George K. Gittes

This is a provisional file, not the final article pathogenesis of diabetes mellitus is fundamentally linked to a decline in pancreatic β-31 cell mass (1). Replacing these insulin-producing cells remains a central goal of regenerative medicine, pursued through two primary avenues: guiding the differentiation human pluripotent 33 stem cells in vitro or harnessing the innate regenerative capacity of the pancreas in vivo (2, 3 meticulous re-analysis of single-cell datasets, they report that the transcriptional signature a 74 previously described adult progenitor population is not found in standard adult islet 75 preparations, but is instead identified in a subset of mesothelial cells of the embryonic pancreas in 76 both mice and humans. This work further identifies transcriptomic and epigenomic connections 77 between these embryonic cells and ductal/endocrine progenitors. Notably, a PROCR-like cell 78 subcluster was also found to be spontaneously co-specified during the differentiation of human iPSCs 79 into pancreatic progenitor cells. These compelling findings encourage a reinterpretation of Procr+ 80 progenitor cell origin, suggesting a potential developmental link to embryonic mesothelium and 81 highlighting the critical importance of cellular context in regenerative biology. 82 83The collective works in this Research Topic underscore key challenges for translational progress. A 85 priority is ensuring the long-term stability and function of new β-cells, which must fully integrate 86 into physiological glucose regulation without loss of identity or viability. This demands rigorous, 87 long-term validation in disease-relevant models. 88 Furthermore, the field must decisively bridge the gap between rodent models and human biology (7,89 8). Advancing human model systems-such as complex organoids and humanized mouse models-90 alongside the integration of human genomic data, will be essential to prioritize clinically relevant 91 targets (9-12). 92From a therapeutic standpoint, achieving safe and targeted delivery of regenerative cues, whether 93 pharmacological or genetic, is paramount. Strategies must minimize off-target effects, potentially 94 leveraging emerging delivery technologies. Furthermore, the success of these approaches, especially 95 in T1D, will hinge on modulating the pancreatic niche to create a supportive microenvironment for 96 new β-cells to mimic in vivo physiological settings. 97In conclusion, this collection highlights the multifaceted path toward β-cell regeneration. By 98 deepening our understanding of developmental principles, cellular plasticity, and niche interactions, 99 the prospect of a curative therapy for diabetes continues to gain momentum. 100 101The authors declare that they have no competing interests. 103

Preclinical gene therapy research, particularly in rodent and large animal models, necessitates the production of AAV vectors with high yield and purity. Traditional approaches in research laboratories often involve extensive use of cell culture dishes to cultivate HEK293T cells, a process that can be both laborious and problematic. Here, a unique in-house method is presented, which simplifies this process with a specific cell factory (or cell stacks, CF10) platform. An integration of polyethylene glycol/aqueous two-phase partitioning with iodixanol gradient ultracentrifugation improves both the yield and purity of the generated AAV vectors. The purity of the AAV vectors is verified through SDS-PAGE and silver staining, while the ratio of full to empty particles is determined using transmission electron microscopy (TEM). This approach offers an efficient cell factory platform for the production of AAV vectors at high yields, coupled with an improved purification method to meet the quality demands for in vivo studies.

Pulmonary hypertension associated with congenital diaphragmatic hernia (CDH) is a critical factor in determining prognosis. We propose that intra-amniotic sildenafil administration is an effective prenatal therapy for CDH-induced pulmonary hypertension. To assess the efficacy of this treatment, we administered sildenafil to nitrofen-induced congenital diaphragmatic hernia fetuses and control fetuses via an intra-amniotic injection after a laparotomy on the pregnant dam at either E13.5 or E15.5. Intra-amniotic sildenafil treatment attenuated peripheral vascular muscularization, enhanced pulmonary blood flow, and increased the ratio of pulmonary artery size to aortic size in congenital diaphragmatic hernia fetuses after both E13.5 and E15.5 treatments. E13.5-treated congenital diaphragmatic hernia fetuses showed a higher and more prolonged expression of cyclic guanosine monophosphate (cGMP)-dependent protein kinase and more production of vascular endothelial growth factor, resulting in a significant improvement in lung architecture. The E13.5-treated congenital diaphragmatic hernia fetuses also had an increase in lung weight-to-body weight ratio and an improved fetal survival. Intra-amniotic sildenafil treatment did not show any detectable negative effects in control fetuses. Intra-amniotic sildenafil treatment for rats attenuates CDH-induced pulmonary hypertension and enhanced peripheral pulmonary blood flow. Moreover, early intervention may be preferable to better accelerate lung development and improve prognosis. Direct sildenafil administration via an intra-amniotic injection may be a promising option in congenital diaphragmatic hernia prenatal therapy.

