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

Vrije Universiteit Brussel

Direct reprogramming of somatic cells into induced hepatocytes

Rombaut, Matthias; Boeckmans, Joost; Rodrigues, Robim M; van Grunsven, Leo A;

Vanhaecke, Tamara; De Kock, Joery

Published in:

Journal of Hepatology

DOI:

10.1016/j.jhep.2021.04.048

Publication date:

2021

License:

CC BY-NC-ND

Document Version:

Final published version Link to publication

Citation for published version (APA):

Rombaut, M., Boeckmans, J., Rodrigues, R. M., van Grunsven, L. A., Vanhaecke, T., & De Kock, J. (2021).

Direct reprogramming of somatic cells into induced hepatocytes: Cracking the Enigma code. Journal of Hepatology, 75(3), 690-705. https://doi.org/10.1016/j.jhep.2021.04.048

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Summary

There is an unmet need for functional primary human hepatocytes to support the pharmaceutical and (bio)medical demand. The unique discovery, a decade ago, that somatic cells can be drawn out of their apparent biological lockdown to reacquire a pluripotent state has revealed a completely new avenue of possibilities for generating surrogate human hepatocytes. Since then, the number of papers reporting the direct conversion of somatic cells into induced hepatocytes (iHeps) has burgeoned. A hepatic cell fate can be established via the ectopic expression of native liver-enriched transcription factors in somatic cells, thereby bypassing the need for an intermediate (pluripotent) stem cell state. That said, understanding and eventually controlling the processes that give rise to functional iHeps remains challenging. In this review, we provide an overview of the state-of-the-art reprogramming cocktails and techniques, as well as their corresponding conversion efficiencies. Special attention is paid to the role of liver-enriched transcription factors as hepatogenic reprogramming tools and small molecules as facilitators of hepat- ic transdifferentiation. To conclude, we formulate recommendations to optimise, standardise and enrich the in vitro production of iHeps to reach clinical standards, and propose minimal criteria for their characterisation.

© 2021 The Author(s). Published by Elsevier B.V. on behalf of European Association for the Study of the Liver.

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.

0/).

Introduction

Globally, end-stage liver disease is an important cause of death for which orthotopic liver trans- plantation is often the only curative treatment.¹ The demand for donor livers therefore far exceeds the current supply. Alternatively, primary human hepatocytes (PHHs) can be transplanted, but diffi- culties in achieving effective ex vivo expansion while maintaining functionality, present a major obstacle to their large-scale application.²To over- come this hurdle, increasing efforts have been directed towards generating surrogate hepatocytes from other cell sources. The introduction of human induced pluripotent stem (hiPSC) technology by Takahashi & Yamanaka revolutionised the field of regenerative and personalised medicine, chal- lenging the traditional view that a cell’s identity is sealed after undergoing development. Since this discovery, it is possible to reprogramme a patient’s somatic cells to a pluripotent stem cell-state with unlimited proliferative capacity.³ Human hepatocyte-like cells have been derived from hiPSCs (hiPSC-HLCs) by mimicking liver develop- mentin vitro(extensively reviewed by Heslop and Duncan⁴). These hiPSC-HLCs are widely employed for in vitro drug testing and hepatotoxicity screening,^5,6and could be used for liver cell ther- apy in the near future.¹

In the spirit of changing the cell fate of somatic cells, in 2011, two research groups demonstrated that it is possible to draw mousefibroblasts out of their biological lockdown and directly convert them to induced hepatocytes (iHeps). They ach- ieved this by ectopic expression of defined liver- enriched transcription factors (LETFs), namely GATA4, hepatocyte nuclear factor (HNF) 1A, and forkhead box (FOX) A3,⁷ or HNF4A plus FOXA1, FOXA2 or FOXA3.⁸It was only a few years later that hepatic transdifferentiation was established on a human level, albeit with a different hepatic reprogramming cocktail.⁹ By interacting with the transcriptional machinery, LETFs can gradually activate the silenced liver-specific gene expression programme, while silencing the somatic gene regulatory network. Furthermore, LETFs can interact with other transcription factors (TFs) and multiple co-activators or co-repressors, allowing for the formation of a platform for recruitment of a transcriptional complex, ultimately governing the tissue-restricted expression of liver-specific genes at specific stages of liver development and ho- meostasis (excellently reviewed by Lau et al.,¹⁰ Schremet al.¹¹and Lemaigre¹²), and orchestrating direct hepatic reprogramming.

1Department ofIn Vitro Toxicology and Dermato- Cosmetology, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel, Laarbeeklaan 103, B- 1090 Brussels, Belgium;

2Liver Cell Biology Research Group, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel, Laarbeeklaan 103, B-1090 Brussels, Belgium

* Corresponding authors.

Address: Departmentof In Vitro Toxicology and Dermato- Cosmetology, Faculty of Medi- cine and Pharmacy, Vrije Uni- versiteit Brussel (VUB), Laarbeeklaan 103, B-1090 Brussels, Belgium; Tel.: +32 (0)2 477 45 17.

E-mail addresses:Matthias.

Rombaut@vub.be(M. Rom- baut),Joery.De.Kock@vub.be(J.

De Kock).

https://doi.org/10.1016/

j.jhep.2021.04.048

Direct reprogramming of somatic cells into induced hepatocytes:

Cracking the Enigma code

Matthias Rombaut

*, Joost Boeckmans

, Robim M. Rodrigues

, Leo A. van Grunsven

, Tamara Vanhaecke

, Joery De Kock

*

Keywords: Direct hepatic reprogramming; induced hepatocytes; liver-enriched transcription factor; bench-to- bedside.

Received 12 February 2021;

received in revised form 29 April 2021; accepted 30 April 2021;

available online 11 May 2021

Key point

Human hepatocyte-based therapies provide a valid alternative to orthotopic liver transplantation for many life-threatening liver diseases, but are hampered by primary cell shortage, ineffectiveex vivoexpan- sion and immunogenicity of allogeneic donor cells.

