IntroductionAdult pancreatic tissue contains cell populations with latent regenerative potential, but the processes governing their expansion and differentiation into endocrine lineages remain unclear.MethodsAdult human pancreatic cells obtained from donor tissue were isolated and expanded and analyzed for lineage potential using single-cell RNA sequencing, flow cytometry, and functional assays. A CD9+ PROCR+ RGS16+ subpopulation, termed islet progenitor-like cells (IPCs), was evaluated for proliferative capacity and differentiation potential.ResultsIPCs exhibited robust proliferative capacity and, upon differentiation, formed insulin- and glucagon-secreting organoids. Treatment of IPCs with the small molecule ISX9 induced expression of key transcription factors RFX6 and NEUROD1 through calcium-dependent chromatin remodeling mediated by NFAT recruitment of p300 and displacement of histone deacetylases (HDAC1-3). Pharmacologic inhibition of HDACs further enhanced IPC maturation and glucose-stimulated insulin secretion.DiscussionThese findings define the molecular and epigenetic mechanisms driving the expansion and differentiation of adult IPCs into functional islet-like organoids, providing a foundation for future regenerative approaches using adult pancreatic tissue as a renewable source for endocrine cell replacement.

Diabetes mellitus remains a leading cause of morbidity worldwide, driven in type 1 diabetes by autoimmune destruction of pancreatic β-cells and in advanced type 2 diabetes by progressive β-cell dysfunction and failure. Diabetes affects around 830 million people globally, with the vast majority residing within low- and middle-income nations. Over the last few decades, the numbers of people who have diabetes and those with untreated diabetes have consistently increased. Although current pharmacologic therapies improve glycemic control, they do not restore functional β-cell mass. Consequently, strategies aimed at protecting, regenerating, or replacing insulin-producing cells have emerged as a major focus of regenerative medicine. Stem cell-based approaches offer the potential to generate renewable sources of glucose-responsive β-like cells, but challenges remain in achieving full functional maturation, immune protection, scalable manufacturing, and durable clinical engraftment. This review examines advances in engineering stem cell-derived insulin-producing cells for islet replacement therapy, with an emphasis on differentiation strategies, immunoprotective approaches, and the translational barriers that must be addressed for durable β-cell replacement.

Total pancreatectomy and islet autotransplantation (TPIAT) is a specialized procedure for patients with chronic (CP) and recurrent acute pancreatitis (RAP) experiencing intractable pain and reduced quality of life. Hospitalizations for pancreatitis are common before TPIAT. However, data comparing hospitalization patterns before and after TPIAT remain limited. This multicenter, prospective observational study of TPIAT (POST) enrolled 380 patients (age 26 (15, 43 years), 34

Introduction and Objective: Pancreatogenic diabetes (T3cDM) is common in chronic pancreatitis, partially caused by beta cell loss and dysfunction due to fibrosis and inflammation. However, islet and pancreatic tissue pathology is rarely available to study. Methods: We analyzed fibrosis and glycemic and islet parameters in pancreas tissue biopsies from patients with recurrent acute or chronic pancreatitis undergoing total pancreatectomy with islet autotransplant (TPIAT) in the multicenter “POST” study. Standardized data for diabetes status and lab results, pancreatitis details, islet mass, and fibrosis were analyzed in a subset (n=84, age 35.38 +/- 15.44) representing male and female participants with normoglycemia, pre-diabetes mellitus (pre-DM), or diabetes mellitus (DM) (n=14 each). Percent fibrosis was calculated based on H&E staining and ImageJ software. Statistical analyses were performed using Pearson's correlation and two-way ANOVA. Data reported as mean +/- SEM. Results: We observed a modest positive association of percent fibrosis with pre-surgery HbA1c (r=0.335, p =0.0019), and negative association with islet equivalents transplanted per kilogram (r=-0.474, p<0.0001), but no association with C-peptide (p=0.15) or fasting glucose (p=0.10). Males (65.79% +/- 3.883) had a larger percentage of fibrosis area compared with females (48.00% +/- 3.664) (p=0.0013). DM participants had more fibrosis than normoglycemia group (65.92% +/- 4.769 vs 49.35% +/- 4.962) (p=0.019). Conclusion: At time of TPIAT surgery, patients with greater fibrosis had impaired glycemic control and lower islet mass. Future work will evaluate for tissue markers of ER stress, autophagy, and bihormonality in CP beta cells with or without diabetes. Disclosure G. McFarlane: None. A. Eaton: None. S.A. Ahmad: None. G. Beilman: None. T.B. Gardner: None. K. Morgan: None. J.D. Nathan: None. B. Naziruddin: None. V. Singh: Advisory Panel; Current; Amgen Inc. Speaker's Bureau; Current; Amgen Inc. Advisory Panel; Current; Zenas BioPharma. Advisory Panel; Ended; Ionis Pharmaceuticals. Stock/Shareholder; Current; Kyttaro, Origin Endoscopy, Solv Endotherapy. G. Trikudanathan: Consultant; Current; Boston Scientific Corporation. M. Wijkstrom: Consultant; Current; Vertex Pharmaceuticals Incorporated. E. Alejandro: None. M.D. Bellin: Other - DSMB member; Current; Vertex Pharmaceuticals Incorporated. Advisory Panel; Current; Novo Nordisk, Sana Biotechnology Inc., Sernova, Corp. Research Support; Current; Dexcom, Inc. Funding National Institute of Diabetes and Digestive and Kidney Diseases(5R01DK109124)Department of Defense(HT9425-23-1-0307)National Institute of Diabetes and Digestive and Kidney Diseases(3R25DK140753-02S1)

