: Principles and applications of microfluidics in the life sciences,
Microfabrication technologies, Barcelona, Spain; ProteinChip SELDI-ToF MS Training Course,
Malvern, USA; In vivo confocal microscopy training, Mavig, Bucharest, Romania; In 2009 - Flow
Cytometer FACS CANTO II Training Course, Heildelberg, Germany; In 2010-2011
xCELLIgence Users Meeting, Munchen, Germany, Intensive Educational Course in Clinical
Immunology, Centre de Recherche des Cordelieres, Paris; 2011 Course “Characterizing and
applying physiologically-based pharmacokinetic models in risk assessment” – WHO Paris.
We have constant young Bachelor of Science degree personnel that perform their diplomas in
the framework of the mentioned domains. The Laboratories involved are constantly hosting in
PhD students or post-doctoral fellows in the framework of the mentioned international
collaborations.
The laboratories involved in the Assay development team have high-throughput technology
with specialized software. Although the equipment is recently purchased (2007-2011) de level of
exploitation by the described team is over 75%.
Domain
Proteomics
Cellular physiology
testing
Equipment
2D electrophoresis
2D-DIGE (Typhoon 9000)
SELDI-ToF-MS
Western blotting
Multiplex xMAP® technology
Protein microarray
Complete ELISA lines
xCELLigence impedance measurement equipment
Confocal microscopy
Varioskan Multimode Reader for time-resolved
chemiluminescence
Complete unit for cell culture manipulation and storage
fluorescence,
Appended publications to the team: A7, A27, A29, A31, A41, A49, A51, A55, A84, A91, A98
with a cumulated rAIS of 8,864 and 20 ISI citations
Projects that funded the mentioned research directions CF3, CF4, CF12, CF13 have a total
budget of 600,000 Euro and F27, F28, F29, F35, F43, F45, F46, F109, F110, F112, F113
850,000 Euro
37
4. Representative project
38
TELOCYTES - A NEW TYPE OF INTERSTITIAL CELLS
‘Victor Babeş’ National Institute of Pathology, Bucharest, Romania
TELOCYTE - A NEW TYPE OF INTERSTITIAL CELL
http://www.telocytes.com
KEY PERSONS INVOLVED IN TEOCYTE PROJECT
Team leader: Acad. Laurentiu M. POPESCU
Senior researchers: Hinescu ME, Ardeleanu C, Mandache E
PostDoc: Gherghiceanu M, Suciu LC, Popescu BO, Cismaşiu V, Cretoius S
PhD students: Manole CG, Nicolescu MI, Cretoiu D
INTERNATIONAL COLLABORATION
• Prof. Maria-Simonetta Faussone-Pellegrini and Prof. Daniele Bani from Department of Anatomy,
Histology and Forensic Medicine, University of Florence, Italy
• Prof. Sawa Kostin from Max-Planck Institute for Heart and Lung Research, Franz Groedel Institute,
Bad Nauheim, Germany
• Prof. Shengshou Hu from the Center for Cardiovascular Regenerative Medicine, Peking Union
Medical College, Chinese Academy of Medical Sciences, Beijing, China
• Prof. Changyong Wang from the Tissue Engineering Research Center, Academy of Military Medical
Sciences, Beijing, China
TELOCYTES - A CASE OF SERENDIPITY: the winding way from Interstitial Cells of Cajal (ICC), via
Interstitial Cajal-Like Cells (ICLC) to TELOCYTES
The history of TC is recent since these cells have been discovered only a few years ago. However, the
growth of knowledge on TC has been exponential from the beginning and we already have much information.
TC were discovered in 2005 when L.M. Popescu’s group from Bucharest, Romania, described a new type of
cell that resides in the stroma of several organs, which became known as interstitial Cajal-like cells (ICLC).
This group named these cells ICLC because of their apparent similarity with the canonical gastrointestinal
interstitial cells of Cajal (ICC), the gut pacemaker cells. A few years later, in 2008, M.S. Faussone-Pellegrini
and her team from Florence, Italy, described ICLC in the muscle coat of the human gut and noticed they
consistently differed from the ICC in both ultrastructure and immunophenotype. In 2010, after confirming the
presence of this peculiar cell type in the stroma of many organs and characterized it by
immunohistochemistry and electron microscopy, the two groups agreed they were describing a ‘novel’ cell
type and that the name ICLC had to be changed with a more appropriate one. From then on, this novel cell
type became known as the telocyte.
1
TELOCYTES (TC) - NEW TYPE OF INTERSTITIAL CELLS WITH LONG PROLONGATIONS NAMED
TELOPODES (Tp).
MORPHOLOGY OF TELOCYTES
To characterize these cells, many different techniques have been used: in vitro, isolated cells in culture; in
situ, observation on fixed specimens; light and fluorescence microscopy; transmission electron microscopy;
scanning electron microscopy; electron tomography
All these techniques have shown that TC are cells with a small body and a variable number of Tp. The shape
of the cell body depends on the number of Tp and can be piriform/spindle/triangular/ stellate. The nucleus is
oval, with a moderately dense chromatin, and has no obvious nucleolus. The cytoplasm surrounding the
nucleus is scarce and contains a small Golgi apparatus, some mitochondria, and few cisternae of rough and
smooth endoplasmic reticulum. Telocytes are certainly defined by their ultrastructural features. Usually, a TC
has extremely long Tp with moniliform aspect generated by alternating podoms and podomers.
Telopodes have particular characteristics:
1. Number : 1–5/cell, usually 1-3
2. Branching: dichotomous pattern
3. Length: tens up to hundred micrometres
4. Aspect moniliform - podomeres alternating with podoms
5. Podomers - 50-100 nm thin segments; usually < 0.2 μm, below the resolving power of light
microscopy
6. Podoms - dilated segments accommodating mitochondria, ER and caveolae (‘Ca2+ release units’)
7. Connected each other by junctions form an interstitial network
Immunohistochemistry
To know the chemical code of TC is of fundamental importance since it allows their unequivocal identification
and also helps evaluate their size, shape, number, and, eventually, movements, migration, and pathological
changes. Although we made many reliable attempts testing an enormous variety of antibodies, a single
marker that can be considered specific for this cell type or, at least, specific for the TC of a given organ has
not been found. TC might show different immunohistochemical profiles among organs and even in the same
organ examined. However, at present, CD34 labeling remains the best available choice for TC identification,
possibly in combination with c-kit and vimentin labeling. Due to these important differences in TC
immunolabelling and since none of the markers tested are ‘specific’, we need to solve this issue and perform
further immunohistochemical techniques, including immunoelectron microscopy.
Distribution
TC have been found in a large variety of cavitary organs: [heart (endo-, myo-, and pericardium); stomach
and intestine, with mesentery; gallbladder; uterus and Fallopian tube] and non-cavitary organs [lungs and
pleura; pancreas (exocrine); mammary gland; placenta].
All the cells identified as TC were:
- organized in a 3D network, dispersed in the extracellular substance, and intermingled with resident
(fibroblasts, mast cells, adipocytes) and nonresident (macrophages, immune cells, granulocytes) cells
- localized at the connective border of various tissues (epithelial, muscular, and nerve tissues) lining them
and around blood vessels.
2
ROLES OF TELOCYTES
Several roles have been suggested for TC, most of which are believable and not mutually:
- key players in organ specific renewing (heart, lung, striated muscle) and could act as stromal support
cells for stem cells. Ultrastructural analysis proved that TCs cardiac network could integrate the
overall ‘information’ from vascular system (endothelial cells and pericytes), nervous system
(Schwann cells), immune system (macrophages, mast cells), interstitium (fibroblasts, extracellular
matrix), stem cells, progenitors and working cardiomyocytes. Generally, heterocellular contacts occur
by means of minute junctions (point contacts, nanocontacts and plane contacts) and the mean
intermembrane distance is often within the range of tens of nm (10-30 nm) which fits in the molecular
interactions domain. Our study showed that homotropic and heterotropic ultrastructural interactions of
TCs in adult heart form an integrative interstitial system. Possibly, TCs network assure physiological
coordination of multicellular signals, essential for stem cells (resident or circulating) decision to
proliferate, differentiate and mature into new cardiomyocytes or other cardiac cell types.
- sustain myocardial tissue organization in developing and adult heart
- in immune surveillance (stromal synapse with mononuclear cells, granulocytes, mast cells,
macrophages)
- TC could be mesenchymal stem cells (MSC); in vivo identity of MSC is still unknown (TC and MSC
are CD34+ cells)
- tensional integration of the tissue, considering their characteristic ultrastructure (extremely long and
contorted processes with intermediate filaments and microtubules parallel to the long axis of the cell,
attachment plaques connecting it to the extracellular matrix)
- in neurotransmission in the gut, possibly contributing to spread the slow waves generated by the ICC.
Even the TC are not fully characterized and their roles are speculative, recent studies showed that TC may
be involved in a few important pathologies:
- isolated atrial amyloidosis and atrial fibrillation;
- neoangiogenesis in cardiac recovery after experimental myocardial infarction;
- PEComas -perivascular epithelioid cell tumours;
- GISTs-gastro-intestinal and extra-gastrointestinal stromal tumours.
