School of Biochemistry, University of Bristol, Bristol, BS8 1TD, UK.
Department of Biochemistry, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, 10700, Thailand.
Stem Cell Res Ther. 2019 Apr 29;10(1):130. doi: 10.1186/s13287-019-1231-z.
Pluripotent stem cells are attractive progenitor cells for the generation of erythroid cells in vitro as have expansive proliferative potential. However, although embryonic (ESC) and induced pluripotent (iPSC) stem cells can be induced to undergo erythroid differentiation, the majority of cells fail to enucleate and the molecular basis of this defect is unknown. One protein that has been associated with the initial phase of erythroid cell enucleation is the intermediate filament vimentin, with loss of vimentin potentially required for the process to proceed.
In this study, we used our established erythroid culture system along with western blot, PCR and interegation of comparative proteomic data sets to analyse the temporal expression profile of vimentin in erythroid cells differentiated from adult peripheral blood stem cells, iPSC and ESC throughout erythropoiesis. Confocal microscopy was also used to examine the intracellular localisation of vimentin.
We show that expression of vimentin is turned off early during normal adult erythroid cell differentiation, with vimentin protein lost by the polychromatic erythroblast stage, just prior to enucleation. In contrast, in erythroid cells differentiated from iPSC and ESC, expression of vimentin persists, with high levels of both mRNA and protein even in orthochromatic erythroblasts. In the vimentin-positive iPSC orthochromatic erythroblasts, F-actin was localized around the cell periphery; however, in those rare cells captured undergoing enucleation, vimentin was absent and F-actin was re-localized to the enucleosome as found in normal adult orthrochromatic erythroblasts.
As both embryonic and adult erythroid cells loose vimentin and enucleate, retention of vimentin by iPSC and ESC erythroid cells indicates an intrinsic defect. By analogy with avian erythrocytes which naturally retain vimentin and remain nucleated, retention in iPSC- and ESC-derived erythroid cells may impede enucleation. Our data also provide the first evidence that dysregulation of processes in these cells occurs from the early stages of differentiation, facilitating targeting of future studies.
多能干细胞是体外生成红细胞的有吸引力的祖细胞，因为它们具有广泛的增殖潜力。然而，尽管胚胎（ESC）和诱导多能（iPSC）干细胞可以被诱导进行红细胞分化，但大多数细胞不能去核，并且这个缺陷的分子基础尚不清楚。一种与红细胞去核的初始阶段相关的蛋白质是中间丝波形蛋白，波形蛋白的丢失可能是该过程进行所必需的。
在这项研究中，我们使用我们建立的红细胞培养系统以及 Western blot、PCR 和比较蛋白质组学数据集的整合，分析了从成人外周血干细胞、iPSC 和 ESC 分化的红细胞在整个红细胞生成过程中波形蛋白的时空表达谱。共聚焦显微镜也用于检查波形蛋白的细胞内定位。
我们表明，在正常成人红细胞分化过程中，波形蛋白的表达在早期就被关闭，在多色性红细胞阶段丢失波形蛋白蛋白，就在去核之前。相比之下，在从 iPSC 和 ESC 分化的红细胞中，波形蛋白的表达持续存在，即使在正染红细胞中，mRNA 和蛋白水平都很高。在阳性 iPSC 正染红细胞中，F-肌动蛋白定位于细胞周围；然而，在那些罕见的正在进行去核的细胞中，波形蛋白缺失，F-肌动蛋白重新定位于核小体，如正常成人正染红细胞中一样。
由于胚胎和成人红细胞都丢失波形蛋白并去核，因此 iPSC 和 ESC 红细胞保留波形蛋白表明存在内在缺陷。与自然保留波形蛋白并保持有核的禽类红细胞类似，iPSC 和 ESC 衍生的红细胞中保留波形蛋白可能会阻碍去核。我们的数据还首次提供了证据表明这些细胞中的分化过程从早期就出现失调，为未来的研究提供了靶向目标。
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