Browsing by Author "Belecky-Adams, Teri"
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Item Analyses of the development and function of stem cell derived cells in neurodegenerative diseases(2022-12) Lavekar, Sailee Sham; Meyer, Jason; Canfield, Scott; Belecky-Adams, Teri; Mastracci, Teresa; Perrin, BenjaminHuman pluripotent stem cells (hPSCs) are an attractive tool for the study of different neurodegenerative diseases due to their potential to form any cell type of the body. Due to their versatility and self-renewal capacity, they have different applications such as disease modeling, high throughput drug screening and transplantation. Different animal models have helped answer broader questions related to the physiological functioning of various pathways and the phenotypic effects of a particular neurodegenerative disease. However, due to the lack of success recapitulating some targets identified from animal models into successful clinical trials, there is a need for a direct translational disease model. Since their advent, hPSCs have helped understand various disease effectors and underlying mechanisms using genetic engineering techniques, omics studies and reductionist approaches for the recognition of candidate molecules or pathways required to answer questions related to neurodevelopment, neurodegeneration and neuroregeneration. Due to the simplified approach that iPSC models can provide, some in vitro approaches are being developed using microphysiological systems (MPS) that could answer complex physiological questions. MPS encompass all the different in vitro systems that could help better mimic certain physiological systems that tend to not be mimicked by in vivo models. In this dissertation, efforts have been directed to disease model as well as to understand the intrinsic as well as extrinsic cues using two different MPS. First, we have used hPSCs with Alzheimer’s disease (AD)-related mutations to differentiate into retinal organoids and identify AD related phenotypes for future studies to identify retinal AD biomarkers. Using 5 month old retinal organoids from AD cell lines as well as controls, we could identify retinal AD phenotypes such as an increase in Aβ42:Aβ40 ratio along with increase in pTau:Tau. Nanostring analyses also helped in identification of potential target genes that are modulated in retinal AD that were related to synaptic dysfunction. Thus, using retinal organoids for the identification of retinal AD phenotypes could help delve deeper into the identification of future potential biomarkers in the retina of AD patients, with the potential to serve as a means for early identification and intervention for patients. The next MPS we used to serve to explore non-cell autonomous effects associated with glaucoma to explore the neurovascular unit. Previous studies have demonstrated the degeneration of RGCs in glaucoma due to a point mutation OPTN(E50K) that leads to the degeneration of RGCs both at morphological and functional levels. Thus, using the previous studies as a basis, we wanted to further unravel the impact of this mutation using the different cell types of the neurovascular unit such as endothelial cells, astrocytes and RGCs. Interestingly, we observed the barrier properties being impacted by the mutation present in both RGCs and astrocytes demonstrated through TEER, permeability and transcellular transport changes. We also identified a potential factor TGFβ2 that was observed to be overproduced by the OPTN E50K astrocytes to demonstrate similar effects with the exogenous addition of TGFβ2 on the barrier. Furthermore, the inhibition of TGFβ2 helped rescue some of the barrier dysfunction phenotypes. Thus, TGFβ2 inhibition can be used as a potential candidate that can be used to further study its impact in in vivo models and how that can be used in translational applications. Thus, MPS systems have a lot of applications that can help answer different physiologically relevant questions that are hard to approach using in vivo models and the further development of these systems to accentuate the aspects of neural development and how it goes awry in different neurodegenerative diseases.Item An Animal Model of Combined Pituitary Hormone Deficiency Disease(2010-08) Colvin, Stephanie C.; Konieczny, Stephen F.; Rhodes, Simon J.; Walvoord, Emily C.; Belecky-Adams, Teri; Herring, B. Paul; Roper, RandallLHX3 is a LIM-homeodomain transcription factor that has essential roles in