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Cited by 4Mesenchymal stem cell researchLiver physiology and pathologyPluripotent Stem Cells Research

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Induction of the differentiation of porcine bone marrow mesenchymal stem cells into premature hepatocyte-like cells in an indirect coculture system with primary hepatocytes

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Ullah, Imran, Seo, Kangmin, Wi, Hayeon, Kim, Youngim, Lee, Seunghoon, Ock, Sun A·2020·Animal Cells and Systems
DOI 10.1080/19768354.2020.1823473PMC PMC7646558
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Abstract

Liver transplantation is currently the only option for patients with end-stage liver disease. Thus, other alternate therapeutic strategies are needed. Bone marrow mesenchymal stem cells (BM-MSCs) are nonhematopoietic cells present in the bone marrow stroma that serve as precursors cells for various other cells. In this study, we evaluated the differentiation of porcine BM-MSCs into hepatocyte-like cells using three types of culture systems: hepatic induction medium (HIM), HIM/primary hepatocyte culture supernatant (HCS; 1:1 ratio), and a hepatocyte coculture system (HCCS; primary hepatocytes in the upper chamber, and BM-MSCs in the lower chamber). Primary hepatocytes were isolated from anesthetized healthy 1-month-old pigs by enzymatic digestion. Hepatic-specific marker expression (albumin [ALB], transferrin [TF], α-fetoprotein [AFP]), glycogen storage, low-density lipoprotein, and indocyanine green uptake were evaluated. Upregulation of hepatic-specific markers (ALB, TF, and AFP) was observed by real-time polymerase chain reaction in the HCCS group. Periodic acid-Schiff staining revealed enhanced glycogen storage in hepatocyte-like cells from the HCCS group compared with that from the HIM/HCS group. Furthermore, hepatocyte like-cells in the HCCS group showed improved LDL and ICG uptake than those in the other groups. Overall, our current study revealed that indirect coculture of primary hepatocytes and BM-MSCs enhanced the differentiation efficacy of BM-MSCs into hepatocyte-like cells by unknown useful soluble factors, including paracrine factors.

