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Maryada Sharma, Rajendra Kumar, Jagat Ram, Manni Luthra-Guptasarma; Establishment and characterization of a novel Serine Protease Induced Reprograming (SPIR) method with applications in ocular tissue regeneration. Invest. Ophthalmol. Vis. Sci. 2017;58(8):1426.
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© ARVO (1962-2015); The Authors (2016-present)
Personalized medicine employing stem cells has immense potential in the treatment of ocular disorders; however, even a decade after the discovery of iPSCs, the precise mechanism is ill-understood. We have serendipitously discovered a novel in-vitro culture method, associated with induction, establishment and enrichment of stem cells, using a serine protease; this serine protease induced reprograming (SPIR) method was used for ocular tissue reprograming.
Human corneal/limbal cells (HCCs) cultured using this method, were evaluated for expression of stem cell markers by immunofluorescence; the secretome was analyzed by multiplex CBA-bead array. HCCs were subjected to the SPIR reprograming; use of these cells along with synthetic collagen resulted in creation of cornea-like tissue (CLT) ex-vivo. CLT was characterized by optical coherence tomography (OCT) and scanning electron microscopy (SEM).
We found that cellular reprograming is associated with induction of a low molecular weight serine protease in the culture system; further, exogenous addition of this protease alone, followed by its interaction with the cognate receptor is necessary and sufficient for induction of reprograming in ocular cells. SPIR-culturing of HCCs resulted in “stemness” marked by upregulation of Nanog, Sox-2, Ki67, β-catenin, CD73, CD90, CD105 and ICAM-1, and increased expression of the serine protease receptor. The secretome revealed significant upregulation of bFGF and IL-1β. Use of SPIR-reprogramed HCCs, along with synthetic collagen gave rise to nearly convex, transparent CLT, akin to native cornea (500-1000µm, as determined by OCT) that integrated completely into human donor-derived corneo-scleral rims, facilitating the desirable sizing (10-11 mm) for therapeutic and aesthetic corneal/limbal transplantations.
Our discovery of “SPIR” is biologically and therapeutically significant. Its “pro-proteolytic” nature activates autocrine growth factors/cytokines, thereby generating a “self-niche”, favoring cellular reprograming, with clinically translatable potential in the context of corneal/limbal disorders and retinal degenerative diseases. SPIR addresses the inherent bottlenecks (complicated protocols, genetic manipulations, time, etc.) associated with current reprograming methods.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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