|Cancer||Cell death||Cell cycle||Cytoskeleton||Exo/endocytosis||Differentiation||Division||Organelles||Signalling||Stem cells||Trafficking|
Cell Biology International (2008) 32, 724732 (Printed in Great Britain)
Characterization of mesenchymal stem cells isolated from the human umbilical cord
Snejana Kestendjievaa*, Dobroslav Kyurkchievb, Gergana Tsvetkovaa, Tzvetozar Mehandjievc, Angel Dimitrovc, Assen Nikolovc and Stanimir Kyurkchieva
aDepartment of Molecular Immunology, Institute of Biology and Immunology of Reproduction, Bulgarian Academy of Sciences, 73 Tzarigradsko shosse, 1113 Sofia, Bulgaria
bLaboratory of Clinical Immunology, University Hospital ‘Sv.I.Rilski’, Medical University Sofia, Sofia, Bulgaria
cUniversity Ob/Gyn Hospital ‘Maichin Dom’, Medical University Sofia, Sofia, Bulgaria
Numerous papers have reported that mesenchymal stem cells (MSCs) can be isolated from various sources such as bone marrow, adipose tissue and others. Nonetheless it is an open question whether MSCs isolated from different sources represent a single cell lineage or if cells residing in different organs are separate members of a family of MSCs. Subendothelial tissue of the umbilical cord vein has been shown to be a promising source of MSCs.
The aim of this study was to isolate and characterize cells derived from the subendothelial layer of umbilical cord veins as regards their clonogenicity and differentiation potential. The results from these experiments show that cells isolated from the umbilical cord vein displayed fibroblast-like morphology and grew into colonies. Immunophenotyping by flow cytometry revealed that the isolated cells were negative for the hematopoietic line markers HLA-DR and CD34 but were positive for CD29, CD90 and CD73. The isolated cells were also positive for survivin, Bcl-2, vimentin and endoglin, as confirmed by RT-PCR and immunofluorescence. These cells can be induced to differentiate into osteogenic and adipogenic cells, but a new finding is that these cells can be induced to differentiate into endothelial cells expressing CD31, vWF and KDR-2, and also form vessel-like structures in Matrigel. The differentiated cells stopped expressing survivin, thus showing a diminished proliferative potential. It can be assumed that the subendothelial layer of the umbilical cord vein contains a population of cells with the overall characteristics of MSCs, with the additional capability to transform into endothelial cells.
Keywords: Umbilical cord, Mesenchymal stem cells, Differentiation potential, Endothelial-like cells, Survivin down regulation.
*Corresponding author. Tel.: +359 2 8723890.
Mesenchymal stem cells (MSCs) have the capability for self-renewal and differentiation into various lineages of mesenchymal origin (adipocytes, osteocytes, chondrocytes, and tenocytes) and even astrogenic, myogenic, cardiomyogenic and nerve cells (Lakshmipathy and Verfaillie, 2005; Minguell et al., 2001; Quesenberry et al., 2004). Mesenchymal stem cells can be isolated and expanded ex vivo without any apparent modification in the phenotype or loss of function. Because of these basic characteristics MSCs are considered to be very important for the development of cell-based therapies and tissue repair in regenerative medicine.
To date the most common source of MSCs has been bone marrow (BM) (Conget and Minguell, 1999; Deans and Mosely, 2000; Minguell et al., 2000). However, aspirating bone marrow from the patient is an invasive procedure; in addition it has been demonstrated that the number and the differentiating potential of bone marrow MSCs decreases with age (D'Ippolito et al., 1999; Rao and Mattson, 2001). Therefore the search for alternative sources of MSCs is a promising subject for research, with efforts focused on tissues containing cells with higher proliferative potency and differentiation capacity as well as a lower risk for viral contamination. MSCs have been isolated from various organs and from the circulating blood of preterm fetuses, where they circulate together with hematopoietic stem cells (Campagnoli et al., 2001; Erices et al., 2000). The presence of MSCs in the umbilical cord blood of term infants is still questionable for some authors. Recently, several groups succeeded in isolating MSCs from umbilical cord blood (Goodwin and Bicknese, 2001; Hou et al., 2002; Rosada et al., 2003); at the same time, controversial results have been obtained by others who suggest that cord blood is not a source for MSCs (Mareschi and Biasin, 2001; Wexler et al., 2003).