pituitary and nervous system development in mammals. Children who are homozygous for recessive mutations in the LHX3 gene present with combined pituitary hormone deficiency disease (CPHD) characterized by deficits of multiple anterior pituitary hormones. Most LHX3 patients also present with additional defects associated with the nervous system including a characteristic limited head rotation and sometimes deafness. However, of the 10 types of LHX3 mutation described to date, one mutation type (W224ter) does not result in the limited head rotation, defining a new form of the disease. W224ter patients have CPHD but do not have nervous system symptoms. Whereas other mutations in LHX3 cause loss of the entire protein or its activity, the W224ter mutation causes specific loss of the carboxyl terminal of the LHX3 protein—a region that we have shown to contain critical regulatory domains for pituitary gene activation. To better understand the molecular and cellular etiology of CPHD associated with LHX3 gene mutations, I have generated knock-in mice that model the human LHX3 W224ter disease. The resulting mice display marked dwarfism, thyroid disease, female infertility, and reduced male fertility. Immunohistochemistry, real-time quantitative polymerase chain reaction (PCR), and enzyme-linked immunosorbant assays (ELISA) were used to measure hormones and regulatory factor protein and RNA levels, an approach which is not feasible with human patients. We have generated a novel mouse model of human pediatric CPHD. Our findings are consistent with the hypothesis that the actions of the LHX3 factor are molecularly separable in the nervous system and pituitary gland.Item Axonal Outgrowth and Pathfinding of Human Pluripotent Stem Cell-Derived Retinal Ganglion Cells(2020-08) Fligor, Clarisse; Meyer, Jason; Marrs, James; Belecky-Adams, Teri; Jones, Kathryn; Baucum, AJRetinal ganglion cells (RGCs) serve as a vital connection between the eye and the brain with damage to their axons resulting in loss of vision and/or blindness. Reti- nal organoids are three-dimensional structures derived from human pluripotent stem cells (hPSCs) which recapitulate the spatial and temporal differentiation of the retina, providing a valuable model of RGC development in vitro. The working hypothesis of these studies is that hPSC-derived RGCs are capable of extensive outgrowth and display target specificity and pathfinding abilities. Initial efforts focused on charac- terizing RGC differentiation throughout early stages of organoid development, with a clearly defined RGC layer developing in a temporally-appropriate manner express- ing a compliment of RGC-associated markers. Beyond studies of RGC development, retinal organoids may also prove useful to investigate and model the extensive axonal outgrowth necessary to reach post-synaptic targets. As such, additional efforts aimed to elucidate factors promoting axonal outgrowth. Results demonstrated significant enhancement of axonal outgrowth through modulation of both substrate composi- tion and growth factor signaling. Furthermore, RGCs possessed guidance receptors that are essential in influencing outgrowth and pathfinding. Subsequently, to de- termine target specificity, aggregates of hPSC-derived RGCs were co-cultured with explants of mouse lateral geniculate nucleus (LGN), the primary post-synaptic target of RGCs. Axonal outgrowth was enhanced in the presence of LGN, and RGCs dis- played recognition of appropriate targets, with the longest neurites projecting towards LGN explants compared to control explants or RGCs grown alone. Generated from xvii the fusion of regionally-patterned organoids, assembloids model projections between distinct regions of the nervous system. Therefore, final efforts of these studies focused upon the generation of retinocortical assembloids in order to model the long-distance outgrowth characteristic of RGCs. RGCs displayed extensive axonal outgrowth into cortical organoids, with the ability to respond to environmental cues. Collectively, these results establish retinal organoids as a valuable tool for studies of RGC develop- ment, and demonstrate the utility of organoid-derived RGCs as an effective platform to study factors influencing outgrowth as well as modeling long-distance projections and pathfinding abilities.Item BMP Pathway and Reactive Retinal Gliosis(2013-03-06) Dharmarajan, Subramanian; Belecky-Adams, Teri; Skalnik, David Gordon; Zhang, Xin; Atkinson, SimonReactive