| Abstract |
Introduction
The recognition of the role of the mesenchymal stromal cells (MSC) in hematopoiesis, as part of the bone
marrow microenvironment, has renewed the interest in the ex vivo expansion of the cord blood (CB) as a
source of hematopoietic stem cell (HSC) for transplantation. Children’s MSC appear to have different
biological properties compared to the adult counterpart. The cord blood is a rich source of stem cells,
whose characterization and isolation requires both specific indicators - such as the surface antigen CD34
and the corresponding CD133 - as well as reliable and reproducible protocols.
In the present study we initially assessed the isolation protocol of CB-CD133+ cells. Furthermore we
evaluated the role of MSC from children’s bone marrow in the ex vivo expansion of haematopoietic stem
cells (CB and BM).
Materials and Methods
The iosolation of CD34+ cells was performed according to an immunomagnetic method following
manufacturer’s instructions. The CD133+ isolation using the same methodology had no reproducibility
and different experimental approaches were applied in order to improve the method.
MSC cells were isolated and cultured from childrens’ bone marrow; their characterization included the
evaluation of CFU-F colonies and the differentiation towards osteo-, lipo-, chondrocytes under
appropriate culture conditions.
The co-culture assay of CD34+ in the presence of MSC was supplemented with 2 combinations of growth
factors: a) GF1: SCF, Intereukin-3 (IL-3), G-CSF, GM-CSF and b) GF2: SCF, thrombopoietin (TPO),
Intereukin-6 (IL-6), Flt-3 ligand, growth factors considered to affect more primitive cell populations. The
identification of the cell subpopulations was achieved by immunophenotypic analysis.
The evaluation of the clonogenic capacity of the expanded hematopoietic population was performed by
assessing the development CFU-GM colonies in methylcellulose cultures.
The SDF-1 and Ang-1 levels in the supernatant of the cultures were determine by immunoenzymatic
ELISA method and cDNA samples were tested by polymerase chain reaction for the expression of Oct-4,
Sox2, and Nanog genes.
Results
Our study showed that a feasible and reliable isolation of CB-CD133+ cells was not achievable even
when modified protocols applied. No sufficient population to further study the co-culture with MSC could
be obtained. As a result the research was focused on the CD34+ cell population.
Characterization of the BM-MSC. The MSC developed in the first and maintained in subsequent
passages both the characteristic fibroblast-like shape and the corresponding surface markers.
CFU-F assay. The MSC fraction of the mononuclear fraction yielded 5.68±0.9 colonies. In P1 the total
CFU-F colonies were 38.75±4.77 and decreased in the successive passages.
CD34 and CD133 isolation. The isolation of the CD34+ population by immunomagnetic method yielded a
highly purified population (93.0±1.06%) while the CD133+ isolation resulted in a population of cells with a
CD133 expression ranging from 10 to 85% (median 60%).
Ex vivo expanded CD34+ cells and total population of HSC. The presence of MSC with a combination of
growth factors yielded the highest increase of both the total cell number and the CD34+ population. The
two different combinations of GF appeared to exert similar effects on the expansion of CD34+, whereas
the presence of MSC alone in the culture of both CB and BM-CD34+ increased their number by
1.58±0.59 and 2.07±0.78 times respectively. However, this condition showed the lowest expansion of
CD34+ cells as compared to the condition where only growth factors were added. The comparison
between CB- and BM-CD34, showed greater expansion capacity of the CB-CD34 regarding both the
total cell number and the CD34+ population.
Immunophenotypic characteristics of the expanded populations. The percentage of CD34+ cells was
higher in the absence of growth factors, regardless of the source of HSC. The preservation of primitive
cell characteristics was further supported by the evaluation of the CD34+38- and CD34+33- cell
subpopulations the expression of which was higher in the condition where hematopoietic cells were
cultivated in the presence of MSC alone.
CFU-GM development. Although the presence of MSC alone in the culture did not particularly increase
the number of CD34+ cells, the population at the end of the co-culture was characterize by greater
clonogenic capacity.
Expression of Oct-4, Sox2 and Nanog. The MSC showed lower expression of Oct-4 and Sox2. Nanog
was expressed in all the various conditions studied.
Evaluation of SDF-1 and Ang-1 levels (ELISA). Both SDF1a and Angio-1 were expressed in the
supernatant especially in the presence of MSC which appears to be their main source. A higher
expression of SDF1a was observed in co-culture of BM-CD34 cells with MSC either alone or in
combination with growth factors, compared to that of CB-CD34 with MSC cells.
