Direct reprogramming of somatic cells into induced hepatocytes: Cracking the Enigma code

Rombaut, Matthias; Boeckmans, Joost; Rodrigues, Robim Marcelino; Grunsven, Leo A. van; Vanhaecke, Tamara; Kock, Joery De

doi:10.1016/j.jhep.2021.04.048

Cited by 20 publications

(16 citation statements)

References 95 publications

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“…However, this step is challenging for hepatocytes as liver development is not initiated by a single and specific master regulator, and the factors driving functional maturation of hepatocytes remain to be fully uncovered. To bypass these limitations, we decided to focus on the LETFs which are known to control the induction of the hepatic program during foetal development and have been tested in somatic cell conversion (Rombaut et al, 2021). The coding sequence of 4 LETFs ( HNF4A , HNF1A , HNF6 and FOXA3 ) was cloned into the OPTi-OX system (Figure 1A) and the resulting inducible cassette was targeted into the AAVS1 gene safe harbour (Bertero et al, 2016; Pawlowski et al, 2017).…”

Section: Resultsmentioning

confidence: 99%

“…As an alternative, overexpression of transcription factors has been explored as a way to improve functionality of in vitro generated hepatocytes (Boon et al, 2020; Nakamori et al, 2016; Zhao et al, 2013). Moreover, transdifferentiation of somatic cells into liver cells has been achieved by overexpression of liver-enriched transcription factors (LETFs) in mouse and human fibroblasts (Rombaut et al, 2021). Importantly, these LETFs comprise the HNF1, HNF3 (FOXA), HNF4 and HNF6 (ONECUT) families all of which play key roles in coordinating liver development (Gordillo et al, 2015; Lau et al, 2018; Schrem et al, 2002).…”

Section: Introductionmentioning

confidence: 99%

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Generation of functional hepatocytes by forward programming with nuclear receptors

Tomaz

Zacharis

Bachinger

et al. 2022

Preprint

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Production of large quantities of hepatocytes remains a major challenge for a number of clinical applications in the biomedical field. Directed differentiation of human pluripotent stem cells (hPSC) into hepatocyte-like cells (HLCs) provides an advantageous solution and a number of protocols have been developed for this purpose. However, these methods usually follow different steps of liver development in vitro which is time consuming and requires complex culture conditions. In addition, HLCs lack the full repertoire of functionalities characterising primary hepatocytes. Here, we explore the interest of forward programming to generate hepatocytes from hPSCs and to bypass these limitations. This approach relies on the overexpression of 3 hepatocyte nuclear factors (HNF1A, HNF6 and FOXA3) in combination with different nuclear receptors expressed in the adult liver using the OPTi-OX platform. Forward programming allows for the rapid production of hepatocytes (FoP-Heps) with functional characteristics using a simplified process. We also uncovered that the overexpression of nuclear receptors such as RORc can enhance specific functionalities of FoP-Heps thereby validating its role in lipid/glucose metabolism. Together, our results show that forward programming could offer a versatile alternative to direct differentiation for generating hepatocytes in vitro.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Generation of functional hepatocytes by forward programming with nuclear receptors

Tomaz

Zacharis

Bachinger

et al. 2022

Preprint

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“…Direct lineage reprogramming can achieve direct induction of functional cell types from one lineage to another, without cells passing through an intermediate pluripotent stage [ 21 ]. This technique has been successfully used to regenerate various cells, such as cardiomyocytes and hepatocytes [ 22 , 23 ]. Therefore, direct lineage reprogramming may provide an alternative method to produce SG cells.…”