Journal of Hepatology2021vol. 75j690–705

Review

A decade after the introduction of direct hepatic reprogramming, we critically discuss the latest direct hepatic reprogramming cocktails and the minimal combination of LETFs required to convert somatic cells towards a hepatic cell fate. To conclude, we discuss recommendations to further optimise, standardise and enrich thein vitropro- duction of human surrogate hepatocytes by direct hepatic reprogramming and propose minimal criteria for their characterisation.

Protagonists of the direct hepatic reprogramming cocktail

LETFs as key drivers for direct hepatic conversion

During hepatic transdifferentiation the somatic identity is downregulated while the endogenous interconnected hepatic cross-regulatory network is gradually activated. The mechanisms underlying direct hepatic reprogramming were elucidated by Lim and colleagues.¹³ They showed a sequential transition initiated by extinguishing the somatic fibroblast gene expression programme, followed by an activation of the mesenchymal-to-epithelial (MET) programme and the hepatic gene expres- sion profile, respectively.¹³ InTable 1, we provide an overview of the direct hepatic reprogramming cocktails, their corresponding conversion efficiency and kinetics, and a characterisation of the iHeps they generate. Studies are primarily divided by cell source of origin (mouse and human), and are re- ported in chronological order.

Synergy between HNF1A, HNF4A and the FOXA family As can be seen inTable 1, HNF1A and/or HNF4A are usually present in direct hepatic reprogramming cocktails, mainly in combination with a member of the FOXA family. HNF4A initiates the hepatic dif- ferentiation cascade during liver development and it co-regulates the transcription of 1,575 hepatic genes throughout adulthood.^14,15 The role of HNF1A in liver development is not entirely clear, yet it can be classified as a rather mature LETF that binds to at least 222 hepatocyte-specific genes in mature hepatocytes, all of which are involved in crucial hepatic pathways.¹⁵ A highly synergistic mechanism is conceivable between HNF1A and HNF4A as they can both bind to the same sets of hepatic genes. This suggests that mature hepatic gene expression depends on multi-input motifs regulated by multiple LETFs.¹⁵ In non-hepatic so- matic cells, most genes involved in hepatic meta- bolism are typically embedded in heterochromatin and therefore not accessible for LETFs to engage in gene transcription.¹⁶ Pioneer factors (e.g. GATA &

FOXA family) can recognise and bind to target DNA sequences in heterochromatin, enabling a remod- elling of the adjoining epigenetic landscape that provides access to LETFs, chromatin modifiers and chromatin remodellers.¹⁷ As such, they establish competence for lineage conversion and are seen as

crucial in direct hepatic reprogramming cocktails.¹⁸ Overexpression of epigenetic modifiers that pro- mote an open chromatin configuration, such as histone demethylases (KDM2B¹⁹), also facilitate hepatic transdifferentiation. The first two reports of direct hepatic reprogramming on mousefibro- blasts showed that single expression of HNF1A⁷or HNF4A⁸ in combination with a member of the FOXA family was sufficient to generate iHeps that can mature to functional hepatocytes in vivo.⁸ Strikingly, Huanget al.found that upon screening of different LETFs involved in liver development and hepatic differentiation, the removal of HNF4A in their direct reprogramming cocktail promoted the formation of epithelial colonies, the first key step in direct hepatic reprogramming. Further removal of a single LETF out of their 3TF reprog- ramming cocktail (GATA4, HNF1A, FOXA3) reduced the possibility of generating epithelial colonies.

They also found that the replacement of FOXA3 by FOXA2 resulted in less induction of hepatic gene expression and epithelial colony formation.⁷On a side note, Horisawa and colleagues showed that all FOXA family members bind to regions distal to the transcription start site. Yet, only FOXA3 transferred to proximal regions and was able to bind RNA po- lymerase II, ultimately inducing the lineage con- version.²⁰ Sekiya and Suzuki showed that the removal of HNF4A from the viral pool resulted in a reduction of albumin and alfa-fetoprotein expres- sion, whereas the expression level of E-cadherin was hardly affected by the removal of any of the LETFs. Consequently, expression of HNF4A with different LETFs was investigated and they showed that combined expression of HNF4A and any member of the FOXA family resulted in expression of hepatic markers.⁸Lim and colleagues compared epithelial formation in response to HNF1A or HNF4A overexpression in mouse fibroblasts and showed that HNF1A is better at inducing MET (14.5%vs.1.4% E-cadherin⁺colonies).¹³That HNF1A plays a more influential role than HNF4A during direct hepatic reprogramming in mousefibroblasts was also confirmed by Rezvani et al.²¹As can be seen in Table 1, direct hepatic reprogramming cocktails for hepatic transdifferentiation of human fibroblasts are more complex and generally require the combined expression of HNF1A and HNF4A with other LETFs. This is supported by thefinding that HNF1A and HNF4A both bind to the same sets of human hepatic genes and both play a central role in the complex interconnected hepatic cross- regulatory circuitry.^15,22 Hwang et al. obtained a metastable state by only overexpressing HNF1A, suggesting that it is not possible to achieve a stable reprogrammed hepatic identity contingent on ectopic expression of a single LETF in human fi- broblasts.²³This shows that the defined minimal LETF combinations in mice are notably different from the extensive direct hepatic reprogramming cocktails needed to direct a hepatic cell fate

Key point

Pioneer factors establish competence for lineage conversion by making epigenetic modifications that are required to start the liver programme.

Therefore, they are pin- pointed as indispensable for direct hepatic reprog- ramming cocktails.

Journal of Hepatology2021vol. 75j690–705 691

Table 1. An overview of the state-of-the-art direct hepatic reprogramming cocktails and reprogramming techniques to generate iHeps, and their corresponding characterisation.

