4. Biomaterials and Tissue Engineering 4.2.1. The use of

10.2.2015
Prague
4. Biomaterials and Tissue Engineering
4.2.1. The use of stem cells and biomaterials in cell therapy
Stem Cells and Biomaterials in Regenerative Medicine
Pavla Jendelová
Institute of Experimental Medicine AS CR, Prague
Biocev Team
Institute of Experimental Medicine, ASCR
Group Leaders: Eva Syková, Šárka Kubinová, Vladimír Holáň, Pavla
Jendelová
Researchers: Amemori, Vacková, Machová-Urdzíková, Erceg,
Jiráková, Forostyak
Collaborations
Institute of Macromolecular Chemistry, ASCR
Institute of Physics, AVCR
Institute of Clinical and Experimental Medicine
New York Medical College
Cambridge Brain Research Center
Norwegian University of Science and Technology
McGowan Institute for Regenerative Medicine University of Pittsburgh, USA
Recent projects
• The comparative study on use of different stem cells in spinal cord
injury treatment
• The development of isolation, cultivation and expansion protocols of
human umbilical cord mesenchymal stromal cells
• The use of mesenchymal stromal cells in the treatment of ocular
injury
• The development and use of nanoparticles for safe cell labelling
• The use of stem cells in neurodegenerative diseases (ALS, AD)
• The development and use of cell-polymer constructs based on
decellularized tissue
Cells used for CNS Tissue Engineering
Advantages
Mesenchymal stem cells
(MSCs)
•
•
•
Bone marrow
Adipose tissue
Umbilical cord tissue
Induced pluripotent stem
cells (iPSC)
•
•
•
•
•
•
•
•
•
•
•
Human fetal neural
precursor cells (SPC-1)
c-myc 4-hydroxytamoxifen
conditionally immortalized
cell line
•
•
•
Disadvantages
•
Autologous transplantation, no
immune-suppressive therapy necessary
Low immunogenicity
No ethical objections
Multipotent
Easily obtainable,
Clinical application already realized
Unlimited proliferation
Pluripotent
Can be created from the tissue of the
same patient that will receive the
transplantation
Lack of ethical implications
Differentiation and good intergration
into the host tissue
Unlimited proliferation
Neural differentiation
Robust survival after transplantation
•
•
Lack of specific identification
markers
Limited proliferation
No transdifferentiation
•
•
Low efficiency of the technology
Risk of tumour formation
•
Immunosuppressive therapy
necessary
Bad interaction with the host cells
Differentiation into glia cells
•
•
Development of Hydrogels for SCI Repair
Synthetic nondegradable
poly(hydroxyethyl
methacrylate) - PHEMA
MOETACl
positive charge – cell adhesion
poly[N-(2-hydroxypropyl)
methacrylamide] - PHPMA
RGD (Arg-Gly-Ser)
cell adhesive peptide sequence
Natural degradable
ECM hydrogels
Porcine spinal cord matrix (SCM)
IKVAV (Ile-Lys-Val-Ala-Val)
Cell adhesive peptide sequence,
neuronal differentiation
Porcine brain matrix (BM)
Cholesterol
Cell adhesion
Porcine urinary bladder matrix
(UBM)
Fibronectin
Cell adhesion
Hyaluronic acid/RGD
Serotonin
Neural differentiation
L-dopamine, serotonin,
tryptamin, carbachol
Release of neurotransmitters
L-dopamine, serotonin,
tryptamin, carbachol
Release of neurotransmitters
Hemisection
Transection
Balloon compression lesion
Hydrogel implantation
Hydrogel injection
Stem cells in SCI
Experimental design and methods
Cell cultures of SPC-01, iPS-NPs or MSCs
o
o
o
o
FACS analysis of cellular markers
Surgery (Brain and Spinal cord lesions)
Transplantation procedure
Behavioral testing (BBB test, Rotarod,
Flat beam test, Motorater, Plantar test
o Immunohistochemical a histological
evaluation
o Gene expression by RT PCR
o Cytokine levels by Luminex (NYMC)
For detailed results see Poster
Development of isolation and expansion protocols for UT MSCs
Umbilical cord tissue mesenchymal stem cells (UT-MSC)
- Comparison of cell isolation methods (explant culture x
enzymatic isolation)
- Cell isolation from fresh x frozen tissue
- Culture conditions – influence of FBS x platelet lysate
- Differentiation into adipocytes, osteocytes and
chondrocytes
- CD marker monitoring at passage 3 and 10
- Monitoring of population doublings (PD) during in vitro
culture
- The use of UT-MSC in in vitro experiments with
decellularized matrix – monitoring of cell proliferation
and migration; monitoring of the effect of laser irradiation
on cell proliferation
- In vivo injection of UT-MSC into spinal cord lesion in rats
Biologic ECM Hydrogels for SCI Repair
•
Mimics the complex composition of ECM (collagens, fibronectin, laminin,
proteoglycans)
•
Tissue specificity – homologous tissue
•
Biologic activity – residual growth factors
•
Injectability – in situ polymerization
•
Degradability
Decellularization of:
 Porcine spinal cord matrix (SCM)
 Porcine brain matrix (BM)
 Porcine urinary bladder matrix (UBM)
 Human umbilical cord tissue
SCM
Medberry,et al., Biomaterials 34, 2013, 1033-1040.
BM
UBM
Method of porcine spinal cord/brain decellularization:
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
Freezing (>16 h at 80oC) and thawing
Cutting to pieces (<3 cm)
Deionized water soak (18-24 h)
0.025% Trypsin (1 h)
3% Triton 100 (1 h)
1 M Sucrose (30 min.)
Deionized water soak (30 min.)
4% Deoxycholic acid (1 h)
0.01% Peracetic acid
Lyophilization and grinding.
Method of porcine urinary bladder decellularization:
1.
2.
3.
4.
5.
Freezing (>16 h at 80oC) and thawing
Mechanical delamination
Deionized water soak (18-24 h)
0.01% Peracetic acid
Lyophilization and grinding
Matrix solubilization:
ECM solubilization (8mg/ml) in 0.01 N HCl, digestion in 1 mg/mL pepsin
Polymerization at pH 7.4 and 37 o C
Medberry, et al., Biomaterials 34, 2013, 1033-1040.
In vitro proliferation of human WJ-MSC on ECM hydrogels
Chemotactic effects of ECM hydrogels on human WJ-MSC
0,5
0,4
0,3
0,2
0,1
0
negative
control
SC-ECM
Brain-ECM
UB-ECM
positive
control SDF-1
ECM Hydrogels Injected into Spinal Cord Hemisection
Neurofilaments – NF160
Axonal growth into the lesion
SCM
2nd week
UBM
500m
m
500m
m
4th week
SCM
Growth of neurofilaments into the lesion
25
control
20
area (%)
500m
m
8th week
SCM
UBM
SC
15
10
5
0
500m
m
50mm
2weeks
4weeks
8weeks
Conclusions
ECM hydrogels prepared by tissue decellularization are biocompatible and
promote in vitro cell growth and migration, and in vivo tissue regeneration
Optimization of ECM hydrogel degradation rate is necessary for the
constructive remodelling of CNS tissue.
CZF nanoparticles are neither cytotoxic nor genotoxic and due to their
relaxivity they represent a very promising contrast agent suitable for cell
labelling.
UT MSCs possess all the same characteristics as BM MSCs and can be
easily expanded in platelet lysate.
NPs or MSCs applied to rat SCI improves lomomotor abilities, spares W
and G matter, produces GF, and reduces apoptosis. MSCs, unlike NPs
reduce levels of inflammatory cytokines
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