Browsing by Author "Hara, Hidetaka"

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    Comparison of porcine corneal decellularization methods and importance of preserving corneal limbus through decellularization
    (PLOS, 2021-03-05) Isidan, Abdulkadir; Liu, Shaohui; Chen, Angela M.; Zhang, Wenjun; Li, Ping; Smith, Lester J.; Hara, Hidetaka; Cooper, David K. C.; Ekser, Burcin; Surgery, School of Medicine
    Background: The aim of this study is to compare the three previously applied, conventional porcine corneal decellularization methods and to demonstrate the importance of preserving the corneal limbus through decellularization. Methods: Fresh, wild-type (with or without) limbus porcine corneas were decellularized using three different methods, including (i) sodium dodecyl sulfate (SDS), (ii) hypertonic saline (HS), and (iii) N2 gas (NG). Post-treatment evaluation was carried out using histological, residual nuclear material, and ultrastructural analyses. Glycerol was used to help reduce the adverse effects of decellularization. The corneas were preserved for two weeks in cornea storage medium. Results: All three decellularization methods reduced the number of keratocytes at different rates in the stromal tissue. However, all methods, except SDS, resulted in the retention of large numbers of cells and cell fragments. The SDS method (0.1% SDS, 48h) resulted in almost 100% decellularization in corneas without limbus. Low decellularization capacity of the NG method (<50%) could make it unfavorable. Although HS method had a more balanced damage-decellularization ratio, its decellularization capacity was lower than SDS method. Preservation of the corneoscleral limbus could partially prevent structural damage and edema, but it would reduce the decellularization capacity. Conclusion: Our results suggest that SDS is a very powerful decellularization method, but it damages the cornea irreversibly. Preserving the corneoscleral limbus reduces the efficiency of decellularization, but also reduces the damage.
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    Decellularization methods for developing porcine corneal xenografts and future perspectives
    (Wiley, 2019-11) Isidan, Abdulkadir; Liu, Shaohui; Li, Ping; Lashmet, Matthew; Smith, Lester J.; Hara, Hidetaka; Cooper, David K. C.; Ekser, Burcin; Ophthalmology, School of Medicine
    Corneal transplantation is the only option to cure corneal opacities. However, there is an imbalance between supply and demand of corneal tissues in the world. To solve the problem of corneal shortage, corneal xenotransplantation studies have been implemented in the past years using porcine corneas. The corneal xenografts could come from (a) wild-type pigs, (b) genetically engineered pigs, (c) decellularized porcine corneas, and (d) decellularized porcine corneas that are recellularized with human corneal cells, eventually with patients' own cells, as in all type of xenografts. All approaches except, the former would reduce or mitigate recipient immune responses. Although several techniques in decellularization have been reported, there is still no standardized protocol for the complete decellularization of corneal tissue. Herein, we reviewed different decellularization methods for porcine corneas based on the mechanism of action, decellularization efficacy, biocompatibility, and the undesirable effects on corneal ultrastructure. We compared 9 decellularization methods including: (a) sodium dodecyl sulfate, (b) triton x-100, (c) hypertonic saline, (d) human serum with electrophoresis, (e) high hydrostatic pressure, (f) freeze-thaw, (h) nitrogen gas, (h) phospholipase A2 , and (i) glycerol with chemical crosslinking methods. It appears that combined methods could be more useful to perform efficient corneal decellularization.
