Department of Bioengineering, Faculty of Engineering, National University of Singapore, Singapore.
J Tissue Eng Regen Med. 2012 Nov;6(10):e74-86. doi: 10.1002/term.1560. Epub 2012 Jul 3.
Stable pluripotent feeder-free propagation of human embryonic stem cells (hESCs) prior to their therapeutic applications remains a major challenge. Matrigel™ (BD Singapore) is a murine sarcoma-derived extracellular matrix (ECM) widely used as a cell-free support combined with conditioned or chemically defined media; however, inherent xenogenic and immunological threats invalidate it for clinical applications. Using human fibrogenic cells to generate ECM is promising but currently suffers from inefficient and time-consuming deposition in vitro. We recently showed that macromolecular crowding (MMC) accelerated ECM deposition substantially in vitro. In the current study, we used dextran sulfate 500 kDa as a macromolecular crowder to induce WI-38 fetal human lung fibroblasts at 0.5% serum condition to deposit human ECM in three days. After decellularization, the generated ECMs allowed stable propagation of H9 hESCs over 20 passages in chemically-defined medium (mTEsR1) with an overall improved outcome compared to Matrigel in terms of population doubling while retaining teratoma formation and differentiation capacity. Of significance, only ECMs generated by MMC allowed the successful propagation of hESCs. ECMs were highly complex and in contrast to Matrigel, contained no vitronectin but did contain collagen XII, ig-h3 and novel for hESC-supporting human matrices, substantial amounts of transglutaminase 2. Genome-wide analysis of promoter DNA methylation states revealed high overall similarity between human ECM- and Matrigel-cultured hESCs; however, distinct differences were observed with 49 genes associated with a variety of cellular functions. Thus, human ECMs deposited by MMC by selected fibroblast lines are a suitable human microenvironment for stable hESC propagation and clinically translational settings.
在将人类胚胎干细胞（hESC）用于治疗之前，稳定的多能无饲养层传代仍然是一个主要挑战。Matrigel™（BD 新加坡）是一种广泛用作无细胞支持物的鼠肉瘤衍生细胞外基质（ECM），与条件培养基或化学定义的培养基结合使用；然而，其固有的异种和免疫原性威胁使其无法用于临床应用。使用人成纤维细胞产生 ECM 很有前景，但目前在体外沉积效率低且耗时。我们最近表明，大分子拥挤（MMC）可大大加速 ECM 的体外沉积。在本研究中，我们使用葡聚糖硫酸盐 500 kDa 作为大分子拥挤剂，在 0.5%血清条件下诱导 WI-38 胎儿人肺成纤维细胞在三天内沉积人 ECM。脱细胞后，生成的 ECM 允许 H9 hESC 在化学定义培养基（mTEsR1）中稳定传代 20 代以上，与 Matrigel 相比，在倍增方面总体上具有更好的结果，同时保持畸胎瘤形成和分化能力。值得注意的是，只有 MMC 生成的 ECM 才能成功传代 hESC。ECM 非常复杂，与 Matrigel 不同，不含有纤连蛋白，但含有胶原蛋白 XII、ig-h3 和新型支持 hESC 的人基质，大量的转谷氨酰胺酶 2。启动子 DNA 甲基化状态的全基因组分析表明，在人类 ECM 和 Matrigel 培养的 hESC 之间存在高度相似性；然而，观察到 49 个与各种细胞功能相关的基因存在明显差异。因此，由选定的成纤维细胞系通过 MMC 沉积的人 ECM 是稳定 hESC 繁殖和临床转化的合适人微环境。
