Expansion, in vivo-ex vivo cycling, and genetic manipulation of primary human hepatocytes

Eleftherios Michailidis; Koen Vercauteren; Liliana Mancio-Silva; Linda Andrus; Cyprien Jahan; Inna Ricardo-Lax; Chenhui Zou; Mohammad Kabbani; Paul Park; Corrine Quirk; Christina Pyrgaki; Brandon Razooky; Lieven Verhoye; Irene Zoluthkin; Wei-Yu Lu; Stuart J Forbes; Luis Chiriboga; Neil D Theise; Roland W Herzog; Hiroshi Suemizu; William M Schneider; Amir Shlomai; Philip Meuleman; Sangeeta N Bhatia; Charles M Rice; Ype P de Jong

doi:10.1073/pnas.1919035117

Expansion, in vivo-ex vivo cycling, and genetic manipulation of primary human hepatocytes

Eleftherios Michailidis, Koen Vercauteren, Liliana Mancio-Silva, Linda Andrus, Cyprien Jahan, Inna Ricardo-Lax, Chenhui Zou, Mohammad Kabbani, Paul Park, Corrine Quirk, Christina Pyrgaki, Brandon Razooky, Lieven Verhoye, Irene Zoluthkin, Wei-Yu Lu, Stuart J Forbes, Luis Chiriboga, Neil D Theise, Roland W Herzog, Hiroshi SuemizuWilliam M Schneider, Amir Shlomai, Philip Meuleman, Sangeeta N Bhatia, Charles M Rice, Ype P de Jong

Clinical Virology

Research output: Contribution to journal › A1: Web of Science-article › peer-review

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Abstract

Primary human hepatocytes (PHHs) are an essential tool for modeling drug metabolism and liver disease. However, variable plating efficiencies, short lifespan in culture, and resistance to genetic manipulation have limited their use. Here, we show that the pyrrolizidine alkaloid retrorsine improves PHH repopulation of chimeric mice on average 10-fold and rescues the ability of even poorly plateable donor hepatocytes to provide cells for subsequent ex vivo cultures. These mouse-passaged (mp) PHH cultures overcome the marked donor-to-donor variability of cryopreserved PHH and remain functional for months as demonstrated by metabolic assays and infection with hepatitis B virus and Plasmodium falciparum mpPHH can be efficiently genetically modified in culture, mobilized, and then recultured as spheroids or retransplanted to create highly humanized mice that carry a genetically altered hepatocyte graft. Together, these advances provide flexible tools for the study of human liver disease and evaluation of hepatocyte-targeted gene therapy approaches.

Original language	English
Journal	Proceedings of the National Academy of Sciences of the United States of America
Volume	117
Issue number	3
Pages (from-to)	1678-1688
Number of pages	11
ISSN	0027-8424
DOIs	https://doi.org/10.1073/pnas.1919035117
Publication status	Published - 2020

Keywords

Animals
Cell Transplantation
Chimera
Disease Models, Animal
Female
Genetic Therapy
Hepatitis B
Hepatitis B virus
Hepatocytes/drug effects
Homeodomain Proteins/genetics
Humans
Hydrolases/genetics
Interleukin Receptor Common gamma Subunit/genetics
Liver/pathology
Liver Diseases/genetics
Malaria
Male
Mice
Mice, Inbred NOD
Mice, Knockout
Plasmodium falciparum
Pyrrolizidine Alkaloids/pharmacology

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10.1073/pnas.1919035117Licence: CC BY-NC-ND

2020pnas1678Final published version, 1.44 MBLicence: CC BY-NC-ND

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Michailidis, E., Vercauteren, K., Mancio-Silva, L., Andrus, L., Jahan, C., Ricardo-Lax, I., Zou, C., Kabbani, M., Park, P., Quirk, C., Pyrgaki, C., Razooky, B., Verhoye, L., Zoluthkin, I., Lu, W.-Y., Forbes, S. J., Chiriboga, L., Theise, N. D., Herzog, R. W., ... de Jong, Y. P. (2020). Expansion, in vivo-ex vivo cycling, and genetic manipulation of primary human hepatocytes. Proceedings of the National Academy of Sciences of the United States of America, 117(3), 1678-1688. https://doi.org/10.1073/pnas.1919035117

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abstract = "Primary human hepatocytes (PHHs) are an essential tool for modeling drug metabolism and liver disease. However, variable plating efficiencies, short lifespan in culture, and resistance to genetic manipulation have limited their use. Here, we show that the pyrrolizidine alkaloid retrorsine improves PHH repopulation of chimeric mice on average 10-fold and rescues the ability of even poorly plateable donor hepatocytes to provide cells for subsequent ex vivo cultures. These mouse-passaged (mp) PHH cultures overcome the marked donor-to-donor variability of cryopreserved PHH and remain functional for months as demonstrated by metabolic assays and infection with hepatitis B virus and Plasmodium falciparum mpPHH can be efficiently genetically modified in culture, mobilized, and then recultured as spheroids or retransplanted to create highly humanized mice that carry a genetically altered hepatocyte graft. Together, these advances provide flexible tools for the study of human liver disease and evaluation of hepatocyte-targeted gene therapy approaches.",

