In Vitro Expansion of Human Mesenchymal Stem Cells: Choice of Serum Is a Determinant of Cell Proliferation, Differentiation, Gene Expression
http://www.100md.com
《干细胞学杂志》
a Institute of Immunology,
b Centre for Occupational and Environmental Medicine, and
c Institute and Department of Pathology, Rikshospitalet University Hospital and University of Oslo, Norway
Key Words. Human mesenchymal stem cells ? Autologous serum ? Fetal bovine serum ? Cell culture ? Gene expression ? In vitro differentiation ? Microarray
Correspondence: J.E. Brinchmann, M.D., Ph.D., Institute of Immunology, Rikshospitalet University Hospital, 0027 Oslo, Norway. Telephone: 47-23-07-37-66; Fax: 47-23-07-38-22; e-mail: j.e.brinchmann@medisin.uio.no
ABSTRACT
Human bone marrow mesenchymal stem cells (hMSCs) represent an appealing source of adult stem cells for cell therapy and tissue engineering. Because hMSCs are present at low frequency in the bone marrow, expansion is necessary before performing clinical studies. Recently, techniques for isolation and extensive subcultivation of hMSCs have been developed. In vitro, culture-expanded hMSCs are capable of differentiation along osteogenic, chondrogenic, and adipogenic lineages . In vivo, several studies in a variety of animal models have shown that hMSCs may be useful in the repair or regeneration of cartilage , damaged bone , tendon , and meniscus . In vitro and in vivo, studies have indicated that hMSCs are capable of differentiation also into cardiomyocytes , skeletal muscle , and neural precursors . hMSCs have been used in clinical trials in children with osteogenesis imperfecta and to promote engraftment and prevent or treat severe graft-versus-host disease (GVHD) in allogeneic stem cell transplantation .
In practically all studies using in vitro–expanded hMSCs, the cell culture medium has been supplemented with fetal bovine serum (FBS). This is also true for human clinical trials approved by the U.S. Food and Drug Administration . The risk of transmission of prion diseases and zoonoses from the use of FBS is considered to be small . A greater risk associated with the use of hMSCs expanded in FBS seems to be the immunogenicity of the xenogeneic FBS proteins. Recently, it was shown that a single preparation of 108 hMSCs grown under standard condition in FBS would carry with it approximately 7–30 mg of FBS proteins . The full clinical impact of this observation remains to be investigated, but the use of autologous serum (AS) instead of FBS was recently shown to prevent life-threatening arrhythmias after cellular cardiomyoplasty . Thus, it seems likely that in the future both clinical and regulatory issues will motivate the use of serum supplements other than FBS. We have therefore examined the possibility of using AS or allogeneic human serum (alloHS) rather than FBS for in vitro expansion of hMSCs. In this study, we present the results of experiments comparing proliferation, phenotype, differentiation capability, and gene expression of hMSCs expanded in different serum preparations.
MATERIALS AND METHODS
hMSCs Are Efficiently Expanded In Vitro in AS and FBS but Not in alloHS
To compare the effect of different serum preparations on the proliferative capability of hMSCs, 108 bone marrow mononuclear cells (BMMCs) depleted of CD14+ cells were established in parallel cultures supplemented with one of three different preparations of FBS, AS, or alloHS. Of the three FBS preparations, FBS (Gibco) consistently yielded the highest cell counts (data not shown). Thus, FBS (Gibco) was selected for all further analyses. A comparison of calculated cumulative cell counts between cells expanded in FBS and AS for four donors is shown in Figure 1. Extrapolation of growth curves suggests that there were approximately 105 hMSCs within the 108 CD14– BMMCs on day 0, giving a precursor frequency of one hMSC in 103 CD14– BMMCs. For each of the donors, the population doubling time was always shorter in hMSCs in AS (median for the four donors, 53 hours; range, 41–54 hours) compared with hMSCs in FBS (median, 84 hours; range, 76–89 hours). In cultures supplemented with alloHS, the number of adherent cells on day 1 was always lower than for cultures with other serum supplements (data not shown). The adherent cells in cultures supplemented with alloHS spread and formed small colonies, but proliferation soon ceased, and as cells cultured in alloHS did not survive (beyond passage 1), cell counts are not entered into Figure 1.