Reprogramming

cocktail Cell type

Gene transfer method

Conversion efficiency

Conver- sion kinetics

In vitrocharacterisation In vivocharacterisation

Other tested TF(s) and

combinations Ref.

Genes involved in direct hepatic reprogramming

In vitro

functionality Mouse model

In vivo

functionality Mouse

GATA4, HNF1A, FOXA3

Mouse embry- onic & adult fibroblasts

Lentiviral Alb⁺(+/-23%) 21 days TJP1, CDH1, ALB, TTR, TF, AFP, CK19, HNF4A and CK18

Glycogen storage, LDL uptake, CYP activity, ICG uptake (20%), albumin secretion, drug metabolism (phenacetin, testosterone and diclofenac)

Fah^-/-/Rag2^-/- mouse

5/12 survived 8 weeks, showed increased body weight and 5-80%

Fah⁺iHep engraftment

Screening by withdrawal (FOXA2, FOXA3, HNF1A, HNF4A, HNF6, GATA4 FOXA1, and HLF)

7

HNF4A, FOXA1 Mouse embry- onic & adult fibroblasts

Retroviral Morphological conversion to iHeps (0,3%), thereof +/-85%

E-cad⁺and Alb⁺

n.s. MRP2, MRP4, ZO-1, ALB, AFP, COMT1, NAT2, MAOA, MAOB, TPMT, GS, GSTA4

Glycogen storage (80%), LDL uptake, albumin secretion, urea production, triglycerides syn- thesis, CYP activ- ity, ICG uptake, drug metabolism

Fah^-/-mouse 40% survived longer than 10 weeks, decrease in ALT, ALP and bilirubin

Screening by withdrawal of 12 transcription factors

8 HNF4A, FOXA2

HNF4A, FOXA3

HNF1B, FOXA3 Mouse embry- onic & adult fibroblasts

Lentiviral, followed by directed hepatic differentiation

E-cad⁺(0,4%) 15 days ALB, TTR, HNF4A, CK8, CK18, CK19, AFP, SOX9, EPCAM, DLK1, PAN-CK, LGR5

n.a. Fah^-/-mouse;

DDC-induced mouse

4/11 survived 8 weeks, 11,6% repo- pulation, Alb expression, decrease in ALT, AST and bilirubin

20 candidate factors

41

56,4% 12 days ALB, HNF4A, SER-

PINA1, GJB1, CYP3A11, CYP7A1, G6P

Glycogen storage, G6P activity, albu- min secretion

n.a. n.a.

GATA4, HNF1A, FOXA3

Mouse embry- onic & adult fibroblasts

oriP/EBNA1- based episomal system

E-cad⁺(0,12%) 30 days AFP, ALB HNF1A, FOXA3, GATA4, CEBPA, HNF4A, TTR, CK8, CK18, CLDN2, CDH, APOA1, CYP39A1, ZO-1, CROT, and AKR1C13

Glycogen storage (>70%), ICG uptake (>50%), LDL uptake, albumin secretion, urea production, upregulation CYPs after treat- ment with inducer (CYP1A1, CYP1A2, CYP2A5, CYP2D22 and CYP3A13)

FRG mouse Survival beyond 45 days

HNF4A, FOXA1;

HNF4A, FOXA3

54

OCT4, SOX2, KLF4, MYC, HNF4A, CEBPA, NR1I2

Mouse embry- onic

fibroblasts

Lentiviral Thy1^-/Alb⁺ (+/- 17%);

Thy1^-/ CYP7A1⁺ (+/- 17%)

25 days ALB, DLK1, KRT18, TAT and CYP7A1

glycogen storage, LDL uptake, ICG uptake, albumin secretion, urea production

CCl4-injured mouse

Engraftable Screening (GATA4, GATA6, FOXA1, FOXA2, FOXA3, HNF1A, HNF1B, HHEX, TBX3, PROX1, HNF4A, OC1, OC2, NR1I2, UPF1, CREB1, USF1, RXRA, CEBPA, CEBPB)

28

(continued on next page)

692JournalofHepatology2021vol.75j690–705

R evie w

Table 1.(continued)

Reprogramming

cocktail Cell type

Gene transfer method

Conversion efficiency

Conver- sion kinetics

In vitrocharacterisation In vivocharacterisation

Other tested TF(s) and

combinations Ref.

Genes involved in direct hepatic reprogramming

In vitro

functionality Mouse model

In vivo

functionality MYC, KLF4,

HNF4A, FOXA1

Mouse embry- onic

fibroblasts

Retroviral n.s. 15 days FOXA2,

HNF4A,HNF1A, C-MET, TTR, ALB, CK18, CYP1A2, CDH, ZO-1, CYP1A1

Urea production, albumin secretion, glycogen storage, LDL uptake, ICG uptake, accumula- tion lipids

CCl4-injured

mouse; FRG

mouse

infiltration of reactive HSCs and macrophages increased,fibrin clearance increased, AST and ALT decreased;

Prolonged survival

KLF4, HNF4A, FOXA1; MYC, HNF4A, FOXA1;

HNF4A, FOXA1;

HNF1A; HNF4A

13

MYC, KLF4, HNF4A, FOXA3

Mouse embry- onic

fibroblasts

Retroviral n.s. 15 days FOXA2,

HNF4A,HNF1A, C-MET, TTR, ALB, CK18, CYP1A2, CDH, ZO-1, CYP1A1, CYP3A44

Urea production, albumin secretion, glycogen storage, LDL uptake, ICG uptake, accumula- tion lipids

CCl4-injured

mouse; FRG

mouse

infiltration of reactive HSCs and macrophages increased,fibrin clearance increased, AST and ALT decreased;

Prolonged survival

HNF4A, FOXA3;

HNF4A, FOXA3 + 1TF (CEBPA, DBP, FOXA2, GATA4, OC1)

