Attenuation of extrinsic signaling reveals the importance of matrix remodeling on maintenance of embryonic stem cell self-renewal
Attenuation of extrinsic signaling reveals the importance of matrix remodeling on maintenance of embryonic stem cell self-renewal
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DOI：
10.1073/pnas.1103100109
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发表时间：
2012-01-17
影响因子：
11.1
通讯作者：
Voldman, Joel
中科院分区：
文献类型：
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作者：
Przybyla, Laralynne M.;Voldman, Joel
自胚胎干细胞（embryonic stem cells，ESCs）分离以来，外源性因子在维持ESCs自我更新中的作用已被广泛研究，但细胞分泌因子在ESCs自我更新中的必要性至今仍未明确。虽然普遍认为，白血病抑制因子（LIF）与血清或骨形态发生蛋白4（BMP 4）一起加入足以维持小鼠ESC（mESC）处于自我更新状态，但这并不排除还需要自分泌因子的可能性。在这里，我们利用微流体灌注装置，该装置能够通过施加连续的介质流来耗尽细胞分泌的因子，从而在全局上减少可扩散的自分泌信号。我们证明了mESC培养数天连续微流体灌注下，并显示细胞分泌的因子被删除，并可以回收下游。我们发现，干扰细胞分泌的信号导致mESC退出其稳定的自我更新状态，在定义的条件下，通常支持自我更新，并表现出外胚层细胞的特性。这种状态变化不是由于已知的自分泌分化诱导剂成纤维细胞生长因子4的存在，但值得注意的是，它可以通过细胞外基质（ECM）的整体重塑来防止。我们还发现细胞分泌的基质重塑蛋白在灌注下被去除，并且细胞外基质重塑的抑制导致mESC分化。总之，我们的数据表明LIF和BMP 4不足以维持自我更新，并且细胞分泌的因子对于持续重塑ECM并从而防止分化是必要的，揭示了通过使用微流体灌注技术的先前未描述的mESC调节水平。
The role of extrinsic factors in maintaining self-renewal of embryonic stem cells (ESCs) has been extensively studied since the cells' isolation, but the necessity for cell-secreted factors in self-renewal has remained undefined to date. Although it is generally accepted that addition of leukemia inhibitory factor (LIF) together with either serum or bone morphogenetic protein 4 (BMP4) is sufficient to maintain mouse ESCs (mESCs) in a self-renewing state, this does not preclude the possibility that autocrine factors are also required. Here we make use of a microfluidic perfusion device that is able to globally diminish diffusible autocrine signaling by applying continuous media flow to deplete cell-secreted factors. We demonstrate mESC culture for several days under continuous microfluidic perfusion and show that cell-secreted factors are removed and can be recovered downstream. We find that perturbing cell-secreted signaling causes mESCs to exit their stable self-renewing state in defined conditions that normally support self-renewal and to exhibit properties characteristic of epiblast cells. This state change is not due to the presence of the known autocrine differentiation inducer fibroblast growth factor 4, but, remarkably, it can be prevented by global remodeling of the extracellular matrix (ECM). We also find that cell-secreted matrix remodeling proteins are removed under perfusion and that inhibition of extracellular matrix remodeling causes mESCs to differentiate. Taken together, our data indicate that LIF and BMP4 are not sufficient to maintain self-renewal and that cell-secreted factors are necessary to continuously remodel the ECM and thereby prevent differentiation, revealing a previously undescribed level of mESC regulation through the use of microfluidic perfusion technology.