Figure 1. Proliferation of human marrow mesenchymal stem cells (hMSCs). Calculated cumulative cell counts in cultures of hMSCs from four different donors are plotted with filled symbols for hMSCs expanded in autologous serum and open symbols for hMSCs expanded in fetal bovine serum. Identically shaped symbols (e.g., open and filled squares) represent results from the same donor.
hMSCs Cultured in AS Are Morphologically and Phenotypically Similar to hMSCs Cultured in FBS
From initial colony formation until senescence, hMSCs showed a fibroblastoid appearance, with no discernible morphological differences between cells expanded in AS and FBS at any time (supplemental online Fig. 1). However, we observed that hMSCs grown in FBS required prolonged exposure to trypsin to detach from the plastic surface (data not shown). This could not be explained, as determined by flow cytometry, by differential expression of the adhesion molecules tested. Indeed, these were almost equally expressed on the cells regardless of the serum supplement (supplemental online Table 1). As expected, hMSCs expressed HLA class I, CD13, CD44, CD90, and CD105 (SH2) .
Table 1. Relative expression of molecules characteristic for different lineages
Choice of Serum Impacts on the Rate of Differentiation of hMSCs
The multilineage differentiation capability of hMSCs expanded in FBS or AS was examined by culturing cells collected at passage 4 under conditions favorable for chondrogenic, osteogenic, and adipogenic differentiation . Examples of staining assays obtained after 3 or 4 (chondrogenic differentiation) weeks of culture in induction medium are shown in Figure 2. At this time, staining for fat globules (adipogenic differentiation) and mineralization (osteogenic differentiation) was observed in cells from all donors and both serum preparations. However, for cells in adipogenic induction medium, fat globules could be observed under the light microscope already after 3–4 days in hMSCs in FBS, whereas fat globules appeared 8–12 days later in hMSCs in AS (data not shown). The same tendency was observed in chondrogenic-induced cultures. Here, cells were cultured in pellets that lost attachment and appeared as spheres. For hMSCs in FBS, the spheres increased in size, indicating production of extracellular matrix (ECM), from approximately 2 weeks of culture. A similar increase in size occurred later (or not at all) for hMSCs in AS. After toluidine blue staining, all spheres of hMSCs in FBS stained positive, whereas spheres from only one out of three of the donor’s hMSCs cultured in AS stained positive (Fig. 2A).
Figure 2. Differentiation of hMSCs. Results of staining assays on hMSCs induced to differentiate in chondrogenic direction (A, stained with toluidine blue), osteogenic direction (B, stained with alzarin red), and adipogenic direction (C, stained with oil-red O) are shown. Left, cells expanded in FBS before differentiation; right, cells expanded in AS. Within each of these categories, differentiated cells are shown in the left panels and undifferentiated cells are shown in the right panels. All images magnified x200. Abbreviations: AS, autologous serum; FBS, fetal bovine serum; hMSCs, human bone marrow mesenchymal stem cells.
Observations from real-time PCR analyses were in line with the results from the staining assays and our observations of the cells in differentiation cultures (Table 1). Collagen type II mRNA was present only in cells undergoing chondrogenic differentiation and at higher levels in cells grown in FBS than in AS. Aggrecan mRNA was detected at much higher levels after chondrogenic differentiation than in other cultures and to a higher extent in cells expanded in FBS than in AS. Collagen type I mRNA was present at relatively high levels in all cultures, upregulated upon chondrogenic differentiation relative to undifferentiated cells and downregulated in osteogenic and adipogenic differentiation. Osteomodulin mRNA appeared most strongly in cultures after chondrogenic or osteogenic induction. PPAR-2 mRNA was detected in cells induced toward adipogenic differentiation at much higher levels in cells expanded in FBS than in AS. Surprisingly, PPAR-2 mRNA was observed also in AS cells induced toward chondrogenic differentiation.
Serum Supplement Is a Determinant of Gene Expression
To assess whether the serum supplement used for hMSC culture affected their gene expression, we performed a series of microarray analyses on cells at passage 4. Out of 22,284 probes expressed on the chips, only a few hundred probes represented genes that were differentially expressed depending on the serum supplement. Although some of these differences in expression were donor-specific, many were shared between the three donors (supplemental online Fig. 2). Using twofold upregulation as cut-off, 79 probes representing 59 genes were upregulated in hMSCs in FBS compared with hMSCs in AS. Some of the most highly overexpressed genes, together with genes found to be functionally related to other observations in this study, are presented in Table 2. A detailed list of all the genes overexpressed in hMSCs cultured in FBS is presented in supplemental online Data 1.
