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Connective Tissue Growth Factor Promotes Efficient Generation of Human Induced Pluripotent Stem Cell-Derived Choroidal Endothelium.

Sun, 2019-06-30 05:28
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Connective Tissue Growth Factor Promotes Efficient Generation of Human Induced Pluripotent Stem Cell-Derived Choroidal Endothelium.

Stem Cells Transl Med. 2017 06;6(6):1533-1546

Authors: Songstad AE, Worthington KS, Chirco KR, Giacalone JC, Whitmore SS, Anfinson KR, Ochoa D, Cranston CM, Riker MJ, Neiman M, Stone EM, Mullins RF, Tucker BA

Abstract
Age-related macular degeneration (AMD) is a leading cause of irreversible blindness in the Western world. Although, the majority of stem cell research to date has focused on production of retinal pigment epithelial (RPE) and photoreceptor cells for the purpose of evaluating disease pathophysiology and cell replacement, there is strong evidence that the choroidal endothelial cells (CECs) that form the choriocapillaris vessels are the first to be lost in this disease. As such, to accurately evaluate disease pathophysiology and develop an effective treatment, production of patient-specific, stem cell-derived CECs will be required. In this study, we report for the first time a stepwise differentiation protocol suitable for generating human iPSC-derived CEC-like cells. RNA-seq analysis of the monkey CEC line, RF/6A, combined with two statistical screens allowed us to develop media comprised of various protein combinations. In both screens, connective tissue growth factor (CTGF) was identified as the key component required for driving CEC development. A second factor tumor necrosis factor (TNF)-related weak inducer of apoptosis receptor was also found to promote iPSC to CEC differentiation by inducing endogenous CTGF secretion. CTGF-driven iPSC-derived CEC-like cells formed capillary tube-like vascular networks, and expressed the EC-specific markers CD31, ICAM1, PLVAP, vWF, and the CEC-restricted marker CA4. In combination with RPE and photoreceptor cells, patient-specific iPSC derived CEC-like cells will enable scientists to accurately evaluate AMD pathophysiology and develop effective cell replacement therapies. Stem Cells Translational Medicine 2017;6:1533-1546.

PMID: 28474838 [PubMed - indexed for MEDLINE]

Development of a Molecularly Stable Gene Therapy Vector for the Treatment of RPGR-associated X-linked Retinitis Pigmentosa.

Sat, 2019-05-25 20:49
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Development of a Molecularly Stable Gene Therapy Vector for the Treatment of RPGR-associated X-linked Retinitis Pigmentosa.

Hum Gene Ther. 2019 May 20;:

Authors: Giacalone JC, Andorf JL, Zhang Q, Burnight E, Ochoa D, Reutzel AJ, Collins MM, Sheffield V, Mullins RF, Han IC, Stone E, Tucker BA

Abstract
In a screen of 1000 consecutively ascertained families we recently found that mutations in the gene RPGR are the 3rd most common cause of all inherited retinal disease. As the two most frequent disease-causing genes, ABCA4 and USH2A, are far too large to fit into clinically relevant AAV vectors, RPGR is an obvious early target for AAV based ocular gene therapy. In generating plasmids for this application, we discovered that those containing wild-type RPGR sequence, which includes the highly repetitive low complexity region ORF15, were extremely unstable (i.e., they showed consistent accumulation of genomic changes during plasmid propagation). To develop a stable RPGR gene transfer vector we used a bioinformatics approach to identify predicted regions of genomic instability within ORF15 (i.e. potential non-B DNA conformations). Synonymous substitutions were made in these regions to reduce the repetitiveness and increase the molecular stability while leaving the encoded amino acid sequence unchanged. The resulting construct was subsequently packaged into AAV serotype 5 and the ability to drive transcript expression and functional protein production was demonstrated via subretinal injection in rat and pull down assays respectively. By making synonymous substitutions within the repetitive region of RPGR we were able to stabilize the plasmid and subsequently generate a clinical grade gene transfer vector (IA-RPGR). Following subretinal injection in rat, we demonstrated that the augmented transcript was expressed at levels similar to wild-type constructs. By performing in vitro pull down experiments we were able to show that IA-RPGR protein product retained normal protein binding properties (i.e. analysis revealed normal binding to PDE6D, INPP5E and RPGRIP1L). In summary, we have generated a stable RPGR gene transfer vector capable of producing functional RPGR protein, which will facilitate safety and toxicity studies required for progression to an IND application.

PMID: 31106594 [PubMed - as supplied by publisher]

Generation of an immortalized human choroid endothelial cell line (iChEC-1) using an endothelial cell specific promoter.

Sat, 2019-05-25 20:49
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Generation of an immortalized human choroid endothelial cell line (iChEC-1) using an endothelial cell specific promoter.

