Human mesenchymal stem cells possess different biological characteristics but do not change their therapeutic potential when cultured in serum free medium

Abstract

Introduction: Mesenchymal stem cells (MSCs) are widely investigated in clinical researches to treat various diseases. Classic culture medium for MSCs, even for clinical use, contains fetal bovine serum. The serum-containing medium (SCM) seems a major obstacle for MSCs-related therapies due to the risk of contamination of infectious pathogens. Some studies showed that MSCs could be expanded in serum free medium (SFM); however, whether SFM would change the biological characteristics and safety issues of MSCs has not been well answered.

Methods: Human umbilical cord mesenchymal stem cells (hUC-MSCs) were cultured in a chemical defined serum free medium. Growth, multipotency, surface antigen expression, telomerase, immunosuppressive ability, gene expression profile and genomic stability of hUC-MSCs cultured in SFM and SCM were analyzed and compared side by side.

Results: hUC-MSCs propagated more slowly and senesce ultimately in SFM. SFM-expanded hUC-MSCs were different from SCM-expanded hUC-MSCs in growth rate, telomerase, gene expression profile. However, SFM-expanded hUC-MSCs maintained multipotency and the profile of surface antigen which were used to define human MSCs. Both SFM- and SCM-expanded hUC-MSCs gained copy number variation (CNV) in long-term in vitro culture.

Conclusion: hUC-MCSs could be expanded in SFM safely to obtain enough cells for clinical application, meeting the basic criteria for human mesenchymal stem cells. hUC-MSCs cultured in SFM were distinct from hUC-MSCs cultured in SCM, yet they remained therapeutic potentials for future regenerative medicine.

Figure 1

In vitro growth characteristics of human umbilical cord mesenchymal stem cells cultured in serum-free medium. Both in SCM (a) and in SFM (b), human umbilical cord mesenchymal stem cells (hUC-MSCs) maintained fibroblast-like morphology (40×). (c) Calculated population-doubling time. Open boxes, hUC-MSCs expanded in SFM; filled boxes, hUC-MSCs expanded in serum-containing medium (SCM). SFM-expanded hUC-MSCs possessed a much longer calculated population-doubling time. (d) In vitro lifespan of hUC-MSCs derived from five different donors. (e) Paired t test was used to compare the lifespan of hUC-MSCs cultured in SFM and SCM. A significant different lifespan between SFM-derived and SCM-derived hUC-MSCs was observed. (f) Senescence-associated β-galactosidase activity analysis of SFM-expanded hUC-MSCs at late passage. Blue stain shows senescent cells (×200).

Figure 2

Induced differentiation, flow cytometric and immunosuppressive ability analysis of human umbilical cord mesenchymal stem cells expanded in serum-free medium. After differentiation induction, (a) osteogenesis was confirmed by Alizarin Red (×40), (b) adipogenesis was stained by Oil Red O (×200) and (c) chondrogenesis was analyzed by Toluidine Blue (×100). (d) Serum-free medium (SFM)-expanded human umbilical cord mesenchymal stem cells (hUC-MSCs) at the 10th passage were labeled with antibodies against human antigens CD14-PE, CD19-PE, CD34-FITC, CD45-PE, CD73-PE, CD90-PE, CD105-PE, HLA-ABC-FITC, HLA-DR-PE and Nestin-PE. (e) Expression of hTERT in hUC-MSCs. Graph shows the level of hTERT transcripts of hUC-MSCs cultured in serum-containing medium (SCM) and SFM (n = 5). Values presented as ratio of positive control (HeLa cells). Immunosuppressive ability of hUC-MSCs was evaluated by co-culturing with human peripheral blood mononuclear cells (hPBMCs). (f) Proliferation of hPBMCs was quantified based the measurement of BrdU incorporation during DNA synthesis. (g) Level of interferon gamma (IFN-γ) in the supernatant was determined by ELISA.

Figure 3

Array-based comparative genomic hybridization analysis of human umbilical cord mesenchymal stem cells expanded in serum-free medium and serum-containing medium. Huge unbalanced genome alteration was not obvious in the single-panel rainbow plot, in which each chromosome was differentiated by color. (a) Sample 2 in serum-free medium (SFM). (b) Sample 2 in serum-containing medium (SCM). (c) Sample 4 in SFM. (d) Sample 4 in SCM. (e) A copy number variation (CNV) segment (chr3:181315609 to 181344028), which was observed in both of the SFM-expanded human umbilical cord mesenchymal stem cells (hUC-MSCs; red arrow), was not observed in either of the SCM-expanded samples (blue arrow). (f) CNV identified by array-based comparative genomic hybridization in culture. Amplifications and deletions were mapped onto the human genome for four hUC-MSC clones. Each individual CNV is marked: red circle, amplification; green circle, deletion.

Figure 4

Clustering of gene expression for serum-free medium-cultured and serum-containing medium-cultured human umbilical cord mesenchymal stem cells. Two different clusters (expanded in serum-free medium and in serum-containing medium) were quite obvious. Green, downregulation; red, upregulation.

Figure 5

Upregulated and downregulated genes in the cell cycle pathway after expansion in serum-free medium. BUB1, BUB1B, CCNA2, CCNB1, CCNB2, CCNE2, Cdc20, Cdc23, Cdc25A, Cdc25C, Cdc45L, Cdc6, Cdc2, CDK2, CDKN2C, CHEK1, ESPL1, MAD2L1, MCM2, MCM7, MCM3, MCM4, MCM5, MCM7, ORC1L, PLK1, PTTG1, SKP2, SMAD3, TFDP1, TGFB1, TGFB2, WEE1, YWHAH and YWHAZ were downregulated after culture in serum-free medium (SFM). Only Cdc14 was upregulated during expansion in SFM. Red, genes that were upregulated; green, genes that were downregulated. MAPK, mitogen-activated protein kinase; MCM, Mini-Chromosome Maintenance.

Figure 6

Validation of the mRNA chip by real-time PCR. Relative expression levels were calculated by log₂(fold change): log₂(fold change) > 1, increasing at least twice; log₂(fold change) < −1, decreasing at least twice. Among the 12 genes we validated, only PRKCA showed an inconsistent expression profile. When analyzed by mRNA chip, the expression of PRKCA was downregulated more than twice. However, in the real-time PCR (RT-PCR) analysis, the expression of PRKCA did not show a significant difference during the process of long-term culture in serum-free medium.