13,53

HNF1A, A-83-01, CHIR99021, BMP-4

Mouse embry- onic

fibroblasts

Retroviral E-cad⁺(26,2%);

Alb⁺(8,2%);

Aat⁺(5,4%)

35 days ALB, SERPINA1, MRP2, MRP3, MAOB, MGST1, SULT1A1, CK18, ZO1

Albumin secretion, CYP activity, LDL uptake, glycogen storage, ICG up- take, accumula- tion lipids

FRG mouse Prolonged survival HNF4A, A-83-01, CHIR99021;

HNF1A, A-83-01, CHIR99021

13,53

HNF4A, FOXA3, KDM2B

Mouse embry- onic

fibroblasts

Lentiviral n.s. 11 days TTR, TAT, ASGR, G6P, GATA4, CDH1, ALB, CYP2B9, CYP3A4, HNF4A, LGR5, DLK1, EPCAM

Glycogen storage, ICG uptake, albu- min secretion, LDL uptake, CYP3A4 activity

CCl₄-injured mouse

survival rate: 100%

iHeps vs. 80% fi- broblasts, engraft- able iHeps, albumin expression

HNF4A, FOXA3 19

FOXA3, GATA4, HNF1A, HNF4A

In vitro: myo- fibroblasts derived from primary mouse hepatic stellate cells In vivo: Mouse myofibroblasts

In vitro:

lentiviral (polycistronic system) In vivo:

adenoviral (polycistronic system)

In vitro:

Alb⁺(+/-12%) In vivo: < 4%

In vitro:

21 days In vivo:

30 days

ALB, SERPINA1, APOA1, CK18, FOXA1, FOXA2, GJB1, ACTB

Glycogen storage, LDL uptake, albu- min secretion, CYP1A2 and CYP3A activity

CCl₄-injured mouse

<4% reprogram- ming efficiency, albumin secretion, urea production, glycogen storage, ICG & LDL uptake, accumulation lipids, CYP activity (CYP3A, 1A1, 2C9, and 1A2), upregu- lation CYP1A1, UGT1A1, ABCC2, and OATP after induction

Screening by withdrawal (FOXA1, FOXA2, FOXA3, GATA4, HNF1A, HNF4A and CEBPA)

57

FOXA1, FOXA2, FOXA3, GATA4, HNF1A, HNF4A

Mouse myofibroblasts

In vivo: adeno- associated viral

0.87% of all hepatocytes in the liver

n.s. n.a. n.a. CCl4-injured

mouse

normal albumin secretion, CYP ac- tivity and urea production

n.a. 21

(continued on next page) JournalofHepatology2021vol.75j690–705693

Table 1.(continued)

Reprogramming

cocktail Cell type

Gene transfer method

Conversion efficiency

Conver- sion kinetics

In vitrocharacterisation In vivocharacterisation

Other tested TF(s) and

combinations Ref.

Genes involved in direct hepatic reprogramming

In vitro

functionality Mouse model

In vivo

functionality HNF4A, FOXA3 Mouse

mesenchymal stem cells

PiggyBac transposon (polycistronic system)

E-cad⁺(25,1%) 12 days CDH, ALB, CK18, G6P, AFP, CK19, ACTB, CYP3A11, CYP2E1, CYP1A2, TAT, TTR, SERPINA1

Urea production, accumulation lipids, glycogen storage, ICG uptake, albumin secretion

n.a. n.a. n.a. 58

FOXA1 + CRVPTD Mouse embryonic fibroblasts &

adult fibroblasts

Retroviral E-cad⁺ (+/-55%);

Alb⁺(+/-36%)

18 days AFP, ALB, CK8, TTR, CDH, VTN, CLDN3, HNF4A, TF, CK18, CK19, EPCAM

Glycogen storage, LDL uptake, CYP activity (CYP1A1, CYP1A2, CYP2B10, CYP2C29, CYP2C38, CYP2D22, CYP2E1, CYP3A11 and CYP3A13), drug metabolisation (phenacetin, tolbutamide, testosterone, diclofenac)

Fah^-/-mouse 4/12 survived 8 weeks & livers were as normal as these of the sur- viving hepatocyte- transplanted mice, ALB & FAH expression, ALT, AST, ALP and total bilirubin markedly reduced

n.a. 31

FOXA2 + CRVPTD FOXA3 + CRVPTD

FOXA3, HNF4A Mouse embryonic fibroblasts

Lipofectamine 2000 (mRNA)

n.s. 12 days ALB, CDH, AFP,

HNF4A, CK18, ASGR1, NR1I2 and CYP1A2

Glycogen storage (>70%), ICG uptake, albumin secretion, xenobi- otic metabolic Activity (>50%)

Alb-TRECK SCID mouse; Fah^1RTyr^c/ RJ

Albumin positive;

Fah production

n.a. 72

FOXA3, HNF1A, GATA4

Mouse embryonic fibroblasts

Lentiviral (polycistronic system)

n.s. 16 days ALB, CDH1,

CYP1A2, CYP3A11, MRP2, AFP, HNF4A, TTR, TF, SOX9, HNF1B, TJP1

AST and LDH activity, CYP3A activity, albumin secretion

n.a. n.a. n.a. 52

Human FOXA2, HNF4A, CEBPB, MYC

Human neonatal &

adult fibroblasts

Retroviral n.s. 20 days ALB, CYP3A4,

SERPINA1,GAPDH

Albumin secretion, glycogen storage, ICG uptake

n.a. n.a. All combinations

of FOXA2 with MYC, CEBPB or HNF4A

9

FOXA3, HNF1A, HNF4A

Human foetal, adult fibroblasts &

mesenchymal stem cells

Lentiviral foetal: Alb⁺/ Aat⁺

(+/-20%);

adult:

Alb⁺/Aat⁺ (+/-10%)

