It therefore remains uncertain whether hPSCs undergo a decrease in proliferation and increase in size with induced differentiation, or if changes in cell proliferation and size (biomass) are coordinated with early hPSC differentiation programs (5). To date, hPSC size measurements have Trimebutine been imprecise and used to document extreme changes in cross-sectional area by microscopy (3). the differentiation of pluripotent stem cells (PSCs). This impasse exists because PSCs grow in tight clusters or colonies, precluding most quantifying approaches. Here, we investigate live cell interferometry as an approach to quantify the biomass and growth of HSF1 human PSC colonies before and during retinoic acid-induced differentiation. We also provide an approach for measuring the rate and coordination of intracolony mass redistribution in HSF1 clusters using live cell interferometry images. We show that?HSF1 cells grow at a consistent, exponential rate regardless of colony size and display coordinated intracolony movement that ceases with the onset of differentiation. By contrast, growth and proliferation rates show a decrease of only 15% decrease during early?differentiation despite global changes in gene expression and previously reported changes in energy metabolism. Overall, these results suggest that cell biomass and proliferation are regulated independent of pluripotency during early differentiation, which is distinct from what occurs with successful reprogramming. Introduction Human pluripotent stem cells (hPSCs) generate all embryonic cell types and can be grown for prolonged periods in?culture, with their pluripotency/self-renewal program controlled by chromatin structure and a core transcription factor network (1). hPSCs are also small, replicate rapidly, and grow in tight colonies. It remains unknown whether the size and growth rate of hPSCs are regulated by the program that regulates pluripotency/self-renewal, a question that has important implications for our understanding of the relationship between growth rate controls and early differentiation. Interestingly, key features of hPSCs can be reestablished in somatic cells by reprogramming factors (2) and recent, single-cell imaging studies have shown that fibroblasts reprogrammed to hPSCs undergo an increase in?proliferation and reduction Trimebutine in area/size within the first cell division (3). This work identified replicative and size Trimebutine barriers that must be rapidly overcome for successful reprogramming. Reprogramming to, and differentiating from, pluripotency are overtly similar but opposite processes (4), and it is unclear whether analogous barriers or checkpoints also exist early during hPSC differentiation. Unfortunately, the growth of hPSCs in tight, multicellular clumps has blocked direct, in?situ measurements of cell growth, size, and other fundamental properties, such as intracolony motion during early hPSC differentiation. It therefore remains uncertain whether hPSCs undergo a decrease in proliferation and increase in size with induced differentiation, or if changes in cell proliferation and size (biomass) are coordinated with early hPSC differentiation programs (5). To date, hPSC size measurements have been imprecise and used to document extreme changes in cross-sectional area by microscopy (3). hPSC growth is typically measured as time to division and not as changes in cell biomass, which is Trimebutine the most direct measure of net cellular growth but requires detection at <5% mass change per hour. Existing optical approaches have also revealed that individual PSC colonies are highly motile, a property that may skew clonogenic counting assays through colony merging (6). Coordinated cell movement within hPSC colonies could provide a basis for colony migration and merging, although this too remains unknown for hPSCs grown in culture (6C8). In general, cell size has been inferred from the radius or?projected area for cells with fixed geometries, such as bacteria, yeasts, and spherical lymphocytes. However, most animal cells, or cells that grow in colonies, have irregular and often changing shapes, resulting in poor estimates of cell biomass based on measurements of projected area (see Fig.?S1 in the Supporting Material). Additionally, changes in cell volume or area may result from changes in solute concentrations or flattening against a substrate rather than from changes in biomass. Flow cytometry measures material density gradients and aqueous content to estimate cell size, SNX25 but cannot quantify the continuous growth of hPSC colonies (9). More promising are micro-electromechanical system microresonators, which can accurately measure the mass of single cells instantaneously and over time (10,11). However, for sufficient sensitivity a resonator must be microns or smaller, making continuous measurements of motile, growing hPSC colonies practically.