Differentiation of human embryonic stem cells derived mesenchymal stem cells into corneal epithelial cells after being seeded on decellularized SMILE-derived lenticules

AIM: To evaluate the feasibility of mesenchymal stem cells(MSCs) to differentiate into corneal epithelial cells after being seeded on the decellularized small incision lenticule extraction(SMILE)-derived lenticules. METHODS: The fresh lenticules procured from patients undergoing SMILE for the correction of myopia were decellularized. The MSCs were subsequently cultivated on those denuded lenticules. The MSCs without lenticules were used as a control. The proliferation activity of the MSCs after seeding 24 h was quantitatively determined with the Cell Counting Kit-8(CCK-8) assay. Immunofluorescence staining and quantitative reverse transcription polymerase chain reaction(qRT-PCR) were used to assess the marker expression in differentiated MSCs. RESULTS: The data showed that both fresh and decellularized lenticules could significantly promote the proliferation of MSCs, compared to that in control(P=0.02 for fresh lenticules, P=0.001 for decellularize ones, respectively). The MSCs seeded on both lenticules were positive for cytokeratin 3(CK3) staining. The expression of CK3 increased 5-fold in MSCs seeded on fresh lenticules and 18-fold on decellularized ones, compared to that in control. There was a significant difference in the expression of CK3 in MSCs seeded on fresh and decellularized lenticules(P<0.001). The expression of CK8 and CK18 was similar in pure MSCs and MSCs seeded on fresh lenticules(P>0.05), while the expression of these markers was decreased in MSCs seeded on decellularized ones. CONCLUSION: These results suggest that the decellularized lenticules might be more suitable for MSCs to differentiate into corneal epithelial cells, which offersthe prospect of a novel therapeutic modality of SMILEderived lenticules in regenerative corneal engineering.

Comparison of two methods used to culture and purify rat retinal Mller cells

AIM:To study two methods for culturing and purifying Sprague-Dawley(SD)rat retinal Muller cells and determine which one is better.METHODS:The passage culture method of Muller cells was respectively carried out by complete pancreatic enzyme digestion method and repeated incomplete pancreatic enzyme digestion method.After culturing retinal cells for one month through these two methods,fluorescence-activated cell sorter(FACS),RT-PCR,and immunohistochemistry technology were performed to examine the enrichment and purity of Muller glial cells,and carried out two-sample approximate t test using SSPS 13.0 to further compare the Muller cell positive rate in both methods.RESULTS:The statistical results showed that the purity of Muller cells was 83.2%±5.16%in group A,and the purity was 98.5%±1.08%in group B.The two-sample approximate t test analysis demonstrated that the difference between group A and group B was statistically significant(t=-9.178,P【0.005).The results clearly exhibited a difference between the purity of Muller cells cultured by the complete pancreatic enzyme digestion method(group A)and the repeated incomplete pancreatic enzyme digestion method(group B).CONCLUSION:Compared with the complete pancreatic enzyme digestion method,this novel method was more efficient and a higher purity of Muller cells could be obtained using this approach.

Promotion on the differentiation of retinal Müller cells into retinal ganglion cells by Brn-3b

AIM： To investigate the role of Brn-3b in differentiation process of stem cells derived from retinal Muller cells into the ganglion cell. METHODS： The passage culture method of Muller cells from retina of newborn Sprague Dawley rats was carried out by repeated incomplete pancreatic enzyme digestion method. The cells were detected by fluorescence- activated ceil sorter （FACS）, immunohistochemistry technology and reverse transcription-polymerase chain reaction （RT-PCR） to determine the purity. The third passage of cells was induced in the serum-free dedifferentiation medium. The expression of the specific markers Ki-67 and nestin of retinal stem cells was measured by RT-PCR and Western blot. The cell proliferation of retinal stem cells was detected by 5- Ethynyl-2＇-deoxyuridine （Edu） staining. The cells were randomly divided into 5 groups as follows： group A： Brn-3bsiRNA group; group B： Brn-3b control siRNA group; group C： pGC-Brn-3b-green fluorescent protein （GFP） group; group D： pGC-GFP group; group E： control group （without any handling）. The purified Muller cells were cultured for 3-7d, then, the percentage of ganglion cells was counted by immunofluorescence staining. RESULTS： FACS demonstrated the purity of retinal Muller cells was more 97.44%. A few spherical cell spheres appeared. Immunofluorescence staining showed that stem cells within the spheres were positive for retinal stem cell-specific markers nestin （red fluorescence, 92.94%±6.48%） and Ki-67 （green fluorescence, 85.96%± 6.04% ）. Meanwhile, RT-PCR analysis showed cell spheres in the culture to have expressed a battery of transcripts characteristic of stem cells such as nestin and Ki-67, which were absent in the Muller cells. Western blot analysis further confirmed the expression of nestin and Ki-67 in the cell spheres but not in the Muller cells. Edu staining showed most of the nuclei within the cell spheres were stained red （82.80%±6.65%）, suggesting the new cell spheres had the capacity for effective proliferation. The statistics result showed the difference between Brn-3bsiRNA group and Brn-3b control siRNA group or the control group was significant （F=15, P〈 0.05）, while the difference between Brn-3b control siRNA group or the control group was not statistically significant （P 〉0.05）. CONCLUSION： The repeated incomplete pancreatic enzyme digestion method is an efficient and practical method to purify retinal Muller cells. Retinal stem cells were successfully cloned in the dedifferentiational medium. Retinal Muller cells are accessible sources of retinal stem cells. Brn-3b is an important regulatory gene in stem cells differentiated into retinal ganglion cell.