14 days ALB, TTR, ASGR1, TF, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP3A4, MDR1, MRP2, MRP3, BSEP, NTCP, OATP1, OATP2, OATPB, RXRB, GR, RXRG

Albumin and Aat secretion, glycogen storage, LDL uptake, ICG uptake, accumula- tion lipids, upre- gulation CYPs after treatment with inducer (CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9), drug metabo- lisation (phenac- etin, coumarin, dextrometorphan)

Con A-injured

mouse; FRG

mouse

5/14 completely recovered, normal AST & ALT levels;

5/15 survived 9 weeks, 0,3-4,2%

repopulation

Screening (FOXA3, GATA4, HNF1B, HNF4A, HHEX, PROX1, CEBPB, KLF4)

26,93,94

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R evie w

Table 1.(continued)

Reprogramming

cocktail Cell type

Gene transfer method

Conversion efficiency

Conver- sion kinetics

In vitrocharacterisation In vivocharacterisation

Other tested TF(s) and

combinations Ref.

Genes involved in direct hepatic reprogramming

In vitro

functionality Mouse model

In vivo

functionality HNF1A, HNF4A,

HNF6, ATF5, PROX1, CEBPA

Human embryonic &

adult fibroblasts

Viral Alb⁺ (90%);

Aat⁺ (+/-100%)

25 days CYP3A4, CYP1A2, CYP2B6, CYP2C9, CYP2C19, CDH, FOXA1, FOXA2, FOXA3, ALB, LRH1, UGT1A1, UGT2B7, UGT2B15, MGST1, NNMT, MRP6

Glycogen storage, LDL uptake, ICG uptake, albumin secretion, accu- mulation lipids, no Afp secretion

Tet-uPA/Rag2^-/- cc^-/-mouse

repopulate 30%, 313l^{g/ml human} albumin, expres- sion of CYP3A4, CYP2C9, CYP1A2, CYP2E1, CYP2C19, and CYP2D6

- each transcrip- tion factor;

HNF4A, HNF1A and HNF6

35

HNF1A + 2TFs (FOXA1 or FOXA3 or HNF4A)

Human embryonic fibroblasts

Synthetic modified mRNA (daily cationic lipid transfection)

n.s. 5 days ALB, AFP Albumin and Afp

secretion

n.a. n.a. n.a. 25

FOXA2, GATA4, FOXA1, FOXA3, HNF4A, HNF1A

Human embryonic fibroblasts

Synthetic modified mRNA (cationic lipid transfection)

n.s. 5 days ALB, AFP, TLR3,

APOA1, APOH, FGB, SERPINA1, CXCL9, CXCL10, ODC1, miR-122, miR-145, miR-192, miR-194

Albumin and Afp secretion

n.a. n.a. 11 TFs (+CEBPA,

GATA6, HHEX, HNF1B, and HNF6A)

25

ATF5, PROX1, FOXA2, FOXA3, HNF4A

Human foetal fibroblasts (MRC5)

Lentiviral Alb⁺/Aat⁺ (+/-27%);

Asgr1⁺ (+/-22%)

28 days ALB, AFP, CYP3A7, SERPINA1, CYP1A2, CYP2C19, CYP3A4, CYP2C9, CYP2D6, UGT1A1, NTCP, ATF5, PROX1, FOXA2, and FOXA3

Albumin secretion, CYP1A2 and CYP3A4 activity

n.a. n.a. Screening by

withdrawal (ATF5, CEBPA, PROX1, FOXA2, FOXA3, HNF1A, HNF4A, HNF6, and GATA4)

27

FOXA3 + CRVPTD Human urine- derived epithelial-like cells

Lentiviral Alb⁺(+/-34%) 24 days ALB, ASGR1, CK18, HNF4A, TTR, TF, ZO- 1, GJB1, CYP1A2, CYP3A4, CYP2B6, CYP2D6, CYP2C8, CYP2C9, NTCP, MRP2, AHR, PXR, RXRA and RXRB

Albumin and Aat secretion, glycogen storage, LDL uptake, accu- mulation lipids

Con A-injured mouse

3 of 12 mice sur- vived 7 days, ALT, AST, and total bili- rubin gradually decreased

n.a. 34

HNF1A + CRVPTD HNF4A + CRVPTD

FOXA3, HNF1A, HNF4A

Human skin fibroblasts

Lentiviral n.s. 10 days CAR, FXR, HNF1A,

ALB, MRP2, SERPINA1, GGT, UGT1A1, ASGR1, TF, HNF4A

n.a. n.a. n.a. n.a. 32

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Table 1.(continued)

Reprogramming

cocktail Cell type

Gene transfer method

Conversion efficiency

Conver- sion kinetics

In vitrocharacterisation In vivocharacterisation

Other tested TF(s) and

combinations Ref.

Genes involved in direct hepatic reprogramming

In vitro

functionality Mouse model

In vivo

functionality HNF1A, FOXA3,

HNF4A

Human hepa- toma (HCCLM3 &

Huh-7)

Adenoviral n.s. 14 days CYP1A2, CYP2C19,

CYP3A4, ALB, PXR, GR, RXRB, PEPCK, GS, AAT, G6P, TF, ALDOB, MRP2, NTCP, OATPB

Glycogen storage, LDL uptake, urea production, albu- min secretion, CYP activity (CYP1A2, CYP2C19, CYP3A4), upregu- lation CYPs after inducer (CYP1A2, CYP2C19, CYP2C8, CYP3A4), drug metabolism (phenacetin, testosterone)

Fah^-/-/Rag2^-/- mouse

Human Fah, Alb and Aat, 4,69%

repopulation, bet- ter survival rate with iHeps than with PHH

n.a. 64

HNF4A, HNF6A, GATA4, FOXA2, HHEX, c-MYC

Human embryonic and neonatal fibroblasts

Lentiviral, fol- lowed by directed hepatic differentiation

Alb⁺(2,7%);

