Epithelial Stem Cell Transplantation Research Articles

Limbal Epithelial Stem Cells Transplantation in Cases with Unilateral Limbal Stem Cell Deficiency Syndrome

This review presents well-known surgical techniques aimed at the treatment of unilateral limbal insufficiency syndrome. Among the many available techniques, one should especially highlight the so-called “simple limbal epithelial transplantation”, which has proven to be an effective, safe and generally available method of treating unilateral limbal insufficiency syndrome (SLN). It should be noted that the risks of recurrence of SLN remain significant, and the mechanisms of engraftment of transplanted limbal epithelial stem cells on the recipient’s eye are not fully understood. The review also considers options for new promising approaches to the possible solution of existing problems in the transplantation of limbal epithelial stem cells.

Understanding Ocular Surface Inflammation in Tears Before and After Autologous Cultivated Limbal Epithelial Stem Cell Transplantation.

We aimed to determine the expression of inflammatory cytokines in the tears of patients with unilateral total limbal stem cell deficiency (TLSCD) caused by chemical burns before and after autologous cultivated limbal epithelial stem cell transplantation (CLET). Tear samples were collected from both eyes of 23 patients with unilateral TLSCD and 11 healthy controls, at fixed timepoints before and after CLET. Dissolved molecules were extracted from Schirmer's strips using a standardised method and analysed on an array plate of ten inflammatory cytokines (V-Plex Proinflammatory Panel 1 Human Kit, MSD). IL1β expression was significantly elevated in the TLSCD eye compared with the unaffected eye at baseline (p < 0.0001) but decreased to normal 3months post-CLET (p = 0.22). IL6 and IL8 were unaffected at baseline but significantly elevated in the TLSCD eyes at 1month post-CLET (p = 0.001 and p < 0.0001, respectively). IL6 returned to normal at 3months and IL8 at 6months post-CLET. There was a significant renewed increase in IL1β, IL6 and IL8 expression at 12months post-CLET (p < 0.0001, p = 0.0001 and p = 0.0003, respectively). IFNγ, IL10 and IL12p70 expression were significantly reduced in both eyes of patients with unilateral TLSCD at all timepoints. IL1β is a specific marker of inflammation in TLSCD eyes that could be therapeutically targeted pre-CLET to improve stem cell engraftment. At 12months post-CLET the spike in levels of IL1β, IL6 and IL8 coincides with cessation of topical steroids, suggesting ongoing subclinical inflammation. We therefore recommend not discontinuing topical steroid treatment in cases where penetrating keratoplasty is indicated; however, further investigation is needed to ascertain this. European Union Drug Regulating Authorities Clinical Trials Database (EuDRACT 2011-000608-16); ISRCTN (International Standard Randomised Controlled Trial Number (isrctn51772481).

A novel technique of amniotic membrane suturing for ex vivo cultivated limbal epithelial stem cell expansion increases the number of progenitor cells

AbstractPurpose: Human limbal epithelial stem cells (HLESCs) reside in limbal epithelial crypts and continuously compensate for the loss of cells in the corneal epithelium. The lack or malfunction of HLESCs leads to a severe ocular surface disease called limbal epithelial stem cell deficiency (LSCD). Cultivated limbal epithelial stem cell transplantation (CLET) of expanded autologous limbal tissue on a human amniotic membrane (HAM) carrier is one of the most common methods for monocular LSCD. We hereby present a novel suturing technique of HAM prior to limbal biopsy expansion and cultivation that mimics natural limbal crypts.Methods: HLESCs derived from limbal biopsies were cultured ex vivo in complex media (COM) on flat or crypt‐like sutured HAMs for 3 weeks. The samples were then processed for immunohistochemistry (IHC) and analysed for progenitor markers: p63α and SOX9; markers for quiescence: CEBPD; proliferation: Ki‐67; differentiation: CX43 and CK3/12; transmembrane markers: E‐cadherin and N‐cadherin. The positivity of markers was counted and compared in cells cultivated on flat vs. crypt‐like sutured HAMs.Results: HLESCs cultured on folded, crypt‐like sutured HAMs had significantly more positivity for p63α, SOX9 (progenitor markers) and Ki‐67 (proliferation marker) compared to those cultured on flat HAMs. Fewer cells were positive for CK3/12 (differentiation marker) in cultures on folded compared to flat HAMs. The number of cells positive for CEBPD (quiescence marker), CX43 and E‐cadherin (differentiation markers) were equal in both groups (flat vs. folded HAMs).Conclusions: A novel technique of HAM suturing that forms crypt‐like undulations mimicking limbal epithelial crypts increases the number of early progenitor cells and could hence increase the quality of the ex vivo expanded limbal epithelial tissue and success for transplantation.