gliosis is known to have a beneficial and a degenerative effect following injury to neurons. Although many factors have been implicated in reactive gliosis, their role in regulating this change is still unclear. We investigated the role of bone morphogenetic proteins in reactive gliosis in vivo and in vitro. In vivo, IHC analysis indicated reactive gliosis in the 6 week Ins2Akita mouse and WPK rat retinas. Expression of BMP7 was upregulated in these models, leading to an increase in the phosphorylation of downstream SMAD1. In vitro, treatment of murine retinal astrocyte cells with a strong oxidizing agent such as sodium peroxynitrite regulated RNA levels of various markers, including GFAP, CSPGs, MMPs and TIMPs. BMP7 treatment also regulated RNA levels to a similar extent, suggesting reactive gliosis. Treatment with high glucose DMEM and BMP4, however, did not elicit increase in levels to a similar degree. Increase in SMAD levels and downstream targets of SMAD signaling such as ID1, ID3 and MSX2 was also observed following treatment with sodium peroxynitrite in vitro and in the 6 week Ins2Akita mouse retinas in vivo. These data concur with previously established data which show an increase in BMP7 levels following injury. It also demonstrates a role for BMP7 in gliosis following disease. Further, it suggests SMAD signaling to play a role in initiating reactivity in astrocytes as well as in remodeling the extracellular matrix following injury and in a disease condition.Item BMP PATHWAY AND RETINAL ASTROGLIOSIS(Office of the Vice Chancellor for Research, 2012-04-13) Dharmarajan, Subramanian; Sheibani, Nadeer; Belecky-Adams, TeriAstrocytes are the star shaped glial (non-neural) cells present in the cen-tral nervous system. The astrocytes perform a wide range of functions in-cluding: serving as an energy source, aiding neuronal development, main-taining homeostasis and detoxification. Astrocytes are also present in the eye, constituting the majority of the glial cell population and are found in the retina. Any injury to nearby neurons makes the astrocytes reactive, altering their function and morphology. One of the features of reactive astrocytes is to form a physical barrier around the injury site, called the glial scar. One group of molecules thought to play a role is the bone morphogenic proteins (BMPs). Although, the BMPs have been found to increase in CNS following injury, their role in making the retinal astrocytes reactive is not yet known. Our purpose in performing these studies was to clarify the role of BMP7 (a type of the BMP) in retinal astrocytes reactive gliosis. Mouse retinal astrocyte cells were incubated with sodium peroxynitrite (a strong oxidizing agent, which has been previously shown to make astrocytes reactive) or different cocncentrations of BMP7 for different time periods. Cells were lysed and total protein or total RNA was isolated to analyze the protein and gene expression levels of different markers. Treatment with peroxynitrite led to statistically significant increase in levels of expression in including astrocyte specific markers and certain in-hibitory molecules. In the BMP7 treated samples, similar increases in the levels of expression for the astrocyte specific markers and the inhibitory molecules. A comparison between the expression profiles of peroxynitrite- and BMP7-treated cells showed largely (but not completely) overlapping pro-files of expression. The work done here helps ascertain the role of BMP7 in reactive gliosis in vitro. Further, the observation of distinct reactivity profiles under different conditions is indicative of involvement of additional pathways.Item Characterization of Ethanol-induced Effects on Zebrafish Retinal Development: Mechanistic Perspective and Therapeutic Strategies(2016) Muralidharan, Pooja; Marrs, James A.; Leung, Yuk Fai; Belecky-Adams, Teri; Meyer, Jason; Anderson, Ryan M.; Randall, Stephen K.Fetal alcohol spectrum disorder (FASD) is a result of prenatal alcohol exposure, producing a wide range of defects including craniofacial, sensory, motor and cognitive deficits. Many ocular abnormalities are frequently associated with FASD including microphthalmia, optic nerve hypoplasia, and cataracts. FASD is highly prevalent in low socioeconomic populations, where it is also accompanied by higher rates of malnutrition and alcoholism. Using zebrafish as a model to study FASD retinal defects has been extremely insightful in understanding