Alb⁺(+/-75%)

15 days; 40 days

ALB, AFP, EPCAM, CK8, CK18, HNF1B, DLK1

n.a. Tet-uPA/Rag2^-/-

cc^-/-mouse;

NPG mouse

+/-50% human Alb⁺, CYP3A4, CYP2E1, CYP2D6, CYP2C9, CYP2C8, CYP1A2, CYP2C19, NTCP, MRP2;

No tumour development

n.a. 36

Alb⁺(>90%) 25 days ALB, CDH1, HNF1A, CEBPA, AAT, LXR, FXR, PXR, PPARA

LDL uptake, albu- min secretion, accumulation lipids, glycogen storage, CYP3A4, CYP2D6, CYP2C9, CYP2C19, CYP1A2, CYP2B6 and CYP2C8, UGTs, NTCP, steatosis, phospholipidosis

n.a. n.a.

HNF4A, FOXA3, HNF1A

Human neonatal dermal fibroblasts

Inducible polycistronic lentiviral

n.s. 10 days GLS2, HGD,

CYP7A1, GPT1, ALB, SERPINA1, ALDH4A1, GPT1

Glycogen storage, glutamine/gluta- mate conversion and secretion, al- bumin secretion

CB17/Icr-Prkdc scid/Crl mice

Human albumin Individual TFs 59

HNF1A, GATA4, FOXA3

human urinary epithelial cells

Lentiviral n.s. 20 days ALB, CK8, CK18, TTR, ASGPR1, SER- PINA1, CYP2B6, CYP2A9, CYP3A4, CYP2C19, CYP1A2

Glycogen storage, accumulation lipids, ICG uptake, upregulation CYPs after inducer (CYP2B6, CYP2C9, CYP3A4, CYP2C19, CYP1A2)

n.a. n.a. Screening (FOXA2,

FOXA3, GATA4, HNF1A, HNF1B, HNF4A)

48

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conversion in human somatic cells and that the extrapolation of mouse to man is rather unreliable, as the DNA binding sites and the interconnected hepatic cross-regulatory circuitry differ in human and mouse liver cells.^24,25 Huang and colleagues showed that in their 3TF hepatic reprogramming cocktail (HNF1A, FOXA3, HNF4A) the concomitant expression of HNF1A is mandatory to induce the hepatic reprogramming of humanfibroblasts.²⁶In addition, Simeonovet al.demonstrated that HNF1A is a mandatory LETF to induce a direct hepatic conversion in human fibroblasts.²⁵ Contrarily, Nakamori and colleagues showed that with- drawing HNF1A from their direct hepatic reprog- ramming cocktail did not change hepatic marker expression, suggesting that HNF1A might not play an important role in human hepatic cell fate con- version. Furthermore, they confirmed that HNF4A plays a crucial role in their direct hepatic reprog- ramming cocktail as hepatic markers drastically decreased upon withdrawal of HNF4A.²⁷ These discrepancies clearly highlight that there is no consensus regarding whether HNF1A or HNF4A plays the more important role in direct hepatic conversion. Yet, it is clear that mouse fibroblasts exhibit a higher propensity for lineage conversion than humanfibroblasts, which are seemingly in a stronger biological lockdown.

Pluripotency-promoting factors improve and accel- erate iHep generation

Concomitant expression of pluripotency-promoting TFs (“OSKM”)^9,28,29makes the somatic cells convert towards a temporary stem cell-like state that allows them to proliferate before reaching hepatic maturity.

Furthermore, it has been shown that these TFs accelerate the sequential cell fate transition.¹³Even though the presence of MET activators Kruppel like factor 4 (KLF4) and c-MYC accelerates the conversion kinetics and, consequently, enhances the efficiency of iHep generation,¹³ these 2 activators must be excluded from direct hepatic conversion cocktails because of their oncogenic properties.^29,30 Over- expression of SV40 large T antigen can bypass the inability of iHeps to proliferate.^26,31–34Yet, this forced proliferation is at the expense of hepatic function- ality. The knockdown of p53 with siRNA has been shown to be a valid alternative for growing iHeps in vitro^35,36and has been shown to increase pluripo- tent reprogramming efficiencies.³⁷However, it is also well-known that diminishing the function of p53 may cause the induction of genomic mutations, as p53 is prominently involved in DNA repair following damage.^38,39To be clinically relevant, cells must be prevented from acquiring the ability to proliferate uncontrollably, as seen in iPSC reprogramming.²³Yet, these iHeps can still be used in preclinical hepato- toxicity screenings and extracorporeal bio-artificial liver devices for acute liver failure.³³

Hepatic maturation factors enhance functionality of obtained iHeps

To further improve the direct conversion towards more mature iHeps, it is opportune to include he- patic maturation factors in the direct hepatic reprogramming cocktail. Hepatic maturation fac- tors are defined as LETFs that are differentially expressed between foetal liver cells and freshly isolated PHHs.³⁵ Duet al. showed that activating transcription factor (ATF) 5, prospero homeobox transcription factor (PROX) 1 and CCAAT/enhancer binding protein (CEBP) A are maturation factors that are crucial for the metabolic maturation of hepatic pathways in iHeps.³⁵ CEBPB⁹and nuclear receptor subfamily 1 group I member 2 (NR1I2)²⁸ are also potent hepatic maturation factors. NR1I2 regulates a plethora of genes involved in phase I, phase II and phase III detoxification reactions. As the metabolism of xenobiotics largely defines a functional hepatocyte, the incorporation of hepatic maturation factors in direct hepatic reprogram- ming cocktails is of great interest.