Section: Discussionmentioning

confidence: 99%

Small molecules facilitate single factor-mediated sweat gland cell reprogramming

Zhou

Sun

et al. 2022

Military Med Res

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Background Large skin defects severely disrupt the overall skin structure and can irreversibly damage sweat glands (SG), thus impairing the skin’s physiological function. This study aims to develop a stepwise reprogramming strategy to convert fibroblasts into SG lineages, which may provide a promising method to obtain desirable cell types for the functional repair and regeneration of damaged skin. Methods The expression of the SG markers cytokeratin 5 (CK5), cytokeratin 10 (CK10), cytokeratin 18 (CK18), carcino-embryonic antigen (CEA), aquaporin 5 (AQP5) and α-smooth muscle actin (α-SMA) was assessed with quantitative PCR (qPCR), immunofluorescence and flow cytometry. Calcium activity analysis was conducted to test the function of induced SG-like cells (iSGCs). Mouse xenograft models were also used to evaluate the in vivo regeneration of iSGCs. BALB/c nude mice were randomly divided into a normal group, SGM treatment group and iSGC transplantation group. Immunocytochemical analyses and starch-iodine sweat tests were used to confirm the in vivo regeneration of iSGCs. Results EDA overexpression drove HDF conversion into iSGCs in SG culture medium (SGM). qPCR indicated significantly increased mRNA levels of the SG markers CK5, CK18 and CEA in iSGCs, and flow cytometry data demonstrated (4.18 ± 0.04)% of iSGCs were CK5 positive and (4.36 ± 0.25)% of iSGCs were CK18 positive. The addition of chemical cocktails greatly accelerated the SG fate program. qPCR results revealed significantly increased mRNA expression of CK5, CK18 and CEA in iSGCs, as well as activation of the duct marker CK10 and luminal functional marker AQP5. Flow cytometry indicated, after the treatment of chemical cocktails, (23.05 ± 2.49)% of iSGCs expressed CK5+ and (55.79 ± 3.18)% of iSGCs expressed CK18+, respectively. Calcium activity analysis indicated that the reactivity of iSGCs to acetylcholine was close to that of primary SG cells [(60.79 ± 7.71)% vs. (70.59 ± 0.34)%, ns]. In vivo transplantation experiments showed approximately (5.2 ± 1.1)% of the mice were sweat test positive, and the histological analysis results indicated that regenerated SG structures were present in iSGCs-treated mice. Conclusion We developed a SG reprogramming strategy to generate functional iSGCs from HDFs by using the single factor EDA in combination with SGM and small molecules. The generation of iSGCs has important implications for future in situ skin regeneration with SG restoration.

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“…Human stem cell-derived hepatocyte-like cells and induced hepatocytes might therefore represent valid alternatives. Although fully mature induced hepatocytes are difficult to obtain for clinical applications due to safety issues, their implementation in basic and translational pharmacological in vitro research seems eminent [ 153 ]. The use of iPSC- or other stem cell-derived hepatic cells obtained from donors with different MAFLD etiologies might enable the construction of tailored in vitro models during early drug development, anticipating patient stratification in clinical trials, and hence reduce failures during late-phase clinical studies.…”

Section: Discussionmentioning

confidence: 99%

From NAFLD to MAFLD: Aligning Translational In Vitro Research to Clinical Insights

et al. 2022

Self Cite

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Although most same-stage non-alcoholic fatty liver disease (NAFLD) patients exhibit similar histologic sequelae, the underlying mechanisms appear to be highly heterogeneous. Therefore, it was recently proposed to redefine NAFLD to metabolic dysfunction-associated fatty liver disease (MAFLD) in which other known causes of liver disease such as alcohol consumption or viral hepatitis do not need to be excluded. Revised nomenclature envisions speeding up and facilitating anti-MAFLD drug development by means of patient stratification whereby each subgroup would benefit from distinct pharmacological interventions. As human-based in vitro research fulfils an irrefutable step in drug development, action should be taken as well in this stadium of the translational path. Indeed, most established in vitro NAFLD models rely on short-term exposure to fatty acids and use lipid accumulation as a phenotypic benchmark. This general approach to a seemingly ambiguous disease such as NAFLD therefore no longer seems applicable. Human-based in vitro models that accurately reflect distinct disease subgroups of MAFLD should thus be adopted in early preclinical disease modeling and drug testing. In this review article, we outline considerations for setting up translational in vitro experiments in the MAFLD era and allude to potential strategies to implement MAFLD heterogeneity into an in vitro setting so as to better align early drug development with future clinical trial designs.

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Direct reprogramming of somatic cells into induced hepatocytes: Cracking the Enigma code

Cited by 20 publications

References 95 publications

Generation of functional hepatocytes by forward programming with nuclear receptors

Generation of functional hepatocytes by forward programming with nuclear receptors

Small molecules facilitate single factor-mediated sweat gland cell reprogramming

From NAFLD to MAFLD: Aligning Translational In Vitro Research to Clinical Insights

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