the ethanol-induced retinal defects at the cellular level. Zebrafish embryos treated with ethanol from mid-blastula transition through somitogenesis (2-24 hours post fertilization; hpf) showed defects similar to human ocular deficits including microphthalmia, optic nerve hypoplasia, and photoreceptor differentiation defects. Ethanol exposure altered critical transcription factor expression involved in retinal cell differentiation. Retinoic acid (RA) and folic acid (FA) nutrient co-supplementation rescued optic nerve and photoreceptor differentiation defects. Ethanol exposure during retinal morphogenesis stages (16-24 hpf), produced retinal defects like those seen with ethanol exposure between 2-24 hpf. Significantly, during ethanol-sensitive time window (16-24 hpf), RA co-supplementation moderately rescued these defects, whereas, FA cosupplementation showed significant rescue of optic nerve and photoreceptor differentiation. RA, but not FA, supplementation after ethanol exposure could restore ethanol-induced optic nerve and photoreceptor differentiation defects. Ethanol exposure did not affect timing of retinal cell differentiation induction, but later increased retinal cell death and proliferation. Ethanol-treated embryos showed increased retinal proliferation in the outer nuclear layer (ONL), inner nuclear layer (INL), and ciliary marginal zone (CMZ) at 48 hpf and 72 hpf. In order to identify the genesis of ethanol-induced persistent retinal defects, ethanol effects on retinal stem cell populations in the CMZ and the Müller glial cells (MGCs) were examined. Ethanol treated retinas had an expanded CMZ indicated by histology and Alcama, a retinal stem cell marker, immunolabeling, but reduced expression of rx1 and the cell cycle exit marker, cdkn1c. Ethanol treated retinas also showed reduced MGCs. At 72 hpf, ONL of ethanol exposed fish showed fewer photoreceptors expressing terminal differentiation markers. Importantly, these poorly differentiated photoreceptors co-expressed the basic helix-loop-helix (bHLH) proneural differentiation factor, neurod, indicating that ethanol exposure produced immature and undifferentiated photoreceptors. Reduced differentiation along with increased progenitor marker expression and proliferation suggest cell cycle exit failure due to ethanol exposure. These results suggested that ethanol exposure disrupted stem cell differentiation progression. Wnt, Notch and proneural gene expression regulate retinal stem cell proliferation and transition into progenitor cells. Ethanol exposure disrupted Wnt activity in the CMZ as well as Notch activity and neurod gene expression in the retina. RA and FA co-supplementation were able to rescue Wnt activity in the CMZ and rescue downstream Notch activity. To test whether the rescue of these Wnt-active cells could restore the retinal cell differentiation pathways, ethanol treated embryos were treated with Wnt agonist. This treatment could restore Wnt-active cells in the CMZ, Notch-active cells in the retina, proliferation, and photoreceptor terminal differentiation. We conclude that ethanol exposure produced persistent defects in the stem cell Wnt signaling, a critical pathway in retinal cell differentiation. Further analysis of underlying molecular mechanisms will provide insight into the embryonic origins of ethanol-induced retinal defects and potential therapeutic targets to cure this disorder.Item Characterizing Changes in the Brain During Hydrocephalic Development and Exploring Potential Treatment Strategies(2024-05) Reed, Makenna M.; Blazer-Yost, Bonnie; Belecky-Adams, Teri; Cummins, Theodore; Baucum, A. J.; Jantzie, LaurenA neurological disorder, hydrocephalus, has an estimated global pediatric prevalence of 380,000 new cases each year [1]. It is a family of diseases that can occur at any age when cerebrospinal fluid builds up within the ventricles of the brain. Thus, the only available treatments are surgical, invasive, and prone to complications. There is a global need for successful treatment strategies without brain surgery. Choroid plexus epithelial cells (CPEC) are responsible for production of cerebrospinal fluid (CSF). Ependymal cells line the ventricles and play roles in CSF maintenance and waste clearance. Astrocytes perform various functions, one being blood-brain barrier (BBB) maintenance. Collectively these cells contribute to brain fluid/electrolyte regulation and barrier integrity. Increased glial fibrillary acidic