Eric Benedetti

Role of the retinal vascular endothelial cell in ocular disease

Retinal endothelial cells line the arborizing microvasculature that supplies and drains the neural retina. The anatomical and physiological characteristics of these endothelial cells are consistent with nutritional requirements and protection of a tissue critical to vision. On the one hand, the endothelium must ensure the supply of oxygen and other nutrients to the metabolically active retina, and allow access to circulating cells that maintain the vasculature or survey the retina for the presence of potential pathogens. On the other hand, the endothelium contributes to the blood-retinal barrier that protects the retina by excluding circulating molecular toxins, microorganisms, and pro-inflammatory leukocytes. Features required to fulfill these functions may also predispose to disease processes, such as retinal vascular leakage and neovascularization, and trafficking of microbes and inflammatory cells. Thus, the retinal endothelial cell is a key participant in retinal ischemic vasculopathies that include diabetic retinopathy and retinopathy of prematurity, and retinal inflammation or infection, as occurs in posterior uveitis. Using gene expression and proteomic profiling, it has been possible to explore the molecular phenotype of the human retinal endothelial cell and contribute to understanding of the pathogenesis of these diseases. In addition to providing support for the involvement of well-characterized endothelial molecules, profiling has the power to identify new players in retinal pathologies. Findings may have implications for the design of new biological therapies. Additional progress in this field is anticipated as other technologies, including epigenetic profiling methods, whole transcriptome shotgun sequencing, and metabolomics, are used to study the human retinal endothelial cell.

Generation of islet-like cells from mouse gall bladder by direct ex vivo reprogramming

Cell replacement is an emerging therapy for type 1 diabetes. Pluripotent stem cells have received a lot of attention as a potential source of transplantable β-cells, but their ability to form teratomas poses significant risks. Here, we evaluated the potential of primary mouse gall bladder epithelial cells (GBCs) as targets for ex vivo genetic reprogramming to the β-cell fate. Conditions for robust expansion and genetic transduction of primary GBCs by adenoviral vectors were developed. Using a GFP reporter for insulin, conditions for reprogramming were then optimized. Global expression analysis by RNA-sequencing was used to quantitatively compare reprogrammed GBCs (rGBCs) to true β-cells, revealing both similarities and differences. Adenoviral-mediated expression of NEUROG3, Pdx1, and MafA in GBCs resulted in robust induction of pancreatic endocrine genes, including Ins1, Ins2, Neurod1, Nkx2-2 and Isl1. Furthermore, expression of GBC-specific genes was repressed, including Sox17 and Hes1. Reprogramming was also enhanced by addition of retinoic acid and inhibition of Notch signaling. Importantly, rGBCs were able to engraft long term in vivo and remained insulin-positive for 15weeks. We conclude that GBCs are a viable source for autologous cell replacement in diabetes, but that complete reprogramming will require further manipulations.

Therapeutic Liver Reconstitution With Murine Cells Isolated Long After Death

BACKGROUND & AIMS Due to the shortage of donor organs, many patients needing liver transplantation cannot receive one. For some liver diseases, hepatocyte transplantation could be a viable alternative, but donor cells currently are procured from the same sources as whole organs, and thus the supply is severely limited. METHODS Here, we investigated the possibility of isolating viable hepatocytes for liver cell therapy from the plentiful source of morgue cadavers. To determine the utility of this approach, cells were isolated from the livers of non-heart-beating cadaveric mice long after death and transplanted into fumarylacetoacetate hydrolase-deficient mice, a model for the human metabolic liver disease hereditary tyrosinemia type I and a stringent in vivo model for hepatic cell transplantation. RESULTS Surprisingly, complete and therapeutic liver repopulation could be achieved with hepatocytes derived up to 27 hours post mortem. CONCLUSIONS Competitive repopulation experiments showed that cadaveric liver cells had a repopulation capacity similar to freshly isolated hepatocytes. Importantly, viable hepatocytes also could be isolated from cadaveric primate liver (monkey and human) efficiently. These data provide evidence that non-heart-beating donors could be a suitable source of hepatocytes for much longer time periods than previously thought possible.

