M. Joseph Phillips

iPS cell modeling of Best disease: Insights into the pathophysiology of an inherited macular degeneration

Best disease (BD) is an inherited degenerative disease of the human macula that results in progressive and irreversible central vision loss. It is caused by mutations in the retinal pigment epithelium (RPE) gene BESTROPHIN1 (BEST1), which, through mechanism(s) that remain unclear, lead to the accumulation of subretinal fluid and autofluorescent waste products from shed photoreceptor outer segments (POSs). We employed human iPS cell (hiPSC) technology to generate RPE from BD patients and unaffected siblings in order to examine the cellular and molecular processes underlying this disease. Consistent with the clinical phenotype of BD, RPE from mutant hiPSCs displayed disrupted fluid flux and increased accrual of autofluorescent material after long-term POS feeding when compared with hiPSC-RPE from unaffected siblings. On a molecular level, RHODOPSIN degradation after POS feeding was delayed in BD hiPSC-RPE relative to unaffected sibling hiPSC-RPE, directly implicating impaired POS handling in the pathophysiology of the disease. In addition, stimulated calcium responses differed between BD and normal sibling hiPSC-RPE, as did oxidative stress levels after chronic POS feeding. Subcellular localization, fractionation and co-immunoprecipitation experiments in hiPSC-RPE and human prenatal RPE further linked BEST1 to the regulation and release of endoplasmic reticulum calcium stores. Since calcium signaling and oxidative stress are critical regulators of fluid flow and protein degradation, these findings likely contribute to the clinical picture of BD. In a larger context, this report demonstrates the potential to use patient-specific hiPSCs to model and study maculopathies, an important class of blinding disorders in humans.

Blood-Derived Human iPS Cells Generate Optic Vesicle–Like Structures with the Capacity to Form Retinal Laminae and Develop Synapses

PURPOSE We sought to determine if human induced pluripotent stem cells (iPSCs) derived from blood could produce optic vesicle-like structures (OVs) with the capacity to stratify and express markers of intercellular communication. METHODS Activated T-lymphocytes from a routine peripheral blood sample were reprogrammed by retroviral transduction to iPSCs. The T-lymphocyte-derived iPSCs (TiPSCs) were characterized for pluripotency and differentiated to OVs using our previously published protocol. TiPSC-OVs were then manually isolated, pooled, and cultured en masse to more mature stages of retinogenesis. Throughout this stepwise differentiation process, changes in anterior neural, retinal, and synaptic marker expression were monitored by PCR, immunocytochemistry, and/or flow cytometry. RESULTS TiPSCs generated abundant OVs, which contained a near homogeneous population of proliferating neuroretinal progenitor cells (NRPCs). These NRPCs differentiated into multiple neuroretinal cell types, similar to OV cultures from human embryonic stem cells and fibroblast-derived iPSCs. In addition, portions of some TiPSC-OVs maintained their distinctive neuroepithelial appearance and spontaneously formed primitive laminae, reminiscent of the developing retina. Retinal progeny from TiPSC-OV cultures expressed numerous genes and proteins critical for synaptogenesis and gap junction formation, concomitant with the emergence of glia and the upregulation of thrombospondins in culture. CONCLUSIONS We demonstrate for the first time that human blood-derived iPSCs can generate retinal cell types, providing a highly convenient donor cell source for iPSC-based retinal studies. We also show that cultured TiPSC-OVs have the capacity to self-assemble into rudimentary neuroretinal structures and express markers indicative of chemical and electrical synapses.

Functional Analysis of Serially Expanded Human iPS Cell-Derived RPE Cultures

PURPOSE To determine the effects of serial expansion on the cellular, molecular, and functional properties of human iPS cell (hiPSC)-derived RPE cultures. METHODS Fibroblasts obtained from four individuals were reprogrammed into hiPSCs and differentiated to RPE cells using previously described methods. Patches of deeply pigmented hiPSC-RPE were dissected, dissociated, and grown in culture until they re-formed pigmented monolayers. Subsequent passages were obtained by repeated dissociation, expansion, and maturation of RPE into pigmented monolayers. Gene and protein expression profiles and morphological and functional characteristics of hiPSC-RPE at different passages were compared with each other and to human fetal RPE (hfRPE). RESULTS RPE from all four hiPSC lines could be expanded more than 1000-fold when serially passaged as pigmented monolayer cultures. Importantly, expansion of hiPSC-RPE monolayers over the first three passages (P1-P3) resulted in decreased expression of pluripotency and neuroretinal markers and maintenance of characteristic morphological features and gene and protein expression profiles. Furthermore, P1 to P3 hiPSC-RPE monolayers reliably demonstrated functional tight junctions, G-protein-coupled receptor-mediated calcium transients, phagocytosis and degradation of photoreceptor outer segments, and polarized secretion of biomolecules. In contrast, P4 hiPSC-RPE cells failed to form monolayers and possessed altered morphological and functional characteristics and gene expression levels. CONCLUSIONS Highly differentiated, pigmented hiPSC-RPE monolayers can undergo limited serial expansion while retaining key cytological and functional attributes. However, passaging hiPSC-RPE cultures beyond senescence leads to loss of such features. Our findings support limited, controlled passaging of patient-specific hiPSC-RPE to procure cells needed for in vitro disease modeling, drug screening, and cellular transplantation.

