1 Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, New York.
2 Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, New York.
Stem Cells Dev. 2019 Feb 15;28(4):278-289. doi: 10.1089/scd.2018.0197. Epub 2019 Jan 28.
There is a critical need to generate functional hepatocytes to aid in liver repair and regeneration upon availability of a renewable, and potentially personalized, source of human hepatocytes (hHEP). Currently, the vast majority of primary hHEP are obtained from human tissue through cadavers. Recent advances in stem cell differentiation have opened up the possibility to obtain fully functional hepatocytes from embryonic or induced pluripotent stem cells, or adult stem cells. With respect to the latter, human bone marrow mesenchymal stromal cells (hBMSCs) can serve as a source of autogenetic and allogenic multipotent stem cells for liver repair and regeneration. A major aspect of hBMSC differentiation is the extracellular matrix (ECM) composition and, in particular, the role of glycosaminoglycans (GAGs) in the differentiation process. In this study, we examine the influence of four distinct culture conditions/protocols (T1-T4) on GAG composition and hepatic markers. α-Fetoprotein and hepatocyte nuclear factor-4α were expressed continually over 21 days of differentiation, as indicated by real time quantitative PCR analysis, while albumin (ALB) expression did not begin until day 21. Hepatocyte growth factor (HGF) appears to be more effective than activin A in promoting hepatic-like cells through the mesenchymal-epithelial transition, perhaps due to the former binding to the HGF receptor to form a unique complex that diversifies the biological functions of HGF. Of the four protocols tested, uniform hepatocyte-like morphological changes, ALB secretion, and glycogen storage were found to be highest with protocol T2, which involves both early- and late-stage combinations of growth factors. The total GAG profile of the hBMSC ECM is rich in heparan sulfate (HS) and hyaluronan, both of which fluctuate during differentiation. The GAG profile of primary hHEP showed an HS-rich ECM, and thus, it may be possible to guide hBMSC differentiation to more mature hepatocytes by controlling the GAG profile expressed by differentiating cells.
在获得可再生的、具有潜在个性化的人类肝细胞(hHEP)来源时，迫切需要生成功能性肝细胞以辅助肝脏修复和再生。目前，绝大多数原发性 hHEP 是通过尸体从人体组织中获得的。干细胞分化的最新进展为从胚胎或诱导多能干细胞或成体干细胞获得完全功能性肝细胞开辟了可能性。就后者而言，人类骨髓间充质基质细胞(hBMSC)可作为肝脏修复和再生的自体和同种异体多能干细胞的来源。hBMSC 分化的一个主要方面是细胞外基质(ECM)组成，特别是糖胺聚糖(GAG)在分化过程中的作用。在这项研究中，我们研究了四种不同的培养条件/方案(T1-T4)对 GAG 组成和肝标志物的影响。实时定量 PCR 分析表明，α-胎蛋白和肝细胞核因子-4α在分化的 21 天内持续表达，而白蛋白(ALB)表达直到第 21 天才开始。肝细胞生长因子(HGF)似乎比激活素 A 更有效地通过间质上皮转化促进类肝细胞，这可能是由于前者与 HGF 受体结合形成独特的复合物，从而使 HGF 的生物学功能多样化。在所测试的四种方案中，发现方案 T2 具有最高的均一的类肝细胞形态变化、ALB 分泌和糖原储存，该方案涉及早期和晚期生长因子的组合。hBMSC ECM 的总 GAG 谱富含肝素硫酸盐(HS)和透明质酸，这两种物质在分化过程中都有波动。原发性 hHEP 的 GAG 谱显示富含 HS 的 ECM，因此，通过控制分化细胞表达的 GAG 谱，可能可以指导 hBMSC 分化为更成熟的肝细胞。