Covas et al. (2003) and Romanov et al. (2003) reported the isolation and characterization of MSCs from the umbilical cord vein (UCV). Mesenchymal stem cells derived from the umbilical cord vein (UC-MSCs) are functionally similar to BM-MSCs. Moreover the procedure for their isolation is not invasive and since the cells are of fetal origin, their proliferative and differentiation potential could be better than that of MSCs from other sources. Furthermore, Baksh et al. (2007) reported comparative studies showing that UC-MSCs isolated from umbilical cord vein had a higher proliferative and differentiation potential when compared to MSCs isolated from bone marrow. Thus the umbilical cord vein is thought to be a promising source of MSCs.
The aim of this study was to isolate MSCs from the umbilical cord vein and to characterize these cells analyzing their clonogenicity, expression of surface markers and differentiation potential.
2 Materials and methods
2.1 Isolation and culture of cells
Umbilical cords (n
2.2 Immunophenotyping of MSC-like cells by flow cytometry
For flow cytometry analysis, cells were harvested by treatment with 0.25% trypsin (PAA Laboratories GmbH, Pashing, Austria), washed with PBS (pH
Cells grown on cover slips were fixed with 4% paraformaldehyde for 10
2.4 SDS-PAGE and Western blotting
Mesenchymal stem cells (1
2.5 Total RNA isolation and RT-PCR
By reverse transcription-polymerase chain reaction, the expression of the anti-apoptotic protein survivin and human telomerase reverse transcriptase (hTERT) was assessed, as well as the reference housekeeping gene β-actin.
Total RNA was extracted from 1
Specific primers for hTERT, survivin and β-actin cDNA
RT-PCR was done following the two-step protocol for the GeneAmp Gold RNA PCR Reagent Kit (Applied Biosystems, Foster City, CA, USA). Briefly, reverse transcription reactions were performed at 25
2.6 In vitro differentiation into osteogenic cells
The differentiation of MSC cells into osteogenic cells was assessed using the 4th to 5th passage cultures. The cells were cultured in DMEM supplemented with 10% FCS (PAA Laboratories GmbH, Pashing, Austria), 0.1
2.7 In vitro differentiation into adipogenic cells
To induce adipogenic differentiation, 4th to 5th passage cells were treated with adipogenic medium for three weeks with medium changes twice a week. The adipogenic medium consisted of DMEM (PAA) supplemented with 1
2.8 In vitro differentiation into endothelial cells
To analyze in vitro endothelial differentiation, a 6-well cell culture dish was coated with Matrigel (Becton Dickinson, Temse, Belgium). MSCs were trypsinized, washed in PBS (pH
As soon as the collected umbilical cords were delivered to the laboratory, they were processed as described above; the time from delivery to processing in the laboratory did not exceed 3–4
The morphology of umbilical-cord-vein-derived cells. (A) After 4 days of cultivation two types of adherent cells were observed: a more numerous cell population consisting of small flattened cells morphologically similar to the endothelial cells (HUVEC) – black arrows; and a population consisting of a few spindle-shape fibroblast-like cells – white arrows. (B) After 2 weeks of culturing these fibroblast-like cells became the predominant cell type. (C, D) The cells displayed a fibroblast-like morphology and grew into colonies. Scale bar – 200
The isolated clonogenic cells were analyzed by the flow cytometry analysis and gated for granularity, size and surface markers. The gated cells were analyzed for the expression of cell membrane proteins markers and found to be negative for the expression of hematopoietic markers such as CD45, CD14, CD3, CD19, CD16/56 and also HLA-DR (MHC II) and CD34 (endothelial/hematopoietic stem cell markers), but were positive for CD29, CD73 and CD90, which are generally considered for markers of mesenchymal stem cells (Fig. 2). These data confirmed that the isolated cells were mesenchymal stem cells and that the culture was homogeneous. Further, the cells were found to be positive for the expression of endoglin, vimentin (Fig. 3A and B), but negative for S100A1, S100A13, MMP3 and cytokeratin when analyzed by indirect immunofluorescence (data not shown). The expression of these proteins was confirmed also by Western blot analysis (Fig. 3C and D). The morphological and immunophenotype characteristics of the isolated cells gave us grounds to assume that a population of umbilical cord mesenchymal stem cells (UC-MSCs) was isolated in these experiments.