protein (GFAP) fluorescence, a marker of activated astrocytes, appeared in hydrocephalic (Tmem67-/-) animals by immunohistochemistry as early as postnatal day (P)10. The tight junction proteins expressed in choroid plexus (CP); claudin-1 (Cl-1) and zona occludin 1 (ZO-1) fluorescent intensity increased in P15 hydrocephalic animals compared to wildtype (Tmem67+/+). These cells also contain aquaporins (AQP), aquaporin-1 (AQP1) and aquaporin-4 (AQP4), important in regulating CSF and interstitial fluid (ISF). Increased fluorescent intensity of AQP4 in the subventricular zone and increased AQP1 apical localization and protein amount in the CP was observed in hydrocephalic animals at postnatal day (P)15. Many of these may be targeted for the treatment of hydrocephalus. However, there is no consensus in pathological findings between models of hydrocephalus and these finding may not translate to common pharmacological targets. A transient receptor potential cation channel, subfamily vanilloid, member 4 (TRPV4) antagonist (RN1734) ameliorates hydrocephalus in a rat model of congenital hydrocephalus (Tmem67 model). It was hypothesized that targeting this mechanosensitive ion channel may slow production of CSF by targeting the CP. However, hydrocephalus pathology can have various effects on the brain. Astrocytes were visualized using fluorescent immunohistochemistry of glial fibrillary acidic protein (GFAP) and RN1734 did not seem to change immunoreactivity to wildtype untreated levels. Increased immunoreactivity of TRPV4 and AQP1 was observed in CP of untreated and RN1734 treated Tmem67-/- rats. AQP4 and TRPV4 immunoreactivity increased in the subventricular zone and periventricular white matter (WM) of hydrocephalic rats. With RN1734, TRPV4 immunoreactivity, but not AQP4, had similar immunoreactivity to wildtype untreated. Increased GFAP and AQP immunoreactivity may indicate residual inflammation in the Tmem67-/- rats. More experiments must be done to further elucidate TRPV4’s role in hydrocephalus pathology. Serum and glucocorticoid-regulated kinase 1 (SGK1) is a kinase implicated in cell volume regulation and CSF production. SI113, an SGK1 inhibitor, ameliorates hydrocephalus in the Tmem67 rodent model. The goal of this study was to determine if SI113 could be used with a new solvent other than dimethyl sulfoxide (DMSO), which can have possible toxic effects. 1-methyl-2-pyrrolidinone (NMP) has high solubility and ability to cross the BBB. These studies showed that NMP as a solvent did not have adverse effects on body weight, however thus far, it has not ameliorated hydrocephalus significantly at the concentration used in this study. There is a possibility that the concentration in NMP that we used was not efficacious enough. CSF and blood plasma samples from animals treated with SI113 24 hours and 30 minutes before euthanasia will be used to investigate the concentration of SI113 that remains in the circulation and the amount that crosses the BBB and blood-cerebrospinal fluid (BCSFB) barriers. We hope that the results will inform dosage for our future studies. Future studies may also examine SI113 mechanism of action in hydrocephalus. This thesis addresses hydrocephalus cell and molecular pathology in the Tmem67 model and examines potential treatment strategies. Future directions include comparing models of hydrocephalus to find common treatment strategies in the hope to find pharmaceutical strategies to better manage human hydrocephalus.Item Development of Therapies to Treat Polycystic Kidney Disease(2013-03-06) Flaig, Stephanie Marge; Blazer-Yost, Bonnie; Gattone II, Vincent H.; Belecky-Adams, TeriPolycystic kidney diseases (PKD) are genetic disorders characterized by fluid filled cysts in the kidney tubules and liver bile ducts. There are two forms of PKD, autosomal dominant polycystic kidney disease (ADPKD) and autosomal recessive polycystic kidney disease (ARPKD). The focus of the studies in this thesis has been on ADPKD. The disease progresses slowly and the fluid-filled cysts grow in size due to increased rates of cell proliferation and fluid secretion into the cyst lumen. The expanding cysts compromise the normal kidney function and result in a decrease of renal function to the point of end-stage renal failure in midlife. Cyst enlargement is due, at least in part, to chloride secretion via the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. Currently therapy is limited to renal cyst