Small molecule-mediated reprogramming of human hepatocytes into bipotent progenitor cells

Currently, much effort is directed to the development of new cell sources for clinical therapy using cell fate conversion approaches by small molecules. Direct lineage reprogramming to a progenitor state has been reported in terminally differentiated rodent hepatocytes, yet remains a challenge in human hepatocytes. Human hepatocytes were isolated from healthy and diseased donor livers and reprogrammed into progenitor cells by two small molecules, A83-01 and CHIR99021 (AC), in the presence of EGF and HGF. The stemness properties of human chemically derived hepatic progenitors (hCdHs) were tested by standard in vitro and in vivo assays and transcriptome profiling. We developed a robust culture system for generating hCdHs with therapeutic potential. The use of HGF proved to be an essential determinant of fate conversion process. Based on functional evidence, activation of HGF/MET signal transduction system collaborated with A83-01 and CHIR99021 to allow a rapid expansion of progenitor cells through activation of ERK pathway. hCdHs expressed hepatic progenitor marker genes and proteins, and could self-renew for at least 10 passages while retaining normal karyotype and potential to differentiate into functional hepatocytes and biliary epithelial cells in vitro. RNASeq gene expression profiling confirmed transcriptional reprogramming of hCdHs toward a progenitor state and suppression of mature hepatocyte transcripts. Upon intrasplenic transplantation into immunocompromised mice with acute liver injury, hCdHs effectively repopulated damaged parenchyma. Our study is a first report of successful reprogramming of human hepatocytes to a population of proliferating bipotent cells with regenerative potential. hCdHs may provide a nove tool that permits expansion and genetic manipulation of patient-specific progenitors to study regeneration and repair of diseased liver. Overall design: Transcriptome analysis for reprogrammed progenitor like cells

当前，学界正致力于通过小分子介导的细胞命运转化策略，开发用于临床治疗的新型细胞来源。已有研究报道，可通过直接谱系重编程将终末分化的啮齿类肝细胞转化为祖细胞状态，但在人类肝细胞中实现该过程仍是一项挑战。本研究从健康及病变供体肝脏中分离人类肝细胞，并在表皮生长因子（Epidermal Growth Factor, EGF）与肝细胞生长因子（Hepatocyte Growth Factor, HGF）存在的条件下，通过两种小分子A83-01与CHIR99021（简称AC）将其重编程为祖细胞。我们通过标准体外、体内实验与转录组分析，对人类化学诱导肝祖细胞（human chemically derived hepatic progenitors, hCdHs）的干性特征进行了验证。本研究成功构建了一套稳定的培养体系，可用于获取具有治疗潜力的hCdHs。实验证实，HGF的添加是细胞命运转化过程的关键决定因素。功能实验结果表明，HGF/MET信号转导系统的激活与A83-01、CHIR99021协同作用，通过激活细胞外调节蛋白激酶（Extracellular Regulated Protein Kinases, ERK）通路，实现祖细胞的快速扩增。hCdHs可表达肝祖细胞标志物基因与蛋白，且能够在至少10次传代过程中维持自我更新能力，同时保留正常核型，并具备体外分化为功能性肝细胞与胆管上皮细胞的潜能。RNA测序（RNA Sequencing, RNASeq）基因表达谱分析证实，hCdHs发生了向祖细胞状态的转录重编程，且成熟肝细胞相关转录本的表达受到抑制。将hCdHs通过脾内移植至急性肝损伤免疫缺陷小鼠体内后，其可有效重建受损的肝实质组织。本研究首次成功将人类肝细胞重编程为具有再生潜能的增殖性双潜能细胞群体。hCdHs可作为一种新型工具，实现患者特异性祖细胞的扩增与遗传操作，用于研究病变肝脏的再生与修复机制。实验整体设计：重编程类祖细胞的转录组分析