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A Method for Sectioning and Immunohistochemical Analysis of Stem Cell-Derived 3-D Organoids.

Sat, 2017-09-02 18:41
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A Method for Sectioning and Immunohistochemical Analysis of Stem Cell-Derived 3-D Organoids.

Curr Protoc Stem Cell Biol. 2016 May 12;37:1C.19.1-1C.19.11

Authors: Wiley LA, Beebe DC, Mullins RF, Stone EM, Tucker BA

Abstract
This unit describes a protocol for embedding, sectioning, and immunocytochemical analysis of pluripotent stem cell-derived 3-D organoids. Specifically, we describe a method to embed iPSC-derived retinal cups in low-melt agarose, acquire thick sections using a vibratome tissue slicer, and perform immunohistochemical analysis. This method includes an approach for antibody labeling that minimizes the amount of antibody needed for individual experiments and that utilizes large-volume washing to increase the signal-to-noise ratio, allowing for clean, high-resolution imaging of developing cell types. The universal methods described can be employed regardless of the type of pluripotent stem cell used and 3-D organoid generated. © 2016 by John Wiley & Sons, Inc.

PMID: 27171793 [PubMed - indexed for MEDLINE]

Generation of Xeno-Free, cGMP-Compliant Patient-Specific iPSCs from Skin Biopsy.

Sat, 2017-08-19 13:09
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Generation of Xeno-Free, cGMP-Compliant Patient-Specific iPSCs from Skin Biopsy.

Curr Protoc Stem Cell Biol. 2017 Aug 14;42:4A.12.1-4A.12.14

Authors: Wiley LA, Anfinson KR, Cranston CM, Kaalberg EE, Collins MM, Mullins RF, Stone EM, Tucker BA

Abstract
This unit describes protocols for the generation of clinical-grade patient-specific induced pluripotent stem cell (iPSC)-derived retinal cells from patients with inherited retinal degenerative blindness. Specifically, we describe how, using xeno-free reagents in an ISO class 5 environment, one can isolate and culture dermal fibroblasts, generate iPSCs, and derive autologous retinal cells via 3-D differentiation. The universal methods described herein for the isolation of dermal fibroblasts and generation of iPSCs can be employed regardless of disease, tissue, or cell type of interest. © 2017 by John Wiley & Sons, Inc.

PMID: 28806854 [PubMed - in process]

Structural and molecular changes in the aging choroid: implications for age-related macular degeneration.

Sat, 2017-07-29 07:21
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Structural and molecular changes in the aging choroid: implications for age-related macular degeneration.

Eye (Lond). 2017 Jan;31(1):10-25

Authors: Chirco KR, Sohn EH, Stone EM, Tucker BA, Mullins RF

Abstract
Age-related macular degeneration (AMD) is a devastating disease-causing vision loss in millions of people around the world. In advanced stages of disease, death of photoreceptor cells, retinal pigment epithelial cells, and choroidal endothelial cells (CECs) are common. Loss of endothelial cells of the choriocapillaris is one of the earliest detectable events in AMD, and, because the outer retina relies on the choriocapillaris for metabolic support, this loss may be the trigger for progression to more advanced stages. Here we highlight evidence for loss of CECs, including changes to vascular density within the choriocapillaris, altered abundance of CEC markers, and changes to overall thickness of the choroid. Furthermore, we review the key components and functions of the choroid, as well as Bruch's membrane, both of which are vital for healthy vision. We discuss changes to the structure and molecular composition of these tissues, many of which develop with age and may contribute to AMD pathogenesis. For example, a crucial event that occurs in the aging choriocapillaris is accumulation of the membrane attack complex, which may result in complement-mediated CEC lysis, and may be a primary cause for AMD-associated choriocapillaris degeneration. The actions of elevated monomeric C-reactive protein in the choriocapillaris in at-risk individuals may also contribute to the inflammatory environment in the choroid and promote disease progression. Finally, we discuss the progress that has been made in the development of AMD therapies, with a focus on cell replacement.

