TY - JOUR
T1 - Gene Regulatory Network Analysis and Engineering Directs Development and Vascularization of Multilineage Human Liver Organoids
AU - Velazquez, Jeremy J.
AU - LeGraw, Ryan
AU - Moghadam, Farzaneh
AU - Tan, Yuqi
AU - Kilbourne, Jacquelyn
AU - Maggiore, Joseph C.
AU - Hislop, Joshua
AU - Liu, Silvia
AU - Cats, Davy
AU - Chuva de Sousa Lopes, Susana M.
AU - Plaisier, Christopher
AU - Cahan, Patrick
AU - Kiani, Samira
AU - Ebrahimkhani, Mo R.
N1 - Funding Information:
We thank all members of Ebrahimkhani and Kiani laboratories for discussions and assistance in this study. We thank the Mayo Clinic Histology Core Laboratory for assistance with histology as well as the ASU Department of Animal Care and Technologies for mouse husbandry. We also thank the UCLA Technology Center for Genomics and Bioinformatics, Novogene, and Genewiz for RNA sequencing. This work was supported by an R01 from the National Institute of Biomedical Imaging and Bioengineering (EB028532), an R01 from the National Heart Lung and Blood Institute (HL141805), and the New Investigator Award from Arizona Biomedical Research Council (ADHS16-162402) to M.R.E. as well as support from the Pittsburgh Liver Research Center (NIH-NIDDK P30DK120531). The research reported in this publication was also partly supported by an R01 from the National Institute of Biomedical Imaging and Bioengineering (EB024562) to S.K. It was also partly supported by the National Institute of General Medical Sciences Award Number T32GM008208. P.C. was supported by R35GM124725.The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Conceptualization, M.R.E. J.J.V. and R.L.; Methodology, M.R.E. J.J.V. R.L. S.K. F.M. P.C. Y.T. C.P. and J.K.; Software, P.C. Y.T. S.M.C.d.S.L, D.C. S.L, J.C.M. and J.J.V.; Formal Analysis, J.J.V. R.L. P.C. Y.T. S.M.C.d.S.L, D.C. J.C.M. S.L. and M.R.E.; Investigation: J.J.V, R.L. F.M. and J.K.; Resources, M.R.E. S.K. P.C. and S.M.C.d.S.L.; Data Curation, J.V. S.L. P.C. and Y.T.; Writing – Original Draft, M.R.E. R.L. J.J.V. and S.K.; Writing – Review & Editing, J.H. F.M. P.C. Y.T. D.C. S.M.C.d.S.L. S.L. and C.P.; Supervision, M.R.E. S.K. and P.C.; Funding Acquisition, M.R.E. S.K. and P.C. M.R.E, S.K. P.C. J.J.V. and R.L. have submitted a patent (WO2019237124) for the work included in this publication.
Funding Information:
We thank all members of Ebrahimkhani and Kiani laboratories for discussions and assistance in this study. We thank the Mayo Clinic Histology Core Laboratory for assistance with histology as well as the ASU Department of Animal Care and Technologies for mouse husbandry. We also thank the UCLA Technology Center for Genomics and Bioinformatics, Novogene, and Genewiz for RNA sequencing. This work was supported by an R01 from the National Institute of Biomedical Imaging and Bioengineering ( EB028532 ), an R01 from the National Heart Lung and Blood Institute ( HL141805 ), and the New Investigator Award from Arizona Biomedical Research Council ( ADHS16-162402 ) to M.R.E. as well as support from the Pittsburgh Liver Research Center ( NIH-NIDDK P30DK120531 ). The research reported in this publication was also partly supported by an R01 from the National Institute of Biomedical Imaging and Bioengineering ( EB024562 ) to S.K. It was also partly supported by the National Institute of General Medical Sciences Award Number T32GM008208 . P.C. was supported by R35GM124725 .The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Publisher Copyright:
© 2020 The Authors
PY - 2021/1/20
Y1 - 2021/1/20
N2 - Pluripotent stem cell (PSC)-derived organoids have emerged as novel multicellular models of human tissue development but display immature phenotypes, aberrant tissue fates, and a limited subset of cells. Here, we demonstrate that integrated analysis and engineering of gene regulatory networks (GRNs) in PSC-derived multilineage human liver organoids direct maturation and vascular morphogenesis in vitro. Overexpression of PROX1 and ATF5, combined with targeted CRISPR-based transcriptional activation of endogenous CYP3A4, reprograms tissue GRNs and improves native liver functions, such as FXR signaling, CYP3A4 enzymatic activity, and stromal cell reactivity. The engineered tissues possess superior liver identity when compared with other PSC-derived liver organoids and show the presence of hepatocyte, biliary, endothelial, and stellate-like cell populations in single-cell RNA-seq analysis. Finally, they show hepatic functions when studied in vivo. Collectively, our approach provides an experimental framework to direct organogenesis in vitro by systematically probing molecular pathways and transcriptional networks that promote tissue development. Informed by computational analyses, Velazquez et al, exercised overexpression of ATF5, PROX1 transcription factors, and CRISPR-mediated activation of CYP3A4 to advance the maturity and vascularity of human iPSC-derived fetal liver organoids in vitro. The findings highlight the importance of integrative systems and synthetic biology to engineer multicellular systems.
AB - Pluripotent stem cell (PSC)-derived organoids have emerged as novel multicellular models of human tissue development but display immature phenotypes, aberrant tissue fates, and a limited subset of cells. Here, we demonstrate that integrated analysis and engineering of gene regulatory networks (GRNs) in PSC-derived multilineage human liver organoids direct maturation and vascular morphogenesis in vitro. Overexpression of PROX1 and ATF5, combined with targeted CRISPR-based transcriptional activation of endogenous CYP3A4, reprograms tissue GRNs and improves native liver functions, such as FXR signaling, CYP3A4 enzymatic activity, and stromal cell reactivity. The engineered tissues possess superior liver identity when compared with other PSC-derived liver organoids and show the presence of hepatocyte, biliary, endothelial, and stellate-like cell populations in single-cell RNA-seq analysis. Finally, they show hepatic functions when studied in vivo. Collectively, our approach provides an experimental framework to direct organogenesis in vitro by systematically probing molecular pathways and transcriptional networks that promote tissue development. Informed by computational analyses, Velazquez et al, exercised overexpression of ATF5, PROX1 transcription factors, and CRISPR-mediated activation of CYP3A4 to advance the maturity and vascularity of human iPSC-derived fetal liver organoids in vitro. The findings highlight the importance of integrative systems and synthetic biology to engineer multicellular systems.
KW - CRISPR
KW - Cas9
KW - gene circuit
KW - gene regulatory network
KW - genetic engineering
KW - induced pluripotent stem cells
KW - liver
KW - maturation
KW - organoids
KW - synthetic biology
UR - http://www.scopus.com/inward/record.url?scp=85098492977&partnerID=8YFLogxK
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U2 - 10.1016/j.cels.2020.11.002
DO - 10.1016/j.cels.2020.11.002
M3 - Article
C2 - 33290741
AN - SCOPUS:85098492977
SN - 2405-4712
VL - 12
SP - 41-55.e11
JO - Cell Systems
JF - Cell Systems
IS - 1
ER -