Modeling Human Retinal Development with Patient‐Specific Induced Pluripotent Stem Cells Reveals Multiple Roles for Visual System Homeobox 2

Human induced pluripotent stem cells (hiPSCs) have been shown to differentiate along the retinal lineage in a manner that mimics normal mammalian development. Under certain culture conditions, hiPSCs form optic vesicle‐like structures (OVs), which contain proliferating progenitors capable of yielding all neural retina (NR) cell types over time. Such observations imply conserved roles for regulators of retinogenesis in hiPSC‐derived cultures and the developing embryo. However, whether and to what extent this assumption holds true has remained largely uninvestigated. We examined the role of a key NR transcription factor, visual system homeobox 2 (VSX2), using hiPSCs derived from a patient with microphthalmia caused by an R200Q mutation in the VSX2 homeodomain region. No differences were noted between (R200Q)VSX2 and sibling control hiPSCs prior to OV generation. Thereafter, (R200Q)VSX2 hiPSC‐OVs displayed a significant growth deficit compared to control hiPSC‐OVs, as well as increased production of retinal pigmented epithelium at the expense of NR cell derivatives. Furthermore, (R200Q)VSX2 hiPSC‐OVs failed to produce bipolar cells, a distinctive feature previously observed in Vsx2 mutant mice. (R200Q)VSX2 hiPSC‐OVs also demonstrated delayed photoreceptor maturation, which could be overcome via exogenous expression of wild‐type VSX2 at early stages of retinal differentiation. Finally, RNAseq analysis on isolated hiPSC‐OVs implicated key transcription factors and extracellular signaling pathways as potential downstream effectors of VSX2‐mediated gene regulation. Our results establish hiPSC‐OVs as versatile model systems to study retinal development at stages not previously accessible in humans and support the bona fide nature of hiPSC‐OV‐derived retinal progeny. Stem Cells 2014;32:1480–1492

Differential Expression of Neuronal Genes in Müller Glia in Two- and Three-Dimensional Cultures

PURPOSE Müller glia in the mammalian retina have some stem cell-like characteristics, although their capacity for neurogenesis remains limited both in vivo and in vitro. In vitro studies to date have used traditional two-dimensional (2D) cell culture to assess neuronal differentiation of Müller glia. The purpose of this study was to compare the effects of 2D and three-dimensional (3D) environments on Müller glial gene expression after growth factor stimulation. METHODS Conditionally immortalized mouse Müller glia cells (ImM10) were cultured under nonimmortalizing conditions with EGF/FGF2 to generate spheres that were differentiated in vitro on uncoated culture dishes (2D) or encapsulated in self-assembling, RADA-16 peptide hydrogels (3D) under identical media and growth factor supplementation conditions. Gene expression was analyzed using quantitative RT-PCR and immunocytochemistry. Cellular morphology was analyzed with light and confocal microscopy; sphere ultrastructure was analyzed with transmission electron microscopy. RESULTS ImM10 Müller cells express numerous genes associated with neural stem cells and retinal progenitors in both normal growth conditions and sphere-forming conditions. When encapsulated in the 3D hydrogel, cells can migrate and send processes into the hydrogel. Many genes associated with neurogenesis, as well as retinal neuron-specific genes, are differentially expressed in 2D and 3D differentiation conditions. CONCLUSIONS ImM10 Müller glia upregulate genes characteristic of retinal neurons after growth factor stimulation in vitro, and gene expression patterns are altered in 3D hydrogel cultures.

Modeling retinal degenerative diseases with human iPS-derived cells: current status and future implications

David M Gamm, Department of Ophthalmology and Visual Sciences, McPherson Eye Research Institute, Waisman Center Stem Cell Research Program, University of Wisconsin School of Medicine and Public Health, Waisman Center, Room T609, 1500 Highland Ave, Madison, WI 53705, USA M Joseph Phillips, and Waisman Center Stem Cell Research Program, University of Wisconsin School of Medicine and Public Health, Waisman Center, Room T609, 1500 Highland Ave, Madison, WI 53705, USA Ruchira Singh Waisman Center Stem Cell Research Program, University of Wisconsin School of Medicine and Public Health, Waisman Center, Room T609, 1500 Highland Ave, Madison, WI 53705, USA