Effect of Human Amniotic Epithelial Stem Cell Transplantation on Preterm Premature Rupture of Fetal Membrane Using the Amniotic Pore Culture Technique in vitro

Objectives: The objective of this study was to evaluate the efficacy of cell therapy using human amniotic epithelial stem cells (hAESCs) for the treatment of premature rupture of membranes (PROM) in vitro. Design: Using the amniotic pore culture technique (APCT), we mimicked the environment of PROM in vitro, thus enabling the observation of the healing process of hAESC-treated amniotic membranes. Materials: Amniotic membrane samples were collected from placentas of pregnant women who underwent elective cesarean sections. APCT model and isolated hAESCs were used in this study. All patients who participated in this study provided their written informed consent prior to the commencement of the study. Settings: To create the APCT model in vitro, isolated amniotic membranes were punched to create 5 mm diameter circles and re-punched to form a 1-mm pore at the center. Membranes were cultured in α-minimal essential medium, and the hAESCs were collected and cultured as well. Subsequently, the APCT models were divided into two groups: hAESC treated and control. Methods: Within the culture period, pore sizes were calculated to evaluate the degree of tissue regeneration in both groups. We then evaluated the histology, cell density, and epithelial thickness of the regenerated tissues. Statistical analyses were performed using SPSS software ver. 20.0 (IBM, Armonk, NY, USA) with repeated-measures one-way analysis of variance or paired samples t test. The significance level was set at p < 0.05. Results: As per the evaluation of the APCT model in vitro, the pore size in the hAESC-treated group reduced by 62.2% on day 6 (62.2 ± 0.19, n = 24), whereas in the control group, it shrank by only 36.8% (p < 0.05) (36.8 ± 0.19, n = 24). Furthermore, the epithelial thickness in the amniotic epithelial stem cell-treated group (10.08 ± 1.26 μm, n = 8) was significantly higher than that in the control group (5.87 ± 0.94 μm, n = 8). Cell density in the regenerated tissue in the amniotic epithelial stem cell-treated group (57 ± 2.77, n = 8) was significantly higher than that in the control group (49 ± 2.23, n = 8). Limitations: In this study, we did not explore the molecular mechanisms by which hAESCs participate in membrane healing in the APCT model. Although our results showed a significant difference, this difference was not too obvious. Therefore, further research on the mechanisms of hAESCs is needed, with more amniotic tissues and APCT samples being tested. Conclusions: We developed an APCT model to investigate the PROM conditions in vitro. By implanting donor hAESCs in the pores of the APCT model, we observed that hAESCs seeding accelerated pore healing in vitro. Thus, hAESCs may be a valuable source of cells for cell therapies in regenerative medicine.

Cell jamming, stratification and p63 expression in cultivated human corneal epithelial cell sheets

Corneal limbal epithelial stem cell transplantation using cultivated human corneal epithelial cell sheets has been used successfully to treat limbal stem cell deficiencies. Here we report an investigation into the quality of cultivated human corneal epithelial cell sheets using time-lapse imaging of the cell culture process every 20 minutes over 14 days to ascertain the level of cell jamming, a phenomenon in which cells become smaller, more rounded and less actively expansive. In parallel, we also assessed the expression of p63, an important corneal epithelial stem cell marker. The occurrence of cell jamming was variable and transient, but was invariably associated with a thickening and stratification of the cell sheet. p63 was present in all expanding cell sheets in the first 9 days of culture, but it’s presence did not always correlate with stratification of the cell sheet. Nor did p63 expression necessarily persist in stratified cell sheets. An assessment of cell jamming, therefore, can shed significant light on the quality and regenerative potential of cultivated human corneal epithelial cell sheets.

