Cell-based therapies require large numbers of cells with the necessary characteristics for successful implantation, engraftment, and function. For cartilage tissue engineering, for example, approximately 8–10
cells/mL of tissue are required as starting material.38
shows the projected cell requirements for a variety of repair diameters and thicknesses, assuming disc-shaped defects. The current state-of-the-art in hMSC technology does not consistently achieve this degree of proliferation without partial or total loss of chondrogenic potential. This loss3,5,10,12
limits the applicability of hMSC-based therapies.
As we have reported previously, exposure of hMSCs to FGF-2 during expansion increases the cell yield and enhances their chondrogenic potential. By first passage, the FGF-treated cells average three population doublings more than the control cells: The FGF-treated cells are smaller than their control counterparts, allowing more cells per cm2
at the same level of confluence.18
In addition, the FGF-treated hMSCs exhibit enhanced chondrogenic potential. After four passages, the FGF-treated cells have doubled eight times more than the control (a 256-fold increase in yield). The degree of differentiation, reflective of the interindividual variability among cell preparations, was variable. Nonetheless, they retain a chondrogenic potential which is very diminished or lost entirely in the control cells. After seven passages, no control cells from any of the cell preparations tested retained any chondrogenic potential, whereas some of the FGF-treated cells did. This is consistent with our previous findings of stimulatory effects of FGF-2 on the proliferation and differentiation of bone-marrow-derived progenitors18
and are confirmed by other groups.13,15–17
However, in these reports, bone-marrow-derived progenitor cells exhibited differentiation potential only when expanded in the presence of FGF-2 or cultured at low density.16
In our hands, early-passage hMSCs expanded under control conditions do exhibit chondrogenic potential, which is enhanced in the FGF-treated hMSCs. These differences might be explained by our rigorous screening and testing of serum lots to select those that support the growth of hMSCs and maintain their multipotentiality. This is an important and documented element of our technology,20
as the use of suboptimal serum results in the rapid loss of multipotentiality and slow expansion of hMSCs. Subtle differences in the protocols (seeding density of primary and subsequent cultures, or formulation of the base media) may also play a role in the lack of differentiation potential reported by others for cells grown without FGF.13,15,16
Analysis of DNA and GAG content suggests that the larger size of the aggregates from FGF-treated cells is because of the increased matrix production, and not because of the presence of more cells in the aggregates. Histologic analyses of the pellets revealed qualitative differences between the treatment groups beyond the difference in the size of the pellets.
Clearly, if hMSCs are to be of value to clinical tissue engineering, it will be critical to understand how their proliferation and differentiation potentials are regulated or by what mechanism FGF-2 supplementation enhances their proliferative and chondrogenic potentials. In this study, we take a first step toward this goal and present gene expression signatures of hMSCs with enhanced chondrogenic potential. As in most microarray studies, our dataset presents some sample-to-sample variability39
; the genes identified here as differentially expressed met very stringent selection criteria, increasing our confidence in the data. The analysis revealed differences due to both time in culture and culture conditions. The effects of time in culture in control cells were rapid, with most of the expression changes occurring during the initial passages, and the cells then settling into a fairly stable phenotype with only very few changes occurring later on. Many of these changes might be part of the adaptation of the cells to the monolayer culture conditions. But, in conjunction with the rapid sequential loss of differentiation potentials, this suggests that, under ordinary culture conditions, chondrogenic potential and, perhaps, true “stemness” is a very transient property of hMSCs.
Unsurprisingly, given the mitogenic effects of the treatment, cell signaling, metabolism, and cell-cycle-related genes figure prominently in the list of genes differentially expressed in the FGF-treated cells compared with their control counterparts. We also observed robust changes in the ECM-related gene expression, which trend toward reduced expression of differentiation-related ECM molecules and enhanced expression of ECM catabolism-related genes. The pervasiveness of this pattern suggests that it is more than merely related to the enhanced cell division, but rather represents a fundamental shift in the phenotype of the cells. This is supported by the fact that merely delivering a mitogenic stimulus via platelet-derived growth factor-beta polypeptide (PDGF-BB) had a similar effect on cell division, but failed to improve the maintenance of chondrogenic potential (data not shown). In future experiments, we plan to use PDGF-BB-stimulated cells in the expression array experiments to tighten the focus on nonmitogenic FGF-2 effects.
Our previous gene expression studies in first passage cells18
suggested that FGF-2 might be mediated through mitogen-activated protein kinase (MAPK) and Wnt signaling pathways.40–44
Here, we focused on the differences between hMSC preparations that can successfully differentiate into chondrocytes and those that cannot. Some differences were consistent with those reported previously between the first passage control and FGF-treated cells.18
We found differences in Wnt signaling between chondrogenic and nonchondrogenic cells with upregulation of secreted frizzled-related protein 1 and downregulation of pregnancy-specific beta-1-glycoprotein 1 in chondrogenic cells. The effects on MAPK signaling were not through elements of the feedback loop as previously reported,18
but rather through the upstream regulators of MAPK signaling such as angiopoietin 1 and midkine, which are upregulated in cells with chondrogenic potential. These transcripts may have a role not only in the maintenance of the chondrogenic potential but also in the overall physiology of hMSCs; angiopoietin 1 has been implicated in the paracrine tissue repair activity displayed by MSCs45–47
and as an antiapoptotic factor48
; secreted frizzled-related protein 1 has also been reported to play a role in the tissue repair activity of MSCs49,50
and in controlling osteogenic differentiation.51–53
Additionally, a recent report indicates that six transmembrane epithelial antigen of the prostate 1, also upregulated in cells with chondrogenic potential, may be a marker for MSCs.54
Our data here must now be validated at the level of protein expression and signaling pathway activation to verify whether the different gene expression profiles detected translate into functional differences.
In summary, the chondrogenic potential of MSCs is vulnerable to cell expansion; the expanded cells may exhibit weak or no chondrogenic potential, rendering them suboptimal or useless for the intended application. Supplementation of the growth medium with FGF-2 not only allows investigators to reach target cell numbers quicker but also extends the level of expansion within which these cells are still useful for tissue-engineered cartilage repair. The FGF-treated cells remain chondrogenic after 30 population doublings, whereas control cells are no longer chondrogenic after approximately 20 population doublings, that is over 1000-fold difference in the number of cells. Taken together, our results indicate that we must be extremely careful when these cells are extensively expanded to achieve a target cell number for cartilage tissue engineering applications in particular, but perhaps also when used in other applications.