Table 2. Selected genes upregulated in human marrow mesenchymal stem cells maintained in fetal bovine serum versus autologous serum at passage 4
Consistent with their slower rate of proliferation, hMSCs in FBS overexpressed genes associated with prolongation of the cell cycle, that is, growth arrest–specific 1 and antiproliferative protein 1. Several genes coding for constituents of the ECM were also upregulated in hMSCs in FBS. Chondrocyte differentiation– related genes, including ECM genes, cytokine receptor-like factor 1 , leptin receptor , and ectonucleotide pyrophosphatase/phosphodiesterase 2 , were overexpressed as well. Genes connected to the chondrocyte-inducting factors transforming growth factor-? and BMP-6 signaling pathways, such as SMAD6 and OLF-1/EBF-associated zinc finger gene, were also upregulated. Moreover, genes related to differentiation into osteoblasts were expressed at higher levels in hMSCs grown in FBS such as cytokine receptor–like factor 1 and glycoprotein (transmembrane) nmb . Other upregulated genes were associated with adipogenesis, such as the leptin receptor , inhibitor of DNA binding 4 , and members of the complement system . Finally, genes related to the synthesis of prostaglandins were also upregulated in hMSCs expanded in FBS. Importantly, many of these genes were overexpressed in FBS also at passage 10.
Considerably fewer genes were consistently upregulated in hMSCs expanded in AS compared with hMSCs in FBS. The entire list is presented in Table 3 (for details, see supplemental online Data 2). Among these genes, angiopoietin-like 4 was previously shown to inhibit apoptosis in endothelial cells and thereby to contribute to the increased cell number observed in these cell cultures . In addition, angiopoietin-like 4 is a target gene for PPARs and thus likely to be involved in adipogenesis. Another gene, ectonucleotide pyrophosphatase/phosphodiesterase 1, has been shown to act as an antagonist of bone mineralization . As for hMSCs in FBS, many of the differentially regulated genes in AS at passage 4 were differentially expressed also at passage 10.
Table 3. Genes upregulated in human marrow mesenchymal stem cells maintained in autologous serum versus fetal bovine serum at passage 4
Serum Supplement Is a Determinant of Transcriptome Stability
To be safe for use in therapeutic protocols, in vitro–expanded hMSCs should not change in the course of cell culture. To determine the effect of serum supplement on transcriptome stability in ex vivo–expanded hMSCs, we compared transcript expression of hMSCs derived from two donors expanded in AS or FBS at passage 4 versus passage 10. Between these time points, the cells underwent approximately 13.5 population doublings, corresponding to approximately 104-fold expansion. Scatter plot analyses of the results are shown in online Figure 3. Although the numbers of differentially expressed genes varied between donors, they were consistently much higher for hMSCs expanded in FBS. For hMSCs in FBS, 46 genes, listed in Table 4 and described in supplemental online Data 3, were differentially expressed over time in both donors. The most striking changes were observed for genes involved in the cell cycle. In passage 10 cells, which were close to proliferative senescence, several genes with essential functions for the normal progression through cell cycle were dramatically downregulated. These genes included topoisomerase (DNA) II alpha (–66-fold), cell division cycle 2 (–30-fold), ribonucleotide reductase M2 polypeptide (–30-fold), Asp (abnormal spindle)-like, microcephaly associated (–24-fold), and others. The effect of FBS on the production of constituents of the ECM and organization of the cytoskeleton was underscored by the fact that a large number of genes important for the cytoskeleton and ECM were upregulated. Moreover, several genes associated with organ specification, including neuronal tissues (neurotrypsin, neurotrophic tyrosine kinase receptor, myelin basic protein, nerve growth factor beta, neurophilin, Eph5A, and tetraspan 3), vessels (prostaglandin 12, tumor endothelial marker 8, neurotrypsin, and Nur77), and bone (osteoprotegrin and oxytocin receptor) were also differentially expressed.
Table 4. Changes in gene expression in human marrow mesenchymal stem cells maintained in fetal bovine serum at passage 10 versus passage 4
In contrast, only six genes, listed in Table 5 and described in supplemental online Data 4, were transcriptionally altered in hMSCs in AS in both donors after 12 population doublings. These were all downregulated at passage 10 and were predominantly genes associated with the ECM and cytoskeleton.
