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Cell Biology International (2008) 32, 724–732 (Printed in Great Britain)
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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

Abstract

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.

1 Introduction

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=10, gestational age – 39–40 weeks) were collected after normal deliveries; each mother signed a consent form according to a protocol approved by the Research Ethics Committee of University Hospital of Obstetrics and Gynecology ‘Maichin Dom’. Each cord was washed out with 70% ethanol, and then the cord vein was catheterized and washed twice internally with PBS (pH=7.4) and its distal end was clamped. The vessel was filled with 2μg/ml collagenase (Sigma) in a Hanks' balanced salt solution (PAA, Austria) and then was incubated for 10min while the cord was gently massaged. After this, the suspension containing endothelial and subendothelial cells was collected and centrifuged for 10min at 1300rpm. After counting the cell numbers, the cell suspension was seeded in 35-mm 6-well plates with a density of approximately 103cells/cm2, cultured in a DMEM-LG medium (PAA Laboratories GmbH, Pashing, Austria) containing 10% FCS (PAA Laboratories GmbH, Pashing, Austria) and maintained at 37°C, 5% CO2. Culture medium was changed every 3rd day. Passaging was carried out using 0.25% trypsin (PAA Laboratories GmbH, Pashing, Austria) and cells from the 3rd to 4th passage were used in our experiments.

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=7.4) and incubated for 30min at RT in the dark with the following antibodies: CD3-FITC, CD45-FITC, CD19-PE, CD14-PE, CD16/CD56-PE, CD34-PE, HLA-DR-PE, CD90-FITC, CD29-PE and CD73-PE (Becton Dickinson, Temse, Belgium). After that the cells were washed in PBS (pH=7.4) and fixed with an FIX solution (Becton Dickinson, Temse, Belgium). The specific fluorescence of 20,000 cells was analyzed on FACScalibur (Becton Dickinson, Temse, Belgium) using CellQuest software.

2.3 Immunofluorescence

Cells grown on cover slips were fixed with 4% paraformaldehyde for 10min at RT, washed with PBS (pH=7.4) and permeabilized with 0.1% Triton-X 100 in PBS (pH=7.4). After several washes with PBS (pH=7.4) cells were incubated overnight at RT with the following antibodies against: S100A1 (rabbit polyclonal antiserum, Molecular Immunology, IBIR), S100A13 (rabbit polyclonal antiserum, Molecular Immunology, IBIR), endoglin (CD105/SH3) (rabbit polyclonal antiserum, Molecular Immunology, IBIR), von Willebrand factor (rabbit polyclonal antiserum, DAKO, Glostrup, Denmark), MMP3 (mouse monoclonal antibody, DAKO, Glostrup, Denmark), vimentin (mouse monoclonal antibody, DAKO, Glostrup, Denmark), cytokeratin (mouse monoclonal antibody, (DAKO, Glostrup, Denmark), KDR (VEGF-R2) (mouse monoclonal antibody, R&D Systems, Minneapolis, MN, USA), CD31(PECAM-1) (sheep IgG antibody, R&D Systems, Minneapolis, MN USA), Bcl-2 (goat antibody, R&D Systems, Minneapolis, MN, USA), and survivin (rabbit antibody, R&D Systems, Minneapolis, MN, USA). The corresponding secondary FITC-conjugated sera (SAPU, Lanarkshire, Scotland) diluted 1:100 in PBS (pH=7.4) were applied for 1h at RT. Monoclonal antibodies of irrelevant specificity were used as negative controls. After the incubation period the cells were washed extensively in PBS (pH=7.4) and mounted with Mowiol mounting medium (Hoechst, Frankfurt, Germany). Cells were observed under an epi-fluorescent microscope (Leitz, Germany).

2.4 SDS-PAGE and Western blotting

Mesenchymal stem cells (1×107 cells) were trypsinized, washed in PBS (pH=7.4); the cell pellets were resuspended in 500μl 2× SDS-PAGE sample buffer (Laemmli, 1970) and the cells were lysed by several cycles of freezing and thawing. Cell lysates with protein contents 50μg/start were separated by SDS-PAGE (12% gels) and transferred to nitrocellulose membranes (Hybond-P, Amersham Biosciences, Buckinghamshire, UK). The membranes were blocked for 12h at RT with 3% nonfat milk in PBS (pH=7.4) containing 0.1% Tween 20. Membranes were incubated with primary antibodies (anti-S100A1, anti-S100A13, anti-endoglin, anti-vimentin, anti-cytokeratin and anti-MMP3) for 2h at RT and after three washes were incubated for 1h at RT with the appropriate horseradish-peroxidase-conjugated secondary antibody. After four additional washes with 0.1% Tween/PBS (pH=7.4), blots were visualized using enhanced chemiluminescence (ECL) reagents (Amersham Biosciences, Buckinghamshire, UK). Blots were exposed to Hyperfilm-ECL (Amersham Biosciences, Buckinghamshire, UK). The molecular weight of the analyzed proteins was estimated using Kaleidoscope Prestained Standards (Bio-Rad Laboratories, Hercules, CA, USA).

