References and Notes Supporting Online Material Materials and Methods Figs. S1 to S11 References Movie S1 Optimally Interacting Minds R�ports-猫眼课题宝

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M. Cornelison;H. M. Filla;A. Stanley;B. B. Rapraeger-B.;Olwin;A Sacco;R. Doyonnas;P. Kraft;S. Vitorovic;H. Blau;W D Callister;M P Lutolf;K. Havenstrite;K. Koleckar;F Le Grand;A. Jones;V. Seale;A. Scimè;Martina Rudnicki;J. Sly;R. D. Miller;S. Kobal;S. Gobaa;A. Barron;F. Rossi;J. Och;Karin Engbloms;Stipendiefond E G M k;Bio;B. Bahrami;Karsten Olsen;P. Latham;A. Roepstorff;G. Rees;C. Frith

A和B，以及图S11）。相比之下，塑料微孔中只有6%的双联体具有这种基因表达模式，这表明柔顺的底物能够使MuSC扩增。虽然基因表达数据是暗示性的，但有必要进行体内功能测定，以明确得出结论，即在培养中发生了自我更新分裂事件。我们最终表明，干细胞自我更新发生在体内功能测定。在群体水平上移植MuSC证明了植入（图1A和1B）。2和3），但并没有明确表明，自我更新分裂发生在文化中，因为人口可能包括非分裂细胞，保持干细胞的特性。因此，在该实验中，我们将MuSC接种在水凝胶微孔阵列中，并在接种后立即和培养后2至3天获得图像，以鉴定仅含有一个双联体的微孔。挑取来自5个微孔的双联体，并使用显微操作器合并，每只小鼠共移植10个细胞（图4A）。可检测到的BLI信号指示由自我更新分裂事件引起的植入，所述自我更新分裂事件必须发生在五个移植的双联体中的至少一个中。值得注意的是，25 μ g（12只中的3只）移植有在软基质上培养的双联体的小鼠表现出可检测的植入（图4C）和对再生肌纤维的贡献（图4D，顶部），提供了体内功能证据，表明MuSC自我更新分裂事件发生在柔顺基质上的培养物中。相比之下，在刚性塑料微孔上生长的双联体在移植后从未表现出植入（14个中的0个）（图4C），表明它们的再生潜力迅速丧失。MuSC在柔顺水凝胶上的自我更新甚至在多次分裂后发生。我们移植了从经历3到5次分裂的单个细胞产生的克隆。值得注意的是，12/8（1/8）移植了单个克隆的小鼠显示出移植，表明MuSC自我更新能力即使在多次分裂后也保留在柔顺的基质上（图4，C和D，底部）。在这里，我们提供了深入了解组织硬度的潜力，骨骼肌微环境的生物物理特性，对干细胞命运的调节。使用单细胞跟踪算法在单细胞水平上询问MuSC行为，我们证明软基质增强MuSC存活，防止分化，并促进干细胞。小鼠中的功能测定最终证明，柔顺底物允许MuSC在培养中自我更新。虽然潜在的机制仍有待阐明，但我们假设刚度降低通过改变细胞形状来保持干性，导致细胞骨架重排和信号传导改变，如图所示。

A and B, and fig. S11). In contrast, only 6� of doublets in plastic microwells have this gene expression pattern, suggesting that a pliant substrate enables MuSC expansion. Although gene expression data are suggestive, an in vivo functional assay is necessary to conclude definitively that a self-renewal division event occurred in culture. We show conclusively that stem cell self-renewal occurs using an in vivo functional assay. The transplantation of MuSCs at a population level demonstrates engraftment (Figs. 2 and 3) but does not definitively show that self-renewal divisions occurred in culture, because the population could include nondividing cells that maintained stem cell properties. Accordingly, in this experiment, we plated MuSCs in hydrogel microwell arrays and obtained images immediately after plating and 2 to 3 days after culturing to identify microwells that contained only one doublet. Doublets from 5 microwells were picked and pooled using a micromanipulator, and 10 cells total were transplanted per mouse (Fig. 4A). A detectable BLI signal indicates engraftment resulting from a self-renewal division event that must have occurred in at least one of the five transplanted doublets. Notably, 25� (3 of 12) of mice transplanted with doublets cultured on soft substrates demonstrate detectable engraftment (Fig. 4C) and contribution to regenerating myofibers (Fig. 4D, top), providing in vivo functional evidence that MuSC self-renewal division events occur in culture on pliant substrates. In contrast, doublets grown on rigid plastic microwells never exhibit engraftment after transplantation (0 of 14) (Fig. 4C), indicating that their regenerative potential is rapidly lost. MuSC self-renewal on pliant hydrogel occurs even after multiple divisions. We transplanted clones that arose from a single cell that underwent 3 to 5 divisions. Remarkably, 12� (1 of 8) of mice transplanted with a single clone show en-graftment, demonstrating that MuSC self-renewal capacity is retained on pliant substrates even after multiple divisions (Fig. 4, C and D, bottom). Here, we provide insight into the potency of tissue rigidity, a biophysical property of the skeletal muscle microenvironment, on stem cell fate regulation. Using a single-cell tracking algorithm to interrogate MuSC behaviors at the single-cell level, we demonstrate that soft substrates enhance MuSC survival, prevent differentiation, and promote stemness. Functional assays in mice demonstrate conclusively that pliant substrates permit MuSC self-renewal in culture. Although the underlying mechanisms remain to be elucidated, we hypothesize that decreased rigidity preserves stemness by altering cell shape, resulting in cyto-skeletal rearrangements and altered signaling, as shown …

References and Notes Supporting Online Material Materials and Methods Figs. S1 to S11 References Movie S1 Optimally Interacting Minds R�ports

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