Flow cytometric histograms showing the immunophenotype of umbilical vein mesenchymal stem cells. The cells were analyzed by their physical parameters: granularity and size. The gated cells were negative for the hematopoietic line markers CD45, CD14, CD3, CD19, CD16/56, and for HLA-DR and CD34. Analyzed cells were positive for CD29, CD73 and CD90, which are considered to be markers of mesenchymal stem cells. Isotype controls show non-specific fluorescence recorded lower than 102 region and for that reason only fluorescence above 102 was read as specific.
Immunofluorescence and Western blotting analysis of cells isolated from the umbilical cord vein. Indirect immunofluorescence staining of MSCs derived from the umbilical cord vein was positive for endoglin (A) and vimentin (B). Scale bar – 20
In experiments to further characterize the isolated UC-MSCs, they were tested for their potential to differentiate into osteogenic or adipogenic cells in the presence of specific inducing factors; as the experiments were performed after the 3rd to 4th passage, i.e., the homogeneous cell populations were differentiated. Osteogenic differentiation was induced for 3 weeks with DMEM-LG supplemented with 10% FCS, 0.1
Differential potential of mesenchymal stem cells derived from the human umbilical vein. Osteogenic differentiation – non-stimulated (A) and stimulated cells (B) – stained by the von Kossa method for Ca2+ deposition in the extracellular matrix. Adipogenic differentiation – non-stimulated (C) and stimulated cells (D) – stained with oil red O for visualization of intracytoplasmic lipid droplets. Scale bar – 200
To assess adipogenic differentiation, the 3rd to 4th passage cells that reached almost 100% confluence were cultured in an adipogenic medium consisting of DMEM supplemented with 1
To induce UC-MSCs cells into endothelial-like cells in vitro, they were cultured in DMEM-LG supplemented with 3%FCS, 50
Endothelial differentiation of mesenchymal stem cells derived from the human umbilical vein. (a) Morphological changes during endothelial differentiation of UC-MSCs on the zero th hour, 3rd hour, 6th hour, 12th hour, 24th hour and 48th hour after seeding. Scale bar – 200
To evaluate the endothelial differentiation of UC-MSCs, cells were detached from the Matrigel by collagenase treatment and the retrieved cells were allowed to attach to gelatin coated coverslips for 4
In a separate series of experiments the UC-MSCs and differentiated cells were compared for expression of survivin by RT-PCR. Using RT-PCR, the presence of mRNA for survivin (a marker for rapidly proliferating cells) (Fig. 6C) and hTERT (a marker for stem cells) (Fig. 6D) were detected in undifferentiated UC-MSCs. The expression of Bcl-2 (Fig. 6A) and survivin (Fig. 6B) at protein level was confirmed by immunofluorescence on undifferentiated UC-MSCs. Differentiated cells were negative for survivin and hTERT at both the protein (data not shown) and mRNA levels (Fig. 6E), which meant that during differentiation of the cells the expression of these markers was downregulated. So, the phenotypic changes during differentiation of cells observed included both the cell morphology and the expression of markers involved in the regulation of cell cycle.