Microvasc Res. 2019 05;123:50-57

Authors: Giacalone JC, Miller MJ, Workalemahu G, Reutzel AJ, Ochoa D, Whitmore SS, Stone EM, Tucker BA, Mullins RF

Abstract
Age-related macular degeneration (AMD) is a common cause of blindness worldwide. While recent studies have revealed that the loss of choroidal endothelial cells (ChECs) is critical to the disease pathogenesis of dry AMD, in vitro studies are needed to fully elucidate the disease mechanism. However, these studies remain hindered due to the lack of publically available human ChEC lines. To address this need, ChECs were harvested form donor tissue and enriched for by using magnetic cell separation using anti-CD31 conjugated microbeads. Next, lenti-viral vectors with endothelial-specific promoters driving genes necessary for immortalization, CDH5p-hTERT and CDH5p TAg, were generated. Stable integration of both gene cassettes allowed cells to maintain their proliferative state and yielded an immortalized cell line (iChEC-1). Immunocytochemical analysis of iChEC-1 confirmed the expression of important ChEC markers such as CA4, a marker of choriocapillaris endothelial cells, CDH5, and CD34, pan-endothelial cell markers. qRT-PCR analysis of expanded clones from iChEC-1 further showed that the line maintained expression of other important endothelial markers, vWF, PECAM1, and PLVAP, similar to primary cells. Functional responses were characterized by tube-forming assays and repopulation of decellularized choroid with the immortalized cell line. In conclusion, the iChEC-1 line presents a suitable immortalized human ChEC line for future in vitro studies of AMD.

PMID: 30571950 [PubMed - indexed for MEDLINE]

Evaluation of serum and ocular levels of membrane attack complex and C-reactive protein in CFH-genotyped human donors.

Sat, 2019-05-18 16:56
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Evaluation of serum and ocular levels of membrane attack complex and C-reactive protein in CFH-genotyped human donors.

Eye (Lond). 2018 11;32(11):1740-1742

Authors: Chirco KR, Flamme-Wiese MJ, Wiley JS, Potempa LA, Stone EM, Tucker BA, Mullins RF

Abstract
BACKGROUND: There is a considerable body of evidence demonstrating a link between the membrane attack complex (MAC) and age-related macular degeneration (AMD), and between C-reactive protein (CRP) and AMD. Both the MAC and the monomeric form of CRP (mCRP) accumulate within the choriocapillaris in AMD. However, the precise contribution of these species to AMD pathophysiology has not been fully elucidated.
METHODS: We sought to directly assess CRP and MAC levels between human serum and ocular tissues from the same CFH Y402H genotyped donors using ELISA of serum and RPE/choroid proteins.
RESULTS: The Y402H polymorphism was associated with significantly increased MAC in RPE/choroid samples, but not in the serum, in a previously unstudied cohort. While MAC levels in the choroid were independent of circulating levels, choroidal CRP was correlated to serum levels.
CONCLUSIONS: These data provide further evidence for local activation of complement within the choriocapillaris in AMD.

PMID: 30013157 [PubMed - indexed for MEDLINE]

Molecular characterization of foveal versus peripheral human retina by single-cell RNA sequencing.

Sat, 2019-05-11 13:34
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Molecular characterization of foveal versus peripheral human retina by single-cell RNA sequencing.

Exp Eye Res. 2019 May 07;:

Authors: Voigt AP, Whitmore SS, Flamme-Wiese MJ, Riker M, Wiley LA, Tucker BA, Stone EM, Mullins RF, Scheetz TE

Abstract
The human retina is a complex tissue responsible for detecting photons of light and converting information from these photons into the neurochemical signals interpreted as vision. Such visual signaling not only requires sophisticated interactions between multiple classes of neurons, but also spatially-dependent molecular specialization of individual cell types. In this study, we performed single-cell RNA sequencing on neural retina isolated from both the fovea and peripheral retina in three human donors. We recovered a total of 8,217 cells, with 3,578 cells originating from the fovea and 4,639 cells originating from the periphery. Expression profiles for all major retinal cell types were compiled, and differential expression analysis was performed between cells of foveal versus peripheral origin. Globally, mRNA for the serum iron binding protein transferrin (TF), which has been associated with age-related macular degeneration pathogenesis, was enriched in peripheral samples. Cone photoreceptor cells were of particular interest and formed two predominant clusters based on gene expression. One cone cluster had 96% of cells originating from foveal samples, while the second cone cluster consisted exclusively of peripherally isolated cells. A total of 148 genes were differentially expressed between cones from the fovea versus periphery. Interestingly, peripheral cones were enriched for the gene encoding Beta-Carotene Oxygenase 2 (BCO2). A relative deficiency of this enzyme may account for the accumulation of carotenoids responsible for yellow pigment deposition within the macula. Overall, this data set provides rich expression profiles of the major human retinal cell types and highlights transcriptomic features that distinguish foveal and peripheral cells.

