Manipal Institute of Regenerative Medicine, MAHE, Bangalore, India.
Principal Scientist, Stempeutics Research Pvt. Ltd, Bangalore, India.
Int J Biochem Cell Biol. 2022 Aug;149:106256. doi: 10.1016/j.biocel.2022.106256. Epub 2022 Jun 27.
Pluripotent stem cell derived-hepatocytes depict fetal -hepatocyte characteristics/maturity and are immunogenic limiting their applications. Attempts have been made to derive hepatocytes from mesenchymal stem cells using developmental cocktails, epigenetic modulators and small molecules. However, achieving a stable terminally differentiated functional state had been a challenge. Inefficient hepatic differentiation could be due to lineage restrictions set during development. Hence a novel lineage reprogramming approach has been utilized to confer competence to adipose-mesenchymal stem cells (ADMSCs) to efficiently respond to hepatogenic cues and achieve a stable functional hepatic state. Lineage reprogramming involved co-transduction of ADMSCs with hepatic endoderm pioneer Transcription factor (TF)-FOXA2, HHEX-a homeobox gene and HNF4α-master TF indispensable for hepatic state maintenance. Lineage priming was evidenced by endogenous HFN4α promoter demethylation and robust responsiveness to minimal hepatic maturation cues. Induced hepatocytes (i-Heps) exhibited mesenchymal-to-epithelial transition and terminal hepatic signatures. Functional characterisation of i-Heps for hepatic drug detoxification systems, xenobiotic uptake/clearance, metabolic status and hepatotropic virus entry validated acquisition of stable hepatic state and junctional maturity Exhaustive analysis of MSC memory in i-Heps indicated loss of MSC-immunophenotype and terminal differentiation to osteogenic/adipogenic lineages. Importantly, i-Heps suppressed phytohemagglutinin-induced T-cell blasts, inhibited allogenic mixed-lymphocyte reactions (MLRs) and secreted immunomodulatory- indoleamine 2,3-dioxygenase in T-cell blast co-cultures akin to native ADMSCs. In a nutshell, the present study identifies a novel cocktail of TFs that reprogram ADMSCs to stable hepatic state. i-Heps exhibit adult hepatocyte functional maturity with robust immune-modulatory abilities rendering suitability for rigorous drug testing, hepatocyte-pathogen interaction studies and transplantation in allogenic settings.
多能干细胞衍生的肝细胞表现出胎儿样肝细胞的特征/成熟度，并且具有免疫原性，限制了它们的应用。已经尝试使用发育鸡尾酒、表观遗传调节剂和小分子从间充质干细胞中诱导产生肝细胞。然而，实现稳定的终末分化功能状态一直是一个挑战。低效的肝分化可能是由于发育过程中设定的谱系限制。因此，采用了一种新的谱系重编程方法，使脂肪间充质干细胞（ADMSC）获得能力，以有效地响应肝发生线索，并实现稳定的功能性肝状态。谱系重编程涉及共转导 ADMSC 与肝内胚层先驱转录因子（TF）-FOXA2、同源盒基因 HHEX-a 和维持肝状态所必需的主 TF-HNF4α。谱系启动的证据是内源性 HFN4α 启动子去甲基化和对最小肝成熟线索的强烈反应。诱导的肝细胞（i-Heps）表现出间充质到上皮的转变和终末肝特征。i-Heps 的肝药物解毒系统、外源性摄取/清除、代谢状态和嗜肝病毒进入的功能特征验证了获得稳定的肝状态和连接成熟。对 i-Heps 中的 MSC 记忆进行详尽分析表明，MSC 免疫表型丧失和向成骨/脂肪细胞谱系的终末分化。重要的是，i-Heps 抑制植物血凝素诱导的 T 细胞母细胞，抑制同种混合淋巴细胞反应（MLR），并在 T 细胞母细胞共培养物中分泌免疫调节吲哚胺 2,3-双加氧酶，类似于天然 ADMSC。简而言之，本研究确定了一种新的 TF 鸡尾酒，可将 ADMSC 重编程为稳定的肝状态。i-Heps 表现出成人肝细胞的功能成熟度，具有强大的免疫调节能力，适合严格的药物测试、肝细胞-病原体相互作用研究和同种异体移植。
