Stem cell sources and characterization in the development of cell-based products for treating retinal disease: An NEI Town Hall report.

National Eye Institute recently issued a new Strategic Plan outlining priority research areas for the next 5 years. Starting cell source for deriving stem cell lines is as an area with gaps and opportunities for making progress in regenerative medicine, a key area of emphasis within the NEI Strategic Plan. There is a critical need to understand how starting cell source affects the cell therapy product and what specific manufacturing capabilities and quality control standards are required for autologous vs allogeneic stem cell sources. With the goal of addressing some of these questions, in discussion with the community-at-large, NEI hosted a Town Hall at the Association for Research in Vision and Ophthalmology annual meeting in May 2022. This session leveraged recent clinical advances in autologous and allogeneic RPE replacement strategies to develop guidance for upcoming cell therapies for photoreceptors, retinal ganglion cells, and other ocular cell types. Our focus on stem cell-based therapies for RPE underscores the relatively advanced stage of RPE cell therapies to patients with several ongoing clinical trials. Thus, this workshop encouraged lessons learned from the RPE field to help accelerate progress in developing stem cell-based therapies in other ocular tissues. This report provides a synthesis of the key points discussed at the Town Hall and highlights needs and opportunities in ocular regenerative medicine.

Combined Inhibition of DYRK1A, SMAD, and Trithorax Pathways Synergizes to Induce Robust Replication in Adult Human Beta Cells.

Small-molecule inhibitors of dual-specificity tyrosine-regulated kinase 1A (DYRK1A) induce human beta cells to proliferate, generating a labeling index of 1.5%-3%. Here, we demonstrate that combined pharmacologic inhibition of DYRK1A and transforming growth factor beta superfamily (TGFßSF)/SMAD signaling generates remarkable further synergistic increases in human beta cell proliferation (average labeling index, 5%-8%, and as high as 15%-18%), and increases in both mouse and human beta cell numbers. This synergy reflects activation of cyclins and cdks by DYRK1A inhibition, accompanied by simultaneous reductions in key cell-cycle inhibitors (CDKN1C and CDKN1A). The latter results from interference with the basal Trithorax- and SMAD-mediated transactivation of CDKN1C and CDKN1A. Notably, combined DYRK1A and TGFß inhibition allows preservation of beta cell differentiated function. These beneficial effects extend from normal human beta cells and stem cell-derived human beta cells to those from people with type 2 diabetes, and occur both in vitro and in vivo.

A randomized clinical trial of recombinant human hyaluronidase-facilitated subcutaneous versus intravenous rehydration in mild to moderately dehydrated children in the emergency department.

BACKGROUND: Alternative treatment of dehydration is needed when intravenous (IV) or oral rehydration therapy fails. Subcutaneous (SC) hydration facilitated by recombinant human hyaluronidase offers an alternative treatment for dehydration. This clinical trial is the first to compare recombinant human hyaluronidase-facilitated SC (rHFSC) rehydration with standard IV rehydration for use in dehydrated children. OBJECTIVE: This Phase IV noninferiority trial evaluated whether rHFSC fluid administration can be given safely and effectively, with volumes similar to those delivered intravenously, to children who have mild to moderate dehydration. METHODS: The study included mild to moderately dehydrated children (Gorelick dehydration score) aged 1 month to 10 years. They were randomized to receive 20 mL/kg of isotonic fluids using rHFSC or IV therapy over 1 hour and then as needed until clinically rehydrated. The primary outcome was total volume of fluid administered (emergency department [ED] plus inpatient hospitalization). Secondary outcomes included mean volume infused in the ED alone, postinfusion dehydration scores and weight changes, line placement success and time, safety, and provider and parent/guardian questionnaire. RESULTS: 148 patients (mean age, 2.3 [1.91] years]; white, 53.4%; black, 31.8%) were enrolled in the intention-to-treat population (73 rHFSC; 75 IV). The primary outcome, mean total volume infused, was 365.0 (324.6) mL in the rHFSC group over 3.1 hours versus 455.8 (597.4) mL in the IV group over 6.6 hours (P = 0.51). The secondary outcome of mean volume infused in the ED alone was 334.3 (226.40) mL in the rHFSC group versus 299.6 (252.33) mL in the IV group (P = 0.03). Dehydration scores and weight changes postinfusion were similar. Successful line placement occurred in all 73 rHFSC-treated patients and 59 of 75 (78.7%) IV-treated patients (P < 0.0001). All IV failures occurred in patients aged <3 years; rHFSC rescue was successful in all patients in whom it was attempted. Both treatments were well tolerated. Clinicians rated fluid administration as easy to perform in 94.5% (69 of 73) of the rHFSC group versus 65.3% (49 of 75) of the IV group (P < 0.001). Parents/caregivers were satisfied or very satisfied with fluid administration in 94.5% (69 of 73) of rHFSC-treated patients and 73.3% (55 of 75) of IV-treated patients. CONCLUSIONS: In mild to moderately dehydrated children, rHFSC was inferior to IV hydration for the primary outcome measure. However, rHFSC was noninferior in the ED phase of hydration. Additional benefits of rHFSC included time and success of line placement, ease of use, and satisfaction. SC hydration facilitated with recombinant human hyaluronidase represents a reasonable addition to the treatment options for children who have mild to moderate dehydration, especially those with difficult IV access. ClinicalTrials.gov identifier: NCT00773175.