Oxymetholone Therapy of Fanconi Anemia Suppresses Osteopontin Transcription and Induces Hematopoietic Stem Cell Cycling

Summary Androgens are widely used for treating Fanconi anemia (FA) and other human bone marrow failure syndromes, but their mode of action remains incompletely understood. Aged Fancd2−/− mice were used to assess the therapeutic efficacy of oxymetholone (OXM) and its mechanism of action. Eighteen-month-old Fancd2−/− mice recapitulated key human FA phenotypes, including reduced bone marrow cellularity, red cell macrocytosis, and peripheral pancytopenia. As in humans, chronic OXM treatment significantly improved these hematological parameters and stimulated the proliferation of hematopoietic stem and progenitor cells. RNA-Seq analysis implicated downregulation of osteopontin as an important potential mechanism for the drug’s action. Consistent with the increased stem cell proliferation, competitive repopulation assays demonstrated that chronic OXM therapy eventually resulted in stem cell exhaustion. These results expand our knowledge of the regulation of hematopoietic stem cell proliferation and have direct clinical implications for the treatment of bone marrow failure.

Fancd2 and p21 function independently in maintaining the size of hematopoietic stem and progenitor cell pool in mice.

Fanconi anemia patients suffer from progressive bone marrow failure. An overactive p53 response to DNA damage contributes to the progressive elimination of Fanconi anemia hematopoietic stem and progenitor cells (HSPC), and hence presents a potential target for therapeutic intervention. To investigate whether the cell cycle regulatory protein p21 is the primary mediator of the p53-dependent stem cell loss, p21/Fancd2 double-knockout mice were generated. Surprisingly double mutant mice displayed even more severe loss of HSPCs than Fancd2(-/-) single mutants. p21 deletion did not rescue the abnormal cell cycle profile and had no impact on the long-term repopulating potential of Fancd2(-/-) bone marrow cells. Collectively, our data indicate that p21 has an indispensable role in maintaining a normal HSPC pool and suggest that other p53-targeted factors, not p21, mediate the progressive elimination of HSPC in Fanconi anemia.

Evaluation of resveratrol and N-acetylcysteine for cancer chemoprevention in a Fanconi anemia murine model

Fanconi anemia (FA) patients suffer from progressive bone marrow failure and often develop cancers. Previous studies showed that antioxidants tempol and resveratrol (RV) delayed tumor onset and reduced hematologic defects in FA murine models, respectively. Here we tested whether antioxidants N‐acetylcysteine (NAC) or RV could delay cancer in tumor prone Fancd2−/−/Trp53+/− mice. Unlike tempol, neither compound had any significant chemopreventive effect in this model. We conclude that not all anti‐oxidants are chemopreventive in FA. In addition, when given to Fancd2−/− mice, NAC helped maintain Fancd2−/− KSL cells in quiescence while tempol did not. The mechanisms behind the different actions of these antioxidants await further investigation. Pediatr Blood Cancer 2014;61:740–742.

Reprogramming human gallbladder cells into insulin-producing β-like cells

The gallbladder and cystic duct (GBCs) are parts of the extrahepatic biliary tree and share a common developmental origin with the ventral pancreas. Here, we report on the very first genetic reprogramming of patient-derived human GBCs to β-like cells for potential autologous cell replacement therapy for type 1 diabetes. We developed a robust method for large-scale expansion of human GBCs ex vivo. GBCs were reprogrammed into insulin-producing pancreatic β-like cells by a combined adenoviral-mediated expression of hallmark pancreatic endocrine transcription factors PDX1, MAFA, NEUROG3, and PAX6 and differentiation culture in vitro. The reprogrammed GBCs (rGBCs) strongly induced the production of insulin and pancreatic endocrine genes and these responded to glucose stimulation in vitro. rGBCs also expressed an islet-specific surface marker, which was used to enrich for the most highly reprogrammed cells. More importantly, global mRNA and microRNA expression profiles and protein immunostaining indicated that rGBCs adopted an overall β-like state and these rGBCs engrafted in immunodeficient mice. Furthermore, comparative global expression analyses identified putative regulators of human biliary to β cell fate conversion. In summary, we have developed, for the first time, a reliable and robust genetic reprogramming and culture expansion of primary human GBCs—derived from multiple unrelated donors—into pancreatic β-like cells ex vivo, thus showing that human gallbladder is a potentially rich source of reprogrammable cells for autologous cell therapy in diabetes.