aspiration, dialysis, and eventually renal transplantation after organ failure, thus it has critical to determine possible drug therapies for the treatment of PKD. Previous studies showed that cyst fluid caused a secretory response in cells lining the cysts. We hypothesized that once the cyst have expanded and become so large that they burst or leak, which could also occur due to renal injury or aging, the cyst fluid may stimulate additional cyst growth. Lysophosphatidic Acid (LPA) was determined to be the active component of human cyst fluid, and we investigated the LPA stimulated signaling pathway. Our data suggest that the LPA stimulates chloride and fluid secretion by a combination of CFTR and Calcium-Activated chloride channels (CaCC) and that the two channels may functionally be linked to each other. The secretion is not occurring through a cAMP stimulated pathway, and it is possible that TMEM16A, a CaCC, plays a larger role than previously expected. Previous studies demonstrated that PPARγ agonists, insulin sensitizing drugs used to treat diabetes, inhibit chloride secretion by the collecting duct principal cells by decreasing CFTR synthesis. It was logical therefore to considered PPARγ agonists as long-term treatment for PKD. The first preclinical studied showed that high (20 mg/kg BW) dose pioglitazone, a PPARγ agonist, inhibited cyst growth in the PCK rat model, a slow progressing model, of PKD. To continue to look at the effects of the PPARγ agonists another preclinical study was completed, which tested if there was a class action of PPARγ agonists and if a lower dose was effective in treating the cystic burden. Using the PCK rat model, and another PPARγ agonist, rosiglitazone, a 24 week study was completed using 3 doses (4, 0.4, and 0.04 mg/kg BW). 4 mg/kg BW rosiglitazone is analogous to 20 mg/kg BW pioglitazone. The data indicated that the rosiglitazone is effective in lowering the cystic burden, and importantly the low dose proved to be effective. An additional rat model, the W-WPK rapidly progressing model was used to determine efficacy across multiple models, and to determine if there was a way to track the progress of the disease in a manner analogous to that used in human patients. The animals were treated with pioglitazone using 2 doses (2 and 20 mg/kg BW), and were imaged using CT scans to track the progress of the disease. The data suggest that pioglitazone was not as effective in the W-WPK rat model as it was the PCK rat model. There was a trend however, that low dose PPARγ agonist was as effective ad high dose. Even more important, the CT scans proved to be an effective way to track the progress of the disease in animal models.Item Developmental Differences and Altered Gene Expression in the Ts65Dn Mouse Model of Down Syndrome(2012-03-20) Billingsley, Cherie Nicole; Roper, Randall J.; Chernoff, Ellen; Belecky-Adams, TeriTrisomy 21 occurs in approximately 1 out of 750 live births and causes brachycephaly, a small oral cavity, a shortened mid-face, and mental impairments in individuals with Down syndrome (DS). Craniofacial dysmorphology occurs in essentially all individuals with trisomy 21 and causes functional difficulties. Mouse models are commonly used to study the etiology of human disorders because of the conserved phenotypes between species. The Ts65Dn Down syndrome mouse model has triplicated homologues for approximately half the genes on human chromosome 21 and exhibits many phenotypes that parallel those found in individuals with DS. Specifically, newborn and adult Ts65Dn mice display similar craniofacial defects as humans with DS. Ts65Dn embryos also exhibit smaller mandibular precursors than their euploid littermates at embryonic day 9.5 (E9.5). Furthermore, Ts65Dn mice exhibit reduced birth weight which suggests a possible generalized delay in overall embryonic growth. Based on previous research at E9.5, it was hypothesized that Ts65Dn E13.5 embryos would have reduced mandibular precursors with altered gene expression. It was also hypothesized that other neural crest derived structures would be reduced in trisomic embryos. Using morphological measurements it was determined that the mandible, Meckel’s cartilage, and hyoid cartilage were significantly reduced in E13.5 trisomic embryos. The tongue was of similar size in trisomic and euploid embryos while cardiac and brain tissue volumes were not significantly different between genotypes. Analysis of total embryonic size at E9.5 and