PMID: 27716746 [PubMed - indexed for MEDLINE]

Choroidal γδ T cells in protection against retinal pigment epithelium and retinal injury.

Sat, 2017-07-22 06:42
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Choroidal γδ T cells in protection against retinal pigment epithelium and retinal injury.

FASEB J. 2017 Jul 20;:

Authors: Zhao Z, Liang Y, Liu Y, Xu P, Flamme-Wiese MJ, Sun D, Sun J, Mullins RF, Chen Y, Cai J

Abstract
γδ T cells located near the epithelial barrier are integral components of local inflammatory and innate immune responses. We have previously reported the presence of choroidal γδ T cells in a model of chronic degeneration of the retinal pigment epithelium (RPE). The goals of the current study were to further define the functions of choroidal γδ T cells and to explore the underlying mechanisms of their action. Our data demonstrate that choroidal γδ T cells are activated by RPE injury in response to NaIO3 treatment, and that they express genes that encode immunosuppressive cytokines, such as IL-4 and IL-10. γδ T cell-deficient mice developed profound RPE and retinal damage at doses that caused minimal effects in wild-type mice, and adoptive transfer of γδ T cells prevented sensitization. Intravitreal injection of IL-4 and IL-10 ameliorated RPE toxicity that was induced by NaIO3Ex vivo coculture of γδ T cells with RPE explants activated the production of anti-inflammatory cytokines via an aryl hydrocarbon receptor (AhR)-dependent mechanism. AhR deficiency abolished the protective effects of γδ T cells after adoptive transfer. Collectively, these findings define important roles for choroid γδ T cells in maintaining tissue homeostasis in the outer retina.-Zhao, Z., Liang, Y., Liu, Y., Xu, P., Flamme-Wiese, M. J., Sun, D., Sun, J., Mullins, R. F., Chen, Y., Cai, J. Choroidal γδ T cells in protection against retinal pigment epithelium and retinal injury.

PMID: 28729290 [PubMed - as supplied by publisher]

Using CRISPR-Cas9 to Generate Gene-Corrected Autologous iPSCs for the Treatment of Inherited Retinal Degeneration.

Fri, 2017-06-23 23:59
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Using CRISPR-Cas9 to Generate Gene-Corrected Autologous iPSCs for the Treatment of Inherited Retinal Degeneration.

Mol Ther. 2017 Jun 12;:

Authors: Burnight ER, Gupta M, Wiley LA, Anfinson KR, Tran A, Triboulet R, Hoffmann JM, Klaahsen DL, Andorf JL, Jiao C, Sohn EH, Adur MK, Ross JW, Mullins RF, Daley GQ, Schlaeger TM, Stone EM, Tucker BA

Abstract
Patient-derived induced pluripotent stem cells (iPSCs) hold great promise for autologous cell replacement. However, for many inherited diseases, treatment will likely require genetic repair pre-transplantation. Genome editing technologies are useful for this application. The purpose of this study was to develop CRISPR-Cas9-mediated genome editing strategies to target and correct the three most common types of disease-causing variants in patient-derived iPSCs: (1) exonic, (2) deep intronic, and (3) dominant gain of function. We developed a homology-directed repair strategy targeting a homozygous Alu insertion in exon 9 of male germ cell-associated kinase (MAK) and demonstrated restoration of the retinal transcript and protein in patient cells. We generated a CRISPR-Cas9-mediated non-homologous end joining (NHEJ) approach to excise a major contributor to Leber congenital amaurosis, the IVS26 cryptic-splice mutation in CEP290, and demonstrated correction of the transcript and protein in patient iPSCs. Lastly, we designed allele-specific CRISPR guides that selectively target the mutant Pro23His rhodopsin (RHO) allele, which, following delivery to both patient iPSCs in vitro and pig retina in vivo, created a frameshift and premature stop that would prevent transcription of the disease-causing variant. The strategies developed in this study will prove useful for correcting a wide range of genetic variants in genes that cause inherited retinal degeneration.

PMID: 28619647 [PubMed - as supplied by publisher]

Association of reduced Connexin 43 expression with retinal vascular lesions in human diabetic retinopathy.