Parathyroid hormone-related protein enhances human ß-cell proliferation and function with associated induction of cyclin-dependent kinase 2 and cyclin E expression.

OBJECTIVE: Inducing human ß-cell growth while enhancing function is a major goal in the treatment of diabetes. Parathyroid hormone-related protein (PTHrP) enhances rodent ß-cell growth and function through the parathyroid hormone-1 receptor (PTH1R). Based on this, we hypothesized that PTH1R is expressed in human ß-cells and that PTHrP has the potential to enhance human ß-cell proliferation and/or function. RESEARCH DESIGN AND METHODS: PTH1R expression, ß-cell proliferation, glucose-stimulated insulin secretion (GSIS), and expression of differentiation and cell-cycle genes were analyzed in human islets transduced with adenoviral PTHrP constructs or treated with PTHrP peptides. The effect of overexpression of late G1/S cell cycle molecules was also assessed on human ß-cell proliferation. RESULTS: We found that human ß-cells express PTH1R. More importantly, overexpression of PTHrP causes a significant approximately threefold increase in human ß-cell proliferation. Furthermore, the amino terminus PTHrP(1-36) peptide is sufficient to increase replication as well as expression of the late G1/S cell-cycle proteins cyclin E and cyclin-dependent kinase 2 (cdk2) in human islets. Notably, PTHrP(1-36) also enhances GSIS. Finally, overexpression of cyclin E alone, but not cdk2, augments human ß-cell proliferation, and when both molecules are expressed simultaneously there is a further marked synergistic increase in replication. CONCLUSIONS: PTHrP(1-36) peptide enhances human ß-cell proliferation as well as function, with associated upregulation of two specific cell-cycle activators that together can induce human ß-cell proliferation several fold. The future therapeutic potential of PTHrP(1-36) for the treatment of diabetes is especially relevant given the complementary therapeutic efficacy of PTHrP(1-36) in postmenopausal osteoporosis.

Safety and pharmacokinetics of subcutaneous ceftriaxone administered with or without recombinant human hyaluronidase (rHuPH20) versus intravenous ceftriaxone administration in adult volunteers.

OBJECTIVE: To compare pharmacokinetics and safety of recombinant human hyaluronidase (rHuPH20)-facilitated subcutaneous (SC) ceftriaxone administration versus SC ceftriaxone preceded by SC saline placebo or intravenous (IV) ceftriaxone administration. RESEARCH DESIGN AND METHODS: This Phase I, two-part, placebo-controlled, crossover study was conducted in 54 healthy volunteers. In Part 1 (N = 24), subjects received 1 mL rHuPH20 (150 USP units) or placebo (0.9% sodium chloride) SC, followed by 1 or 2 g ceftriaxone (10-350 mg/mL). In Part 2 (N = 30), subjects received 1 g ceftriaxone at the Part 1 maximum tolerated concentration (MTC) administered either SC - preceded by SC rHuPH20 or placebo - or IV. Subjects were monitored for adverse events (AEs); blood samples were obtained (Part 2 only) during 48 hours post-dosing for ceftriaxone bioanalysis. MAIN OUTCOME MEASURES: Part 1 primary endpoint was the SC ceftriaxone (with or without rHuPH20) MTC. Pharmacokinetic parameters were determined in Part 2. Bioequivalence was based on maximum concentration (C(max)) and area under plasma concentration-time curve (AUC). RESULTS: The highest SC ceftriaxone concentration tested in Part 1 (350 mg/mL) was selected as the Part 2 MTC. In Part 2, median time to maximum concentration (t(max)) was 1 hour earlier (P < 0.0001), and C(max) was 12% higher (P < 0.0001) for ceftriaxone (350 mg/mL) administered via rHuPH20-facilitated SC versus SC preceded by placebo. IV ceftriaxone led to higher C(max) and shorter t(max) values than either SC treatment. Ceftriaxone exposure (AUC) was comparable among all three treatments. At least 1 AE was experienced by 100% of subjects after SC ceftriaxone and 76% after IV; most commonly reported AEs were infusion-site reactions. CONCLUSIONS: Ceftriaxone AUC did not differ significantly between the three administration routes. C(max) was higher and t(max) shorter with rHuPH20-facilitated SC than SC preceded by placebo. rHuPH20-facilitated SC ceftriaxone was generally well tolerated. This study is limited by evaluation of healthy adults and absence of repeated-dose groups.