Comparative study of undifferentiated and differentiated UC-MSCs. Indirect immunofluorescence staining of undifferentiated UC-MSCs was positive for Bcl-2 (A) and survivin (B). Scale bar – 20μm (A, B). UC-MSCs also expressed survivin at mRNA level (C, lane 1) and hTERT (D, lane1) analyzed by RT-PCR. Differentiated osteogenic cells did not express survivin (E, lane 2) compared to control undifferentiated UC-MSCs (E, lane1). Lane 2 (C, D) and lane 3 (E) are showing molecular weight markers containing 10 bands ranging from 100
It should be mentioned that UC-MSCs isolated from all the separate samples (n
In the present study we have described the characterization of a cell population derived from the subendothelium of human umbilical vein using assays for clonogenicity, expression of specific markers by flow cytometry, immunofluorescence and RT-PCR. Under adipogenic- and osteogenic-inducing conditions, these cells were able to differentiate in osteogenic and adipogenic cells. The immunophenotypical, morphological profile and differentiation potential of these cells are similar to that of MSCs isolated from BM (Conget and Minguell, 1999; Majumdar et al., 1998) and of MSCs derived from umbilical cord veins described previously by Covas et al. (2003) and Romanov et al. (2003). In this study for the first time we have demonstrated that, in the presence of factors inducing specific differentiation, UC-MSCs differentiated into endothelial cells. Cells cultured on the Matrigel formed polygonal vessel-like structures. The cells which formed vessel-like structures expressed vWF, PECAM-1 and KDR, which are specific endothelial markers. These data substantiate the reports of Panepucci et al. (2004) regarding active genes in UC-MSCs that participate in pathways related to matrix remodeling and angiogenesis. Taken together these data would suggest that UC-MSCs could be appropriate for the therapies aiming at increased revascularization.
In the present study UC-MSCs were analyzed for the expression and shown to be positive for survivin, hTERT by RT-PCR and survivin and Bcl-2 by indirect immunofluorescence. Survivin, a 16.5-kd cytoplasmic protein, exhibits functions of both an apoptosis inhibitor and a regulator of cell division (Reed and Bischoff, 2000). Survivin that has been linked to unchecked proliferation in cancer cells may play an important role in regulating cell cycle entry and cell division in normal hematopoietic stem cells (Fukuda et al., 2002) and regulate mechanisms of tissue and organ differentiation during the human embryogenesis (Colette et al., 1998). The expression of survivin from UC-MSCs at both protein and mRNA levels is proof confirming the differentiation potential and proliferative capacity of these cells. It is well known that hTERT expression is under specific stringent regulation during human development and differentiation (Yashima et al., 1998) and during activation and development of lymphocytes (Weng et al., 1997); its expression is upregulated in tumor cells as compared to normal cells of the same tissue (Avilion et al., 1996). Bcl-2 family genes are involved in regulation of apoptosis, as Bcl-2 itself is a promoter of cell survival because it inhibits the activation of caspases (Adams and Cory, 1998). In experiments in vitro it has been demonstrated that higher expression of Bcl-2 promotes the survival and growth of cells dependent on growth factor in its absence (Vaux et al., 1988). Generally Bcl-2 is considered to be a major anti-apoptotic regulator of the cell cycle. Expression of survivin, hTERT and Bcl-2 by undifferentiated UC-MSCs is intimately related to their survival and proliferation in vitro and their potential to form single cell colonies. The down regulation of survivin expression during the differentiation of UC-MSCs induced in vitro demonstrates that the cells lost their high proliferative capacity after becoming differentiated. These data demonstrate an important general feature of the MSCs which is their controlled proliferation after differentiation, which would prevent the uncontrolled proliferation of the cells after infusion in the organism.
In a rat myocardial infarction model Wu et al. (2007a,b) transplanted umbilical-cord-derived stem cells and about 2 weeks later detected the expression of cardiac troponin, von Willebrand factor and smooth muscle actin by some of the transplanted cells. These findings would suggest the differentiation of the umbilical-cord-derived stem cells in the corresponding cell lineages.
In conclusion, a clonogenic cell population of UC-MSCs was isolated from an umbilical cord vein subendothelial layer that can be grown in vitro, form cell colonies and differentiate into osteogenic, adipogenic cells. The data support the reports of Romanov et al. (2003) and Covas et al. (2003). The new findings in these experiments is the differentiation of human UC-MSCs into endothelial-like cells capable of forming capillary structures and expressing markers specific to endothelial lineage, and the finding that UC-MSCs, upon differentiation, downregulate the expression of survivin, which is a regulator of the cell cycle.
