in vitro human pluripotent stem cell differentiation into insulin-producing pancreatic cells

Differentiating Human Pluripotent Stem Cells into Pancreatic ?-Cells

Human pluripotent stem cells (hPSCs) have the unique ability to differentiate into various cell types, making them a viable alternative to human pancreatic &#946;-cells1. These hPSCs are categorized into two types: embryonic stem cells (hESCs), derived from the blastocyst2, and induced pluripotent cells (hiPSCs), generated by reprogramming somatic cells directly3. The development of techniques to differentiate hPSCs into &#946;-cells, has important implications for both fundamental research and clinical practice1,4. Diabetes mellitus is a chronic disease affecting &#62;400 million people worldwide and results from the inability of the body to regulate glycemia due to malfunction or loss of pancreatic &#946;-cells5. The limited availability of pancreatic islet cells for transplantation has hindered the development of cell replacement therapies for diabetes2,4,6,7. The ability to generate glucose-responsive insulin-secreting cells using hPSCs serves as a useful cellular model for studying human islet development and function. It can also be used to test potential therapeutic candidates for diabetes treatment in a controlled environment. Moreover, hPSCs have the potential to produce pancreatic islet cells that are genetically identical to the patient, reducing the risk of immune rejection after transplantation2,4,7.
In recent years, there have been significant advancements in the refinement of hPSC culture and differentiation protocols, resulting in increased efficiency and reproducibility of the differentiation process toward generating pancreatic &#946;-cells8,9.
The following protocol outlines the essential stages of directed differentiation of pancreatic &#946;-cells. It involves the regulation of specific cell signaling pathways at distinct time points. It is based on the protocol developed by Sui L. et al.10 (2018) for the generation of hPSCs into pancreatic &#946;-cells. The protocol was adjusted to recent updates from Sui L. et al.11 (2021), as the latest research emphasizes the significance of using aphidicolin (APH) treatment to enhance the differentiation of &#946;-cells. The current protocol includes the addition of APH to the medium during the later stages of the process. Furthermore, modifications have been made to the composition of the medium during the early stages of differentiation compared to the initial protocol. A notable change is the addition of Keratinocyte Growth Factor (KGF) on Day 6 and continuing until Day 8. The keratinocyte growth factor (KGF) is introduced from day 6 to day 8, which slightly differs from the initial protocol10, where KGF was not included in the stage 4 medium.
The first and essential step in the generation of &#946;-cell-like cells is the directed differentiation of hPSCs into definitive endoderm (DE), a primitive germ layer that gives rise to the epithelial lining of various organs, including the pancreas. After the formation of DE, the cells undergo differentiation into the primitive gut tube, which is followed by the specification of the posterior foregut fate. The posterior foregut then develops into pancreatic progenitor cells, which have the potential to differentiate into all cell types of the pancreas, including the endocrine and exocrine cells. The subsequent stage in the process involves pancreatic endocrine progenitors giving rise to the hormone-secreting cells found in the islets of Langerhans. In the end, the differentiation process reaches its final stage by producing fully functional pancreatic &#946;-cell like cells9,10. It is important to note that this process is complex and often requires optimization of the culture conditions, such as specific growth factors and extracellular matrix components, to improve the efficiency and specificity of differentiation9,10. Furthermore, generating functional &#946;-cell like cells from hPSCs in vitro is still a major challenge. Ongoing research focuses on improving differentiation protocols and enhancing the maturation and function of the resulting &#946;-cells9.
In this protocol, the use of gentle cell dissociation during the culture and passage of hPSCs is essential to maintain cell viability and pluripotency, significantly improving the efficiency of differentiation into pancreatic &#946;-cells. Additionally, each stage-specific medium has been meticulously optimized following the protocol developed by Sui L. et al.10 to promote a high yield of insulin-secreting cells in clusters that closely resemble the human islet.

Author Spotlight: Advancements and Challenges in &amp;#946;-Cells Differentiation from Pluripotent Stem Cells

Optimized Protocol for Generating Functional Pancreatic Insulin-secreting Cells from Human Pluripotent Stem Cells

Attaining a high and consistent efficiency in the differentiation process for converting pluripotent stem cells into human beta cells is a notable challenge. This challenge is influenced by the precision and the consistency with which the experimenter follows the protocol. Moreover, the amount of beta cells obtained in each experiment is quite variable, which may result in the reduced function of beta cells across multiple experiments.In the context of high leptin beta cell research in diabetes, the availability of cadaveric eyelet donors is quite limited. Consequently, the use of stem cell differentiated beta cells is offering an unlimited and abundant supply of insulin secreting cells. Beta cell differentiation is a pivotal advancement in our field, offering numerous benefits.It allows us to explore the development and function of beta cells, leading to a deeper understanding of diabetes causes and beta cell dysfunction. We aim to use this technique for our research focusing on the genetics of diabetes. Our primary objective is to focus on mutation of certain genes that can influence beta cells and eyelet function.So this protocol will facilitate the access to human eyelet derived from pre-potent stem cells with mutations of interest.

Watch this Scientific Journal Video about In Vitro Human Pluripotent Stem Cell Differentiation into Insulin-Producing Pancreatic Cells at JoVE.com

In Vitro Human Pluripotent Stem Cell Differentiation into Insulin-Producing Pancreatic Cells

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Research

JoVE Journal

Biology

338 visualizações.  Cell Biology and Functional Genomics. Antes da passagem celular. revestir uma placa de seis poços com solução de revestimento a frio e incubar a 37 graus Celsius com 5% de dióxido de carbono por uma hora, aspirar o meio de células-tronco da placa de seis poços contendo células-tronco pluripotentes humanas e adicionar um mililitro de DPBS livre de cálcio e magnésio em cada poço. Após a segunda lavagem, adicionar 500 microlitros de solução de dissociação em cada poço e incubar por dois a cinco minutos à temperatura...

Vídeo: Diferenciação in vitro de células-tronco pluripotentes humanas em células pancreáticas produtoras de insulina

Human pluripotent stem cells (hPSCs) can differentiate into any kind of cell, making them an excellent alternative source of human pancreatic &#946;-cells. hPSCs can either be embryonic stem cells (hESCs) derived from the blastocyst or induced pluripotent cells (hiPSCs) generated directly from somatic cells using a reprogramming process. Here a video-based protocol is presented to outline the optimal culture and passage conditions for hPSCs, prior to their differentiation and subsequent generation of insulin-producing pancreatic cells. This methodology follows the six-stage process for &#946;-cell directed differentiation, wherein hPSCs differentiate into definitive endoderm (DE), primitive gut tube, posterior foregut fate, pancreatic progenitors, pancreatic endocrine progenitors, and ultimately pancreatic &#946;-cells. It is noteworthy that this differentiation methodology takes a period of 27 days to generate human pancreatic &#946;-cells. The potential of insulin secretion was evaluated through two experiments, which included immunostaining and glucose-stimulated insulin secretion.

This article presents a protocol for directed differentiation and functional analysis of &#946;-cell like cells. We describe optimal culture conditions and passages for human pluripotent stem cells before generating insulin-producing pancreatic cells. The six-stage differentiation progresses from definitive endoderm formation to functional &#946;-cell like cells secreting insulin in response to glucose.