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Michailidis, E, Vercauteren, K, Mancio-Silva, L, Andrus, L, Jahan, C, Ricardo-Lax, I, Zou, C, Kabbani, M, Park, P, Quirk, C, Pyrgaki, C, Razooky, B, Verhoye, L, Zoluthkin, I, Lu, W-Y, Forbes, SJ, Chiriboga, L, Theise, ND, Herzog, RW, Suemizu, H, Schneider, WM, Shlomai, A, Meuleman, P, Bhatia, SN, Rice, CM & de Jong, YP 2020, 'Expansion, in vivo-ex vivo cycling, and genetic manipulation of primary human hepatocytes', Proceedings of the National Academy of Sciences of the United States of America, vol. 117, no. 3, pp. 1678-1688. https://doi.org/10.1073/pnas.1919035117

Expansion, in vivo-ex vivo cycling, and genetic manipulation of primary human hepatocytes. / Michailidis, Eleftherios; Vercauteren, Koen; Mancio-Silva, Liliana et al.
In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 117, No. 3, 2020, p. 1678-1688.

Research output: Contribution to journal › A1: Web of Science-article › peer-review

TY - JOUR

T1 - Expansion, in vivo-ex vivo cycling, and genetic manipulation of primary human hepatocytes

AU - Michailidis, Eleftherios

AU - Vercauteren, Koen

AU - Mancio-Silva, Liliana

AU - Andrus, Linda

AU - Jahan, Cyprien

AU - Ricardo-Lax, Inna

AU - Zou, Chenhui

AU - Kabbani, Mohammad

AU - Park, Paul

AU - Quirk, Corrine

AU - Pyrgaki, Christina

AU - Razooky, Brandon

AU - Verhoye, Lieven

AU - Zoluthkin, Irene

AU - Lu, Wei-Yu

AU - Forbes, Stuart J

AU - Chiriboga, Luis

AU - Theise, Neil D

AU - Herzog, Roland W

AU - Suemizu, Hiroshi

AU - Schneider, William M

AU - Shlomai, Amir

AU - Meuleman, Philip

AU - Bhatia, Sangeeta N

AU - Rice, Charles M

AU - de Jong, Ype P

N1 - FTX; (CC BY-NC-ND 4.0)

PY - 2020

Y1 - 2020

N2 - Primary human hepatocytes (PHHs) are an essential tool for modeling drug metabolism and liver disease. However, variable plating efficiencies, short lifespan in culture, and resistance to genetic manipulation have limited their use. Here, we show that the pyrrolizidine alkaloid retrorsine improves PHH repopulation of chimeric mice on average 10-fold and rescues the ability of even poorly plateable donor hepatocytes to provide cells for subsequent ex vivo cultures. These mouse-passaged (mp) PHH cultures overcome the marked donor-to-donor variability of cryopreserved PHH and remain functional for months as demonstrated by metabolic assays and infection with hepatitis B virus and Plasmodium falciparum mpPHH can be efficiently genetically modified in culture, mobilized, and then recultured as spheroids or retransplanted to create highly humanized mice that carry a genetically altered hepatocyte graft. Together, these advances provide flexible tools for the study of human liver disease and evaluation of hepatocyte-targeted gene therapy approaches.

AB - Primary human hepatocytes (PHHs) are an essential tool for modeling drug metabolism and liver disease. However, variable plating efficiencies, short lifespan in culture, and resistance to genetic manipulation have limited their use. Here, we show that the pyrrolizidine alkaloid retrorsine improves PHH repopulation of chimeric mice on average 10-fold and rescues the ability of even poorly plateable donor hepatocytes to provide cells for subsequent ex vivo cultures. These mouse-passaged (mp) PHH cultures overcome the marked donor-to-donor variability of cryopreserved PHH and remain functional for months as demonstrated by metabolic assays and infection with hepatitis B virus and Plasmodium falciparum mpPHH can be efficiently genetically modified in culture, mobilized, and then recultured as spheroids or retransplanted to create highly humanized mice that carry a genetically altered hepatocyte graft. Together, these advances provide flexible tools for the study of human liver disease and evaluation of hepatocyte-targeted gene therapy approaches.

KW - Animals

KW - Cell Transplantation

KW - Chimera

KW - Disease Models, Animal

KW - Female

KW - Genetic Therapy

KW - Hepatitis B

KW - Hepatitis B virus

KW - Hepatocytes/drug effects

KW - Homeodomain Proteins/genetics

KW - Humans

KW - Hydrolases/genetics

KW - Interleukin Receptor Common gamma Subunit/genetics

KW - Liver/pathology

KW - Liver Diseases/genetics

KW - Malaria

KW - Male

KW - Mice

KW - Mice, Inbred NOD

KW - Mice, Knockout

KW - Plasmodium falciparum

KW - Pyrrolizidine Alkaloids/pharmacology

U2 - 10.1073/pnas.1919035117

DO - 10.1073/pnas.1919035117

M3 - A1: Web of Science-article

C2 - 31915293

SN - 0027-8424

VL - 117

SP - 1678

EP - 1688

JO - Proceedings of the National Academy of Sciences of the United States of America

JF - Proceedings of the National Academy of Sciences of the United States of America

IS - 3

ER -

Expansion, in vivo-ex vivo cycling, and genetic manipulation of primary human hepatocytes

Abstract

Keywords

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