The retinoblastoma protein and its homolog p130 regulate the G1/S transition in pancreatic beta-cells.

OBJECTIVE: The retinoblastoma protein family (pRb, p130, p107) plays a central role in the regulation of cell cycle progression. Surprisingly, loss of pRb in the beta-cell has no discernible effect on cell cycle control. Therefore, we explored the effects of individual loss of either p130 or p107 in addition to the simultaneous loss of both pRb/p130 on the beta-cell. RESEARCH DESIGN AND METHODS: Adult mice deficient in either p130 or p107 or both pRb/p130 were examined for effects on beta-cell replication, function, and survival. The Cre-Lox system was also used to inactivate pRb in wild-type and p130-deficient beta-cells in vitro. RESULTS: In vivo loss of either p107 or p130 did not affect beta-cell replication or function. Combined pRb/p130 loss, however, resulted in dramatically accelerated proliferation as well as apoptotic cell death. Pancreas and beta-cell mass were significantly reduced in double mutants. Despite this, overall glucose tolerance was normal, except for mild postprandial hyperglycemia. Ex vivo, acute deletion of pRb in p130-deficient beta-cells also caused a striking increase in proliferation. The combined deletion of pRb/p130 upregulated islet expression of E2F2 but not E2F1. CONCLUSIONS: These studies define an essential role for the pocket proteins in controlling the G(1)/S transition in beta-cells. When deficient in both pRb and p130, beta-cells undergo unrestrained cell cycle reentry and activation of apoptosis. These studies underscore the central role of the pRb pathway in controlling beta-cell turnover and provide new cellular targets for beta-cell regeneration.

Marginal mass islet transplantation with autologous mesenchymal stem cells promotes long-term islet allograft survival and sustained normoglycemia.

Allogeneic islet transplantation is an option to treat diabetes however there are obstacles that are limiting its clinical use. We have examined whether mesenchymal stem cells (MSC) improve islet graft survival and whether such therapy allows for better graft acceptance with reduced requirement for immunosuppression. In vitro-expanded syngeneic bone marrow-derived MSC were co-transplanted with islets into omental pouch in a rat model of streptozotocin-induced diabetes. Marginal mass syngeneic islet transplantation into the omentum with MSC promoted sustained normoglycemia. Interestingly, allogeneic islets +MSC, but not islets alone, with short-term use of immunosuppression enhanced long-term islet graft survival, insulin expression in the grafts and induced normal serum insulin levels and normoglycemia. T cells from recipients transplanted with allogeneic islets +MSC produced low levels of IFN-gamma and TNF-alpha upon ex-vivo activation, and this transplantation protocol promoted the generation of IL-10-secreting CD4(+) T cells. These data encourage further preclinical and eventually, clinical MSC-based islet transplantation to improve the outcome of allogeneic islet transplantation in the treatment of diabetes.

Survey of the human pancreatic beta-cell G1/S proteome reveals a potential therapeutic role for cdk-6 and cyclin D1 in enhancing human beta-cell replication and function in vivo.

