Establishment of a rapid and footprint-free protocol for differentiation of human embryonic stem cells into pancreatic endocrine cells with synthetic mRNAs encoding transcription factors
Abstract Background Transplantation of pancreatic β cells generated in vitro from pluripotent stem cells (hPSCs) such as embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) has been proposed as an alternative therapy for diabetes. Though many differentiation protocols have been dev...
| Main Authors: | , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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BMC
2018-10-01
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| Series: | Stem Cell Research & Therapy |
| Subjects: | |
| Online Access: | http://link.springer.com/article/10.1186/s13287-018-1038-3 |
| _version_ | 1828826050496299008 |
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| author | Hideomi Ida Tomohiko Akiyama Keiichiro Ishiguro Sravan K. Goparaju Yuhki Nakatake Nana Chikazawa-Nohtomi Saeko Sato Hiromi Kimura Yukihiro Yokoyama Masato Nagino Minoru S. H. Ko Shigeru B. H. Ko |
| author_facet | Hideomi Ida Tomohiko Akiyama Keiichiro Ishiguro Sravan K. Goparaju Yuhki Nakatake Nana Chikazawa-Nohtomi Saeko Sato Hiromi Kimura Yukihiro Yokoyama Masato Nagino Minoru S. H. Ko Shigeru B. H. Ko |
| author_sort | Hideomi Ida |
| collection | DOAJ |
| description | Abstract Background Transplantation of pancreatic β cells generated in vitro from pluripotent stem cells (hPSCs) such as embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) has been proposed as an alternative therapy for diabetes. Though many differentiation protocols have been developed for this purpose, lentivirus-mediated forced expression of transcription factors (TF)—PDX1 and NKX6.1—has been at the forefront for its relatively fast and straightforward approach. However, considering that such cells will be used for therapeutic purposes in the future, it is desirable to develop a procedure that does not leave any footprint on the genome, as any changes of DNAs could potentially be a source of unintended, concerning effects such as tumorigenicity. In this study, we attempted to establish a novel protocol for rapid and footprint-free hESC differentiation into a pancreatic endocrine lineage by using synthetic mRNAs (synRNAs) encoding PDX1 and NKX6.1. We also tested whether siPOU5F1, which reduces the expression of pluripotency gene POU5F1 (also known as OCT4), can enhance differentiation as reported previously for mesoderm and endoderm lineages. Methods synRNA-PDX1 and synRNA-NKX6.1 were synthesized in vitro and were transfected five times to hESCs with a lipofection reagent in a modified differentiation culture condition. siPOU5F1 was included only in the first transfection. Subsequently, cells were seeded onto a low attachment plate and aggregated by an orbital shaker. At day 13, the degree of differentiation was assessed by quantitative RT-PCR (qRT-PCR) and immunohistochemistry for endocrine hormones such as insulin, glucagon, and somatostatin. Results Both PDX1 and NKX6.1 expression were detected in cells co-transfected with synRNA-PDX1 and synRNA-NKX6.1 at day 3. Expression levels of insulin in the transfected cells at day 13 were 450 times and 14 times higher by qRT-PCR compared to the levels at day 0 and in cells cultured without synRNA transfection, respectively. Immunohistochemically, pancreatic endocrine hormones were not detected in cells cultured without synRNA transfection but were highly expressed in cells transfected with synRNA-PDX1, synRNA-NKX6.1, and siPOU5F1 at as early as day 13. Conclusions In this study, we report a novel protocol for rapid and footprint-free differentiation of hESCs to endocrine cells. |
| first_indexed | 2024-12-12T14:30:55Z |
| format | Article |
| id | doaj.art-2d82226ca15a41c395522aebc3a4c712 |
| institution | Directory Open Access Journal |
| issn | 1757-6512 |
| language | English |
| last_indexed | 2024-12-12T14:30:55Z |
| publishDate | 2018-10-01 |
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| spelling | doaj.art-2d82226ca15a41c395522aebc3a4c7122022-12-22T00:21:32ZengBMCStem Cell Research & Therapy1757-65122018-10-019111010.1186/s13287-018-1038-3Establishment of a rapid and footprint-free protocol for differentiation of human embryonic stem cells into pancreatic endocrine cells with synthetic mRNAs encoding transcription factorsHideomi Ida0Tomohiko Akiyama1Keiichiro Ishiguro2Sravan K. Goparaju3Yuhki Nakatake4Nana Chikazawa-Nohtomi5Saeko Sato6Hiromi Kimura7Yukihiro Yokoyama8Masato Nagino9Minoru S. H. Ko10Shigeru B. H. Ko11Department of Systems Medicine, Keio University School of MedicineDepartment of Systems Medicine, Keio University School of MedicineDepartment of Systems Medicine, Keio University School of MedicineDepartment of Systems Medicine, Keio University School of MedicineDepartment of Systems Medicine, Keio University School of MedicineDepartment of Systems Medicine, Keio University School of