E13.5 revealed smaller trisomic embryos with developmental attenuation that was not related to maternal trisomy. A microarray analysis performed on the mandibular precursor revealed 155 differentially expressed non-trisomic genes. Sox9 was of particular interest for its role in cartilage condensation and endochondral ossification. It was hypothesized that the overexpression of Sox9 in the developing mandible would be localized to Meckel’s and hyoid cartilages. Immunohistochemistry performed on the mandibular precursor confirmed an overexpression of Sox9 in both Meckel’s and the hyoid cartilages. This research provides further insight into the development of trisomic tissues, both neural crest and non-neural crest-derived, and also the specific molecular mechanisms that negatively affect mandibular development in Ts65Dn mice and presumably individuals with Down syndrome.Item Differentiation and characterization of cell types associated with retinal degenerative diseases using human induced pluripotent stem cells(2014-07-31) Gupta, Manav; Meyer, Jason S.; Belecky-Adams, Teri; Randall, Stephen Karl, 1953-Human induced pluripotent stem (iPS) cells have the unique ability to differentiate into 200 or so somatic cell types that make up the adult human being. The use of human iPS cells to study development and disease is a highly exciting and interdependent field that holds great promise in understanding and elucidating mechanisms behind cellular differentiation with future applications in drug screening and cell replacement studies for complex and currently incurable cellular degenerative disorders. The recent advent of iPS cell technology allows for the generation of patient-specific cell lines that enable us to model the progression of a disease phenotype in a human in vitro model. Differentiation of iPS cells toward the affected cell type provides an unlimited source of diseased cells for examination, and to further study the developmental progression of the disease in vitro, also called the “disease-in-a-dish” model. In this study, efforts were undertaken to recapitulate the differentiation of distinct retinal cell affected in two highly prevalent retinal diseases, Usher syndrome and glaucoma. Using a line of Type III Usher Syndrome patient derived iPS cells efforts were undertaken to develop such an approach as an effective in vitro model for studies of Usher Syndrome, the most commonly inherited disorder affecting both vision and hearing. Using existing lines of iPS cells, studies were also aimed at differentiation and characterization of the more complex retinal cell types, retinal ganglion cells (RGCs) and astrocytes, the cell types affected in glaucoma, a severe neurodegenerative disease of the retina leading to eventual irreversible blindness. Using a previously described protocol, the iPS cells were directed to differentiate toward a retinal fate through a step-wise process that proceeds through all of the major stages of neuroretinal development. The differentiation process was monitored for a period of 70 days for the differentiation of retinal cell types and 150 days for astrocyte development. The different stages of differentiation and the individually derived somatic cell types were characterized by the expression of developmentally associated transcription factors specific to each cell type. Further approaches were undertaken to characterize the morphological differences between RGCs and other neuroretinal cell types derived in the process. The results of this study successfully demonstrated that Usher syndrome patient derived iPS cells differentiated to the affected photoreceptors of Usher syndrome along with other mature retinal cell types, chronologically analogous to the development of the cell types in a mature human retina. This study also established a robust method for the in vitro derivation of RGCs and astrocytes from human iPS cells and provided novel methodologies and evidence to characterize these individual somatic cell types. Overall, this study provides a unique insight into the application of human pluripotent stem cell biology by establishing a novel platform for future studies of in vitro disease modeling of the retinal degenerative diseases: Usher syndrome and glaucoma. In downstream applications of this study, the disease relevant cell types derived from human iPS cells can be used as tools to further study disease progression, drug screening and cell replacement strategies.