Department of Pathology, College of Basic Medical Sciences, Qingdao University, 308 Ningxia Road, Qingdao, 266071, Shandong, China.
Department of Critical Care Medicine, Qingdao Center Hospital, affiliated with Qingdao University, 127 Siliunan Road, Qingdao, 266042, Shandong, China.
Stem Cell Res Ther. 2020 Oct 27;11(1):454. doi: 10.1186/s13287-020-01976-1.
Human hair follicle mesenchymal stem cells (hHFMSCs) isolated from hair follicles possess multilineage differentiation potential. OCT4 is a gene critically associated with pluripotency properties. The cell morphology and adhesion of hHFMSCs significantly changed after transduction of OCT4 and two subpopulations emerged, including adherent cells and floating cell. Floating cells cultured in hematopoietic induction medium and stimulated with erythropoetic growth factors could transdifferentiate into mature erythrocytes, whereas adherent cells formed negligible hematopoietic colonies. The aim of this study was to reveal the role of cell morphology and adhesion on erythropoiesis induced by OCT4 in hHFMSCs and to characterize the molecular mechanisms involved.
Floating cell was separated from adherent cell by centrifugation of the upper medium during cell culture. Cell size was observed through flow cytometry and cell adhesion was tested by disassociation and adhesion assays. RNA sequencing was performed to detect genome-wide transcriptomes and identify differentially expressed genes. GO enrichment analysis and KEGG pathway analysis were performed to analysis the functions and pathways enriched by differentially expressed genes. The expression of tight junction core members was verified by qPCR and Western blot. A regulatory network was constructed to figure out the relationship between cell adhesin, cytoskeleton, pluripotency, and hematopoiesis.
The overexpression of OCT4 influenced the morphology and adhesion of hHFMSCs. Transcripts in floating cells and adherent cells are quite different. Data analysis showed that upregulated genes in floating cells were mainly related to pluripotency, germ layer development (including hematopoiesis lineage development), and downregulated genes were mainly related to cell adhesion, cell junctions, and the cytoskeleton. Most molecules of the tight junction (TJ) pathway were downregulated and molecular homeostasis of the TJ was disturbed, as CLDNs were disrupted, and JAMs and TJPs were upregulated. The dynamic expression of cell adhesion-related gene E-cadherin and cytoskeleton-related gene ACTN2 might cause different morphology and adhesion. Finally, a regulatory network centered to OCT4 was constructed, which elucidated that he TJ pathway critically bridges pluripotency and hematopoiesis in a TJP1-dependent way.
Regulations of cell morphology and adhesion via the TJ pathway conducted by OCT4 might modulate hematopoiesis in hHFMSCs, thus developing potential mechanism of erythropoiesis in vitro.
从毛囊中分离出来的人毛囊间充质干细胞（hHFMSCs）具有多能性分化潜能。OCT4 是一个与多能性特性密切相关的基因。OCT4 转导后，hHFMSCs 的细胞形态和黏附性发生显著变化，出现两种亚群，包括贴壁细胞和悬浮细胞。悬浮细胞在造血诱导培养基中培养，并在促红细胞生成素的刺激下可分化为成熟红细胞，而贴壁细胞形成的造血集落可忽略不计。本研究旨在揭示 OCT4 诱导 hHFMSCs 红细胞生成过程中细胞形态和黏附的作用，并探讨其相关的分子机制。
细胞培养过程中，通过离心上层培养基分离出悬浮细胞。通过流式细胞术观察细胞大小，通过解离和黏附实验检测细胞黏附。进行 RNA 测序以检测全基因组转录组并鉴定差异表达基因。GO 富集分析和 KEGG 通路分析用于分析差异表达基因富集的功能和通路。通过 qPCR 和 Western blot 验证紧密连接核心成员的表达。构建调控网络以了解细胞黏附、细胞骨架、多能性和造血之间的关系。
OCT4 的过表达影响 hHFMSCs 的形态和黏附。悬浮细胞和贴壁细胞中的转录本差异很大。数据分析显示，悬浮细胞中上调的基因主要与多能性、胚层发育（包括造血谱系发育）有关，而下调的基因主要与细胞黏附、细胞连接和细胞骨架有关。紧密连接（TJ）途径的大多数分子下调，TJ 分子的稳态受到干扰，CLDNs 破坏，JAMs 和 TJPs 上调。细胞黏附相关基因 E-cadherin 和细胞骨架相关基因 ACTN2 的动态表达可能导致不同的形态和黏附。最后，构建了以 OCT4 为中心的调控网络，阐明 TJ 途径通过 TJP1 依赖性方式将多能性和造血联系起来。
OCT4 通过 TJ 途径调节细胞形态和黏附，可能调节 hHFMSCs 中的造血，从而为体外红细胞生成提供潜在的机制。
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