Concise Review: When Colonies Are Not Clones: Evidence and Implications of Intracolony Heterogeneity in Mesenchymal Stem Cells

Abstract

The emergence of heterogeneity in putative mesenchymal stem cell (MSC) populations during in vitro expansion is not appreciated fully by the various communities who study, engineer, and use such stem cells. However, this functional diversity holds direct implications for basic research and therapeutic applications of MSCs that require predictable phenotypic function and efficacy. Despite numerous clinical trials pursuing MSC therapies, the in vitro expansion of homogeneous populations to therapeutically relevant quantities remains an elusive goal. Variation in MSC cultures has been noted not only among donors and within populations expanded from the same donor, but also debatably within single-cell-derived colonies. The potential for even intracolony heterogeneity suggests that any purified subpopulation will inevitably become heterogeneous upon further expansion under current culture conditions. Here, we review the noted or retrospective evidence of intracolony MSC heterogeneity, to facilitate discussion of its possible causes and potential solutions to its mitigation. This analysis suggests that functional diversity within an MSC colony must be considered in design of experiments and trials for even nonclonal stem cell populations, and can be mitigated or even exploited when the mechanisms of onset are better understood. STEM CELLS 2016;34:1135-1141 SIGNIFICANCE STATEMENTThe emergence of heterogeneity in putative mesenchymal stem cell (MSC) populations during in vitro expansion is not fully appreciated; however, it holds direct implications for basic research and therapeutic applications of such stem cells. In this concise review, data documenting heterogeneity even within single-cell-derived colonies are highlighted, illustrating that any purified subpopulation will become heterogeneous upon further expansion under current culture conditions and that the common, underlying assumption of MSC clonal purity should be reexamined. The impact of recognizing the existence of such inevitable heterogeneity could lead to studies aimed toward elucidating its causes and engineering its mitigation for improved clinical outcomes.

High fidelity visualization of cell-to-cell variation and temporal dynamics in nascent extracellular matrix formation

“…Continuous labeling between days 0–9 showed that matrix protein accumulation by MSCs lagged behind that of chondrocytes, and differed in its spatial organization. Notably, there was high MSC-to-MSC variation in the labeled extracellular protein present (Figs 6a,c and S8a,b), consistent with the heterogeneous nature of this cell population222324. This heterogeneity manifested in two ways: in the intensity of the labeled matrix proteins and in the pattern of matrix protein distribution.…”

Morphological features of IFN-γ–stimulated mesenchymal stromal cells predict overall immunosuppressive capacity

“…Improvements on single-cell morphological analysis using high-dimensional singlecell classification techniques such as SPADE or viSNE could allow for identification of morphologically distinct subpopulations of MSCs that could be enriched for immunotherapies (52). These approaches could also provide insight into the progression and establishment of MSC heterogeneity as it relates to differences in donor (or tissue) source and in manufacturing (40,53). Advancements in live-cell tracking and monitoring in vitro would further allow for direct correlation of early morphological phenotypes with assay outcomes (15,54).…”

Application of platelet-rich plasma with stem cells in bone and periodontal tissue engineering

“…In fact, these challenges and benefits from addressing emergent population heterogeneity are not limited to MSCs, and they can be considered for other tissue-derived stem cells and induced pluripotent cell populations and can be overcome by sorting by label-free biophysical markers or by biophysical or other characteristics. 47 Because of the extremely low yields of BMSC progenitors (typically 0.001%–0.01%) obtained from bone marrow aspirates, large quantities of bone marrow must be procured, which can cause additional donor site morbidity. 48 In addition, several passages are required in order to separate the hematopoietic population and other cell types from the pure BMSCs.…”