Abstract TMP33: Repair of Ischemic Intestinal Epithelial Stem Cells: Potential Therapy to Improve Stroke Outcomes

Background: Nearly 50% of all stroke patients experience “leaky” gut, gut hemorrhage and gut epithelium damage. Gut leakiness may increase circulating inflammatory cytokines and other gut products such as endotoxins, which can impair stroke recovery. Here we tested the hypothesis that normalizing gut function via transplantation of intestinal epithelial stem cells (IESC) after stroke may stimulate repair of gut structures and improve stroke outcomes. Methods: Reproductive senescent female Sprague-Dawley rats used for this study and assigned to the following groups: Control (no stroke); stroke with sham transplant (vehicle); stroke with IESC transplantation. Rats were subjected to stereotaxic surgery to occlude the left middle cerebral artery by using Endothelin-1. Primary IECs were isolated from young female rats to prepare organoids cultures. Dissociated organoids were labeled with PKH26 and injected iv either once (48h after stroke) or 3 times (4h/24h/48h after stroke). Behavioral assays and saphenous blood draws were performed pre-stroke, 2d and 5d after stroke. Trunk blood, brain tissue and a segment of small intestine was collected at termination and processed for the expression of the stem cell marker Lgr5+, Na/K ATPase-α, and tight junction proteins. Results: Significant deterioration of the gut architecture was observed after stroke, including blunted or absent villi and irregular crypts. In animals that received PKH26-labeled organoid tranplants at 48h post stroke, labeled cells were seen in the center of the villus and a few organoid cells were immunositve for Lgr5+. Animals that received 3 organoid injections showed PKH26-labeled cells incorporated in both the villus and crypts. In these animals, villus were well formed and appeared no different from non-stroke controls. Sensory motor function assessed by adhesive removal test on the side contralateral to the infarction was severely impaired in the stroke/no transplant animals (120+secs/timed out), while this impairment was attenuated in the group that received 3 organoid injections (31±11 secs). Conclusion: These data suggest that transplantation of IESC after stroke may promote repair of gut villus and crypts, with a concomitant improvement in sensory motor function.

Optimization of Human Limbal Stem Cell Culture by Replating a Single Limbal Explant.

Cultured limbal epithelial stem cell transplantation is a clinical procedure used to regenerate the corneal epithelium in patients with limbal stem cell deficiency. The protocols used to expand limbal epithelial cells in vitro need to be optimized, since the scarcity of human ocular tissue donors is limiting the potential use of this procedure. Here, we describe a method to consecutively expand a single human limbal explant. With this method it is possible to obtain up to three limbal epithelial primary cultures from the same explant, thus increasing the efficiency of the in vitro cell culture.

Xenofree generation of limbal stem cells for ocular surface advanced cell therapy

BackgroundLimbal stem cells (LSC) sustain the corneal integrity and homeostasis. LSC deficiency (LSCD) leads to loss of corneal transparency and blindness. A clinical approach to treat unilateral LSCD comprises autologous cultured limbal epithelial stem cell transplantation (CLET). CLET uses xenobiotic culture systems with potential zoonotic transmission risks, and regulatory guidelines make necessary to find xenofree alternatives.MethodsWe compared two xenofree clinical grade media and two feeder layers. We used CnT07, a defined commercial medium for keratinocytes, and a modified xenofree supplemented hormonal epithelial medium with human serum (XSHEM). Optimal formulation was used to compare two feeder layers: the gold standard 3T3 murine fibroblasts and human processed lipoaspirate cells (PLA). We tested the expressions of ΔNp63α and cytokeratin 3 and 12 by qPCR and immunofluorescence. Morphology, viability, clonogenicity, proliferation, and cell growth assays were carried out. We also evaluated interleukin 6 (IL-6) and stromal-derived factor 1 (SDF-1) by qPCR and ELISA.ResultsXSHEM maintained better LSC culture viability and morphology than CnT07. Irradiated PLA feeder cells improved the undifferentiated state of LSC and enhanced their growth and clonogenicity stimulating IL-6 secretion and SDF-1 expression, as well as increased proliferation and cell growth when compared with irradiated 3T3 feeder cells.ConclusionsThe combination of XSHEM and PLA feeder cells efficiently sustained LSC xenofree cultures for clinical application. Moreover, PLA feeder layers were able to improve the LSC potential characteristics. Our results would have direct clinical application in CLET for advanced therapy.Graphical abstract

Short- and Long-Term Results of Xenogeneic-Free Cultivated Autologous and Allogeneic Limbal Epithelial Stem Cell Transplantations.

