Developmental Stage-Specific Hepatocytes Induce Maturation of HepG2 Cells by Rebuilding the Regulatory Circuit

Li, Yanning; Liu, Demei; Zong, Yanhong; Qi, Jinsheng; B, Li; Liu, Kun; Xiao, Hui

doi:10.2119/molmed.2014.00173

Cited by 8 publications

(5 citation statements)

References 47 publications

(59 reference statements)

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“…Amongst these were NOTCH, P53, HEDGEHOG and WNT pathway (CCND2, AXIN2, MYC, WISP1). This is all in agreement with previous studies that have found expression of these genes to be correlated with hepatic maturation (Chen, et al , 2016, Li, et al , 2015b, Matsui, et al , 2002, Tanimizu and Miyajima, 2004). These results further suggest that while collagen plays a role in hepatic differentiation, other components of the ECM, which remain to be identified, play additive roles, highlighting the importance of using complex ECM signals in differentiation protocols.…”

Section: Resultssupporting

confidence: 93%

Decellularized human liver extracellular matrix (hDLM)‐mediated hepatic differentiation of human induced pluripotent stem cells (hIPSCs)

Jaramillo

Yeh

Yarmush

et al. 2018

J Tissue Eng Regen Med

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Liver tissue engineering has emerged as a promising approach in organ transplantation but has been hampered by the lack of a reliable and readily available cell source. Human induced pluripotent stem cells hiPSCs have been highlighted as a desirable source, due to their differentiation potential, ability to self-renew, and the possibility of making patient-specific cells. We developed a decellularization protocol that efficiently removes cellular material while retaining extracellular matrix components. Subsequently, hiPSCs were differentiated on decellularized human liver extracellular matrix (hDLM) scaffolds using an established hepatic differentiation protocol. We demonstrate that using hDLM leads to upregulation markers of hepatic functions when compared with standard differentiation conditions. In addition, expression of a number of hepatic transcription and nuclear factors were found to be within levels comparable with those of primary human adult hepatocytes. Analysis of progression of differentiation on hDLM demonstrated that hepatic developmental marker expression was consistent with hepatic development. The hDLM-derived cells exhibited key hepatic characteristics that were comparable with those observed in primary neonatal human hepatocytes. We investigated the optimal timing of the introduction of hDLM into the differentiation protocol and found that the best results are obtained when cells are plated on hDLM since the earliest stages and accompanied by a progressive loss of sensitivity to substrate composition at later stages. The significance of this work is that it allows for the development of differentiation protocols that take into account signals from extracellular matrix, closely recapitulating of the in vivo micro-environment and resulting in cells that are phenotypically closer to mature hepatocytes.

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Section: Resultssupporting

confidence: 93%

Decellularized human liver extracellular matrix (hDLM)‐mediated hepatic differentiation of human induced pluripotent stem cells (hIPSCs)

Jaramillo

Yeh

Yarmush

et al. 2018

J Tissue Eng Regen Med

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“…HepG2 cells cultured in a three-dimensional in vitro model using extracellular matrix–based hydrogel for long-term culture stop proliferating, self-organize, and differentiate to form multiple polarized spheroids 31 . Li et al reported that co-culture with mouse embryonic hepatocytes promotes the maturation of HepG2, and that upregulation of HNF4α is an important mechanism 32 . Thus, it is possible that HepG2 cells have strong properties as hepatic progenitor cells and consequently have low function as mature hepatocytes.…”

Section: Discussionmentioning

confidence: 99%

DNMT1/PKR double knockdowned HepG2 (HepG2-DP) cells have high hepatic function and differentiation ability

