Multipotent MSC clones in this study had a significantly higher proliferation potential than their lineage-committed counterparts, as measured by colony-forming efficiency and growth kinetics during ex vivo expansion. For the latter, inocula of OAC clones produced cultures with statistically greater cell accumulation, faster growth rates and less apoptotic cells than inocula of O clones. We did not detect significant differences in proliferation potential between OA and OC clones. These data were obtained with early passage MSCs harvested from a single bone marrow donor. The variance in proliferation among clones of a given potency is consistent with the broad range in the extent of differentiation within a potency group (Fig. 2). The proliferative heterogeneity observed in our clones demonstrates the stochastic variability inherent to the process of cell growth and differentiation, and development of a predictive model of potency on the basis of proliferation parameters would clearly need to account for such variation.
Results from this study agree with preliminary data from our laboratory on the proliferation potential of MSCs from a different donor. The median MSC concentration in clonal cultures after 6 days of ex vivo expansion decreased significantly with lineage commitment (Russell et al., 2010). Similar data were obtained before and after freezing the clones. Combined results from our two studies suggest that the observed loss of proliferation potential with lineage commitment was not donor specific, nor an artifact of cryopreservation.
The findings presented here on colony-forming efficiency aid in interpreting data from our high-capacity assay on MSC heterogeneity. The assay quantifies the percentage of colony-forming cells of a given potency in heterogeneous MSC cultures. OAC clones account for ∼50% of colony-forming MSCs from healthy donors examined to date, as compared with 10–20% for O clones (Russell et al., 2010). The prevalence of multipotent MSCs may be due, in part, to their greater colony-forming efficiency relative to more lineage-committed clones.
The correlation between MSC proliferation and potency detected here with clonal analysis is consistent with diminished growth and differentiation of heterogeneous MSC cultures with serial passage. After passage 4–6, the colony-forming efficiency of MSCs and their accumulation during ex vivo expansion is significantly lower than at early passage (Bruder et al., 1997; DiGirolamo et al., 1999). This decrease in proliferation in heterogeneous MSC cultures is accompanied by a substantial reduction, if not complete loss, in adipogenic and chondrogenic potential (DiGirolamo et al., 1999; Kretlow et al., 2008).
Our research provides insight into conflicting reports on the relationship between MSC proliferation and potency obtained by clonal analysis. Consistent with our findings on MSCs from healthy donors, Mareddy et al. (2007) observed that most of their fast-growing MSC clones from bone marrow of osteoarthritic patients exhibited an OAC phenotype; whereas, the majority of the slow-growing clones were more lineage committed. In contrast, a similar investigation was unable to detect a dependence of proliferation on MSC potency (Karystinou et al., 2009). In this case, analysis was confined to OAC and OC clones of MSCs from osteoarthritic and healthy donors. Our results reconcile these discrepancies: we observed significant differences in proliferation potential between OAC and O clones, but not among OAC, OA, and OC clones. Lee et al. (2010) reported that highly proliferative MSC clones were multipotent, as in our research; however, they had insufficient quantities of slow-growing clones for a detailed analysis of potency. Our research emphasizes the importance of including lineage-committed clones in the analysis of MSC properties as a function of potency.
The difference in proliferation potential between OAC and O clones is relevant to the cultivation of MSCs and prediction of their therapeutic efficacy. We detected faster growth rates for OAC clones, suggesting that low inoculation densities may enrich the content of this cell population in heterogeneous MSC cultures during ex vivo expansion. This agrees with the inverse correlation of the yield of multipotent MSCs to the inoculation density previously observed (Sekiya et al., 2002; Sotiropoulou et al., 2006). Also, our research demonstrated that OAC clones are statistically more clonogenic than O clones. This supports the use of colony-forming efficiency as a simple measurement to monitor the content of multipotent cells in MSC preparations from different donors (DiGirolamo et al., 1999). The content of fast-growing, multipotent cells profoundly affects the therapeutic efficacy of MSCs. For example, a population of rapidly proliferating MSCs exhibited preferential tissue engraftment relative to more slowly proliferating MSCs (Lee et al., 2006). As such, colony-forming efficiency could be a predictive measure of the efficacy of MSC therapies.
O clones expressed a characteristic phenotype of senescence: slow growth rate, large cell size, and elevated β-galactosidase activity at pH 6.0 in subconfluent cultures. In agreement with our data, there are reports of fast-growing MSCs with a small, spindle-shaped morphology and slow-growing MSCs that were large, flat, and cuboid (Colter et al., 2001; Tormin et al., 2009). The yield of small MSCs (classified as recycling stem (RS) cells and type I cells) in heterogeneous cultures is inversely related to the incubation time of each passage (Sekiya et al., 2002). The fraction of large, flat MSCs (also known as type II and mature cells) in culture increases upon serial passage, particularly at high plating densities (Mets and Verdonk, 1981; Neuhuber et al., 2008). Cultures enriched for small MSCs are multipotent; whereas, larger MSCs have diminished differentiation potential (Neuhuber et al., 2008; Sekiya et al., 2002). Furthermore, Mareddy et al. (2007) detected senescence-associated β-galactosidase activity in slow-growing MSCs with limited potency and negligible activity in fast-growing, multipotent MSCs.
These findings have ramifications for the preparation of MSC therapies for donors with appreciable concentrations of senescent cells, particularly for patients with non-union bone fractures and the elderly. MSCs harvested from non-union bone fractures have a greater content of senescent cells and limited osteogenic potential relative to marrow-derived MSCs (Bajada et al., 2009). Similarly, our senescent O clones exhibit limited mineralization during osteogenesis. Recent data suggest that senescent MSCs accumulate in the marrow of the elderly (Wagner et al., 2009; Zhou et al., 2008). For these patients, the loss of proliferation and osteogenic potential in senescent MSCs may compromise the efficacy of autologous stem cell therapies.
Enrichment of robust multipotent cells from heterogeneous MSC cultures containing senescent cells may improve the treatment outcome of autologous MSC therapies by increasing both cell yield after ex vivo expansion and enhancing the integrity of regenerated tissue. As mentioned in the Introduction, an immunophenotype of multipotent MSCs remains poorly defined. High-throughput clonal analysis can be helpful in identifying molecular markers that can distinguish MSCs with different differentiation potential. To this end, we have identified CD146 as a possible biomarker of MSC potency with our high-capacity assay: this cell adhesion molecule is more abundant on the surface of OAC clones than on the parent MSCs from which the clones were derived (Russell et al., 2010). Identification of potency markers may enable consistent and rapid production of efficacious MSC therapies from numerous donors.