Funding from research grants: NUCLEU PN 06.26/2005-2008; CEEX 112/2006-2008; NUCLEU PN
PN09_33/2009-2011
FUTURE RESEARCH DIRECTIONS
-
Telocytes - specific markers
Telocytes - origin and lineage tracing
Role of telocytes in physiology and pathology
ANNEX 1 - Reprint of “Faussone-Pellegrini MS, Popescu LM (2011) TELOCYTES. BioMolecular Concepts
DOI: 10.1515/BMC.2011.039”
ANNEX 2 - Reprint of “Popescu LM, Faussone-Pellegrini MS (2010) TELOCYTES – a case of serendipity:
the winding way from Interstitial Cells of Cajal (ICC), via Interstitial Cajal-Like Cells (ICLC) to
TELOCYTES. J Cell Mol Med 14: 729–740 (24 ISI citations)
3
ONGOING PROJECT:
‘TELOCYTES IN HEART RENEWAL’
project coordinated by Acad. L.M. Popescu (348.837 euro)
IDEI-PCE 2011 competition for 3 years (2011-2014)
Stem cell therapy for cardiac diseases has been started before an intrinsic regenerative capacity of heart to
be proved and accepted. The dogma that mammalian heart is terminally differentiated organ has been
challenged by the reports of few types of resident cardiac stem or progenitor cells. Moreover, a new type of
interstitial cell – telocyte - has been described in the adult heart and one important role seems to be nursing
stem cells and progenitors in the cardiac stem cell niches. We plan to study the cellular and developmental
biology of cardiac stem niches and their involvement in cardiac renewal considering that basic mechanisms
governing its physiology are still unknown. By extensive ultrastructural investigation (electron tomography
included), confocal microscopy and miRNAs detection we plan a basic research of telocytes and cardiac
stem cell niches in normal, ageing and diseased mammalian heart. We also will run a comparative study of
regeneration in mammalian and zebrafish (known to have high regenerative capacity) injured hearts. We will
try to answer major question: there are one or more types of cardiac stem cell; which cells are mandatory for
cardiac renewal; which factors are most important in stem cell differentiation; how newly formed
cardiomyocytes are integrated in contractile myocardium; how all these are challenged in diseased heart? All
these questions must be answered before an effective cell therapy could be envisaged.
4
Visibility
5
Article in press - uncorrected proof
Page 2 of 16
BioMol Concepts, Vol. xx (2011), pp. xxx-xxx • Copyright by Walter de Gruyter • Berlin • Boston. DOI 10.1515/BMC.2011.039
Review
Telocytes
Maria-Simonetta Faussone Pellegrini1,* and
¸ M. Popescu2,3
Laurentiu
1
Section of Histology, Department of Anatomy, Histology
and Forensic Medicine, University of Florence, Viale G.
Pieraccini 6, I-50139 Florence, Italy
2
Q1:
Department of Cellular and Molecular Medicine ‘Carol
Please
Davila’ University of Medicine and Pharmacy, Bucharest,
supply the
Romania
postal code
3
Department of Advanced Studies, ‘Victor Babeş’ National
for affiliation
¸
Institute
of Pathology, 99-101 Splaiul Independentei,
2
RO-050096 Bucharest, Romania
* Corresponding author
e-mail: [email protected]
Abstract
Here, we review the history, morphology, immunohistochemical phenotype, and presumptive roles of a new type of
interstitial tissue cells, formerly called interstitial Cajal-like
cells (ICLC) and by 2010 named ‘telocytes’ (TC). Many
different techniques have been used to characterize TC and
provide their unequivocal identification: (i) in vitro, cultures
and isolated cells; (ii) in situ, fixed specimens examined by
light and fluorescence microscopy, transmission (TEM) and
scanning electron microscopy, and electron tomography.
TEM allowed sure identification and characterization of the
most peculiar feature of TC: the long, thin, and convoluted
prolongations named ‘telopodes’. An enormous variety of
antibodies have been tested, but presently none are reliable
to specifically label TC. TC have a mesenchymal origin and
are resident connective tissue (stromal) cells. Possible identification with ‘already identified’ stromal cell types (fibroblasts, fibrocytes, fibroblast-like cells, and mesenchymal
stromal cells) is discussed. We conclude that in adulthood,
most of the TC have the morphology of fibrocytes. Apparently, immunocytochemistry suggests that a variety of TC
populations showing different, likely organ-specific, immunophenotypes might exist. Several roles have been hypothesized for TC: mechanical roles, intercellular signaling,
guiding and nursing of immature cells during organogenesis,
and being themselves a pool of precursors for many of the
mesenchyme-derived cells in adulthood; however, none of
these roles have been proven yet. On the basis of the available data, we propose TC may be key players in organ regeneration and repair.
Keywords: fibroblasts, interstitial Cajal-like cells (ICLC),
interstitial cells of Cajal (ICC), mesenchymal cells,
telopodes.
Introduction: a short history
The history of telocytes (TC) is recent since these cells have
been discovered only a few years ago. However, the growth
of knowledge on TC has been exponential from the beginning and we already have much information. TC were discovered in 2005 when L.M. Popescu’s group from Bucharest,
Romania, described a new type of cell that resides in the
stroma of several organs (1–7), which became known as
interstitial Cajal-like cells (ICLC). This group named these
cells ICLC because of their apparent similarity with the
canonical gastrointestinal interstitial cells of Cajal (ICC), the
gut pacemaker cells (8–11). A few years later, in 2008, M.S.
Faussone-Pellegrini and her team from Florence, Italy,
described ICLC in the muscle coat of the human gut and
noticed they consistently differed from the ICC in both
ultrastructure and immunophenotype (12). In 2010, after
confirming the presence of this peculiar cell type in the stroma of many organs and characterized it by immunohistochemistry and electron microscopy, the two groups agreed
they were describing a ‘novel’ cell type and that the name
ICLC had to be changed with a more appropriate one (13).
From then on, this novel cell type became known as the
telocyte (13).
Rationale for the term ‘telocyte’
The interstitial tissue making up the stroma of an organ is
the connecting ‘device’ for the specific structures of the
organ and the resident connective tissue cells are usually
named ‘stromal cells’. However, according to some authors
and depending on the organ where these cells reside, they
have received a variety of other names: fibroblasts, fibrocytes, fibroblast-like cells, myofibroblasts, mesenchymal
cells, interstitial cells, and ICLC. Under transmission electron microscopic (TEM) examination, the cells formerly
called ICLC reveal all their characteristics that are unique,
unequivocal, and not yet described for any other cell type.
To avoid further confusion and to give a precise identity to
these cells, Bucharest’s team coined for them the term ‘telocyte’ wscell bearing long prolongations (13)x on the basis of
their most peculiar feature: the presence of prolongations that
are extremely long (tens to up to hundreds of micrometers,
as measured on TEM images), thin (mostly below 0.2 mm),
and with a moniliform aspect (Figures 1–5). The concept of
TC was promptly adopted by this group and several other
laboratories (14–24).
2011/0038
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short general
title/caption
for Figures 1
and 4
Page 3 of 16
2 M.-S.F. Pellegrini and L.M. Popescu
Figure 1 (A and B) CD34-immunoreactivity; submucosa of human stomach. (A) The CD34-positive cells (in brown) have a triangular or
ovoid body and a variable number of thin and long prolongations that give a stellate shape to the whole cell. These processes have knobs
along their length and a dichotomous branching. Bar, 20 mm. wWith permission from (12).x (B) A detail of the body and prolongations of
a CD34-positive cell. Bar, 15 mm. (C) Human nonpregnant myometrium in cell culture, day 3, first passage. Giemsa staining. One telocyte
establishing contact with a smooth muscle cell (myocyte) by a cell process (telopode) of about 65 mm long. Photographic composition of
four serial phase contrast images; original magnification, 40=. In the red rectangles, a higher magnification clearly shows the moniliform
aspect of the telopode; at least 40 specific dilations (podoms) of the telopode, interconnected by thin segments (podomeres), are visible in
a ‘beadlike’ fashion. Original magnification, 40= wWith permission from (3).x (D) Human resting mammary gland stroma, TEM. One
telopode, which appears very long and convoluted, with intercalated podomeres and podoms. Note the homocellular junctions marked by
red circles, as well as shed vesicles (SV, blue) and an exosome (violet). coll, collagen. wReproduced with permission from (1).x
Figure 2 TEM imaging.
(A) Rat mesentery. One TC with a small nucleated body and three long telopodes, all of them having a sinuous trajectory and forming an
interstitial complicated 3D network. (B) Human exocrine pancreas. The TC form with their typical long telopodes a network around the
acini.
Page 4 of 16
Telocytes 3
Figure 3 Electron tomography (thick section of about 300 nm)
showing nanostructures connecting one telopode with two adjacent
cardiomyocytes in adult mouse heart.
The bridging structures (encircled) are 10–15 nm and suggest a
molecular interaction between the telopode and the cardiomyocytes.
The dilated segment of this telopode contains a mitochondrion (m).
wReproduced with permission from (25).x
Morphology
To characterize these cells, many different techniques have
been used: (i) in vitro, isolated cells in culture (Figure 1C);
(ii) in situ, observation on fixed specimens; (iii) light (Figure
1A,B) and fluorescence microscopy (Figure 4A); (iv) transmission electron microscopy (Figures 1D, 2A,B, 4B–D and
5); (v) scanning electron microscopy; (vi) electron tomography (Figure 3).
