Directed differentiation of human iPSC into insulin producing cells is improved by induced expression of PDX1 and NKX6.1 factors in IPC progenitors

Journal of Translational Medicine, Dec 2016

Background Induced pluripotent stem cells (iPSC) possess an enormous potential as both, scientific and therapeutic tools. Their application in the regenerative medicine provides new treatment opportunities for numerous diseases, including type 1 diabetes. In this work we aimed to derive insulin producing cells (IPC) from iPS cells established in defined conditions. Methods We optimized iPSC generation protocol and created pluripotent cell lines with stably integrated PDX1 and NKX6.1 transgenes under the transcriptional control of doxycycline-inducible promoter. These cells were differentiated using small chemical molecules and recombinant Activin A in the sequential process through the definitive endoderm, pancreatic progenitor cells and insulin producing cells. Efficiency of the procedure was assessed by quantitative gene expression measurements, immunocytochemical stainings and functional assays for insulin secretion. Results Generated cells displayed molecular markers characteristic for respective steps of the differentiation. The obtained IPC secreted insulin and produced C-peptide with significantly higher hormone release level in case of the combined expression of PDX1 and NKX6.1 induced at the last stage of the differentiation. Conclusions Efficiency of differentiation of iPSC to IPC can be increased by concurrent expression of PDX1 and NKX6.1 during progenitor cells maturation. Protocols established in our study allow for iPSC generation and derivation of IPC in chemically defined conditions free from animal-derived components, which is of the utmost importance in the light of their prospective applications in the field of regenerative medicine.

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Directed differentiation of human iPSC into insulin producing cells is improved by induced expression of PDX1 and NKX6.1 factors in IPC progenitors

Walczak et al. J Transl Med Directed differentiation of human iPSC into insulin producing cells is improved by induced expression of PDX1 and NKX6.1 factors in IPC progenitors Maciej P. Walczak 0 3 Anna M. Drozd 0 3 Ewelina Stoczynska‑Fidelus 0 2 3 Piotr Rieske 0 1 2 3 Dawid P. Grzela 0 3 0 Department of Research and Development, Celther Polska Ltd. , Milionowa 23, 93‐193 Łódź , Poland 1 Research and Development Unit, Personather Ltd. , Milionowa 23, 93‐193 Łódź , Poland 2 Department of Tumor Biology, Medical University of Łódź , Żeligowskiego 7/9, 90‐752 Łódź , Poland 3 Department of Research and Development, Celther Polska Ltd. , Milionowa 23, 93‐193 Łódź , Poland Background: Induced pluripotent stem cells (iPSC) possess an enormous potential as both, scientific and therapeutic tools. Their application in the regenerative medicine provides new treatment opportunities for numerous diseases, including type 1 diabetes. In this work we aimed to derive insulin producing cells (IPC) from iPS cells established in defined conditions. Methods: We optimized iPSC generation protocol and created pluripotent cell lines with stably integrated PDX1 and NKX6.1 transgenes under the transcriptional control of doxycycline‑ inducible promoter. These cells were differentiated using small chemical molecules and recombinant Activin A in the sequential process through the definitive endoderm, pancreatic progenitor cells and insulin producing cells. Efficiency of the procedure was assessed by quantitative gene expression measurements, immunocytochemical stainings and functional assays for insulin secretion. Results: Generated cells displayed molecular markers characteristic for respective steps of the differentiation. The obtained IPC secreted insulin and produced C‑ peptide with significantly higher hormone release level in case of the combined expression of PDX1 and NKX6.1 induced at the last stage of the differentiation. Conclusions: Efficiency of differentiation of iPSC to IPC can be increased by concurrent expression of PDX1 and NKX6.1 during progenitor cells maturation. Protocols established in our study allow for iPSC generation and derivation of IPC in chemically defined conditions free from animal‑ derived components, which is of the utmost importance in the light of their prospective applications in the field of regenerative medicine. Defined culture conditions; Diabetes; Differentiation; Induced pluripotent stem cells; Insulin producing cells; NKX6; 1; PDX1; Reprogramming - Type 1 diabetes (T1D) is one of the most frequent chronic autoimmune diseases diagnosed among juveniles, and its global incidence continues to rise [1]. This condition is characterized by pancreatic beta cell damage leading to insufficient insulin production and altered carbohydrate metabolism. It develops at an early age and requires constant treatment, which generates substantial costs and lowers quality of life. Commonly, therapy of type 1 diabetes is based on supplementation of deficient hormone in form of regular injections. However, this method does not address the cause of the disease, regarded as a lack of functional intrinsic mechanisms providing carbohydrate homeostasis. Pancreatic islet transplantation is another approach to T1D treatment. Its application is however limited due to complex medical procedure and shortage in the number of islets donors. Moreover, it is not a permanent solution since patients who are forced to undergo several © The Author(s) 2016. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. procedures eventually need to receive insulin injections due to the destruction of the graft by the immune system [2]. A concept of a therapy based on differentiated induced pluripotent stem cells (iPSC) raises great promises in the field of T1D treatment. These cells are generated from somatic cells by forced expression of transcription factors characteristic for embryonic stem cells [3]. Numerous investigations have been conducted to confirm resemblance between iPSC and embryonic stem cells (ESC) isolated from the inner cell mass of developing embryo [4, 5]. These cells are characterized by features of great importance in terms of disease treatment, such as the fact that they are obtained from patient’s own cells. This trait eliminates risk of potential stem cell-based graft rejection [6]. A number of research teams are focused on generation of functional insulin producing cells (IPC) that could (...truncated)


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Maciej Walczak, Anna Drozd, Ewelina Stoczynska-Fidelus, Piotr Rieske, Dawid Grzela. Directed differentiation of human iPSC into insulin producing cells is improved by induced expression of PDX1 and NKX6.1 factors in IPC progenitors, Journal of Translational Medicine, 2016, pp. 341, 14, DOI: 10.1186/s12967-016-1097-0