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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]

Bulk and single-cell gene expression analyses reveal aging human choriocapillaris has pro-inflammatory phenotype.

Wed, 2020-06-17 13:20
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Bulk and single-cell gene expression analyses reveal aging human choriocapillaris has pro-inflammatory phenotype.

Microvasc Res. 2020 Jun 09;:104031

Authors: Voigt AP, Whitmore SS, Mulfaul K, Chirco KR, Giacalone JC, Flamme-Wiese MJ, Stockman A, Stone EM, Tucker BA, Scheetz TE, Mullins RF

Abstract
The human choroidal vasculature is subject to age-related structural and gene expression changes implicated in age-related macular degeneration (AMD). In this study, we performed both bulk and single-cell RNA sequencing on infant (n = 4 for bulk experiments, n = 2 for single-cell experiments) and adult (n = 13 for bulk experiments, n = 6 for single-cell experiments) human donors to characterize how choroidal gene expression changes with age. Differential expression analysis revealed that aged choroidal samples were enriched in genes encoding pro-inflammatory transcription factors and leukocyte transendothelial cell migration adhesion proteins. Such genes were observed to be differentially expressed specifically within choroidal endothelial cells at the single-cell level. Immunohistochemistry experiments support transcriptional findings that CD34 is elevated in infant choriocapillaris endothelial cells while ICAM-1 is enriched in adults. These results suggest several potential drivers of the pro-inflammatory vascular phenotype observed with advancing age.

PMID: 32531351 [PubMed - as supplied by publisher]

Visualization of Mouse Choroidal and Retinal Vasculature Using Fluorescent Tomato Lectin Perfusion.

Wed, 2020-06-10 10:56
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Visualization of Mouse Choroidal and Retinal Vasculature Using Fluorescent Tomato Lectin Perfusion.

Transl Vis Sci Technol. 2020 Jan;9(1):1

Authors: Jiao C, Adler K, Liu X, Sun W, Mullins RF, Sohn EH

Abstract
Purpose: To develop a reliable and simplified method to assess choroid and retinal vasculature on whole mount and cross sections in mice using tomato lectin (TL; Lycopersicon esculentum).
Methods: Albino mice (n = 27) received 1 mg/mL of TL (conjugated to Dylight-594) intravascularly through the tail vein, jugular vein, or cardiac left ventricle. Whole mounts of the retina and choroid were evaluated using fluorescence microscopy. Perfusion with GSL-IB4 conjugated to Dylight-594 and fluorescein isothiocyanate was performed to compare against labeling with TL. Co-labeling of choroidal endothelial cells with perfused TL on cross-sections with antibodies directed against the choriocapillaris-restricted endothelial cell marker CA4 was performed. The percentage of perfused choroidal and retinal vessels was assessed semiquantitatively. One mouse was subjected to thermal laser damage before perfusion to cause retinal and choroidal vasculature ablation.
Results: Intravascular injection of TL led to consistent, robust labeling of retinal and choroidal vascular walls. On cross-sections, choriocapillaris was co-labeled with CA4 and TL. On flat mount, TL perfusion resulted in better labeling of choroidal vessels using tail/jugular vein injection compared with cardiac perfusion (P < .01). More consistent labeling of the choroidal and retinal vascular trees was observed with TL than with GSL-IB4. Vascular damage caused by laser ablation was detected readily using this method.
Conclusions: TL injection intravascularly can reliably label normal and ablated choroid and retinal vasculature in mouse in a quick, simple manner.
Translational Relevance: These data will help to facilitate modeling in rodents for diseases such as age-related macular degeneration, diabetes, and other ischemic/angiogenic processes that can also be used for treatment evaluation.

PMID: 32509436 [PubMed]

Optimizing Donor Cellular Dissociation and Subretinal Injection Parameters for Stem Cell-Based Treatments.

Wed, 2020-06-10 10:56
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Optimizing Donor Cellular Dissociation and Subretinal Injection Parameters for Stem Cell-Based Treatments.