ABSTRACT Background and Aims Adult pancreas-derived islet progenitor cells (IPCs) have recently been shown to expand in culture and differentiate into endocrine-like organoids. However, translation of this approach to a clinically compatible workflow requires cell enrichment strategies and validation using tissue obtained during real-world clinical procedures. Here, we adapted our previously described IPC platform to non-endocrine pancreatic tissue fractions generated during clinical islet isolation procedures and evaluated their capacity to generate functional islet organoids. Methods Non-endocrine pancreatic tissue fractions obtained during clinical islet isolation were expanded ex vivo and enriched using fluorescence-activated cell sorting (FACS) for CD81 and CD9, surface markers previously identified in IPC populations. Sorted cells were expanded, induced to form IPC clusters, and differentiated with ISX9 to generate islet organoids. Differentiation was assessed by gene expression analysis, flow cytometry, immunofluorescence, calcium flux assays, glucose-stimulated insulin and glucagon secretion, and single-cell RNA sequencing. Results Clinically derived non-endocrine cell fractions yielded expandable IPC populations expressing progenitor-associated markers. FACS-purified and expanded CD81 + /CD9 + IPCs were enriched with BMPR1A and P2RY1. Sorted cells generated three-dimensional BMPR1A + and RGS16 + IPC clusters. IPC clusters differentiated into islet organoids with upregulated expression of canonical beta-and alpha-cell transcription factors. Single-cell transcriptomic profiling revealed activation of coordinated endocrine gene programs and alignment with reference human islet endocrine signatures, while the undifferentiated IPC compartment was marked by enrichment of PTX3, FST, CEMIP, and GREM1. Terminally differentiated cells exhibited depolarization-induced calcium influx and glucose-regulated insulin and glucagon secretion. Conclusions These findings establish an adaptable workflow for expansion and production of functional islet organoids recovered from clinically derived pancreatic tissue. This strategy may provide an unlimited autologous source of adult progenitor-derived islets for future islet cell replacement therapies in diabetes.

Background. In total pancreatectomy with islet autotransplantation (TPIAT), a greater number of islets transplanted produces more favorable outcomes. We aimed to determine predictors of islet isolation outcomes. Methods. We investigated factors associated with islet isolation outcomes expressed as islet number (IN), islet equivalents (IEQ; standardized to an islet with 150 mu m diameter), IN/kg, or IEQ/kg using data from the multicenter Prospective Observational Study of TPIAT. Single-predictor linear regression was used to estimate the association of individual patient and disease characteristics with islet isolation outcomes, and augmented backward elimination was used to select variables to include in multivariable analyses. Results. In multivariable analyses, only elevated hemoglobin A1c was associated with worse outcomes for all measures (P < 0.001 for all). Total IEQ obtained for transplant was higher for participants with Hispanic ethnicity (P = 0.002) or overweight status pre-TPIAT (P < 0.001) and lower with non-White race (P = 0.03), genetic pancreatitis (P = 0.02), history of lateral pancreaticojejunostomy (P = 0.03), and presence of atrophy (P = 0.006) or ductal changes (P = 0.014) on imaging. IEQ/kg was higher in females (P = 0.01) and Hispanic participants (P = 0.046) and generally lower with older age (nonlinear association, P < 0.001) and pancreatic atrophy (P < 0.001) on imaging. Total IN and IN/kg showed trends similar, but not identical, to IEQ and IEQ/kg, respectively. Conclusions. Patient demographics and certain pancreatic disease features were associated with outcomes from islet isolation. Hemoglobin A1c before TPIAT was the metabolic testing measure most strongly associated with islet isolation results.

Multipotent progenitor-like cells have been identified in the adult pancreas under various physiological and pathological conditions. Here, we identify and characterize a subset of adult pancreas-derived cells, termed islet pancridia cells (IPCs), that can be expanded in vitro and retain the potential for endocrine differentiation and islet cell function. Single-cell RNA sequencing of expanded pancridia revealed transcriptomic profiles resembling immature beta cells, enriched with markers of epithelial-mesenchymal transition. A CD9⁺, PROCR⁺ subpopulation of IPCs formed IPC clusters marked by restricted expression of RGS16, a known islet progenitor marker. In vivo, co-transplantation of expanded IPCs with a subtherapeutic dose of islets significantly improved graft function and partially restored native pancreatic endocrine activity in streptozotocin-induced diabetic mice. In vitro, treatment of RGS16⁺ IPC clusters with the small molecule ISX9 induced differentiation into islet organoids that co-expressed and secreted insulin and glucagon. ISX9-mediated differentiation was driven by calcineurin/NFAT-dependent recruitment of the histone acetyltransferase p300 and displacement of histone deacetylases (HDACs) at the RFX6 and NEUROD1 promoters. Pre-treatment with the HDAC inhibitor ITF2357 further enhanced islet cell differentiation by promoting chromatin remodeling and facilitating NFAT-targeted recruitment of p300. These findings uncover calcium-dependent and epigenetic mechanisms that regulate the differentiation of multipotent CD9⁺, PROCR⁺, RGS16⁺ IPCs into functional islet organoids and offer potential strategies for regenerating islet cell mass to treat diabetes. ### Competing Interest Statement A patent application related to the cell isolation and differentiation methods described in this manuscript has been filed by Baylor Scott & White Research Institute and is currently under review by the U.S. Patent and Trademark Office. Baylor Healthcare System Foundation

