|
1
|
Torizal FG, Utami T, Lau QY, Inamura K, Nishikawa M, Sakai Y. Dialysis based-culture medium conditioning improved the generation of human induced pluripotent stem cell derived-liver organoid in a high cell density. Sci Rep 2022; 12:20774. [PMID: 36456801 PMCID: PMC9715714 DOI: 10.1038/s41598-022-25325-9] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/01/2022] [Accepted: 11/28/2022] [Indexed: 12/03/2022] Open
Abstract
Human pluripotent stem cell-derived liver organoids (HLOs) have recently become a promising alternative for liver regenerative therapy. To realize this application, a large amount of human-induced pluripotent stem cells (hiPSCs) derived-liver cells are required for partial liver replacement during transplantation. This method requires stepwise induction using costly growth factors to direct the hiPSCs into the hepatic lineage. Therefore, we developed a simple dialysis-based medium conditioning that fully utilized growth factors accumulation to improve hepatic differentiation of hiPSCs at a high cell density. The results demonstrated that the dialysis culture system could accumulate the four essential growth factors required in each differentiation stage: activin A, bone morphogenetic protein 4 (BMP4), hepatocyte growth factor (HGF), and oncostatin M (OSM). As a result, this low lactate culture environment allowed high-density bipotential hepatic differentiation of up to 4.5 × 107 cells/mL of human liver organoids (HLOs), consisting of hiPSC derived-hepatocyte like cells (HLCs) and cholangiocyte like-cells (CLCs). The differentiated HLOs presented a better or comparable hepatic marker and hepatobiliary physiology to the one that differentiated in suspension culture with routine daily medium replacement at a lower cell density. This simple miniaturized dialysis culture system demonstrated the feasibility of cost-effective high-density hepatic differentiation with minimum growth factor usage.
Collapse
Affiliation(s)
- Fuad Gandhi Torizal
- grid.26999.3d0000 0001 2151 536XDepartment of Bioengineering, Graduate School of Engineering, The University of Tokyo, Bunkyo-ku, Tokyo, Japan ,grid.26999.3d0000 0001 2151 536XDepartment of Chemical Systems Engineering, Graduate School of Engineering, The University of Tokyo, Bunkyo-ku, Tokyo, Japan
| | - Tia Utami
- grid.26999.3d0000 0001 2151 536XDepartment of Bioengineering, Graduate School of Engineering, The University of Tokyo, Bunkyo-ku, Tokyo, Japan
| | - Qiao You Lau
- grid.26999.3d0000 0001 2151 536XDepartment of Bioengineering, Graduate School of Engineering, The University of Tokyo, Bunkyo-ku, Tokyo, Japan
| | - Kousuke Inamura
- grid.26999.3d0000 0001 2151 536XDepartment of Chemical Systems Engineering, Graduate School of Engineering, The University of Tokyo, Bunkyo-ku, Tokyo, Japan
| | - Masaki Nishikawa
- grid.26999.3d0000 0001 2151 536XDepartment of Chemical Systems Engineering, Graduate School of Engineering, The University of Tokyo, Bunkyo-ku, Tokyo, Japan
| | - Yasuyuki Sakai
- grid.26999.3d0000 0001 2151 536XDepartment of Bioengineering, Graduate School of Engineering, The University of Tokyo, Bunkyo-ku, Tokyo, Japan ,grid.26999.3d0000 0001 2151 536XDepartment of Chemical Systems Engineering, Graduate School of Engineering, The University of Tokyo, Bunkyo-ku, Tokyo, Japan
| |
Collapse
|
|
2
|
Toba Y, Deguchi S, Mimura N, Sakamoto A, Harada K, Hirata K, Takayama K, Mizuguchi H. Comparison of commercially available media for hepatic differentiation and hepatocyte maintenance. PLoS One 2020; 15:e0229654. [PMID: 32106262 PMCID: PMC7046223 DOI: 10.1371/journal.pone.0229654] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/13/2019] [Accepted: 02/12/2020] [Indexed: 11/19/2022] Open
Abstract
Human hepatocytes are essential materials in pharmaceutical researches. Not only primary human hepatocytes (PHH) but also human iPS cell-derived hepatocyte-like cells (human iPS-HLCs) are expected to be applied as materials for pharmaceutical researches. To date, several culture media have been developed for culturing human hepatocytes. However, there have been no reports comparing these media to determine which is most suitable for culturing human hepatocytes. In this study, we compared five commercial media (Hepatocyte Culture Medium (HCM), HepatoZYME-SFM, Cellartis Power Primary HEP Medium, DMEM/F12, and William’s E Medium (WEM)) to determine which is most suitable for culturing PHH and human iPS-HLCs. In hepatic differentiation of human iPS cells (day 14–25 of differentiation), albumin (ALB) and urea secretion abilities and CYP2C9, CYP2C19, and CYP3A4 activities were the highest when using HCM or WEM. During maintenance of human iPS-HLCs, ALB and urea producing abilities and CYP2C9, CYP2C19, and CYP3A4 activities were the highest when using HCM. Importantly, we found that human iPS-HLCs cultured in HCM were maintained for 3 weeks or more without impairment of their hepatic functions. These results suggest that it is necessary to select an optimal medium for hepatic differentiation and maintenance of human iPS-HLCs. In the case of PHH culture, there was little difference in hepatic functions among the five media. However, the CYP2C9, CYP2C19, and CYP3A4 activities were the highest when using HCM and WEM. In conclusion, it is important to select the optimal medium for specific application when carrying out pharmaceutical researches using human hepatocytes.
