ArticleStem cell research & therapy2018
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.
Article in Stem cell research & therapy, 2018. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 8 papers.
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Who cites it
8 citing papers in PubMed, 13 citations in OpenAlex.
- Epigenetic control of PDX1 and NGN3 by a computationally designed PRC2 inhibitor enforces pancreatic endocrine differentiation from pluripotent stem cells.Research square · 2026Article
- Oxygenated Scaffolds for Pancreatic Endocrine Differentiation from Induced Pluripotent Stem Cells.Advanced healthcare materials · 2024Article
- Making human pancreatic islet organoids: Progresses on the cell origins, biomaterials and three-dimensional technologies.Theranostics · 2022Review
- A simple method of hiPSCs differentiation into insulin-producing cells is improved with vitamin C and RepSox.PloS one · 2021Article
- Signaling Molecules Regulating Pancreatic Endocrine Development from Pluripotent Stem Cell Differentiation.International journal of molecular sciences · 2020Review
- An Efficient and Footprint-Free Protocol for the Transdifferentiation of Hepatocytes Into Insulin-Producing Cells With IVT mRNAs.Frontiers in genetics · 2020Article
- Directed Differentiation of Pluripotent Stem Cells by Transcription Factors.Molecules and cells · 2019Review
- Stem cells as a potential therapy for diabetes mellitus: a call-to-action in Latin America.Diabetology & metabolic syndrome · 2019Review
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Authors and funding
12 authors at 2 institutions in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
backgroundTransplantation 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.
methodssynRNA-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.
resultsBoth 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.
conclusionsIn this study, we report a novel protocol for rapid and footprint-free differentiation of hESCs to endocrine cells.
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