All these techniques have shown that TC are cells with a
small body and a variable number of long prolongations
named telopodes (Tp). The shape of the cell body depends
on the number of Tp and can be piriform/spindle/triangular/
stellate (Figures 1A,B, 2A,B and 4A). The nucleus is oval,
with a moderately dense chromatin, and has no obvious
nucleolus. The cytoplasm surrounding the nucleus is scarce
and contains a small Golgi apparatus, some mitochondria,
and few cisternae of rough and smooth endoplasmic reticulum (Figure 2A,B). Average dimensions of the TC body are
9.3"3.2 mm (min. 6.3 mm; max. 16.4 mm). Mitochondria
represent 2% of cell body volume. Each TC can have
1–5 Tp. However, frequently only 2–3 Tp are observed on
a single section depending on site and angle of the section
since their three-dimensional (3D) convolutions prevent them
from being observed at their full length in a 2D thin section.
Convolutions of the Tp, however, are not always present and
have variable extent and complexity depending on the organ
where TC are located. The Tp moniliform aspect (Figure
1C,D) is due to an alternation of thin segments, podomeres
(whose caliber is below the resolving power of light microscopy, 0.1"0.05 mm; min. 0.003 mm, max. 0.24 mm) and
dilated segments, podoms, which accommodate mitochondria, rough and smooth endoplasmic reticulum, and
caveolae – the so-called Ca2q uptake/release units. Tp establish several types of homo- and heterocellular junctions (Figure 3) (25), release shed vesicles and exosomes (Figure 1D)
(26), have a dichotomous branching pattern forming a 3D
Figure 4 (A) Human stomach. Double CD34/c-kit labeling. CD34 positivity is represented in red and c-kit positivity in green. The CD34and c-kit-positive cells are often very close to each other but none of them are double labeled. Bar, 25 mm. (B and C) CD34 immunoelectrolabeling. (B) Mouse small intestine. CD34 positivity is present along the plasma membrane of a long, thin process of a TC, while ICC,
nerve fibers, and smooth muscle cells (SMC) are CD34 negative. Bar, 1 mm. (C) Mouse stomach. Detail of a CD34 immunoelectrolabeled
telopode. CD34 positivity clearly appears as electron-dense spherules regularly distributed on the telopode plasma membrane. Bar, 0.4 mm.
Page 5 of 16
Figure 5 Schema and electron micrograph illustrating the relation of several telocytes (blue) with a column of cardiomyocyte progenitors
(CMP, brown) in an epicardial stem cell niche of adult mouse.
The telopodes run parallel to the main axis of the CMP column and seem to establish the direction of development. wReproduced with
permission from (27).x
labyrinthine network (Figures 1A and 2A), and might show
adhesion plaques with the extracellular matrix (27).
Electron microscopy
Scanning electron microscopy provides good images of presumptive TC (13), and electron tomography provides images
of cell-to-cell contacts (25). However, TEM alone allows
sure identification of TC, evaluation of the cell-to-cell interrelationships, and a detailed description of the Tp.
Vital staining, cultures
In cell culture, TC shape and, in particular, Tp length can be
easily evaluated since in these conditions Tp are not convoluted (Figure 1C). Moreover, MitoTracker Green FM, a
lipophylic selective dye that becomes fluorescent once it
accumulates in the lipid environment of mitochondria, confirms these organelles are present in the TC body and at the
level of Tp dilations (5, 28). Immunohistochemistry performed on cultured or isolated TC from determined organs
does not always give the same results as those obtained on
the corresponding TC in situ.
Immunohistochemistry
To know the chemical code of TC is of fundamental importance since it allows their unequivocal identification and also
helps evaluate their size, shape, number, and, eventually,
movements, migration, and pathological changes. Unfortunately, although Bucharest’s group made many reliable
attempts in testing an enormous variety of antibodies (14,
29, 30), a single marker that can be considered specific for
this cell type or, at least, specific for the TC of a given organ
has not been found. Indeed, TC might show different immunohistochemical profiles among organs and even in the same
organ examined. In the chorial villi (14), some TC are c-kit
positive and some CD34 positive; all of the CD34-positive
TC express vimentin and caveolin-1, and some of them also
c-kit (14). In cultured cells from the same placental villi,
some TC are double positive for c-kit and iNOS and others
for c-kit and VEGF (14). Skeletal muscle TC are c-kit,
caveolin-1, and CD34 positive, and have been found to
secrete VEGF (18). In the mouse heart, most of the TC are
CD34 positive and a few are c-kit positive (31). Presently,
only one study, performed by Florence’s group on both light/
fluorescence and ultrastructural detection of CD34 and c-kit
in the human gut, is available (12). In this study, immuno-
Page 6 of 16
Telocytes 5
histochemistry combined with TEM allowed to put into light
that the enteric TC are CD34-positive and c-kit-negative
cells, and also to exclude ICC and TC as being the same cell
type (Figure 4A,B,D). Notably, the TC located in the mucosa
are neither CD34 nor c-kit positive (12). Briefly, CD34 labeling does not allow an unequivocal TC identification since it
does not label all TC, at least in some organs we.g., gut,
placenta, and striated muscle (12, 14, 18)x or during embryonic life we.g., in the heart (20) and gut (Faussone-Pellegrini,
personal observations)x. However, at present, CD34 labeling
remains the best available choice for TC identification, possibly in combination with c-kit and vimentin labeling.
In conclusion, due to these important differences in TC
immunolabeling and since none of the markers tested are
‘specific’, it would be desirable if more laboratories other
than those in Bucharest and Florence will study this issue
and perform further immunohistochemical techniques,
including immunoelectron microscopy.
Distribution
TC have been found in a large variety of cavitary and noncavitary organs (Table 1). Therefore, we would reasonably
conclude these cells are ubiquitous. All the cells identified
as TC were located within the connective tissue and could
be (i) organized in a 3D network, dispersed in the extracellular substance, and intermingled with resident (fibroblasts,
mast cells, adipocytes) and nonresident (macrophages,
immune cells, granulocytes) cells, or (2) at the connective
border of various tissues (epithelial, muscular, and nerve tissues) lining them and around blood vessels; these TC are
likely organized in a 2D network. Those located around
blood vessels presumably correspond to the adventitial cells
and those located around myenteric plexus ganglia and nerve
strands correspond to the covering cells (12, 38).
Possible TC identification with ‘already
identified’ stromal cell types
Usually, connective tissue cells are perceived as being mainly
(or even only) fibroblasts and/or fibrocytes. Importantly, it
has to be noted that across different countries and laboratories, there is great confusion between the terms fibroblast and
fibrocyte. In some European countries and outside of Europe,
only the term fibroblasts is used; however, two types of
Table 1 TC distribution.
Cavitary organs
Noncavitary organs
Heart (endo-, myo-, and pericardium)
(6, 16, 19, 20, 25) (26–31)
Stomach and intestine (12, 21)
Gallbladder (32)
Uterus (3, 33, 34)
Fallopian tube (5, 35)
Blood vessels (36)
Lung and pleura (17)
Exocrine pancreas (2)
Mammary gland (1)
Placenta (14)
Skeletal muscle (18)
Mesentery (37)
fibroblasts are recognized, the active and the quiescent fibroblasts. Conversely, in some other European countries (Italy,
Germany, Romania, etc.) both fibroblasts and fibrocytes are
recognized as distinct cell types. There is a general agreement that cells called fibroblasts correspond to the active
connective tissue cells involved in synthesis and organization
of extracellular components (ground substance and fibers).
These cells are, therefore, obviously present during development and whenever there is a need for renewal or repair
of extracellular components. The so-called fibrocytes conceivably correspond to the quiescent fibroblasts, which are
typical of connective tissues during adult life. The distinction
between fibrocytes and fibroblasts is based on their markedly
different ultrastructural features; these differences are commonly reported in histology textbooks and can be summarized as follows.
The (active) fibroblast body is large and pleomorphic; the
nucleus is typically euchromatic and has one to two nucleoli;
the Golgi complex is prominent; and the rough endoplasmic
reticulum is well developed (about 5–12% of cell volume).
Cell processes are few, short, and of large caliber, thus being
easily appreciable under a light microscope. These cells are
markedly different from TC, but some of the cells labeled
as ‘fibroblasts’ in the figures reported in histology textbooks
and other literature show the morphology of TC and not that
of active fibroblasts. Recently, some markers have been tested to differentiate cardiac TC from fibroblasts (which are
c-kit negative) (39). Noteworthy, microRNA expression
(e.g., miR-193) clearly differentiated TC from fibroblasts and
in culture also allowed to discriminate between TC and other
stromal cells (39).
The body of fibrocyte (or quiescent fibroblast) cells is
small and oval; the nucleus is moderately heterochromatic
and the nucleolus is difficult to locate; the Golgi complex is
small; and the rough endoplasmic reticulum is scarce. Cell
prolongations are few, long, and thin (usually described as
tapering, slender processes). Intriguingly, the body, nucleus,
and some of the ultrastructural characteristics of fibrocytes
are the same as those reported for the TC, with the exception
of the extension, convolution, and moniliform aspect of processes. The morphology of the cells labeled as ‘fibrocytes’
in figures reported in histology textbooks and other literature
is very similar to that of the TC. In figure 1 of a recent
review (40), the cells located in the connective tissue have
the typical features of both fibrocytes and TC. These latter
cells are named fibrocytes and considered to be circulating
mesenchymal progenitor cells that participate in tissue
responses to injury and invasion (40).
