Characterisation of corneas following different time and storage methods for their use as a source of stem-like limbal epithelial cells.

The transplantation of expansions of limbal epithelial stem cells (LESC) remains one of the most efficient therapies for the treatment of limbal stem cell deficiency (LSCD) to date. However, the available donor corneas are scarce, and the corneas conserved for long time, under hypothermic conditions (after 7 days) or in culture (more than 28 days), are usually discarded due to poor viability of the endothelial cells. To establish an objective criterion for the utilisation or discarding of corneas as a source of LESC, we characterized, by immunohistochemistry analysis, donor corneas conserved in different conditions and for different periods of time. We also studied the potency of LESCs isolated from these corneas and maintained in culture up to 3 cell passages. We hoped that the study of markers of LESCs present in both the corneoscleral histological sections and the cell cultures would show the adequacy of the methods used for cell isolation and how fit the LESC enrichment of the obtained cell populations to be expanded was. Thus, the expressions of markers of the cells residing in the human limbal and corneal epithelium (cytokeratin CK15 and CK12, vimentin, Collagen VII, p63α, ABCG2, Ki67, Integrin ß4, ZO1, and melan A) were analysed in sections of corneoscleral tissues conserved in hypothermic conditions for 2-9 days with post-mortem time (pmt) < 8 h or for 1 day with pmt > 16 h, and in sclerocorneal rims maintained in an organ culture medium for 29 days. Cell populations isolated from donor corneoscleral tissues were also assessed based on these markers to verify the adequacy of isolation methods and the potential of expanding LESCs from these tissues. Positivity for several putative stem cell markers such as CK15 and p63α was detected in all corneoscleral tissues, although a decrease was recorded in the ones conserved for longer times. The barrier function and the ability to adhere to the extracellular matrix were maintained in all the analysed tissues. In limbal epithelial cell cultures, a simultaneous decrease in the melan A melanocyte marker and the putative stem cell markers was detected, suggesting a close relationship between the melanocytes and the limbal stem cells of the niche. Holoclones stained with putative stem cell markers were obtained from long-term, hypothermic, stored sclerocorneal rims. The results showed that the remaining sclerocorneal rims after corneal transplantation, which were conserved under hypothermic conditions for up to 7 days and would have been discarded at a first glance, still maintained their potential as a source of LESC cultures.

Plasma-Based Bioinks for Extrusion Bioprinting of Advanced Dressings.

Extrusion bioprinting based on the development of novel bioinks offers the possibility of manufacturing clinically useful tools for wound management. In this study, we show the rheological properties and printability outcomes of two advanced dressings based on platelet-rich plasma (PRP) and platelet-poor plasma (PPP) blended with alginate and loaded with dermal fibroblasts. Measurements taken at 1 h, 4 days, and 18 days showed that both the PRP- and PPP-based dressings retain plasma and platelet proteins, which led to the upregulation of angiogenic and immunomodulatory proteins by embedded fibroblasts (e.g., an up to 69-fold increase in vascular endothelial growth factor (VEGF), an up to 188-fold increase in monocyte chemotactic protein 1 (MCP-1), and an up to 456-fold increase in hepatocyte growth factor (HGF) 18 days after printing). Conditioned media harvested from both PRP and PPP constructs stimulated the proliferation of human umbilical vein endothelial cells (HUVECs), whereas only those from PRP dressings stimulated HUVEC migration, which correlated with the VEGF/MCP-1 and VEGF/HGF ratios. Similarly, the advanced dressings increased the level of interleukin-8 and led to a four-fold change in the level of extracellular matrix protein 1. These findings suggest that careful selection of plasma formulations to fabricate wound dressings can enable regulation of the molecular composition of the microenvironment, as well as paracrine interactions, thereby improving the clinical potential of dressings and providing the possibility to tailor each composition to specific wound types and healing stages.

Cytocompatibility and Suitability of Protein-Based Biomaterials as Potential Candidates for Corneal Tissue Engineering.

