ABSTRACT

Stem cell and tissue engineering-based therapies for acute liver failure (ALF) have been limited by the lack of an optimal cell source. We aimed to determine the suitability of human parthenogenetic embryonic stem cells (hPESCs) for the development of strategies to treat ALF. We studied the ability of human parthenogenetic embryonic stem cells (hPESCs) with high whole-genome SNP homozygosity, which were obtained by natural activation during in vitro fertilization (IVF), to differentiate into functional hepatocyte-like cells in vitro by monolayer plane orientation. hPESCs were induced on a single-layer flat plate for 21 d in complete medium with the inducers activin A, FGF-4, BMP-2, HGF, OSM, DEX, and B27. Polygonal cell morphology and binuclear cells were observed after 21 d of induction by using an inverted microscope. RT-qPCR results showed that the levels of hepatocyte-specific genes such as AFP, ALB, HNF4a, CYP3A4, SLCO1B3, and ABCC2 significantly increased after induction. Immunocytochemical assay showed CK18 and Hepa expression in the induced cells. Indocyanine green (ICG) staining showed that the cells had the ability to absorb and metabolize dyes. Detection of marker proteins and urea in cell culture supernatants showed that the cells obtained after 21 d of induction had synthetic and secretory functions. The typical ultrastructure of liver cells was observed using TEM after 21 d of induction. The results indicate that naturally activated hPESCs can be induced to differentiate into hepatocellular cells by monolayer planar induction.

摘要

由于缺乏最佳细胞来源，基于干细胞和组织工程的急性肝衰竭 (ALF) 疗法受到限制。我们旨在确定人类孤雌生殖胚胎干细胞 (hPESCs) 是否适用于制定治疗 ALF 的策略。我们研究了在体外受精 (IVF) 过程中通过自然激活获得的具有高全基因组 SNP 纯合度的人类孤雌生殖胚胎干细胞 (hPESC) 通过单层平面定向在体外分化为功能性肝细胞样细胞的能力。hPESCs 在单层平板上用诱导剂激活素 A、FGF-4、BMP-2、HGF、OSM、DEX 和 B27 在完全培养基中诱导 21 天。诱导21 d后倒置显微镜观察多角形细胞形态和双核细胞。RT-qPCR结果显示，AFP、ALB、HNF4a、CYP3A4、SLCO1B3、ABCC2等肝细胞特异性基因水平在诱导后显着升高。免疫细胞化学分析显示诱导细胞中 CK18 和 Hepa 表达。吲哚菁绿 (ICG) 染色显示细胞具有吸收和代谢染料的能力。细胞培养上清液中标记蛋白和尿素的检测表明，诱导21 d后获得的细胞具有合成和分泌功能。诱导21 d后用透射电镜观察肝细胞的典型超微结构。结果表明，通过单层平面诱导可以诱导天然活化的hPESCs分化为肝细胞。ABCC2 诱导后显着升高。免疫细胞化学分析显示诱导细胞中 CK18 和 Hepa 表达。吲哚菁绿 (ICG) 染色显示细胞具有吸收和代谢染料的能力。细胞培养上清液中标记蛋白和尿素的检测表明，诱导21 d后获得的细胞具有合成和分泌功能。诱导21 d后用透射电镜观察肝细胞的典型超微结构。结果表明，通过单层平面诱导可以诱导天然活化的hPESCs分化为肝细胞。ABCC2 诱导后显着升高。免疫细胞化学分析显示诱导细胞中 CK18 和 Hepa 表达。吲哚菁绿 (ICG) 染色显示细胞具有吸收和代谢染料的能力。细胞培养上清液中标记蛋白和尿素的检测表明，诱导21 d后获得的细胞具有合成和分泌功能。诱导21 d后用透射电镜观察肝细胞的典型超微结构。结果表明，通过单层平面诱导可以诱导天然活化的hPESCs分化为肝细胞。细胞培养上清液中标记蛋白和尿素的检测表明，诱导21 d后获得的细胞具有合成和分泌功能。诱导21 d后用透射电镜观察肝细胞的典型超微结构。结果表明，通过单层平面诱导可以诱导天然活化的hPESCs分化为肝细胞。细胞培养上清液中标记蛋白和尿素的检测表明，诱导21 d后获得的细胞具有合成和分泌功能。诱导21 d后用透射电镜观察肝细胞的典型超微结构。结果表明，通过单层平面诱导可以诱导天然活化的hPESCs分化为肝细胞。