Collapse
Affiliation(s)
- Yukiko Toba
- Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan
- Laboratory of Hepatocyte Regulation, National Institutes of Biomedical Innovation, Health and Nutrition, Osaka, Japan
| | - Sayaka Deguchi
- Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan
- Laboratory of Hepatocyte Regulation, National Institutes of Biomedical Innovation, Health and Nutrition, Osaka, Japan
| | - Natsumi Mimura
- Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan
| | - Ayaka Sakamoto
- Laboratory of Hepatocyte Regulation, National Institutes of Biomedical Innovation, Health and Nutrition, Osaka, Japan
| | - Kazuo Harada
- Laboratory of Applied Environmental Biology, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan
| | - Kazumasa Hirata
- Laboratory of Applied Environmental Biology, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan
| | - Kazuo Takayama
- Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan
- Laboratory of Hepatocyte Regulation, National Institutes of Biomedical Innovation, Health and Nutrition, Osaka, Japan
- PRESTO, Japan Science and Technology Agency, Saitama, Japan
- * E-mail: (KT); (HM)
| | - Hiroyuki Mizuguchi
- Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan
- Laboratory of Applied Environmental Biology, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan
- Global Center for Medical Engineering and Informatics, Osaka University, Osaka, Japan
- Integrated Frontier Research for Medical Science Division, Institute for Open and Transdisciplinary Research Initiatives (OTRI), Osaka University, Osaka, Japan
- * E-mail: (KT); (HM)
| |
Collapse
|
|
3
|
Tomizawa M, Shinozaki F, Motoyoshi Y, Sugiyama T, Yamamoto S, Ishige N. Hepatocyte selection medium-enriched hepatocellular carcinoma cells are positive for α-fetoprotein and CD44. Oncol Lett 2017; 14:899-902. [PMID: 28693249 PMCID: PMC5494728 DOI: 10.3892/ol.2017.6239] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/04/2016] [Accepted: 03/03/2017] [Indexed: 11/06/2022] Open
Abstract
Tissues surrounding hepatocellular carcinomas (HCCs) lack glucose. Hepatocyte selection medium (HSM) is deficient in glucose and is supplemented with galactose. HCC cells were cultured in HSM to investigate the stem cell markers α-fetoprotein (AFP) and cluster of differentiation 44 (CD44). HCC cells (HLF and PLC/PRF/5 cells) were cultured in HSM. Viable cell numbers were determined on days 0 and 7 following culture in HSM. RNA was isolated and subjected to reverse transcription-quantitative PCR (RT-qPCR) to analyze the mRNA expression levels of AFP and CD44. Immunostaining was performed to analyze the protein levels of AFP and CD44. The number of viable cells was significantly decreased on day 7 following culture in HSM. The expression levels of AFP and CD44 increased on day 7 as assessed using RT-qPCR. Immunostaining confirmed the results of RT-qPCR analysis. The number of viable HCC cells was decreased in HSM, whereas the expression levels of AFP and CD44 increased. Therefore, HSM is potentially useful for the enrichment of HCC cells with cancer stem cell characteristics.
Collapse
Affiliation(s)
- Minoru Tomizawa
- Department of Gastroenterology, National Hospital Organization, Shimoshizu Hospital, Yotsukaido, Chiba 284-0003, Japan
| | - Fuminobu Shinozaki
- Department of Radiology, National Hospital Organization, Shimoshizu Hospital, Yotsukaido, Chiba 284-0003, Japan
| | - Yasufumi Motoyoshi
- Department of Neurology, National Hospital Organization, Shimoshizu Hospital, Yotsukaido, Chiba 284-0003, Japan
| | - Takao Sugiyama
- Department of Rheumatology, National Hospital Organization, Shimoshizu Hospital, Yotsukaido, Chiba 284-0003, Japan
| | - Shigenori Yamamoto
- Department of Pediatrics, National Hospital Organization, Shimoshizu Hospital, Yotsukaido, Chiba 284-0003, Japan
| | - Naoki Ishige
- Department of Neurosurgery, National Hospital Organization, Shimoshizu Hospital, Yotsukaido, Chiba 284-0003, Japan
| |
Collapse
|
|
4
|
Tomizawa M, Shinozaki F, Motoyoshi Y, Sugiyama T, Yamamoto S, Ishige N. Oncostatin M in William's E medium is suitable for initiation of hepatocyte differentiation in human induced pluripotent stem cells. Mol Med Rep 2017; 15:3088-3092. [PMID: 28358419 DOI: 10.3892/mmr.2017.6406] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/29/2016] [Accepted: 02/20/2017] [Indexed: 11/06/2022] Open
Abstract
William's E (WE) is a suitable medium for the differentiation of human induced pluripotent stem (iPS) cells to the hepatocyte lineage. The aim of the present study was to investigate various growth factors in their ability to promote hepatocyte differentiation of iPS cells in WE medium. Human iPS 201B7 cells were cultured in WE medium supplemented with growth factors, and mRNA expression levels and promoter activities of α‑fetoprotein (AFP) and albumin were examined by reverse transcription‑quantitative polymerase chain reaction and luciferase assay, respectively. In addition, time course analysis of AFP mRNA expression was performed in 201B7 cells cultured in WE medium supplemented with oncostatin M. The results demonstrated that mRNA expression levels of AFP were significantly elevated by most growth factors tested as supplements in WE medium, except all‑trans retinoic acid, compared with cells cultured in ReproFF (a medium that maintains pluripotency). The highest increase in AFP mRNA expression levels was observed by oncostatin M stimulation. Albumin mRNA expression levels were increased by all‑trans retinoic acid and insulin‑transferrin‑selenium supplementation in WE medium compared with cells cultured in ReproFF. Oncostatin M supplementation significantly stimulated the promoter activity of the AFP gene, but no growth factor tested significantly stimulated the promoter activity of the albumin gene. By time course analysis, significant increase of AFP mRNA expression was observed on the sixth day post‑stimulation, compared with cells cultured in WE medium alone. In conclusion, the present study demonstrated that oncostatin M supplementation in WE medium was sufficient to initiate hepatocyte differentiation in iPS cells.
Collapse
Affiliation(s)
- Minoru Tomizawa
- Department of Gastroenterology, National Hospital Organization, Shimoshizu Hospital, Yotsukaido, Chiba 284‑0003, Japan
| | - Fuminobu Shinozaki
- Department of Radiology, National Hospital Organization, Shimoshizu Hospital, Yotsukaido, Chiba 284‑0003, Japan
| | - Yasufumi Motoyoshi
- Department of Neurology, National Hospital Organization, Shimoshizu Hospital, Yotsukaido, Chiba 284‑0003, Japan
| | - Takao Sugiyama
- Department of Rheumatology, National Hospital Organization, Shimoshizu Hospital, Yotsukaido, Chiba 284‑0003, Japan
| | - Shigenori Yamamoto
- Department of Pediatrics, National Hospital Organization, Shimoshizu Hospital, Yotsukaido, Chiba 284‑0003, Japan
| | - Naoki Ishige
- Department of Neurosurgery, National Hospital Organization, Shimoshizu Hospital, Yotsukaido, Chiba 284‑0003, Japan
| |
Collapse
|
|
5
|
Tomizawa M, Shinozaki F, Motoyoshi Y, Sugiyama T, Yamamoto S, Ishige N. Proliferation of sphere-forming hepatocellular carcinoma cells is suppressed in a medium without glucose and arginine, but with galactose and ornithine. Oncol Lett 2017; 13:1264-1268. [PMID: 28454244 PMCID: PMC5403635 DOI: 10.3892/ol.2017.5565] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/21/2015] [Accepted: 08/25/2016] [Indexed: 01/04/2023] Open
Abstract
Resistance to sorafenib in hepatocellular carcinoma (HCC) cells exhibiting stemness was evaluated using a sphere formation assay. A hepatocyte selection medium (HSM) deficient in glucose and arginine was used to suppress the proliferation of cell spheres composed of HLF and PLC/PRF/5 HCC cells, which were subjected to a sphere formation assay. Cell spheres were cultured with sorafenib and subjected to a cell proliferation assay and the expression levels of cytochrome P450 (CYP3A4) were analyzed in RNA extracted from sphere-forming cells using reverse transcription-quantitative polymerase chain reaction. Sphere-forming PLC/PRF/5 cells were more resistant to sorafenib, as compared with control cells, exhibiting higher expression levels of CYP3A4. When cultured in HSM, suppressed proliferation was observed in the sphere-forming PLC/PRF/5 cells and in the control cells, with no significant variation between them. The results suggest that deprivation of glucose and arginine is a potential novel treatment for HCC.
