Identification of subpopulations in mesenchymal stem cell-like cultures from human umbilical cord

Abstract

Background: A variety of cell types can be identified in the adherent fraction of bone marrow mononuclear cells including more primitive and embryonic-like stem cells, mesenchymal stem cells (MSC), lineage-committed progenitors as well as mature cells such as osteoblasts and fibroblasts. Different methods are described for the isolation of single bone marrow stem cell subpopulations - beginning from ordinary size sieving, long term cultivation under specific conditions to FACS-based approaches. Besides bone marrow-derived subpopulations, also other tissues including human umbilical cord (UC) have been recently suggested to provide a potential source for MSC. Although of clinical importance, these UC-derived MSC populations remain to be characterized. It was thus the aim of the present study to identify possible subpopulations in cultures of MSC-like cells obtained from UC. We used counterflow centrifugal elutriation (CCE) as a novel strategy to successfully address this question.

Results: UC-derived primary cells were separated by CCE and revealed differentially-sized populations in the fractions. Thus, a subpopulation with an average diameter of about 11 mum and a small flat cell body was compared to a large sized subpopulation of about 19 mum average diameter. Flow cytometric analysis revealed the expression of certain MSC stem cell markers including CD44, CD73, CD90 and CD105, respectively, although these markers were expressed at higher levels in the small-sized population. Moreover, this small-sized subpopulation exhibited a higher proliferative capacity as compared to the total UC-derived primary cultures and the large-sized cells and demonstrated a reduced amount of aging cells.

Conclusion: Using the CCE technique, we were the first to demonstrate a subpopulation of small-sized UC-derived primary cells carrying MSC-like characteristics according to the presence of various mesenchymal stem cell markers. This is also supported by the high proliferative capacity of these MSC-like cells as compared to whole primary culture or other UC-derived subpopulations. The accumulation of a self-renewing MSC-like subpopulation by CCE with low expression levels of the aging marker senescence-associated beta-galactosidase provides a valuable tool in the regenerative medicine and an alternative to bone-marrow-derived MSC.

Figure 1

Cell size distribution in the UC-derived primary culture (a, b, c) and in the subpopulations of small-sized (d, e, f) and large-sized (g, h, i) cells obtained after CCE. Cell size was investigated using Vi-CELL Series Cell Viability Analyzer (a, d, g) and flow cytometry using FSC signals as a measure of cell size (c, f, i). Images of the corresponding cell cultures were taken on the next day after cell seeding using phase contrast microscopy (total magnification 100 ×) (b, e, h). Exemplary results of one representative experiment are presented (n = 3).

Figure 2

Immunophenotype of cells obtained from human umbilical cord tissue. A. Flow cytometric analysis of surface antigen expression in the UC-derived cultures was performed using the labelled antibody anti CD44-PE; anti CD73-PE; anti CD90-FITC; anti CD105-R-PE. At least 10,000 events are displayed. B. Exemplary CD90 and CD73 expression normalized on cell size: comparison of subpopulations of small- (filled histogram) and large-sized cells (unfilled histogram). The fluorescence of the conjugated monoclonal antibodies (anti CD90-FITC and anti CD73-PE) as well as the forward scatter (FSC) signals were measured on linear scale. The ratios were calculated by the EPICS XL/MCL flow cytometer (Beckman Coulter).

Figure 3

Cell proliferative activity in cultures generated from the UC-derived primary cultures and from subpopulations of small- and large-sized cells. Proliferation was measured by counting the total number of obtained intact cells. Following CCE, cells were seeded at a density of 500 cells/cm² and cultivated in αMEM containing 10% human serum over 4 passages (P5–P8) in quadruplicates. Student's t-tests were performed for the recognition of the significant differences (marked with asterisks) in comparison to UC-derived primary cell population.

Figure 4

SA-β-gal-positive cells in the UC-derived primary cell population (A) and in the CCE-derived subpopulations of small- (B) and large-sized (C) cells. Cells were cultured for 6 days after elutriation. Following subculture, the cells were seeded at the density of 6,000 cells/cm² and cultured for further 48 h in complete medium. A relative high portion of binucleated cells (arrows) were detectable in the subpopulation of the large-sized cells. Student's t-tests were performed for the recognition of the significant differences (marked with asterisks) in comparison to UC-derived primary cell population.