Cells having the ultrastructure of active fibroblasts have
been described both in normal and pathological conditions
by many laboratories, but there is a general agreement that
they are not to be considered as true fibroblasts: thus, these
cells were named fibroblast-like cells. No specific function
has been attributed to them. These cells do not resemble TC.
According to histologists and pathologists, mesenchymal
stromal cells are cells still present in adulthood that are mostly arranged along blood vessels, particularly along capillaries
(41). In usual histology textbooks, these cells are never
Q3:
Please check
the edits in
the sentence
‘According
to
histologists
...’
Page 7 of 16
shown, but their description corresponds to that of small
fibroblasts. On the contrary, embryonic mesenchymal cells
are shown in many embryology textbooks and other literature. These cells have a round body filled with free ribosomes, a small Golgi complex, a nucleus with clear
chromatin, and a large nucleolus. Cell processes can be
absent, or when present are usually long and thin. TC
morphology, therefore, does not correspond to that of mesenchymal cells, at least not those described during embryonic
life.
Pre- and postnatal differentiation
Studies aimed at gaining information on pre- and postnatal
TC differentiation have not been performed, and little information is currently available. Such studies would give an
answer on which is the origin of TC and on whether these
cells are differentiated or immature cells. The knowledge of
TC morphology and immunophenotype during their maturative steps could be of great help in identifying all TC variations during development and in recognizing them at every
age. Noteworthy, it is possible that, according to the organ
where they reside, TC could show different degrees of differentiation. This possibility is suggestive for the presence of
TC subtypes in adulthood, which differ from each other in
morphology, immunophenotype, and name. Also, studies
aimed at investigating whether TC can retain the capability
to further differentiate, are committed to one or more specific
cell lineages, and are able to differentiate spontaneously or
after injury, would be welcome.
At present, Florence’s team indirectly provided some evidences for TC differentiation. Results obtained by studying
the developing mouse heart, as well as primary cultures of
neonatal mouse cardiac cells (20, 42), showed that at earlier
embryonic stages wembryonic day 14 (E14)x, all the interstitial cells had typical mesenchymal ultrastructural features
and none of them were CD34 positive. However, by E17,
some of them acquired CD34 positivity and, at birth (P0),
also fibroblast-like features. After birth (by P6), the putative
TC showed their typical ultrastructural features while CD34
positivity became uncertain or limited to few of them. Of
note, the acquisition of the immuno- and the ultrastructural
phenotypes are not synchronized. In studies on the human
small intestine from fetal life to birth and on ICC plasticity
(43, 44), mesenchymal cells were seen to become ICC and
smooth muscle cells passing through an intermediate cell
type having fibroblast-like features. TC precursors also have
fibroblast-like features; however, at variance with the ICC
that were c-kit positive and already differentiated in fetuses
at term (45), TC are never c-kit positive and acquire their
typical CD34 positivity and ultrastructure only after birth
(Faussone-Pellegrini, unpublished data). According to some
recent data (46, 47), the CD34-positive cells present in these
fetuses might be in fact immature ICC.
On the basis of the available information, we can reasonably conclude that TC are mesenchymal in origin, are resident connective tissue cells from their earliest developmental
steps, and during their differentiation share fibroblast-like
features with immature ICC, smooth muscle cells, and true
fibroblasts. An obvious question arises when studying TC
differentiation: does the TC correspond to fibroblast-like
cells? In adulthood, most if not all of the TC have the morphology of cells called fibrocytes; however, presently we
have no information on whether different TC populations
exist in the various organs and on whether some of them
maintain fibroblastic-like appearance also in adulthood.
Moreover, it cannot be excluded that TC will acquire this
feature in the presence of stimuli to tissue renewal or repair
contributing to (i) new synthesis of the extracellular components, or (ii) differentiation of new cells to replace the
dead ones, or (iii) spatial reorganization of the organ.
We would like to hypothesize TC are ‘progenitor cells’
more or less committed according to the organs and still able
to further differentiate. This hypothesis opens a wide and
fascinating field for future researches that will surely provide
results that are surprising and of high impact.
Roles
Several roles have been suggested for TC, most of which are
believable and not mutually exclusive. However, none of
them have been proven yet.
TC might have a role as a mechanical support. The TC of
the rat mesentery form a 3D network hypothesized to be at
the same time resistant and deformable following stretches
consequent to gut movements, mainly directed to avoid
blood vessel closure (37). The TC located in the gut muscle
coat, in particular those at the myenteric plexus level, also
form a 3D network that is likely resistant to and deformable
following intestinal movements (12).
TC might guide the migration of other cells to define the
final organization of an organ or its repair or renewal.
According to a recent study (48), TC guide the migration of
mesenchymal cells into the mesothelial layer of the epicardium, thus being involved in mesothelial renewal. Cardiac
TC should guide myocardial precursors to form the correct
3D tissue pattern and contribute to compaction of the embryonic myocardial trabeculae. Indeed, cardiomyoblasts and TC
were seen to form stem cell (SC) niches in the subepicardial
region of the adult mouse heart (Figure 5) (27, 42, 49), to
migrate during development from the epicardium, from
where they presumably originated, and to form an extended
network of Tp closely embracing the growing cardiomyocytes (20). Results obtained in studies of co-cultures of TC
and cardiomyoblasts confirmed that TC can intervene in the
aggregation of cardiomyocyte clusters (20).
An immune surveillance role was suggested for the network of CD34-positive interstitial cells, further identified as
TC, located in human fallopian tube (50).
Intriguingly, it has also been suggested that TC might play
a role in neurotransmission in the gut, possibly contributing
to spread the slow waves generated by the ICC. Indeed, the
intramuscular ICC and TC seem to be part of a unique network, in which, however, only ICC are innervated (12).
Page 8 of 16
Telocytes 7
TC might be involved in intercellular signaling. The cardiac TC have been hypothesized to play a nursing role (27)
and those in the oviduct and myometrium to be sensors for
steroid hormones (34, 35). Significantly, the Tp establish
homo- and heterocellular junctions (25); release shed vesicles and exosomes (26); and show paracrine secretion of IL6,
VEGF, and NO. Thus TC, by sending macromolecular signals to neighboring cells, could influence their transcriptional
activity.
Finally, TC might represent a pool of cell precursors for
a variety of cell types with common mesenchymal origin.
Up to now, this role has been proposed for the placental (14)
and enteric TC (12). The latter might be ICC precursors that
renew ICC undergoing apoptosis (51), thus keeping the ICC
number constant throughout life. Recently, a nonsatellite resident progenitor cell niche (presumably made by TC) was
described in the striated muscle (18). In cultures of this tissue, TC (but not satellite cells) were seen to emerge from
muscle explants and form cell networks, suggesting a key
role in muscle regeneration and repair (18).
Pathology
Information on pathological TC would be of high interest.
At present, only one published paper deals with TC involvement in heart amyloidosis in patients with atrial fibrillation
(52). By TEM, amyloid deposits were located in interstitial
recesses surrounded by long and slender TC processes, likely
limiting the spread of deposits into the interstitium. Interstitial cells, likely TC, have been characterized in the upper
lamina propria of bladders of patients with neurogenic detrusor overactivity and bladder pain syndrome. These cells were
seen to shift toward a fibroblast phenotype (53).
The study of TC in mutant animals is also a tantalizing
challenge. Presently, the only available information indicates
the absence of caveolae in the TC from the myocardium (54)
and gut muscle coat (55) of Cav-1 knockout mice. This finding remains unexplained, mostly because of the lack of physiological data.
Perspectives: regenerative medicine
It is tempting to speculate that TC, as progenitor and/or guiding and nursing cells, are a novel, possible target for therapeutic strategies (56–58). The challenge of using muscle
progenitor cells for skeletal and cardiac muscle reconstruction in animal models or humans has not been solved to date,
mainly due to scarce graft cell survival explained by a lack
of adequate paracrine factors, tissue guidance, and blood
vessel scaffold (59–64). Therefore, new attempts aimed at
potentiating cardiac repair and regeneration after ischemic
injury received great momentum from the hypothesis of a
coexistence and cooperation of organ-specific TC and SC
(20). Briefly, TC and SC can be seen as working in tandem,
representing a better option for therapy rather than SC alone
(65). An important goal would be to ascertain whether such
TC–SC cooperation requires homologous TC and SC from
the same organ or whether TC from any organ source can
be used for cell therapy. In the first case, TC of a given organ
should be committed to differentiate or cooperate with the
cells specific to this organ, likely because of their similar
embryologic origin (e.g., from the epicardium, the mesothelium, or even the endoderma, ectoderma and both intra- and
extraembryonic mesoderma). In the second case, TC extracted from any organ could correctly function even when grafted into organs of a different embryologic origin, with
obvious advantages in terms of availability and plasticity.
For more information on telocytes, see the papers and
images at http://www.telocytes.com.
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volume and
page
numbers for
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53 and 55
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2007; 1101: 139–65.