Collapse
Affiliation(s)
- Minoru Tomizawa
- Department of Gastroenterology, National Hospital Organization, Shimoshizu Hospital, Yotsukaido, Chiba 284-0003, Japan
| | - Fuminobu Shinozaki
- Department of Radiology, National Hospital Organization, Shimoshizu Hospital, Yotsukaido, Chiba 284-0003, Japan
| | - Yasufumi Motoyoshi
- Department of Neurology, National Hospital Organization, Shimoshizu Hospital, Yotsukaido, Chiba 284-0003, Japan
| | - Takao Sugiyama
- Department of Rheumatology, National Hospital Organization, Shimoshizu Hospital, Yotsukaido, Chiba 284-0003, Japan
| | - Shigenori Yamamoto
- Department of Pediatrics, National Hospital Organization, Shimoshizu Hospital, Yotsukaido, Chiba 284-0003, Japan
| | - Naoki Ishige
- Department of Neurosurgery, National Hospital Organization, Shimoshizu Hospital, Yotsukaido, Chiba 284-0003, Japan
| |
Collapse
|
|
6
|
Tomizawa M, Shinozaki F, Motoyoshi Y, Sugiyama T, Yamamoto S, Ishige N. Proliferation and motility of hepatocellular, pancreatic and gastric cancer cells grown in a medium without glucose and arginine, but with galactose and ornithine. Oncol Lett 2017; 13:1276-1280. [PMID: 28454246 PMCID: PMC5403308 DOI: 10.3892/ol.2017.5568] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/04/2015] [Accepted: 11/10/2016] [Indexed: 12/14/2022] Open
Abstract
Human primary hepatocytes are able to survive in a medium without glucose and arginine, but supplemented with galactose and ornithine (hepatocyte selection medium; HSM). To address the possibility of the application of HSM in cancer therapy, hepatocellular carcinoma cells, pancreatic cancer cells and gastric cancer cells were cultured in HSM. Cell proliferation was analyzed using an MTS assay. Morphological changes were analyzed using hematoxylin and eosin staining. Apoptosis was analyzed using a TUNEL assay and cell motility was assessed with a scratch assay. Cell proliferation was significantly suppressed in cell lines grown in HSM (P<0.01 in all the cell lines). Hematoxylin and eosin staining revealed pyknotic nuclei, suggesting that these cells had undergone apoptosis. The number of TUNEL-positive cells was significantly increased in HSM. In the scratch assay, the distance between the growing edge and the scratched edge was significantly lower (P<0.01 in all the cell lines) in cells cultured in HSM, compared with those grown in Dulbecco's modified Eagle's medium or RPMI-1640. Therefore, the proliferation and motility of hepatocellular carcinoma cells, pancreatic cancer cells and gastric cancer cells was suppressed, and these cells subsequently underwent apoptosis in a medium without glucose and arginine, but containing galactose and ornithine.
Collapse
Affiliation(s)
- Minoru Tomizawa
- Department of Gastroenterology, National Hospital Organization, Shimoshizu Hospital, Yotsukaido, Chiba 284-0003, Japan
| | - Fuminobu Shinozaki
- Department of Radiology, National Hospital Organization, Shimoshizu Hospital, Yotsukaido, Chiba 284-0003, Japan
| | - Yasufumi Motoyoshi
- Department of Neurology, National Hospital Organization, Shimoshizu Hospital, Yotsukaido, Chiba 284-0003, Japan
| | - Takao Sugiyama
- Department of Rheumatology, National Hospital Organization, Shimoshizu Hospital, Yotsukaido, Chiba 284-0003, Japan
| | - Shigenori Yamamoto
- Department of Pediatrics, National Hospital Organization, Shimoshizu Hospital, Yotsukaido, Chiba 284-0003, Japan
| | - Naoki Ishige
- Department of Neurosurgery, National Hospital Organization, Shimoshizu Hospital, Yotsukaido, Chiba 284-0003, Japan
| |
Collapse
|
|
7
|
Tomizawa M, Shinozaki F, Motoyoshi Y, Sugiyama T, Yamamoto S, Ishige N. Cell death in a co-culture of hepatocellular carcinoma cells and human umbilical vascular endothelial cells in a medium lacking glucose and arginine. Oncol Lett 2017; 13:258-262. [PMID: 28123551 PMCID: PMC5245067 DOI: 10.3892/ol.2016.5454] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/19/2015] [Accepted: 05/18/2016] [Indexed: 12/14/2022] Open
Abstract
Human primary hepatocytes are able to survive in a medium without glucose and arginine that is instead supplemented with galactose and ornithine (hepatocyte selection medium; HSM). This is because the cells produce glucose and arginine by the action of galactokinase (GALK) and ornithine carbamoyltransferase (OTC), respectively. It was expected that hepatocellular carcinoma (HCC) cells do not survive in HSM. In the current study, HCC cell lines (namely HLE, HLF, PLC/PRL/5, Hep3B and HepG2) and human umbilical vascular endothelial cells (HUVECs) were cultured in HSM, and the expression levels of GALK1, GALK2 and OTC were analyzed by reverse transcription-quantitative polymerase chain reaction. HLE, HLF and PLC/PRL/5 cells died on day 11, while Hep3B, HepG2 and HUVECs died on day 7. HLF cells were further analyzed as these cells had lower expression levels of GALK1, GALK2 and OTC compared with adult liver cells, and survived until day 11. In these cells, the expression levels of GALK1, GALK2 and OTC did not change on days 3 and 7 as compared to day 0. In addition, a co-culture of HLF cells with HUVECs was established and the medium was changed to HSM. It was observed that HLF cells and HUVECs in co-culture were damaged in HSM. In summary, HCC cells and HUVECs died in a medium without glucose and arginine that was supplemented with galactose and ornithine. HCC cells and HUVECs were damaged in HSM, suggesting a potential application for treatment with the medium.