Towards expandable hepatoblasts with hepatic pro- genitor factors

Yu and colleagues were thefirst to incorporate a LETF of early hepatogenesis, namely HNF1B, in their reprogramming cocktail. Since HNF1B is responsible for normal hepatic bud formation and gut regionalisation during early hepatic develop- ment,⁴⁰it will induce and maintain expression of early hepatic TFs and immature hepatic markers.⁴¹ As such, they managed to reprogrammefibroblasts into bipotential hepatic stem cells with the ability to proliferate. Another study based a direct hepatic reprogramming cocktail on the concept of tissue generation in lower animals.⁴² This gene expres- sion plasticity inspired Xie et al. to generate expandable induced hepatic progenitor cells that could then be coaxed into abundant competent human induced hepatocytes (hiHeps) by hepatic differentiation. They included HHEX in the direct hepatic reprogramming cocktail, which is highly expressed in human foetal liver cells, to simulate the earlier stages of hepatogenesis. Global gene expression analysis showed that the human induced hepatic progenitor cells they obtained share a similar gene expression pattern with hu- man foetal liver cells. Furthermore, genes enriched in human hepatic progenitors are greatly upregu- lated in the human-induced hepatic progenitors.

After propagation of up to 30 passages, maturation was obtained by culturing the human-induced hepatic progenitors in a hepatocyte medium that has shown good results for the cultivation of PHHs.

As such, the obtained hiHeps showed upregulation of crucial and mature hepatic markers, reaching similar levels as in freshly isolated PHHs and adult liver tissue.³⁶

Key point

Inclusion of maturation factors in the direct hepatic reprogramming cocktail enhances the hepatic func- tionality of iHeps. These factors are defined as LETFs that differ between foetal and adult liver cells.

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Small molecules to facilitate direct hepatic conversion

Small molecules used to improve hiPSC-HLC gen- eration could be applied to increase the efficiency of direct hepatic conversion and possibly, in the future, to generate chemically iHeps without the need for gene transfer. Hou and colleagues showed that iPSCs can be generated from mouse somatic cells by using a combination of only 7 small mol- ecules: CRFVPTD (CHIR99021, RepSox, Forskolin, Valproic acid, Parnate, TTNPB, DZNep).⁴³ This breakthrough triggered Guoet al.to translate this process to direct hepatic reprogramming. They report the direct conversion of mouse embryonic fibroblasts into iHeps by a chemical cocktail (CRVPTD) in combination with a single hepatocyte- specific pioneer factor (FOXA1, FOXA2 or FOXA3).³¹ The same research group managed to repeat the same process on a human cell level by over- expressing FOXA3, HNF1A or HNF4A.³⁴ The addi- tion of CHIR99021 potentiates self-renewal of pluripotent stem cells and rapid proliferation of somatic cells by inhibition of glycogen synthase kinase (GSK) 3, which mediates activation of the Wnt/b-catenin pathway. Yet, it has also been shown that the small molecule facilitates somatic reprogramming, and thus the transition of mesenchyme to epithelia.^13,44Parnate (lysine-spe- cific demethylase 1 inhibitor),⁴⁴ DZNep (histone methylation inhibitor)⁴⁵and valproic acid (histone deacetylase inhibitor)⁴⁴ are epigenetic modifiers that modulate the chromatin structure by creating an open, transcriptionally active euchromatin configuration at gene coding and regulatory re- gions. As such, they facilitate gene transcription towards the hepatic lineage. RepSox is an activin receptor-like kinase (ALK) inhibitor that – by inhibiting the canonical activin/nodal/transforming growth factor (TGF)-b pathway – inhibits TGF-b- induced epithelial-to-mesenchymal transition (EMT) and enhances MET.⁴⁶By activating the reti- noic acid receptor with the small molecule TTNPB, hepatic nuclear receptor-mediated pathways are modulated, as such promoting hepatic lineage conversion.⁴⁷Limet al. showed that 2 small mol- ecules (A-83-01 & CHIR99021) could replace the pluripotent reprogramming TFs KLF4 and c-MYC in the direct hepatic reprogramming cocktail.¹³ Wu et al. also enriched their direct hepatic reprog- ramming cocktail with A-83-01 and CHIR99021⁴⁸ and Zakikhan et al. with A-83-01 alone.¹⁹ As A- 83-01 is an ALK 4/5/7 inhibitor, it induces MET and promotes direct hepatic conversion.⁴⁶

From a tissue regeneration point of view, Xie et al. aspired to generate proliferating human induced hepatic progenitors that become func- tionally competent hiHeps after an additional maturation step. In afirst hepatic conversion step, they used CHIR99021, lysophosphatidic acid, SB431542, and sphingosine-1-phosphate to generate induced hepatic progenitors; in thefinal

maturation step the medium is supplemented with forskolin and SB431542.³⁶ Lysophosphatidic acid and sphingosine-1-phosphate are known inducers of proliferation and play a key role in hepatic tissue regeneration.⁴⁹Similar to A-83-01, SB431542 sup- presses the canonical activin/nodal/TGF-bpathway, which enhances MET, and is both beneficial for hepatic progenitor generation and for hepatocyte maturation.⁵⁰ To potentiate the directed hepatic differentiation process, a cAMP-dependent protein kinase A (PKA) activator, like the small molecule forskolin, can be added to the culture medium.

Importantly, Boonet al.showed that high levels of extracellular amino acids induce a metabolic- competent state in hiPSCs-HLCs and HepG2 cells, even reaching similar levels to those in PHHs.⁵¹ Supplementing the rich direct hepatic reprogram- ming medium with a supraphysiological concen- tration of amino acids might thus push hepatic transdifferentiation towards an unprecedented level of hepatic maturity (Fig. 1).

Minimal criteria for iHep characterisation and reporting

Phenotypic features of iHeps can be definedin vitro based on 4 levels: i) morphology, ii) gene expres- sion, iii) protein expression and iv) hepatic func- tionality and should always be compared to PHHs as the golden standard. In addition, iHep func- tionality can be evaluated in an in vivo setting, when therapeutic evaluation is envisaged.