Tanaka‐Yachi

Aizawa

Shimizu

et al. 2022

Sci Rep

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HepG2 cells are widely used as a human hepatocytes model, but their functions, including drug metabolism, are inferior to primary hepatocytes. We previously reported that the hepatic gene expressions in HepG2 cells were upregulated by treatment with zebularine, which is an inhibitor of DNA methylation, through the inhibition of both DNA methyltransferase 1 (DNMT1) and double-stranded RNA-dependent protein kinase (PKR). In this study, we established a new HepG2 cell subline, HepG2-DP cells, by stable double knockdown of DNMT1 and PKR and evaluated its function. Albumin production, expression of CYP1A2 genes, and accumulation of lipid droplets were increased in HepG2-DP cells compared with the original HepG2 cells. Comprehensive gene expression analysis of transcription factors revealed that the expression of important genes for hepatic function, such as HNF1β, HNF4α, ONECUT1, FOXA1, FOXA2, FOXA3, and various nuclear receptors, was upregulated in HepG2-DP cells. These results indicate that the newly established HepG2-DP cells are a highly functional hepatocyte cell line. In addition, we investigated whether HepG2-DP cells are able to mature by differentiation induction, since HepG2 cells are derived from hepatoblastoma. The gene expression of major CYPs and Phase II, III drug-metabolizing enzyme genes was significantly increased in HepG2-DP cells cultured in differentiation induction medium. These results suggest that HepG2-DP cells can be further matured by the induction of differentiation and could therefore be applied to studies of drug metabolism and pharmacokinetics.

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“…2 (see section on supplementary data given at the end of this article). Binding of SMAD3 to its sites was analyzed by chromatin immunoprecipitation (ChIP), performed as reported (Li et al 2015). Briefly, anti-SMAD3 antibody (CST) or control rabbit IgG (Santa Cruz) was added, and then protein G-agarose beads (Santa Cruz) were used.…”

Section: Chromatin Immunoprecipitationsmentioning

confidence: 99%

C-peptide prevents SMAD3 binding to alpha promoters to inhibit collagen type IV synthesis

Zhong

Gong

et al. 2018

Journal of Molecular Endocrinology

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Activation of transforming growth factor β1 (TGFB1)/SMAD3 signaling may lead to additional synthesis of collagen type IV (COL4), which is a major contributor to extracellular matrix (ECM) accumulation in diabetic nephropathy (DN). C-peptide can attenuate fibrosis to have unique beneficial effects in DN. However, whether and how C-peptide affects TGFB1/SMAD3-activated COL4 synthesis is unclear. In this study, pathological changes, expression of COL4 a1-a5 chains (), COL4 distribution and protein and TGFB1 and SMAD3 protein were first assessed in a rat model of diabetes. Then, rat mesangial cells were treated with high glucose (HG) and/or C-peptide to investigate the underlying mechanism. expression, COL4 protein and secretion, TGFB1 protein, SMAD3 nuclear translocation and binding of SMAD3 to its cognate sites in the promoters of, and were measured. It was found that C-peptide attenuated glomerular pathological changes and suppressed renal mRNA expression, COL4 protein content and TGFB1 protein content. C-peptide had a dose-dependent effect to inhibit mRNA expression, COL4 protein content and secretion, in HG-stimulated mesangial cells. In addition, the HG-induced increase in TGFB1 protein content was significantly reduced by C-peptide. Although not apparently affecting SMAD3 nuclear translocation, C-peptide prevented SMAD3 from binding to its sites in the , and promoters in HG-stimulated mesangial cells. In conclusion, C-peptide could prevent SMAD3 from binding to its sites in the, and promoters, to inhibit COL4 generation. These results may provide a mechanism for the alleviation of fibrosis in DN by C-peptide.

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Developmental Stage-Specific Hepatocytes Induce Maturation of HepG2 Cells by Rebuilding the Regulatory Circuit

Cited by 8 publications

References 47 publications

Decellularized human liver extracellular matrix (hDLM)‐mediated hepatic differentiation of human induced pluripotent stem cells (hIPSCs)

Decellularized human liver extracellular matrix (hDLM)‐mediated hepatic differentiation of human induced pluripotent stem cells (hIPSCs)

DNMT1/PKR double knockdowned HepG2 (HepG2-DP) cells have high hepatic function and differentiation ability

C-peptide prevents SMAD3 binding to alpha promoters to inhibit collagen type IV synthesis

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