Medical Biotechnology Centre, Department of Endocrinology, University Hospital of Odense, University of South Denmark, Odense, Denmark.
J Bone Miner Res. 2010 Jun;25(6):1216-33. doi: 10.1002/jbmr.34.
Directing differentiation of human embryonic stem cells (hESCs) into specific cell types using an easy and reproducible protocol is a prerequisite for the clinical use of hESCs in regenerative-medicine procedures. Here, we report a protocol for directing the differentiation of hESCs into mesenchymal progenitor cells. We demonstrate that inhibition of transforming growth factor beta (TGF-beta)/activin/nodal signaling during embryoid body (EB) formation using SB-431542 (SB) in serum-free medium markedly upregulated paraxial mesodermal markers (TBX6, TBX5) and several myogenic developmental markers, including early myogenic transcriptional factors (Myf5, Pax7), as well as myocyte-committed markers [NCAM, CD34, desmin, MHC (fast), alpha-smooth muscle actin, Nkx2.5, cTNT]. Continuous inhibition of TGF-beta signaling in EB outgrowth cultures (SB-OG) enriched for myocyte progenitor cells; markers were PAX7(+) (25%), MYOD1(+) (52%), and NCAM(+) (CD56) (73%). DNA microarray analysis revealed differential upregulation of 117 genes (>2-fold compared with control cells) annotated to myogenic development and function. Moreover, these cells showed the ability to contract (80% of the population) and formed myofibers when implanted intramuscularly in vivo. Interestingly, SB-OG cells cultured in 10% fetal bovine serum (FBS) developed into a homogeneous population of mesenchymal progenitors that expressed CD markers characteristic of mesenchymal stem cells (MSCs): CD44(+) (100%), CD73(+) (98%), CD146(+) (96%), and CD166(+) (88%) with the ability to differentiate into osteoblasts, adipocytes, and chondrocytes in vitro and in vivo. Furthermore, microarray analysis of these cells revealed downregulation of genes related to myogenesis: MYH3 (-167.9-fold), ACTA1 (-161-fold), MYBPH (-139-fold), ACTC (-100.3-fold), MYH8 (-45.5-fold), and MYOT (-41.8-fold) and marked upregulation of genes related to mesoderm-derived cell lineages. In conclusion, our data provides a simple and versatile protocol for directing the differentiation of hESCs into a myogenic lineage and then further into mesenchymal progenitors by blocking the TGF-beta signaling pathway.
使用易于复制的方案指导人类胚胎干细胞（hESC）分化为特定细胞类型，是 hESC 在再生医学程序中临床应用的前提。在这里，我们报告了一种指导 hESC 分化为间充质祖细胞的方案。我们证明，在无血清培养基中使用 SB-431542（SB）抑制胚胎体（EB）形成过程中的转化生长因子β（TGF-β）/激活素/ nodal 信号，可显著上调轴旁中胚层标志物（TBX6、TBX5）和几个肌发生发育标志物，包括早期肌源性转录因子（Myf5、Pax7），以及肌细胞定向标志物[NCAM、CD34、结蛋白、MHC（快）、α-平滑肌肌动蛋白、Nkx2.5、cTNT]。在 EB 外植体培养物（SB-OG）中持续抑制 TGF-β 信号，可富集肌细胞祖细胞标志物；标记物为 PAX7（+）（25%）、MYOD1（+）（52%）和 NCAM（+）（CD56）（73%）。DNA 微阵列分析显示，117 个基因的表达水平差异上调（与对照细胞相比超过 2 倍），这些基因注释为肌发生和功能。此外，这些细胞具有收缩的能力（人群的 80%），并且在体内肌肉内植入时形成肌纤维。有趣的是，在 10%胎牛血清（FBS）中培养的 SB-OG 细胞发育为表达间充质干细胞（MSC）特征性 CD 标记的同质间充质祖细胞群：CD44（+）（100%）、CD73（+）（98%）、CD146（+）（96%）和 CD166（+）（88%），具有体外和体内分化为成骨细胞、脂肪细胞和成软骨细胞的能力。此外，对这些细胞的微阵列分析显示，与肌发生相关的基因下调：MYH3（-167.9 倍）、ACTA1（-161 倍）、MYBPH（-139 倍）、ACTC（-100.3 倍）、MYH8（-45.5 倍）和 MYOT（-41.8 倍），与中胚层来源细胞谱系相关的基因显著上调。总之，我们的数据提供了一种简单而通用的方案，通过阻断 TGF-β 信号通路，指导 hESC 分化为肌源性谱系，然后进一步分化为间充质祖细胞。
Differentiation. 2007-6
Front Oncol. 2024-6-26
Front Bioeng Biotechnol. 2024-6-20
Cell Stem Cell. 2024-6-6
Zotero 插件