Survival of aging CD264⁺ and CD264⁻ populations of human bone marrow mesenchymal stem cells is independent of colony‐forming efficiency

Abstract

In vivo mesenchymal stem cell (MSC) survival is relevant to therapeutic applications requiring engraftment and potentially to nonengraftment applications as well. MSCs are a mixture of progenitors at different stages of cellular aging, but the contribution of this heterogeneity to the survival of MSC implants is unknown. Here, we employ a biomarker of cellular aging, the decoy TRAIL receptor CD264, to compare the survival kinetics of two cell populations in human bone marrow MSC (hBM‐MSC) cultures. Sorted CD264+ hBM‐MSCs from two age‐matched donors have elevated β‐galactosidase activity, decreased differentiation potential and form in vitro colonies inefficiently relative to CD264− hBM‐MSCs. Counterintuitive to their aging phenotype, CD264+ hBM‐MSCs exhibited comparable survival to matched CD264− hBM‐MSCs from the same culture during in vitro colony formation and in vivo when implanted ectopically in immunodeficient NIH III mice. In vitro and in vivo survival of these two cell populations were independent of colony‐forming efficiency. These findings have ramifications for the preparation of hBM‐MSC therapies given the prevalence of aging CD264+ cells in hBM‐MSC cultures and the popularity of colony‐forming efficiency as a quality control metric in preclinical and clinical studies with MSCs.

A cautionary tale about the use of colony-forming efficiency as a proxy for the survival of mesenchymal stem cells

“…CFU, colony-forming unit; hBMSCs, human bone marrow mesenchymal stem cells; Luc, luciferase CD264 + cells (Fig. 1b) [4]. These findings cast doubt on the utility of colony-forming efficiency as a metric of in vivo survival.…”

“…Our findings do not support this supposition. In the study described above, we compared the survival of CD264 +/− hBMSCs attached to ceramic scaffolds, which were implanted subcutaneously in immunodeficient mice [4]. Bioluminescence imaging revealed that matched implants of CD264 − and CD264 + hBMSCs from the same culture had a similar in vivo half-life despite a lower colony-forming efficiency for the aging Fig. Illustration of the discrepancy between the colony-forming efficiency and survival of CD264 +/− populations of hBMSCs.…”

“…In this scenario, colony-forming efficiency underestimates the percentage of surviving cells. Recently, my research group published an illustrative example with human bone marrow MSCs (hBMSCs) [ 4 ]. Each culture was separated into two populations by fluorescence-activated cell sorting based on surface expression of CD264.…”

“…As discussed below, the underlying assumption of equivalence between these two parameters is invalid in some instances. A recently published study from my laboratory provides illustrative examples [4]. In such cases, measurements of colony-forming efficiency can lead to misinterpretation of experimental data and spurious conclusions about MSC survival.…”

Senescence in Mesenchymal Stem Cells: Functional Alterations, Molecular Mechanisms, and Rejuvenation Strategies

“…Furthermore, low but not high CD146 expression was associated with a senescent phenotype in MSCs (Jin et al, 2016), and CD146 + MSCs showed increased migratory potential toward degenerating tissues (Wangler et al, 2019). CD264 is another surface marker of in vitro aging in MSCs that is unrelated to the chronologic age of the donor (Madsen et al, 2017); cells expressing this protein exhibit increased senescence-associated β-galactosidase (SA-β-gal) activity and reduced differentiation potential and colony-forming efficiency compared to CD264 − MSCs (Madsen et al, 2020). Other surface markers that show altered expression with in vitro/in vivo aging are summarized in Table 2.…”