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Hemogenic Reprogramming of Human Fibroblasts by Enforced Expression of Transcription Factors
Published on: November 4, 2019
[Analysis of transcriptional factors expression profile during dedifferentiation of liver cell cultured in vitro]
Ruiyang Li1, Yunzhi Wang1, Rui Ge1
1School of Life Sciences, Fudan University, Shanghai 200438, China.
Abstract:
Primary hepatocytes are widely used in drug metabolism and toxicity assessment. As the culture of primary hepatocytes in vitro is a process of dedifferentiation, hepatocytes lose normal metabolic detoxification function gradually. The mechanism of hepatocyte dedifferentiation has been not clear so far. TFs play an important role in the dedifferentiation and non-parenchymal cells can maintain the function of hepatocytes in vitro. However, the current methods cannot be used in effective identification and quantitative analysis of a large number of TFs. In this paper, the mo-culture system (only primary hepatocytes) and co-culture system (primary hepatocytes and non-parenchymal cells) were established. The cells were cultured for 24 h, 48 h, 72 h as monolayer. The changes of TFs during the culture were obtained by TOT (Transcription factor response elements on tip) transcription factor enrichment method and mass spectrometry. A total of 219 TFs were identified in three individual replicates. The result revealed that up-regulated TFs were enriched in cell proliferation, death and immune response pathways, and down-regulated TFs were involved in metabolism pathway. The establishment of such culture-TFs identification system is of great significance to reveal the mechanism of primary hepatocyte dedifferentiation and crosstalk between hepatocytes and non-parenchymal cells.
Insights
Hepatocyte dedifferentiation in culture impairs drug metabolism function. This study identifies transcription factors (TFs) involved, revealing pathways affected and enabling better in vitro models for toxicity assessment.
Area of Science:
- Hepatocyte biology
- Cellular differentiation
- Drug metabolism and toxicology
Background:
- Primary hepatocytes are crucial for drug metabolism and toxicity studies.
- Hepatocyte dedifferentiation in vitro leads to loss of normal function.
- Mechanisms underlying hepatocyte dedifferentiation remain unclear.
Purpose of the Study:
- To investigate the role of transcription factors (TFs) in hepatocyte dedifferentiation.
- To compare TF changes in primary hepatocyte monoculture versus co-culture systems.
- To establish a system for identifying and quantifying TFs during hepatocyte culture.
Main Methods:
- Established monoculture and co-culture systems of primary hepatocytes.
- Cultured cells as monolayers for 24, 48, and 72 hours.
- Utilized Transcription factor response elements on tip (TOT) and mass spectrometry for TF identification.
Main Results:
- Identified a total of 219 transcription factors across three replicates.
- Observed enrichment of up-regulated TFs in cell proliferation, death, and immune response pathways.
- Found down-regulated TFs were primarily involved in metabolism pathways.
Conclusions:
- The developed culture-TF identification system is significant for understanding hepatocyte dedifferentiation.
- This system aids in elucidating the crosstalk between hepatocytes and non-parenchymal cells.
- Findings provide insights into maintaining hepatocyte function in vitro for drug assessment.
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