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Gradient Fractionated Separation of Chondrogenically Committed Cells Derived from Human Embryonic Stem Cells

机译：源自人类胚胎干细胞的软骨形成细胞的梯度分级分离

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Cartilage regeneration is a fast growing field that combines biotechnology and molecular techniques in creating new tissue mimicking the native microenvironment. Human embryonic stem cells (hESCs) are a highly potent cell source for cartilage regeneration owing to their infinite proliferation capacity and pluripotency. Thus, lineage-specific differentiation of hESCs often results in populations with cellular heterogeneity. Chondrogenesis was induced through high-density micromass culture of hESCs and by addition of chondrogenic medium; 1:100 ITS+, 100 nM dexamethasone, 40 μg/ml l-proline, 50 μg/mL ascorbic acid-2-phosphate, 1:100 Knockout serum, and 10 ng/mL TGFβ3. At day 14 micromasses were dissociated and chondrogenically committed cell separated in a fraction-based discontinuous density gradient. After fractionation the chondrogenically committed cells were analyzed with regard to embryonic- and chondrogenic gene expression and fraction F3 and F4 with histology. In general, we found that the chondrogenic condition compared with the control condition had a significant effect on the following gene expression levels: NANOG, OCT4, SOX5, SOX9, ACAN, and COL2A1 in all fractions. Furthermore, we found in the chondrogenic condition that NANOG, OCT4, and SOX9 were significantly higher in F4 compared with F3, whereas COL2A1 and the ratio COL2A1:COL1A1 were significantly lower. Additionally, toluidine blue pH 4 stains of pellet cultures of F3 and F4 revealed that cells from F3 were more homogenous in morphology than F4. In conclusion, we propose a simple strategy to obtain more homogenous population of chondrogenically committed cells from hESCs using micromass culture and discontinuous density gradient separation.
机译：软骨再生是一个快速发展的领域，结合了生物技术和分子技术来创建模仿天然微环境的新组织。人类胚胎干细胞（hESCs）由于其无限的增殖能力和多能性，是软骨再生的高效细胞来源。因此，hESCs的谱系特异性分化通常会导致群体具有细胞异质性。软骨形成是通过hESCs的高密度微团培养和添加软骨形成培养基诱导的。 1：100 ITS + ，100 nM地塞米松，40μg/ ml脯氨酸，50μg/ ml抗坏血酸-2-磷酸酯，1：100敲除血清和10μng/ mLTGFβ3。在第14天，解离微团，并以基于分数的不连续密度梯度分离成软骨细胞。分级分离后，通过组织学分析软骨生成的细胞的胚胎和软骨生成基因表达以及级分F3和F4。通常，我们发现软骨形成条件与对照条件相比对以下基因表达水平有显着影响：所有部分中的NANOG，OCT4，SOX5，SOX9，ACAN和COL2A1。此外，我们发现，在成软骨条件下，F4中的NANOG，OCT4和SOX9显着高于F3，而COL2A1和COL2A1：COL1A1的比率显着较低。另外，F3和F4的沉淀培养物的甲苯胺蓝pH 4染色显示，来自F3的细胞在形态上比F4更均一。总之，我们提出了一种简单的策略，可以使用微质量培养和不连续的密度梯度分离技术从hESC中获得更具同质性的成软骨细胞。

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