Purpose:To evaluate the short- and long-term success rates of xenogeneic-free cultivated limbal epithelial stem cell transplantation (CLET) for the treatment of limbal stem cell deficiency (LSCD).Methods:Thirteen patients with LSCD underwent an autologous (n = 9) or allogeneic (n = 4) CLET. The primary end point was to assess the long-term anatomical success rate of transplanted grafts at a follow-up of at least 3 years, in comparison with the short-term outcomes. Secondary end points involved reviewing functional improvement, patient-reported symptoms, and change in percentage area of corneal vascularization in both short-term and long-term.Results:The mean short- and long-term follow-up periods were 2.1 ± 0.38 years and 6.7 ± 1.81 years, respectively. The total anatomical success rate was 46.1% in the short-term, but it decreased to 23.1% in the long-term. A partial success rate of 30.8% was observed in both short- and long-term, and the failure rate increased from 23.1% to 46.1%. The mean percentage of vessel area decreased from 12.11% ± 5.29% preoperatively to 7.82% ± 6.70% in the short-term and increased to 8.70% ± 6.32% in the long-term. There was a significant improvement in best-corrected visual acuity (P = 0.044) in the short-term although not in the long-term (P = 0.865).Conclusions:This study shows that anatomical and functional success rates of CLET decrease over time. We believe that the decline of success is related to the extent of disease, cell origin, and lack of niche protection because subtotal LSCD and autologous donor cells confer a higher chance of success in the long-term.

Экспериментальная трансплантация лимбальных эпителиальных стволовых клеток на фибриновом скаффолде

Экспериментальная трансплантация лимбальных эпителиальных стволовых клеток на фибриновом скаффолде

Limbal stem cell deficiency: etiology, pathogenesis, priniciples and prospects of surgical treatment

The review is focused on the modern view of the etiology and pathogenesis of limbal stem cells deficiency. The history of development of tissue and ex-vivo transplantation of limbal epithelial stem cells is presented. Certain promising directions of the treatment of patients with limbal stem cells deficiency are presented.

How to improve the prevention and treatment of ocular chemical burns in China: important elements

Ocular chemical burns are common and serious ocular emergencies which require immediate and intensive evaluation and treatment. Loss of vision and disfigurement affect the victims, bringing great sufferings to themselves and their families. China is the biggest developing country in the world with a large number of such cases. The prevention of ocular chemical burns is emphasized in different aspects. After emergency treatment, proper care of chemical burns is started by control of inflammation with corticosteroids. Topical and systemic ascorbic acid supplement is important. Re-epithelialization is critical to stabilize the ocular surface and to prevent corneal ulceration and melting. The goal of treatment is mainly to restore the ocular structure and function. Neuroprotection is important during the treatment course for control of both glaucoma and inflammation. Prognosis depends on the degree of limbal, corneal and conjunctival involvement at the time of injury as well as the management. Medical treatments only or with combination of surgical procedures, including amniotic membrane transplantation, epithelial or limbal stem cell transplantation, tenonplasty, keratoplasty and keratoprosthesis, are according to the classification of ocular chemical burns and the phases. Further investigations should be done in the future in both prevention and management of ocular chemical burns in China. (Chin J Ophthalmol, 2018, 54: 401-405).

Outcomes of Penetrating Keratoplasty Following Autologous Cultivated Limbal Epithelial Stem Cell Transplantation.

The purpose of this study is to investigate the outcomes of penetrating keratoplasty (PKP) following autologous cultivated limbal epithelial stem cell transplantation (CLET). A prospective, single center, interventional cohort study investigating patients with unilateral total limbal stem cell deficiency (LSCD) treated with CLET who underwent PKP. Patients with confirmed corneal re-epithelialization > 6 months post-CLET, and with best-corrected visual acuity (BCVA) <0.3 logMAR were offered PKP. CLET survival assessed by slit lamp, corneal impression cytology (CIC), and in vivo confocal microscopy. Confirmation of corneal re-epithelialization by histological and immunocytochemical (ICC) examination of trephined corneal buttons. Mean change in best-corrected visual acuity (logMAR) following PKP and PKP survival at 12 months were calculated. Twenty patients underwent PKP. Mean time of PKP was 19 months (range 11-41 months, SD 7.26) post-CLET. Median follow-up time post-PKP was 15 months (range 1-32, SD 10.2). CIC and ICC of all corneas confirmed corneal re-epithelialization before PKP. Mean pre-PKP BCVA was 1.46 (range 0.3-2.7, SD 0.94) improving to a mean post-PKP BCVA of 0.74 (range 0-2.7, SD 0.87); mean improvement in BCVA post-PKP of 36 letters (95% CI 15.0-57.1, p = .002). Kaplan-Meier mean graft survival was 90.9% (95% CI 50.8-98.7) at 12 months. We recommend a two-stage approach with CLET followed by PKP >12 months later. Patients experienced a significant improvement in BCVA following PKP. PKP did not have a detrimental effect on CLET survival. PKP survival post-CLET is better than that reported for high risk PKP. Stem Cells 2018;36:925-931.

