Identification of subpopulations in mesenchymal stem cell-like cultures from human umbilical cord
© Majore et al; licensee BioMed Central Ltd. 2009
Received: 18 February 2009
Accepted: 20 March 2009
Published: 20 March 2009
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.
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 μm and a small flat cell body was compared to a large sized subpopulation of about 19 μm 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.
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 β-galactosidase provides a valuable tool in the regenerative medicine and an alternative to bone-marrow-derived MSC.
MSC were first identified in the bone marrow  and characterized as a population of non-hematopoetic multipotent stem cells. Similar to other stem cell types MSC possess the potential for self-renewal and for differentiation into highly specialized cells upon appropriate stimulation. For example, MSC differentiation into cell types of the mesodermal lineage has been extensively investigated [2, 3]. Moreover a variety of studies have demonstrated that MSC may also generate mature cells typically arisen from endoderm [4–6] or ectoderm [7–9] suggesting that cultures of bone marrow MSC may represent an admixture of phenotypically, functionally and biochemically different cells [10–12]. Indeed, besides MSC a variety of different cell types of predominantly mesodermal origin could be identified in the adherent fraction of bone marrow mononuclear cells including more primitive and embryonic-like stem cells, lineage-committed progenitors as well as mature cells such as osteoblasts and fibroblasts [13–16]. Therefore bone marrow MSC cultures appear to provide a broad spectrum of stem cells with various differentiation potential. However, the amount of primitive stem cells in these cultures is rare and can vary depending on the age of donor, method of cell isolation or cultivation respectively [17, 18].
The research over the last decade has demonstrated that bone marrow is not the exclusive source for MSC. Cells with similar characteristics can be extracted from virtually all post-natal  as well as extra-embryonic tissues such as amniotic membrane , placenta  and UC [22–24]. However, the in vivo immunophenotype of MSC and distinct unique surface markers for the exact identification of MSC in the various tissues remains unclear . In 2004, the International Society for Cellular Therapy appointed a set of standard criteria to facilitate a more uniform characterization of MSC. This current statement corroborates the prevalent opinion that the simultaneous expression of cell surface markers including CD44, CD73, CD90 and CD105 with a concomitant absence of CD45 and CD34 expression represents a specific phenotype for cultured MSC .
Different methods are described for the isolation of single bone marrow stem cell subpopulations – beginning from ordinary size sieving [26, 27], long term cultivation under specific conditions [15, 28, 29] to FACS-based approaches [30, 31] and previous work has suggested certain differentially-sized subpopulations of small, rapidly proliferating cells with high differentiation capacity [16, 30]. In this context, it was the aim of the present study to identify possible subpopulations in cultures of MSC-like cells obtained from human UC and we are the first using CCE as a novel strategy to successfully address this question.
MSC-like cell isolation from umbilical cord tissue
Human umbilical cords were obtained from consenting patients (n = 3) delivering full-term (38–40 weeks) infants by Cesarean section. The use of this material has been approved by the Institutional Review Board, project #3037 in an extended permission on 17th June, 2006. After removing the blood cells from the UC with PBS (phosphate buffered saline) enriched with 5 g/l glucose (Sigma Aldrich Chemie, Deisenhofen, Germany), 50 μg/ml gentamicin (PAA Laboratories GmbH, Pasching, Austria), 2.5 μg/ml amphotericin B (Sigma), 100 U/ml penicillin and 100 μg/ml streptomycin (PAA Laboratories GmbH), the UC tissue was cut into approx. 0.5 cm3 large pieces and then incubated in αMEM (Invitrogen GmbH, Karlsruhe, Germany) reinforced with 15% of allogous human serum (kindly provided by the Division of Transfusion Medicine, Medical University Hannover, Germany) and 50 μg/ml gentamicin at 37°C in a humidified atmosphere with 5% CO2. The medium was changed every second day. A beginning outgrowth of an adherent cell layer from single tissue pieces was observed after approx. 10 days. After 2 weeks the UC tissue was removed and the adherent cells were harvested by accutase treatment according to the manufacturer's protocol (PAA Laboratories GmbH) for 5 min at 37°C. The obtained cell suspension was centrifuged at 200 × g for 5 min and the cells were resuspended in αMEM supplemented with 10% human serum and 50 μg/ml gentamicin and subcultured at a density of 4,000 cells/cm2. Following the second subconfluent passage, cells were harvested for the following characterization experiments or cryopreserved. Cryoconservation was performed with about 1.5 × 106 cells/ml in αMEM containing 10% (v/v) DMSO (Sigma) and 80% of human serum in liquid nitrogen.