Collapse
Affiliation(s)
- Minoru Tomizawa
- Department of Gastroenterology, National Hospital Organization Shimoshizu Hospital, Yotsukaido, Chiba 284-0003, Japan
| | - Fuminobu Shinozaki
- Department of Radiology, National Hospital Organization Shimoshizu Hospital, Yotsukaido, Chiba 284-0003, Japan
| | - Yasufumi Motoyoshi
- Department of Neurosurgery, National Hospital Organization Shimoshizu Hospital, Yotsukaido, Chiba 284-0003, Japan
| | - Takao Sugiyama
- Department of Rheumatology, National Hospital Organization Shimoshizu Hospital, Yotsukaido, Chiba 284-0003, Japan
| | - Shigenori Yamamoto
- Department of Pediatrics, National Hospital Organization Shimoshizu Hospital, Yotsukaido, Chiba 284-0003, Japan
| | - Naoki Ishige
- Department of Internal Medicine, National Hospital Organization Shimoshizu Hospital, Yotsukaido, Chiba 284-0003, Japan
| |
Collapse
|
|
8
|
Kilberg MS, Terada N, Shan J. Influence of Amino Acid Metabolism on Embryonic Stem Cell Function and Differentiation. Adv Nutr 2016; 7:780S-9S. [PMID: 27422515 PMCID: PMC4942862 DOI: 10.3945/an.115.011031] [Citation(s) in RCA: 36] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have promise in regenerative medicine because of their ability to differentiate into all 3 primary germ layers. This review describes recent advances in the understanding of the link between the metabolism of ESCs/iPSCs and their maintenance/differentiation in the cell culture setting, with particular emphasis on amino acid (AA) metabolism. ESCs are endowed with unique metabolic features with regard to energy consumption, metabolite flux through particular pathways, and macromolecular synthesis. Therefore, nutrient availability has a strong influence on stem cell growth, self-renewal, and lineage specification, both in vivo and in vitro. Evidence from several laboratories has documented that self-renewal and differentiation of mouse ESCs are critically dependent on proline metabolism, with downstream metabolites possibly serving as signal molecules. Likewise, catabolism of either threonine (mouse) or methionine (human) is required for growth and differentiation of ESCs because these AAs serve as precursors for donor molecules used in histone methylation and acetylation. Epigenetic mechanisms are recognized as critical steps in differentiation, and AA metabolism in ESCs appears to modulate these epigenetic processes. Recent reports also document that, in vitro, the nutrient composition of the culture medium in which ESCs are differentiated into embryoid bodies can influence lineage specification, leading to enrichment of a specific cell type. Although research designed to direct tissue specification of differentiating embryoid bodies in culture is still in its infancy, early results indicate that manipulation of the nutrient milieu can promote or suppress the formation of specific cell lineages.
Collapse
Affiliation(s)
| | - Naohiro Terada
- Pathology, Immunology, and Laboratory Medicine, University of Florida College of Medicine, Gainesville, FL
| | - Jixiu Shan
- Departments of Biochemistry and Molecular Biology and
| |
Collapse
|
|
9
|
Tomizawa M, Shinozaki F, Motoyoshi Y, Sugiyama T, Yamamoto S, Ishige N. Improved Survival and Initiation of Differentiation of Human Induced Pluripotent Stem Cells to Hepatocyte-Like Cells upon Culture in William's E Medium followed by Hepatocyte Differentiation Inducer Treatment. PLoS One 2016; 11:e0153435. [PMID: 27073925 PMCID: PMC4830564 DOI: 10.1371/journal.pone.0153435] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/12/2015] [Accepted: 03/29/2016] [Indexed: 12/18/2022] Open
Abstract
BACKGROUND Hepatocyte differentiation inducer (HDI) lacks both glucose and arginine, but is supplemented with galactose and ornithine, and is added together with other reagents such as apoptosis inhibitor and oncostatin M. Although human induced pluripotent stem (iPS) cells initiate hepatocyte differentiation, most die within 7 days. In this study, we investigated both HDI and conventional media for their potential to improve cell survival. MATERIALS AND METHODS 201B7 iPS cells were cultured in conventional media. This consisted of three cycles of 5-day culture in William's E (WE) medium, followed by a 2-day culture in HDI. RESULTS Expression levels of α-feto protein (AFP) were higher in cells cultured in WE and in Dulbecco's Modified Eagle's Medium/Nutrient F-12 Ham (DF12). 201B7 cells expressed the highest AFP and albumin (ALB) when cultured in HDI for 2 days following 7-day culture in WE. After three cycles of 5-day culture in WE followed by 2 days in HDI, 201B7 cells expressed AFP and ALB 54 ± 2.3 (average ± standard deviation) and 73 ± 15.1 times higher, respectively, than those cultured in ReproFF (feeder-free condition). CONCLUSION 201B7 cells survived culture in WE for 7 days followed HDI for 2 days. After three cycles of culture under these conditions, hepatocyte differentiation was enhanced, as evidenced by increased AFP and ALB expression.
Collapse
Affiliation(s)
- Minoru Tomizawa
- Department of Gastroenterology, National Hospital Organization, Shimoshizu Hospital, 934–5 Shikawatashi, Yotsukaido City, Chiba 284–0003, Japan
| | - Fuminobu Shinozaki
- Department of Radiology, National Hospital Organization, Shimoshizu Hospital, 934–5 Shikawatashi, Yotsukaido City, Chiba 284–0003, Japan
| | - Yasufumi Motoyoshi
- Department of Neurology, National Hospital Organization, Shimoshizu Hospital, 934–5 Shikawatashi, Yotsukaido City, Chiba 284–0003, Japan
| | - Takao Sugiyama
- Department of Rheumatology, National Hospital Organization, Shimoshizu Hospital, 934–5 Shikawatashi, Yotsukaido City, Chiba 284–0003, Japan
| | - Shigenori Yamamoto
- Department of Pediatrics, National Hospital Organization, Shimoshizu Hospital, 934–5 Shikawatashi, Yotsukaido City, Chiba 284–0003, Japan
| | - Naoki Ishige
- Department of Neurosurgery, National Hospital Organization, Shimoshizu Hospital, 934–5 Shikawatashi, Yotsukaido City, Chiba 284–0003, Japan
| |
Collapse
|
|
10
|
Tomizawa M, Shinozaki F, Motoyoshi Y, Sugiyama T, Yamamoto S, Ishige N. Hepatocyte selection medium eliminating induced pluripotent stem cells among primary human hepatocytes. World J Methodol 2015; 5:108-114. [PMID: 26413482 PMCID: PMC4572022 DOI: 10.5662/wjm.v5.i3.108] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/10/2015] [Revised: 05/22/2015] [Accepted: 08/30/2015] [Indexed: 02/06/2023] Open
Abstract
Hepatic insufficiency is a fatal liver disease with a significant decrease in functioning hepatocytes. If hepatocytes could be generated from human induced pluripotent stem (hiPS) cells and transplanted into patients with hepatic insufficiency, the disease may become curable. However, a major limitation to this therapeutic strategy is due to the tumorigenicity of hiPS cells and their ability to form cancer. Current methods for eliminating unwanted hiPS cells use genetic manipulation or reagents that are potentially hazardous for hepatocytes; therefore, revised methods are necessary and anticipated. Glucose and arginine are essential cell culture medium ingredients for the survival of most cells, including hiPS cells. However, hepatocytes can produce its own glucose and arginine through galactokinase and ornithine transcarbamylase, respectively. Therefore, it was hypothesized that unwanted hiPS cells could be eliminated in a medium without glucose and arginine, and supplemented with galactose and ornithine instead. This modified medium has been established as hepatocyte selection medium (HSM). So far, attempts to generate a pure colony of mature hepatocytes from hiPS cells have not been successful. After establishment of co-culture in HSM, primary human hepatocytes survive while hiPS cells die within three days. Our latest results regarding a modification of HSM will be introduced in this manuscript.
