Generation of Hepatic Progenitor Cells from the Primary Hepatocytes of Nonhuman Primates Using Small Molecules

Abstract

Background:

Since primates have more biological similarities to humans than do other animals, they are a valuable resource in various field of research, including biomedicine, regenerative medicine, and drug discovery. However, there remain limitations to maintenance and expansion of primary hepatocytes derived from nonhuman primates. To overcome these limitations, we developed a novel culture system for primate cells.

Methods:

Primary hepatocytes from Macaca fascicularis (mf-PHs) were isolated from hepatectomized liver. To generate chemically derived hepatic progenitor cells (mf-CdHs), mf-PHs were cultured with reprogramming medium containing A83-01, CHIR99021, and hepatocyte growth factor (HGF). The bi-potent differentiation capacity of mf-CdHs into hepatocytes and biliary epithelial cells was confirmed by treatment with hepatic differentiation medium (HDM) and cholangiocytic differentiation medium (CDM), respectively.

Results:

mf-PHs cultured with reprogramming medium showed rapid proliferation capacity in vitro and expressed progenitor-specific markers. Moreover, when cultured in HDM, these progenitor cells stably differentiated into hepatocyte-like cells expressing the mature hepatic markers. On the other hand, when cultured in CDM, the differentiated biliary epithelial cells expressed mature cholangiocyte characteristics.

Conclusion:

The results of the present study demonstrate that we successfully induced the formation of hepatic progenitor cells from mf-PHs by culturing them with a combination of small molecules, including growth factors. These results offer a means of expanding nonhuman primate hepatocytes without genetic manipulation for cellular resource, preclinical applications and regenerative medicine for the liver.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

References

  1. 1.

    Trefts E, Gannon M, Wasserman DH. The liver. Curr Biol. 2017;27:R1141–55.

    Article  Google Scholar 

  2. 2.

    Xiang C, Du Y, Meng G, Yi L, Sun S, Song N, et al. Long-term functional maintenance of primary human hepatocytes in vitro. Science. 2019;364:399–402.

    CAS  Article  Google Scholar 

  3. 3.

    Katsuda T, Kawamata M, Inoue A, Yamaguchi T, Abe M, Ochiya T. Long-term maintenance of functional primary human hepatocytes using small molecules. FEBS Lett. 2020;594:114–25.

    CAS  Article  Google Scholar 

  4. 4.

    Fu GB, Huang WJ, Zeng M, Zhou X, Wu HP, Liu CC, et al. Expansion and differentiation of human hepatocyte-derived liver progenitor-like cells and their use for the study of hepatotropic pathogens. Cell Res. 2019;29:8–22.

    CAS  Article  Google Scholar 

  5. 5.

    Du C, Feng Y, Qiu D, Xu Y, Pang M, Cai N, et al. Highly efficient and expedited hepatic differentiation from human pluripotent stem cells by pure small-molecule cocktails. Stem Cell Res Ther. 2018;9:58.

    CAS  Article  Google Scholar 

  6. 6.

    Woodworth CD, Kreider JW, Mengel L, Miller T, Meng YL, Isom HC. Tumorigenicity of simian virus 40-hepatocyte cell lines: effect of in vitro and in vivo passage on expression of liver-specific genes and oncogenes. Mol Cell Biol. 1988;8:4492–501.

    CAS  Article  Google Scholar 

  7. 7.

    Kim Y, Kang K, Lee SB, Seo D, Yoon S, Kim SJ, et al. Small molecule-mediated reprogramming of human hepatocytes into bipotent progenitor cells. J Hepatol. 2019;70:97–107.

    CAS  Article  Google Scholar 

  8. 8.

    Gurung S, Werkmeister JA, Gargett CE. Inhibition of transforming growth factor-β receptor signaling promotes culture expansion of undifferentiated human endometrial mesenchymal stem/stromal cells. Sci Rep. 2015;5:15042.

    CAS  Article  Google Scholar 

  9. 9.

    Laco F, Woo TL, Zhong Q, Szmyd R, Ting S, Khan FJ, et al. Unraveling the inconsistencies of cardiac differentiation efficiency induced by the GSK3b inhibitor CHIR99021 in human pluripotent stem cells. Stem Cell Reports. 2018;10:1851–66.

    CAS  Article  Google Scholar 

  10. 10.

    Nakamura T, Mizuno S. The discovery of hepatocyte growth factor (HGF) and its significance for cell biology, life sciences and clinical medicine. Proc Jpn Acad Ser B Phys Biol Sci. 2010;86:588–610.

    CAS  Article  Google Scholar 

  11. 11.