This study examines extracellular matrix (ECM) protein (ECM) expression in chronic pancreatitis (CP) patients and its correlation with graft function after total pancreatectomy with islet autologous transplantation (TPIAT). Pancreatic sections from 29 CP patients undergoing TPIAT were analyzed for ECM including pan-laminin, laminin alpha 5 (LAMA5), collagen IV, and Perlecan by immunohistochemistry and scored by the percentage positive staining area within the whole tissue area. Graft function was monitored by blood glucose and C-peptide levels. Laminin alpha 5 levels in blood plasma were greater in CP. Laminin alpha 5 expression was significantly higher in all CP patient etiological categories including alcoholic, hereditary, idiopathic, Oddi dysfunction, and pancreatic divisum compared to healthy controls. The overall expression of LAMA5 positively correlated with expression of the ECM proteins pan-laminin ( R = 0.63, P < 0.001), collagen IV ( R = 0.67, P < 0.001), and Perlecan ( R = 0.71, P < 0.001). Increased LAMA5 expression was observed within islet endothelial vascular tissue and the peri-islet basal membrane. Increased LAMA5 expression in the pancreas correlated with poor islet isolation yield and posttransplant islet function after 3 months. Increased endothelial expression of LAMA5 and ECM proteins is indicative of progressive damage to the pancreas and correlates with poor graft function after TPIAT.

Background:Liver transplantation remains the optimal treatment for end-stage liver disease; however, the selection of suitable donors is often reliant on tissue biopsies that are invasive and reflective of only a portion of the liver. Prediction of posttransplant graft function or complications by molecular analyses of RNA profiles released by the liver during preservation would provide a more comprehensive and timely intervention to optimize recipient selection and survival. Methods:We collected exosomes from liver preservation fluid and identified differentially expressed small RNA profiles for different donor types and recipient outcomes. Our cohort consisted of donors after circulatory death or brain death, as well as living donors. Significant changes were determined by statistical analysis. Results:We demonstrate a successful method to collect exosomes from preservation fluid and isolate sufficient small RNA for sequencing analysis. MicroRNA (miRNA) profiles identified distinct groupings of living donors and donors after circulatory death types, with donor after circulatory death miRNA profiles providing a set of stress-induced biomarkers that project a greater propensity for poor function based on bioinformatic algorithms. Similarly, distinct miRNA profiles were expressed in different posttransplant complication groups. Conclusions:This method of exosomal collection for predictive miRNA signatures supports a promising technique for noninvasive sample collection to support patient-centered therapy after liver transplantation by identifying biomarkers for transplant outcomes.

BACKGROUND & AIMS:Total pancreatectomy with islet autotransplantation (TPIAT) is an emerging but controversial therapy for patients with intractably painful chronic pancreatitis (CP) and recurrent acute pancreatitis (RAP). Outcomes of TPIAT have not been validated in multicenter studies. METHODS:The study prospectively enrolled 384 participants, who were a mean age of 30 years (standard deviation [SD],17 years), 34% pediatric, and 62% female, with painful CP or RAP undergoing TPIAT at 12 centers. Numeric pain scores, opioid use, and health-related quality of life (QOL), including physical component summary and mental component summary scores, were collected at baseline, at 6 months, and 1 year post-TPIAT. Predictors of outcomes were evaluated with regression modeling. RESULTS:Opioid use, assessed over a 14-day interval, decreased from 61% before to 24% at 1 year after TPIAT (P < .001). Daily abdominal pain decreased from 65% to 23%, whereas the mean pain score decreased from 4.9 (SD, 2.3) to 2.3 (SD, 2.5; both P < .001). In a multivariable model, opioid use at 1 year was associated with older age and opioid use pre-TPIAT (odds ratio, 9.21; 95% confidence interval, 3.93-25.7). Physical component summary and mental component summary scores improved by ≥10 points in 58% and 35%, respectively. Higher QOL scores at 1 year were associated with higher scores before surgery and younger age. Mean hemoglobin A1c was 7% (SD, 1.9%) with 20% insulin independent at 1 year. Duration of disease, pancreas morphology, and etiology did not predict response to TPIAT. CONCLUSION:In this prospective multicenter study of patients with intractably painful CP or RAP, TPIAT significantly reduced pain burden and improved QOL. Although young age predicted better outcomes, duration and etiology of disease did not.