Advanced therapies push regulatory boundaries: Novel therapeutic approaches require more regulatory flexibility and transparency.

The regulation of new medical applications has always been lagging behind the science that enabled these therapies in the first place, but emerging therapeutic approaches are pushing the boundaries harder than ever. Although fundamental principles around safety, efficacy and quality are cast in stone, new interpretations and greater flexibility are required for the clinical use of advanced medicines and treatments without introducing unacceptable risks. There are also ethical considerations that, although not new, are amplified by the potential of emerging therapies to address previously untreatable conditions. All major regulatory bodies, such as the US Food and Drug Administration (FDA) and the European Medical Agency (EMA), have for some years been aware of the challenges posed in particular by Advanced Therapy Medicinal Products (ATMPs). These roughly include three categories: gene therapy medicinal products (GTMPs), somatic cell therapy medicinal products (SCTMPs) and tissue‐engineered products (TEPs), many of which have emerged from the “omics” revolution and stem cell research. > All major regulatory bodies […] have for some years been aware of the challenges posed in particular by Advanced Therapy Medicinal Products ### Advanced Therapy Medicinal Products Both GTMPs and SCTMPs involve ex vivo manipulation of cells, but with a crucial difference in terms of manufacture and function. GTMPs deliver recombinant DNA to specific target cells in the body, whereas SCTMPs are whole cells or tissues genetically manipulated to change their biological characteristics or functions for preventing, diagnosing or treating disease. These cells can be autologous, coming from the patient, allogeneic from another human being or xenogeneic from animals. One recent example of GMTP is recombinant viral vaccines against cancer engineered from an immunogenic virus particle to express tumour antigens, cytokines or both [1]. SCTMPs were the first AMPs to be tested clinically in 1997 for corneal epithelial stem cell transplantation. Since then, the technique has been …

Poly(ethylene glycol)-modified silk fibroin membrane as a carrier for limbal epithelial stem cell transplantation in a rabbit LSCD model

BackgroundLimbal epithelial stem cells (LESCs) play important roles in corneal epithelial homeostasis and regeneration, and damage to the limbus will lead to limbal stem cell deficiency (LSCD), with conjunctivalization and even visual impairment. Cultured LESCs have been used for ocular surface reconstruction, and silk fibroin (SF) membranes have shown potential as a substrate for LESC cultivation. Both culture methods and the carriers of LESCs affect outcomes following LESC transplantation.MethodsRabbit LESCs were cultured from tissue explant, single cell-suspension, and cell cluster culture methods. Ratios of p63α and/or ABCB5-positive LESCs, differentiated corneal epithelial cells (CK12 staining), and corneal tight junction formation (Claudin-1 staining) were examined to choose the most applicable LESC cultures. SF membranes were prepared and modified by 400-Da poly(ethylene glycol) (PEG). The characteristics of stem cells and normal corneal differentiation of LESCs cultured on PEG-modified SF membranes were further examined by immunofluorescence staining and flow cytometric analysis. LESCs cultured on PEG-modified SF membranes (LESC/SF grafts) and PEG-modified SF membranes (SF grafts) were transplanted onto rabbit corneas with total LSCD. New blood vessels, corneal epithelial defects, and cornea clarity were examined after transplantation. Furthermore, corneal epithelial thickness, stromal thickness, and the percentage area of CK12-positive corneal epithelium were quantified 4 months after transplantation.ResultsTissue explant and single cell-suspension cultures harvested more p63α and/or ABCB5-positive LESCs, generated more CK12-positive corneal epithelial cells, and formed more corneal tight junctions than cell cluster cultures. Prepared PEG-modified SF membranes were transparent, flexible, and sturdy enough for surgical manipulation. LESCs cultured on PEG-modified SF membranes maintained characteristics of stem cells and normal corneal differentiation. LESC/SF grafts inhibited new blood vessels and rescued corneal epithelial defects in the rabbit total LSCD model. In addition, LESC/SF grafts repopulated the limbus and increased corneal epithelial thickness, stromal thickness, and the area percentage of CK12-positive corneal epithelium.ConclusionsLESCs from tissue explant and single cell-suspension cultures were more applicable corneal epithelial cells for ocular surface reconstruction. LESC/SF grafts repaired corneal epithelial defects and reversed LSCD, and PEG-modified SF membranes were suitable to be a carrier for LESC transplantation.