Fri, 2017-06-23 23:59
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Association of reduced Connexin 43 expression with retinal vascular lesions in human diabetic retinopathy.

Exp Eye Res. 2016 May;146:103-6

Authors: Tien T, Muto T, Zhang J, Sohn EH, Mullins RF, Roy S

Abstract
Connexin 43 (Cx43) downregulation promotes apoptosis in retinal vascular cells of diabetic animal models; however, its relevance to human diabetic retinopathy has not been established. In this study, we investigated whether diabetes alters Cx43 expression and promotes retinal vascular lesions in human retinas. Diabetic human eyes (aged 64-94 years) and non-diabetic human eyes (aged 61-90 years) were analyzed in this study. Retinal protein samples and retinal capillary networks were assessed for Cx43 level by Western blot (WB) analysis and immunostaining. In parallel, retinal capillary networks were stained with hematoxylin and periodic acid Schiff to determine the extent of pericyte loss (PL) and acellular capillaries (AC) in these retinas. Cx43 protein expression was significantly reduced in the diabetic retinas compared to non-diabetic retinas as indicated by WB analysis (81 ± 11% of control). Additionally, a significant decrease in the number of Cx43 plaques per unit length of vessel was observed in the diabetic retinas compared to those of non-diabetic retinas (62 ± 10% of control; p < 0.005). Importantly, a strong inverse relationship was noted between Cx43 expression and the relative number of AC (r = -0.89; p < 0.0005), and between Cx43 expression and number of pericyte loss (r = -0.88; p < 0.0005). Overall, these results show that Cx43 expression is reduced in the human diabetic retinas and Cx43 reduction is associated with increased vascular cell death. These findings suggest that diabetes decreases retinal Cx43 expression and that the development of PL and AC is associated with reduced Cx43 expression in human diabetic retinopathy.

PMID: 26738943 [PubMed - indexed for MEDLINE]

Clinically Focused Molecular Investigation of 1000 Consecutive Families with Inherited Retinal Disease.

Fri, 2017-06-02 19:37
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Clinically Focused Molecular Investigation of 1000 Consecutive Families with Inherited Retinal Disease.

Ophthalmology. 2017 May 27;:

Authors: Stone EM, Andorf JL, Whitmore SS, DeLuca AP, Giacalone JC, Streb LM, Braun TA, Mullins RF, Scheetz TE, Sheffield VC, Tucker BA

Abstract
PURPOSE: To devise a comprehensive multiplatform genetic testing strategy for inherited retinal disease and to describe its performance in 1000 consecutive families seen by a single clinician.
DESIGN: Retrospective series.
PARTICIPANTS: One thousand consecutive families seen by a single clinician.
METHODS: The clinical records of all patients seen by a single retina specialist between January 2010 and June 2016 were reviewed, and all patients who met the clinical criteria for a diagnosis of inherited retinal disease were included in the study. Each patient was assigned to 1 of 62 diagnostic categories, and this clinical diagnosis was used to define the scope and order of the molecular investigations that were performed. The number of nucleotides evaluated in a given subject ranged from 2 to nearly 900 000.
MAIN OUTCOME MEASURES: Sensitivity and false genotype rate.
RESULTS: Disease-causing genotypes were identified in 760 families (76%). These genotypes were distributed across 104 different genes. More than 75% of these 104 genes have coding sequences small enough to be packaged efficiently into an adeno-associated virus. Mutations in ABCA4 were the most common cause of disease in this cohort (173 families), whereas mutations in 80 genes caused disease in 5 or fewer families (i.e., 0.5% or less). Disease-causing genotypes were identified in 576 of the families without next-generation sequencing (NGS). This included 23 families with mutations in the repetitive region of RPGR exon 15 that would have been missed by NGS. Whole-exome sequencing of the remaining 424 families revealed mutations in an additional 182 families, and whole-genome sequencing of 4 of the remaining 242 families revealed 2 additional genotypes that were invisible by the other methods. Performing the testing in a clinically focused tiered fashion would be 6.1% more sensitive and 17.7% less expensive and would have a significantly lower average false genotype rate than using whole-exome sequencing to assess more than 300 genes in all patients (7.1% vs. 128%; P < 0.001).
CONCLUSIONS: Genetic testing for inherited retinal disease is now more than 75% sensitive. A clinically directed tiered testing strategy can increase sensitivity and improve statistical significance without increasing cost.