34. Cretoiu D, Ciontea SM, Popescu LM, Ceafalan L, Ardeleanu
C. Interstitial Cajal-like cells (ICLC) as steroid hormone sensors in human myometrium: immunocytochemical approach. J
Cell Mol Med 2006; 10: 789–95.
35. Cretoiu SM, Cretoiu D, Suciu L, Popescu LM. Interstitial Cajallike cells of human Fallopian tube express estrogen and progesterone receptors. J Mol Histol 2009; 40: 387–94.
36. Gherghiceanu M, Hinescu ME, Andrei F, Mandache E, Macarie
CE, Faussone-Pellegrini MS, Popescu LM. Interstitial Cajallike cells (ICLC) in myocardial sleeves of human pulmonary
veins. J Cell Mol Med 2008; 12: 1777–81.
37. Hinescu ME, Popescu LM, Gherghiceanu M, Faussone-Pellegrini MS. Interstitial Cajal-like cells in rat mesentery: an ultrastructural and immunohistochemical approach. J Cell Mol Med
2008; 12: 260–70.
38. Gabella G. Innervation of the gastrointestinal tract. Int Rev
Cytol 1979; 59: 129–93.
39. Cismasiu
¸ ¸ VB, Radu E, Popescu LM. miR-193 expression differentiates telocytes from other stromal cells. J Cell Mol Med
2011; 15: 1071–4.
40. Herzog EL, Bucala R. Fibrocytes in health and disease. Exp
Hematol 2010; 38: 548–56.
41. Marchand F. Die örtlichen reaktiven Vorgänge (Lehre von der
Entzündung). In: Krehl L and Marchand F, editors: Handbuch
der allgemeinen pathologie, vol. 4, part 1. Leipzig, 1924; 78.
42. Faussone-Pellegrini MS, Bani D. Relationships between telocytes and cardiomyocytes during pre- and post-natal life. J Cell
Mol Med 2010; 14: 1061–3.
43. Faussone-Pellegrini MS. Cytodifferentiation of the interstitial
cells of Cajal of mouse colonic circular muscle layer. An E.M.
study from fetal to adult life. Acta Anat 1987; 128: 98–109.
44. Faussone-Pellegrini MS, Vannucchi MG, Ledder O, Tian-Ying
Huang T-Y, Hanani M. Plasticity of interstitial cells of Cajal: a
study of mouse colon. Cell Tissue Res 2006; 325: 211–7.
45. Faussone-Pellegrini MS, Vannucchi MG, Alaggio R, Strojna A,
Midrio P. Morphology of the interstitial cells of Cajal of the
human ileum from foetal to neonatal life. J Cell Mol Med 2007;
11: 482–94.
46. Lorincz A, Redelman D, Horváth VJ, Bardsley MR, Chen H,
Ordög T. Progenitors of interstitial cells of Cajal in the postnatal
murine stomach. Gastroenterology 2008; 134: 1083–93.
47. Huizinga JD, White EJ. Progenitor cells of interstitial cells of
Cajal: on the road to tissue repair. Gastroenterology 2008; 134:
1252–4.
48. Gherghiceanu M, Popescu LM. Human epicardium: ultrastructural ancestry of mesothelium and mesenchymal cells. J Cell
Mol Med 2009; 13: 2949–51.
49. Gherghiceanu M, Popescu LM. Cardiomyocyte precursors and
telocytes in epicardial stem cell niche: electron microscope
images. J Cell Mol Med 2010; 14: 871–7.
Q5:
Please check
the editor
names and
add the
publisher’s
name for
Ref. 41
Page 10 of 16
Telocytes 9
50. Yamazaki K, Eyden BP. Gap junctions and nerve terminals
among stromal cells in human fallopian tube ampullary mucosa.
J Submicrosc Cytol Pathol 1998: 30: 399–408.
51. Gibbons SJ, De Giorgio R, Faussone-Pellegrini MS, GarrityPark MM, Miller SM, Schmalz PF, Young-Fadok TM, Larson
DW, Dozois EJ, Camilleri M, Stanghellini V, Szurszewski JH,
Farrugia G. Apoptotic cell death of human interstitial cells of
Cajal. Neurogastroenterol Motil 2009; 21: 85–93.
52. Mandache E, Gherghiceanu M, Macarie C, Kostin S, Popescu
LM. Telocytes in human isolated atrial amyloidosis: ultrastructural remodelling. J Cell Mol Med 2010; 14: 2739–47.
53. Gevaert T, De Vos R, Everaerts W, Libbrecht L, Van Der Aa
F, van den Oord J, Roskams T, De Ridder D. Characterization
of upper lamina propria interstitial cells in bladders from
patients with neurogenic detrusor overactivity and bladder pain
syndrome. J Cell Mol Med 2011; Jan 20. doi: 10.1111/j.15824934.2011.01262.x.
54. Gherghiceanu M, Hinescu M, Popescu LM. Myocardial interstitial Cajal-like cells (ICLC) in caveolin-1 KO mice. J Cell
Mol Med 2009; 13: 202–6.
55. Cipriani G, Serboiu CS, Gherghiceanu M, Faussone-Pellegrini
MS, Vannucchi MG. NK-receptors, Substance P, Ano1 expression and ultrastructural features of the muscle coat in Cav-1-/mouse ileum. J Cell Mol Med 2011. May 2. doi: 10.1111/
j.1582-4934.2011.01333.x.
56. Klumpp D, Horch RE, Kneser U, Beier JP. Engineering skeletal
muscle tissue—new perspectives in vitro and in vivo. J Cell
Mol Med 2010; 14: 2622–9.
57. Polykandriotis E, Popescu LM, Horch RE. Regenerative medicine: then and now—an update of recent history into future
possibilities. J Cell Mol Med 2010; 14: 2350–8.
Maria-Simonetta FaussonePellegrini, Professor Emeritus
of Histology and Embriology
at the Faculty of Medicine of
the University of Florence, is
member of the Editorial
Board of the Journal of Cellular and Molecular Medicine, Assistant Editor of the
Italian Journal of Anatomy
and Embryology, Honorary
Foreign Member of the
Romanian Academy of Medical Science. Her main researches deal on the interstitial cells of Cajal that in 1977 she
successfully proposed to be the intestinal pacemaker cells.
She also studies enteric and cerebral neuronal and glial cells,
gastrointestinal muscles, stromal cells formerly named interstitial Cajal-like cells and now telocytes. All these topics
have been studied by electron microscope and histochemistry
in normal and pathological conditions and during differentiation. Her publications are about 200, most of which in
Journals with IF.
58. Popescu LM, Gherghiceanu M, Kostin S, Faussone-Pellegrini
MS. Telocytes and heart renewing. In: Wang P, Kuo CH, Takeda
N, Singal PK, editors. Adaptation biology and medicine, vol 6.
Cell adaptations and challenges. New Delhi: Narosa, 2010:
17–39.
59. Gittenberger-de Groot AC, Winter EM, Poelmann R. Epicardium-derived cells (EPDCs) in development, cardiac disease
and repair of ischemia. J Cell Mol Med 2010; 14: 1056–60.
60. Li TS, Cheng K, Lee ST, Matsushita S, Davis D, Malliaras K,
Zhang Y, Matsushita N, Smith RR, Marbán E. Cardiospheres
recapitulate a niche-like microenvironment rich in stemness and
cell-matrix interactions, rationalizing their enhanced functional
potency for myocardial repair. Stem Cells 2010; 28: 2088–98.
61. Rupp H, Rupp TP, Alter P, Jung N, Pankuweit S, Maisch B.
Intrapericardial procedures for cardiac regeneration by stem
cells: need for minimal invasive access (Attach Lifter) to the
normal pericardial cavity. Herz 2010; 35: 458–65.
62. Limana F, Capogrossi MC, Germani A. The epicardium in cardiac repair: from the stem cell view. Pharmacol Ther 2011; 129:
82–96.
63. Kostin S. Types of cardiomyocyte death and clinical outcomes
in patients with heart failure. J Am Coll Cardiol 2011; 57:
1532–4.
64. Russell JL, Goetsch SC, Gaiano NR, Hill JA, Olson EN,
Schneider JW. A dynamic notch injury response activates epicardium and contributes to fibrosis repair. Circ Res 2011; 108:
51–9.
65. Popescu LM. The tandem: telocytes—stem cells. Int J Biol Biomed Eng 2011; 2/5: 83–92.
Received June 28, 2011; accepted August 3, 2011
L.M. Popescu, MD, PhD,
Dr. h.c.mult., is currently
Professor of Cellular and
Molecular Medicine, ‘Davila’ University of Medicine,
Bucharest, Romania and
Head of the National Institute
of Pathology, Bucharest,
Romania. He is fellow of the
National Academy of Sciences and the President of
Academy of Medical Sciences. Recently, he became
President Elect of the Federation of European Academies of
Medicine, and of the International Society for Adaptive Medicine. He published over 100 scientific articles in international peer-review journals and is cited more than 1500
times. He has a Hirsch Index of about 30. Professor Popescu
is Editor-in-Chief (and founder) of the Journal of Cellular
and Molecular Medicine (Wiley/Blackwell), with a 5-year IF
of 5. He is credited with the discovery of Telocytes.
