Ophthalmic indications of amniotic membrane transplantation in Mexico: an eight years Amniotic Membrane Bank experience.

Amniotic membrane, the inner layer of the placenta, has biological properties (e.g. promotes epithelization, reduces fibrosis, secretes antimicrobial products and inhibits immune responses) which make it a useful option for several ophthalmologic procedures, especially those involving the ocular surface. Its use in eye surgery has been reported by other authors. To our knowledge, there is a lack of descriptive studies on surgical indications using amniotic membrane in Mexican population. Here we describe the eight years Amniotic Membrane Bank experience in Mexico, including a detailed protocol of the donors selection, tissue harvesting, preparation, storage and distribution of amniotic membrane since its establishment in 2007. Moreover, we describe the Ophthalmological indications of amniotic membrane transplantation of the total of 1686 amniotic membranes fragments used during eight years. The five most common indications for amniotic membrane transplantation were pterygium (46 %), corneal ulcers (12.6 %), conjunctival surface repair (11.1 %), neoplasms (7.4 %), and persistent epithelial defects (7.3 %). In addition, we compared the indications of amniotic membrane use in two different types of Institutions: general hospitals and ophthalmologic reference hospitals. We found interesting differences between the indications and use rates between these institutions, although pterygium was the most frequent pathology that amniotic membrane fragments were used in both institutions, there was up to a five-fold increase in the use of amniotic membrane for correction of persistent epithelial defects in reference hospitals which could be explained due to the more complex and severe ophthalmological pathologies admitted in reference hospitals. In conclusion, Amniotic Membrane is used in a numerous ocular pathologies and especially on pterygium in our Mexican population.

Stem cells isolated from the human stromal limbus possess immunosuppressant properties.

PURPOSE: Mesenchymal stromal stem cells (MSC) are non-hemopoietic cells with the capacity to self-renewal and to differentiate into various cell lineages of mesenchymal origin. More recently, the immune regulatory potential of MSC has been focused on. Furthermore, mesenchymal stem cells obtained from diverse tissues possess immunomodulatory properties and inhibit proinflammatory immune reactions. The aim of this study was to determine the immunosuppressive characteristics of mesenchymal stem cells isolated from human limbal (L-MSC) tissue. METHODS: L-MSC were enzymatically obtained from cadaveric sclero-corneal rims and expanded in vitro. The cells were characterized by flow cytometry using specific antibodies to mesenchymal stem cells markers. Clonogenic and tissue transdifferentiation in vitro assays were performed. The effect of L-MSC soluble factors on T cell proliferation was determined by flow cytometry. Cytokines such as transforming growth factor-b1 (TGF-ß1) and Interleukin-10 (IL-10) on supernatants from L-MSC were identified by enzyme-linked immunosorbent assay (ELISA). RESULTS: Herein, we described that L-MSC cells in vitro-expanded were positive for the expression of vimentin, CD29, CD34, CD39, CD73 and CD105 mesenchymal stem cells markers; meanwhile, this cell population was negative to CD45 and HLA-DR hemopoietic markers as well as to cytokeratin expression. Clonogenic assays showed that these cells were able to form colonies. In addition, this L-MSC population had the ability to transdifferentiate into neurons and chondrocytes and to form tubular networks on matrigel in the presence of vascular endothelial growth factor (VEGF). These results indicated that these cells were stem cells. Additionally, soluble factors secreted by L-MSC were capable of mediating the suppression of T-cell receptor (TCR)-engagement lymphocyte proliferation. In an attempt to identify the possible immunosuppressive factors secreted by L-MSC, TGFß1 and IL-10 cytokines were determined in the L-MSC supernatants by ELISA; interestingly, TGFß1 was constitutively secreted by this cell population; in contrast, IL-10 was not detectable. Moreover, TGFßRII neutralizing antibodies were able to revert the TCR-engagement lymphocyte proliferation inhibition mediated by L-MSC. Thus, TGFß1 secreted by L-MSC was able to suppress T cell proliferation. CONCLUSIONS: Taken together these results, explain in part the immunosuppressive features of this cell population obtained from the human limbus. All these characteristics make this cell population an excellent source to be used in the regenerative medicine.

