Yu Bing, Li Hengyu, Chen Jie, He Zhiying, Sun Haixiang, Yang Guangshun, Shang Changzhen, Wang Xin, Li Chuanjiang, Chen Yajin, Hu Yiping
Department of Cell Biology, Center for Stem Cell and Medicine, Navy Medical University (Second Military Medical University), Shanghai, P. R. China.
Department of Hepatic Surgery V, Eastern Hepatobiliary Surgery Hospital, Navy Medical University (Second Military Medical University), Shanghai, P.R. China.
Liver Int. 2020 Sep;40(9):2293-2304. doi: 10.1111/liv.14509. Epub 2020 Jun 8.
BACKGROUND & AIM: Shortage of donor hepatocytes limits hepatocyte transplantation for clinical application. Induced hepatic stem cells (iHepSCs) have capacities of self-renewal and bipotential differentiations. Here, we investigated whether iHepSCs could be extensively expanded, and whether they could differentiate into sufficient functional hepatocytes as donors for transplantation therapy after their extensive expansions.
Murine extensively expanded iHepSCs (50-55 passages) were induced to differentiate into iHepSC-Heps under a chemically defined condition. iHepSC-Heps were proved for carrying morphological hepatocyte characters and hepatocytic functions including low-density lipoprotein uptake, glycogen storage, CLF secretion, ICG uptake and release, Alb secretion, urea synthesis and metabolism-relative gene expressions respectively. Next, both iHepSCs and iHepSC-Heps were transplanted into Fah-/- mice respectively. Both liver repopulation and alleviation of liver function were compared between two transplantation groups.
Murine iHepSCs still maintained the capacities of self-renewal and bipotential differentiations after extensive expansion. The efficiency for the functional hepatocyte differentiation from extensively expanded iHepSCs reached to 72.64%. Transplantations of both extensively expanded iHepSCs and iHepSC-Heps resulted in liver engraftment in Fah-/- mice. Survival rate of Fah-/- mice recipients and level of liver repopulation were 50% and 20.32 ± 4.58% respectively in iHepSC-Heps group, while 33% and 10.4 ± 4.3% in iHepSCs group.
Extensively expanded iHepSCs can efficiently differentiate into hepatocytes in chemical defined medium. Transplantation of iHepSC-Heps was more effective and more efficient than transplantation of iHepSCs in Fah-/- mice. Our results suggested an innovative system to obtain sufficient hepatocytes through hepatic differentiation of iHepSCs generated by lineage reprogramming.
供体肝细胞的短缺限制了肝细胞移植在临床中的应用。诱导肝干细胞（iHepSCs）具有自我更新和双向分化的能力。在此，我们研究了iHepSCs是否能够大量扩增，以及在大量扩增后它们是否能够分化为足够数量的功能性肝细胞作为移植治疗的供体。
在化学限定条件下，诱导小鼠大量扩增的iHepSCs（传代50 - 55次）分化为iHepSC - Heps。分别从形态学肝细胞特征以及肝细胞功能方面对iHepSC - Heps进行验证，这些功能包括低密度脂蛋白摄取、糖原储存、CLF分泌、ICG摄取与释放以及白蛋白分泌、尿素合成和代谢相关基因表达。接下来，将iHepSCs和iHepSC - Heps分别移植到Fah - / -小鼠体内。比较两个移植组之间的肝脏再植情况和肝功能改善情况。
小鼠iHepSCs在大量扩增后仍保持自我更新和双向分化的能力。大量扩增的iHepSCs向功能性肝细胞分化的效率达到72.64%。将大量扩增的iHepSCs和iHepSC - Heps移植到Fah - / -小鼠体内均导致肝脏植入。在iHepSC - Heps组中，Fah - / -小鼠受体的存活率和肝脏再植水平分别为50%和20.32±4.58%，而在iHepSCs组中分别为33%和10.4±4.3%。
在化学限定培养基中，大量扩增的iHepSCs能够高效分化为肝细胞。在Fah - / -小鼠中，iHepSC - Heps的移植比iHepSCs的移植更有效且更高效。我们的结果提示了一种创新系统，可通过谱系重编程产生的iHepSCs的肝分化来获得足够的肝细胞。