Page 11 of 16
Table 1. Telocytes distribution.
cavitary organs
non-cavitary organs
heart (endo-, myo-, and peri-cardium)
(6,16,19,20,25-29,30,31)
lung and pleura (17)
stomach and intestine (12,21)
exocrine pancreas (2)
gallbladder (32)
mammary gland (1)
uterus (3,33,34)
placenta (14)
Fallopian tube (5,35)
skeletal muscle (18)
blood vessels (36)
mesentery (37)
Page 12 of 16
Figure 1. A and B. CD34-immunoreactivity; submucosa of human stomach. A: the CD34-positive
cells (in brown) have a triangular or ovoid body and a variable number of thin and long
prolongations that give a stellate shape to the whole cell. These processes have knobs along their
length and a dichotomous branching. Bar: 20 µm. (with permission from ref. 12). B: a detail of the
body and prolongations of a CD34-positive cell. Bar = 15 µm. C: human non-pregnant myometrium
in cell culture, day 3, the first passage. Giemsa staining. One Telocyte establishing contacts with a
smooth muscle cell (myocyte) by a cell process (telopode) of about 65 µm long. Photographic
composition of 4 serial phase contrast images; original magnification 40x. In red rectangles, a
higher magnification clearly shows the moniliform aspect of the telopode; at least 40 specific
dilations (podoms) of the telopode, interconnected by thin segments (podomeres), are visible in a
‘beadlike’ fashion. Original magnification 40x. (with permission from ref. 3). D: human resting
mammary gland stroma, transmission electron microscopy. One telopode, that appears very long
and convoluted, with intercalated podomeres and podoms. Note homocellular junctions marked by
red circles, as well as shed vesicles (SV, blue) and an exosome (violet). coll = collagen. (with
permission from ref. 1).
170x108mm (300 x 300 DPI)
Page 13 of 16
Figure 2. A and B: Transmission electron microscope. A: rat mesentery. One telocyte (TC) with a
small nucleated body and three long telopodes, all of them having a sinuous trajectory and forming
an interstitial complicated 3D network. B: human exocrine pancreas. The telocytes (TC) form with
their typical long telopodes a network around the acini.
180x124mm (300 x 300 DPI)
Page 14 of 16
Figure 3. This electron tomography (thick section of about 300 nm) shows nanostructures
connecting one telopode with two adjacent cardiomyocytes in adult mouse heart. The bridging
structures (encircled) have 10-15 nm and suggest a molecular interaction between the telopode and
the cardiomyocytes. The dilated segment of this telopode contains a mitochondrion (m). (with
permission from ref. 25).
87x52mm (300 x 300 DPI)
Page 15 of 16
Figure 4. A: human stomach. Double CD34/c-kit labelling. CD34-positivity is in red and c-kitpositivity in green. The CD34- and c-kit-positive cells are often very close to each other but none of
them is double labelled. Bar: 25 µm. B and C. CD34-immunoelectro-labelling. B: mouse small
intestine. CD34-positivity is present along the plasma membrane of a long, thin process of a
telocyte (TC), while interstitial cells of Cajal (ICC), nerve fibers and smooth muscle cells (SMC) are
CD34-negative. Bar: 1 µm, C: mouse stomach. Detail of a CD34-immunoelectro-labelled telopode.
CD34-positivity clearly appears as electron-dense spherules regularly distributed on the telopode
plasma membrane. Bar: 0.4 µm.
180x122mm (300 x 300 DPI)
Page 16 of 16
Figure 5. Schema and electron micrograph illustrating the relationships several telocytes (blue)
have with a column of cardiomyocyte progenitors (CMP, brown) in an epicardial stem cell niche of
adult mouse. The telopodes run parallel to the main axis of the CMP column and seem to establish
the direction of development. (with permission from ref. 27).
180x134mm (300 x 300 DPI)
J. Cell. Mol. Med. Vol 14, No 4, 2010 pp. xxx-yyy
Editorial
(Review-Article)
TELOCYTES - A case of serendipity:
the winding way from Interstitial Cells of Cajal (ICC),
via Interstitial Cajal-Like Cells (ICLC) to
TELOCYTES
L. M. Popescu
a
a, b,*,
Maria-Simonetta Faussone-Pellegrini
c, *
Department of Cellular and Molecular Medicine, “Carol Davila” University of Medicine and Pharmacy, Bucharest, Romania
b "Victor Babeș" National Institute of Pathology, Bucharest, Romania
c Department of Anatomy, Histology and Forensic Medicine, Section of Histology, University of Florence, Florence, Italy
Accepted: February 25, 2010
Abstract
Ramon y Cajal discovered a particular cell type in the gut, which he named "interstitial neurons" more that 100 years ago. In the
early 1970s, electron microscope (EM) studies showed that indeed a special interstitial cell type corresponding to the cells discovered by Cajal is localized in the gut muscle coat, but it became obvious that they were not neurons. Consequently, they were renamed
"Interstitial Cells of Cajal" (ICC) and considered to be pace-makers for gut motility.
For the past 10 years many groups were interested in whether or not ICC are present outside the gastrointestinal tract, and
indeed, peculiar interstitial cells were found in: upper and lower urinary tracts, blood vessels, pancreas, male and female reproductive tracts, mammary gland, placenta, and, recently, in the heart as well as in the gut. Such cells, now mostly known as Interstitial
Cajal-Like Cells (ICLC), were given different and confusing names. Moreover, ICLC are only apparently similar to canonical ICC. In
fact, EM and cell cultures revealed very particular features of ICLC, which unequivocally distinguishes them from ICC and all other
interstitial cells: the presence of 2–5 cell body prolongations that are very thin (less than 0.2 mm, under resolving power of light
microscopy), extremely long (tens to hundreds of mm), with a moniliform aspect (many dilations along), as well as caveolae .
Given the unique dimensions of these prolongations (very long and very thin) and to avoid further confusion with other interstitial cell types (e.g. fibroblast, fibrocyte, fibroblast-like cells, mesenchymal cells), we are proposing the term TELOCYTES for them,
and TELOPODES for their prolongations, by using the Greek affix "telos".
Keywords: telocytes
myocardium
•
• interstitial cells of Cajal (ICC) • interstitial Cajal-like cells (ICLC) • interstitial cells • telopodes
myometrium • mammary gland • genitourinary tract • digestive tract • pancreas • stromal cells •
regenerative medicine
* Correspondence to: L.M. PoPesCu, M.D., Ph.D.
Department of Cellular and Molecular Medicine,
“Carol Davila” university of Medicine and Pharmacy,
P.o. Box 35-29, Bucharest 35, Romania.
e-mail: [email protected]
DoI: 10.1111/j.1582-4934.2010.001059.x
•
**
*
*
*
1.6 mm
Fig. 1 Rabbit colon; circular muscle layer. one interstitial cell of
Cajal (ICC) with an elongated body and three processes located in
the connective interstitium. This cell has close cell-to-cell contact
between both body and processes and two smooth muscle cells
(sMC, arrows) and nerve endings (N, double arrows). This cell has
the typical features of an intramuscular ICC: conspicuous Golgi
apparatus and several rough endoplasmic reticulum cisternae; basal
lamina is thin and discontinous.
*
4 mm
Fig. 2 Human stomach; circular muscle layer. An interstitial cell of
Cajal (ICC) located in the connective interstitium between the crosssectioned bundles of smooth muscle cells (sMC), with which ICC
establishes numerous cell-to-cell contacts (asterisks). Note the proxAt first there was…….What was at the beginning? The answer imity of a large nerve bundle (N). The ICC body is spindle-shaped and
is: the cells that s. Ramon y Cajal discovered in the muscle the cytoplasm is rich in filaments. The basal lamina is thick but discoat of the gut and called “interstitial neurons” more than continuous.
Interstitial Cells of Cajal (ICC)
100 years ago [1]. He gave them this name since these cells
i) looked like nerve cells, ii) were identifiable through staining
techniques which specifically labelled neurons (e.g. methylene
blue or silver impregnation), and iii) were located in the
interstitium between nerve endings and smooth muscle cells.
However, unbelievably, their “existence” was more or less
rejected by the scientific community of that time. After about
half a century, examination of the gut muscle coat under the
electron microscope revealed cells probably corresponding to
the so-called Cajal’s “interstitial neurons.” Among the pioneers
of this “re-discovery” were M.s. Faussone-Pellegrini [2] and,
independently, L. Thuneberg [3]: it was immediately clear that
these cells were not neurons. Therefore, scientists labelled
them Interstitial Cells of Cajal (acronym: ICC). From then the
“interstitial neurons” of Cajal were buried with all the honours
they deserved and “Interstitial Cells of Cajal (ICC)” were born
and grew (Figs. 1 & 2).
At present, scientific papers (e.g. [4, 5]) are available for:
a) a complete map of the distribution of ICC within the muscle
coat of the gut of several mammals, including humans, b) a
detailed description of their ultrastructural features which is of
great help in their identification, c) molecules expressed by
2
ICC, considered to be markers for these cells (c-kit) and their
role(s) (NK2r). It has also been demonstrated that ICC origin
is mesenchymal, thus further confirming that they are not
neuronal cells [6].
