Human mesenchymal stem cells/multipotent stromal cells consume accumulated autophagosomes early in differentiation

Abstract

IntroductionBone marrow mesenchymal stem cells/multipotent stromal cells (MSCs) are recruited to sites of injury and subsequently support regeneration through differentiation or paracrine activity. During periods of stress such as wound site implant or differentiation, MSCs are subjected to a variety of stressors that might activate pathways to improve cell survival and generate energy. In this study, we monitored MSC autophagy in response to the process of differentiation.MethodsMSC autophagosome structures were observed by using transmission electron microscopy and a tandem green fluorescent protein-red fluorescent protein autophagic flux reporter to monitor the mammalian microtubule-associated protein-1 light chain 3 (LC3) turnover in real time. MSCs were differentiated by using standard osteogenic and adipogenic media, and autophagy was examined during short-term and long-term differentiation via immunoblots for LC3I and II. Autophagy was modulated during differentiation by using rapamycin and bafilomycin treatments to disrupt the autophagosome balance during the early stages of the differentiation process, and differentiation was monitored in the long term by using Von Kossa and Oil Red O staining as well as quantitative polymerase chain reaction analysis of typical differentiation markers.ResultsWe found that undifferentiated MSCs showed an accumulation of a large number of undegraded autophagic vacuoles, with little autophagic turnover. Stimulation of autophagy with rapamycin led to rapid degradation of these autophagosomes and greatly increased rough endoplasmic reticulum size. Upon induction of osteogenic differentiation, MSC expression of LC3II, a common autophagosome marker, was lost within 12 hours, consistent with increased turnover. However, during adipogenic differentiation, drug treatment to alter the autophagosome balance during early differentiation led to changes in differentiation efficiency, with inhibited adipocyte formation following rapamycin treatment and accelerated fat accumulation following autophagosome blockade by bafilomycin.ConclusionsOur findings suggest that MSCs exist in a state of arrested autophagy with high autophagosome accumulation and are poised to rapidly undergo autophagic degradation. This phenotype is highly sensitive, and a balance of autophagy appears to be key in efficient MSC differentiation and function, as evidenced by our results implicating autophagic flux in early osteogenesis and adipogenesis.

Mesenchymal stem cells/multipotent stromal cells (MSCs) are glycolytic and thus glucose is a limiting factor of in vitro models of MSC starvation

“…Briefly, we found rapid uptake of glucose in MSC cultures, coinciding with a glycolytic MSC phenotype that suggests a key role for glucose in implant sites or approaches to extending MSC lifespan. We also found that MSC autophagy, which we have previously found is a unique and important process in MSC function [4], responded rapidly to changes in glucose concentration. Interestingly, in a separate series of experiments, oxygen deprivation did not increase autophagy, and our calculations suggested that only in near anoxic conditions (<1%) would this be rate limiting; thus, we did not isolate this nutrient in these in vitro manipulations.…”

“…We have previously found that MSC autophagy is intimately tied to normal MSC function in differentiation through a unique phenotype of accumulated autophagosomes in vitro [4]. This could explain how the cells survive the depletion of glucose, both in vitro and in vivo.…”

“…Several studies from our group and others have outlined the key role of autophagy in MSC stress responses, including differentiation, death resistance, and others [3, 4, 21]. As implanted MSCs are immediately subject to a lack of available nutrients, the rapid autophagic response seen in MSCs exposed to changing glucose concentrations further outlines the crucial nature of basal autophagy and autophagosome cycling in the MSC response to changing conditions and stressors.…”

“…One major challenge faced by these cells that are present in all tissues is that wounding disrupts the blood supply that brings nutrients. A key response to cellular starvation is autophagy, which has been recently reported to occur in MSCs at the start of differentiation in a manner that enhances the efficiency [3, 4]. Thus, to contribute to repair, the MSCs must survive and subsequently differentiate or secrete beneficial factors in harsh environments.…”

Equine metabolic syndrome impairs adipose stem cells osteogenic differentiation by predominance of autophagy over selective mitophagy

“…Our data stand with a good agreement with Nuschke et al . who showed that human MSCs consume accumulated autophagosomes early in differentiation confirming autophagy key role in that process. Moreover, it was described that hypoxia‐mediated differentiation of RAW264.7 depends on autophagy activated via HIF‐1‐α .…”