Jessica K. Fox

Enrichment of human embryonic stem cell-derived NKX6.1-expressing pancreatic progenitor cells accelerates the maturation of insulin-secreting cells in vivo.

Human embryonic stem cells (hESCs) are considered a potential alternative to cadaveric islets as a source of transplantable cells for treating patients with diabetes. We previously described a differentiation protocol to generate pancreatic progenitor cells from hESCs, composed of mainly pancreatic endoderm (PDX1/NKX6.1‐positive), endocrine precursors (NKX2.2/synaptophysin‐positive, hormone/NKX6.1‐negative), and polyhormonal cells (insulin/glucagon‐positive, NKX6.1‐negative). However, the relative contributions of NKX6.1‐negative versus NKX6.1‐positive cell fractions to the maturation of functional β‐cells remained unclear. To address this question, we generated two distinct pancreatic progenitor cell populations using modified differentiation protocols. Prior to transplant, both populations contained a high proportion of PDX1‐expressing cells (∼85%–90%) but were distinguished by their relatively high (∼80%) or low (∼25%) expression of NKX6.1. NKX6.1‐high and NKX6.1‐low progenitor populations were transplanted subcutaneously within macroencapsulation devices into diabetic mice. Mice transplanted with NKX6.1‐low cells remained hyperglycemic throughout the 5‐month post‐transplant period whereas diabetes was reversed in NKX6.1‐high recipients within 3 months. Fasting human C‐peptide levels were similar between groups throughout the study, but only NKX6.1‐high grafts displayed robust meal‐, glucose‐ and arginine‐responsive insulin secretion as early as 3 months post‐transplant. NKX6.1‐low recipients displayed elevated fasting glucagon levels. Theracyte devices from both groups contained almost exclusively pancreatic endocrine tissue, but NKX6.1‐high grafts contained a greater proportion of insulin‐positive and somatostatin‐positive cells, whereas NKX6.1‐low grafts contained mainly glucagon‐expressing cells. Insulin‐positive cells in NKX6.1‐high, but not NKX6.1‐low grafts expressed nuclear MAFA. Collectively, this study demonstrates that a pancreatic endoderm‐enriched population can mature into highly functional β‐cells with only a minor contribution from the endocrine subpopulation. Stem Cells 2013;31:2432–2442

Circulating miR-375 as a Biomarker of β-Cell Death and Diabetes in Mice

Type 1 diabetes is a progressive autoimmune disease that is largely silent in its initial stages. Yet, sensitive methods for detection of β-cell death and prediction and prevention of diabetes are lacking. Micro-RNAs (miRNAs) have been found at high concentrations in body fluids. Here in this study we sought to determine whether an islet enriched miRNA, miR-375, is a suitable blood marker to detect β-cell death and predict diabetes in mice. We measured miR-375 levels by quantitative RT-PCR in plasma samples of streptozotocin (STZ)-treated C57BL/6 mice and nonobese diabetic (NOD) mice. We also measured miR-375 levels in media samples of cytokine- or STZ-treated islets in the presence or absence of cell-death inhibitors. High-dose STZ administration dramatically increased circulating miR-375 levels, prior to the onset of hyperglycemia. Similarly, in the NOD mouse model of autoimmune diabetes, circulating miR-375 levels were significantly increased 2 weeks before diabetes onset. Moreover, cytokine- and STZ-induced cell death in isolated mouse islets produced a striking increase in extracellular miR-375 levels, which was reduced by cell death inhibitors. These data suggest that circulating miR-375 can be used as a marker of β-cell death and potential predictor of diabetes.

