In Vitro Differentiation of Pluripotent Stem Cells into Functional β Islets Under 2D and 3D Culture Conditions and In Vivo Preclinical Validation of 3D Islets

Abstract

Since the advent of pluripotent stem cells, (embryonic and induced pluripotent stem cells), applications of such pluripotent stem cells are of prime importance. Indeed, scientists are involved in studying the basic biology of pluripotent stem cells, but equal impetus is there to direct the pluripotent stem cells into multiple lineages for cell therapy applications. Scientists across the globe have been successful, to a certain extent, in obtaining cells of definitive endoderm and also pancreatic β islets by differentiating human pluripotent stem cells. Pluripotent stem cell differentiation protocols aim at mimicking in vivo embryonic development. As in vivo embryonic development is a complex process and involves interplay of multiple cytokines, the differentiation protocols also involve a stepwise use of multiple cytokines. Indeed the novel markers for pancreas organogenesis serve as the roadmaps to develop new protocols for pancreatic differentiation from pluripotent stem cells. Earliest developed protocols for pancreas differentiation involved "Nestin selection pathway," a pathway common for both neuronal and pancreatic differentiation lead to the generation of cells that were a combination of cells from neuronal lineage. Eventually with the discovery of hierarchy of β cell transcription factors like Pdx1, Pax4, and Nkx2.2, forced expression of such transcription factors proved successful in converting a pluripotent stem cell into a β cell. Protocols developed almost half a decade ago to the recent ones rather involve stepwise differentiations involving various cytokines and could generate as high as 25 % functional insulin-positive cells in vitro. Most advanced protocols for β islet differentiations from human pluripotent stem cells focused on 3D culture conditions, which reportedly produced 60-65 % functional β islet cells. Here, we describe the protocol for differentiation of human pluripotent stem cells into functional β cells under both 2D and 3D culture conditions.

Atrial-like Engineered Heart Tissue: An In Vitro Model of the Human Atrium

“…CMs generated from human induced pluripotent stem cells (hiPSCs) may offer a platform to study disease mechanism and evaluate novel drugs. However, hiPSC-CMs are believed to consist predominantly of ventricular-like cells with a small percentage of atrial-like and nodal-like cells (van den Berg et al., 2016, Blazeski et al., 2012, Ma et al., 2011, Marczenke et al., 2017). Recent developments therefore aim at establishing hiPSC-derived models of predominantly atrial-like myocytes.…”

Pdx1 expression up-regulated by Tet-on system induces the mouse embryonic stem cells to pancreatic-like cells through Notch pathway

“…Our study indicates that Pdx1 can activate downstream target genes to induce differentiation into pancreatic cells. In recent years, ESCs have been induced to differentiate into insulin-secreting cells and islet-like cells [33,34] in vitro. Our results revealed that pancreatic precursor cells (Pdx1 + cells) can differentiate into several types of pancreatic-like cells: exocrine cells and ductal cells during the early stage and endocrine cells at a later stage.…”

Culture medium, extracellular components and differentiation in culturing of human pluripotent stem cells

“…If appropriate facilities, equipment, sterile reagents and aseptic techniques are used, there is no need to use antibiotics during cell structure (single layer /2D vs. suspension/3D), pathway modulator and a transcriptional memory of the cells may also affect differentiation [9][10][11].…”