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    Expression of NeuGc on Pig Corneas and Its Potential Significance in Pig Corneal Xenotransplantation
    (Wolters Kluwer, 2016-01) Lee, Whayoung; Miyagawa, Yuko; Long, Cassandra; Ekser, Burcin; Walters, Eric; Ramsoondar, Jagdeece; Ayares, David; Tector, A. Joseph; Cooper, David K. C.; Hara, Hidetaka; Department of Surgery, IU School of Medicine
    PURPOSE: Pigs expressing neither galactose-α1,3-galactose (Gal) nor N-glycolylneuraminic acid (NeuGc) take xenotransplantation one step closer to the clinic. Our aims were (1) to document the lack of NeuGc expression on corneas and aortas and cultured endothelial cells [aortic endothelial cells (AECs); corneal (CECs)] of GTKO/NeuGcKO pigs, and (2) to investigate whether the absence of NeuGc reduced human antibody binding to the tissues and cells. METHODS: Wild-type (WT), GTKO, and GTKO/NeuGcKO pigs were used for the study. Human tissues and cultured cells were negative controls. Immunofluorescence staining was performed using anti-Gal and anti-NeuGc antibodies, and human IgM and IgG binding to tissues was determined. Flow cytometric analysis was used to determine Gal and NeuGc expression on cultured CECs and AECs and to measure human IgM/IgG binding to these cells. RESULTS: Both Gal and NeuGc were detected on WT pig corneas and aortas. Although GTKO pigs expressed NeuGc, neither humans nor GTKO/NeuGcKO pigs expressed Gal or NeuGc. Human IgM/IgG binding to corneas and aortas from GTKO and GTKO/NeuGcKO pigs was reduced compared with binding to WT pigs. Human antibody binding to GTKO/NeuGcKO AECs was significantly less than that to GTKO AECs, but there was no significant difference in binding between GTKO and GTKO/NeuGcKO CECs. CONCLUSIONS: The absence of NeuGc on GTKO aortic tissue and AECs is associated with reduced human antibody binding, and possibly will provide a better outcome in clinical xenotransplantation using vascularized organs. For clinical corneal xenotransplantation, the absence of NeuGc expression on GTKO/NeuGcKO pig corneas may not prove an advantage over GTKO corneas.
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    Genetic engineering of porcine endothelial cell lines for evaluation of human-to-pig xenoreactive immune responses
    (Springer Nature, 2021-06-23) Li, Ping; Walsh, Julia R.; Lopez, Kevin; Isidan, Abdulkadir; Zhang, Wenjun; Chen, Angela M.; Goggins, William C.; Higgins, Nancy G.; Liu, Jianyun; Brutkiewicz, Randy R.; Smith, Lester J.; Hara, Hidetaka; Cooper, David K.C.; Ekser, Burcin; Surgery, School of Medicine
    Xenotransplantation (cross-species transplantation) using genetically-engineered pig organs offers a potential solution to address persistent organ shortage. Current evaluation of porcine genetic modifications is to monitor the nonhuman primate immune response and survival after pig organ xenotransplantation. This measure is an essential step before clinical xenotransplantation trials, but it is time-consuming, costly, and inefficient with many variables. We developed an efficient approach to quickly examine human-to-pig xeno-immune responses in vitro. A porcine endothelial cell was characterized and immortalized for genetic modification. Five genes including GGTA1, CMAH, β4galNT2, SLA-I α chain, and β2-microglobulin that are responsible for the production of major xenoantigens (αGal, Neu5Gc, Sda, and SLA-I) were sequentially disrupted in immortalized porcine endothelial cells using CRISPR/Cas9 technology. The elimination of αGal, Neu5Gc, Sda, and SLA-I dramatically reduced the antigenicity of the porcine cells, though the cells still retained their ability to provoke human natural killer cell activation. In summary, evaluation of human immune responses to genetically modified porcine cells in vitro provides an efficient method to identify ideal combinations of genetic modifications for improving pig-to-human compatibility, which should accelerate the application of xenotransplantation to humans.
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    PUBLISHER CORRECTION: Genetic engineering of porcine endothelial cell lines for evaluation of human-to-pig xenoreactive immune responses
    (Springer Nature, 2021-08-16) Li, Ping; Walsh, Julia R.; Lopez, Kevin; Isidan, Abdulkadir; Zhang, Wenjun; Chen, Angela M.; Goggins, William C.; Higgins, Nancy G.; Liu, Jianyun; Brutkiewicz, Randy R.; Smith, Lester J.; Hara, Hidetaka; Cooper, David K.C.; Ekser, Burcin; Surgery, School of Medicine