Elevation of glucagon levels and increase in α cell proliferation is associated with states of hyperglycemia in diabetes. A better understanding of the molecular mechanisms governing glucagon secretion could have major implications for understanding abnormal responses to hypoglycemia in patients with diabetes and provide novel avenues for diabetes management. Using mice with inducible induction of Rheb1 in α cells (αRheb ^Tg mice), we showed that short-term activation of mTORC1 signaling is sufficient to induce hyperglucagonemia through increased glucagon secretion. Hyperglucagonemia in αRheb ^Tg mice was also associated with an increase in α cell size and mass expansion. This model allowed us to identify the effects of chronic and short-term hyperglucagonemia on glucose homeostasis by regulating glucagon signaling in the liver. Short-term hyperglucagonemia impaired glucose tolerance, which was reversible over time. Liver glucagon resistance in αRheb ^Tg mice was associated with reduced expression of the glucagon receptor and genes involved in gluconeogenesis, amino acid metabolism, and urea production. However, only genes regulating gluconeogenesis returned to baseline upon improvement of glycemia. Overall, these studies demonstrate that hyperglucagonemia exerts a biphasic response on glucose metabolism: Short-term hyperglucagonemia lead to glucose intolerance, whereas chronic exposure to glucagon reduced hepatic glucagon action and improved glucose tolerance

Chronic pancreatitis is a debilitating disease affecting millions worldwide. These patients suffer from bouts of severe pain that are minimally relieved by pain medications and may necessitate major surgeries with high morbidity and mortality. Previously, we demonstrated that “chemical pancreatectomy,” a pancreatic intraductal infusion of dilute acetic acid solution, ablated the exocrine pancreas while preserving the endocrine pancreas. Notably, chemical pancreatectomy resolved chronic inflammation, alleviated allodynia in the cerulein pancreatitis model, and improved glucose homeostasis. Herein, we extensively tested the feasibility of a chemical pancreatectomy in NHPs and validated our previously published pilot study. We did serial computed tomography (CT) scans of the abdomen and pelvis, analyzed dorsal root ganglia, measured serum enzymes, and performed histological and ultrastructural assessments and pancreatic endocrine function assays. Based on serial CT scans, chemical pancreatectomy led to the loss of pancreatic volume. Immunohistochemistry and transmission electron microscopy demonstrated exocrine pancreatic ablation with endocrine islet preservation. Importantly, chemical pancreatectomy did not increase pro-nociceptive markers in harvested dorsal root ganglia. Also, chemical pancreatectomy improved insulin secretion to supranormal levels in vivo and in vitro. Thus, this study may provide a foundation for translating this procedure to patients with chronic pancreatitis or other conditions requiring a pancreatectomy.

Chronic pancreatitis is a debilitating disease affecting millions worldwide. These patients suffer from bouts of severe pain that are minimally relieved by pain medications and may necessitate major surgeries with high morbidity and mortality. Previously, we demonstrated that “chemical pancreatectomy,” a pancreatic intraductal infusion of dilute acetic acid solution, ablated the exocrine pancreas while preserving the endocrine pancreas. Notably, chemical pancreatectomy resolved chronic inflammation, alleviated allodynia in the cerulein pancreatitis model, and improved glucose homeostasis. Herein, we extensively tested the feasibility of a chemical pancreatectomy in NHPs and validated our previously published pilot study. We did serial computed tomography (CT) scans of the abdomen and pelvis, analyzed dorsal root ganglia, measured serum enzymes, and performed histological and ultrastructural assessments and pancreatic endocrine function assays. Based on serial CT scans, chemical pancreatectomy led to the loss of pancreatic volume. Immunohistochemistry and transmission electron microscopy demonstrated exocrine pancreatic ablation with endocrine islet preservation. Importantly, chemical pancreatectomy did not increase pro-nociceptive markers in harvested dorsal root ganglia. Also, chemical pancreatectomy improved insulin secretion to supranormal levels in vivo and in vitro. Thus, this study may provide a foundation for translating this procedure to patients with chronic pancreatitis or other conditions requiring a pancreatectomy.

Systemic messenger RNA (mRNA) delivery to organs outside the liver, spleen, and lungs remains challenging. To overcome this issue, we hypothesized that altering nanoparticle chemistry and administration routes may enable mRNA-induced protein expression outside of the reticuloendothelial system. Here, we describe a strategy for delivering mRNA potently and specifically to the pancreas using lipid nanoparticles. Our results show that delivering lipid nanoparticles containing cationic helper lipids by intraperitoneal administration produces robust and specific protein expression in the pancreas. Most resultant protein expression occurred within insulin-producing β cells. Last, we found that pancreatic mRNA delivery was dependent on horizontal gene transfer by peritoneal macrophage exosome secretion, an underappreciated mechanism that influences the delivery of mRNA lipid nanoparticles. We anticipate that this strategy will enable gene therapies for intractable pancreatic diseases such as diabetes and cancer.