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 10 Technology 1004 Medical Biotechnology

Hideomi Ida, Tomohiko Akiyama, Keiichiro Ishiguro, Sravan K. Goparaju, Yuki Nakatake, Nana Chikazawa-Nohtomi, Saeko Sato, Hiromi Kimura, Yukihiro Yokoyama, Masato Nagino, Minoru Ko, Shigeru Ko

Research output: Contribution to journalArticle

3 Citations (Scopus)

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.

Original languageEnglish
Article number277
JournalStem Cell Research and Therapy
Volume9
Issue number1
DOIs
Publication statusPublished - 2018 Oct 25

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Endocrine Cells
Biotechnology
Stem cells
Transcription Factors
Cells
Technology
Messenger RNA
Transfection
Genes
Hormones
Insulin
Medical problems
Cultured Cells
Somatostatin
Glucagon
Pancreatic Hormones
Induced Pluripotent Stem Cells
Polymerase Chain Reaction
Lentivirus
Pluripotent Stem Cells

Keywords

  • Embryonic stem cells
  • Endocrine differentiation
  • NKX6.1
  • Pancreatic β cells
  • PDX1
  • siPOU5F1
  • Synthetic mRNAs
  • Transcription factors

ASJC Scopus subject areas

  • Medicine (miscellaneous)
  • Molecular Medicine
  • Biochemistry, Genetics and Molecular Biology (miscellaneous)
  • Cell Biology

Cite this

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 10 Technology 1004 Medical Biotechnology. / Ida, Hideomi; Akiyama, Tomohiko; Ishiguro, Keiichiro; Goparaju, Sravan K.; Nakatake, Yuki; Chikazawa-Nohtomi, Nana; Sato, Saeko; Kimura, Hiromi; Yokoyama, Yukihiro; Nagino, Masato; Ko, Minoru; Ko, Shigeru.

In: Stem Cell Research and Therapy, Vol. 9, No. 1, 277, 25.10.2018.

Research output: Contribution to journalArticle

Ida, Hideomi ; Akiyama, Tomohiko ; Ishiguro, Keiichiro ; Goparaju, Sravan K. ; Nakatake, Yuki ; Chikazawa-Nohtomi, Nana ; Sato, Saeko ; Kimura, Hiromi ; Yokoyama, Yukihiro ; Nagino, Masato ; Ko, Minoru ; Ko, Shigeru. / 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 10 Technology 1004 Medical Biotechnology. In: Stem Cell Research and Therapy. 2018 ; Vol. 9, No. 1.
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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.",
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T1 - 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 10 Technology 1004 Medical Biotechnology

AU - Ida, Hideomi

AU - Akiyama, Tomohiko

AU - Ishiguro, Keiichiro

AU - Goparaju, Sravan K.

AU - Nakatake, Yuki

AU - Chikazawa-Nohtomi, Nana

AU - Sato, Saeko

AU - Kimura, Hiromi

AU - Yokoyama, Yukihiro

AU - Nagino, Masato

AU - Ko, Minoru

AU - Ko, Shigeru

PY - 2018/10/25

Y1 - 2018/10/25

N2 - 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.

AB - 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.

KW - Embryonic stem cells

KW - Endocrine differentiation

KW - NKX6.1

KW - Pancreatic β cells

KW - PDX1

KW - siPOU5F1

KW - Synthetic mRNAs

KW - Transcription factors

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