In Vitro Expansion of Primary Human Hepatocytes with Efficient Liver Repopulation Capacity

Zhang, Kun; Zhang, Ludi; Liu, Wenming; Ma, Xiaolong; Cen, Jin; Sun, Zhen; Wang, Chenhua; Feng, Sisi; Zhang, Zhengtao; Yue, Liyun; Lu, Hui; Zhu, Zhenfeng; Chen, Xiaotao; Feng, Anqi; Wu, Jiaying; Jiang, Zhiwu; Peng, Li; Cheng, Xin; Gao, Dong; Peng, Luying; Hui, Lijian

doi:10.1016/j.stem.2018.10.018

Cited by 161 publications

(203 citation statements)

References 40 publications

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“…Hepatocyte-like cells differentiated from human PSCs are enriched with ammonia because these cells have a high metabolic activity of ammonia that is toxic to mammalian cells, especially neural cells [12]. Hepatocytes can be maintained in vitro without any introduction of any genes [13,14]. In this study, we propagated hepatocytes or hepatocyte-like cells derived from human PSC, i.e.…”

Section: Introductionmentioning

confidence: 99%

Ammonia-based enrichment and long-term propagation of zone I hepatocyte-like cells

Tsuneishi

Saku

Miyata

et al. 2020

Preprint

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Zone I and zone III hepatocytes metabolize ammonia through urea cycle and drug by cytochrome P450, respectively. Ammonia has a cytotoxic effect, and can therefore be used as a selection agent for enrichment of hepatocytes. Besides, isolated hepatocytes from livers can be propagated ex vivo under appropriate condition. However, it has not been investigated so far whether ammonia-treated hepatocyte-like cells are able to proliferate in vitro. In this study, we employed the ammonia selection strategy to purify hepatocyte-like cells that were differentiated from human pluripotent stem cells (PSCs) that are embryonic stem cells (ESCs) and induced pluripotent stem cells. Hepatocyte-like cells after exposure to ammonia highly expressed the CPS1 gene that metabolizes ammonia to carbamoyl phosphate. The resistance to cytotoxicity or cell death by ammonia is probably attributed to the metabolic activity of ammonia in the cells. In addition to the ammonia metabolism-related genes, ammonia-selected PSC-derived hepatocytes increased expression of the CYP3A4 gene, one of the cytochrome P450 genes, that is mainly expressed in zone III hepatocytes. Ammonia-selected hepatocyte-like cells derived from both ESCs and iPSCs can be propagated in vitro up to 30 population doublings for more than 190 days without affecting expression of the liver-associated genes, implying that the ammoniaselected cells have immortality or equivalent life span on the appropriate feeder cells like ESCs and iPSCs. The long-term cultivation of ammonia-selected hepatocyte-like cells resulted in the increased expression of hepatocyte-associated genes such as the CPS1 and CYP3A4 genes. The ammonia selection method to enrich a hepatocyte population was also applicable to immortalized cells from the liver. Ammonia treatment in combination with in vitro propagation will be used to obtain large amounts of hepatocytes or hepatocyte-like cells for pharmacology, toxicology and regenerative medicine.

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Section: Introductionmentioning

confidence: 99%

Ammonia-based enrichment and long-term propagation of zone I hepatocyte-like cells

Tsuneishi

Saku

Miyata

et al. 2020

Preprint

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“…Organoids are an important bridge between two-dimensional cultures and in vivo models because they are more physiologically relevant than monolayer cell culture models while being far more amenable to manipulation of niche components, signaling pathways, and genome editing than in vivo models [1]. Since 2009, many protocols have been implemented for the culture of organoids isolated from various tissues, such as liver [2][3][4][5], pancreas [6][7][8], lung [9], gut [10][11][12], stomach [13,14], and prostate [15]. Consequently, organoids can provide excellent model systems for a wide range of basic research studies, including expression profiling studies and analyses of rare cell lineages that are difficult to access in vivo [16].…”

Section: Introductionmentioning

confidence: 99%

Standardized GMP-compliant scalable production of human pancreas organoids

et al. 2020

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Background: Organoids are three-dimensional in vitro-grown cell clusters that recapitulate key features of native organs. In regenerative medicine, organoid technology represents a promising approach for the replacement of severely damaged organs, such as the pancreas in patients with type 1 diabetes. Isolation human pancreas organoids (hPOs) in chemically defined serum-free culture media would be a major milestone for this approach. Methods: Starting from discarded pancreatic tissues, we developed a large-scale process for obtaining clinically relevant quantities of undifferentiated organoids, obviating enzymatic digestion and operator-dependent pancreatic ducts picking steps. hPO identity was characterized by molecular and flow cytometry analysis.Results: This work demonstrates that it is possible to obtain a large-scale production of organoids. We introduced some innovations in the isolation, expansion, and freezing of hPOs from five donors. First of all, the choice of the starting material (islet-depleted pancreas) that allows obtaining a high quantity of hPOs at low passages. On the other hand, we introduced mechanical dissociation and we eliminated the picking step to exclude the operatordepending steps, without affecting the success of the culture (100% success rate). Another important improvement was to replace R-spondin-1 (Rspo1) conditioned medium with Rspo1 recombinant molecule to obtain a welldefined composition of the expansion medium. Finally, we implemented a GMP-compliant freezing protocol. hPOs showed exponential growth with diameter and area that increased three-and eight-fold in 7 days, respectively. Immunophenotypic profile and gene expression analysis revealed that hPOs were composed of ductal (82.33 ± 8.37%), acinar (2.80 ± 1.25%) cells, and pancreatic progenitors (5.81 ± 2.65%). Conclusion: This work represents a milestone for a GMP-compliance hPO production and, ultimately, their clinical application as a type 1 diabetes therapy.

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“…Nevertheless, these methods do not seem to be promising when applied on human hepatocytes. But recently, a research by Hui et al 10 brings another player to the table: human hepatocyte medium (HM). …”

Section: Introductionmentioning

confidence: 99%

“…Published in Cell Stem Cell , Hui's study 10 reported that they may solve the most critical problems for PHHs in vitro expansion by establishing a new human hepatocyte culture system - with the addition of Wnt3a and removal of Rspo1, Noggin and forskolin - as HM in which HHs can achieve a 10,000-fold expansion ( Figure 1 ). Through examining global genes expression profiling and cell function, they find that the proliferating human hepatocytes (ProliHHs) cultured in this system are at a bi-phenotypic “intermediate” status between mature hepatocytes and liver progenitor cells (LPCs) ( Figure 1 ).…”

Section: Introductionmentioning

confidence: 99%

“…Schematic diagram illustrates the expansion of PHHs from diversified individuals to a large quantity, when cultured in HM established by Hui et al, 10 with the addition of Wnt3a and removal of Rspo1, Noggin and forskolin. The ProliHHs maintained a bi-phenotypic status between primary hepatocytes and liver progenitor cells can mature after inducing re-differentiation by 3D culture and repopulate when transplanted into the liver of immuno-deficient Fah knockout mice indicated.…”

Section: Introductionmentioning

confidence: 99%

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