Human Amniotic Membrane Mesenchymal Stem Cell-Synthesized PGE2 Exerts an Immunomodulatory Effect on Neutrophil Extracellular Trap in a PAD-4-Dependent Pathway through EP2 and EP4.

Human amniotic membrane mesenchymal stem cells (hAM-MSC) secrete a myriad of components with immunosuppressive activities. In the present research, we aimed to describe the effect of prostaglandin E2 (PGE2) secreted by hAM-MSCs on neutrophil extracellular trap (NET) release and to characterize the role of its receptors (EP2/EP4) in PAD-4 and NFκB activity in neutrophils. Human peripheral blood neutrophils were ionomycin-stimulated in the presence of hAM-MSC conditioned medium (CM) treated or not with the selective PGE2 inhibitor MF-63, PGE2, EP2/EP4 agonists, and the selective PAD-4 inhibitor GSK-484. NET release, PAD-4, and NFκB activation were analyzed. Ionomycin induced NET release, which was inhibited in the presence of hAM-MSC-CM, while CM from hAM-MSCs treated with MF-63 prevented NET release inhibition. PGE2 and EP2/EP4 agonists, and GSK-484 inhibited NET release. EP2/EP4 agonists and GSK-484 inhibited H3-citrullination but did not affect PAD-4 protein expression. Finally, PGE2 and EP2/EP4 agonists and GSK-484 increased NFκB phosphorylation. Taken together, these results suggest that hAM-MSC exert their immunomodulatory activities through PGE2, inhibiting NET release in a PAD-4-dependent pathway. This research proposes a new mechanism by which hAM-MSC exert their activities when modulating the innate immune response and inhibiting NET release.

Short-Term Results Analysis in the Allogenic Transplantation of Limbal Stem Cells Expanded on Amniotic Membrane in Patients with Bilateral Limbal Stem Cell Deficiency.

Purpose: The objective of this study was to describe the short-term results of allogenic transplantation of limbal stem cells expanded on amniotic membrane for the ocular surface reconstruction. Methods: Prospective nonrandomized, nonmasked study in a single ophthalmological center. Ten patients with bilateral total limbal stem cell deficiency (LSCD) were included. Expression and presence of ABCB5 and Δp63α in amniotic membrane-cultured limbal epithelial stem cells were analyzed, in relationship with clinical changes after allogenic transplantation. An objective evaluation was performed to determine corneal transparency and superficial vascularization. Results: In a median follow-up time of 11.6 months, 7 patients (70%) were considered as failure compared with the preoperative status. ABCB5 and Δp63α are expressed in similar amount in the limbal epithelial cells expanded in vitro and transplanted in patients with bilateral LSCD. Conclusions: Transplantation of allogenic epithelial limbal cells expanded in amniotic membrane could be considered in patients with LSCD due to burns or congenital etiologies such as aniridia, but its benefit is limited for patients with immunologic diseases.

Neutrophil Extracellular Traps: Current Perspectives in the Eye.

Neutrophil extracellular traps (NETs) have been the subject of research in the field of innate immunity since their first description more than a decade ago. Neutrophils are the first cells recruited at sites of inflammation, where they perform their specific functions, including the release of NETs, which consist of web-like structures composed of granule proteins bound to decondensed chromatin fibres. This process has aroused interest, as it contributes to understanding how pathogenic microorganisms are contained, but they are also associated with pathophysiological processes of a wide range of diseases. Currently, there are growing reports of new molecules involved in the formation and release of NETs. However, whether the release of NETs contributes to eye diseases remains unclear. For this reason, the overall aim of this review is to gather current data of recent research in the ophthalmology field, where there is still much to discover.