Table 5. Changes in gene expression in human marrow mesenchymal stem cells maintained in autologous serum at passage 10 versus passage 4
DISCUSSION
We would like to acknowledge the skilled technical assistance of Siv Haugen Tunheim and Aileen Murdoch Larsen. This work was supported through the Norwegian Center for Stem Cell Research by the Research Council of Norway and Gidske og Peter Jacob S?rensens Foundation for the Promotion of Science.
DISCLOSURES
The authors indicate no potential conflicts of interest.
REFERENCES
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Muraglia A, Cancedda R, Quarto R. Clonal mesenchymal progenitors from human bone marrow differentiate in vitro according to a hierarchical model. J Cell Sci 2000;113:1161–1166.
Wakitani S, Goto T, Pineda SJ et al. Mesenchymal cell-based repair of large, full-thickness defects of articular cartilage. J Bone Joint Surg Am 1994;76:579–592.
Bruder SP, Kurth AA, Shea M et al. Bone regeneration by implantation of purified, culture-expanded human mesenchymal stem cells. J Orthop Res 1998;16:155–162.
De Kok I, Peter SJ, Archambault M et al. Investigation of allogeneic mesenchymal stem cell-based alveolar bone formation: preliminary findings. Clin Oral Implants Res 2003;14:481–489.
Awad HA, Boivin GP, Dressler MR et al. Repair of patellar tendon injuries using a cell-collagen composite. J Orthop Res 2003;21:420–431.
Murphy JM, Fink DJ, Hunziker EB et al. Stem cell therapy in a caprine model of osteoarthritis. Arthritis Rheum 2003;48:3464–3474.
Pittenger MF, Martin BJ. Mesenchymal stem cells and their potential as cardiac therapeutics. Circ Res 2004;95:9–20.
Kawada H, Fujita J, Kinjo K et al. Nonhematopoietic mesenchymal stem cells can be mobilized and differentiate into cardiomyocytes after myocardial infarction. Blood 2004;104:3581–3587.
Wakitani S, Saito T, Caplan AI. Myogenic cells derived from rat bone marrow mesenchymal stem cells exposed to 5-azacytidine. Muscle Nerve 1995;18:1417–1426.
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Horwitz EM, Prockop DJ, Fitzpatrick LA et al. Transplantability and therapeutic effects of bone marrow-derived mesenchymal cells in children with osteogenesis imperfecta. Nat Med 1999;5:309–313.
Horwitz EM, Gordon PL, Koo WK et al. Isolated allogeneic bone marrow- derived mesenchymal cells engraft and stimulate growth in children with osteogenesis imperfecta: implications for cell therapy of bone. Proc Natl Acad Sci U S A 2002;99:8932–8937.
Le Blanc K, Rasmusson I, Sundberg B et al. Treatment of severe acute graft-versus-host disease with third party haploidentical mesenchymal stem cells. Lancet 2004;363:1439–1441.
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Di Niccola M, Carlo-Stella C, Magni M et al. Human bone marrow stromal cells suppress T-lymphocyte proliferation induced by cellular or non-specific mitogenic stimuli. Blood 2002;99:3838–3843.
Krampera M, Glennie S, Dyson J et al. Bone marrow mesenchymal stem cells inhibit the response of naive and memory antigen-specific T cells to their cognate peptide. Blood 2003;101:3722–3729.
Koc ON, Gerson SL, Cooper BW et al. Rapid hematopoietic recovery after coinfusion of autologous-blood stem cells and culture-expanded marrow mesenchymal stem cells in advanced breast cancer patients receiving high-dose chemotherapy. J Clin Oncol 2000;18:307–316.
Doerr HW, Cinatl J, Sturmer M et al. Prions and orthopedic surgery. Infection 2003;31:163–171.