Native and synthetic scaffolds for limbal epithelial stem cell transplantation.

The outcome of patients with corneal blindness that receive stem cell grafts to restore eye health and correct vision varies considerably and may be due to the different biological and synthetic scaffolds used to deliver these cells to the ocular surface. This review will highlight the positive attributes and limitations of the myriad of carriers developed for clinical use as well as those that are being trialled in pre-clinical models. The overall focus is on developing a standardized therapy for patients, however due to the multiple causes of corneal blindness, a personal regenerative medicine approach may be the best option.

Conjunctival-limbal autograft in total unilateral limbal stem cell deficiency

Background : Corneal epithelium is renewed by stem cells (SC) that reside at the corneal limbus. Reduced number of SC or their abnormal function lead to the ocular surface disease called limbal stem cell deficiency (LSCD), characterized by corneal conjunctivalization, vascularization, persistent epithelial defects, chronic inflammation, and loss of vision. In a case of total unilateral LSCD, autologous transplantation of limbal epithelial stem cells (LESC) from the healthy eye is needed. We describe the surgical technique of choice for autologous limbal transplantation, called conjunctival limbal autograft (CLAU) that we combined with amniotic membrane (AM) use. We present the results of CLAU in three patients with total unilateral LSCD due to chemical injury. Methods : Autologous limbal transplantation CLAU begins with the removal of fibrovascular pannus from the diseased corneal surface and the harvesting of two conjunctival-limbal grafts from the healthy eye. The grafts are then transplanted on to the limbal area of the recipient eye. AM is used as a patch to cover the denuded cornea and limbal grafts, as well as a barrier preventing the conjunctival epithelium from encroaching on to the temporal and nasal side of the corneal surface. In the donor eye, AM is used to cover the donor sites. CLAU with the use of AM was performed in 3 patients with unilateral LSCD due to chemical eye injury. In one patient limbal transplantation was combined with symblepharon lysis for entropium repair. In all cases AM was removed 3–6 days postoperatively to assess the growth of new epithelium from the limbal grafts. In all patients the ocular surface was covered with another AM until the cornea was completely epithelized and the new epithelium stable. In one patient the corneal regrafting and cataract removal was performed subsequently. Results : CLAU was successful in 2 patients and partially successful in 1 patient during the follow up. In all cases the growth of new epithelium from the limbal grafts was noted on day 3–6 after CLAU. The cornea was completely epithelized within 2 weeks in 2 patients and after 35 days in one patient. In two patients the corneal epithelium remained clear, smooth and stable during the follow up of 3.5 years and 4 months, respectively. In one patient, uneven epithelium probably representing a mosaic of corneal and conjunctival cells was noted in the central corneal region, where a small corneal ulcer developed 5 months after CLAU. In donor eyes no postoperative complications were noted, the donor sites epithelized within few days. Conclusions : Autologous limbal transplantation according to CLAU surgical technique combined with the use of AM is a successful and safe therapy for restoring corneal surface in total unilateral LSCD after chemical injury. It enables further surgical procedures for restoring the vision such as corneal transplantation and cataract surgery.

