Department of Mechanical Science and Engineering, University of Illinois at Urbana-Champaign, Urbana, Illinois, United States of America.
PLoS One. 2010 Dec 13;5(12):e15655. doi: 10.1371/journal.pone.0015655.
Maintaining undifferentiated mouse embryonic stem cell (mESC) culture has been a major challenge as mESCs cultured in Leukemia Inhibitory Factor (LIF) conditions exhibit spontaneous differentiation, fluctuating expression of pluripotency genes, and genes of specialized cells. Here we show that, in sharp contrast to the mESCs seeded on the conventional rigid substrates, the mESCs cultured on the soft substrates that match the intrinsic stiffness of the mESCs and in the absence of exogenous LIF for 5 days, surprisingly still generated homogeneous undifferentiated colonies, maintained high levels of Oct3/4, Nanog, and Alkaline Phosphatase (AP) activities, and formed embryoid bodies and teratomas efficiently. A different line of mESCs, cultured on the soft substrates without exogenous LIF, maintained the capacity of generating homogeneous undifferentiated colonies with relatively high levels of Oct3/4 and AP activities, up to at least 15 passages, suggesting that this soft substrate approach applies to long term culture of different mESC lines. mESC colonies on these soft substrates without LIF generated low cell-matrix tractions and low stiffness. Both tractions and stiffness of the colonies increased with substrate stiffness, accompanied by downregulation of Oct3/4 expression. Our findings demonstrate that mESC self-renewal and pluripotency can be maintained homogeneously on soft substrates via the biophysical mechanism of facilitating generation of low cell-matrix tractions.
维持未分化的小鼠胚胎干细胞（mESC）培养一直是一个主要的挑战，因为在白血病抑制因子（LIF）条件下培养的 mESC 会自发分化，多能基因和特化细胞的基因表达不稳定。在这里，我们发现，与传统刚性基质上接种的 mESC 形成鲜明对比的是，在与 mESC 固有硬度相匹配的软基质上培养 5 天，并且没有外源 LIF 的情况下，mESC 出人意料地仍然产生了均匀的未分化集落，保持了高水平的 Oct3/4、Nanog 和碱性磷酸酶（AP）活性，并且有效地形成了类胚体和畸胎瘤。在没有外源 LIF 的软基质上培养的不同 mESC 系，在维持具有相对高水平 Oct3/4 和 AP 活性的均匀未分化集落的能力方面，至少保持了 15 代，这表明这种软基质方法适用于不同 mESC 系的长期培养。在没有 LIF 的这些软基质上的 mESC 集落产生的细胞-基质牵引力和刚度较低。随着基质刚度的增加，集落的牵引力和刚度增加，同时 Oct3/4 表达下调。我们的研究结果表明，mESC 自我更新和多能性可以通过促进低细胞-基质牵引力产生的生物物理机制在软基质上均匀地维持。
