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Single-Cell Roadmap of generating human induced hepatocyte-like cells from fibroblasts using two-step lineage reprogramming strategy

ABSTRACT: Single-Cell Roadmap of generating human induced hepatocyte-like cells from fibroblasts using two-step lineage reprogramming strategy

PROVIDER: PRJNA1166183 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR30863958_1.fastq.gz	Fastqsanger.gz
	SRR30863958_2.fastq.gz	Fastqsanger.gz
	SRR30863959_1.fastq.gz	Fastqsanger.gz
	SRR30863959_2.fastq.gz	Fastqsanger.gz
	SRR30863960_1.fastq.gz	Fastqsanger.gz

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Single-Cell Roadmap of generating human induced hepatocyte-like cells from fibroblasts using two-step lineage reprogramming strategy

Project description:Background: Hepatocytes are crucial for drug screening, disease modeling, and clinical transplantation, yet generating large numbers of hepatocytes in vitro faces challenges, particularly with traditional direct reprogramming methods. These methods often result in cells with limited functionality and residual memory of their original state. To address these issues, we have a novel two-step lineage reprogramming strategy, which converts fibroblasts into human induced hepatocytes (hiHeps) via an intermediate hepatic progenitor-like cell stage. hiHeps resembled closely with primary human hepatocytes in molecular and functional identity, showing great potential for hepatocyte-based application, however the mechanism underlying the two-step reprogramming is unclear. Methods: This study employs high-throughput single-cell RNA sequencing (scRNA-seq) to explore the molecular and cellular dynamics of this process. Besides, we applied fluorescence-activated cell sorting, RT-qPCR, and shRNA knockdown to validate the key findings of the scRNA-seq results. Results: The findings reveal that the two-step reprogramming mimics a trajectory of liver development, with similar transcriptional waves of developmental genes during reprogramming process. CD24 and DLK1 were identified as markers for two different hepatic progenitor populations, both contributing to hepatocyte maturation. Notably, Lipid metabolism was found to enhance the maturation of progenitors into functional hepatocytes. Additionally, HNF4A and HHEX play roles in determining cell fate between hepatocytes and intestinal cells in the late reprogramming stages. Conclusions: This research provides a comprehensive single-cell roadmap for generating hiHeps in vitro, enhancing our understanding and optimization of the two-step reprogramming protocol.

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Project description:Hepatocytes hold significant promise for applications in drug screening, disease modeling, and clinical cell transplantation. Generating large amouts of hepatocyte in vitro for those application is still challenging. We have previously established a 5C medium for long-term culture and functional maintanace human primary hepatocytes (PHHs) in vitro. However, 5C condtion could not support hepatocyte expansion, which limited its application. In this study, we established a new hepatocyte expansion medium (NHEM) for efficient hepatocyte expansion, which turned the PHHs into hepatic progenitor-like cells (HPLCs). We also generated an optimized 6C condtion for HPLCs maturation. Overall, this study provide a new strategy to generate large amounts of hepatocytes in vitro.

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