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

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Tissue Engineering and Regenerative Medicine Aims and scope

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.

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Acknowledgement

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

Author information

Author notes

  1. Da Hee Hong and Changhee Lee contributed equally to this work as first authors.

Authors and Affiliations

  1. Department of Surgery, Hanyang University College of Medicine, Seoul, 04763, Republic of Korea

    Da Hee Hong, Changhee Lee, Yohan Kim, Dongho Choi & Jaemin Jeong

  2. HY Indang Center of Regenerative Medicine and Stem Cell Research, Hanyang University, Seoul, 04763, Republic of Korea

    Da Hee Hong, Changhee Lee, Yohan Kim, Dongho Choi & Jaemin Jeong

  3. Hanyang ICT Fusion Medical Research Center, Hanyang University, Seoul, 04763, Republic of Korea

    Da Hee Hong, Changhee Lee, Yohan Kim, Dongho Choi & Jaemin Jeong

  4. Laboratory of Radiation Exposure and Therapeutics, National Radiation Emergency Medical Center, Korea Institute of Radiological and Medical Science, Seoul, 01812, Republic of Korea

    Seung Bum Lee

  5. Korea Institute of Toxicology, Daejeon, 53212, Republic of Korea

    Su-Cheol Han

  6. GenNBio, Inc., Seoul, 06193, Republic of Korea

    Sung Joo Kim & Heung-Mo Yang

  7. Department of Obstetrics and Gynecology, Hanyang University College of Medicine, Seoul, 04763, Republic of Korea

    Kiyoung Ryu

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  1. Da Hee Hong
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  2. Changhee Lee
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Corresponding authors

Correspondence to Jaemin Jeong or Kiyoung Ryu.

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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
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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 18, 305–313 (2021). https://doi.org/10.1007/s13770-020-00327-8

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  • DOI: https://doi.org/10.1007/s13770-020-00327-8

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