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Effect of Molecular Weight and Functionality on Acrylated Poly(caprolactone) for Stereolithography and Biomedical Applications.

Wed, 2018-08-01 11:24
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Effect of Molecular Weight and Functionality on Acrylated Poly(caprolactone) for Stereolithography and Biomedical Applications.

Biomacromolecules. 2018 Jul 25;:

Authors: Green BJ, Worthington KS, Thompson JR, Bunn SJ, Rethwisch M, Kaalberg EE, Jiao C, Wiley LA, Mullins RF, Stone EM, Sohn EH, Tucker BA, Guymon CA

Abstract
Degradable polymers are integral components in many biomedical polymer applications. The ability of these materials to decompose in situ has become a critical component for tissue engineering, allowing scaffolds to guide cell and tissue growth while facilitating gradual regeneration of native tissue. The objective of this work is to understand the role of prepolymer molecular weight and functionality of photocurable poly(caprolactone) (PCL) in determining reaction kinetics, mechanical properties, polymer degradation, biocompatibility, and suitability for stereolithography. PCL, a degradable polymer used in a number of biomedical applications, was functionalized with acrylate groups to enable photopolymerization and 3D printing via stereolithography. PCL prepolymers with different molecular weight and functionality were studied to understand the role of molecular structure on reaction kinetics, mechanical properties and degradation rates. The mechanical properties of photocured PCL were dependent on cross-link density and directly related to the molecular weight and functionality of the prepolymers. High molecular weight, low functionality PCLDA prepolymers exhibited lower modulus and higher strain at break while low molecular weight, high functionality PCLTA prepolymers exhibited lower strain at break and higher modulus. Additionally, degradation profiles of cross-linked PCL followed a similar trend, with low cross-link density leading to degradation times up to 2.5 times shorter than more highly cross-linked polymers. Furthermore, photopolymerized PCL showed biocompatibility both in vitro and in vivo, causing no observed detrimental effects on seeded murine induced pluripotent stem cells or when implanted into pig retinas. Finally, the ability to create 3-dimensional PCL structures is shown by fabrication of simple structures using digital light projection stereolithography. Low molecular weight, high functionality PCLTA prepolymers printed objects with feature sizes near the hardware resolution limit of 50 μm. This work lays the foundation for future work in fabricating micro-scale PCL structures for a wide range of tissue regeneration applications.

PMID: 30044915 [PubMed - as supplied by publisher]

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

Wed, 2018-07-18 06:45
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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 Jul 16;:

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 - as supplied by publisher]

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

Tue, 2018-05-15 14:28
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Using CRISPR-Cas9 to Generate Gene-Corrected Autologous iPSCs for the Treatment of Inherited Retinal Degeneration.

Mol Ther. 2017 Sep 06;25(9):1999-2013

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 - indexed for MEDLINE]

Feeder-free differentiation of cells exhibiting characteristics of corneal endothelium from human induced pluripotent stem cells.

Tue, 2018-05-01 10:45
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Feeder-free differentiation of cells exhibiting characteristics of corneal endothelium from human induced pluripotent stem cells.

Biol Open. 2018 Apr 23;:

Authors: Wagoner MD, Bohrer LR, Aldrich BT, Greiner MA, Mullins RF, Worthington KS, Tucker BA, Wiley LA

Abstract
The purpose of this study was to devise a strategy for the derivation of corneal endothelial cells (CEnCs) from adult fibroblast-derived induced pluripotent stem cells (iPSCs). IPSCs were generated from an adult human with normal ocular history via expression of OCT4, SOX2, KLF4 and c-MYC Neural crest cells (NCCs) were differentiated from iPSCs via addition of CHIR99021 and SB4315542. NCCs were driven toward a CEnC fate via addition of B27, PDGF-BB and DKK-2 to CEnC media. Differentiation of NCCs and CEnCs was evaluated via rt-PCR, morphological and immunocytochemical analysis. At 17 days post-NCC induction, there were notable changes in cell morphology and upregulation of the neural crest lineage transcripts PAX3, SOX9, TFAP2A, SOX10 and p75NTR and the proteins p75/NGFR and SOX10. Exposure of NCCs to B27, PDGF-BB and DKK-2 induced a shift in morphology from a spindle-shaped neural phenotype to a tightly-packed hexagonal appearance and increased expression of the transcripts ATP1A1, COL8A1, COL8A2, AQP1 and CDH2 and the proteins, ZO-1, N-Cad, AQP-1 and Na+/K+ATPase. Replacement of NCC media with CEnC media on day 3, 5 or 8 reduced the differentiation time needed to yield CEnCs. IPSC-derived CEnCs could be used for evaluation of cornea endothelial disease pathophysiology and for testing of novel therapeutics.

PMID: 29685994 [PubMed - as supplied by publisher]

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

Tue, 2018-05-01 10:45
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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 - indexed for MEDLINE]

cGMP production of patient-specific iPSCs and photoreceptor precursor cells to treat retinal degenerative blindness.

Tue, 2018-05-01 10:45
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cGMP production of patient-specific iPSCs and photoreceptor precursor cells to treat retinal degenerative blindness.

Sci Rep. 2016 07 29;6:30742

Authors: Wiley LA, Burnight ER, DeLuca AP, Anfinson KR, Cranston CM, Kaalberg EE, Penticoff JA, Affatigato LM, Mullins RF, Stone EM, Tucker BA

Abstract
Immunologically-matched, induced pluripotent stem cell (iPSC)-derived photoreceptor precursor cells have the potential to restore vision to patients with retinal degenerative diseases like retinitis pigmentosa. The purpose of this study was to develop clinically-compatible methods for manufacturing photoreceptor precursor cells from adult skin in a non-profit cGMP environment. Biopsies were obtained from 35 adult patients with inherited retinal degeneration and fibroblast lines were established under ISO class 5 cGMP conditions. Patient-specific iPSCs were then generated, clonally expanded and validated. Post-mitotic photoreceptor precursor cells were generated using a stepwise cGMP-compliant 3D differentiation protocol. The recapitulation of the enhanced S-cone phenotype in retinal organoids generated from a patient with NR2E3 mutations demonstrated the fidelity of these protocols. Transplantation into immune compromised animals revealed no evidence of abnormal proliferation or tumor formation. These studies will enable clinical trials to test the safety and efficiency of patient-specific photoreceptor cell replacement in humans.

PMID: 27471043 [PubMed - indexed for MEDLINE]