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 1×107 MSCs or differentiated osteogenic cells by using an innuPREP Blood RNA Kit (AJ Roboscreen, Leipzig, Germany) after cell detachment and washing with PBS. Total RNA was reverse transcribed using random hexamer primers and MultiScribe Reverse Transcriptase (GeneAmp RNA PCR Reagent Kit, Applied Biosystems, Foster City, CA, USA). Primer sets used and the sizes of produced fragments are listed in Table 1.

Table 1.

Specific primers for hTERT, survivin and β-actin cDNA

PrimerPrimer motifFragment length (bp)Ta (°C)
hTERT (Invitrogen)Sense 5′-GTGTGCTGCAGCTCCCATTTC-3′26465
Antisense 5′-GCTGCGTCTGGGCTGTCC-3′
Survivin (Invitrogen)Sense 5′-ACAGCATCGAGCCAAGTCAT-3′43160
Antisense 5′-GAGCTGCAGGTTCCTTATC-3′
Actin β (Clontech)Sense 5′-ATCTGGCACCACACCTTCTACAATGAGCTGCG-3′83860
Antisense 5′-CGTCATACTCCTGCTTGCTGATCCACATCTGC-3′

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°C for 10min hybridization and 42°C for 12min reverse transcription. PCR reactions were performed at 95°C for 1min denaturation, primer annealing at 60°C for survivin and β-actin and 65°C for hTERT; primer extension was performed at 72°C for 2min for 34 cycles and 72°C for 10min as an extra cycle of elongation. Amplified products were separated on 2% agarose gels containing ethidium bromide for visualization and photographed under UV light. The size of the analyzed genes was estimated by the DNA Ladder (Sigma Aldrich, St. Louis, MO, USA), which contains 10 bands ranging from 100/150bp to 1000/1050bp in exactly 100bp increments.

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), 0.1μM dexamethasone (Sigma Aldrich, St. Louis, MO, USA), 10μM β-glycerophosphate (Sigma Aldrich, St. Louis, MO, USA) and 50μM ascorbic acid (Sigma Aldrich, St. Louis, MO, USA) for three weeks. The medium was changed twice a week. Osteogenic differentiation was evaluated by calcium deposition staining using the von Kossa method. The cells were fixed for 10min in 4% PFA; after washing they were incubated with 1% silver nitrate for 60min. After several washes with distilled water, any un-reacted silver was removed with 5% sodium thiosulfate for 5min.

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 1μM dexamethasone (Sigma Aldrich, St. Louis, MO, USA), 10μg/ml bovine insulin (Sigma Aldrich, St. Louis, MO, USA), 0.5mM 3-isobutyl-1-methyl-xanthine (IBMX) (Sigma Aldrich, St. Louis, MO, USA), 200μM indomethacin (Sigma Aldrich, St Louis, MO, USA) and 10% FCS (PAA Laboratories GmbH, Pashing, Austria). On the 21st day the cells were fixed in 10% formalin neutral solution (Merck, Darmstadt, Germany) for 30min and stained with fresh 0.6% oil red O solution to show lipid droplets in induced cells.

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=7.4) and suspended in endothelial differentiation medium at a concentration of 1×105/ml; 2ml of cell suspension was added to each well. The endothelial differentiation medium contained LG-DMEM (PAA Laboratories GmbH, Pashing, Austria), 3% FCS (PAA Laboratories GmbH, Pashing, Austria), 50ng/ml VEGF-BPE and 10ng/ml b-FGF (R&D Systems, Minneapolis, MN, USA). Cultures were incubated at 37°C in a 5% CO2 humidified atmosphere for 3 days. Endothelial differentiation was evaluated by indirect immunofluorescence for the expression of the following endothelial markers: vWF rabbit anti-human IgG antibody (DAKO, Glostrup, Denmark), PECAM-1 (CD31) (sheep anti-human IgG antibody, R&D Systems, Minneapolis, MN, USA) and KDR (VEGFR-2) (mouse monoclonal antibody, R&D Systems, Minneapolis, MN, USA).