Collapse
|
|
11
|
Tomizawa M, Shinozaki F, Motoyoshi Y, Sugiyama T, Yamamoto S, Ishige N. An Optimal Medium Supplementation Regimen for Initiation of Hepatocyte Differentiation in Human Induced Pluripotent Stem Cells. J Cell Biochem 2015; 116:1479-1489. [PMID: 25683148 DOI: 10.1002/jcb.25139] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/17/2015] [Accepted: 02/10/2015] [Indexed: 12/22/2022]
Abstract
Human induced pluripotent stem (hiPS) cells are an ideal source for hepatocytes. Glucose and arginine are necessary for cells to survive. Hepatocytes have galactokinase (GALK), which metabolizes galactose for gluconeogenesis, and ornithine transcarbamylase (OTC), which converts ornithine to arginine in the urea cycle. Hepatocyte selection medium (HSM) lacks both glucose and arginine, but contains galactose and ornithine. Although human primary hepatocytes survive in HSM, all the hiPS cells die in 3 days. The aim of this study was to modify HSM so as to initiate hepatocyte differentiation in hiPS cells within 2 days. Hepatocyte differentiation initiating medium (HDI) was prepared by adding oncostatin M (10 ng/ml), hepatocyte functional proliferation inducer (10 nM), 2,2'-methylenebis (1,3-cyclohexanedione) (M50054) (100 μg/ml), 1× non-essential amino acid, 1× sodium pyruvate, nicotinamide (1.2 mg/ml), L-proline (30 ng/ml), and L-glutamine (0.3 mg/ml) to HSM. HiPS cells (201B7 cells) were cultured in HDI for 2 days. RNA was isolated, used as template for cDNA, and subjected to real-time quantitative polymerase chain reaction. Alpha-fetoprotein, γ-glutamyl transpeptidase, and delta-like 1 were upregulated. Expression of albumin was not observed. Expression of transcription factors specific to hepatocytes was upregulated. The expression of GALK2, OTC, and CYP3A4 were increased. In conclusion, differentiation of 201B7 cells to hepatoblast-like cells was initiated in HDI. Limitations were small number of cells were obtained, and the cells with HDI were not mature hepatocytes.
Collapse
Affiliation(s)
- Minoru Tomizawa
- Department of Gastroenterology, National Hospital Organization, Shimoshizu Hospital, Yotsukaido City, Chiba, 284-0003, Japan
| | - Fuminobu Shinozaki
- Department of Radiology, National Hospital Organization, Shimoshizu Hospital, Yotsukaido City, Chiba, 284-0003, Japan
| | - Yasufumi Motoyoshi
- Department of Neurology, National Hospital Organization, Shimoshizu Hospital, Yotsukaido City, Chiba, 284-0003, Japan
| | - Takao Sugiyama
- Department of Rheumatology, National Hospital Organization, Shimoshizu Hospital, Yotsukaido City, Chiba, 284-0003, Japan
| | - Shigenori Yamamoto
- Department of Pediatrics, National Hospital Organization, Shimoshizu Hospital, Yotsukaido City, Chiba, 284-0003, Japan
| | - Naoki Ishige
- Department of Neurosurgery, National Hospital Organization, Shimoshizu Hospital, Yotsukaido City, Chiba, 284-0003, Japan
| |
Collapse
|
|
12
|
Tomizawa M, Shinozaki F, Motoyoshi Y, Sugiyama T, Yamamoto S, Sueishi M. Dual gene expression in embryoid bodies derived from human induced pluripotent stem cells using episomal vectors. Tissue Eng Part A 2014; 20:3154-3162. [PMID: 24980753 PMCID: PMC4259172 DOI: 10.1089/ten.tea.2014.0132] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/02/2014] [Accepted: 05/20/2014] [Indexed: 01/16/2023] Open
Abstract
Transcription factors are essential for the differentiation of human induced pluripotent stem cells (iPS) into specialized cell types. Embryoid body (EB) formation promotes the differentiation of iPS cells. We sought to establish an efficient method of transfection and rotary culture to generate EBs that stably express two genes. The pMetLuc2-Reporter vector was transfected using FuGENE HD (FuGENE), Lipofectamine LTX (LTX), X-tremeGENE, or TransIT-2020 transfection reagents. The media was analyzed using a Metridia luciferase (MetLuc) assay. Transfections were performed on cells adherent to plates/dishes (adherent method) or suspended in the media (suspension method). The 201B7 cells transfected with episomal vectors were selected using G418 (200 μg/mL) or hygromycin B (300 μg/mL). Rotary culture was performed at 2.5 or 9.9 rpm. Efficiency of EB formation was compared among plates and dishes. Cell density was compared at 1.6×10(3),×10(4), and×10(5) cells/mL. The suspended method of transfection using the FuGENE HD reagent was the most efficient. The expression of pEBMulti/Met-Hyg was detected 11 days posttransfection. Double transformants were selected 6 days posttransfection with pEBNK/EGFP-Neo and pEBNK/Cherry-Hyg. Both EGFP and CherryPicker were expressed in all of the surviving cells. EBs were formed most efficiently from cells cultured at a density of 1.6×10(5) cells/mL in six-well plates or 6 cm dishes. The selected cells formed EBs. FuGENE-mediated transfection of plasmids using the suspension method was effective in transforming iPS cells. Furthermore, the episomal vectors enabled us to perform a stable double transfection of EB-forming iPS cells.