PMID: 31075224 [PubMed - as supplied by publisher]

Two-photon Polymerized Poly(caprolactone) Retinal Cell Delivery Scaffolds and their Systemic and Retinal Biocompatibility.

Sat, 2019-05-11 13:34
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Two-photon Polymerized Poly(caprolactone) Retinal Cell Delivery Scaffolds and their Systemic and Retinal Biocompatibility.

Acta Biomater. 2019 May 02;:

Authors: Thompson JR, Worthington KS, Green BJ, Mullin NK, Jiao C, Kaalberg EE, Wiley LA, Han IC, Russell SR, Sohn EH, Guymon CA, Mullins RF, Stone EM, Tucker BA

Abstract
Cell replacement therapies are often enhanced by utilizing polymer scaffolds to improve retention or direct cell orientation and migration. Obstacles to refinement of such polymer scaffolds often include challenges in controlling the microstructure of biocompatible molecules in three dimensions at cellular scales. Two-photon polymerization of acrylated poly(caprolactone) (PCL) could offer a means of achieving precise microstructural control of a material in a biocompatible platform. In this work, we studied the effect of various formulation and two-photon polymerization parameters on minimum laser power needed to achieve polymerization, resolution, and fidelity to a target 3D model designed to be used for retinal cell replacement. Overall, we found that increasing the concentration of crosslink-able groups decreased polymerization threshold and the size of resolvable features while increasing fidelity of the scaffold to the 3D model. In general, this improvement was achieved by increasing the number of acrylate groups per prepolymer molecule, increasing the acrylated PCL concentration, or decreasing its molecular weight. Resulting two-photon polymerized PCL scaffolds successfully supported human iPSC derived retinal progenitor cells in vitro. Sub-retinal implantation of cell free scaffolds in a porcine model of retinitis pigmentosa did not cause inflammation, infection or local or systemic toxicity after one month. In addition, comprehensive ISO 10993 testing of photopolymerized scaffolds revealed a favorable biocompatibility profile. These results represent an important step towards understanding how two-photon polymerization can be applied to a wide range of biologically compatible chemistries for various biomedical applications. STATEMENT OF SIGNIFICANCE: Inherited retinal degenerative blindness results from the death of light sensing photoreceptor cells. To restore high-acuity vision a photoreceptor cell replacement strategy will likely be necessary. Unfortunately, single cell injection typically results in poor cell survival and integration post-transplantation. Polymeric biomaterial cell delivery scaffolds can be used to promote donor cell viability, control cellular polarity and increase packing density. A challenge faced in this endeavor has been developing methods suitable for generating scaffolds that can be used to deliver stem cell derived photoreceptors in an ordered columnar orientation (i.e., similar to that of the native retina). In this study we combined the biomaterial poly(caprolactone) with two-photon lithography to generate a biocompatible, clinically relevant scaffold suitable for retina cell delivery.

PMID: 31055121 [PubMed - as supplied by publisher]

Choriocapillaris Degeneration in Geographic Atrophy.

Sat, 2019-05-11 13:34
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Choriocapillaris Degeneration in Geographic Atrophy.

Am J Pathol. 2019 Apr 30;:

Authors: Sohn EH, Flamme-Wiese MJ, Whitmore SS, Workalemahu G, Marneros AG, Boese EA, Kwon YH, Wang K, Abramoff MD, Tucker BA, Stone EM, Mullins RF

Abstract
Early age-related macular degeneration (AMD) is characterized by degeneration of the choriocapillaris, the vascular supply of retinal photoreceptor cells. We assessed vascular loss during disease progression in the choriocapillaris and larger vessels in the deeper choroid. Human donor maculas from controls (n=99), early AMD (n=35), or clinically diagnosed with geographic atrophy (GA; n=9, collected from outside the zone of retinal pigment epithelium degeneration) were evaluated using Ulex europaeus agglutinin-I labeling to discriminate between vessels with intact endothelial cells and ghost vessels. Morphometric analyses of choriocapillaris density (cross sectional area of capillary lumens divided by length) and of vascular lumen-to-stroma ratio in the outer choroid were performed. Choriocapillaris loss was observed in early AMD (Bonferroni corrected P (pcorr)=0.024) with greater loss in GA (pcorr <10-9) even in areas of intact retinal pigment epithelium. In contrast, changes in lumen-to-stroma ratio in the outer choroid were not found to differ between controls and AMD or GA eyes (P >0.05), suggesting that changes in the choriocapillaris are more prevalent in AMD than those in the outer choroid. In addition, vascular endothelial growth factor-A levels were negatively correlated with choriocapillaris vascular density. These findings support the concept that choroidal vascular degeneration contributes to dry AMD, and that these changes are predominant in the microvasculature. Addressing the capillary loss in AMD remains an important translational target.

PMID: 31051169 [PubMed - as supplied by publisher]