Introduction: Diabetic burn wounds and ulcers are significant complications of diabetic patients. The aim of this study is to investigate the use of platelet rich-plasma (PRP) and/or keratinocyte-like cells (KLCs) in diabetic thermal wound rat model and to evaluate EGF, FGF-2, TGF-β1, COL1α2, MCP-1 and VEGF-α as wound healing markers at gene expression level. Method: In this study, we used adipose tissue as the source of mesenchymal stem cells (MSCs) and differentiated MSCs into KLCs. KLCs were characterized and transferred to the burn areas on the dorsum of streptozotocine (STZ)-induced diabetic rats. We prepared PRP from rat blood and evaluated its effect alone or in combination with KLCs. On 3rd, 7th, 10th and 14th days after treatment, wound areas were measured and biopsy samples were excised from the wound areas of the KLCs and/or PRP-treated and untreated diabetic rats to analyze gene expression levels of wound healing markers by qPCR. Results: We observed that, wound contraction started earlier in the PRP and/or KLCs-treated groups in comparison to the control group. However, PRP and KLCs when applied in combination showed additive affect in wound healing. In all groups treated with KLCs and/or PRP, the gene expression levels of evaluated growth factors and COL1α2 increased, while MCP-1 levels decreased when compared to the untreated diabetic rats. In addition, the most prominent difference in qPCR results belongs to combined PRP and KLCs-treated group. Conclusion: We demonstrated that applying PRP and KLCs in combination has a greater potential for treatment of diabetic burn wounds.

The cornea is the outermost tissue of the eye and it must be transparent for the maintenance of good visual function. The superficial epithelium of the cornea, which is renewed continuously by corneal stem cells, plays a critical role in the permanence of this transparency. These stem cells are localized at the cornea-conjunctival transition zone, referred to as the limbus. When this zone is affected/destroyed, limbal stem cell deficiency ensues. Loss of limbal stem cell function allows colonization of the corneal surface by conjunctival epithelium. Over 6 million people worldwide are affected by corneal blindness, and limbal stem cell deficiency is one of the main causes. Fortunately, it is becoming possible to recover vision by autologous transplantation of limbal cells obtained from the contralateral eye in unilateral cases. Due to the potential risks to the donor eye, only a small amount of tissue can be obtained, in which only 1-2% of the limbal epithelial cells are actually limbal stem cells. Vigorous attempts are being made to expand limbal stem cells in culture to preserve or even enrich the stem cell population. Ex vivo expanded limbal stem cell treatment in limbal stem cell deficiency was first reported in 1997. In the 20 years since, various protocols have been developed for the cultivation of limbal epithelial cells. It is still not clear which method promotes effective stem cell viability and this remains a subject of ongoing research. The most preferred technique for limbal cell culture is the explant culture model. In this approach, a small donor eye limbal biopsy is placed as an explant onto a biocompatible substrate (preferably human amniotic membrane) for expansion. The outgrowth (cultivated limbal epithelial cells) is then surgically transferred to the recipient eye.Due to changing regulations concerning cell-based therapy, the implementation of cultivated limbal epithelial transplantation in accordance with Good Laboratory Practice using xenobiotic-free systems is becoming widely accepted both in Turkey and worldwide.

Aim: The aim of this study is to investigate the effects of miR150 transfection on NK-like cells differentiated from adipose tissue derived mesenchymal stem cells (AD-MSCs). Methods: NK-like cells were differentiated from AD-MSCs and activated by miR150 transfection. Transfected/non-transfected NK-like cells were characterized by immunohistochemical and RTPCR analyzes. Apoptotic efficiency of the transfected/non-transfected NK-like cells on pancreatic cancer cells PANC1 were determined by TUNEL and RT-PCR. Results: In miR150-transfected cells, the increased expression of NK cell-specific genes such as GZMB, KIR2DL2, CD16, CD56, NKG2D, NKp46 and increased immunoreactivity of NK cell-specific surface marker CD314 (NKG2D) were evident. TUNEL assays showed that NK-like cells with/without transfection induced apoptosis in PANC1 cells in the same manner. The decrease in oncogene expression and the increase in the tumor suppressor gene expression in PANC1 cells upon co-culture with NK-like cells differentiated from AD-MSCs were more prominent following miRNA150 transfection. Conclusion: It was shown in vitro that NK-like cells could be obtained by differentiation from AD-MSCs and their efficiency could be increased via miR150 transfection. The results are encouraging for further clinical studies in improvement of immunotherapeutic approaches for cancer therapy.

In this study, we aimed to compare the effects of six different cell culture media and autologous serum (AS) on the phenotypic characteristics of rabbit limbal epithelial stem cells (LESC) cultivated on porous polyethylene terephthalate (PET) membranes. Limbal explants from rabbit corneas were grown on PET membrane inserts in five different media: DMEM-F12 with fetal bovine serum (FBS) (DMEM-F12-FBS), with pluripotin (DMEM-F12-pluripotin) and with autologous serum (DMEM-F12-AS), Epilife, Keratinocyte Serum Free Medium (KSFM) and Defined-Keratinocyte Serum Free Medium. The effects of different media were evaluated by total cell yield from explants, measuring the expression of proteins by immunofluorescence and gene expression by Real Time PCR. In all five media tested, most of the limbal epithelial cells (LEC) which proliferated from explants were positive for cytokeratin (CK) 14 (85-90%), indicating that all five media support the growth of LESC from explants. The expression of differentiation markers; CK 3 and 12 was highest in DMEM-F12-FBS (56%), was lower in Epilife and KSFM (26 and 19%, respectively), with the lowest values (13%) obtained in DMEM-F12-AS. Gene expression of limbal cultures on PET membrane inserts was compared to fresh limbal tissue. In DMEM-F12-FBS, DMEM-F12-pluripotin, and DMEM-F12-AS, expression of potential LESC markers CXCR4 and polycomb complex protein BMI-1 were similar to limbal tissue. DMEM-F12 with 10% AS maintained a higher percentage of potential stem cell marker genes and lower expression of genes involved in differentiation compared to Epilife or KSFM. Our study shows that rabbit LEC can be cultivated on PET inserts using DMEM-F12 with autologous serum without a requirement for amniotic membrane or feeder cells.

Background: The sparsity of established tools for the grading of limbal stem cell deficiency hinder objective assessments of the clinical outcome of cultivated limbal epithelial cell transplantation. To advance towards the development of standards for the comparison of the outcomes of these bio-surgical protocols we have now applied a battery of recognized objective and patient-declared subjective outcome criteria to the autologous modality of cultivated limbal epithelial cell transplantation. Methods: The prospective study involved ten patients (M/F = 9/1; mean age = 42.1 years) displaying overt unilateral limbal stem cell deficiency complying with the inclusion criteria described in Methods. Limbal biopsies were obtained from the contralateral eye and their outgrowths after 2-week cultures were transplanted on the affected eye after pannus resection. Outcomes were followed up for 12 months. The objective tests were scores for bestcorrected visual acuity (BCVA); using the LogMAR scale, a multiparametric ocular surface score (OSS), and the Schirmer's test. Subjective scores were based on patient answers to a) perception of visual improvement/pain; b) the 25-item National Eye Institute Visual Function Questionnaire (NEI-VFQ 25); and c) the 12-item Ocular Surface Disease Index Questionnaire (OSDI). All procedures were performed under good manufacture practices using solely xeno-free reagents. In all cases, a single biopsy was divided into two pieces and they were expanded in order to prevent outgrowth failure. In 5 patients, both biopsies generated healthy culture sheet. In those cases the lesser outgrowth were used for immune-histological characterization.

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