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b Centre for Occupational and Environmental Medicine, and
c Institute and Department of Pathology, Rikshospitalet University Hospital and University of Oslo, Norway
Key Words. Human mesenchymal stem cells ? Autologous serum ? Fetal bovine serum ? Cell culture ? Gene expression ? In vitro differentiation ? Microarray
Correspondence: J.E. Brinchmann, M.D., Ph.D., Institute of Immunology, Rikshospitalet University Hospital, 0027 Oslo, Norway. Telephone: 47-23-07-37-66; Fax: 47-23-07-38-22; e-mail: j.e.brinchmann@medisin.uio.no
ABSTRACT
Human bone marrow mesenchymal stem cells (hMSCs) represent an appealing source of adult stem cells for cell therapy and tissue engineering. Because hMSCs are present at low frequency in the bone marrow, expansion is necessary before performing clinical studies. Recently, techniques for isolation and extensive subcultivation of hMSCs have been developed. In vitro, culture-expanded hMSCs are capable of differentiation along osteogenic, chondrogenic, and adipogenic lineages . In vivo, several studies in a variety of animal models have shown that hMSCs may be useful in the repair or regeneration of cartilage , damaged bone , tendon , and meniscus . In vitro and in vivo, studies have indicated that hMSCs are capable of differentiation also into cardiomyocytes , skeletal muscle , and neural precursors . hMSCs have been used in clinical trials in children with osteogenesis imperfecta and to promote engraftment and prevent or treat severe graft-versus-host disease (GVHD) in allogeneic stem cell transplantation .
In practically all studies using in vitro–expanded hMSCs, the cell culture medium has been supplemented with fetal bovine serum (FBS). This is also true for human clinical trials approved by the U.S. Food and Drug Administration . The risk of transmission of prion diseases and zoonoses from the use of FBS is considered to be small . A greater risk associated with the use of hMSCs expanded in FBS seems to be the immunogenicity of the xenogeneic FBS proteins. Recently, it was shown that a single preparation of 108 hMSCs grown under standard condition in FBS would carry with it approximately 7–30 mg of FBS proteins . The full clinical impact of this observation remains to be investigated, but the use of autologous serum (AS) instead of FBS was recently shown to prevent life-threatening arrhythmias after cellular cardiomyoplasty . Thus, it seems likely that in the future both clinical and regulatory issues will motivate the use of serum supplements other than FBS. We have therefore examined the possibility of using AS or allogeneic human serum (alloHS) rather than FBS for in vitro expansion of hMSCs. In this study, we present the results of experiments comparing proliferation, phenotype, differentiation capability, and gene expression of hMSCs expanded in different serum preparations.
MATERIALS AND METHODS
hMSCs Are Efficiently Expanded In Vitro in AS and FBS but Not in alloHS
To compare the effect of different serum preparations on the proliferative capability of hMSCs, 108 bone marrow mononuclear cells (BMMCs) depleted of CD14+ cells were established in parallel cultures supplemented with one of three different preparations of FBS, AS, or alloHS. Of the three FBS preparations, FBS (Gibco) consistently yielded the highest cell counts (data not shown). Thus, FBS (Gibco) was selected for all further analyses. A comparison of calculated cumulative cell counts between cells expanded in FBS and AS for four donors is shown in Figure 1. Extrapolation of growth curves suggests that there were approximately 105 hMSCs within the 108 CD14– BMMCs on day 0, giving a precursor frequency of one hMSC in 103 CD14– BMMCs. For each of the donors, the population doubling time was always shorter in hMSCs in AS (median for the four donors, 53 hours; range, 41–54 hours) compared with hMSCs in FBS (median, 84 hours; range, 76–89 hours). In cultures supplemented with alloHS, the number of adherent cells on day 1 was always lower than for cultures with other serum supplements (data not shown). The adherent cells in cultures supplemented with alloHS spread and formed small colonies, but proliferation soon ceased, and as cells cultured in alloHS did not survive (beyond passage 1), cell counts are not entered into Figure 1.
Figure 1. Proliferation of human marrow mesenchymal stem cells (hMSCs). Calculated cumulative cell counts in cultures of hMSCs from four different donors are plotted with filled symbols for hMSCs expanded in autologous serum and open symbols for hMSCs expanded in fetal bovine serum. Identically shaped symbols (e.g., open and filled squares) represent results from the same donor.
hMSCs Cultured in AS Are Morphologically and Phenotypically Similar to hMSCs Cultured in FBS
From initial colony formation until senescence, hMSCs showed a fibroblastoid appearance, with no discernible morphological differences between cells expanded in AS and FBS at any time (supplemental online Fig. 1). However, we observed that hMSCs grown in FBS required prolonged exposure to trypsin to detach from the plastic surface (data not shown). This could not be explained, as determined by flow cytometry, by differential expression of the adhesion molecules tested. Indeed, these were almost equally expressed on the cells regardless of the serum supplement (supplemental online Table 1). As expected, hMSCs expressed HLA class I, CD13, CD44, CD90, and CD105 (SH2) .