Treating Diet-Induced Diabetes and Obesity with Human Embryonic Stem Cell-Derived Pancreatic Progenitor Cells and Antidiabetic Drugs

Summary Human embryonic stem cell (hESC)-derived pancreatic progenitor cells effectively reverse hyperglycemia in rodent models of type 1 diabetes, but their capacity to treat type 2 diabetes has not been reported. An immunodeficient model of type 2 diabetes was generated by high-fat diet (HFD) feeding in SCID-beige mice. Exposure to HFDs did not impact the maturation of macroencapsulated pancreatic progenitor cells into glucose-responsive insulin-secreting cells following transplantation, and the cell therapy improved glucose tolerance in HFD-fed transplant recipients after 24 weeks. However, since diet-induced hyperglycemia and obesity were not fully ameliorated by transplantation alone, a second cohort of HFD-fed mice was treated with pancreatic progenitor cells combined with one of three antidiabetic drugs. All combination therapies rapidly improved body weight and co-treatment with either sitagliptin or metformin improved hyperglycemia after only 12 weeks. Therefore, a stem cell-based therapy may be effective for treating type 2 diabetes, particularly in combination with antidiabetic drugs.

Accelerated Maturation of Human Stem Cell-Derived Pancreatic Progenitor Cells into Insulin-Secreting Cells in Immunodeficient Rats Relative to Mice

Summary Pluripotent human embryonic stem cells (hESCs) are a potential source of transplantable cells for treating patients with diabetes. To investigate the impact of the host recipient on hESC-derived pancreatic progenitor cell maturation, cells were transplanted into immunodeficient SCID-beige mice or nude rats. Following the transplant, basal human C-peptide levels were consistently higher in mice compared with rats, but only rats showed robust meal- and glucose-responsive human C-peptide secretion by 19–21 weeks. Grafts from rats contained a higher proportion of insulin:glucagon immunoreactivity, fewer exocrine cells, and improved expression of mature β cell markers compared with mice. Moreover, ECM-related genes were enriched, the collagen network was denser, and blood vessels were more intricately integrated into the engrafted endocrine tissue in rats relative to mice. Overall, hESC-derived pancreatic progenitor cells matured faster in nude rats compared with SCID-beige mice, indicating that the host recipient can greatly influence the fate of immature pancreatic progenitor cells post-transplantation.

Partial ablation of leptin signaling in mouse pancreatic α-cells does not alter either glucose or lipid homeostasis.

The role of glucagon in the pathological condition of diabetes is gaining interest, and it has been recently reported that its action is essential for hyperglycemia to occur. Glucagon levels, which are elevated in some diabetic models, are reduced following leptin therapy. Likewise, hyperglycemia is corrected in type 1 diabetic mice treated with leptin, although the mechanisms have not been fully determined. A direct inhibitory effect of leptin on mouse and human α-cells has been demonstrated at the levels of electrical activity, calcium signaling, and glucagon secretion. In the present study we employed the Cre-loxP strategy to generate Lepr(flox/flox) Gcg-cre mice, which specifically lack leptin receptors in glucagon-secreting α-cells, to determine whether leptin resistance in α-cells contributes to hyperglucagonemia, and also whether leptin action in α-cells is required to improve glycemia in type 1 diabetes with leptin therapy. Immunohistochemical analysis of pancreas sections revealed Cre-mediated recombination in ∼ 43% of the α-cells. We observed that in vivo Lepr(flox/flox) Gcg-cre mice display normal glucose and lipid homeostasis. In addition, leptin administration in streptozotocin-induced diabetic Lepr(flox/flox) Gcg-cre mice restored euglycemia similarly to control mice. These findings suggest that loss of leptin receptor signaling in close to one-half of α-cells does not alter glucose metabolism in vivo, nor is it sufficient to prevent the therapeutic action of leptin in type 1 diabetes.

Hypothyroidism impairs human stem cell-derived pancreatic progenitor cell maturation in mice

