Emergent heterogeneity in putative mesenchymal stem cell colonies: Single-cell time lapsed analysis

Abstract

Bone marrow stromal cells (BMSCs) include a subset of stem cells that are considered promising for developmental studies and therapeutic applications. While it is appreciated generally that BMSC populations can exhibit morphological and functional heterogeneity upon in vitro culture expansion, the potential for heterogeneity within a single colony forming unit–generated ostensibly from a single mother cell–is less explored but is critical to design of both fundamental studies and cell therapy production. Here we observed BMSC colony formation in real time via time lapsed optical imaging and analysis, to quantify whether and how heterogeneity emerged over multiple cell divisions spanning the duration of a typical colony formation unit assay. These analyses demonstrate that such colonies are neither homogeneous subpopulations of stem cells nor necessarily derived from single originating cells. While the mechanisms for and causes of this intracolony heterogeneity are not understood fully, we further demonstrate that extensive cell-cell contacts do not correlate with senescence, but that media exchange was concurrent with diversification in even the most uniform single-cell-derived colonies. These direct quantitative observations and visualizations of colony formation provide new insights that are motivated by significant implications for both basic research and stem cell-based therapies.

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

“…Many stem cell scientists recognize that cell survival is only one of several factors that contribute to colony formation [ 5 ]. Some researchers, however, employ colony formation as an indirect measure of in vitro cell survival [ 6 , 7 ].…”

Cell Heterogeneity, Rather Than Cell Solvents Affects the Behavior of Mesenchymal Stem Cells in Vitro and Vivo

“…Additionally, it is generally believed that cell clusters derived from a single cell should be functionally homogeneous stem cells, but this is not the always case. Single-cell colonies are not necessarily homogeneous subpopulation, and colonies cannot accurately represent their entire putative stem cell subpopulation [20]. Heterogeneity is so pervasive that it is reasonable to infer MSC populations are intrinsically heterogeneous [21].…”

From the Basis of Epimorphic Regeneration to Enhanced Regenerative Therapies

“…Indeed, some authors have considered whether among the heterogenous MSC population they are some subsets of MSC with a higher regenerative potential and whether particular subsets are more regenerative whatever the damaged tissue we are studying (O'Connor, 2019 ). Thus, using single cell RNA sequencing method, MSC heterogeneity has been intensively studied and the existence of several distinct MSC subsets that possess diverse functions has been shown (Huang et al, 2019 ; Rennerfeldt et al, 2019 ; Sun et al, 2020 ). More advances on the identification and characterization of such MSC regenerative subsets are needed for the identification of the most suitable MSC population for a given application and thus significantly improve MSC-based therapy for OA.…”