Department of Intractable Diseases, National Center for Global Health and Medicine, Tokyo, Japan.
Department of Laboratory Animal Medicine, National Center for Global Health and Medicine, Tokyo, Japan.
Hepatol Commun. 2023 Feb 27;7(3):e0051. doi: 10.1097/HC9.0000000000000051. eCollection 2023 Mar 1.
Cells of interest can be prepared from somatic cells by forced expression of lineage-specific transcription factors, but it is required to establish a vector-free system for their clinical use. Here, we report a protein-based artificial transcription system for engineering hepatocyte-like cells from human umbilical cord-derived mesenchymal stem cells (MSCs).
MSCs were treated for 5 days with 4 artificial transcription factors (4F), which targeted hepatocyte nuclear factor (HNF)1α, HNF3γ, HNF4α, and GATA-binding protein 4 (GATA4). Then, engineered MSCs (4F-Heps) were subjected to epigenetic analysis, biochemical analysis and flow cytometry analysis with antibodies to marker proteins of mature hepatocytes and hepatic progenitors such as delta-like homolog 1 (DLK1) and trophoblast cell surface antigen 2 (TROP2). Functional properties of the cells were also examined by injecting them to mice with lethal hepatic failure.
Epigenetic analysis revealed that a 5-day treatment of 4F upregulated the expression of genes involved in hepatic differentiation, and repressed genes related to pluripotency of MSCs. Flow cytometry analysis detected that 4F-Heps were composed of small numbers of mature hepatocytes (at most 1%), bile duct cells (19%) and hepatic progenitors (50%). Interestingly, ~20% of 4F-Heps were positive for cytochrome P450 3A4, 80% of which were DLK1-positive. Injection of 4F-Heps significantly increased survival of mice with lethal hepatic failure, and transplanted 4F-Heps expanded to more than 50-fold of human albumin-positive cells in the mouse livers, well consistent with the observation that 4F-Heps contained DLK1-positive and/or TROP2-positive cells.
Taken together with observations that 4F-Heps were not tumorigenic in immunocompromised mice for at least 2 years, we propose that this artificial transcription system is a versatile tool for cell therapy for hepatic failures.
通过强制表达谱系特异性转录因子，可以从体细胞中制备感兴趣的细胞，但需要建立一个无载体系统，以便将其用于临床。在这里，我们报告了一种基于蛋白质的人工转录系统，用于从人脐带间充质干细胞（MSC）中工程化肝细胞样细胞。
用 4 种人工转录因子（4F）处理 MSC 5 天，这 4 种转录因子靶向肝细胞核因子（HNF）1α、HNF3γ、HNF4α 和 GATA 结合蛋白 4（GATA4）。然后，用成熟肝细胞和肝祖细胞如 delta 样同源物 1（DLK1）和滋养细胞表面抗原 2（TROP2）的标记蛋白的抗体对工程 MSC（4F-Heps）进行表观遗传学分析、生化分析和流式细胞术分析。还通过将细胞注射到具有致命性肝衰竭的小鼠中来检查细胞的功能特性。
表观遗传学分析显示，4F 的 5 天处理上调了参与肝分化的基因的表达，并抑制了与 MSC 多能性相关的基因。流式细胞术分析检测到 4F-Heps 由少量成熟肝细胞（最多 1%）、胆管细胞（19%）和肝祖细胞（50%）组成。有趣的是，~20%的 4F-Heps 对细胞色素 P450 3A4 呈阳性，其中 80%为 DLK1 阳性。4F-Heps 的注射显著提高了致命性肝衰竭小鼠的存活率，并且移植的 4F-Heps 在小鼠肝脏中扩增到超过 50 倍的人白蛋白阳性细胞，与观察到的 4F-Heps 包含 DLK1 阳性和/或 TROP2 阳性细胞一致。
综合观察到 4F-Heps 在免疫功能低下的小鼠中至少 2 年没有致瘤性，我们提出该人工转录系统是肝衰竭细胞治疗的通用工具。
