To understand organ function, it is important to have an inventory of its cell types and of their corresponding marker genes. This is a particularly challenging task for human tissues like the pancreas, because reliable markers are limited. Hence, transcriptome-wide studies are typically done on pooled islets of Langerhans, obscuring contributions from rare cell types and of potential subpopulations. To overcome this challenge, we developed an automated platform that uses FACS, robotics, and the CEL-Seq2 protocol to obtain the transcriptomes of thousands of single pancreatic cells from deceased organ donors, allowing in silico purification of all main pancreatic cell types. We identify cell type-specific transcription factors and a subpopulation of REG3A-positive acinar cells. We also show that CD24 and TM4SF4 expression can be used to sort live alpha and beta cells with high purity. This resource will be useful for developing a deeper understanding of pancreatic biology and pathophysiology of diabetes mellitus.

Loss of pancreatic islet β-cell mass and β-cell dysfunction are central in the development of type 2 diabetes (T2DM). We recently showed that mature human insulin-containing β-cells can convert into glucagon-containing α-cells ex vivo. This loss of β-cell identity was characterized by the presence of β-cell transcription factors (Nkx6.1, Pdx1) in glucagon⁺ cells. Here, we investigated whether loss of β-cell identity also occurs in vivo and whether it is related to the presence of (pre)diabetes in humans and non-human primates. We observed an 8x increased frequency of insulin⁺ cells co-expressing glucagon in diabetic donors. Up to 5% of the cells that were Nkx6.1⁺ but insulin^- co-expressed glucagon, which represents a 5x increased frequency compared to the control group. This increase in bihormonal and Nkx6.1⁺glucagon⁺insulin^- cells was also found in islets of diabetic macaques. The higher proportion of bihormonal cells and Nkx6.1⁺glucagon⁺insulin^- cells in diabetic macaques and humans was correlated with the presence and extent of islet amyloidosis. These data indicate that loss of β-cell identity occurs in T2DM and could contribute to the decrease of functional β-cell mass. Maintenance of β-cell identity is a potential novel strategy to preserve β-cell function in diabetes.

Generating an unlimited source of human insulin-producing cells is a prerequisite to advance β cell replacement therapy for diabetes. Here, we describe a 3D culture system that supports the expansion of adult human pancreatic tissue and the generation of a cell subpopulation with progenitor characteristics. These cells display high aldehyde dehydrogenase activity (ALDHhi), express pancreatic progenitors markers (PDX1, PTF1A, CPA1, and MYC), and can form new organoids in contrast to ALDHlo cells. Interestingly, gene expression profiling revealed that ALDHhi cells are closer to human fetal pancreatic tissue compared with adult pancreatic tissue. Endocrine lineage markers were detected upon in vitro differentiation. Engrafted organoids differentiated toward insulin-positive (INS+) cells, and circulating human C-peptide was detected upon glucose challenge 1 month after transplantation. Engrafted ALDHhi cells formed INS+ cells. We conclude that adult human pancreatic tissue has potential for expansion into 3D structures harboring progenitor cells with endocrine differentiation potential.

Obesity is associated with many serious afflictions such as cardiovascular disease, cancer, and diabetes. One of the main cellular systems used to study the underlying physiological and biological processes is the 3T3-L1 preadipocyte differentiation model. However, studies on 3T3-L1 adipocytes are hampered by the fact that genetic modification of mature adipocytes is notoriously difficult. In this report, we evaluated the use of lentivirus-mediated gene transfer into 3T3-L1 mature adipocytes. We demonstrate that quiescent, fully differentiated 3T3-L1 adipocytes as well as 3T3-L1 preadipocytes can be efficiently transduced with HIV-1-derived lentiviral vectors. Upon transduction using LV-PGK-GFP lentiviral vector at 100 ng p24 per 10(5) cells, more than 95% of the 3T3-L1 adipocytes in the culture expressed the GFP reporter gene. There were no overt signs of toxicity or cytopathogenicity in the cultures. Furthermore, modification of undifferentiated preadipocytes did not affect their capacity to differentiate. In addition, insulin-induced glucose uptake was not affected by the procedure. In contrast, adenoviral-mediated gene transfer into 3T3-L1 adipocytes is associated with marked cytopathogenicity. From these data, we conclude that lentiviral vectors are the gene-transfer system of choice for genetic modification of mature adipocytes. The availability of an efficient vector system may stimulate the use of adipose tissue as a target for gene therapy in obesity and other disorders.

High-fat, low-carbohydrate ketogenic diets (KD) are used for weight loss and for treatment of refractory epilepsy. Recently, short-time studies in rodents have shown that, besides their beneficial effect on body weight, KD lead to glucose intolerance and insulin resistance. However, the long-term effects on pancreatic endocrine cells are unknown. In this study we investigate the effects of long-term KD on glucose tolerance and β- and α-cell mass in mice. Despite an initial weight loss, KD did not result in weight loss after 22 wk. Plasma markers associated with dyslipidemia and inflammation (cholesterol, triglycerides, leptin, monocyte chemotactic protein-1, IL-1β, and IL-6) were increased, and KD-fed mice showed signs of hepatic steatosis after 22 wk of diet. Long-term KD resulted in glucose intolerance that was associated with insufficient insulin secretion from β-cells. After 22 wk, insulin-stimulated glucose uptake was reduced. A reduction in β-cell mass was observed in KD-fed mice together with an increased number of smaller islets. Also α-cell mass was markedly decreased, resulting in a lower α- to β-cell ratio. Our data show that long-term KD causes dyslipidemia, a proinflammatory state, signs of hepatic steatosis, glucose intolerance, and a reduction in β- and α-cell mass, but no weight loss. This indicates that long-term high-fat, low-carbohydrate KD lead to features that are also associated with the metabolic syndrome and an increased risk for type 2 diabetes in humans.