Collapse
Affiliation(s)
- Minoru Tomizawa
- Department of Gastroenterology, National Hospital Organization, Shimoshizu Hospital, Yotsukaido City, Japan
| | - Fuminobu Shinozaki
- Department of Radiology, National Hospital Organization, Shimoshizu Hospital, Yotsukaido City, Japan
| | - Yasufumi Motoyoshi
- Department of Neurology, National Hospital Organization, Shimoshizu Hospital, Yotsukaido City, Japan
| | - Takao Sugiyama
- Department of Rheumatology, National Hospital Organization, Shimoshizu Hospital, Yotsukaido City, Japan
| | - Shigenori Yamamoto
- Department of Pediatrics, National Hospital Organization, Shimoshizu Hospital, Yotsukaido City, Japan
| | - Makoto Sueishi
- Department of Rheumatology, National Hospital Organization, Shimoshizu Hospital, Yotsukaido City, Japan
| |
Collapse
|
|
13
|
Kondo Y, Yoshihashi S, Mimori K, Ogihara R, Kanehama Y, Maki Y, Enosawa S, Kurose K, Iwao T, Nakamura K, Matsunaga T. Selective culture method for hepatocyte-like cells differentiated from human induced pluripotent stem cells. Drug Metab Pharmacokinet 2014; 29:407-13. [PMID: 24785642 DOI: 10.2133/dmpk.dmpk-14-rg-022] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
This study aimed to establish culture conditions which are able to give the differentiation of induced pluripotent (iPS) cells to hepatocytes. To this end, we examined the usefulness of a culture medium containing the components involved in the intermediary metabolism in the liver. More specifically, we examined the effect of the "modified L-15 medium" containing galactose, phenylalanine and ornitine, but deprived of glucose, tyrosine, arginine and pyruvic acid. The medium was altered according to changes in the expression of enzymes that participate in liver-specific pathways. After 25 days of differentiation, the differentiated cells expressed hepatocyte markers and drug-metabolizing enzymes. These expression levels were increased using modified L-15 medium. The survival of human fetal liver cells and the death of human fibroblasts were observed during culture in modified L-15 medium. Most of the cells that differentiated from human iPS cells using modified L-15 medium were stained by anti-human albumin antibody. These results suggest that iPS cells can be converted to high purity-differentiated hepatocytes by cultivating them in modified L-15 medium.
Collapse
Affiliation(s)
- Yuki Kondo
- Department of Clinical Pharmacy, Graduate School of Pharmaceutical Sciences, Nagoya City University
| | | | | | | | | | | | | | | | | | | | | |
Collapse
|
|
14
|
Cardinale V, Carpino G, Reid LM, Gaudio E, Alvaro D. Notch2 signaling and undifferentiated liver cancers: evidence of hepatic stem/progenitor cell origin. Hepatology 2013; 58:1188. [PMID: 23359130 DOI: 10.1002/hep.26280] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/25/2012] [Accepted: 11/27/2012] [Indexed: 12/14/2022]
Affiliation(s)
- Vincenzo Cardinale
- Department of Medico-Surgical Sciences and Biotechnologies; Polo Pontino; Italy
| | | | - Lola M. Reid
- Department of Cell Biology and Physiology; Program in Molecular Biology and Biotechnology; University of North Carolina School of Medicine; Chapel Hill; NC
| | - Eugenio Gaudio
- Department of Anatomical, Histological, Forensic Medicine, and Orthopedics Sciences Sapienza University of Rome; Rome; Italy
| | | |
Collapse
|
|
15
|
Tomizawa M, Shinozaki F, Sugiyama T, Yamamoto S, Sueishi M, Yoshida T. Survival of primary human hepatocytes and death of induced pluripotent stem cells in media lacking glucose and arginine. PLoS One 2013; 8:e71897. [PMID: 23967260 PMCID: PMC3743790 DOI: 10.1371/journal.pone.0071897] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/01/2013] [Accepted: 07/05/2013] [Indexed: 12/16/2022] Open
Abstract
BACKGROUND Tumorigenicity is an associated risk for transplantation of hepatocytes differentiated from human induced pluripotent stem (hiPS) cells. Hepatocytes express the enzymes galactokinase and ornithine transcarbamylase (OTC) to aid in their own survival. However, hiPS cells do not express these enzymes, and therefore, are not be expected to survive in a medium containing galactose and ornithine and lacking glucose and arginine. MATERIALS AND METHODS Real-time quantitative polymerase chain reaction (PCR) was performed to analyze the expression of galactokinase 1 (GALK1)1 and GALK2, ornithine carbamyltransferase, and phenylalanine hydroxylase (PAH). The hiPS cell line 201B7 was cultured in hepatocyte selection medium (HSM), which lacks glucose and arginine but contains galactose and ornithine. Furthermore, microscopic analysis of the cultured cells was performed after hematoxylin and eosin (H&E) staining, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL). The hiPS cells were immunostained to assess their pluripotency in HSM. In addition, the primary human hepatocytes were cultured with or without hiPS cells in HSM. RESULTS The expression levels of GALK1, GALK2, OTC, and PAH in 201B7 were 22.2±5.0 (average ± standard deviation), 14.2% ±1.1%, 1.2% ±0.2%, and 8.4% ±0.7% respectively, compared with those in the adult liver. The hiPS cell population diminished when cultured in HSM and completely disappeared after 3 days. The cultured cells showed condensation or fragmentation of their nuclei, thereby suggesting apoptosis. TUNEL staining confirmed that the cells had undergone apoptosis. The 201B7 cells were positive for Nanog, SSEA-4, and TRA-1-60. The primary human hepatocytes survived when cultured alone in HSM and when co-cultured with hiPS cells. CONCLUSION Therefore, HSM is and ideal medium for eliminating hiPS cells and purifying hepatocytes without inducing any damage.
Collapse
Affiliation(s)
- Minoru Tomizawa
- Department of Gastroenterology, National Hospital Organization, Shimoshizu Hospital, Yotsukaido City, Chiba, Japan.
| | | | | | | | | | | |
Collapse
|
|
16
|
Shan J, Hamazaki T, Tang TA, Terada N, Kilberg MS. Activation of the amino acid response modulates lineage specification during differentiation of murine embryonic stem cells. Am J Physiol Endocrinol Metab 2013; 305:E325-35. [PMID: 23736538 PMCID: PMC4116408 DOI: 10.1152/ajpendo.00136.2013] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/05/2023]
Abstract
In somatic cells, a collection of signaling pathways activated by amino acid limitation have been identified and referred to as the amino acid response (AAR). Despite the importance of possible detrimental effects of nutrient limitation during in vitro culture, the AAR has not been investigated in embryonic stem cells (ESC). AAR activation caused the expected increase in transcription factors that mediate specific AAR pathways, as well as the induction of asparagine synthetase, a terminal AAR target gene. Neither AAR activation nor stable knockdown of activating transcription factor (Atf) 4, a transcriptional mediator of the AAR, adversely affected ESC self-renewal or pluripotency. Low-level induction of the AAR over a 12-day period of embryoid body differentiation did alter lineage specification such that the primitive endodermal, visceral endodermal, and endodermal lineages were favored, whereas mesodermal and certain ectodermal lineages were suppressed. Knockdown of Atf4 further enhanced the AAR-induced increase in endodermal formation, suggesting that this phenomenon is mediated by an Atf4-independent mechanism. Collectively, the results indicate that, during differentiation of mouse embryoid bodies in culture, the availability of nutrients, such as amino acids, can influence the formation of specific cell lineages.