Avtologni veznično-limbalni presadek pri enostranskem popolnem pomanjkanju limbalnih epitelnih matičnih celic

Background: Corneal epithelium is renewed by stem cells (SC) that reside at the corneal limbus. Reduced number of SC or their abnormal function lead to the ocular surface disease called limbal stem cell deficiency (LSCD), characterized by corneal conjunctivalization, vascularization, persistent epithelial defects, chronic inflammation, and loss of vision. In a case of total unilateral LSCD, autologous transplantation of limbal epithelial stem cells (LESC) from the healthy eye is needed. We describe the surgical technique of choice for autologous limbal transplantation, called conjunctival limbal autograft (CLAU) that we combined with amniotic membrane (AM) use. We present the results of CLAU in three patients with total unilateral LSCD due to chemical injury.Methods: Autologous limbal transplantation CLAU begins with the removal of fibrovascular pannus from the diseased corneal surface and the harvesting of two conjunctival-limbal grafts from the healthy eye. The grafts are then transplanted on to the limbal area of the recipient eye. AM is used as a patch to cover the denuded cornea and limbal grafts, as well as a barrier preventing the conjunctival epithelium from encroaching on to the temporal and nasal side of the corneal surface. In the donor eye, AM is used to cover the donor sites. CLAU with the use of AM was performed in 3 patients with unilateral LSCD due to chemical eye injury. In one patient limbal transplantation was combined with symblepharon lysis for entropium repair. In all cases AM was removed 3–6 days postoperatively to assess the growth of new epithelium from the limbal grafts. In all patients the ocular surface was covered with another AM until the cornea was completely epithelized and the new epithelium stable. In one patient the corneal regrafting and cataract removal was performed subsequently.Results: CLAU was successful in 2 patients and partially successful in 1 patient during the follow up. In all cases the growth of new epithelium from the limbal grafts was noted on day 3–6 after CLAU. The cornea was completely epithelized within 2 weeks in 2 patients and after 35 days in one patient. In two patients the corneal epithelium remained clear, smooth and stable during the follow up of 3.5 years and 4 months, respectively. In one patient, uneven epithelium probably representing a mosaic of corneal and conjunctival cells was noted in the central corneal region, where a small corneal ulcer developed 5 months after CLAU. In donor eyes no postoperative complications were noted, the donor sites epithelized within few days.Conclusions: Autologous limbal transplantation according to CLAU surgical technique combined with the use of AM is a successful and safe therapy for restoring corneal surface in total unilateral LSCD after chemical injury. It enables further surgical procedures for restoring the vision such as corneal transplantation and cataract surgery.

Transplantation of Lung Epithelial Progenitors Pre-differentiated Amniotic Fluid Mesenchymal Stem Cells Attenuated Elastase-Induced Pulmonary Emphysema

Transplantation of Lung Epithelial Progenitors Pre-differentiated Amniotic Fluid Mesenchymal Stem Cells Attenuated Elastase-Induced Pulmonary Emphysema

Limbal and Conjunctival Epithelial Cell Cultivation on Contact Lenses-Different Affixing Techniques and the Effect of Feeder Cells.

Corneal blindness due to limbal stem-cell deficiency can be treated by transplantation of cultivated limbal epithelial stem cells (LESCs). We examined LESC cultivation on a contact lens (CL) carrier. Our goal was to optimize explant affixation and assess the possible benefit of 3T3 feeder cells. Human cadaver limbal and conjunctival explants were allowed to attach to CLs under the airflow of the laminar box (dried group) or affixed on CLs using suturing (sutured group) or tissue adhesives (glued group), then cultivated with or without 3T3 feeder cells. Outgrowth efficiency was statistically analyzed. CEBPδ, p63, CK3/12, and CK13 were detected by immunofluorescence in expanded cells. Suturing and gluing provided excellent sample attachment, whereas drying was less effective. Cell expansion was better in sutured than in dried or glued samples. Presence of 3T3 feeder resulted in significantly better cell growth (P=0.048), most importantly in dried samples (P=0.008). Stepwise regression analysis indicated that cell expansion was dependent on the affixing method (P<0.001) and the presence of feeder layer (P=0.003). Expanded cells maintained their CK expression profiles and expressed putative stem-cell markers p63 and CEBPδ. The 3T3 feeder did not influence the expression of putative LESC markers or growth rate. Suturing is an effective way to fasten explants to CLs. 3T3 fibroblasts are not necessary in this system, although they may enhance cell outgrowth when samples are exposed to stress. However, once cells begin to expand, neither expression of putative stem-cell markers nor growth rate is influenced by feeder cells.