In Vitro High-Capacity Assay to Quantify the Clonal Heterogeneity in Trilineage Potential of Mesenchymal Stem Cells Reveals a Complex Hierarchy of Lineage Commitment

TLDR

Bone marrow mesenchymal stem cells are a promising source for diverse cellular therapies, and their heterogeneity’s impact on therapeutic efficacy is highlighted. The study develops a high‑capacity assay to quantify clonal heterogeneity in MSC potency. The assay uses a 96‑well format to classify MSC clones by colony‑forming efficiency and trilineage differentiation (adipogenic, chondrogenic, osteogenic) while preserving a frozen template of known‑potency clones. The assay revealed that normal bone marrow MSCs exhibit all eight trilineage potential categories, with 55–62 % colony‑forming efficiency and nearly half of colonies being tripotent, indicating a more complex lineage hierarchy, higher proliferation for multipotent clones, and CD146 as a potential multipotency marker, underscoring the assay’s broad applicability.

Abstract

Abstract In regenerative medicine, bone marrow is a promising source of mesenchymal stem cells (MSCs) for a broad range of cellular therapies. This research addresses a basic prerequisite to realize the therapeutic potential of MSCs by developing a novel high-capacity assay to quantify the clonal heterogeneity in potency that is inherent to MSC preparations. The assay utilizes a 96-well format to (1) classify MSCs according to colony-forming efficiency as a measure of proliferation capacity and trilineage potential to exhibit adipo-, chondro-, and osteogenesis as a measure of multipotency and (2) preserve a frozen template of MSC clones of known potency for future use. The heterogeneity in trilineage potential of normal bone marrow MSCs is more complex than previously reported: all eight possible categories of trilineage potential were detected. In this study, the average colony-forming efficiency of MSC preparations was 55–62%, and tripotent MSCs accounted for nearly 50% of the colony-forming cells. The multiple phenotypes detected in this study infer a more convoluted hierarchy of lineage commitment than described in the literature. Greater cell amplification, colony-forming efficiency, and colony diameter for tri- versus unipotent clones suggest that MSC proliferation may be a function of potency. CD146 may be a marker of multipotency, with ∼2-fold difference in mean fluorescence intensity between tri- and unipotent clones. The significance of these findings is discussed in the context of the efficacy of MSC therapies. The in vitro assay described herein will likely have numerous applications given the importance of heterogeneity to the therapeutic potential of MSCs.