Collapse
Affiliation(s)
- Jixiu Shan
- Department of Biochemistry and Molecular Biology, McKnight Brain Institute, Shands Cancer Center, and Center for Nutritional Sciences, University of Florida College of Medicine, Gainesville, Florida, USA
| | | | | | | | | |
Collapse
|
|
17
|
Tomizawa M, Shinozaki F, Sugiyama T, Yamamoto S, Sueishi M, Yoshida T. Activin A is essential for Feeder-free culture of human induced pluripotent stem cells. J Cell Biochem 2013; 114:584-588. [PMID: 22991093 DOI: 10.1002/jcb.24395] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/09/2012] [Accepted: 09/07/2012] [Indexed: 01/12/2023]
Abstract
Feeder-free culture of human induced pluripotent stem (hiPS) cells is necessary for their clinical application to avoid adverse effects of foreign proteins. hiPS cells were cultured with combinations of activin (A), CHIR99021 (C), basic fibroblast growth factor (F), and leukemia inhibitory factor (L) under feeder-free conditions. Culture was terminated after 12 passages or when the cell morphology changed from pluripotency. Pluripotency was analyzed by alkaline phosphatase (ALP) staining and immunostaining with antibodies to Oct3/4, Nanog, SSEA4, and TRA-1-60. SB431542 (SB), an activin inhibitor, was added to the culture, and the morphology of the cells was observed. hiPS cells cultured with A, AC, and ACL after 12 passages were positive for ALP staining. Oct3/4 was positive in hiPS cells cultured with A, AC, and ACL. hiPS cells were positive for Nanog when cultured with A and AC; however, Nanog signal was weaker in cells cultured with ACL. SSEA4 was positive in hiPS cells cultured with A and AC but almost negative in those cultured with ACL. hiPS cells were positive for TRA-1-60 when cultured with A, AC, and ACL. hiPS cells lose their undifferentiated morphology at six passages when cultured with A + SB, five passages with AC + SB, and nine passages with ACL. We conclude that feeder-free culture of hiPS cells requires A or AC to maintain pluripotency.
Collapse
Affiliation(s)
- Minoru Tomizawa
- Department of Gastroenterology, National Hospital Organization Shimoshizu Hospital, 934-5 Shikawatashi, Yotsukaido City, Chiba 284-0003, Japan.
| | | | | | | | | | | |
Collapse
|
|
18
|
TOMIZAWA MINORU, SHINOZAKI FUMINOBU, SUGIYAMA TAKAO, YAMAMOTO SHIGENORI, SUEISHI MAKOTO, YOSHIDA TAKANOBU. Single-step protocol for the differentiation of human-induced pluripotent stem cells into hepatic progenitor-like cells. Biomed Rep 2013; 1:18-22. [PMID: 24648886 PMCID: PMC3956730 DOI: 10.3892/br.2012.2] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/28/2012] [Accepted: 07/31/2012] [Indexed: 11/05/2022] Open
Abstract
Induced pluripotent stem (iPS) cells are ideal sources of hepatocyte for transplantation into patients experiencing hepatic failure. Growth and transcription factors were analyzed to design a single-step protocol for the differentiation of iPS cells into hepatocytes. The expression of transcription factors was analyzed using reverse transcription-polymerase chain reaction (RT-PCR) and compared among iPS cells, as well as fetal and adult liver cells. iPS cells were cultured with growth factors and RT-PCR was performed to analyze the expression of transcription factors. iPS cells were introduced with transcription factors, cultured with growth factors and subjected to real-time quantitative PCR. Indocyanine green (ICG) was added to the medium as a hepatocyte marker. Sox17, GATA4, GATA6, FoxA2, HEX, HNF4α and C/EBPα were expressed in fetal and adult liver cells, but not in iPS cells. Sox17, GATA6 and HNF4α were expressed after exposure a combination of oncostatin M, epidermal growth factor, retinoic acid, dexamethasone and ITS (OERDITS). When iPS cells were introduced with FoxA2, GATA4, HEX and C/EBPα and cultured with OERDITS for 8 days, the cells expressed α-fetoprotein, δ-like (Dlk)-1 and γ-glutamyl transpeptidase (GTP), and ICG uptake was observed. Exposure to FoxA2, GATA4, HEX and C/EBPα and culturing with OERDITS supplementation potentially serves as a single-step inducer for the differentiation of iPS cells into hepatic progenitor-like cells within 8 days.
Collapse
Affiliation(s)
| | | | | | | | | | - TAKANOBU YOSHIDA
- Internal Medicine, National Hospital Organization Shimoshizu Hospital, Yotsukaido, Chiba 284-0003,
Japan
| |
Collapse
|
|
19
|
Abstract
Regenerative medicine using stem cells has attracted much attention, since stem cells are responsible for highly proliferative activity and multipotential ability of differentiation. Induced pluripotent stem cells and embryonic stem cells or the adult stem cells such as bone marrow-derived stem cells and adipose tissue-derived stem cells have been expected as a cell source of regenerative medicine. Since differentiating methods of human stem cells into the defined lineage of cells remains to be developed, we focus on the differentiating strategies of pluripotent stem cells and mesenchymal stem cells into liver lineage, especially on cytokine function and gene expression during hepatic differentiation. The survey of previously published papers discloses that the protocols that mimic the liver developmental process seem to be effective in obtaining functional hepatocytes. However, in order to develop hepatic regenerative medicine that is useful in a clinical setting, more effective and potent strategies that obtain mature hepatocytes are required.
Collapse
Affiliation(s)
- Goshi Shiota
- Division of Molecular and Genetic Medicine, Department of Genetic Medicine and Regenerative Therapeutics, Graduate School of Medicine, Tottori University, Yonago, Japan
| | | |
Collapse
|
|
20
|
TOMIZAWA MINORU, SHINOZAKI FUMINOBU, SUGIYAMA TAKAO, YAMAMOTO SHIGENORI, SUEISHI MAKOTO, YOSHIDA TAKANOBU. Activin A maintains pluripotency markers and proliferative potential of human induced pluripotent stem cells. Exp Ther Med 2011; 2:405-408. [PMID: 22977517 PMCID: PMC3440779 DOI: 10.3892/etm.2011.219] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/07/2010] [Accepted: 02/07/2011] [Indexed: 12/31/2022] Open
Abstract
To investigate the role of Activin A in the embryoid bodies (EBs) of human induced pluripotent stem (iPS) cells, EBs were transferred onto dishes coated with Matrigel after 4 days of incubation with Activin A and observed under a microscope. Alkaline phosphatase staining and immunostaining were performed to analyze the pluripotency of the cells, and the MTS assay was performed to analyze their proliferative potential. Fourteen days after EB formation, cells cultured with Activin A (100 ng/ml) showed no morphological alterations. Cells cultured with 10-100 ng/ml of Activin A were positive for alkaline phosphatase staining, while cells cultured with 0-3 ng/ml showed negative staining. Cells cultured with 10 ng/ml of Activin A were positive for Oct3/4, Nanog, SSEA-4 and TRA-1-60, while cells cultured with 0 ng/ml Activin A were negative. Cells cultured with 3-30 ng/ml of Activin A maintained their proliferative potential, while loss of proliferative potential was observed in cells cultured with 100 ng/ml Activin A. In conclusion, Activin A maintained pluripotency markers in human iPS cells cultured as EBs with Activin A.
