Initial WNT/β-Catenin Activation Enhanced Mesoderm Commitment, Extracellular Matrix Expression, Cell Aggregation and Cartilage Tissue Yield From Induced Pluripotent Stem Cells.
Initial WNT/β-Catenin Activation Enhanced Mesoderm Commitment, Extracellular Matrix Expression, Cell Aggregation and Cartilage Tissue Yield From Induced Pluripotent Stem Cells.
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DOI：
10.3389/fcell.2020.581331
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发表时间：
2020
影响因子：
5.5
通讯作者：
Diederichs S
中科院分区：
文献类型：
--
作者：
Kreuser U;Buchert J;Haase A;Richter W;Diederichs S
诱导多能干细胞（iPSCs）在体外中胚层分化并随后分化为软骨细胞等中胚层衍生物，目前受到大量细胞损失的困扰，严重限制了组织产量。目前需要更多关于调节iPSCs中胚层分化的关键因素的知识，以驱动所有细胞进入所需的谱系，并克服当前对中间细胞选择步骤的需求，以去除错误分化的细胞。利用两个独立的人类iPSC系，我们报告了由小分子CHIR99021诱导的短初始WNT/β-catenin脉冲（24小时）增强了中胚层标志物（PDGFRα、HAND1、KDR和GATA4）的表达，支持了多能性的退出（减少了OCT4、SOX2和LIN28A），抑制了外胚层的错误分化（减少了PAX6、TUBB3和NES）。重要的是，初始CHIR脉冲使细胞增殖增加到第14天（5倍），调节了黏附相关基因的表达（CDH3上调，CDH6下调），并增加了细胞外基质（ECM）相关基因的表达（COL6, COL1, COL3, COL5， DCN， NPNT， LUM， MGP， MATN2和VTN），从而产生了更多具有高聚集能力的基质相互作用祖细胞。与对照组相比，对软骨颗粒形成的增强贡献使8周后的细胞产量增加了200倍。CHIR组II型胶原和蛋白聚糖阳性区增大，表明软骨形成细胞数量增加。最后，短暂的初始WNT激活改善了中胚层的承诺，我们的数据首次证明，通过刺激细胞增殖、ECM表达和细胞聚集，WNT脉冲是使软骨形成前的细胞选择步骤过时的关键步骤。这种对WNT/β-catenin功能的深入了解，是人类多能干细胞稳健高效地生成高质量中胚层祖细胞，以及在随后的体外软骨形成过程中挽救低组织产量的重要一步，这在临床软骨再生、疾病建模和药物筛选中是非常需要的。
Mesodermal differentiation of induced pluripotent stem cells (iPSCs) in vitro and subsequent specification into mesodermal derivatives like chondrocytes is currently afflicted with a substantial cell loss that severely limits tissue yield. More knowledge on the key players regulating mesodermal differentiation of iPSCs is currently needed to drive all cells into the desired lineage and to overcome the current need for intermediate cell selection steps to remove misdifferentiated cells. Using two independent human iPSC lines, we here report that a short initial WNT/β-catenin pulse induced by the small molecule CHIR99021 (24 h) enhanced expression of mesodermal markers (PDGFRα, HAND1, KDR, and GATA4), supported the exit from pluripotency (decreased OCT4, SOX2, and LIN28A) and inhibited ectodermal misdifferentiation (reduced PAX6, TUBB3, and NES). Importantly, the initial CHIR pulse increased cell proliferation until day 14 (five-fold), adjusted expression of adhesion-related genes (CDH3 up, CDH6 down) and increased extracellular matrix (ECM)-related gene expression (COL6, COL1, COL3, COL5, DCN, NPNT, LUM, MGP, MATN2, and VTN), thus yielding more matrix-interacting progenitors with a high aggregation capability. Enhanced contribution to chondrogenic pellet formation increased the cell yield after eight weeks 200-fold compared to controls. The collagen type II and proteoglycan-positive area was enlarged in the CHIR group, indicating an increased number of cartilage-forming cells. Conclusively, short initial WNT activation improved mesoderm commitment and our data demonstrated for the first time to our knowledge that, acting via stimulation of cell proliferation, ECM expression and cell aggregation, WNT pulsing is a key step to make cell selection steps before chondrogenesis obsolete. This advanced understanding of the WNT/β-catenin function is a major step toward robust and efficient generation of high-quality mesodermal progenitors from human iPSCs and toward rescuing low tissue yield during subsequent in vitro chondrogenesis, which is highly desired for clinical cartilage regeneration, disease modeling and drug screening.
DOI：
10.1083/jcb.73.3.736
发表时间：
1977-06
期刊：
The Journal of cell biology
影响因子：
--
作者：
von der Mark K;von der Mark H
通讯作者：
von der Mark H