Table 1. Relative expression of molecules characteristic for different lineages
Choice of Serum Impacts on the Rate of Differentiation of hMSCs
The multilineage differentiation capability of hMSCs expanded in FBS or AS was examined by culturing cells collected at passage 4 under conditions favorable for chondrogenic, osteogenic, and adipogenic differentiation . Examples of staining assays obtained after 3 or 4 (chondrogenic differentiation) weeks of culture in induction medium are shown in Figure 2. At this time, staining for fat globules (adipogenic differentiation) and mineralization (osteogenic differentiation) was observed in cells from all donors and both serum preparations. However, for cells in adipogenic induction medium, fat globules could be observed under the light microscope already after 3–4 days in hMSCs in FBS, whereas fat globules appeared 8–12 days later in hMSCs in AS (data not shown). The same tendency was observed in chondrogenic-induced cultures. Here, cells were cultured in pellets that lost attachment and appeared as spheres. For hMSCs in FBS, the spheres increased in size, indicating production of extracellular matrix (ECM), from approximately 2 weeks of culture. A similar increase in size occurred later (or not at all) for hMSCs in AS. After toluidine blue staining, all spheres of hMSCs in FBS stained positive, whereas spheres from only one out of three of the donor’s hMSCs cultured in AS stained positive (Fig. 2A).
Figure 2. Differentiation of hMSCs. Results of staining assays on hMSCs induced to differentiate in chondrogenic direction (A, stained with toluidine blue), osteogenic direction (B, stained with alzarin red), and adipogenic direction (C, stained with oil-red O) are shown. Left, cells expanded in FBS before differentiation; right, cells expanded in AS. Within each of these categories, differentiated cells are shown in the left panels and undifferentiated cells are shown in the right panels. All images magnified x200. Abbreviations: AS, autologous serum; FBS, fetal bovine serum; hMSCs, human bone marrow mesenchymal stem cells.
Observations from real-time PCR analyses were in line with the results from the staining assays and our observations of the cells in differentiation cultures (Table 1). Collagen type II mRNA was present only in cells undergoing chondrogenic differentiation and at higher levels in cells grown in FBS than in AS. Aggrecan mRNA was detected at much higher levels after chondrogenic differentiation than in other cultures and to a higher extent in cells expanded in FBS than in AS. Collagen type I mRNA was present at relatively high levels in all cultures, upregulated upon chondrogenic differentiation relative to undifferentiated cells and downregulated in osteogenic and adipogenic differentiation. Osteomodulin mRNA appeared most strongly in cultures after chondrogenic or osteogenic induction. PPAR-2 mRNA was detected in cells induced toward adipogenic differentiation at much higher levels in cells expanded in FBS than in AS. Surprisingly, PPAR-2 mRNA was observed also in AS cells induced toward chondrogenic differentiation.
Serum Supplement Is a Determinant of Gene Expression
To assess whether the serum supplement used for hMSC culture affected their gene expression, we performed a series of microarray analyses on cells at passage 4. Out of 22,284 probes expressed on the chips, only a few hundred probes represented genes that were differentially expressed depending on the serum supplement. Although some of these differences in expression were donor-specific, many were shared between the three donors (supplemental online Fig. 2). Using twofold upregulation as cut-off, 79 probes representing 59 genes were upregulated in hMSCs in FBS compared with hMSCs in AS. Some of the most highly overexpressed genes, together with genes found to be functionally related to other observations in this study, are presented in Table 2. A detailed list of all the genes overexpressed in hMSCs cultured in FBS is presented in supplemental online Data 1.
Table 2. Selected genes upregulated in human marrow mesenchymal stem cells maintained in fetal bovine serum versus autologous serum at passage 4
Consistent with their slower rate of proliferation, hMSCs in FBS overexpressed genes associated with prolongation of the cell cycle, that is, growth arrest–specific 1 and antiproliferative protein 1. Several genes coding for constituents of the ECM were also upregulated in hMSCs in FBS. Chondrocyte differentiation– related genes, including ECM genes, cytokine receptor-like factor 1 , leptin receptor , and ectonucleotide pyrophosphatase/phosphodiesterase 2 , were overexpressed as well. Genes connected to the chondrocyte-inducting factors transforming growth factor-? and BMP-6 signaling pathways, such as SMAD6 and OLF-1/EBF-associated zinc finger gene, were also upregulated. Moreover, genes related to differentiation into osteoblasts were expressed at higher levels in hMSCs grown in FBS such as cytokine receptor–like factor 1 and glycoprotein (transmembrane) nmb . Other upregulated genes were associated with adipogenesis, such as the leptin receptor , inhibitor of DNA binding 4 , and members of the complement system . Finally, genes related to the synthesis of prostaglandins were also upregulated in hMSCs expanded in FBS. Importantly, many of these genes were overexpressed in FBS also at passage 10.
