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Wed, 2020-10-21 05:00

Retina. 2020 Nov;40(11):e68-e69. doi: 10.1097/IAE.0000000000002958.

NO ABSTRACT

PMID:33086372 | DOI:10.1097/IAE.0000000000002958

Stepwise differentiation and functional characterization of human induced pluripotent stem cell-derived choroidal endothelial cells

Thu, 2020-09-24 05:00

Stem Cell Res Ther. 2020 Sep 23;11(1):409. doi: 10.1186/s13287-020-01903-4.

ABSTRACT

BACKGROUND: Endothelial cells (ECs) are essential regulators of the vasculature, lining arteries, veins, and capillary beds. While all ECs share a number of structural and molecular features, heterogeneity exists depending on their resident tissue. ECs lining the choriocapillaris in the human eye are lost early in the pathogenesis of age-related macular degeneration (AMD), a common and devastating form of vision loss. In order to study the mechanisms leading to choroidal endothelial cell (CEC) loss and to develop reagents for repairing the choroid, a reproducible in vitro model, which closely mimic CECs, is needed. While a number of protocols have been published to direct induced pluripotent stem cells (iPSCs) into ECs, the goal of this study was to develop methods to differentiate iPSCs into ECs resembling those found in the human choriocapillaris specifically.

METHODS: We transduced human iPSCs with a CDH5p-GFP-ZEO lentiviral vector and selected for transduced iPSCs using blasticidin. We generated embryoid bodies (EBs) from expanded iPSC colonies and transitioned from mTESR™1 to EC media. One day post-EB formation, we induced mesoderm fate commitment via addition of BMP-4, activin A, and FGF-2. On day 5, EBs were adhered to Matrigel-coated plates in EC media containing vascular endothelial cell growth factor (VEGF) and connective tissue growth factor (CTGF) to promote CEC differentiation. On day 14, we selected for CECs using either zeocin resistance or anti-CD31 MACS beads. We expanded CECs post-selection and performed immunocytochemical analysis of CD31, carbonic anhydrase IV (CA4), and RGCC; tube formation assays; and transmission electron microscopy to access vascular function.

RESULTS: We report a detailed protocol whereby we direct iPSC differentiation toward mesoderm and utilize CTGF to specify CECs. The CDH5p-GFP-ZEO lentiviral vector facilitated the selection of iPSC-derived ECs that label with antibodies directed against CD31, CA4, and RGCC; form vascular tubes in vitro; and migrate into empty choroidal vessels. CECs selected using either antibiotic selection or CD31 MACS beads showed similar characteristics, thereby making this protocol easily reproducible with or without lentiviral vectors.

CONCLUSION: ECs generated following this protocol exhibit functional and biochemical characteristics of CECs. This protocol will be useful for developing in vitro models toward understanding the mechanisms of CEC loss early in AMD.

PMID:32967716 | DOI:10.1186/s13287-020-01903-4

Retinal Tropism and Transduction of Adeno-Associated Virus (AAV) Varies by Serotype and Route of Delivery (Intravitreal, Subretinal or Suprachoroidal) in Rats

Sat, 2020-09-19 05:00

Hum Gene Ther. 2020 Sep 18. doi: 10.1089/hum.2020.043. Online ahead of print.

ABSTRACT

Viral-mediated gene augmentation offers tremendous promise for the treatment of inherited retinal diseases. The development of effective gene therapy requires an understanding of the vector's tissue-specific behavior, which may vary depending on serotype, route of delivery, or target species. Using an ex vivo organotypic explant system, we previously demonstrated that retinal tropism and transduction of adeno-associated virus type 2 (AAV2) varies significantly depending on serotype in human eyes. However, the ex vivo system has limited ability to assess route of ocular delivery, and relatively little literature exists on tropic differences between serotypes and routes of delivery in vivo. In this study, we demonstrate that retinal tropism and transduction efficiency of five different AAV2 serotypes (AAV2/1, AAV2/2, AAV2/6, AAV2/8, AAV2/9) expressing eGFP driven by a cytomegalovirus promoter vary greatly depending on serotype and route of delivery (intravitreal, subretinal, or suprachoroidal) in rats. With subretinal delivery, all serotypes successfully transduced the retinal pigmented epithelium and outer nuclear layer (ONL), with AAV2/1 displaying the highest transduction efficiency and AAV2/2 and AAV2/6 showing lower ONL transduction. There was minimal transduction of the inner retina via subretinal delivery for any serotype. Tropism by suprachoroidal delivery mirrored that of subretinal delivery for all AAV serotypes but resulted in a wider distribution and greater ONL transduction. With intravitreal delivery, retinal transduction was seen primarily in the inner retina (retinal nerve fiber, ganglion cell, and inner nuclear layers) for AAV2/1 and AAV2/6, with AAV2/6 showing the highest transduction. When compared to data from human explant models, there are substantial differences in tropism and transduction that are important to consider when using rats as preclinical models for the development of ocular gene therapies for humans.

PMID:32948113 | DOI:10.1089/hum.2020.043

Spectacle: An interactive resource for ocular single-cell RNA sequencing data analysis

Thu, 2020-09-10 05:00

Exp Eye Res. 2020 Sep 7:108204. doi: 10.1016/j.exer.2020.108204. Online ahead of print.

