To facilitate the transition of human MSC biology from basic studies to clinical application, advances in the reagents used to expand these therapeutically relevant cells has become an absolute necessity. One of the most important achievements in this area will be the transition from SCM to a better-defined SFM. Although recent efforts have shown that human placenta and bone marrow-derived MSCs can be isolated and expanded over the short term in serum-free medium [14
], no published work had shown the ability to expand human MSCs under serum-free conditions for the long term in culture. To achieve such a feat, the optimized serum-free medium StemPro®
MSC SFM was developed [10
]. Perhaps one of the most important features of this serum-free formulation was the optimization of the growth factors required for optimal human MSC expansion. Three of the most important growth factors described as playing a role in both human MSC proliferation and differentiation are PDGF-BB, bFGF, and TGF-β1 [10
]. Whereas PDGF-BB and bFGF had positive effects on cell growth individually, TGF-β1 appeared to provide no enhancement of cell proliferation on its own. Interestingly, although combinations of any two growth factors appeared to provide minimal or no significant enhancement of cell proliferation compared with single factors, the combination of all three factors provided an obvious synergistic effect.
To test the ability of the optimized SFM to support continual propagation, established MSC cultures in SCM were transitioned directly into SFM. It is important to note the difference in morphology of cells in SFM versus SCM. On removing from serum, cells grown in SFM adopted a more spindle-shaped morphology with a distinct cell-growth pattern. This morphology allowed cells in SFM to be grown at much higher densities compared with cells grown in SCM. As a result, this morphologic change caused cells cultured in SCM versus SFM to require different seeding densities or passage frequencies or both. Also, unlike cells grown in SCM, which can be grown optimally at very low seeding densities, cells in SFM appeared to perform better at higher seeding densities (~1 × 104 cells/cm2), facilitating optimal cell expansion and continual propagation. The ability to expand human MSCs at high densities with retained multipotency provides a beneficial approach.
As the MSC field moves toward the establishment of more-robust and efficient clinical protocols, the ability to expand large numbers of cells at high density quickly by using fewer culture wares and reagents may prove very useful. It is noteworthy that cells transitioned directly from SCM to SFM may have shown enhanced proliferation for the first one to two passages directly out of SCM. After two passages, SFM cultures provided an average expansion rate (doubling time; average for eight passages) similar to that achieved with SCM (46.3 ± 7.1 versus 52.5 ± 6.9 (SD) hours, respectively). In addition, similar to that described recently [15
], our optimized SFM was capable of supporting expansion of MSCs directly from primary human bone marrow. Unlike typical observations for primary cultures in SCM, those in SFM did not result in fibroblast colonies, but rather generated somewhat evenly distributed proliferative MSC-like cells throughout the culture flask. The reason for this different growth pattern is unknown, but may be a result of the substrate (human plasma fibronectin or CELLstart™) required for MSC growth in SFM or the presence of recombinant human growth factors (PDGF-BB, bFGF, and TGF-α1), which have been described as having a migratory effect on MSCs [16
It is important to note that the described multipassage expansion experiments were conducted by using (a) pooled passage 4 human MSCs isolated in SCM from four individual donors and transitioned to SFM; (b) single-donor human MSCs isolated in SCM and transitioned to SFM; and (c) primary human MSCs isolated directly in SFM from a single donor. Although the presented data do not directly address donor-to-donor variability and the efficacy for human MSCs isolated in SCM versus SFM, it is noteworthy that Agata et al
] directly addressed these questions. In this work, the authors concluded that the efficacy of primary cultures was greater in SFM compared with SCM and that this effect was independent of specific donors.
The use of pooled human MSCs in our study was intended to overcome donor-to-donor variability and provide a reliable and consistent population of MSCs for experimentation. Although this approach certainly provides a consistent average population of cells for relatively short-term cultures, it is possible during long-term culture that human MSCs from a single donor (or donors) with faster growth rates may overtake the cultures and therefore provide a less-average and more-optimal result.
Despite the nontraditional growth patterns, morphology, and expansion protocols required for growing human MSCs in SFM, expanded MSCs in this medium still displayed multipotentiality with successful differentiation to adipocytes, osteoblasts, and chondrocytes. As the chondrogenic potential of cultured MSCs is often the first differentiation capacity lost during in vitro
expansion (unpublished results), the apparent retained robust chondrogenic differentiation potential shown through pellet cultures and Toludine Blue O staining is a promising characteristic provided by this serum-free medium. As described by Agata et al
], human bone marrow-derived MSCs isolated and expanded in SFM still retained the competence for osteogenic induction and ectopic bone formation.
As proposed previously by the International Society for Cell Therapy [13
], a population of multipotent human MSCs must possess a specific cell-surface antigen expression profile. In accordance with that defined by this group, cells grown in SFM displayed positive expression of CD73, CD90, and CD105 and negative expression of CD11b, CD14, CD19, CD34, CD45, and CD79a, as detected by quantitative real-time PCR and flow cytometry. Interestingly, cells grown in SFM displayed significantly enhanced expression of the intermediate filament nestin. Whereas nestin was originally identified as a marker for neural stem cells [20
], it has since been reported to be present in numerous cell types including human MSCs [21
]. As pericytes have been suggested the in vivo
origin of in vitro
expanded human MSCs [22
] and have been reported to express nestin in vivo
], it may be possible that nestin expression represents a more in vivo
phenotype for expanded MSCs.
After an analysis of gene-expression data generated on SFM versus SCM cultures, dendrogram results reveal that, over multiple passages, cells grown in SFM clustered together and cells in SCM cluster together. Despite this clustering, the pairwise comparison of SFM versus SFM global gene expression (passage 2; R2 = 0.9188) suggests a close correlation between the expression pattern of MSCs grown in SCM and SFM. In addition, cells grown in either medium for additional passages continue to show close correlation with earlier passaged cells in the same medium, suggesting minimal genotypic changes. Although differences in gene-expression analysis between MSCs grown in SFM and SCM do not seem to have an impact on either proliferation or multipotency, a more-detailed analysis on the effect these differentially expressed genes may play on overall MSC biology must be further explored. It is important to note that the bulk of the characterization data provided in this work was conducted on cells isolated by using SCM and thereafter transitioned to SFM and further expanded under such conditions. As the possibility exists that the standard SCM isolation procedure may result in the selection of a certain population of primary cells (MSCs) with a distinct phenotype, it cannot be ruled out that such characterization experiments (that is, gene-array studies) may differ when comparing cells isolated under SCM versus SFM conditions.
In addition to the data presented here focusing on the expansion of human bone marrow-derived MSCs, it has also been observed that the described SFM may be used for expansion of human MSCs from other sources including umbilical cord-derived MSCs and adipose-derived stem cells (ADSCs) (unpublished data). Despite the preliminary data suggesting the ability to expand these additional MSC populations in SFM, future studies will be necessary to determine whether additional supplementation is necessary most efficiently to expand these populations.