After the first pioneers, who were morphologists, other
morphologists consider ICC as interstitial cells possibly
forming a functional network, while physiologists see them
as pace-maker cells. All together these researchers agreed
with and confirmed the role proposed for these cells by
Cajal: to be actively involved in the regulation of
gastrointestinal motility, and not only as pace-maker cells
but also in neurotransmission and stretch sensing (see
[7–9] for reviews). Moreover, pathologists view them as the
origin of Gastro-Intestinal Stromal Tumors (GIST) and
being primarily involved in several gastrointestinal
disorders [10]. Apparently, ICC are “great expectations” for
pharmacologists and / or some drug companies, because it
is reasonable to assume that there are millions of cases of
motility disorders of the digestive tract, although there are
no available statistics.
J. Cell. Mol. Med. Vol 14, No 4, 2010
Serendipity ...
from ICLC to telocytes
During the last decade, new players appeared in the
ICC field. However, we are faced with a perfect
example of serendipity (see ref. [11]). The term, coined
almost 250 years ago by Horace Walpole, an english
novelist, defines serendipity as the ability of making
fortunate and unexpected discoveries by accident
andsagacity. Louis Pasteur once said, “In the field of
observation, chance only favors the prepared mind.”
There are plenty of instances of serendipity in many
scientific domains, from the discovery of quinine,
vaccines, mast cells, penicillin or X-rays in medical
field, to the detection of uranus in astronomy and even
Teflon ®, cellophane and microwaves ....
Let us see what has happened and what might
happen. Fascinated by ICC, Popescu and his team
looked for cells located in pancreas [12, 13] (see
Figs. 3–5), where Cajal also saw his “interstitial
neurons”. The Bucharest team extended their studies
to other organs: myometrium (see Figs. 6 & 7),
fallopian tube, placenta, mammary gland, gall
bladder, mesentery, pulmonary veins ([14–22]),
demonstrating the frequency and the ubiquity of the
cell type they found. Noteworthy, a series of
publications was dedicated to the presence and
significance of Interstitial Cajal-like Cells (ICLC) in
mammalian and human myocardium and epicardium
[23–33]; see Figs. 8–10. Many authors also described
cells they were considering more or less ICC and/or
ICLC at least in the upper and lower urinary tracts
([34–38], for reviews see [39]), blood vessels [40–44],
lymphatics [45], as well as male and female
reproductive systems ([46 - 49], for reviews see [50]).
Popescu et al. performed an eM examination, as well as
staining techniques used by Cajal to label his “interstitial
neurons” and the immunohistochemical methods supposed
to specifically label the ICC, and observed cells with a
typical interstitial location and a phenotype more or less
similar to that of the ICC. The existence of a (“new”) not yet
described cell type became increasingly clear to both: the
Bucharest experts and others. However, the name chosen
was not so (much) different from ICC: interstitial Cajal-like
cells (ICLC) (first used in ref. [15]), since at that time the
aim was to stress how similar these cells were to ICC.
Incidentally, this was not a good idea because, in fact, there
were only a few similarities, not to mention the peculiar
morphology of the ICLC, a morphology that is unique
among all other interstitial cell types.
TC
TC
TC
TC
Fig. 3 Rat exocrine pancreas. Non-conventional light microscopy;
objective 100x. Tissue fixed with glutaraldehyde and postfixed in oso4.
Thin section of epon-embedded material (~ 1 mm) was stained with toluidin blue; cap = capillary; ven = venule. At least 4 telocytes (TC) are present in the interstitium among acini (a). Note the cell bodies of TC and
the emerging prolongations - telopodes (dashed lines). The length of the
telopodes is very impressive: tens of mm (!); they are very thin (less than
0.5 mm). Reproduced with permission from [13].
Fig. 4. Immunohistochemistry: Telocytes in human pancreas. Paraffinembedded pancreas sections were incubated with polyclonal antibodies
against CD 117. Nuclei were counterstained with Mayer hematoxylin.
Telocytes (arrows) with fusiform body can be seen. having typical long,
moniliform cytoplasmic processes that ‘touch’ the acini. original magnification: 100x, oil immersion.
Reproduced with permission from [12].
Fig. 5 Human
exocrine pancreas.Positive
immunostaining
of telocytes for
CD34 (arrows),
counterstained
with Mayer’s
hematoxylin,
40x.
Reproduced
with permission
from [13].
3
TeloCyTe
telocyte
Electron microscopy
Cell culture
Fig. 6 Digitally-colored EM image of a telocyte in rat myometrium: telocyte (blue), smooth muscle cells - sMC (sienna-brown); N = Nuclei.
Note three long, moniliform processes that encircle bundles of cross-cut smooth muscle cells. original magnification x 6,800. Inset: Human
pregnant myometrium. Primary confluent culture (day 8) showing a telocyte with at least 3 prolongations with several ‘beads’ along telopodes.
Reproduced with permission from [14] .
Fig. 7 EM of human non-pregnant uterus. Note the telocyte covering
smooth muscle cells (M). The telopode is digitally coloured in blue, marked
with asterisks. Image obtained in 2006.
Courtesy of Prof. M.Taggart (Newcastle University, UK) and Dr. CarolynJ.P.Jones (Manchester University, UK)
TeloCyTe
(Telopode)
M
4
M
500 nm
TeloCyTeS
Telopode
TeloCyTe
Fig. 8. EM image of
telocytes (rat) in the
right atrial interstitium; telocytes are indicated by arrows. Note
the characteristic
aspects of telopodes:
very long and very thin
cellular elongations,
with uneven calibre
(moniliform aspect).
Rectangles show portions of telocyte body,
containing (abundant)
rough endoplasmic
reticulum (reR).
Telo
pode
TeloCyTe
2 mm
Telopode
Fig. 9. Electron micrograph from cardiac stem cells niche
(subepicardium) illustrating the relationships of the telocyte (digitally blue coloured) with cardiomyocyte progenitors - CMP, (brown).
The telocyte processes (telopodes) run parallel with the main axis of
the CMP and seem to establish their direction of development.
5
TEloCyTE
telopode
telopode
Fig. 10. Representative scanning electron micrograph. Monkey left ventricular myocardium. The image shows a typical telocyte located across the cardiomyocytes. Another (possible) telocyte appears located near the cardiomyocytes (upper left). The three-dimensional view
reveals close interconnections of ICLCs with cardiomyocytes and capillaries (cap).
ICLC continue to be studied with eM and
immunohistochemistry. In particular, in order to best characterize
them and understand their role(s), Popescu’s team tested many
markers. At present, however, only eM gives an unequivocal and
conclusive answer. Immunohistochemistry, on the contrary, has
given answers most of which are confusing because the positivity
to the markers tested is different between organs and animal
species (Fig. 11). Moreover, most of the markers expressed by
the ICLC are in common with several cell types (see CD34 which
labels ICLC and endothelium, c-kit which labels ICC and some
ICLC, etc.). surely ICLC share the same mesenchymal origin with
all these cells, but this origin is a terribly vague marker.
TeloCyTe
6
Fig. 11 Human fallopian tube; subconfluent primary culture.
Double immunofluorescent labeling of an ‘octopus’-like telocyte:
vimentin (green) and CD117/ckit (red). Vimentin reactivity is mainly localized within the cell processes, and CD117/c-kit has a
patchy pattern. The cell nucleus is shown in blue (Hoechst 33342);
original magnification 60x.
TeloCyTeS
2 mm
Fig 12. Rabbit colon. Two telocytes (asterisks) close to the submucosal border of the circular muscle layer. These cells have a small oval body,
mainly occupied by the nucleus, and extremely thin and long processes extend beyond the cellular body, curving repeatedly. No basal lamina is
present around these cells. sMC = smooth muscle cells.
using electron microscopy (Fig. 12), and immunoelectron microscopy, Faussone-Pellegrini et al. (Fig. 13) reexamined the muscle coat of the gut. This is a region where
these authors and many other researchers have seen,
described and studied the ICC from all possible angles,
without, however, paying attention to the existence of cells
like ICLC. The results were particularly intriguing: ICLC are
telopode
present in the gut muscle coat in great quantities (Fig. 12),
coexist with the ICC and often share an identical
distribution. one of the conclusions of that study was that
ICLC and ICC are two different cell types. Another
collateral conclusion is that we will never know whether the
“interstitial neurons” described by Cajal correspond to the
ICC, to the ICLC or encompass both.
TeloCyTe
0.5 mm
Fig. 13 CD34-immunoelectro-labeling: small intestine. CD34-immunoelectro-labeling is present on the surface of a telocyte. The labelling
appears as an electron-dense material distributed all along the plasma membrane, from which spherules protrude outside.
Reproduced with kind permission from [52].
7
Topic: Interstitial Cajal-like Cells (now Telocytes)
published
articles
citations
Fig. 14 The growing scientific interest in “Interstitial Cajal-like Cells” (now TELOCYTES). Charts based on data released by Web of
science (Thomson Reuters ISI Web of Knowledge)
TELOCYTES and TELOPODES
obviously, the cells that we named “ICLC” are different from
ICC. Therefore, it is reasonable (even mandatory) to give them
a different name that refers to them, only. As the “interstitial
neurons” became known as “Interstitial Cells of Cajal”, we think
that the “Interstitial Cajal-Like Cells” (ICLC) should be called
“TeLoCYTes” from now on, by using the Greek affix “Telos”.
Aristotle believed that Telos (Telos) was an object’s or
individual’s greatest potential [11]. The initial meaning of the
word was “burden”, and the most probable semantic
development was from “duty”/“task” to “execution of task”,
“completeness”, and most important, “power to decide” [51].