OBJECTIVES: To comprehensively inventory the proteins that control the G1/S cell cycle checkpoint in the human islet and compare them with those in the murine islet, to determine whether these might therapeutically enhance human beta-cell replication, to determine whether human beta-cell replication can be demonstrated in an in vivo model, and to enhance human beta-cell function in vivo. RESEARCH DESIGN AND METHODS: Thirty-four G1/S regulatory proteins were examined in human islets. Effects of adenoviruses expressing cdk-6, cdk-4, and cyclin D1 on proliferation in human beta-cells were studied in both in vitro and in vivo models. RESULTS: Multiple differences between murine and human islets occur, most strikingly the presence of cdk-6 in human beta-cells versus its low abundance in the murine islet. Cdk-6 and cyclin D1 in vitro led to marked activation of retinoblastoma protein phosphorylation and cell cycle progression with no induction of cell death. Human islets transduced with cdk-6 and cyclin D1 were transplanted into diabetic NOD-SCID mice and markedly outperformed native human islets in vivo, maintaining glucose control for the entire 6 weeks of the study. CONCLUSIONS: The human G1/S proteome is described for the first time. Human islets are unlike their rodent counterparts in that they contain easily measurable cdk-6. Cdk-6 overexpression, alone or in combination with cyclin D1, strikingly stimulates human beta-cell replication, both in vitro as well as in vivo, without inducing cell death or loss of function. Using this model, human beta-cell replication can be induced and studied in vivo.

Lessons from the first comprehensive molecular characterization of cell cycle control in rodent insulinoma cell lines.

OBJECTIVE: Rodent insulinoma cell lines may serve as a model for designing continuously replicating human beta-cell lines and provide clues as to the central cell cycle regulatory molecules in the beta-cell. RESEARCH DESIGN AND METHODS: We performed a comprehensive G1/S proteome analysis on the four most widely studied rodent insulinoma cell lines and defined their flow cytometric profiles and growth characteristics. RESULTS: 1) Despite their common T-antigen-derived origins, MIN6 and BTC3 cells display markedly different G1/S expression profiles; 2) despite their common radiation origins, RINm5F and INS1 cells display striking differences in cell cycle protein profiles; 3) phosphorylation of pRb is absent in INS1 and RINm5F cells; 4) cyclin D2 is absent in RINm5F and BTC3 cells and therefore apparently dispensable for their proliferation; 5) every cell cycle inhibitor is upregulated, presumably in a futile attempt to halt proliferation; 6) among the G1/S proteome members, seven are pro-proliferation molecules: cyclin-dependent kinase-1, -2, -4, and -6 and cyclins A, E, and D3; and 7) overexpression of the combination of these seven converts arrested proliferation rates in primary rat beta-cells to those in insulinoma cells. Unfortunately, this therapeutic overexpression appears to mildly attenuate beta-cell differentiation and function. CONCLUSIONS: These studies underscore the importance of characterizing the cell cycle at the protein level in rodent insulinoma cell lines. They also emphasize the hazards of interpreting data from rodent insulinoma cell lines as modeling normal cell cycle progression. Most importantly, they provide seven candidate targets for inducing proliferation in human beta-cells.

Elevation in intracellular long-chain acyl-coenzyme A esters lead to reduced beta-cell excitability via activation of adenosine 5'-triphosphate-sensitive potassium channels.

Closure of pancreatic beta-cell ATP-sensitive potassium (K(ATP)) channels links glucose metabolism to electrical activity and insulin secretion. It is now known that saturated, but not polyunsaturated, long-chain acyl-coenyzme A esters (acyl-CoAs) can potently activate K(ATP) channels when superfused directly across excised membrane patches, suggesting a plausible mechanism to account for reduced beta-cell excitability and insulin secretion observed in obesity and type 2 diabetes. However, reduced beta-cell excitability due to elevation of endogenous saturated acyl-CoAs has not been confirmed in intact pancreatic beta-cells. To test this notion directly, endogenous acyl-CoA levels were elevated within primary mouse beta-cells using virally delivered overexpression of long-chain acyl-CoA synthetase-1 (AdACSL-1), and the effects on beta-cell K(ATP) channel activity and cell excitability was assessed using the perforated whole-cell and cell-attached patch-clamp technique. Data indicated a significant increase in K(ATP) channel activity in AdACSL-1-infected beta-cells cultured in medium supplemented with palmitate/oleate but not with the polyunsaturated fat linoleate. No changes in the ATP/ADP ratio were observed in any of the groups. Furthermore, AdACSL-1-infected beta-cells (with palmitate/oleate) showed a significant decrease in electrical responsiveness to glucose and tolbutamide and a hyperpolarized resting membrane potential at 5 mm glucose. These results suggest a direct link between intracellular fatty ester accumulation and K(ATP) channel activation, which may contribute to beta-cell dysfunction in type 2 diabetes.