Considerably fewer genes were consistently upregulated in hMSCs expanded in AS compared with hMSCs in FBS. The entire list is presented in Table 3 (for details, see supplemental online Data 2). Among these genes, angiopoietin-like 4 was previously shown to inhibit apoptosis in endothelial cells and thereby to contribute to the increased cell number observed in these cell cultures . In addition, angiopoietin-like 4 is a target gene for PPARs and thus likely to be involved in adipogenesis. Another gene, ectonucleotide pyrophosphatase/phosphodiesterase 1, has been shown to act as an antagonist of bone mineralization . As for hMSCs in FBS, many of the differentially regulated genes in AS at passage 4 were differentially expressed also at passage 10.
Table 3. Genes upregulated in human marrow mesenchymal stem cells maintained in autologous serum versus fetal bovine serum at passage 4
Serum Supplement Is a Determinant of Transcriptome Stability
To be safe for use in therapeutic protocols, in vitro–expanded hMSCs should not change in the course of cell culture. To determine the effect of serum supplement on transcriptome stability in ex vivo–expanded hMSCs, we compared transcript expression of hMSCs derived from two donors expanded in AS or FBS at passage 4 versus passage 10. Between these time points, the cells underwent approximately 13.5 population doublings, corresponding to approximately 104-fold expansion. Scatter plot analyses of the results are shown in online Figure 3. Although the numbers of differentially expressed genes varied between donors, they were consistently much higher for hMSCs expanded in FBS. For hMSCs in FBS, 46 genes, listed in Table 4 and described in supplemental online Data 3, were differentially expressed over time in both donors. The most striking changes were observed for genes involved in the cell cycle. In passage 10 cells, which were close to proliferative senescence, several genes with essential functions for the normal progression through cell cycle were dramatically downregulated. These genes included topoisomerase (DNA) II alpha (–66-fold), cell division cycle 2 (–30-fold), ribonucleotide reductase M2 polypeptide (–30-fold), Asp (abnormal spindle)-like, microcephaly associated (–24-fold), and others. The effect of FBS on the production of constituents of the ECM and organization of the cytoskeleton was underscored by the fact that a large number of genes important for the cytoskeleton and ECM were upregulated. Moreover, several genes associated with organ specification, including neuronal tissues (neurotrypsin, neurotrophic tyrosine kinase receptor, myelin basic protein, nerve growth factor beta, neurophilin, Eph5A, and tetraspan 3), vessels (prostaglandin 12, tumor endothelial marker 8, neurotrypsin, and Nur77), and bone (osteoprotegrin and oxytocin receptor) were also differentially expressed.
Table 4. Changes in gene expression in human marrow mesenchymal stem cells maintained in fetal bovine serum at passage 10 versus passage 4
In contrast, only six genes, listed in Table 5 and described in supplemental online Data 4, were transcriptionally altered in hMSCs in AS in both donors after 12 population doublings. These were all downregulated at passage 10 and were predominantly genes associated with the ECM and cytoskeleton.
Table 5. Changes in gene expression in human marrow mesenchymal stem cells maintained in autologous serum at passage 10 versus passage 4
DISCUSSION
We would like to acknowledge the skilled technical assistance of Siv Haugen Tunheim and Aileen Murdoch Larsen. This work was supported through the Norwegian Center for Stem Cell Research by the Research Council of Norway and Gidske og Peter Jacob S?rensens Foundation for the Promotion of Science.
DISCLOSURES
The authors indicate no potential conflicts of interest.
REFERENCES
Pittenger MF, Mackay AM, Beck SC et al. Multilineage potential of adult human mesenchymal stem cells. Science 1999;284:143–147.
Muraglia A, Cancedda R, Quarto R. Clonal mesenchymal progenitors from human bone marrow differentiate in vitro according to a hierarchical model. J Cell Sci 2000;113:1161–1166.
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