At present, one could easily see the fast-rate ascending
trend of the interest in “Interstitial Cajal-like Cells” (or ICLC)
during the last 5 years (see Fig. 14). Note the visible difference
between the growing rate of the two parameters (published
items vs. citations), showing a progressive interest in these
cells, which are actually telocytes.
Between 1991 and 2009, a PubMed search of the
Medline database retrieved over 250 records on topic of
Interstitial Cajal-like Cells and more than 7400 citations, with
an average of almost 30 citations per item, consequently
leading to a Hirschindex of 43.
Telocytes [refs. 12–33]
General aspect of the telocyte is of a small, oval-shaped cellular
body, containing a nucleus, surrounded by a small amount of
8
cytoplasm. The cellular body average dimensions are, as
measured on eM images, 9.39 mm ± 3.26 mm (min = 6.31 mm;
max = 16.42 mm). The nucleus occupies about 25% of the cell
volume and contains clusters of heterochromatin attached to the
nuclear envelope.
The perinuclear cytoplasm is rich in mitochondria (which occupy
about 5% of the cell body), contains a small Golgi complex, as well
as elements of rough and smooth endoplasmic reticulum and
cytoskeletal elements (thin and intermmediate filaments).
The cell periphery is represented by an usual plasmalemma,
with no (or thin and discontinuous) basal lamina, and many
caveolae (about 2–3% of cytoplasmic volume; ~0.5 caveolae/ mm
of cell membrane length).
The shape of the telocytes is according to the number of their
telopodes: piriform for one prolongation, spindle for two
telopodes, triangular for three, stellate etc. Presumably, their
spatial appearance would be that of a polyhedron with a different
number of vertices, depending on their telopode number.
Telopodes [refs. 12-33, 52]
since we are thinking that telopodes are distinctive for telocytes,
we would like to emphasize at least the following characteristics:
1. number: one to five, frequently only 2–3 telopodes are
observed on a single section, depending on site and angle of
section, since their 3D convolutions impede them to be
observed at their full length in a 2D very thin section;
2. length: tens – up to hundreds of mm, as measured on eM
images (Figs. 6–9, 12, 13). However, under favorable condition
in cell cultures, their entire length can be captured (Fig. 15);
3. thickness: uneven caliber, mostly below 0.2 mm (resolving power of
light microscopy), visible under electron microscopy, only 0.10 mm
± 0.05 mm (min = 0.03 mm; max = 0.24 mm; see Figs. 6–9, 12, 13);
4. moniliform aspect, with many dilations along (e.g. Fig. 15);
5. presence of “Ca2+-release units” at the level of the dilations,
accommodating i) mitochondria (as seen by vital staining using
Janus Green B or MitoTraker Green FM, as well as by eM); ii)
elements of endoplasmic reticulum and iii) caveolae;
6. branching, with a dichotomous pattern;
7. organization in a network – forming a labyrinthine system by
tridimensional convolution and overlapping, communicating
through gap junctions.
This characteristic feature makes telopode clearly different from
neuronal dendrites, processes of antigen-presenting dendritic cells or
fibroblasts and myofibroblasts. All the previously mentioned cell
processes (except telopodes) have a thick emergence from the cell
body, followed by gradual thinning.
Noteworthy, except the axons of some type of neurons, telopodes
of telocytes are probably the longest cellular prolongations in the
human body! Furthermore, we have to emphasize that telopodes are
completely different from nerve cells axons or dendrites.
Telocytes have "strategic" positioning in a tissue, in between blood
capillaries and their specific target cells (e.g. smooth muscle cells,
cardiomyocytes) (see Fig. 10 for a typical topography of telopodes) and
in close contact with nerve endings. The distance between telopodes
and myocytes is within the range of tens of nm, which fits the domain of
macromolecular interactions.
Last but not least, to underline that telopodes could establish close
contacts, like synapses, with immunoreactive cells, in various organs,
we called such "connective connections" as stromal synapses [53]
(see Fig. 16).
TeloCyTe
Why telocytes were not described so far
as a distinct cell type ?
Naturally, a question may arise: why so many scientists ignored
telocytes? It is out of question that a cell could be seen under the
microscope, only. Presumably, telocytes were mainly neglected due
to the physical constraints of light and/or electron microscopy
methodology.
Fig. 15 Human non-pregnant myometrium in cell culture; day 3; the 1st
passage. Giemsa staining. A telocyte establishing contacts with a myocyte by
a telopode of about 65 mm long. Photographic composition of 4 serial phase
contrast images, original magnification 40x. In red rectangles, a higher magnification clearly shows the moniliform aspect; at least 40 specific dilations are
visible in a ‘bead-like’ fashion.
9
TeloCyTe
a) Light microscopy. The usual stain of H & e could not allow the
differentiation of a telocyte cell body and the cell body of a fibrocyte
/ fibroblast. on the other hand, the area of the microscopic field is
too small to get a clear and complete image of telopodes, which are
anyway below the resolving power (0.2 mm) of light microscopy. In
addition, even the best possible sections obtained using a
microtome (2–4 mm thickness) cannot offer a very good resolution.
In light microscopy, using an oil immersion objective with a
10x eyepiece, the diameter of area of the section is less than
100 mm wide, which will correspond, for instance, to no more
than a row of 4–5 hepatocytes. Therefore, it would be a matter
of luck to catch an entire telocyte with its long and convoluted
telopodes.
b) Electron microscopy uses ultrathin sections of about
60–80 nm. At 2,000x magnification, a regular telocyte
appears too small to be clearly observed. successively
increasing the magnification, in spite of improving the
accuracy of the ultrastructural details, the entire structure fails
to be included in the observation field. A magnification of
about 7,000x shows only a small fragment of the telocyte. At
28,000x magnification, usually, the limited field under
observation does not allow recognition of a telocyte.
Instead of conclusions:
why have telocytes?
usualy, people are looking at interstitial cells as being mainly
fibroblasts. However, fibroblasts have the function of generating
connective tissue matrix, specifically collagen. The distinctions
between fibroblasts and telocytes becomes important as their
functions should be mostly different. The expected progress in
knowledge for interstitial cells will show that not all the cells
present in the interstitium, apparently fibroblasts / fibrocytes,
should be labelled as fibroblasts. In other words, other types of
Fig. 20 electron micrographs show details of telopodes
from
mouse
epicardium.
A.
The
sinuous
and
moniliform
t etelopodes
l oaccommodate
p o dmitochondria
e (m), caveolae and eR interstitial cells, mainly telocytes, should not be ignored as they
cisternae. Calcium release units (arrows) could be seen at have been in the past.
this level. Note close vicinity of telopodes with nerves. B.
shed vesicles (arrow) emerge from telopodes in the interstiIt may be possible that different locations of telopodes could
tial space. C. Gap junction (arrow) between two telopodes.
be associated with different roles. Anyway, the expression of c-kit
receptors differs between telocyte populations (possible sitetelopode
dependent?). Intriguingly, for researchers accustomed with the
pace-maker role of ICC, telocytes presumably exercise other
functions, since they have also been found in non-cavitary
organs, such as pancreas [12, 13], mesentery [21] and even
placenta [20]. Moreover, cardiac telocytes do not function as
pace-maker, but they may influence the rate and rhythm
generated by nodal system.
Fig. 17. Electron micrographs show details of telopodes
Hypotethically, many roles were ascribed to telocytes (formerly,
from mouse epicardium. shed vesicles (arrowhead) emerge
ICLC). However, there is no reasonable evidence to support them.
from telopodes in the interstitial space.
Fig. 16. Rat myometrium: TEM; original magnification 5,700x;
image digitally colored. A multicontact synapse (Ms) between an
eosinophil (green) and a telocyte (violet) indicated by cassette. An
unmyelinated nerve bundle (vermilion) appears in proximity of telocyte,
but has no direct contact with it. Apparently, the telocyte ‘prefers’ the
eosinophil
10
In our opinion telocytes are involved in intercellular signaling,
taking into account the 3D network of telopodes and their strategic
position in between target cells, blood capillary and nerve ending.
At least two mechanisms could be considered: i) a paracrine
and/or juxtacrine secretion of small signal molecules and ii)
shedding microvesicles (see Fig. 17), which play unique roles in
horizontal transfer of important macromolecules among
neighbouring cells (e.g. proteins or RNAs, microRNA included).
such a mechanism, via shed vesicles, may serve to rapid
phenotype adjusment in a variety of conditions [54].
An important role, which could be attributed to telocytes
in the heart is that of being active players in cardiac
renewing, since they are “nursing” cardiomyocyte
progenitors in epicardial stem cell niches [31, 32]. A strong
argument is provided by comparative micro-anatomy: the
newt heart is a model for adult heart regeneration, as newts
can functionally regenerate their heart after amputation of
the apex of the ventricle [55]. In this regenerative process,
a supporting network of stromal cells is primarily developed
and these cells, which fulfill all ultrastructural criteria for
telocytes, are present in large number [56]. Therefore,
apparently an important goal for regenerative medicine
would be to find some factors to stimulate telocytes, as
autologuous in situ cells ...
Epilogue
"There are no small problems. Problems that appear small
are large problems that are not understood”.
RamonyCajal[57]
Forsupplementarymaterialsontelocytespleasesee
www.telocytes.com
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