Counterflow Centrifugal Elutriation (CCE)
Parameters for CCE, cell size distribution and cell viability in the obtained CCE fractions and in the UC-derived primary cell population
11.1 ± 1.3
65.0 ± 15.3
12.4 ± 1.1
88.3 ± 0.7
14.0 ± 1.9
94.5 ± 3.9
14.3 ± 1.0
86.9 ± 9.4
15.4 ± 1.1
80.7 ± 10.8
2.8 (without centrifugation)
19.1 ± 3.1
75.1 ± 9.4
Primary cell population (control)
15.0 ± 1.8
83.9 ± 5.2
Phenotypic analysis by flow cytometry
MSC were harvested by use of accutase for 5 min at 37°C, recovered by centrifugation at 200 × g for 5 min, washed twice in ice-cold PBS supplemented with 2% FCS (PAA Laboratories GmbH) and resuspended to a concentration of about 105 cells/antibody test. Thus, 20 μL of a pre-diluted PE-conjugated mouse anti-human CD44, a PE-conjugated mouse anti-human CD73, a FITC-conjugated mouse anti-human CD90 antibody (all from BD Biosciences, Heidelberg, Germany) and a R-PE-conjugated mouse anti-human CD105 antibody (Invitrogen GmbH, Karlsruhe, Germany) was used, respectively. Negative control staining was performed using a FITC-conjugated mouse IgG1 κ isotype, a PE-conjugated mouse IgG1 kappa isotype (all BD Biosciences) and a R-PE-conjugated mouse IgG1 isotype antibody (Invitrogen), respectively.
After storage for 20 minutes at room temperature in the dark, 400 μL of PBS supplemented with 2% FCS were added and analyzed in the EPICS XL/MCL flow cytometer (Beckman Coulter GmbH). Living cells were gated in a dot plot of forward versus side scatter signals acquired on linear scale. At least, 10,000 gated events were acquired on a LOG fluorescence scale. Positive staining was defined as the emission of a fluorescence signal that exceeded levels obtained by >99% of cells from the control population stained with matched isotype antibodies. For the antigen expression which was normalized to cell size, the fluorescence of the conjugated monoclonal antibodies as well as the forward scatter signals were measured on linear scale. The ratios of fluorescence signals versus scatter signals were calculated by the EPICS XL/MCL flow cytometer (Beckman Coulter). Histograms were generated using the software WinMDI 2.8 (Joseph Trotter).
Determination of cell proliferative activity
Immediately after CCE, the small-sized population (cells of the elutriation fraction 1), the large-sized population (cells of the elutriation fraction 6) and the UC-derived primary control population before elutriation was seeded at a density of 500 cells/cm2 and cultivated in 25 cm2 cell culture flasks (Sarstedt, Nuembrecht, Germany) in culture medium containing 10% human serum over 4 passages (P5–P8). After approx. 90% of confluency in the most rapidly proliferating subculture, cells from all three cultures were simultaneously harvested and replated at the same cell density. The cell number within the individual passages was determinate by the use of phase-contrast microscopy and trypan blue exclusion test.
Determination of cell senescence
The amount of senescent cells was determined in the small-sized population (cells of the elutriation fraction 1), the large-sized population (cells of the elutriation fraction 6) and the UC-derived primary control population by the use of the Senescence β-Galactosidase Staining Kit (Cell Signaling Technology, Danvers, USA) and DAPI (4',6-Diamidin-2'-phenylindoldihydrochlorid) (Roche Diagnostics GmbH, Mannheim, Germany) fluorescence counterstain in accordance to the manufacturers' instructions. Thus, the 3 different populations were cultured for 6 days after elutriation, passaged and seeded at a density of 6,000 cells/cm2 for 48 h before senescence-associated β-galactosidase (SA-β-gal) staining. After completion of the staining procedures, 4 representative images were taken from diverse areas of each cell culture using phase-contrast microscopy, fluorescence microscopy and CellBImaging Software (Olympus GmbH, Hamburg, Germany). For the calculation of the percentage of senescent cells the total number of cell nuclei and number of cell nuclei surrounded by cyan dye were enumerated.