Currently, it is extremely difficult to unambigu- ously compare iHep study outcomes, because no reporting standards have been defined. It is clear that in many studies thein vitrocharacterisation of iHeps is limited to gene and protein expression (Table 1). Often, the expression of hepatic markers is only compared to the original cell source (e.g.

fibroblasts) and not PHHs. Therefore, we strive for a consensus that scientific papers about iHeps should at least report on a predefined set of hepatic markers and compare their expression to PHHs.

Importantly, Nakamori et al. showed that hiHeps still retain expression offibroblast markers, while hepatic markers are strongly upregulated.²⁷ It is therefore of equal relevance to report on the downregulation of markers of the somatic cell source.7,13,27,28,35,36,52–54Better-characterised iHeps will help us uncover important shortcomings that can be acted upon to generate fully functional and metabolically competent iHeps in the future. Ana- lysing the transcriptome and/or proteome of single cells can aid with this, as it enables the reliable identification of different constituent cell types.

This will give us a better understanding of the direct hepatic reprogramming efficiency and the presence of non-hepatic or inefficiently reprog- rammed cell types. Transcriptomic and proteomic data, generated during the phenotypic characteri- sation of iHeps, should be deposited in publicly Key point

Expandable induced he- patic progenitor cells can be obtained by including hepatic progenitor factors in the direct hepatic reprogramming cocktail.

These factors are highly expressed in human foetal liver cells and mimic the earlier stages of hepatogenesis.

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accessible repositories, to significantly improve reporting transparency and to allow for the phenotypic comparison of different iHeps, PHHs and other hepatocyte-like cells. Ultimately, the hepatic functionality of iHeps should be reported and compared to PHHs. Defining the phase I and II biotransformation capacity and other functional hepatic traits (e.g. glycogen storage, LDL uptake, albumin secretion, urea production) should be standard criteria for evaluating iHeps. We propose that all studies should report absolute hepatic functionality data and not only relative fold changes to the non-hepatic cell of origin (e.g.

fibroblast). This would allow us to unambiguously compare the functionality of the generated iHeps to other hepatocyte-like cells and PHHs. In case a therapeutic evaluation is envisaged, the function- ality of the obtained iHeps should be tested in a relevantin vivoanimal model, with– at least–a report on their ability to engraft and attenuate liver damage parameters including alanine amino- transferase (ALT), aspartate aminotransferase (AST), and bilirubin when compared to PHHs.

Altogether, we propose a set of minimal criteria to characterise iHeps for both laboratory-based sci- entific investigations and for (pre-)clinical studies (Box 1). The aim of this proposition is solely to provide unambiguous characterisation criteria of iHeps at 5 different levels, based on the currently available knowledge.

Recommendations for generation of iHeps in view of potential clinical applications Optimising viral-induced direct hepatic reprogramming

Most of the current direct hepatic reprogramming protocols overexpress several LETFs, yet they are expressed by individual viral vectors carrying a single transgene. Hence, a heterogenous cell pop- ulation is obtained with inconsistent transductions of LETFs, subsequently resulting in variable hepatic conversion efficiencies. By combining several

transgenes into 1 construct, a multicistronic vector system is obtained (Fig. 1). The traditional way to co-express multiple genes in a single mRNA is by including an internal ribosome entry site (IRES) sequence or a 2A oligopeptide sequence in be- tween open reading frames. Yet, over the years, the 2A co-expression system has shown to be more reliable for stoichiometric expression,⁵⁵ since expression of the transgene before the IRES is generally significantly higher than that of the sec- ond transgene.⁵⁶ Furthermore, 2A sequences only comprise 60–80 bp, which enables a multicistronic sequence of several transgenes, whereas the number of transgenes is limited when using IRES (588 bp).⁵² By constructing an all-in-one poly- cistronic hepatic reprogramming cassette,^52,57–59 cells reprogramme synchronously and a homoge- nous cell population is obtained as viral trans- duction results in co-expression of the direct hepatic reprogramming cocktail. Also, implement- ing a gene-inducible system^23,59allows for prolif- eration prior to initiating direct conversion, making it possible to generate a cell line and subsequently facilitating high-throughput screening and gener- ation of good manufacturing practice-grade iHeps.

Besides this, these systems make it possible to temporarily and quantitatively activate or suppress transgenes, as well as exhibiting a higher efficiency and less side effects than the traditional over- expression of transgenes.⁶⁰To minimise the risks linked to viral gene transfer, so-called “safe harbour”loci may be used in the future in order to direct cell fate conversions (Fig. 1). These safe harbour sites can be targeted using genome editing technology, resulting in stable transgene expres- sion with minimal adverse effects on global or local gene expression.⁶¹ Yet, several articles warn of variable or inhibited transgene expression and therefore safe harbour loci should be carefully assessed.^62,63 Another way to circumvent the problem of transgene-integrated cytotoxicity is by using non-integrating viral vectors. Adenoviral Direct hepatic reprogramming

Small molecules

Small molecules

mRNA transfection mRNA transfection

Integrating viral vector Polycistronic

viral vector Non-integrating

viral vector

Safe harbour locus

Non-integrating viral vector Non-integrating

viral vector

Safe harbour locus

Polycistronic viral vector Integrating

viral vector Fibroblasts

iHeps Clinical applicability

Conversion efficiency

Fig. 1. Fundamental issue of directly converting somatic cells to iHeps.Conversion methods that result in a good conversion efficiency are difficult to translate to the clinic and transdifferentiation methods that are compatible with clinical applications result in a low conversion efficiency. iHeps, induced hepatocytes.

Key point To date, unambiguous comparison among study outcomes is difficult, because the characterisa- tion of iHeps has not been standardised. A set of min- imal criteria to report on iHeps for both laboratory- based scientific investiga- tions and for (pre-)clinical studies adds transparency to this researchfield and will help move human iHeps from bench-to- bedside.

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