PMID: 28559085 [PubMed - as supplied by publisher]

Rapid Communication: Solution for the MEEK Glue Transfer Problem.

Fri, 2017-06-02 19:37
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Rapid Communication: Solution for the MEEK Glue Transfer Problem.

J Burn Care Res. 2017 May 23;:

Authors: Culnan DM, Craft-Coffman B, Bitz G, Mullins RF

Abstract
Meek micrografting permits wide expansion of skin grafts in true ratios from 3:1 to 9:1, as well as the utilization of poor donor sites. The proprietary glue critical to successful skin transference is unavailable in the United States. While the technique is widely employed worldwide, alternative glues resulted in poor skin transfer and frustrated use in American burn centers. The authors present their protocol resulting in effective MEEK skin transfer using Mastisol® adhesive: "The Rule of Sevens." 1) Soak the corks in normal saline for 7 minutes. 2) Then spread the grafts on the corks and mince with the MEEK machine. 3) Spray the epidermal surface of the micrograft-covered corks thoroughly with 7 pumps of Mastisol® from a distance of 7 inches (17.7 cm). 4) Allow the Mastisol® to dry for 7 minutes on the micrografts. 5) Apply the corks with the Mastisol®-imbued skin to the gauzes. Press firmly for 7 seconds. 6) Allow the skin to transfer from cork to gauze undisturbed for 7 minutes. Next, carefully remove the corks and expand the gauzes. Apply the micrograft-covered gauzes to excised and prepared wound beds and staple into position. 7) After 7 days, remove the gauzes, though the authors have left them in place for up to 21 days. This novel protocol provides reliable skin transfer and permits the modified MEEK technique to be a consistent part of our practice. The authors present this rapid communication to allow others to utilize this technique without the frustration of adhesive failure resulting in lost grafts.

PMID: 28557867 [PubMed - as supplied by publisher]

West Nile Virus Infection in Human and Mouse Cornea Tissue.

Fri, 2017-06-02 19:37
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West Nile Virus Infection in Human and Mouse Cornea Tissue.

Am J Trop Med Hyg. 2016 Nov 02;95(5):1185-1191

Authors: Blitvich BJ, Wang T, Saxena V, Zeng S, Harmon KM, Raymond MD, Goins KM, Reed CR, Mullins RF, Greiner MA

Abstract
The purpose of this study was to determine the in vitro and ex vivo susceptibility of human corneal cells to West Nile virus (WNV) infection and evaluate the ability of the virus to disseminate to the corneas of infected mice. Human corneal epithelial cells were challenged with WNV, incubated for 1-6 days, and tested for evidence of WNV infection. Viral RNA and antigen were detected at every time point, and the virus reached a peak titer of 2.5 × 10(7) plaque-forming units (pfu)/mL at 3 days postinoculation (PI). Corneas procured from donors were incubated in culture dishes containing WNV for 1-5 days and tested for evidence of WNV. Viral RNA and antigen were detected, and the virus reached a mean peak titer of 4.9 × 10(4) pfu/mL at 5 days PI. Mice were inoculated intraperitoneally with WNV, and their eyes were harvested at 2, 5, and 8 days PI and tested for evidence of WNV. Viral RNA was detected in corneas of four of nine systemically infected mice as early as 2 days PI. We conclude that human corneal cells support WNV replication in vitro and ex vivo, and WNV may disseminate into the corneas of experimentally infected mice. These findings indicate that corneal transmission cannot be ruled out as a novel mode of human-to-human WNV transmission and additional experiments should be conducted to assess this risk further.

PMID: 27672204 [PubMed - indexed for MEDLINE]