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Genetic Modification of Human Pancreatic Progenitor Cells Through Modified mRNA

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Synthetic mRNA

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1428))

Abstract

In this chapter, we describe a highly efficient genetic modification strategy for human pancreatic progenitor cells using modified mRNA-encoding GFP and Neurogenin-3. The properties of modified mRNA offer an invaluable platform to drive protein expression, which has broad applicability in pathway regulation, directed differentiation, and lineage specification. This approach can also be used to regulate expression of other pivotal transcription factors during pancreas development and might have potential therapeutic values in regenerative medicine.

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References

  1. Warren L, Manos PD, Ahfeldt T et al (2010) Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA. Cell Stem Cell 7(5):618–630. doi:10.1016/j.stem.2010.08.012

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Elcheva I, Brok-Volchanskaya V, Kumar A et al (2014) Direct induction of haematoendothelial programs in human pluripotent stem cells by transcriptional regulators. Nat Commun 5:4372. doi:10.1038/ncomms5372

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Hansson ML, Albert S, Gonzalez Somermeyer L et al (2015) Efficient delivery and functional expression of transfected modified mRNA in human embryonic stem cell-derived retinal pigmented epithelial cells. J Biol Chem 290(9):5661–5672. doi:10.1074/jbc.M114.618835

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Lui KO, Zangi L, Silva EA et al (2013) Driving vascular endothelial cell fate of human multipotent Isl1+ heart progenitors with VEGF modified mRNA. Cell Res 23(10):1172–1186. doi:10.1038/cr.2013.112

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Creusot RJ, Chang P, Healey DG et al (2010) A short pulse of IL-4 delivered by DCs electroporated with modified mRNA can both prevent and treat autoimmune diabetes in NOD mice. Mol Ther 18(12):2112–2120. doi:10.1038/mt.2010.146

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Kormann MS, Hasenpusch G, Aneja MK et al (2011) Expression of therapeutic proteins after delivery of chemically modified mRNA in mice. Nat Biotechnol 29(2):154–157. doi:10.1038/nbt.1733

    Article  CAS  PubMed  Google Scholar 

  7. Zangi L, Lui KO, von Gise A et al (2013) Modified mRNA directs the fate of heart progenitor cells and induces vascular regeneration after myocardial infarction. Nat Biotechnol 31(10):898–907. doi:10.1038/nbt.2682

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Barton FB, Rickels MR, Alejandro R et al (2012) Improvement in outcomes of clinical islet transplantation: 1999-2010. Diabetes Care 35(7):1436–1445. doi:10.2337/dc12-0063

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Pagliuca FW, Millman JR, Gurtler M et al (2014) Generation of functional human pancreatic beta cells in vitro. Cell 159(2):428–439. doi:10.1016/j.cell.2014.09.040

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Leung KK, Liang J, Ma MT et al (2012) Angiotensin II type 2 receptor is critical for the development of human fetal pancreatic progenitor cells into islet-like cell clusters and their potential for transplantation. Stem Cells 30(3):525–536. doi:10.1002/stem.1008

    Article  CAS  PubMed  Google Scholar 

  11. Leung KK, Suen PM, Lau TK et al (2009) PDZ-domain containing-2 (PDZD2) drives the maturity of human fetal pancreatic progenitor-derived islet-like cell clusters with functional responsiveness against membrane depolarization. Stem Cells Dev 18(7):979–990. doi:10.1089/scd.2008.0325

    Article  CAS  PubMed  Google Scholar 

  12. Jennings RE, Berry AA, Kirkwood-Wilson R et al (2013) Development of the human pancreas from foregut to endocrine commitment. Diabetes 62(10):3514–3522. doi:10.2337/db12-1479

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Pan FC, Brissova M (2014) Pancreas development in humans. Curr Opin Endocrinol Diabetes Obes 21(2):77–82. doi:10.1097/MED.0000000000000047

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Benitez CM, Goodyer WR, Kim SK (2012) Deconstructing pancreas developmental biology. Cold Spring Harbor Perspect Biol 4 (6). doi: 10.1101/cshperspect.a012401

    Google Scholar 

  15. Grapin-Botton A, Majithia AR, Melton DA (2001) Key events of pancreas formation are triggered in gut endoderm by ectopic expression of pancreatic regulatory genes. Genes Dev 15(4):444–454. doi:10.1101/gad.846001