This work was supported by Grant No. G4-1/2005 and Grant No. L 1517/2005 by the National Science Fund of the Ministry of Education and Science, Sofia, Bulgaria.
Avilion AA, Piatyszek, MA, Gupta, J, Shay, JW, Bacchetti, S, Greider, CW. Human telomerase RNA and telomerase activity in immortal cell lines and tumor tissues. Cancer Res 1996:56:645-50
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 2007:25:1384-92
Campagnoli C, Roberts, IA, Kumar, S, Bennett, PR, Bellantuono, I, Fisk, NM. Identification of mesenchymal stem/progenitor cells in human first trimester fetal blood, liver and bone marrow. Blood 2001:98:2396-402
Colette A, Crotty, PL, McGrath, J, Berrebi, D, Diebold, J, Altieri, DC. Developmentally regulated expression of the novel cancer anti-apoptosis gene survivin in human and mouse differentiation. Am J Pathol 1998:152:43-9
D'Ippolito G, Schiller, PC, Ricordi, C, Roos, BA, Howard, GA. Age-related osteogenic potential of mesenchymal stromal stem cells from human vertebral bone marrow. J Bone Miner Res 1999:14:1115-22
Fukuda S, Foster, R, Porter, S, Pelus, LM. The anti-apoptosis protein, survivin, regulates cell cycle entry of normal cord blood CD34+ cells and modulates cell cycle and proliferation of mouse hematopoietic progenitor cells. Blood 2002:100:2463-71
Goodwin HS, Bicknese, AR. Multilineage differentiation activity by cells isolated from umbilical cord blood: expression of bone, fat, neural markers. Biol Blood Marrow Transplant 2001:7:581-8
Hou L, Cao, H, Wei, G, Bai, C, Zhang, Y, Wu, Z. Study of in vitro expansion and differentiation into neuron-like cells of human umbilical cord blood mesenchymal stem cells. Zhonghua Xue Ye 2002:23:415-9
Majumdar MK, Thiede, MA, Mosca, JD, Moorman, M, Gerson, SL. Phenotypic and functional comparison of human bone marrow mesenchymal stem cells (MSCs) and stromal cells. J Cell Physiol 1998:176:57-66
Mareschi K, Biasin, E. Isolation of human mesenchymal stem cells: bone marrow versus umbilical cord blood. Hematologica 2001:86:1099-100
Minguell JJ, Erices, A, Conget, P. Mesenchymal stem cells. Exp Biol Med 2001:226:507-20
Panepucci RA, Siufi, JLC, Silva, WA, Proto-Siquiera, R, Neder, L, Orellana, M. Comparison of gene expression of umbilical cord vein and bone marrow-derived mesenchymal stem cells. Stem Cells 2004:22:1263-78
Romanov YA, Svintsitskaya, VA, Smirnov, VN. Searching for alternative source of postnatal human mesenchymal stem cells: candidate MSC-like cells from umbilical cord. Stem Cells 2003:21:105-10
Wexler SA, Donaldson, C, Dening-Kendall, P. Adult bone marrow is a rich source of human mesenchymal stem cells but umbilical cord and mobilized blood are not. Br J Hematol 2003:121:368-74
Wu Kai Hong, Zhou, Bin, Yu, Cun Tao, Cui, Bin, Lu, Shi Hong, Han, Zhong Chao. Therapeutic potential of human umbilical cord derived stem cells in a rat. Myocardial Infarction model. Ann Thorac Surg 2007:83:1491-8
Yashima K, Maitra, A, Rogers, BB, Timmons, CF, Rathi, A, Pinar, H. Expression of the RNA component of telomerase during human development and differentiation. Cell Growth Differ 1998:9:805-13
Received 25 September 2007/1 November 2007; accepted 25 February 2008doi:10.1016/j.cellbi.2008.02.002