Posters
Presenting Author Academic/Professional Position
Daniela Gonzalez
Academic Level (Author 1)
Medical Student
Academic Level (Author 2)
Post-doc
Discipline/Specialty (Author 2)
Human Genetics
Academic Level (Author 3)
Staff
Discipline/Specialty (Author 3)
Human Genetics
Academic Level (Author 4)
Staff
Discipline/Specialty (Author 4)
Human Genetics
Academic Level (Author 5)
Staff
Discipline/Specialty (Author 5)
Human Genetics
Discipline Track
Biomedical Science
Abstract Type
Research/Clinical
Abstract
Background: One of the most common forms of chronic liver disease is nonalcoholic fatty liver disease (NAFLD). NAFLD is a condition of hepatic steatosis, excess fat accumulation in hepatocytes. It affects about 38% adults worldwide and about 50% adults in the Rio Grande Valley, and is a major risk factor for more serious nonalcoholic steatohepatitis (NASH), liver cirrhosis, and hepatocellular carcinoma (HCC). The risk for NAFLD is likely due to a complex interplay of genetic and environmental factors. Other metabolic diseases, such as obesity and type 2 diabetes, that have a high prevalence in the Valley, likely contribute to NAFLD risk. For NAFLD, one of the principal cell types is the hepatocyte, samples of which require invasive liver biopsy and are difficult to obtain. Commonly used human HCC cell lines or immortalized hepatocyte lines tend to accumulate genomic deviations and fail to recapitulate the functional state. This novel approach of using iPSC-generated hepatocytes, which recapitulate the hepatic function and biology of their donors, will enable us to study NAFLD pathophysiology and disease risk without the need to obtain primary hepatocytes through invasive liver biopsy.
Methods: iPSCs were differentiated into mature functional hepatocytes using a three-stage differentiation protocol. First, iPSCs were differentiated into SOX17 and CXCR positive definitive endodermal (DE) cells, followed by the differentiation of DE cells into hepatic precursors. In the final stage, hepatic precursors were seeded onto a collagen-coated cell culture surface and matured into functional hepatocytes. To validate the generated hepatocytes, morphology and hepatic marker expression were assessed through brightfield and immunofluorescence (IF) microscopy. For a comprehensive characterization of the generated hepatocytes, genome-wide gene expression analysis by next-generation mRNA sequencing was performed between the iPSCs and their differentiated hepatocytes. The tissue-specific gene expression and functional states of the generated hepatocytes were assessed by enrichment analyses of the hepatocyte transcriptome against various cell and tissue-specific gene expression data sets.
Results: The generated hepatocytes showed characteristic cobblestone morphology, were flat and polygonal in shape with distinct round nuclei, and expressed hepatic markers: alpha-fetoprotein (AFP), hepatocyte nuclear factor 4 alpha (HNF4α), albumin (ALB), and E-cadherin (E-Cad). A total of 7,970 genes were found significantly differentially expressed (moderated t statistics FDR-corrected p-value ≤ 0.05 and FC absolute (FC abs) ≥ 2.0) between iPSCs and their differentiated hepatocytes. The 4,164 genes that were significantly upregulated constituted the majority of the differentiated hepatocytes transcriptome and showed significant enrichment in GTEx gene sets for adult liver samples, thus validating the similarity of generated cells to primary hepatocytes. The 3,806 genes that were downregulated during the differentiation showed enrichment in iPSC/ESC self-renewal and maintenance-related Gene Ontology (GO) terms.
Conclusions: In this project, we generated iPSC-derived hepatocytes and confirmed their characterization through morphology and hepatic markers expression. The genome-wide gene expression and tissue-specific gene set enrichment analyses showed that the generated hepatocytes demonstrated significant similarities to adult liver samples. These results strongly support the utility of iPSC-generated hepatocytes for in vitro modeling of NAFLD.
Presentation Type
Poster
Recommended Citation
Gonzalez, Daniela; Aceves, Miriam; Guerra, Lorena; Granados, Jose C.; Juarez, Felicia; Novilla, Earl; Leandro, Ana C.; Leandro, Marcelo; Curran, Joanne E.; Blangero, John; Williams-Blangero, Sarah; and Kumar, Satish, "Generation of Functional Hepatocytes from Human Induced Pluripotent Stem Cells" (2025). Research Colloquium. 35.
https://scholarworks.utrgv.edu/colloquium/2025/posters/35
Included in
Generation of Functional Hepatocytes from Human Induced Pluripotent Stem Cells
Background: One of the most common forms of chronic liver disease is nonalcoholic fatty liver disease (NAFLD). NAFLD is a condition of hepatic steatosis, excess fat accumulation in hepatocytes. It affects about 38% adults worldwide and about 50% adults in the Rio Grande Valley, and is a major risk factor for more serious nonalcoholic steatohepatitis (NASH), liver cirrhosis, and hepatocellular carcinoma (HCC). The risk for NAFLD is likely due to a complex interplay of genetic and environmental factors. Other metabolic diseases, such as obesity and type 2 diabetes, that have a high prevalence in the Valley, likely contribute to NAFLD risk. For NAFLD, one of the principal cell types is the hepatocyte, samples of which require invasive liver biopsy and are difficult to obtain. Commonly used human HCC cell lines or immortalized hepatocyte lines tend to accumulate genomic deviations and fail to recapitulate the functional state. This novel approach of using iPSC-generated hepatocytes, which recapitulate the hepatic function and biology of their donors, will enable us to study NAFLD pathophysiology and disease risk without the need to obtain primary hepatocytes through invasive liver biopsy.
Methods: iPSCs were differentiated into mature functional hepatocytes using a three-stage differentiation protocol. First, iPSCs were differentiated into SOX17 and CXCR positive definitive endodermal (DE) cells, followed by the differentiation of DE cells into hepatic precursors. In the final stage, hepatic precursors were seeded onto a collagen-coated cell culture surface and matured into functional hepatocytes. To validate the generated hepatocytes, morphology and hepatic marker expression were assessed through brightfield and immunofluorescence (IF) microscopy. For a comprehensive characterization of the generated hepatocytes, genome-wide gene expression analysis by next-generation mRNA sequencing was performed between the iPSCs and their differentiated hepatocytes. The tissue-specific gene expression and functional states of the generated hepatocytes were assessed by enrichment analyses of the hepatocyte transcriptome against various cell and tissue-specific gene expression data sets.
Results: The generated hepatocytes showed characteristic cobblestone morphology, were flat and polygonal in shape with distinct round nuclei, and expressed hepatic markers: alpha-fetoprotein (AFP), hepatocyte nuclear factor 4 alpha (HNF4α), albumin (ALB), and E-cadherin (E-Cad). A total of 7,970 genes were found significantly differentially expressed (moderated t statistics FDR-corrected p-value ≤ 0.05 and FC absolute (FC abs) ≥ 2.0) between iPSCs and their differentiated hepatocytes. The 4,164 genes that were significantly upregulated constituted the majority of the differentiated hepatocytes transcriptome and showed significant enrichment in GTEx gene sets for adult liver samples, thus validating the similarity of generated cells to primary hepatocytes. The 3,806 genes that were downregulated during the differentiation showed enrichment in iPSC/ESC self-renewal and maintenance-related Gene Ontology (GO) terms.
Conclusions: In this project, we generated iPSC-derived hepatocytes and confirmed their characterization through morphology and hepatic markers expression. The genome-wide gene expression and tissue-specific gene set enrichment analyses showed that the generated hepatocytes demonstrated significant similarities to adult liver samples. These results strongly support the utility of iPSC-generated hepatocytes for in vitro modeling of NAFLD.
