Srp Arh Celok Lek. 2013 Mar-Apr;141(3-4):178-86.

Mesenchymal stem cells isolated from peripheral blood and umbilical cord Wharton’s jelly.

Drenka Trivanović1 Jelena Kocić1, Slavko Mojsilović1, Aleksandra Krstić1, Vesna Ilić2, Ivana Okić Đorđević1, Juan Francisco Santibanez1, Gordana Jovčić1 , Milan Terzić3, Diana Bugarski1

1   Laboratory of Experimental Hematology, Institute for Medical Research, University of Belgrade, Belgrade, Serbia

2    Laboratory of Immunology, Institute for Medical Research, University of Belgrade, Belgrade, Serbia

3   Institute for Gynecology and Obstetrics, Clinical Center of Serbia, Belgrade, Serbia

 

SUMMARY

Introduction Mesenchymal stem cells (MSCs) are a promising tool for regenerative medicine, but due to the heterogeneity of their populations, different sources and isolation techniques, the characteristics defining MSCs are inconsistent.

Objective The aim of this study was to compare the characteristics of MSCs derived from two different human tissues: peripheral blood (PB-MSCs) and umbilical cord Wharton’s Jelly (UC-MSCs).

Methods The PB-MSC and UC-MSC were isolated by adherence to plastic after gradient-density separation or an explant culture method, respectively, and compared regarding their morphology, clonogenic efficiency, proliferating rates, immunophenotype and differentiation potential.

Results The MSCs derived from both sources exhibit similar morphology, proliferation capacity and multilineage (osteogenic, chondrogenic, adipogenic and myogenic) differentiation potential. Differences were observed in the clonogenic capacity and the immunophenotype, since the UC-MSCs showed higher CFU-F (Colony-Forming Units-Fibroblastic) cloning efficiency, as well as higher embryonic markers (Nanog, Sox2, SSEA4) expression. When additional surface antigens were analyzed by flow cytometry (CD44, CD90, CD105, CD33, CD34, CD45, CD11b, CD235a) or immunofluorescent labeling (vimentin, STRO-1 and α-smooth muscle actin), most appeared to have similar epitope profiles irrespective of MSC source.

Conclusion The results obtained demonstrated that both MSCs represent good alternative sources of adult MSCs that could be used in cell therapy applications.

PMID: 23745340

 

SUPPLEMENT:

Due to their indispensable regenerative, reparative, angiogenic and immunosuppressive properties, MSCs have generated increasing interest in a variety of biomedical disciplines and several areas of ongoing clinical applications. However, because of the heterogeneity of the stem cell populations, as well as different sources of their origin and different isolation techniques used among laboratories, the characteristics defining MSCs are inconsistent. Whether these cell populations isolated from diverse sources represent intrinsically similar or different cell types is still largely under debate.

The objective of this study was to compare biological characteristics of MSCs derived from two different human tissues with a common feature that both are discarded after routine medical interventions and therefore are ready available source for MSCs isolation. We chose to compare MSCs derived from one adult tissue, peripheral blood, and one perinatal tissue, umbilical cord Wharton’s Jelly. The comparison was made to describe MSCs behavior in cell culture, which could be useful for their future use in potential medical procedures. After isolation and establishment of long-term cultures, cells were further characterized regarding their morphology, clonogenic efficiency, proliferating rates, immunophenotype and differentiation potential. The data obtained demonstrated that the two MSCs types showed similar morphology, proliferative rates and differentiation capacity, but differed in clonogenic capacities and, in part, in immunophenotype.

The UC-MSCs possessed 2-fold higher colony forming efficiency, similarly to previous reports demonstrating higher proliferation capacities of neonatal tissue-derived MSCs in comparison to the adult tissue-derived MSCs [1, 2, 3]. However, although the colony forming efficiency of the PB-MSC was lower, the proliferation assays demonstrated similar and extremely high proliferation rate of both PB-MSCs and UC-MSCs. The immunophenotyping evidenced the expression of embryonic markers, such as Nanog, Sox2 and SSEA4, in both MSCs types, which is in agreement with findings that human adult MSCs derived from different sources may express embryonic markers [4, 5]. Although these results indicated that both MSCs populations are highly multipotent, higher percentage of cells positive for these markers in UC-MSCs population indicated that postnatal UC-MSCs are more primitive then adult PB-MSCs. Other surface antigens, analyzed by flow cytometry (CD44, CD90, CD105, CD33, CD34, CD45, CD11b, CD235a) or immunofluorescent labeling (vimentin, STRO-1 and α-smooth muscle actin), showed similar expression profiles, irrespective of MSC source. Due to differences in marker expression, the conserved mesodermal differentiation capacity within the investigated MSCs seems to be more relevant for their quality at present, as the multilineage differentiation potential is the hallmark of MSCs.

The vast variability and heterogeneity between various MSCs, reported by different groups as well as in our study, beside other properties, could be influenced by the tissue of their origin and the “age” of MSCs. Of particular interest are the differences and similarities, between the MSCs derived from adult and neonatal tissues, since, as confirmed in this study, MSCs from birth-associated tissues are considered more primitive than those obtained from other tissues, with higher proliferative and expansion potential, as well as greater and much easier accessibility of clinical samples [2]. As for the peripheral blood-derived MSCs, their very low number in human blood under steady-state conditions is potentially limiting issue, in addition to their true tissue origin. Namely, there are speculations that these cells are migrants form the bone marrow or other organs [1, 2, 6, 7], since similar to the ability of hematopoietic stem cells to egress from the marrow and home to other sites, MSCs might possess this feature, too. Additionally, with the analogy to the hierarchical organization of hematopoietic stem cells, in which the multipotency is progressively restricted, the population diversity observed between various MSCs could be due to the isolation of cells representing stem/progenitor cells of different maturity.

In summary, the lack of specific MSC markers makes their identification and study more difficult, but until this issue is resolved, the ease of accessibility for isolation, high expansion potential in culture, presumptive plasticity, immunosuppressive properties, homing to sites of tissue injury and ethical considerations are supporting the use of adult MSCs for clinical application.

 

References:

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Fig1

Figure 1. Embryoinic stem cell markers expression in PB-MSCs and UC-MSCs. Representative flow cytometry histograms show the expresion (unsheded peacs) of selected molecules (SOX2, NANOG and SSEA4) by different MSC populations compared to isotype controls (shaded).

 

Fig2

Figure 2. Differentiation of PB-MSCs and UC-MSCs. (A) Osteogenic differentiation was proved by positive staining for ALP activity; (B) Chondrogenic differentiation of MSCs with positive staining of proteoglycans by Safranin O; (C) Adipogenic differentiation was confirmed by Oil Red O staining of intracytoplasmatic lipid droplets. (D) Myogenic differentiation characterised by the formation of myotubes stained with crystal violet.

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