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Gu G, Brown JR, Melton DA (2003) Direct lineage tracing reveals the ontogeny of pancreatic cell fates during mouse embryogenesis. Mech Dev 120(1):35–43

    Article  CAS  PubMed  Google Scholar 

  17. Swales N, Martens GA, Bonne S et al (2012) Plasticity of adult human pancreatic duct cells by neurogenin3-mediated reprogramming. PLoS One 7(5):e37055. doi:10.1371/journal.pone.0037055

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Van de Casteele M, Leuckx G, Baeyens L et al (2013) Neurogenin 3+ cells contribute to beta-cell neogenesis and proliferation in injured adult mouse pancreas. Cell Death Dis 4:e523. doi:10.1038/cddis.2013.52

    Article  PubMed  PubMed Central  Google Scholar 

  19. Yang K, Wang Y, Du Z et al (2014) Short-reactivation of neurogenin-3 and mesenchymal microenvironment is require for beta-cells differentiation during fetal pancreas development and islet regeneration. Rom J Morphol Embryol 55(2):305–311

    PubMed  Google Scholar 

  20. Chien KR, Zangi L, Lui KO (2015) Synthetic chemically modified mRNA (modRNA): toward a new technology platform for cardiovascular biology and medicine. Cold Spring Harbor Perspect Med 5(1):a014035. doi:10.1101/cshperspect.a014035

    Article  Google Scholar 

  21. Lui KO, Zangi L, Chien KR (2014) Cardiovascular regenerative therapeutics via synthetic paracrine factor modified mRNA. Stem Cell Res 13(3 Pt B):693–704. doi:10.1016/j.scr.2014.06.007

    Article  CAS  PubMed  Google Scholar 

  22. Suen PM, Zou C, Zhang YA et al (2008) PDZ-domain containing-2 (PDZD2) is a novel factor that affects the growth and differentiation of human fetal pancreatic progenitor cells. Int J Biochem Cell Biol 40(4):789–803. doi:10.1016/j.biocel.2007.10.020

    Article  CAS  PubMed  Google Scholar 

  23. Lui KO (2014) VEGF-A: the inductive angiogenic factor for development, regeneration and function of pancreatic beta cells. Curr Stem Cell Res Ther 9(5):396–400

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

We thank Dr. Ke Xu for his advice on molecular cloning. We also thank Dr. Kathy Juan Liang for sharing her experience in hPPC cultures. The authors declare no conflict of interest. This work was supported by Research Grants Council-Early Career Scheme (24110515 to K.O.L.), Health and Medical Research Fund (03140346 to K.O.L.) the Croucher Foundation (K.O.L.), CUHK Strategic Recruitment Grant (K.O.L.), and CUHK Faculty of Medicine Postdoctoral Fellowship (S.L.).

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Authors and Affiliations

  1. Department of Chemical Pathology, The Chinese University of Hong Kong, Princes of Wales Hospital, Shatin, Hong Kong, SAR, China

    Song Lu & Kathy O. Lui

  2. Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong, Princes of Wales Hospital, Shatin, Hong Kong, SAR, China

    Song Lu, Christie C. Chow & Kathy O. Lui

  3. School of Biomedical Sciences, The Chinese University of Hong Kong, Shatin, Hong Kong, SAR, China

    Junwei Zhou, Po Sing Leung & Stephen K. Tsui

Authors
  1. Song Lu
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  2. Christie C. Chow
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  3. Junwei Zhou
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  4. Po Sing Leung
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  5. Stephen K. Tsui
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  6. Kathy O. Lui
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Corresponding author

Correspondence to Kathy O. Lui .

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Editors and Affiliations

  1. Dept of Biochemistry&Molecular Bio, Louisiana State Univ Health Scien center, Shreveport, Louisiana, USA

    Robert E. Rhoads

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Lu, S., Chow, C.C., Zhou, J., Leung, P.S., Tsui, S.K., Lui, K.O. (2016). Genetic Modification of Human Pancreatic Progenitor Cells Through Modified mRNA. In: Rhoads, R. (eds) Synthetic mRNA. Methods in Molecular Biology, vol 1428. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3625-0_21

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  • DOI: https://doi.org/10.1007/978-1-4939-3625-0_21

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-3623-6

  • Online ISBN: 978-1-4939-3625-0

  • eBook Packages: Springer Protocols

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