Prolonged survival of microencapsulated neonatal porcine islets in mice treated with a combination of anti-CD154 and anti-LFA-1 monoclonal antibodies.

BACKGROUND: The aim of this study was to determine whether short-term administration of a combination of anti-CD154 and anti-LFA-1 monoclonal antibodies can prolong the survival of microencapsulated neonatal porcine islets (NPI) in immunocompetent mice. METHODS: Microencapsulated NPI were transplanted into the peritoneal cavity of streptozotocin-induced diabetic B6 mice that received a short-term treatment of a combination of anti-CD154 and anti-LFA-1 monoclonal antibodies. Blood glucose levels of each recipient were measured for more than 100 days posttransplantation or until graft rejection. Microcapsules were recovered to determine the presence of immune cells using immunoperoxidase staining. In addition, the levels of mouse anti-porcine immunoglobulin (Ig) G antibodies in the serum of each recipient were measured by flow cytometry. RESULTS: Short-term administration of a combination of monoclonal antibodies resulted in significant prolongation of microencapsulated NPI xenograft survival. All treated mice (n = 20) achieved normoglycemia within 10-35 days posttransplantation and 11/20 mice remained normoglycemic for more than 100 days posttransplantation. In contrast, only 1/20 of the untreated mice achieved normoglycemia and this mouse became diabetic at 17 days posttransplantation. Histological examination of the recovered microcapsules from long-term surviving treated mice revealed minimal cellular overgrowth containing intact viable islets, whereas several layers of immune cells surrounding the capsules containing nonviable islets were observed in untreated mice. The levels of mouse anti-porcine IgG was also reduced in treated recipients compared to untreated mice. CONCLUSIONS: These data demonstrate that short-term administration of anti-CD154 and anti-LFA-1 monoclonal antibodies can be effective in promoting long-term survival of microencapsulated NPI in immune-competent mice.

Peginterferon Alfa-2a plus ribavirin versus interferon alfa-2a plus ribavirin for chronic hepatitis C in HIV-coinfected persons.

BACKGROUND: Chronic hepatitis C virus (HCV) infection is a cause of major complications in persons who are also infected with the human immunodeficiency virus (HIV). However, the treatment of HCV infection in such persons has been associated with a high rate of intolerance and a low rate of response. We conducted a multicenter, randomized trial comparing peginterferon plus ribavirin with interferon plus ribavirin for the treatment of chronic hepatitis C in persons coinfected with HIV. METHODS: A total of 66 subjects were randomly assigned to receive 180 microg of peginterferon alfa-2a weekly for 48 weeks, and 67 subjects were assigned to receive 6 million IU of interferon alfa-2a three times weekly for 12 weeks followed by 3 million IU three times weekly for 36 weeks. Both groups received ribavirin according to a dose-escalation schedule. At week 24, subjects who did not have a virologic response (those who had an HCV RNA level greater than or equal to 60 IU per milliliter) underwent liver biopsy, and medications were continued in subjects with either a virologic response or histologic improvement. RESULTS: Treatment with peginterferon and ribavirin was associated with a significantly higher rate of sustained virologic response (an HCV RNA level of less than 60 IU per milliliter 24 weeks after completion of therapy) than was treatment with interferon and ribavirin (27 percent vs. 12 percent, P=0.03). In the group given peginterferon and ribavirin, only 14 percent of subjects with HCV genotype 1 infection had a sustained virologic response (7 of 51), as compared with 73 percent of subjects with an HCV genotype other than 1 (11 of 15, P<0.001). Histologic responses were observed in 35 percent of subjects with no virologic response who underwent liver biopsy. CONCLUSIONS: In persons infected with HIV, the combination of peginterferon and ribavirin is superior to the combination of interferon and ribavirin in the treatment of chronic hepatitis C. These regimens may provide clinical benefit even in the absence of virologic clearance. The marked discrepancy in the rates of sustained virologic response between HCV genotypes indicates that strategies are needed to improve the outcome in persons infected with HCV genotype 1.