274. Expansion and Conversion of Patient-Derived Human Gallbladder Cells Into Insulin-Producing Pancreatic Endocrine-Like Type Cells

Islet transplantation is an efficacious therapeutic option for T1D, but, supply of donor pancreata does not meet the demand. Variable successes in generating glucose-responsive insulin-producing beta-like cells from pluripotent and differentiated cells have been reported. We report on genetic reprogramming of highly expandable patient-derived human gallbladder cells (GBCs) to produce insulin for autologous transplant. Using feeder-free and feeder-type culture methods, we expanded GBCs in vitro in the order of 10^8 total cells after 4 passages starting from 10^4 GBCs. Cultures can be maintained for at least 15 passages, albeit with slowdown in growth. Transcriptome profiling revealed GBCs to be deficient in factors important for beta-cell development, specification, and maintenance such as NEUROG3, MAFA, NKX6-1, NKX2-2, PAX4, PAX6, NEUROD1, INSM1, RFX6, GCK, and INS. MNX1 and PDX1 are moderately expressed in cultures but at significantly lower levels found in beta-cells. Viral transduction of PDX1, MAFA, and NEUROG3 (PNM) induced expression of INS at 1/10,000th compared to that of human islet cells. Transduced GBCs began to show morphological changes from a monolayer into 3D clusters as early as 2 days after transduction. Retinoic acid, GLP-1, FGF10, DAPT, T3, Alk5 inhibitor, and B27 in the reprogramming medium and the addition of PAX6 to PNM transduction highly improved both INS and NKX6-1 induction reaching 10% of islet cells. These reprogrammed GBCs also express high levels of NEUROD1, NKX2-2, RFX6, PAX4, MAFB, HOPX, SST, GHRL, CHGA, TMEM27, SYP, KCNJ11, and ABCC8 comparable to islet cells. Average transduction efficiency was 50% yielding 9% C-peptide+ cells by immunostaining. Cellular C-peptide content was equivalent to 0.3% of that of purified beta-cells. At best, we observed 5-fold increase in C-peptide concentration in response to high glucose. We further enriched for INS+ reprogrammed GBCs using a cell surface mAb (HPi1) shown to isolate human pancreatic islet cells. Immunolabeling showed that reprogramming significantly increased the percentage of Hpi1+ GBCs. RT-qPCR of Hpi1+ GBCs showed greater than 1000-fold enrichment in gene expression of INS, NKX6-1, and PCSK1 compared to Hpi1- cells. Likewise, we observed greater than 100-fold enrichment for NEUROD1, SST, GHRL, and RFX6. In addition, we saw significant increase in reprogramming efficiency by adding an isoxazole to the reprogramming medium resulting to 10-fold higher INS expression, 2.5-fold increase in the frequency of C-peptide+ cells, and significant drop in SST and GHRL expression. Reprogrammed GBCs can engraft, survive, and produce C-peptide for at least 2 weeks in under the kidney capsule, mammary fat pad, and dorsal subcutaneous fat in NSG mice. Transplant experiments in diabetic mice are ongoing. In summary, we have transdifferentiated patient-derived adult GBCs into beta-like cells ex vivo, thus showing that GBCs are potential replacement sources of autologous insulin-producing cells.