Introduction: Islets-like clusters derived from pluripotent stem cells can contain an off-target cell type with characteristics of gut enterochromaffin cells. Native insulin-secreting pancreatic beta cells and gut enterochromaffin cells both express the transcription factor Nkx6.1. Alternative stem cell-derived islets lacking expression of Nkx6.1 were developed to improve in vivo potency compared to populations containing Nkx6.1-expressing cells. This novel cell population has not been previously described. Method: Populations of stem-cell derived islets containing abundant Nkx6.1-expressing cells and populations containing few Nkx6.1 expressing cells were transplanted to streptozotocin-induced diabetic rodents. Blood glycemia was monitored over time. Grafts were explanted and analyzed for pancreatic endocrine cell content. Results: Populations of stem-cell derived islets containing few Nkx6.1-expressing cells were more potent at controlling rodent blood glycemia than populations containing abundant Nkx6.1-expressing cells. Grafts from animals with low-nkx6.1 implants displayed higher quantities of cells with a mature pancreatic endocrine phenotype. These cells activated expression of Nkx6.1 after transplant. Conclusion: A novel cell population lacking expression of the marker Nkx6.1 pre-implant exhibits higher potency in vivo compared to a population expressing Nkx6.1, presumably due to elimination of off-target enterochromaffin cells. Higher potency improves therapeutic potential by reduction of effective dose.

Abstract Disclosure: W.L. Rust: Employee; Self; Seraxis Inc. Stock Owner; Self; Seraxis Inc. P.M. Dalal: Employee; Self; Seraxis Inc. Stock Owner; Self; Seraxis Inc. A.P. Koval: Employee; Self; Seraxis Inc. Stock Owner; Self; Seraxis Inc. J.J. Ratiu: Employee; Self; Seraxis Inc. Stock Owner; Self; Seraxis Inc. S.M. Southard: Employee; Self; Seraxis Inc. Stock Owner; Self; Seraxis Inc. P.S. Strumph: Employee; Self; Seraxis Inc. Stock Owner; Self; Seraxis Inc. C.A. Welsch: Employee; Self; Seraxis Inc. Stock Owner; Self; Seraxis Inc. Background and Aims: We report the development of a novel human multipotent immortal stem cell line, SR1423 that efficiently differentiates to clusters of functional endocrine pancreatic cells. These Synthetic Replacement Endocrine (SRE) clusters contain cell types that recapitulate native islet function in vitro and potently control blood glucose in animal models of type 1 diabetes. Detailed characterization reveals significant distinctions between SRE, native islets and SC-beta cells. SRE clusters are manufactured following GMP requirements in closed vessels with large-scale formats and are being developed for near-term clinical studies. Materials and Methods: SRE was generated by transient expression of the Yamanaka factors in human islet cells harvested from a consented donor pancreas. Rather than screen for pluripotency, this cell line was selected for its ability to differentiate to the definitive endoderm in a first screen, and Pdx1+ pancreatic progenitors in a second screen. SRE does not meet criteria for pluripotency as it fails to express the master control gene for mesoderm specification, brachyury, in response to mesoderm-inducing agents. Comparison against a pluripotent stem cell database demonstrates poor overall differentiation capability for the mesoderm lineage. In contrast, SRE responds to basic differentiation protocols that drive endocrine pancreatic fate choice and generates highly pure populations of cells with endocrine cell characteristics. We therefore characterize SR1423 as a multipotent, and not pluripotent stem cell. In a prospectively designed islet replacement study male NSG mice with streptozotocin-induced diabetes were implanted with SRE clusters to the kidney capsule or gonadal fat pad (as a surrogate for the omentum) and disease progression was monitored. Results: SRE resemble human islets in morphology and distribution of hormone expressing cells. In this mouse model of STZ-induced type 1 diabetes (blood glucose > 180 mg/dL), SRE transplantation resulted in euglycemia 3-4 weeks post-implant; 72 mg/dL +/− 12 SEM when implanted to the kidney capsule (n=13), and 79 mg/dL +/− 15 SEM when implanted to the gonadal fat pad (n=10). Control mice (without SRE implant) in this model of type 1 diabetes (n=2) remained hyperglycemic; 570 mg/dL +/− 20 SEM. Conclusion: These data are consistent with demonstration of proof of principle in this model with resolution of hyperglycemia after transplant of SRE. This is the first description of a novel cell type (non-native, non-SC-beta) with potential clinical therapeutic benefit. SRE clusters are currently in preparation for clinical studies. Presentation: Friday, June 16, 2023

This article describes a stem cell line derived by reprogramming of native human islet cells that consistently generates pure populations of endocrine pancreatic clusters following a simple differentiation protocol. Surprisingly, the population of stem cell derived pancreatic endocrine clusters that was most consistently capable of regulating blood glucose in rodent models of diabetes lacked robust expression of the key beta cell maturation-associated factor NKX6-1 but did manifest high expression of other key drivers of endocrine cell specification and maturation, ISL1 and MAFA. These data support the hypothesis that multiple pancreatic profiles can be identified in stem cell derived cultures and that these have disparate in vivo potency. The population with low NKX6-1 and high in vivo potency was further characterized by transcriptome profiling as an endocrine-committed population progressively maturing in vitro to a state proximal to the native islet.

