BM represents the main source of MSCs with a low frequency of 0.001%–0.01%, depending on the age of donors [35, 36]. These elongated fibroblast-like cells have the capacity to self-renew and differentiate into multiple lineages of mesodermal tissues such as cartilage, bone, fat, tendon, muscle, myocardium, and stromal cells. Endodermal (endothelial cells) and ectodermal (neural cells) lineages have also been obtained in vitro after specific induction [5, 33, 37]. Because of the high potential of these cells to be subcultured and differentiated in vitro, they have aroused a great interest in their use for cell and gene therapy. In the present study, we evaluated two other potential sources of mesenchymal cells: MPB and full-term UCB.
UCB and MPB are currently used as a source of hematopoietic cells and progenitor cells after chemotherapy, and their application as an MSC source is still debated. Previous studies have demonstrated that preterm UCB contains more adherent fibroblastoid cells than full-term UCB but that only 25% of UCB harvested gave rise to mesenchymal-like cells [31, 38]. MSCs can also be isolated from the subendothelial layer of the umbilical cord vein but not in full-term UCB [39, 40]. Recently, Lee et al. obtained homogeneous plastic adherent cells from the MNC fractions of cryopreserved UCB . These cells exhibited fibroblast-like morphology and typical mesenchymal phenotype. The cause of these divergent results could be related to differences in culture conditions required for the growth of UCB-MSCs. Recently, critical parameters for the isolation of MSCs from UCB have been described. The time between collection and isolation (<15 hours), the volume of samples, and the count of MNCs seem to be crucial for obtaining MSCs from full-term UCB . In MPB, similar controversial results have been reported about the effective presence of MSCs. MPB-MSCs have been described in blood collections from breast cancer patients and in normal volunteer blood. In these studies, MSCs were also isolated though the plastic adhesion method. After several passages, the adherent cells exhibited the same morphology and phenotype as BM-MSCs. However, using the same procedure, no MSCs have been observed by other groups [12, 27–29]. In our study, we developed different methods for obtaining MSCs from full-term UCB or MPB. In the first, we used the classic plastic adhesion and subcultures. In the second, we added 5% of CM during the first 48 hours of PM. CM was obtained from BM-MSC cultures containing a homogeneous population with more than 90% of cells expressing the putative mesenchymal markers (SH2, SH3). Indeed, MSCs produce their own cytokines and growth factors essential to their development. These findings prompted us to evaluate if the addition of BM-MSC CM can modulate the growth of MSCs in UCB and MPB samples. The third method was the immunoselection of CD133+ cells directly from fresh samples. CD133+ cells are considered as a population of noncommitted early progenitors capable of self-renewing and differentiating into blood cells and other cell types. Thus, CD133 seems to be a marker associated with more primitive stem cell phenotype than CD34 . Using the purified CD133-positive cell fraction, we obtained a large quantity of MSCs after the PM. After four passages, the number of cells harvested was similar to BM-MSCs. These results suggest that the CD133-positive cell fraction contains both hematopoietic and mesenchymal stem cells. Indeed, more than 85% of CD133-positive cells were CD34 positive. The absence of CFU-F in MPB and UCB prior to expansion or selection could have diverse explanations. The MSC frequency in adult BM is one in 3 to 4 × 104 cells, using the CFU-F assay . In similar conditions, UCB and MPB do not produce CFU-F. The frequency of MSCs in UCB and MPB is thus so low that their survival or growth can be affected by culture conditions (serum, pH of medium). The MNC fraction obtained from UCB and MPB yields an adherent layer of heterogeneous cells in the primary culture. We cannot exclude the possibility that accessory cells such as macrophages, lymphocytes, or other hematopoietic cells could inhibit the proliferation of CFU-F. To determine whether MSCs isolated from UCB and MPB, like BM-MSCs, are able to differentiate into multiple cell types, cells were plated into specific induction media for the generation of adipocytes, osteocytes, chondrocytes, and neuronal/glial cells. After 2–3 weeks of culture under these conditions, lipid vacuoles, calcium deposits, chondrogenic matrix, and neuronal/glial cells were observed, which demonstrated the multipotentiality of MSCs. Finally, we observed that MSCs, isolated from the MPB or UCB as embryonic stem cells, express Oct4, a transcriptional factor present in undifferentiated cells with high proliferative potential.