Pancreatic progenitors derived from human embryonic stem cells (hESCs) are a potential source of transplantable cells for treating diabetes and are currently being tested in clinical trials. Yet, how the milieu of pancreatic progenitor cells, including exposure to different factors after transplant, may influence their maturation remains unclear. Here, we examined the effect of thyroid dysregulation on the development of hESC-derived progenitor cells in vivo. Hypothyroidism was generated in SCID-beige mice using an iodine-deficient diet containing 0.15% propyl-2-thiouracil, and hyperthyroidism was generated by addition of L-thyroxine (T4) to drinking water. All mice received macroencapsulated hESC-derived progenitor cells, and thyroid dysfunction was maintained for the duration of the study (“chronic”) or for 4 weeks posttransplant (“acute”). Acute hyperthyroidism did not affect graft function, but acute hypothyroidism transiently impaired human C-peptide secretion at 16 weeks posttransplant. Chronic hypothyroidism resulted in severely blunted basal human C-peptide secretion, impaired glucose-stimulated insulin secretion, and elevated plasma glucagon levels. Grafts from chronic hypothyroid mice contained fewer β-cells, heterogenous MAFA expression, and increased glucagon+ and ghrelin+ cells compared to grafts from euthyroid mice. Taken together, these data suggest that long-term thyroid hormone deficiency may drive the differentiation of human pancreatic progenitor cells toward α- and ε-cell lineages at the expense of β-cell formation.

Leptin Administration Enhances Islet Transplant Performance in Diabetic Mice

Islet transplantation is an effective method to obtain long-term glycemic control for patients with type 1 diabetes, yet its widespread use is limited by an inadequate supply of donor islets. The hormone leptin has profound glucose-lowering and insulin-sensitizing action in type 1 diabetic rodent models. We hypothesized that leptin administration could reduce the dose of transplanted islets required to achieve metabolic control in a mouse model of type 1 diabetes. We first performed a leptin dose-response study in C57Bl/6 mice with streptozotocin (STZ)-induced diabetes to determine a leptin dose insufficient to reverse hyperglycemia. Subsequently, we compared the ability of suboptimal islet transplants of 50 or 125 syngeneic islets to achieve glycemic control in STZ-induced diabetic C57Bl/6 mice treated with or without this dose of leptin. The dose-response study revealed that leptin reverses STZ-induced diabetes in a dose-dependent manner. Supraphysiological leptin levels were necessary to restore euglycemia but simultaneously increased risk of hypoglycemia, and also lost efficacy after 12 days of administration. In contrast, 1 µg/day leptin only modestly reduced blood glucose but maintained efficacy throughout the study duration. We then administered 1 µg/day leptin to diabetic mice that underwent transplantation of 50 or 125 islets. Although these islet doses were insufficient to ameliorate hyperglycemia alone, coadministration of leptin with islet transplantation robustly improved control of glucose and lipid metabolism, without increasing circulating insulin levels. This study reveals that low-dose leptin administration can reduce the number of transplanted islets required to achieve metabolic control in STZ-induced diabetic mice.

Impaired Ca2+ Signaling in β-Cells Lacking Leptin Receptors by Cre-loxP Recombination

Obesity is a major risk factor for diabetes and is typically associated with hyperleptinemia and a state of leptin resistance. The impact of chronically elevated leptin levels on the function of insulin-secreting β-cells has not been elucidated. We previously generated mice lacking leptin signaling in β-cells by using the Cre-loxP strategy and showed that these animals develop increased body weight and adiposity, hyperinsulinemia, impaired glucose-stimulated insulin secretion and insulin resistance. Here, we performed several in vitro studies and observed that β-cells lacking leptin signaling in this model are capable of properly metabolizing glucose, but show impaired intracellular Ca2+ oscillations and lack of synchrony within the islets in response to glucose, display reduced response to tolbutamide and exhibit morphological abnormalities including increased autophagy. Defects in intracellular Ca2+ signaling were observed even in neonatal islets, ruling out the possible contribution of obesity to the β-cell irregularities observed in adults. In parallel, we also detected a disrupted intracellular Ca2+ pattern in response to glucose and tolbutamide in control islets from adult transgenic mice expressing Cre recombinase under the rat insulin promoter, despite these animals being glucose tolerant and secreting normal levels of insulin in response to glucose. This unexpected observation impeded us from discerning the consequences of impaired leptin signaling as opposed to long-term Cre expression in the function of insulin-secreting cells. These findings highlight the need to generate improved Cre-driver mouse models or new tools to induce Cre recombination in β-cells.