Fluorescence imaging based analysis of actin turnover: longitudinal profiling of stem cell phenotypes during differentiation
Creators: name: Mishra, Prakhar
Publisher: name: No Publisher Supplied
Year: 2020

description: Stem cells derived from adults have become the focal point of cell-based regenerative therapies. The most commonly used stem cells, mesenchymal stem cells (abbreviated MSCs), have several traits such as the ability to migrate to injury site, immunomodulation, in vitro expansion and differentiation into a variety of cells. However, the widespread usage of MSCs is hampered by poor characterization, tissue culture induced senescence and innate heterogeneity that makes it challenging to predict their clinical efficacy. The traditional methods for evaluation of stem cells are primarily terminal assays that rely on discrete time-point data sets that offer limited, discontinuous information and often overlook the intrinsic phenotypic diversity of these cells. In this thesis, we address some of these issues by developing a novel platform for live cell imaging which can be used to monitor evolving MSCs by generating continuous data sets over the course of several hours or several days while sparing the cells for multiplexing with other traditional assays. Our imaging-based approach relies on a cell permeable fluorogenic probe, SiR-actin (SA), that becomes fluorescent only when it labels endogenous actin filaments (F-actin). The idea of harnessing actin as a sentinel stem cell reporter is supported from past research conducted by Moghe lab and numerous other research groups, as the cytoskeleton can modulate and display the phenotypic changes associated with lineage commitment. Unlike previous research that focused primarily on morphology of actin cytoskeleton, we focused on utilizing the shifting actin turnover rates in response to extracellular cues as the key reporter metric. After SA labelling, actin reorganization leads to removal of the SA probe from F-actin binding sites and subsequent decline in SA fluorescence. When cells are cultured in differentiation media, the rate of fluorescence loss of SA provided insights about the kinetics of lineage specific change in actin reorganization. We report that initiation of differentiation involves decline in actin turnover during adipogenesis and chondrogenesis within few hours. By combining SA with another F-actin probe, phalloidin, we were able to parse heterogenous single cells across the standard tri-lineages (adipogenic, osteogenic and chondrogenic) within 1 hour of stimulation. In addition, our approach enabled assessment of in vitro aging by demonstrating a slowdown in actin turnover within 1 hour of analysis. Next, to establish the link between actin turnover and stem cell differentiation, we employed immunolabeling to demonstrate co-occurrence of altered actin turnover with differentiation markers in MSCs as well induced pluripotent stem cells (iPSCs). We also propose that inherent actin turnover status of individual cells could be a determinant of their differentiation potential. To this end, we isolated cells based on differential SA expression and found that the actin turnover plays a role in determining the ability to differentiate towards osteogenic or adipogenic fate. In summary, we propose actin turnover as a novel dynamic marker as it provides immediate readouts of changing cell states that could be utilized to forecast stem cell behavior towards regenerative therapies.