Collapse
Affiliation(s)
| | | | | | | | | | - TAKANOBU YOSHIDA
- Internal Medicine, National Hospital Organization Shimoshizu Hospital, Yotsukaido, Chiba 284-0003,
Japan
| |
Collapse
|
|
21
|
Snykers S, De Kock J, Rogiers V, Vanhaecke T. In vitro differentiation of embryonic and adult stem cells into hepatocytes: state of the art. Stem Cells 2009; 27:577-605. [PMID: 19056906 PMCID: PMC2729674 DOI: 10.1634/stemcells.2008-0963] [Citation(s) in RCA: 193] [Impact Index Per Article: 12.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
Stem cells are a unique source of self-renewing cells within the human body. Before the end of the last millennium, adult stem cells, in contrast to their embryonic counterparts, were considered to be lineage-restricted cells or incapable of crossing lineage boundaries. However, the unique breakthrough of muscle and liver regeneration by adult bone marrow stem cells at the end of the 1990s ended this long-standing paradigm. Since then, the number of articles reporting the existence of multipotent stem cells in skin, neuronal tissue, adipose tissue, and bone marrow has escalated, giving rise, both in vivo and in vitro, to cell types other than their tissue of origin. The phenomenon of fate reprogrammation and phenotypic diversification remains, though, an enigmatic and rare process. Understanding how to control both proliferation and differentiation of stem cells and their progeny is a challenge in many fields, going from preclinical drug discovery and development to clinical therapy. In this review, we focus on current strategies to differentiate embryonic, mesenchymal(-like), and liver stem/progenitor cells into hepatocytes in vitro. Special attention is paid to intracellular and extracellular signaling, genetic modification, and cell-cell and cell-matrix interactions. In addition, some recommendations are proposed to standardize, optimize, and enrich the in vitro production of hepatocyte-like cells out of stem/progenitor cells.
Collapse
Affiliation(s)
- Sarah Snykers
- Department of Toxicology, Vrije Universiteit Brussel, Belgium.
| | | | | | | |
Collapse
|
|
22
|
Mansuroglu T, Dudás J, Elmaouhoub A, Joza TZ, Ramadori G. Hepatoblast and mesenchymal cell-specific gene-expression in fetal rat liver and in cultured fetal rat liver cells. Histochem Cell Biol 2009; 132:11-9. [PMID: 19381675 PMCID: PMC2693773 DOI: 10.1007/s00418-009-0596-y] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 03/26/2009] [Indexed: 01/13/2023]
Abstract
The aim of this study was to determine whether passaged rat fetal liver cells are functional hepatoblasts. Hepatocyte/hepatoblast- and liver myofibroblast-gene-expressions were studied in adult and fetal rat liver tissues as well as in primary and passaged cultures of isolated rat fetal liver cells at both the mRNA and protein level. Desmin- and Alpha-Smooth Muscle Actin (SMA)-positive cells were located in the walls of liver vessels, whereas Desmin-positive/SMA-negative cells were distributed within the liver parenchyma. Primary cultures contained Prox1-positive hepatoblasts, Desmin/SMA-positive myofibroblasts and only a few Desmin-positive/SMA-negative cells. Albumin and alpha-fetoprotein (AFP) could be detected in the primary cultures and to a lesser extent after the first passage. The number of Desmin-positive/SMA-negative cells decreased with successive passage, such that after the second passage, only Desmin/SMA-positive cells could be detected. SMA-gene-expression increased during the passages, suggesting that myofibroblasts become the major cell population of fetal liver cell cultures over time. This observation needs to be taken into account, should passaged fetal liver cells be used for liver cell transplantation. Moreover it contradicts the concept of epithelial-mesenchymal transformation and suggests rather that selective overgrowth of mesenchymal cells occurs in culture.
Collapse
MESH Headings
- Actins/metabolism
- Animals
- Antigens, Differentiation/metabolism
- Cells, Cultured
- Desmin/metabolism
- Endothelium, Vascular/embryology
- Endothelium, Vascular/growth & development
- Endothelium, Vascular/metabolism
- Female
- Hepatocytes/cytology
- Hepatocytes/metabolism
- Liver/cytology
- Liver/embryology
- Liver/growth & development
- Liver/metabolism
- Mesoderm/cytology
- Mesoderm/embryology
- Mesoderm/growth & development
- Mesoderm/metabolism
- Muscle, Smooth/cytology
- Muscle, Smooth/embryology
- Muscle, Smooth/growth & development
- Muscle, Smooth/metabolism
- Pregnancy
- Rats
- Rats, Wistar
- alpha-Fetoproteins/metabolism
Collapse
Affiliation(s)
- Tümen Mansuroglu
- Department of Internal Medicine, Section of Gastroenterology and Endocrinology, Georg-August-University Göttingen, Robert-Koch-Strasse 40, 37075 Göttingen, Germany
| | - József Dudás
- Department of Internal Medicine, Section of Gastroenterology and Endocrinology, Georg-August-University Göttingen, Robert-Koch-Strasse 40, 37075 Göttingen, Germany
- Department of Otorhinolaryngology, University Hospital Innsbruck, Anichstrasse 35, 6020 Innsbruck, Austria
| | - Abderrahim Elmaouhoub
- Department of Internal Medicine, Section of Gastroenterology and Endocrinology, Georg-August-University Göttingen, Robert-Koch-Strasse 40, 37075 Göttingen, Germany
| | - Tobias Z. Joza
- Department of Internal Medicine, Section of Gastroenterology and Endocrinology, Georg-August-University Göttingen, Robert-Koch-Strasse 40, 37075 Göttingen, Germany
| | - Giuliano Ramadori
- Department of Internal Medicine, Section of Gastroenterology and Endocrinology, Georg-August-University Göttingen, Robert-Koch-Strasse 40, 37075 Göttingen, Germany
| |
Collapse
|