MedicineDepartment of Systems Medicine, Keio University School of MedicineDepartment of Systems Medicine, Keio University School of MedicineDivision of Surgical Oncology, Department of Surgery, Nagoya University Graduate School of MedicineDivision of Surgical Oncology, Department of Surgery, Nagoya University Graduate School of MedicineDepartment of Systems Medicine, Keio University School of MedicineDepartment of Systems Medicine, Keio University School of MedicineAbstract Background Transplantation of pancreatic β cells generated in vitro from pluripotent stem cells (hPSCs) such as embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) has been proposed as an alternative therapy for diabetes. Though many differentiation protocols have been developed for this purpose, lentivirus-mediated forced expression of transcription factors (TF)—PDX1 and NKX6.1—has been at the forefront for its relatively fast and straightforward approach. However, considering that such cells will be used for therapeutic purposes in the future, it is desirable to develop a procedure that does not leave any footprint on the genome, as any changes of DNAs could potentially be a source of unintended, concerning effects such as tumorigenicity. In this study, we attempted to establish a novel protocol for rapid and footprint-free hESC differentiation into a pancreatic endocrine lineage by using synthetic mRNAs (synRNAs) encoding PDX1 and NKX6.1. We also tested whether siPOU5F1, which reduces the expression of pluripotency gene POU5F1 (also known as OCT4), can enhance differentiation as reported previously for mesoderm and endoderm lineages. Methods synRNA-PDX1 and synRNA-NKX6.1 were synthesized in vitro and were transfected five times to hESCs with a lipofection reagent in a modified differentiation culture condition. siPOU5F1 was included only in the first transfection. Subsequently, cells were seeded onto a low attachment plate and aggregated by an orbital shaker. At day 13, the degree of differentiation was assessed by quantitative RT-PCR (qRT-PCR) and immunohistochemistry for endocrine hormones such as insulin, glucagon, and somatostatin. Results Both PDX1 and NKX6.1 expression were detected in cells co-transfected with synRNA-PDX1 and synRNA-NKX6.1 at day 3. Expression levels of insulin in the transfected cells at day 13 were 450 times and 14 times higher by qRT-PCR compared to the levels at day 0 and in cells cultured without synRNA transfection, respectively. Immunohistochemically, pancreatic endocrine hormones were not detected in cells cultured without synRNA transfection but were highly expressed in cells transfected with synRNA-PDX1, synRNA-NKX6.1, and siPOU5F1 at as early as day 13. Conclusions In this study, we report a novel protocol for rapid and footprint-free differentiation of hESCs to endocrine cells.http://link.springer.com/article/10.1186/s13287-018-1038-3Embryonic stem cellsEndocrine differentiationTranscription factorsSynthetic mRNAsPancreatic β cellsPDX1 |
| spellingShingle | Hideomi Ida Tomohiko Akiyama Keiichiro Ishiguro Sravan K. Goparaju Yuhki Nakatake Nana Chikazawa-Nohtomi Saeko Sato Hiromi Kimura Yukihiro Yokoyama Masato Nagino Minoru S. H. Ko Shigeru B. H. Ko Establishment of a rapid and footprint-free protocol for differentiation of human embryonic stem cells into pancreatic endocrine cells with synthetic mRNAs encoding transcription factors Stem Cell Research & Therapy Embryonic stem cells Endocrine differentiation Transcription factors Synthetic mRNAs Pancreatic β cells PDX1 |
| title | Establishment of a rapid and footprint-free protocol for differentiation of human embryonic stem cells into pancreatic endocrine cells with synthetic mRNAs encoding transcription factors |
| title_full | Establishment of a rapid and footprint-free protocol for differentiation of human embryonic stem cells into pancreatic endocrine cells with synthetic mRNAs encoding transcription factors |
| title_fullStr | Establishment of a rapid and footprint-free protocol for differentiation of human embryonic stem cells into pancreatic endocrine cells with synthetic mRNAs encoding transcription factors |
| title_full_unstemmed | Establishment of a rapid and footprint-free protocol for differentiation of human embryonic stem cells into pancreatic endocrine cells with synthetic mRNAs encoding transcription factors |
| title_short | Establishment of a rapid and footprint-free protocol for differentiation of human embryonic stem cells into pancreatic endocrine cells with synthetic mRNAs encoding transcription factors |
| title_sort | establishment of a rapid and footprint free protocol for differentiation of human embryonic stem cells into pancreatic endocrine cells with synthetic mrnas encoding transcription factors |
| topic | Embryonic stem cells Endocrine differentiation Transcription factors Synthetic mRNAs Pancreatic β cells PDX1 |
| url | http://link.springer.com/article/10.1186/s13287-018-1038-3 |
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