12th Congress of the European Hematology ... - Haematologica

12 th Congress of the European Hematology Association
Stem cell biology and microenvironment
0326
THE SIMILARITIES OF HUMAN MESENCHYMAL STEM CELLS AND PERICYTES
D. Covas, R.A. Panepucci, A.M. Fontes, W.A. Silva-Jr, M.D. Orellana,
L. Neder, A.R.D. Santos, L.C. Perez, M.C. Jamur, M.A. Zago
University of S. Paulo, RIBEIRAO PRETO, Brazil
Background. Multipotent mesenchymal stromal cells (MSC) can be isolated
from a variety of tissues. Their relationship with pericytes and
fibroblasts has not been established thus far, although they share many
functional properties. Aims. To evaluate the relationship between MSC
and pericytes on the basis of gene expression. Materials and Methods.
We analyzed 14 samples of MSCs from adult or fetal tissues, two MSC
cultures differentiated in vitro, and two cultures of retinal pericytes
obtained either by immunological separation with anti-CD146 or adhesion,
respectively. Cells were cultured under appropriate conditions to
promote osteogenic, adipocytic or chondroblastic differentiation, evaluated
both by morphology and by specific histochemical staining. Global
gene expression was analyzed by SAGE for human retinal pericytes
and MSC from BM and from umbilical cord vein. The expression of 39
selected genes was evaluated by serial dilutions of the cDNA in RT-PCR
reactions for 31 different cell cultures including undifferentiated and differentiated
MSC from adult and fetal sources, pericytes, fibroblasts,
endothelial cells, and cells from bone marrow, liver, brain, skeletal muscle,
skin and heart. Real time PCR for five genes confirmed the results
obtained by serial dilution. Cluster analysis of gene expression data was
performed using the Cluster 3.0 software, both for gene expression of
31 cell cultures evaluated by serial dilution and for 17 SAGE libraries
from MSCs, endothelial cells, skeletal and heart muscles, CD34 + cells,
pericytes, skin fibroblasts, stellate hepatic cells and myofibroblasts differentiated
from stellate cells. The use of human cells in these experiments
has been approved by the institutional research review committee.
Results. Cell morphology of MSCs and pericytes were very similar,
both under light and transmission electron microscopy, as well as the
phenotypes defined by 23 markers, especially negativity for CD34,
CD33, CD45, CD14, HLA-DR, KDR and CD31, and positivity for
CD73, CD90, CD29, CD44 and HLA-I. Osteogenic and adipocytic differentiation
was documented for most MSC cultures and for pericytes;
chondrocytic differentiation was positive for the MSCs that were tested.
Cluster analysis of SAGE gene expression profiles showed that MSC,
pericytes and stellate hepatic cells form a consistent group, separated
from another consistent group formed by fibroblasts, smooth muscle
cells and myofibroblasts differentiated from stellate cells. Cluster analysis
of semi quantitative expression data of the 39 selected genes confirmed
that all the MSC lines, pericytes and fibroblast share a common
expression profile distinct from other normal cells. Despite the similarity,
FSP-1 was more frequently expressed on fibroblasts and NG-2 on
pericytes as compared with MSCs. Conclusions. We conclude that human
MSC and pericytes, as operationally defined by culture methods, are
similar cells located in the wall of the vasculature where they function
as a source of precursor cells for repair and tissue maintenance. Additionally,
the close relationship of fibroblasts and smooth muscle cells with
myofibroblast differentiated from stellate cells further indicates that
these functionally compromised cells may be closely related to these precursor
cells, a proposition that has conceptual and practical implications.
0327
STUDY OF FUNCTIONAL AND MOLECULAR CHARACTERISTICS OF THREE DIFFERENT
BONE MARROW CELL POPULATIONS FOR EXPANSION OF MESECHYMAL STEM CELLS
H. Papadaki, M.C. Kastrinaki, A. Damianaki, V.M. Vlahava,
P. Fragioudaki, E. Stavroulaki, G.D. Eliopoulos
University of Crete School of Medicine, VOUTES, HERAKLION, Greece
Background. There is currently great interest in exploring the use of
mesenchymal stem cells (MSCs) for clinical purposes. Identification of
markers and sources for MSC isolation is, therefore, of particular importance.
Aims. To compare the colony/cloning efficiency, expression of
stemness markers and differentiation potential of three different bone
marrow (BM) derived cell populations, namely the CD105 + /CD45 – , the
Glycophorin A (GlycoA) – /CD45 – and BM mononuclear cells (BMMCs).
Methods. Normal human BM cells were isolated from posterior iliac crest
aspirates. A colony forming unit-fibroblast (CFU-F) assay was used for
the determination of the colony efficiency of the BMMC or the
118 | haematologica/the hematology journal | 2007; 92(s1)
immunomagnetically sorted CD105 + /CD45 – and GlycoA-/CD45- BM
cell fractions. CFU-F colonies derived from the three different cell subpopulations
were isolated with clone-rings and further expanded using
a standard MSC culture protocol. Clone efficiency was determined by
evaluating the number of clones survived after full in vitro expansion versus
the number of initially expanded clones. The adipogenic, osteogenic,
and chondrogenic (AOC) differentiation potential of MSCs expanded
from these clones and cultured under defined culture conditions, were
assessed by using Oil red O, Alkaline phosphatase/Von Kossa and Alcian
Blue immunohistochemical stains respectively, as well as by evaluating
the expression of specific differentiation-associated gene expression by
RT-PCR. Finally, the immunomagnetically sorted CD105 + /CD45 – and
GlycoA – /CD45 – cell populations were assessed at day-0 for the expression
of Oct4 and Nanog stemness genes using real-time RT-PCR. Results.
In the CFU-F assay, the colony efficiency was statistically significantly
higher when CD105 + /CD45 – cells were used in the culture (144.8±62.8)
compared to GlycoA – /CD45 – cells (21.5±16.2) or BMMCs (10.9±11.6)
(p