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.

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.

Time-Driven Activity-Based Costing in Interventional Oncology: Cost Measurement and Cost Variability for Hepatocellular Carcinoma Therapies.

PURPOSE: To use time-drive activity-based costing (TDABC) to characterize and compare costs of transarterial chemoembolization (TACE), transarterial radioembolization (TARE), and ablation. METHODS: This three-part study involved (1) prospective observation to record resources used during TACE, TARE, and ablation and statistical evaluation of interobserver and interprocedure variability; (2) Bland-Altman analysis of prospective measurements and medical record time stamps to establish practicality of using retrospective data in place of direct observation; (3) retrospective time stamp assessment for 117 ablations, 61 TACE procedures, and 61 TARE procedures to reveal variability drivers. RESULTS: Ablation costs were lowest ($3,744), which were 74% of TACE costs ($5,089) and 18% of TARE costs ($20,818). Consumables were the greatest cost contributor, accounting for 65% of ablation, 58% of TACE, and 90% of TARE costs. A single consumable contributed to most of the overall costs: the ablation probe (42%), ethiodized oil for TACE (30%), and yttrium-90 microspheres for TARE (80%). Bland-Altman analysis showed agreement between retrospective time stamps and prospective measurements. Ablation costs increased from $3,288 to $4,245 to $4,461 for one, two, or three tumors treated. TACE cost increased from $5,051 to $5,296 for lobar versus selective approaches. CONCLUSION: A bottom-up costing approach using TDABC is feasible to assess true costs of hepatocellular carcinoma treatments and demonstrates ablation costs are significantly less than those of TACE and TARE. Replication of these methods at other institutions can facilitate development of a bundled payment model to promote utilization of locoregional therapies for hepatocellular carcinoma.

Survey of Radon Testing and Mitigation by Wisconsin Residents, Landlords, and School Districts.

INTRODUCTION: Radon is the second-leading cause of lung cancer in the United States, the leading cause of lung cancer in nonsmokers, and is estimated to cause 21,000 deaths every year. Radon is especially prevalent in the upper Midwest. This study aimed to assess radon testing and mitigation practices among residential homeowners, landlords, and school districts in Wisconsin. METHODS: Two survey sample datasets were used to assess radon testing and mitigation in residential homes: the Survey of the Health of Wisconsin (SHOW) and Wisconsin Behavioral Risk Factor Surveillance System (BRFSS) survey. Wisconsin landlords and school administrators were surveyed to assess radon testing and mitigation in rental properties and schools, respectively. RESULTS: Approximately 30% of Wisconsin homeowners (22.1% from SHOW and 39.9% from BRFSS) have tested their properties for radon. Similarly, 31.0% of Wisconsin landlords (40/129) and 35.1% of Wisconsin school districts (78/222) have tested their schools for radon. Of homeowners with elevated radon, about 60% mitigated. School districts whose radon levels tested high most commonly did not mitigate, with costs and/or lack of funding cited as the most common barrier. DISCUSSION/CONCLUSION: Radon testing and mitigation practices are inadequate in Wisconsin, and future work will seek to determine the best methods to increase testing and mitigation and reduce radon-induced lung cancer deaths in Wisconsin.

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.

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.