Discussion
We assessed the effect of children’s BM-MSC and that of recombinant early- and late-acting growth
factors, alone and in combination, on the proliferation, expansion, and differentiation of hematopoietic cell
population derived from cord blood and bone marrow. The role of cytokines without stromal support in the
expansion of HSCs has been extensively studied but results in an increased total cell number with
simultaneous differentiation and maturation of the expanded population, finding confirmed herein. This is
why supplementation of the coculture with MSC, seemed an attractive alternative which would promote
mainly the progenitor cell expansion. We used two growth factor combinations one including mostly late
acting and the second ones considered as more early acting. We observed no difference between the
two GF cocktails in any of the studied parameters possibly indicative of a quite more complex cross talk
among various factors and, for sure, not limited to a small number of cytokines and growth factors. In our
results less differentiation occurred in cells cultured with MSC alone, than with GF or GF+MSC and also
MSC and GF provide more efficient total cell proliferation. A similar finding has been reported by others
who observed a more efficient expansion of CD34+ cells when CB-CD34 were supplemented with
combination of MSC and a variety of growth factors. In the literature, there is a big range of total
nucleated cell expansion reported when both MSC and cytokines were present in culture and there was
no difference to that observed in the presence of cytokines alone. MSC seem to preserve more the
clonogeneic population as the frequency of the developed CFU-GM was the highest in their presence. No
direct comparison can be made with the results of others as the experimental conditions (CB MNC/MSC
co culture) or the result expression (colonies per cell number) are different. The relationships between
HSC and the cells of the microenvironment were implied by the presence of HSC infiltrating the stroma.
The study of the trypsinized cells of the feeder layer, showed an expression of CD34 and CD45 and
CFU-GM development in the corresponding assays. Our results support the report of others who tried to
show the significance of the cell to cell contact between HSC and stromal cells. Further research is
required in order to fully clarify the importance of the intimate relationship between the two populations
on the expansion and “stemness” of HSC. Oct-4, Sox2, and Nanog were selected based on the
knowledge that they are involved in self-renewal and pluripotency of stem cells and are frequently used
as markers for primitive cells. Our results showed that no condition modified the expression of any of the
three transcription factors. Despite the fact that MSC are undifferentiated cells they had the lowest
expression of the Oct4, Sox2, Nanog and were not influenced by the different culture conditions. These
results are in agreement to those who recently reported the expression of Oct4, Nanog mRNA in BMMSCs
to be much lower compared to pluripotent stem cells and Sox2mRNA to be minimally detected in a
number of BM-MSC preparations. Reports from human adipose-derived stem cells found either no
change on the expression of Nanog and Oct4 with low doses of bone morphogenetic protein (BMP) in ex
vivo expansion study or Sox-2 and in addition Rex-1 to remain unaltered in a spinner flask cultivation.
Earlier experiments have shown that the cross-talk between SDF-1a and its ligand participates in the
homing of stem cells in their niches and repopulation of the BM. We found higher SDF-1a levels where
MSC were present and difference in the levels between CB and BM cultures confirming the suggestion
that SDF-1a could be among the factors playing critical role to the delayed engraftment observed in CB
transplantation. It has been also reported that supernatants from in vitro-cultured CD34+ cells contained
substantial amounts of SDF-1a and we indeed detected SDF-1a expression, in the cultures without
MSC. Ang-1 which has been reported that may have a role in maintaining hematopoietic stem cells in a
quiescent state within the BM did not exhibit any difference between our BM and CB cultures and in the
various conditions tested with the requirement though,the presence of MSC in the culture. In other
reports researchers were unable to detect Ang-1 in all their samples. In addition there was discrepancy
with the results of the literature. They attribute them to an age-related post-transcriptional effect on the
expression of proteins and to the exposure of MSC to fetal bovine serum and FGF-b. As long as our MSC
culture protocol includes both FBS and FGF-b and the origin of our BM-MSC is young donors this
rationale, is further supported and seems to add to the notion of age related differences of the cell
performance. In many cases no direct comparison can be made with data from the literature, as there
are many and major differences in the culture conditions applied. This emphasizes the need for more
standardized protocols while, on the other hand, stresses the complexity of the biology even in the limited
ex vivo system and possibly clarifies a very small part of it. We observed that CB-CD34 expansion
produces similar results to BM-CD34 expanded cells when only stromal cells are present and no additive
effect was seen in BM-CD34 when growth factors were added. Biological features of CB hematopoietic
progenitors seem to provide them the potential for more successful ex vivo expansion than their BM
counterparts
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