Loss of pancreatic islet b-cell mass and b-cell dysfunction are central in the development of type 2 diabetes (T2DM). We recently showed that mature human insulincontaining b-cells can convert into glucagon-containing a-cells ex vivo. This loss of b-cell identity was characterized by the presence of b-cell transcription factors (Nkx6.1, Pdx1) in glucagon + cells. Here, we investigated whether the loss of b-cell identity also occurs in vivo, and whether it is related to the presence of (pre)diabetes in humans and nonhuman primates. We observed an eight times increased frequency of insulin + cells coexpressing glucagon in donors with diabetes. Up to 5% of the cells that were Nkx6.1 + but insulin 2 coexpressed glucagon, which represents a five times increased frequency compared with the control group. This increase in bihormonal and Nkx6.1 + glucagon + insulin 2 cells was also found in islets of diabetic macaques. The higher proportion of bihormonal cells and Nkx6.1 + glucagon + insulin 2 cells in macaques and humans with diabetes was correlated with the presence and extent of islet amyloidosis. These data indicate that the loss of b-cell identity occurs in T2DM and could contribute to the decrease of functional b-cell mass. Maintenance of b-cell identity is a potential novel strategy to preserve b-cell function in diabetes.Loss of pancreatic b-cell mass and b-cell dysfunction are central in the development of type 2 diabetes (T2DM) and, in combination with peripheral insulin resistance, lead to hyperglycemia (1). Whereas b-cells, on the one hand, fail to properly secrete insulin at a given glucose level, there is also a progressive decline in the number of b-cells (2,3). Loss of b-cell mass has been ascribed to increased apoptosis in T2DM (4). In patients with T2DM, b-cell mass can be up to 40-60% lower than in healthy control subjects (4-6). In addition, abnormal function of glucagon-producing a-cells leading to hyperglucagonemia is associated with T2DM (7). b-cell dedifferentiation and subsequent transition to other islet cell types were suggested as an alternative explanation for the loss of functional b-cell mass in mice (8,9). In this concept, b-cells lose insulin content and insulin secretory capacity followed by the production of other endocrine hormones such as glucagon (8). We recently showed (10) that loss of b-cell identity with the conversion of b-cells into glucagon-containing a-cells can occur in human pancreatic islets ex vivo.A number of transcription factors have been identified to be essential for the development and maintenance of functional b-cells (11,12). Recent reports (13,14) indicate that a selective loss of transcription factors MafA, Nkx6.1, and Pdx1 is associated with b-cell dysfunction and T2DM. Chronic hyperglycemia in rats is accompanied by the loss of b-cell transcription factors (15). Moreover, mouse b-cells that genetically lack FOXO1 can dedifferentiate in vivo under conditions of metabolic stress and subsequently can convert (or transdifferentiate) into glucagonproducing a-cells...

Conversion of one terminally differentiated cell type into another (or transdifferentiation) usually requires the forced expression of key transcription factors. We examined the plasticity of human insulin-producing β-cells in a model of islet cell aggregate formation. Here, we show that primary human β-cells can undergo a conversion into glucagon-producing α-cells without introduction of any genetic modification. The process occurs within days as revealed by lentivirus-mediated β-cell lineage tracing. Converted cells are indistinguishable from native α-cells based on ultrastructural morphology and maintain their α-cell phenotype after transplantation in vivo. Transition of β-cells into α-cells occurs after β-cell degranulation and is characterized by the presence of β-cell–specific transcription factors Pdx1 and Nkx6.1 in glucagon+ cells. Finally, we show that lentivirus-mediated knockdown of Arx, a determinant of the α-cell lineage, inhibits the conversion. Our findings reveal an unknown plasticity of human adult endocrine cells that can be modulated. This endocrine cell plasticity could have implications for islet development, (patho)physiology, and regeneration.

Clinical islet transplantation is a promising treatment for patients with type 1 diabetes. However, pancreatic islets vary in size and shape affecting their survival and function after transplantation because of mass transport limitations. To reduce diffusion restrictions and improve islet cell survival, the generation of islets with optimal dimensions by dispersion followed by reassembly of islet cells, can help limit the length of diffusion pathways. This study describes a microwell platform that supports the controlled and reproducible production of three-dimensional pancreatic cell clusters of human donor islets. We observed that primary human islet cell aggregates with a diameter of 100–150 μm consisting of about 1000 cells best resembled intact pancreatic islets as they showed low apoptotic cell death (<2%), comparable glucose-responsiveness and increasing PDX1, MAFA and INSULIN gene expression with increasing aggregate size. The re-associated human islet cells showed an a-typical core shell configuration with beta cells predominantly on the outside unlike human islets, which became more randomized after implantation similar to native human islets. After transplantation of these islet cell aggregates under the kidney capsule of immunodeficient mice, human C-peptide was detected in the serum indicating that beta cells retained their endocrine function similar to human islets. The agarose microwell platform was shown to be an easy and very reproducible method to aggregate pancreatic islet cells with high accuracy providing a reliable tool to study cell–cell interactions between insuloma and/or primary islet cells.

BackgroundThe generation of reporter lines for cell identity, lineage, and physiologic state has provided a powerful tool in advancing the dissection of mouse kidney morphogenesis at a molecular level. Although use of this approach is not an option for studying human development in vivo, its application in human induced pluripotent stem cells (iPSCs) is now feasible.MethodsWe used CRISPR/Cas9 gene editing to generate ten fluorescence reporter iPSC lines designed to identify nephron progenitors, podocytes, proximal and distal nephron, and ureteric epithelium. Directed differentiation to kidney organoids was performed according to published protocols. Using immunofluorescence and live confocal microscopy, flow cytometry, and cell sorting techniques, we investigated organoid patterning and reporter expression characteristics.ResultsEach iPSC reporter line formed well patterned kidney organoids. All reporter lines showed congruence of endogenous gene and protein expression, enabling isolation and characterization of kidney cell types of interest. We also demonstrated successful application of reporter lines for time-lapse imaging and mouse transplantation experiments.ConclusionsWe generated, validated, and applied a suite of fluorescence iPSC reporter lines for the study of morphogenesis within human kidney organoids. This fluorescent iPSC reporter toolbox enables the visualization and isolation of key populations in forming kidney organoids, facilitating a range of applications, including cellular isolation, time-lapse imaging, protocol optimization, and lineage-tracing approaches. These tools offer promise for enhancing our understanding of this model system and its correspondence with human kidney morphogenesis.