ABSTRACT

Single-cell RNA sequencing has revolutionized ocular gene expression studies. This technology has enabled researchers to identify expression signatures for rare cell types and characterize how gene expression changes across biological conditions, such as topographic region or disease status. However, sharing single-cell RNA sequencing results remains a major obstacle, particular for individuals without a computational background. To address these limitations, we developed Spectacle, an interactive web-based resource for exploring previously published single-cell RNA sequencing data from ocular studies. Spectacle is powered by a locally developed R package, cellcuratoR, which utilizes the Shiny framework in R to generate interactive visualizations for single-cell expression data. Spectacle contains five pre-processed ocular single-cell RNA sequencing data sets and is accessible via the web at OcularGeneExpression.org/singlecell. With Spectacle, users can interactively identify which cell types express a gene of interest, detect transcriptomic subpopulations within a cell type, and perform highly flexible differential expression analyses. The freely-available Spectacle system reduces the bioinformatic barrier for interacting with rich single-cell RNA sequencing studies from ocular tissues, making it easy to quickly identify cell types that express a gene of interest.

PMID:32910939 | DOI:10.1016/j.exer.2020.108204

Response to letter to the editor by Dhananjay Shukla.

Thu, 2020-08-27 04:56
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Response to letter to the editor by Dhananjay Shukla.

Retina. 2020 Aug 18;:

Authors: Sohn EH, Mullins RF, Eliott D

PMID: 32826793 [PubMed - as supplied by publisher]

Prospective, Single-center, Open-label, Pilot Study Using Cryopreserved Umbilical Tissue Containing Viable Cells in the Treatment of Complex Acute and Chronic Wounds.

Thu, 2020-08-27 04:56
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Prospective, Single-center, Open-label, Pilot Study Using Cryopreserved Umbilical Tissue Containing Viable Cells in the Treatment of Complex Acute and Chronic Wounds.

Wounds. 2020 Jun 21;:

Authors: Mullins RF, Hassan Z, Homsombath B, Fagan S, Craft-Coffman B, Wilson J, Rumbaugh JG, Saunders M, Danilkovitch A

Abstract
INTRODUCTION: Complex wounds with exposed bone, muscle, tendon, or hardware continue to be a therapeutic challenge for wound care providers. Wounds with exposed structures are more susceptible to infection, necrosis, and amputation. As such, rapid granulation to cover exposed deep tissue structures is essential for patient recovery.
OBJECTIVE: In this prospective, pilot study, the authors evaluate the clinical outcomes of a cryopreserved umbilical tissue graft containing viable cells (vCUT) in the treatment of complex wounds.
MATERIALS AND METHODS: Ten patients with 12 wounds each received 1 application of vCUT. Two patients did not complete the study and were removed from the per-protocol population. Data analyses were performed on the remaining 8 patients with 10 wounds. The average wound area was 16.5 cm2 with an average duration of 10 months. Post-application, patients were followed for an additional 4 weeks for granulation, closure, and safety outcomes.
RESULTS: By the end of the study, 8 of 10 (80.0%) vCUT-treated wounds achieved 100% granulation, and 3 wounds (30.0%) went on to achieve complete closure. The median area reduction was 40.5% and the median volume reduction was 59.4%.
CONCLUSIONS: The results of this study suggest vCUT in conjunction with standard of care can be a viable treatment option for acute and chronic lower extremity complex wounds.

PMID: 32813668 [PubMed - as supplied by publisher]

Label-free microfluidic enrichment of photoreceptor cells.

Thu, 2020-08-13 00:56
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Label-free microfluidic enrichment of photoreceptor cells.

Exp Eye Res. 2020 Aug 06;:108166

Authors: Stone NE, Voigt AP, Cooke JA, Giacalone JC, Hanasoge S, Mullins RF, Tucker BA, Sulchek T

Abstract
Inherited retinal degenerative disorders such as retinitis pigmentosa and Usher syndrome are characterized by progressive death of photoreceptor cells. To restore vision to patients blinded by these diseases, a stem cell-based photoreceptor cell replacement strategy will likely be required. Although retinal stem cell differentiation protocols suitable for generating photoreceptor cells exist, they often yield a rather heterogenous mixture of cell types. To enrich the donor cell population for one or a few cell types, scientists have traditionally relied upon the use of antibody-based selection approaches. However, these strategies are quite labor intensive and require animal derived reagents and equipment that are not well suited to current good manufacturing practices (cGMP). The purpose of this study was to develop and evaluate a microfluidic cell sorting device capable of exploiting the physical and mechanical differences between retinal cell types to enrich specific donor cell populations such as RPE cells and photoreceptor cells. Using this device, we were able to separate a mixture of RPE and iPSC-derived photoreceptor precursor cell lines into two substantially enriched fractions. The enrichment factor of the RPE fraction was 2 and that of the photoreceptor precursor cell fraction was 2.7. Similarly, when human retina, obtained from 3 independent donors, was dissociated and passed through the sorting device, the heterogeneous mixture could be reliably sorted into RPE and photoreceptor cell rich fractions. In summary, microfluidic cell sorting is a promising approach for antibody free enrichment of retinal cell populations.

PMID: 32771499 [PubMed - as supplied by publisher]

Two-photon polymerized poly(caprolactone) retinal cell delivery scaffolds and their systemic and retinal biocompatibility.

Thu, 2020-08-13 00:56
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Two-photon polymerized poly(caprolactone) retinal cell delivery scaffolds and their systemic and retinal biocompatibility.

Acta Biomater. 2019 08;94:204-218

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 - indexed for MEDLINE]