Cell fractions obtained during CCE
During this procedure, six separate cell fractions with continuously increasing cell size were obtained (Table 1). Two clearly distinct cell subpopulations consisting of small cells with an average diameter of 11.1 ± 1.3 μm (elutriation fraction 1; figure 1d–f) and large cells with the diameter of 19.1 ± 3.1 μm (elutriation fraction 6; figure 1g–i) were isolated from primary cultures of human umbilical cord tissue. The small-sized subpopulation represented about 4,1% and the large-sized cells about 40% of the entire population. A similar distribution was observed in the UC-derived primary cultures of all 3 patients.
Cell size determination following elutriation was performed via Vi-CELL Series Cell Viability Analyzer (figure 1a, d, g) and revealed an initial viability of 65 ± 15% in the small cell population and 75 ± 9% in the large cell population (table 1). During subsequent culture the viability of these two populations increased to more than 90%, respectively. Analysis of the cultures by flow cytometry confirmed a different size distribution of the CCE-obtained subpopulations in the forward scatter (figure 1c, f, i). Moreover, cell cycle analysis demonstrated no significant differences in the cell cycle distribution of either the small-sized or the large-sized subpopulations with about 90% of cells in the G1/GO phase (data not shown).
Characterization of the immunophenotype
Proliferative activity of elutriated cells
Analysis of senescence in the original UC-derived primary cultures and in the CCE-enriched small- and large-sized subpopulations
Culture of umbilical cord tissue pieces yielded an adherent growing cell population with a diverse morphology of small and large cells. Flow cytometric analysis revealed high levels of CD44, CD73, CD90 and CD105 expression whereas the expression of CD-proteins typical for hematopoietic cells remained undetectable. These findings suggested the presence of mesenchymal stem cell-like cells according to MSC standard criteria of the International Society for Cellular Therapy . Moreover, a distinct subpopulation of the UC-derived primary cells demonstrated the potential to differentiate along the osteogenic pathway as evaluated by increase of alkaline phosphatase activity and increased mineralization (data not shown). This differentiation potential discriminated the UC-derived subpopulation from fibroblasts which are also most likely present in the original primary culture and carry a similar immunophenotype.
We observed marked morphological and cell size differences in the UC-derived primary cultures despite the rather homogeneous immunophenotype. For separation of these morphologically different cells, we applied CCE as a new approach. This technique yielded two subpopulations displaying distinct differences both, in cell size and morphology. Whereas the CCE-derived small-sized subpopulation exhibited the highest proliferative capacity and the most pronounced expression of mesenchymal stem cell markers, similar properties were observed in subpopulations of small-sized and rapidly-growing multipotential stem cells derived from human bone marrow [15, 26, 33].
Together, these findings suggested that the identified small-sized subpopulation of primary UC-derived cells exhibits MSC-like characteristics which may indicate a high relevance for applications in the area of regenerative medicine. This is also supported by the significant lower proliferative activity of the large-sized cells as well as the high portion of senescent cells in these cultures during long-term cultivation. In this context, the long-term expansion of the small-sized subpopulation was associated with a gradual loss of homogeneity displaying an enlarged and more diverse morphology. Accordingly, we speculate that the small cells may represent precursors of the larger, more mature cells, which eventually become senescent.
CCE provides a useful approach to enrich small rapidly-proliferating MSC-like cells in mixed UC-derived primary cultures. However, further characterization of these subpopulations is required with respect to the differentiation capacity and the application potential in regenerative medicine.
(mesenchymal stem cells)
(counterflow centrifugal elutriation)
The authors would like to thank Antonina Lavrentieva for help in evaluation of SA-β-gal staining and are grateful to Martina Weiss for the corroboration in preparing of figures included in the manuscript.
This study was supported by a grant from the German Research Foundation (Project number KA 1784/5-1).