Retinal Function and Structure in Ant1-Deficient Mice

PURPOSE Mutations in ANT, a mitochondrial ATP transporter, are typically associated with myopathy. Because of the high metabolic demands of the retina, the authors examined whether elimination of the Ant1 isoform in a transgenic mouse affects retinal function or morphology. METHODS RT-PCR was used to confirm Ant1 expression in retinas of wild-type (WT) or Ant1(-/-) mice. Full-field ERGs were used to test retinal function under dark- and light-adapted conditions and the recovery of the photoresponse to a bright flash. Using histologic methods, the authors assessed the retinal location of ANT and ANT1-β-gal reporter protein, mitochondrial activity with cytochrome c oxidase (COX) and succinate dehydrogenase (SDH) staining, retinal layer thickness, and bipolar cell types using Chx10 and recoverin. RESULTS Ant1(-/-) mice had supernormal ERG b-waves under both dark- and light-adapted conditions. X-Gal staining was detected in a subset of cells within the inner retina. The following characteristics were normal in Ant1(-/-) mice compared with age-matched WT mice: recovery of the photoresponse, COX and SDH activity, retinal morphology, and bipolar cell morphology. CONCLUSIONS The presence of ANT1 in a subset of inner retinal cells accompanied by supernormal ERG responses suggests that ANT1 may be localized to hyperpolarizing bipolar cells. However, the immunohistochemical techniques used here did not show any differences in bipolar cells. Moderate functional changes coupled with a lack of detectable morphologic changes suggest that ANT1 is not essential for ATP transport in the retina.

Regulation of WNT Signaling by VSX2 During Optic Vesicle Patterning in Human Induced Pluripotent Stem Cells

Few gene targets of Visual System Homeobox 2 (VSX2) have been identified despite its broad and critical role in the maintenance of neural retina (NR) fate during early retinogenesis. We performed VSX2 ChIP‐seq and ChIP‐PCR assays on early stage optic vesicle‐like structures (OVs) derived from human iPS cells (hiPSCs), which highlighted WNT pathway genes as direct regulatory targets of VSX2. Examination of early NR patterning in hiPSC‐OVs from a patient with a functional null mutation in VSX2 revealed mis‐expression and upregulation of WNT pathway components and retinal pigmented epithelium (RPE) markers in comparison to control hiPSC‐OVs. Furthermore, pharmacological inhibition of WNT signaling rescued the early mutant phenotype, whereas augmentation of WNT signaling in control hiPSC‐OVs phenocopied the mutant. These findings reveal an important role for VSX2 as a regulator of WNT signaling and suggest that VSX2 may act to maintain NR identity at the expense of RPE in part by direct repression of WNT pathway constituents. Stem Cells 2016;34:2625–2634

A Novel Approach to Single Cell RNA‐Sequence Analysis Facilitates In Silico Gene Reporting of Human Pluripotent Stem Cell‐Derived Retinal Cell Types

Cell type‐specific investigations commonly use gene reporters or single‐cell analytical techniques. However, reporter line development is arduous and generally limited to a single gene of interest, while single‐cell RNA (scRNA)‐sequencing (seq) frequently yields equivocal results that preclude definitive cell identification. To examine gene expression profiles of multiple retinal cell types derived from human pluripotent stem cells (hPSCs), we performed scRNA‐seq on optic vesicle (OV)‐like structures cultured under cGMP‐compatible conditions. However, efforts to apply traditional scRNA‐seq analytical methods based on unbiased algorithms were unrevealing. Therefore, we developed a simple, versatile, and universally applicable approach that generates gene expression data akin to those obtained from reporter lines. This method ranks single cells by expression level of a bait gene and searches the transcriptome for genes whose cell‐to‐cell rank order expression most closely matches that of the bait. Moreover, multiple bait genes can be combined to refine datasets. Using this approach, we provide further evidence for the authenticity of hPSC‐derived retinal cell types. Stem Cells 2018;36:313–324

Retinal Ganglion Cell Diversity and Subtype Specification from Human Pluripotent Stem Cells

Summary Retinal ganglion cells (RGCs) are the projection neurons of the retina and transmit visual information to postsynaptic targets in the brain. While this function is shared among nearly all RGCs, this class of cell is remarkably diverse, comprised of multiple subtypes. Previous efforts have identified numerous RGC subtypes in animal models, but less attention has been paid to human RGCs. Thus, efforts of this study examined the diversity of RGCs differentiated from human pluripotent stem cells (hPSCs) and characterized defined subtypes through the expression of subtype-specific markers. Further investigation of these subtypes was achieved using single-cell transcriptomics, confirming the combinatorial expression of molecular markers associated with these subtypes, and also provided insight into more subtype-specific markers. Thus, the results of this study describe the derivation of RGC subtypes from hPSCs and will support the future exploration of phenotypic and functional diversity within human RGCs.