    Kim Y, Jeong J, Choi D. Small-molecule-mediated reprogramming: a silver lining for regenerative medicine. Exp Mol Med. 2020;52:213–26.

    CAS  Article  Google Scholar 

  12. 12.

    Jin L, Ji S, Tang X, Guo X, Lu Y, Chen H, et al. Isolation and characterization of liver epithelial progenitor cells from normal adult rhesus monkeys (Macaca mulatta). Cell Res. 2019;19:268–70.

    Article  Google Scholar 

  13. 13.

    Tomioka I, Maeda T, Shimada H, Kawai K, Okada Y, Igarashi H, et al. Generating induced pluripotent stem cells from common marmoset (Callithrix jacchus) fetal liver cells using defined factors, including Lin28. Genes Cells. 2010;15:959–69.

    CAS  Article  Google Scholar 

  14. 14.

    Vondran FW, Katenz E, Schwartlander R, Morgul MH, Raschzok N, Gong X, et al. Isolation of primary human hepatocytes after partial hepatectomy: criteria for identification of the most promising liver specimen. Artif Organs. 2008;32:205–13.

    Article  Google Scholar 

  15. 15.

    Asumda FZ, Hatzistergos KE, Dykxhoorn DM, Jakubski S, Edwards J, Thomas E, et al. Differentiation of hepatocyte-like cells from human pluripotent stem cells using small molecules. Differentiation. 2018;101:16–24.

    CAS  Article  Google Scholar 

  16. 16.

    Siller R, Greenhough S, Naumovska E, Sullivan GJ. Small-molecule-driven hepatocyte differentiation of human pluripotent stem cells. Stem Cell Reports. 2015;4:939–52.

    CAS  Article  Google Scholar 

  17. 17.

    Liang G, Zhang Y. Genetic and epigenetic variations in iPSCs: potential causes and implications for application. Cell Stem Cell. 2013;l3:149–59.

    Article  Google Scholar 

  18. 18.

    Bain J, Plater L, Elliott M, Shpiro N, Hastie CJ, Mclauchlan H, et al. The selectivity of protein kinase inhibitors: a further update. Biochem J. 2007;408:297–315.

    CAS  Article  Google Scholar 

  19. 19.

    Li X, Zuo X, Jing J, Ma Y, Wang J, Liu D, et al. Small-molecule-driven direct reprogramming of mouse fibroblasts into functional neurons. Cell Stem Cell. 2015;17:195–203.

    CAS  Article  Google Scholar 

  20. 20.

    Ladewig J, Mertens J, Kesavan J, Doerr J, Poppe D, Glaue F, et al. Small molecules enable highly efficient neuronal conversion of human fibroblasts. Nat Methods. 2012;9:575–8.

    CAS  Article  Google Scholar 

  21. 21.

    Kunisada Y, Tsubooka-Yamazoe N, Shoji M, Hosoya M. Small molecules induce efficient differentiation into insulin-producing cells from human induced pluripotent stem cells. Stem Cell Res. 2012;8:274–84.

    CAS  Article  Google Scholar 

  22. 22.

    Tojo M, Hamashima Y, Hanyu A, Kajimoto T, Saitoh M, Miyazono K, et al. The ALK-5 inhibitor A-83-01 inhibits Smad signaling and epithelial-to-mesenchymal transition by transforming growth factor-β. Cancer Sci. 2005;96:791–800.

    CAS  Article  Google Scholar 

  23. 23.

    Ichida JK, Blanchard J, Lam K, Son EY, Chung JE, Egli D, et al. A small-molecule inhibitor of tgf-Beta signaling replaces sox2 in reprogramming by inducing nanog. Cell Stem Cell. 2009;5:491–503.

    CAS  Article  Google Scholar 

  24. 24.

    Della Fazia MA, Pettirossi V, Ayroldi E, Riccardi C, Magni MV, Servillo G. Differential expression of CD44 isoforms during liver regeneration in rats. J Hepatol. 2001;34:555–61.

    CAS  Article  Google Scholar 

  25. 25.

    Soncin F, Ward CM. The function of E-cadherin in stem cell pluripotency and self-renewal. Genes (Basel). 2011;2:229–59.

    CAS  Article  Google Scholar 

  26. 26.

    Shafritz DA, Dabeva MD. Liver stem cells and model systems for liver repopulation. J Hepatol. 2002;36:552–64.

    Article  Google Scholar 

  27. 27.

    Han X, Wang Y, Pu W, Huang X, Qiu L, Li Y, et al. Lineage tracing reveals the bipotency of SOX9+ hepatocytes during liver regeneration. Stem Cell Reports. 2019;12:624–38.