Anti-scarring effect of sodium hyaluronate at filtration pathway after filtering surgery in rabbits

AIM: To investigate the anti-scarring effect of sodium hyaluronate(HA) at filtration pathway after filtering surgery in a rabbit model.METHODS: Fifteen healthy adult New Zealand white rabbits were selected for trabeculectomy in both eyes. The right eyes were used as HA group with 0.1 m L HA injected into the anterior chamber at the end of the operation;the left eyes were used with 0.1 m L sodium lactate Ringer’s solution(RS) injected into the anterior chamber as RS group. Intraocular pressure(IOP), filtering blebs morphology, inflammatory reaction and complications were observed at the 7, 60, and 90 d after surgery.RESULTS: One day after surgery, the IOP of HA and RS groups were 12.75±1.92 and 10.50±1.59 mm Hg(P=0.005). At the 7;day postoperative, the filtering blebs of each group were functional type and TGF-β expression was significantly difference in both groups(0.10±0.01 vs 0.14±0.02, P=0.024). After 60 d of the operation, all filtering blebs were scarring and alpha-smooth muscle actin(α-SMA) expression was significantly difference in both groups(0.40±0.04 vs 0.35±0.02, P=0.032). α-SMA positive cells were mainly distributed in the junction of conjunctiva and sclera and around the blood vessels. The collagen volume fraction(CVF) of HA and RS group was(75.49±7.01)% and(79.93±5.35)%(P=0.044). On the 90;day after the operation, CVF was(82.57±5.19)% and(88.08±1.75)% in HA and RS groups(P=0.036). There was no α-SMA positive cell in HA group, while a few positive cells were observed in RS group(P=0.000).CONCLUSION: HA has effect of anti-scar and antiinflammation on filtration pathway after filtering surgery within 3 mo by inhibiting fibroblast proliferation and collagen deposition.

Therapeutic effect analysis on the treatment of congenital glaucoma through modified combined trabeculotomy-trabeculectomy

AIM： To evaluate the therapeutic effect and the safety of the treatment of congenital glaucoma through modified combined trabeculotomy-trabeculectomy. METHODS： The clinical data of 27 cases （altogether 42 eyes）, which included 7 cases of infants （10 eyes） and 20 cases of teenagers （32 eyes）, of congenital glaucoma undertook modified combined trabeculotomy trabeculectomy were analyzed retrospectively. The parameters evaluated included the post operation visual acuity, the anterior chamber, the filtering bleb, the intraocular pressure, the C/D ratio, visual field, the retinal nerve fiber layer changes and the complications. RESULTS： The follow-up period was 1 to 29mo, averaging 13.3 ±7.7mo. Upon the last visit after the operation, functional filtering blebs developed in all the involved eyes. The intraocular pressure was controlled under 21 mm Hg, which was decreased by 60% when compared with that before the operation, without using any medication. There were no significant changes in the post operation visual acuity and the retinal nerve fiber layer thickness before and after the operation in teenager group （P〉0.05）, and both the post operation C/D ratio and the visual field mean defect （MD） were reduced compared with those before the operation （P〈0.05）. There were no severe complications in any of the patients. CONCLUSION： The modified combined trabeculotomy- trabeculectomy can effectively reduce the intraocular pressure and control the development of glaucoma in cases of congenital glaucoma. It is a safe and effective operative method for the treatment of congenital glaucoma