3 Results

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 3–4h. The cells were seeded at concentration of 100cells/cm2; this is the concentration used in cloning experiments. After 3 days of culture the medium was changed for the first time after isolation and two types of adherent cells were observed: one was a cell population consisting of small flattened cells morphologically similar to the endothelial cells (human umbilical vein endothelial cells); the second population consisted of spindle-shape fibroblast-like cells (Fig. 1A). Since no growth factors stimulating the growth of endothelial-like cells were present in the culture medium these cells died in about 7–10 days. The fibroblast-like cells were the only cell types growing under these conditions after 2 weeks of culture (Fig. 1B). After the 2nd passage the culture appeared to be homogeneous and a monolayer formed (Fig. 1C). The cells displayed a fibroblast-like morphology and formed colonies (Fig. 1D). Thus a population of clonogenic cells with fibroblast morphology and proliferative potential was selected from the initially heterogeneous cell population.

Fig. 1

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 – 200μm (A,D), 300μm (B), 400μm (C).

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.

Fig. 2

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.

Fig. 3

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 – 20μm (A, B). These data were confirmed by Western blotting (C, D). Results of Western blotting analysis for UC-MSCs showed a positive reaction for endoglin (C, lane1) and for vimentin (D, lane1). The human fibroblast line HF1 was used as positive control (lane 2 in C, D).

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, 0.1μM dexamethasone, 10μM β-glycerophosphate and 50μM ascorbic acid. In both the control and differentiating cultures the medium was changed twice a week. Cells started to change morphologically as early as 5 days in an inducing culture medium; the cells lost their typical fibroblast appearance turning into wider polygonal cells. These changes became more obvious with time and on the 21st day a von Kossa staining was performed. The effectiveness of differentiation was assessed by histochemical staining for the identification of Ca2+ crystals by von Kossa staining. Intense black staining can be detected between osteogenic cells (Fig. 4B), while control cells without osteogenic stimuli stained in the same manner were negative for Ca2+ depositions (Fig. 4A). Another change observed was that, upon differentiation, the osteogenic cells continued to grow as a monolayer while the cells in control cultures preserved their fibroblast-like morphology and were still able to form colonies.

Fig. 4

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 – 200μm (A,B), 400μm (C, D).

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 1μM dexamethasone, 10μg/ml bovine insulin, 0.5mM IBMX, 200μM indomethacin and 10% FCS for 21 days. After 7–8 days of adipogenic differentiation tiny intracytoplasmic droplets could be observed; after 14 days of cultivation most cells became larger and had a round shape. The effectiveness of differentiation was assessed by histochemical staining for the identification of neutral lipid vacuoles by Oil red O staining. In the adipogenic differentiated cells, red stained intracellular vacuoles (Fig. 4D) can be observed, while in the nondifferentiated control group lipid droplets were not detected (Fig. 4C).

To induce UC-MSCs cells into endothelial-like cells in vitro, they were cultured in DMEM-LG supplemented with 3%FCS, 50ng/ml VEGF (bovine pituitary extract) and 10ng/ml b-FGF on Matrigel coated 6-well dishes. During the first 12h cells spread randomly and started to form small and seldom interconnected clusters (Fig. 5a). After 24h of cultivation the clusters became larger, with thin connections among them, and formed polygonal vessel-like structures on the Matrigel. Two days after plating, interconnections between clusters became thicker. Morphological changes were observed under light microscopy and photographed in vivo (Fig. 5a).

Fig. 5

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 – 200μm (0h, 3h, 6h, 12h and 24h), 400μm (48h). (b) Differentiation into endothelial cells was demonstrated for the expression of endothelial markers vWF (A), KDR (VEGF-R2) (B) and PECAM-1 (C) by indirect immunofluorescence staining. Scale bar – 20μm (A, B and C).

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 4h. The coverslips were then fixed with 4% PFA and stained for the endothelial markers CD31, KDR (VEGF-R2) and vWF. Analyzed cells were positive for the investigated endothelial markers – a fact that represents another evidence for successful endothelial differentiation (Fig. 5b).

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.

Fig. 6

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 100bp to 1000bp in exact 100bp increments.

It should be mentioned that UC-MSCs isolated from all the separate samples (n=10) behaved similarly in the tests for clonogenicity and differentiation assays; exemplary photomicrographs are presented in this paper.

4 Discussion

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.

Acknowledgements

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.

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Received 25 September 2007/1 November 2007; accepted 25 February 2008

doi:10.1016/j.cellbi.2008.02.002
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