Genetic and Chemical Screenings Identify HDAC3 as a Key Regulator in Hepatic Differentiation of Human Pluripotent Stem Cells

Li, Shuang; Li, Mushan; Liu, Xi-Juan; Yang, Yuanyuan; Wei, Yuda; Chen, Yanhao; Qiu, Yan; Zhou, Tingting; Feng, Zhuanghui; Ma, Dongzhu; Fang, Jing; Ying, Hao; Wang, Hui; Musunuru, Kiran; Shao, Zongshu; Zhao, Yongxu; Ding, Qiurong

doi:10.1016/j.stemcr.2018.05.001

Cited by 25 publications

(26 citation statements)

References 29 publications

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“…These models have been sophisticated further using modern genome editing systems. Recently, pluripotent stem cells with an albumin reporter system have facilitated the discovery of key players involved in hepatocyte maturation (Li et al., 2018). In our study, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) endonuclease nickase was employed for hepatocyte nuclear factor-4alpha (HNF4α) genome editing in human pluripotent stem cells.…”

Section: Introductionmentioning

confidence: 99%

Multiomics Analyses of HNF4α Protein Domain Function during Human Pluripotent Stem Cell Differentiation

et al. 2019

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During mammalian development, liver differentiation is driven by signals that converge on multiple transcription factor networks. The hepatocyte nuclear factor signaling network is known to be essential for hepatocyte specification and maintenance. In this study, we have generated deletion and point mutants of hepatocyte nuclear factor-4alpha (HNF4a) to precisely evaluate the function of protein domains during hepatocyte specification from human pluripotent stem cells. We demonstrate that nuclear HNF4a is essential for hepatic progenitor specification, and the introduction of point mutations in HNF4a 0 s Small Ubiquitin-like Modifier (SUMO) consensus motif leads to disrupted hepatocyte differentiation. Taking a multiomics approach, we identified key deficiencies in cell biology, which included dysfunctional metabolism, substrate adhesion, tricarboxylic acid cycle flux, microRNA transport, and mRNA processing. In summary, the combination of genome editing and multiomics analyses has provided valuable insight into the diverse functions of HNF4a during pluripotent stem cell entry into the hepatic lineage and during hepatocellular differentiation.

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Section: Introductionmentioning

confidence: 99%

Multiomics Analyses of HNF4α Protein Domain Function during Human Pluripotent Stem Cell Differentiation

et al. 2019

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“…iPSC lines previously derived from fibroblasts from a non-African ancestry donor (1016SevA; Harvard Stem Cell Institute) (15)(16)(17)(18) and PBMCs from an African ancestry donor (Penn134-61-26; WiCell) were maintained in feederfree culture on 10-cm dishes coated with 0.5% Geltrex (Gibco) in Modified Tenneille's Special Recipe 1 (mTeSR1; STEMCELL Technologies), supplemented with 1% penicillin/streptomycin (Gibco) and 0.02% Plasmocin (Invivogen). iPSCs were confirmed to be mycoplasma-free and below passage 48.…”

Section: Materials and Methods Ipsc Culturementioning

confidence: 99%

Profiling APOL1 Nephropathy Risk Variants in Genome-Edited Kidney Organoids with Single-Cell Transcriptomics

et al. 2020

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Background DNA variants in APOL1 associate with kidney disease, but the pathophysiologic mechanisms remain incompletely understood. Model organisms lack the APOL1 gene, limiting the degree to which disease states can be recapitulated. Here we present single-cell RNA sequencing (scRNA-seq) of genome-edited human kidney organoids as a platform for profiling effects of APOL1 risk variants in diverse nephron cell types. Methods We performed footprint-free CRISPR-Cas9 genome editing of human induced pluripotent stem cells (iPSCs) to knock in APOL1 high-risk G1 variants at the native genomic locus. iPSCs were differentiated into kidney organoids, treated with vehicle, IFN-g, or the combination of IFN-g and tunicamycin, and analyzed with scRNA-seq to profile cell-specific changes in differential gene expression patterns, compared with isogenic G0 controls. Results Both G0 and G1 iPSCs differentiated into kidney organoids containing nephron-like structures with glomerular epithelial cells, proximal tubules, distal tubules, and endothelial cells. Organoids expressed detectable APOL1 only after exposure to IFN-g. scRNA-seq revealed cell type-specific differences in G1 organoid response to APOL1 induction. Additional stress of tunicamycin exposure led to increased glomerular epithelial cell dedifferentiation in G1 organoids. Conclusions Single-cell transcriptomic profiling of human genome-edited kidney organoids expressing APOL1 risk variants provides a novel platform for studying the pathophysiology of APOL1-mediated kidney disease.

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“…Shan et al[38] developed a high-throughput chemical screening platform and identified two different classes of small molecules, which are able to induce functional proliferation of human primary hepatocytes in vitro and improve HLC maturation. By utilizing an established hepatic lineage hPSC reporter line, our laboratory performed genetic and chemical screenings, and identified several modulators involved in hepatic differentiation, and CI-994 compound (histone deacetylase 3 inhibitor) that can promote HLC differentiation at a late stage[39].…”

Section: Derivation Of Human Hlcsmentioning

confidence: 99%

Derivation and applications of human hepatocyte-like cells

Li¹,

Huang²,

Zhao³

et al. 2019

WJSC

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Human hepatocyte-like cells (HLCs) derived from human pluripotent stem cells (hPSCs) promise a valuable source of cells with human genetic background, physiologically relevant liver functions, and unlimited supply. With over 10 years’ efforts in this field, great achievements have been made. HLCs have been successfully derived and applied in disease modeling, toxicity testing and drug discovery. Large cohorts of induced pluripotent stem cells-derived HLCs have been recently applied in studying population genetics and functional outputs of common genetic variants in vitro . This has offered a new paradigm for genome-wide association studies and possibly in vitro pharmacogenomics in the nearly future. However, HLCs have not yet been successfully applied in bioartificial liver devices and have only displayed limited success in cell transplantation. HLCs still have an immature hepatocyte phenotype and exist as a population with great heterogeneity, and HLCs derived from different hPSC lines display variable differentiation efficiency. Therefore, continuous improvement to the quality of HLCs, deeper investigation of relevant biological processes, and proper adaptation of recent advances in cell culture platforms, genome editing technology, and bioengineering systems are required before HLCs can fulfill the needs in basic and translational research. In this review, we summarize the discoveries, achievements, and challenges in the derivation and applications of HLCs.

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Genetic and Chemical Screenings Identify HDAC3 as a Key Regulator in Hepatic Differentiation of Human Pluripotent Stem Cells

Cited by 25 publications

References 29 publications

Multiomics Analyses of HNF4α Protein Domain Function during Human Pluripotent Stem Cell Differentiation

Multiomics Analyses of HNF4α Protein Domain Function during Human Pluripotent Stem Cell Differentiation

Profiling APOL1 Nephropathy Risk Variants in Genome-Edited Kidney Organoids with Single-Cell Transcriptomics

Derivation and applications of human hepatocyte-like cells

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