    CAS  Article  Google Scholar 

  28. 28.

    Nieto-Nicolau N, de la Torre RM, Fariñas O, Savio A, Vilarrodona A, Casaroli-Marano RP. Extrinsic modulation of integrin α6 and progenitor cell behavior in mesenchymal stem cells. Stem Cell Res. 2020;47:101899.

    CAS  Article  Google Scholar 

  29. 29.

    Gires O. EpCAM in hepatocytes and their progenitors. J Hepatol. 2012;56:490–2.

    CAS  Article  Google Scholar 

  30. 30.

    Yu Y, Liu H, Ikeda Y, Amiot BP, Rinaldo P, Duncan SA, et al. Hepatocyte-like cells differentiated from human induced pluripotent stem cells: relevance to cellular therapies. Stem Cell Res. 2012;9:196–207.

    CAS  Article  Google Scholar 

  31. 31.

    DeLaForest A, Nagaoka M, Si-Tayeb K, Noto FK, Konopka G, Battle MA, et al. HNF4A is essential for specification of hepatic progenitors from human pluripotent stem cells. Development. 2011;138:4143–53.

    CAS  Article  Google Scholar 

  32. 32.

    Stockert RJ. The asialoglycoprotein receptor: relationships between structure, function, and expression. Physiol Rev. 1995;75:591–609.

    CAS  Article  Google Scholar 

  33. 33.

    Tabibian JH, Msayuk AI, Masyuk TV, O’Hara SP, LaRusso NF. Physiology of cholangiocytes. Compr Physiol. 2013;3:541–65.

    PubMed  Google Scholar 

  34. 34.

    Park SH, Kang BK, Lee JE, Chun SW, Jang K, Kim YH, et al. Design and Fabrication of a thin-walled free-form scaffold on the basis of medical image data and a 3D printed template: its potential use in bile duct regeneration. ACS Appl Mater Interfaces. 2017;9:12290–8.

    CAS  Article  Google Scholar 

Download references

Acknowledgement

This work was supported by a research fund from Hanyang University (HY-2017).

Author information

Affiliations

Authors

Corresponding authors

Correspondence to Jaemin Jeong or Kiyoung Ryu.

Ethics declarations

Conflicts of interest

The authors declare no conflicts of interest relevant to this article.

Ethical statement

The study was conducted on crab-eating monkeys (cynomolgus monkey, Macaca fascicularis) in Research Center for Animal Model, Korea Institute of Toxicology (KIT), which is a Good Laboratory Practice facility and located at 30 Baek Hak 1-gil, Jeongeup, Jeollabuk-Do, Republic of Korea. The crab-eating monkeys, which are laboratory non-primate from China (Guangxi Grandforest scientific primate company, Ltd., China), are housed in individual cages (510 W × 800 L × 764 H mm) following 30-day quarantine and acclimation. Size of cage was satisfied the requirements for ‘The Guide for the Care and Use of Laboratory Animals (ILAR publication, 2010 National Academy Press. All were males. The room environment was automatically controlled 20–26 °C, relative humidity 50 ± 10%, 12 h light/12 h dark cycle with 150–300 lx, and ventilation 10 –20 times/hour. Temperature and relative humidity were monitored and recorded daily. Animal room and cage cleaning was performed according to the Research Center for Animal Model’s standard operating procedure. The monkeys were provided food, Lab diet ® #5002, PMI Nutrition International, USA) at 9:00 a.m. and 6:00 p.m. and water ad libitum and were fed approximately 60 g (totally 120 g a day, 60 g twice a day of) of food (Certified Primate Diet #5048, PMI nutrition International, Inc.) twice a day. The animals were managed at KIT, an accredited animal facility, complying with the AAALAC International Animal Care Policies. The Animal Care and Use Committee of the KIT reviewed and approved all the study protocols. All animals were deeply anesthetized by an excess amount of thiopental sodium injection and killed after bleeding. The experimental use of nonhuman primate was performed after receiving approval of the Institutional Animal Care and Use Committee (IACUC) of Korea Institute of Toxicology (IACUC KIT-1811-0441). Information on the liver tissues of each subject is given in Table 2.

Table 2 Laboratory characteristics of the three cynomolgus monkeys enrolled in this study

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Hee Hong, D., Lee, C., Kim, Y. et al. Generation of Hepatic Progenitor Cells from the Primary Hepatocytes of Nonhuman Primates Using Small Molecules. Tissue Eng Regen Med (2021). https://doi.org/10.1007/s13770-020-00327-8

Download citation

Keywords

  • Primate
  • Small molecules
  • Hepatic progenitors
  • Reprogramming
  • Stem cells