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Figure 1. Strategies for differentiation of BM-MSCs into hepatocyte-like cells. First, 5 × 104 BM-MSCs were seeded on 35-mm dishes (day 0) and treated with 1 μM 5- Azacytidine on day 1. Cells were then induced using different methods until day 20. All groups were treated with 30 ng/mL Activin A for 24 h on day 3. Furthermore, BM-MSCs were induced into hepatocyte-like cells using stepwise chemical induction medium, such hepatocyte initiation medium (HIM I) and hepatocyte induction medium (HIM II) (group I, HIM) or a mixture (1:1) of HIM and hepatocyte culture supernatant (HCS) (group II, HIM/HCS). HIM/HCS was exchanged with fresh medium after every 3 days. In group III, BM-MSCs were indirectly cultured with hepatocytes in a coculture system (HCCS) (group III, HCCS) using Transwells. The concentration of hepatocytes was the same as that of BM-MSCs. Hepatocytes were exchanged every week.
Figure 1. Strategies for differentiation of BM-MSCs into hepatocyte-like cells. First, 5 × 104 BM-MSCs were seeded on 35-mm dishes (day 0) and treated with 1 μM 5- Azacytidine on day 1. Cells were then induced using different methods until day 20. All groups were treated with 30 ng/mL Activin A for 24 h on day 3. Furthermore, BM-MSCs were induced into hepatocyte-like cells using stepwise chemical induction medium, such hepatocyte initiation medium (HIM I) and hepatocyte induction medium (HIM II) (group I, HIM) or a mixture (1:1) of HIM and hepatocyte culture supernatant (HCS) (group II, HIM/HCS). HIM/HCS was exchanged with fresh medium after every 3 days. In group III, BM-MSCs were indirectly cultured with hepatocytes in a coculture system (HCCS) (group III, HCCS) using Transwells. The concentration of hepatocytes was the same as that of BM-MSCs. Hepatocytes were exchanged every week.CC BY · PubMed Central
Figure 2. Isolation and confirmation of porcine primary hepatocytes. (A) Livers of anesthetized 1-week-old pigs were perfused with liver perfusion medium containing 5% FBS and liver digest medium at 37°C. The extracted liver was minced, dissociated with a tissue grinder, filtered using 100 and 40 μm cell strainers, washed with hepatocyte wash medium, and kept in LN2 after cryopreservation (1 × 107 cells). (B) Fresh porcine primary hepatocytes showed both polygonal cytoplasm and binuclear hepatocytes. (C) Binuclear hepatocytes were observed using anti-E-cadherin antibodies and PI staining (white dot circle and magnified boxes). a and b showed merged images of E-cadherin (green)/PI (red) and E-cadherin (green)/PI (red)/bright field, respectively.
Figure 2. Isolation and confirmation of porcine primary hepatocytes. (A) Livers of anesthetized 1-week-old pigs were perfused with liver perfusion medium containing 5% FBS and liver digest medium at 37°C. The extracted liver was minced, dissociated with a tissue grinder, filtered using 100 and 40 μm cell strainers, washed with hepatocyte wash medium, and kept in LN2 after cryopreservation (1 × 107 cells). (B) Fresh porcine primary hepatocytes showed both polygonal cytoplasm and binuclear hepatocytes. (C) Binuclear hepatocytes were observed using anti-E-cadherin antibodies and PI staining (white dot circle and magnified boxes). a and b showed merged images of E-cadherin (green)/PI (red) and E-cadherin (green)/PI (red)/bright field, respectively.CC BY · PubMed Central
Figure 3. Expression of hepatocyte-specific and apoptosis-related genes in porcine primary hepatocytes cultured in vitro for 1 week. Fresh and frozen/thawed hepatocytes were cultured in hepatocyte maintenance medium (HMM) for 7 days. Fresh hepatocytes without in vitro culture were used as a control for each group. A, B, and C show albumin (ALB), transferrin (TF), and CYP3A29, respectively. D and E show the pro-apoptotic gene BAX and the anti-apoptotic gene BCL2L1, respectively. RQ: relative quantification; error bars represent RQ ± min and RQ ± max; differences were considered significant at p < 0.05. All experiments were performed in triplicate. Letters a, b, c, and d indicate significant differences (p < 0.05) among groups.
Figure 3. Expression of hepatocyte-specific and apoptosis-related genes in porcine primary hepatocytes cultured in vitro for 1 week. Fresh and frozen/thawed hepatocytes were cultured in hepatocyte maintenance medium (HMM) for 7 days. Fresh hepatocytes without in vitro culture were used as a control for each group. A, B, and C show albumin (ALB), transferrin (TF), and CYP3A29, respectively. D and E show the pro-apoptotic gene BAX and the anti-apoptotic gene BCL2L1, respectively. RQ: relative quantification; error bars represent RQ ± min and RQ ± max; differences were considered significant at p < 0.05. All experiments were performed in triplicate. Letters a, b, c, and d indicate significant differences (p < 0.05) among groups.CC BY · PubMed Central
Figure 4. Morphological transition of BM-MSCs into hepatocyte-like cells after 3 weeks. Each group was induced for 21 days and observed under a microscope. A, B, and C show HIM, HIM/HCS, and HCCS groups, respectively. a, b, and c show induction time intervals (day 1, day 13, and day 20, respectively). Scale bars = 200 µm.
Figure 4. Morphological transition of BM-MSCs into hepatocyte-like cells after 3 weeks. Each group was induced for 21 days and observed under a microscope. A, B, and C show HIM, HIM/HCS, and HCCS groups, respectively. a, b, and c show induction time intervals (day 1, day 13, and day 20, respectively). Scale bars = 200 µm.CC BY · PubMed Central
Figure 5. Expression of hepatic-specific markers in hepatocyte-like cells induced from porcine BM-MSCs. The hepatic-specific markers (A) ALB, (B) AFP, and (C) TF were analyzed in HIM, HIM/HCS, and HCCS groups. RQ: relative quantification; error bars represent RQ ± min and RQ ± max; differences were considered significant at p < 0.05. All experiments were performed in triplicate. Letters a, b, c, and d showed significant differences (p < 0.05) among groups.
Figure 5. Expression of hepatic-specific markers in hepatocyte-like cells induced from porcine BM-MSCs. The hepatic-specific markers (A) ALB, (B) AFP, and (C) TF were analyzed in HIM, HIM/HCS, and HCCS groups. RQ: relative quantification; error bars represent RQ ± min and RQ ± max; differences were considered significant at p < 0.05. All experiments were performed in triplicate. Letters a, b, c, and d showed significant differences (p < 0.05) among groups.CC BY · PubMed Central
Figure 6. Hepatocyte function assays in hepatocyte-like cells induced from porcine BM-MSCs. (A) ICG uptake and release assays were performed for evaluation of toxic agent absorption and degrading ability. Uptake tests were performed in hepatocyte-like cells after treatment with ICG for 2 h (A-1), and release tests were performed at 6 h after treatment (A-2). Scale bars = 100 µm. (B) Glycogen storage was analyzed by PAS staining (red). Scale bars = 100 µm. (C) The LDL uptake tests in hepatocyte-like cells. Red fluorescence: LDL; blue fluorescence: DAPI (nuclear staining). Scale bar = 50 µm. a, b, and c represent HIM, HIM/HCS, and HCCS groups, respectively.
Figure 6. Hepatocyte function assays in hepatocyte-like cells induced from porcine BM-MSCs. (A) ICG uptake and release assays were performed for evaluation of toxic agent absorption and degrading ability. Uptake tests were performed in hepatocyte-like cells after treatment with ICG for 2 h (A-1), and release tests were performed at 6 h after treatment (A-2). Scale bars = 100 µm. (B) Glycogen storage was analyzed by PAS staining (red). Scale bars = 100 µm. (C) The LDL uptake tests in hepatocyte-like cells. Red fluorescence: LDL; blue fluorescence: DAPI (nuclear staining). Scale bar = 50 µm. a, b, and c represent HIM, HIM/HCS, and HCCS groups, respectively.CC BY · PubMed Central

