Differential profile of protein expression on human keratocytes treated with autologous serum and plasma rich in growth factors (PRGF)

ABSTRACTPURPOSETo establish an in vitro model of aniridia-related keratopathy (ARK) using CRISPR/Cas9 engineered human keratocytes with mutations in the PAX6 gene, and to study the Notch Homolog 1, Translocation-Associated (Notch1), sonic hedgehog (SHH), mammalian target of rapamycin (mTOR), and Wnt/β-catenin signaling pathways in the PAX6 mutant keratocytes.METHODSPrimary human keratocytes were isolated from healthy corneas. Keratocytes were transduced with Cas9 lentiviral particles in order to create cells stably expressing Cas9 nuclease. Lentiviral particles carrying PAX6 sgRNA were transduced into the Cas9 keratocytes creating mutants. Analysis of signaling pathways was assessed by RT-qPCR for gene expression and western blot for protein expression.RESULTSHuman keratocytes stably expressing Cas9 nuclease were created. Keratocytes carrying PAX6 gene mutation were successfully generated. PAX6 mutant keratocytes showed modified expression patterns of extracellular matrix components such as collagens and fibrotic markers. Analysis of the Notch1, SHH, mTOR, and Wnt/β-catenin signaling pathways in the PAX6 mutant keratocytes revealed altered gene and protein expression of the key players involved in these pathways.CONCLUSIONSA properly functioning PAX6 gene in keratocytes is crucial for the regulation of signaling pathways important for cell fate determination, proliferation, and inflammation. Pax6 mutation in the in vitro settings leads to changes in these pathways which resemble those found in corneas of patients with ARK.

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Stability of freeze-dried plasma rich in growth factors eye drops stored for 3 months at different temperature conditions

European Journal of Ophthalmology ◽

10.1177/1120672120913035 ◽

2020 ◽

pp. 112067212091303 ◽

Cited By ~ 3

Author(s):

Eduardo Anitua ◽

María de la Fuente ◽

Francisco Muruzábal ◽

Jesús Merayo-Lloves

Keyword(s):

Growth Factors ◽

Room Temperature ◽

Storage Condition ◽

Eye Drops ◽

Temperature Conditions ◽

Healthy Donors ◽

Freeze Dried ◽

And Storage ◽

Blood Centrifugation ◽

Human Keratocytes

Purpose: The purpose of this study was to analyze the biological content and activity of freeze-dried plasma rich in growth factors eye drops after their storage at 4°C and at room temperature for 3 months with respect to fresh samples (time 0). Methods: Plasma rich in growth factors was obtained after blood centrifugation from three healthy donors. After platelet activation, the obtained plasma rich in growth factors eye drops were lyophilized alone or in combination with lyoprotectant (trehalose), then they were stored for 3 months at room temperature or at 4°C. Several growth factors were analyzed at each storage time and condition. Furthermore, the proliferative and migratory potential of freeze-dried plasma rich in growth factors eye drops kept for 3 months at different temperature conditions was evaluated on primary human keratocytes. Results: The different growth factors analyzed maintained their levels at each time and storage condition. Freeze-dried plasma rich in growth factors eye drops stored at room temperature or 4°C for 3 months showed no significant differences on the proliferative activity of keratocytes in comparison with fresh samples. However, the number of migratory human keratocytes increased significantly after treatment with lyophilized plasma rich in growth factors eye drops kept for 3 months compared to those obtained at time 0. No significant differences were observed between the freeze-dried plasma rich in growth factors eye drops whether mixed or not with lyoprotectant. Conclusion: Freeze-dried plasma rich in growth factors eye drops preserve the main growth factors and their biological activity after storage at room temperature or 4°C for up to 3 months. Lyophilized plasma rich in growth factors eye drops conserve their biological features even without the use of lyoprotectants for at least 3 months.

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Autologous serum and plasma rich in growth factors in ophthalmology: preclinical and clinical studies

Acta Ophthalmologica ◽

10.1111/aos.12710 ◽

2015 ◽

Vol 93 (8) ◽

pp. e605-e614 ◽

Cited By ~ 55

Author(s):

Eduardo Anitua ◽

Francisco Muruzabal ◽

Ali Tayebba ◽

Ana Riestra ◽

Victor L. Perez ◽

...

Keyword(s):

Growth Factors ◽

Clinical Studies ◽

Autologous Serum ◽

Preclinical And Clinical Studies

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Applications of Autologous Serum Eye Drops in Aniridic Keratopathy

European Ophthalmic Review ◽

10.17925/eor.2009.03.01.51 ◽

2009 ◽

Vol 03 (01) ◽

pp. 51 ◽

Cited By ~ 2

Author(s):

José Santiago López-García ◽

Keyword(s):

Growth Factors ◽

Tear Film ◽

Biomechanical Properties ◽

Autologous Serum ◽

Eye Drops ◽

Impression Cytology ◽

Film Instability ◽

Congenital Aniridia ◽

Corneal Cells ◽

Serum Eye Drops

Aniridia is an uncommon congenital bilateral disease that involves the cornea, anterior chamber, lens, retina and optic nerve. Corneal changes in aniridic keratopathy include recurrent erosions of corneal epithelium, tear film instability, chronic pain, corneal vascularisation, dry eye, progressive corneal opacification and blindness. The autologous serum contains a variety of growth factors, vitamins and immunoglobulins, some of which are in higher concentrations than in natural tears. The growth factors and protein found in the serum may help the proliferation, migration and adhesion of epithelial corneal cells. They are by nature non-allergenic and their biochemical and biomechanical properties are similar to those of normal tears. The ocular surface, including corneal impression cytology and tear film evaluation, of patients with congenital aniridia was studied prior to and after treatment with autologous serum eye drops.

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