autologous tissue repair cells from bone marrow require mac

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autologous tissue repair cells from bone marrow require mac
AUTOLOGOUS TISSUE REPAIR CELLS FROM BONE MARROW REQUIRE MAC-1 INTEGRIN
FUNCTION FOR EXPANSION OF BOTH HEMATOPOIETIC CELLS AND MESENCHYMAL STEM CELLS
J. Osborne, K. Ledford, C. Parrish , A. Wong, F. Zeigler, and R. Bartel
Aastrom Biosciences, Inc. Ann Arbor, MI USA
ABSTRACT
IXMYELOCEL-T is an autologous, bone marrow derived, ex-vivo
cultured, mixed cell product composed of both hematopoietic
cell types and mesenchymal stem cells, which has shown clinical
efficacy in ischemic tissue repair. MAC-1 is an integrin formed
by non-covalent association of αM and β2 subunits, which
are involved in many key functions of hematopoietic cells,
including intercellular adhesion and lymphocyte activation via
the ligands ICAM-1 and CD90/Thy-1. FACS analysis shows that
~50% of the total mixture of TRCs express the Mac-1 integrin
complex, and this expression is limited to hematopoietic cells,
primarily lymphocytic and myeloid lineages; the highest level
of expression is detected by differentiated granulocytes and
macrophages based on co-expression of CD66b and CD14.
Characterization of the TRC manufacturing process has shown
that only the CD14+ monocyte/macrophages and the CD90+
MSCs expand significantly during the ex-vivo culture, and we
hypothesized that MAC-1 expression had functional significance
in the generation of the TRC mixed cell population in vitro. Using
neutralizing antibodies to CD11b, CD18, and the MAC-1 complex
we show that CD14+ macrophage expansion is blocked by as
AASTROM CULTURE PROCESS SUPPORTS A PRODUCT WITH INCREASED SIZE AND
GRANULARITY COMPARED TO BM MNCS
BMMNC
much as 80% when MAC-1 binding is blocked. Interestingly, the
expansion of the CD90+ MSCs which do not express MAC-1,
was also highly dependent on MAC-1 function since as much as
65% of the MSC expansion was blocked by MAC-1 neutralizing
antibodies. Although the magnitude of these effects is donordependent, all donors tested to date display similar dependence
on MAC-1 function. We are currently investigating the
significance of MAC-1 functions in engraftment and efficacy in a
rodent model of hind limb ischemia.
IMMUNOFLUORESCENT STAINING DEMONSTRATES INTERACTION BETWEEN
CD90+ AND CD11B+ PHENOTYPES
IXMYELOCEL-T
AASTROM CULTURE PROCESS EXPANDS CD14+AUTO+ (MACROPHAGE) AND CD90+ (MSC) POPULATIONS
Immunostaining for CD90+ MSCs and CD11b+ macrophages imaged at 10x magnification
NEUTRALIZATION OF CD11B DURING THE CULTURE PROCESS VISIBLY REDUCED ADHESIVE
INTERACTIONS BETWEEN MSCS AND CD11B+ HEMATOPOIETIC CELLS
CD14+ PKH26
CD90+ Dy649
MACROPHAGES ARE CD11B+
A
B
C
Contour density plots generated from
flow cytometric analysis demonstrating
that the CD14+ macrophage
population within the IXMYELOCEL-T
product is CD11b+
A) CD11b+ population (A1)
B) CD14+ population (B4)
C) CD14+CD11b+ (C2)
GRANULOCYTES ARE CD11B+
A
B
C
Contour density plots generated
from flow cytometric analysis
demonstrating that the CD66b+
granulocyte population is
overwhelmingly CD11b+
20x imaging prior to phenotypic analysis demonstrating reduced adhesion to MSCs. BM MNCs were cultured using
Aastrom proprietary process. Aastroms base media was spiked with LEAF pure neutralizing CD11b antibody clone
ICRF44, LEAF pure Isotype Control, or an equal volume of DPBS. Cultures were then grown for 12 days. After the
culture process cells were harvested with trypsin, counted manually by trypan, and stained for flow cytometric
quantification of surface marker expression. N=3 donors.
MAC-1 NEUTRALIZATION
SIGNIFICANTLY REDUCED THE NUMBER
OF CD11B+ CELLS AT HARVEST
MAC-1 NEUTRALIZATION
SIGNIFICANTLY REDUCED THE NUMBER
OF CD90+ CELLS AT HARVEST
A) CD11b+ Population
B) CD66b+ Population
C) Note the CD66b+ population shifts
up when stained for CD11b, indicating
this population is dual positive
CD11b+CD66b+
INTERACTIONS BETWEEN MSCS AND A CD14+ HEMATOPOIETIC COMPONENT
EXIST DURING CULTURE PROCESS
Significant reduction in CD11b+ cell number
resulting from MAC-1 neutralization is observed.
N=3 donors
Immunostaining for CD14+ populations day
7 culture of IXMYELOCEL-T, imaged at 10x
magnfication. Green=CD14+ Blue=Nuclei
Note the interaction between CD14+ and
phase contrast stromal layer
Significant reduction in CD90+ MSC cell number
resulting from MAC-1 neutralization is observed.
N=3 donors
CONCLUSIONS
•Stromal CD90+ and hematopoietic
CD11b+ cells interact during
production of IXMYELOCEL-T
•Neutralization of CD11b significantly
reduces the number of MAC-1+ cells
at harvest.
•Neutralization of CD11b function
during culture reduces this interaction
leading to inhibited CD90+ expansion.
•Interactions facilitated by MAC1 are necessary for expansion of
IXMYELOCEL-T MSC population.

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