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Metadata

Authors
Ullah, I., Seo, K., Wi, H., Kim, Y., Lee, S., & Ock, S. A.
Affiliations
  • Quaid-i-Azam University (PK)
  • Rural Development Administration (KR)
  • Animal Biotechnology Division, National Institute of Animal Science, Rural Development Administration, Wanju-gun, Republic of Korea
Corresponding author
Ock, Sun A
ORCID
Venue
Animal Cells and Systems
Publisher
Informa UK Limited
Published
2020-09-02
Volume
24
Issue
5
Pages
289-298
ISSN
  • 1976-8354 (ISSN-L)
  • 2151-2485
DOI
10.1080/19768354.2020.1823473
PMC
PMC7646558
OpenAlex
W3092516100
Cited by
4
References
0
Citations by year
2025: 1 · 2023: 1 · 2022: 1 · 2021: 1
FWCI
0.39
Type
journal article
Language
en
Open access
Licence
Locations
  • Animal Cells and Systems (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Mesenchymal stem cell research
  • Health Sciences › Medicine › Hepatology › Liver physiology and pathology
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Pluripotent Stem Cells Research
Keywords
bone marrow mesenchymal stem cells, hepatocyte-like cells, stem cell differentiation, primary hepatocytes, pigs, paracrine factors, glycogen storage, liver regeneration

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CoChat. (2026). Citation check: Induction of the differentiation of porcine bone marrow mesenchymal stem cells into premature hepatocyte-like cells in an indirect coculture system with primary hepatocytes. https://app.cochat.ai/papers/10.1080/19768354.2020.1823473
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Ullah, I., Seo, K., Wi, H., Kim, Y., Lee, S., & Ock, S. A. (2020). Induction of the differentiation of porcine bone marrow mesenchymal stem cells into premature hepatocyte-like cells in an indirect coculture system with primary hepatocytes. Animal Cells and Systems. https://doi.org/10.1080/19768354.2020.1823473