- Friedenstein AJ, Chailakhjan RK, Lalykina KS: The development of fibroblast colonies in monolayer cultures of guinea-pig bone marrow and spleen cells. Cell Tissue Kinet. 1970, 3: 393-403.PubMed
- Dennis JE, Merriam A, Awadallah A, Yoo JU, Johnstone B, Caplan AI: A quadripotential mesenchymal progenitor cell isolated from the marrow of an adult mouse. J Bone Miner Res. 1999, 14: 700-709.View ArticlePubMed
- Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, Moorman MA, Simonetti DW, Craig S, Marshak DR: Multilineage potential of adult human mesenchymal stem cells. Science. 1999, 284: 143-147.View ArticlePubMed
- Sato Y, Araki H, Kato J, Nakamura K, Kawano Y, Kobune M, Sato T, Miyanishi K, Takayama T, Takahashi M, Takimoto R, Iyama S, Matsunaga T, Ohtani S, Matsuura A, Hamada H, Niitsu Y: Human mesenchymal stem cells xenografted directly to rat liver are differentiated into human hepatocytes without fusion. Blood. 2005, 106: 756-763.View ArticlePubMed
- Oh SH, Witek RP, Bae SH, Zheng D, Jung Y, Piscaglia AC, Petersen BE: Bone marrow-derived hepatic oval cells differentiate into hepatocytes in 2-acetylaminofluorene/partial hepatectomy-induced liver regeneration. Gastroenterology. 2007, 132: 1077-1087.View ArticlePubMed
- Oh SH, Witek RP, Bae SH, Darwiche HA, Jung Y, Pi L, Brown A, Petersen BE: Detection Of Transketolase In Bone Marrow-Derived Insulin Producing Cells: Benfotiamine Enhances Insulin Synthesis And Glucose Metabolism. Stem Cells Dev. 2008
- Wislet-Gendebien S, Wautier F, Leprince P, Rogister B: Astrocytic and neuronal fate of mesenchymal stem cells expressing nestin. Brain Res Bull. 2005, 68: 95-102.View ArticlePubMed
- Song S, Song S, Zhang H, Cuevas J, Sanchez-Ramos J: Comparison of neuron-like cells derived from bone marrow stem cells to those differentiated from adult brain neural stem cells. Stem Cells Dev. 2007, 16: 747-756.View ArticlePubMed
- Tondreau T, Dejeneffe M, Meuleman N, Stamatopoulos B, Delforge A, Martiat P, Bron D, Lagneaux L: Gene expression pattern of functional neuronal cells derived from human bone marrow mesenchymal stromal cells. BMC Genomics. 2008, 9: 166-PubMed CentralView ArticlePubMed
- Vogel W, Grünebach F, Messam CA, Kanz L, Brugger W, Bühring HJ: Heterogeneity among human bone marrow-derived mesenchymal stem cells and neural progenitor cells. Haematologica. 2003, 88: 126-133.PubMed
- Phinney DG: Biochemical heterogeneity of mesenchymal stem cell populations: clues to their therapeutic efficacy. Cell Cycle. 2007, 6: 2884-2889.View ArticlePubMed
- Jones E, McGonagle D: Human bone marrow mesenchymal stem cells in vivo. Rheumatology (Oxford). 2008, 47: 126-31.View Article
- Jiang Y, Jahagirdar BN, Reinhardt RL, Schwartz RE, Keene CD, Ortiz-Gonzalez XR, Reyes M, Lenvik T, Lund T, Blackstad M, Du J, Aldrich S, Lisberg A, Low WC, Largaespada DA, Verfaillie CM: Pluripotency of mesenchymal stem cells derived from adult marrow. Nature. 2002, 418: 41-49.View ArticlePubMed
- Ratajczak MZ, Kucia M, Majka M, Reca R, Ratajczak J: Heterogeneous populations of bone marrow stem cells – are we spotting on the same cells from the different angles?. Folia Histochem Cytobiol. 2004, 42 (3): 139-146.PubMed
- D'Ippolito G, Howard GA, Roos BA, Schiller PC: Isolation and characterization of marrow-isolated adult multilineage inducible (MIAMI) cells. Exp Hematol. 2006, 34: 1608-1610.View ArticlePubMed
- Ratajczak MZ, Zuba-Surma EK, Wysoczynski M, Ratajczak J, Kucia M: Very small embryonic-like stem cells: characterization, developmental origin, and biological significance. Exp Heamtol. 2008, 36: 742-751.View Article