To survey current practices in clinical islet transplantation to identify the critical factors for designing a successful implant strategy for islets derived from pluripotent stem cells. Of particular importance is the volume of a therapeutic dose and the host sites capable of housing that volume. A calculated dose of surviving islets required for normoglycemia in patients is large, though less than the number of islets present in a healthy pancreas. Great strides have been made developing methods for delivery of a large dose of islets that maintain cell viability and potency. Although many of these endeavor to provide the islets close association to the host vasculature, only some of the strategies maintain the architecture and distribution of the individual islets. The trends in clinical research and in laboratory models suggest that strategies that maintain the architecture and distribution of the individual islets are the most successful. This requirement increases the volume required to deliver a therapeutic dose and limits the anatomical sites available for clinical transplant.

Several beta cell replacement therapies have commenced the preclinical and clinical development. Some of the most advanced technologies are pluripotent cell-derived therapies packaged within immune-protective devices. If clinically validated, and made practical for widespread use, these technologies could upend long-established industries focused on diabetes management and care of diabetes-related morbidity. There is justifiable excitement among researchers and investors, and accelerating participation by established pharmaceutical companies. However, questions relating to the practicality of the therapy and the size of the target patient population, and concerns related to the pricing strategy and regulatory path have tempered optimism among industry watchers. It is unknown whether the technology has advanced enough to cause major transformation in the treatment of diabetes in the short term, or whether a more gradual shift over the long-term should be anticipated.

We examined data and patterns in clinical islet transplant studies registered on ClinicalTrials.gov (CTgov) for treatment of type 1 diabetes (T1D), with a goal of extracting insights to apply in the design of a pluripotent stem cell-derived islet therapy. Clinical islet transplantation, as a cell therapy (rather than solid organ transplant) is a unique precedent for stem cell-based islet therapies. Registration activity shows that the field is not growing significantly, and newer registrations suggest that the reasons for stagnation include need for a more optimal site of infusion/transplantation, and especially a need for better immune protective strategies to advance a more effective and durable therapy for T1D. Stem Cells Translational Medicine 2019;8:209&214.

Induced pluripotent stem cell (iPSC) technology enables the creation and selection of pluripotent cells with specific genetic traits. This report describes a pluripotent cell line created specifically to form replacement pancreatic cells as a therapy for insulin-dependent diabetes. Beginning with primary pancreatic tissue acquired through organ donation, cells were isolated, re-programmed using non-integrating vectors and exposed to a four day differentiation protocol to generate definitive endoderm, a developmental precursor to pancreas. The best performing iPSC lines were then subjected to a 12-day basic differentiation protocol to generate endocrine pancreas precursors. The line that most consistently generated highly pure populations was selected for further development. This approach created an iPSC-variant cell line, SR1423, with a genetic profile correlated with preferential differentiation toward endodermal lineage at the loss of mesodermal potential. This report further describes an improved differentiation protocol that, coupled with SR1423, generated populations of greater than 60% insulin-expressing cells that secrete insulin in response to glucose and are capable of reversing diabetes in rodents. Created and banked following cGMP guidelines, SR1423 is a candidate cell line for the production of insulin-producing cells useful for the treatment of diabetes.

The monoterpene d-limonene exhibits chemotherapeutic and chemopreventive potential in breast cancer patients. D-limonene and its related compounds, perillyl alcohol and perillyl aldehyde, were chosen as candidate drugs for application in a screen for nontoxic inhibitors of cell migration. Using the nontumorigenic human breast cell line MCF-10A, we delineated the toxicity as greatest for the perillyl aldehyde, intermediate for perillyl alcohol, and least for limonene. A noncytotoxic concentration of 0.5 mmol/L perillyl alcohol inhibited the migration, while the same concentration of limonene failed to do so. Adhesion of the MCF-10A cell line and the human breast cancer cell line MDA-MB 435 to fibronectin was unaffected by 1.5 mmol/L perillyl alcohol. 0.4 mmol/L perillyl alcohol inhibited the growth of MDA-MB 435 cells. All migration-inhibiting concentrations of perillyl alcohol for MDA-MB 435 cells proved to be toxic. These results suggest that subtoxic doses of perillyl alcohol may have prophylactic potential in the treatment of breast cancer.

This review will briefly describe integrin function, address why integrins are attractive targets for chemotherapeutic drug design, and discuss some ongoing studies aimed at inhibiting integrin activity. Integrins are cell surface heterodimeric receptors. They modulate many cellular processes including: growth, death (apoptosis), adhesion, migration, and invasion by activating several signaling pathways. Many potential chemotherapeutic agents target integrins directly (eg, polypeptides, monoclonal antibodies, adenovirus vectors). These agents may be clinically useful in controlling the metastatic spread of cancer.