Stem Cells Transl Med. 2019 08;8(8):797-809

Authors: Scruggs BA, Jiao C, Cranston CM, Kaalberg E, Wang K, Russell SR, Wiley LA, Mullins RF, Stone EM, Tucker BA, Sohn EH

Abstract
Subretinal delivery of stem cell-derived retinal cells as a strategy to treat retinal degenerative blindness holds great promise. Currently, two clinical trials are underway in which human fetal retinal progenitor cells (RPCs) are being delivered to patients by intravitreal or subretinal injection to preserve or restore vision, respectively. With the advent of the induced pluripotent stem cell (iPSC), and in turn three-dimensional derivation of retinal tissue, it is now possible to generate autologous RPCs for cell replacement. The purpose of this study was to evaluate the effect of commonly used cell isolation and surgical manipulation strategies on donor cell viability. iPSC-RPCs were subjected to various conditions, including different dissociation and isolation methods, injection cannula sizes, and preinjection storage temperatures and times. The effects of commonly used surgical techniques on both host and donor cell viability were evaluated in Yucatan mini-pigs (n = 61 eyes). We found a significant increase in cell viability when papain was used for RPC isolation. In addition, a significant decrease in cell viability was detected when using the 41G cannula compared with 31G and at storage times of 4 hours compared with 30 minutes. Although 96.4% of all eyes demonstrated spontaneous retinal reattachment following injection, retinal pigment epithelium (RPE) abnormalities were seen more frequently in eyes receiving injections via a 31G cannula; interestingly, eyes that received cell suspensions were relatively protected against such RPE changes. These findings indicate that optimization of donor cell isolation and delivery parameters should be considered when developing a subretinal cell replacement strategy. Stem Cells Translational Medicine 2019;8:797&809.

PMID: 31004408 [PubMed - indexed for MEDLINE]

How much green does it take to be orange? Determining the cost associated with trauma center readiness.

Wed, 2020-06-03 09:12
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How much green does it take to be orange? Determining the cost associated with trauma center readiness.

J Trauma Acute Care Surg. 2019 05;86(5):765-773

Authors: Ashley DW, Mullins RF, Dente CJ, Johns TJ, Garlow LE, Medeiros RS, Atkins EV, Solomon G, Abston D, Ferdinand CH, Georgia Research Institute for Trauma Study Group

Abstract
BACKGROUND: Readiness costs are real expenses incurred by trauma centers to maintain essential infrastructure to provide emergent services on a 24/7 basis. Although the components for readiness are well described in the American College of Surgeons' Resources for Optimal Care of the Injured Patient, the cost associated with each component is not well defined. We hypothesized that meeting the requirements of the 2014 Resources for Optimal Care of the Injured Patient would result in significant costs for trauma centers.
METHODS: The state trauma commission in conjunction with trauma medical directors, program managers, and financial officers of each trauma center standardized definitions for each component of trauma center readiness cost and developed a survey tool for reporting. Readiness costs were grouped into four categories: administrative/program support staff, clinical medical staff, in-house operating room, and education/outreach. To verify consistent cost reporting, a financial auditor analyzed all data. Trauma center outliers were further evaluated to validate variances. All level I/level II trauma centers (n = 16) completed the survey on 2016 data.
RESULTS: Average annual readiness cost is US $10,078,506 for a level I trauma center and US $4,925,103 for level IIs. Clinical medical staff was the costliest component representing 55% of costs for level Is and 64% for level IIs. Although education/outreach is mandated, levels I and II trauma centers only spend approximately US $100,000 annually on this category (1%-2%), demonstrating a lack of resources.
CONCLUSION: This study defines the cost associated with each component of readiness as defined in the Resources for Optimal Care of the Injured Patient manual. Average readiness cost for a level I trauma center is US $10,078,506 and US $4,925,103 for a level II. The significant cost of trauma center readiness highlights the need for additional trauma center funding to meet the requirements set forth by the American College of Surgeons.
LEVEL OF EVIDENCE: Economic and value-based evaluations, level III.

PMID: 30768564 [PubMed - indexed for MEDLINE]