- Wagner W, Ho AD: Mesenchymal stem cell preparations-comparing apples and oranges. Stem Cell Rev. 2007, 3: 239-248.View ArticlePubMed
- Ho AD, Wagner W, Franke W: Heterogeneity of mesenchymal stromal cell preparations. Cytotherapy. 2008, 10: 320-330.View ArticlePubMed
- da Silva Meirelles L, Chagastelles PC, Nardi NB: Mesenchymal stem cells reside in virtually all post-natal organs and tissues. J Cell Sci. 2006, 119: 2204-2213.View ArticlePubMed
- Alviano F, Fossati V, Marchionni C, Arpinati M, Bonsi L, Franchina M, Lanzoni G, Cantoni S, Cavallini C, Bianchi F, Tazzari PL, Pasquinelli G, Foroni L, Ventura C, Grossi A, Bagnara GP: Term Amniotic membrane is a high throughput source for multipotent Mesenchymal Stem Cells with the ability to differentiate into endothelial cells in vitro. BMC Dev Biol. 2007, 7: 11-PubMed CentralView ArticlePubMed
- Fukuchi Y, Nakajima H, Sugiyama D, Hirose I, Kitamura T, Tsuji K: Human placenta-derived cells have mesenchymal stem/progenitor cell potential. Stem Cells. 2004, 22: 649-658.View ArticlePubMed
- Baksh D, Yao R, Tuan RS: Comparison of proliferative and multilineage differentiation potential of human mesenchymal stem cells derived from umbilical cord and bone marrow. Stem Cells. 2007, 25: 1384-1392.View ArticlePubMed
- Can A, Karahuseyinoglu S: Concise review: human umbilical cord stroma with regard to the source of fetus-derived stem cells. Stem Cells. 2007, 25: 2886-2895.View ArticlePubMed
- Ennis J, Sarugaser R, Gomez A, Baksh D, Davies JE: Isolation, characterization, and differentiation of human umbilical cord perivascular cells (HUCPVCs). Methods Cell Biol. 2008, 86: 121-136.View ArticlePubMed
- Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini F, Krause D, Deans R, Keating A, Prockop Dj, Horwitz E: Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy. 2006, 8: 315-7.View ArticlePubMed
- Colter DC, Sekiya I, Prockop DJ: Identification of a subpopulation of rapidly self-renewing and multipotential adult stem cells in colonies of human marrow stromal cells. Proc Natl Acad Sci USA. 2001, 98: 7841-7845.PubMed CentralView ArticlePubMed
- Hung SC, Chen NJ, Hsieh SL, Li H, Ma HL, Lo WH: Isolation and characterization of size-sieved stem cells from human bone marrow. Stem Cells. 2002, 20: 249-258.View ArticlePubMed
- Verfaillie CM, Schwartz R, Reyes M, Jiang Y: Unexpected potential of adult stem cells. Ann N Y Acad Sci. 2003, 996: 231-234.View ArticlePubMed
- Pochampally RR, Smith JR, Ylostalo J, Prockop DJ: Serum deprivation of human marrow stromal cells (hMSCs) selects for a subpopulation of early progenitor cells with enhanced expression of OCT-4 and other embryonic genes. Blood. 2004, 103: 1647-1652.View ArticlePubMed
- Smith JR, Pochampally R, Perry A, Hsu SC, Prockop DJ: Isolation of a highly clonogenic and multipotential subfraction of adult stem cells from bone marrow stroma. Stem Cells. 2004, 22: 823-831.View ArticlePubMed
- Ratajczak MZ, Zuba-Surma EK, Machalinski B, Ratajczak J, Kucia M: Very small embryonic-like (VSEL) stem cells: purification from adult organs, characterization, and biological significance. Stem Cell Rev. 2008, 4: 89-99.View ArticlePubMed
- Hass R, Gunji H, Hirano M, Weichselbaum R, Kufe D: Phorbol ester-induced monocytic differentiation is associated with G2 delay and down-regulation of cdc25 expression. Cell Growth Differ. 1993, 4 (3): 159-166.PubMed
- Colter DC, Class R, DiGirolamo CM, Prockop DJ: Rapid expansion of recycling stem cells in cultures of plastic-adherent cells from human bone marrow. Proc Natl Acad Sci USA. 2000, 97: 3213-3218.PubMed CentralView ArticlePubMed
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