A two-step lineage reprogramming strategy to generate functionally competent human hepatocytes from fibroblasts

Xie, Bingqing; Sun, Dihua; Du, Yuanyuan; Jia, Jinping; Sun, Shicheng; Xu, Jun; Liu, Yifang; Xiang, Chengang; Chen, Sitong; Xie, Huangfan; Wang, Qiming; Li, Guang‐Ya; Lyu, Xuehui; Shen, Hui; Li, Shiyu; Wu, Min; Zhang, Xiaonan; Pu, Ying; Xiang, Kuanhui; Lai, Weifeng; Yuan, Zhenghong; Cheng, Li; Shi, Yan; Lu, Shichun; Deng, Hongkui

doi:10.1038/s41422-019-0196-x

Cited by 44 publications

(55 citation statements)

References 41 publications

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“…Importantly, these cells failed to mature in vivo in a transgenic mouse model designed for liver Lineage conversion by forced transcription factor expression has challenged the concepts of cell plasticity since its pioneer description, in 1987 [15]. In the past years, different combinations of transcription factors, soluble factors, and small molecules were shown to be effective in generating functional iHEPs [1,2,4,[16][17][18][19][20][21][22][23][24][25]. Here, we employed the combination of Foxa2 and Hnf4a, which reproducibly and effectively led to the generation of iHEP colonies.…”

Section: Discussionmentioning

confidence: 99%

Phenotype instability of hepatocyte-like cells produced by direct reprogramming of mesenchymal stromal cells

et al. 2020

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Background: Hepatocyte-like cells (iHEPs) generated by transcription factor-mediated direct reprogramming of somatic cells have been studied as potential cell sources for the development of novel therapies targeting liver diseases. The mechanisms involved in direct reprogramming, stability after long-term in vitro expansion, and safety profile of reprogrammed cells in different experimental models, however, still require further investigation.Methods: iHEPs were generated by forced expression of Foxa2/Hnf4a in mouse mesenchymal stromal cells and characterized their phenotype stability by in vitro and in vivo analyses.Results: The iHEPs expressed mixed hepatocyte and liver progenitor cell markers, were highly proliferative, and presented metabolic activities in functional assays. A progressive loss of hepatic phenotype, however, was observed after several passages, leading to an increase in alpha-SMA + fibroblast-like cells, which could be distinguished and sorted from iHEPs by differential mitochondrial content. The resulting purified iHEPs proliferated, maintained liver progenitor cell markers, and, upon stimulation with lineage maturation media, increased expression of either biliary or hepatocyte markers. In vivo functionality was assessed in independent pre-clinical mouse models. Minimal engraftment was observed following transplantation in mice with acute acetaminophen-induced liver injury. In contrast, upon transplantation in a transgenic mouse model presenting host hepatocyte senescence, widespread engraftment and uncontrolled proliferation of iHEPs was observed, forming islands of epithelial-like cells, adipocytelike cells, or cells presenting both morphologies. Conclusion: The results have significant implications for cell reprogramming, suggesting that iHEPs generated by Foxa2/Hnf4a expression have an unstable phenotype and depend on transgene expression for maintenance of hepatocyte-like characteristics, showing a tendency to return to the mesenchymal phenotype of origin and a compromised safety profile.

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Section: Discussionmentioning

confidence: 99%

Phenotype instability of hepatocyte-like cells produced by direct reprogramming of mesenchymal stromal cells

et al. 2020

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“…Next, we aimed to generate functional EPS-derived hepatocytes (EPS-Heps) from the EPS-HPLCs using combination of a cAMP activator and a TGFβ inhibitor (hepatocyte maturation medium, HMM) that we recently developed to mature hepatic progenitors and maintain mature hepatocyte function. 12,13 After maturation in HMM for 2 weeks, EPS-Heps showed a polygonal morphology that was similar to primary hepatocytes ( Supplementary Information, Fig. S1i), which suggested that EPS-Heps with mature hepatic identity were generated.…”

Section: Dear Editormentioning

confidence: 95%

“…These offtarget identities could be altered with further optimization of differentiation protocol. Additionally, using CellNet, we analyzed reprogrammed human induced hepatocytes (hiHeps) 12 and public RNA-seq data of conventional iPSC-derived hepatocytes (GSE103078 and GSE98710). The results showed that the EPS-Heps more closely resembled F-PHHs than hepatocytes generated from conventional human pluripotent stem cells or from lineage reprogramming ( Fig.…”

Section: Dear Editormentioning

confidence: 99%

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Generation of human hepatocytes from extended pluripotent stem cells

et al. 2020

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“…Human embryonic skin fibroblasts (HEFs) were obtained as previously described. 64 Briefly, human embryonic skin tissues, obtained from aborted tissues with informed patient consent, were minced with forceps and incubated in 1 mg/mL collagenase IV for 1-2 h at 37°C. After enzyme treatment, cells were collected by centrifugation and resuspended in HEF medium (DMEM containing 10% FBS, 1% GlutaMAX, 1% NEAA and 1% penicillin/ streptomycin).…”

Section: Cell Culturementioning

confidence: 99%

Elimination of senescent cells by β-galactosidase-targeted prodrug attenuates inflammation and restores physical function in aged mice

et al. 2020

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Cellular senescence, a persistent state of cell cycle arrest, accumulates in aged organisms, contributes to tissue dysfunction, and drives age-related phenotypes. The clearance of senescent cells is expected to decrease chronic, low-grade inflammation and improve tissue repair capacity, thus attenuating the decline of physical function in aged organisms. However, selective and effective clearance of senescent cells of different cell types has proven challenging. Herein, we developed a prodrug strategy to design a new compound based on the increased activity of lysosomal β-galactosidase (β-gal), a primary characteristic of senescent cells. Our prodrug SSK1 is specifically activated by β-gal and eliminates mouse and human senescent cells independently of senescence inducers and cell types. In aged mice, our compound effectively cleared senescent cells in different tissues, decreased the senescence-and age-associated gene signatures, attenuated low-grade local and systemic inflammation, and restored physical function. Our results demonstrate that lysosomal β-gal can be effectively leveraged to selectively eliminate senescent cells, providing a novel strategy to develop anti-aging interventions.Cell Research (2020) 0:1-16; https://doi.

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A two-step lineage reprogramming strategy to generate functionally competent human hepatocytes from fibroblasts

Cited by 44 publications

References 41 publications

Phenotype instability of hepatocyte-like cells produced by direct reprogramming of mesenchymal stromal cells

Phenotype instability of hepatocyte-like cells produced by direct reprogramming of mesenchymal stromal cells

Generation of human hepatocytes from extended pluripotent stem cells

Elimination of senescent cells by β-galactosidase-targeted prodrug attenuates inflammation and restores physical function in aged mice

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