The vision impairments suffered by millions of people worldwide and the shortage of corneal donors show the need of substitutes that mimic native tissue to promote cell growth and subsequent tissue regeneration. The current study focused on the in vitro assessment of protein-based biomaterials that could be a potential source for corneal scaffolds. Collagen, soy protein isolate (SPI), and gelatin films cross-linked with lactose or citric acid were prepared and physicochemical, transmittance, and degradation measurements were carried out. In vitro cytotoxicity, cell adhesion, and migration studies were performed with human corneal epithelial (HCE) cells and 3T3 fibroblasts for the films' cytocompatibility assessment. Transmittance values met the cornea's needs, and the degradation profile revealed a progressive biomaterials' decomposition in enzymatic and hydrolytic assays. Cell viability at 72 h was above 70% when exposed to SPI and gelatin films. Live/dead assays and scanning electron microscopy (SEM) analysis demonstrated the adhesion of both cell types to the films, with a similar arrangement to that observed in controls. Besides, both cell lines were able to proliferate and migrate over the films. Without ruling out any material, the appropriate optical and biological properties shown by lactose-crosslinked gelatin film highlight its potential for corneal bioengineering.

Nucleoporins redistribute inside the nucleus after cell cycle arrest induced by histone deacetylases inhibition.

Nucleoporins are the main components of the nuclear-pore complex (NPC) and were initially considered as mere structural elements embedded in the nuclear envelope, being responsible for nucleocytoplasmic transport. Nevertheless, several recent scientific reports have revealed that some nucleoporins participate in nuclear processes such as transcription, replication, DNA repair and chromosome segregation. Thus, the interaction of NPCs with chromatin could modulate the distribution of chromosome territories relying on the epigenetic state of DNA. In particular, the nuclear basket proteins Tpr and Nup153, and the FG-nucleoporin Nup98 seem to play key roles in all these novel functions. In this work, histone deacetylase inhibitors (HDACi) were used to induce a hyperacetylated state of chromatin and the behavior of the mentioned nucleoporins was studied. Our results show that, after HDACi treatment, Tpr, Nup153 and Nup98 are translocated from the nuclear pore toward the interior of the cell nucleus, accumulating as intranuclear nucleoporin clusters. These transitory structures are highly dynamic, and are mainly present in the population of cells arrested at the G0/G1 phase of the cell cycle. Our results indicate that the redistribution of these nucleoporins from the nuclear envelope to the nuclear interior may be implicated in the early events of cell cycle initialization, particularly during the G1 phase transition.

Histone deacetylase inhibitors induce invasion of human melanoma cells in vitro via differential regulation of N-cadherin expression and RhoA activity.

BACKGROUND: Histone deacetylase inhibitors (HDACi) exert multiple cytotoxic actions on cancer cells. Currently, different synthetic HDACi are in clinical use or clinical trials; nevertheless, since both pro-invasive and anti-invasive activities have been described, there is some controversy about the effect of HDACi on melanoma cells. METHODS: Matrigel and Collagen invasion assays were performed to evaluate the effect of several HDACi (Butyrate, Trichostatin A, Valproic acid and Vorinostat) on two human melanoma cell line invasion (A375 and HT-144). The expression of N- and E-Cadherin and the activity of the RhoA GTPase were analyzed to elucidate the mechanisms involved in the HDACi activity. RESULTS: HDACi showed a pro-invasive effect on melanoma cells in vitro. This effect was accompanied by an up-regulation of N-cadherin expression and an inhibition of RhoA activity. Moreover, the down-regulation of N-cadherin through blocking antibodies or siRNA abrogated the pro-invasive effect of the HDACi and, additionally, the inhibition of the Rho/ROCK pathway led to an increase of melanoma cell invasion similar to that observed with the HDACi treatments. CONCLUSION: These results suggest a role of N-cadherin and RhoA in HDACi induced invasion and call into question the suitability of some HDACi as antitumor agents for melanoma patients.

The spermatogonial stem cell niche in testicular germ cell tumors.

Spermatogonial stem cells (SSCs) are pluripotent elements found in the adult seminiferous epithelium between Sertoli cells and a basal lamina which covers the multilayered external wall of peritubular myoid cells. The microenvironment of this pluripotent stem cell niche creates the complex and dynamic system that is necessary for the initiation of spermatogenesis, but this system also contains factors which can potentially collaborate in the progression of testicular germ cell tumors (TGCTs). In this review, we summarize our current knowledge about some important structural and molecular features related to the SSC niche, including growth factors, adhesion molecules, extracellular matrix, mechanical stress and vascularization. We discuss their possible collaborative effects on the generation and progression of TGCTs, which are a type of cancer representing the most frequent neoplasia among young men and whose incidence has grown very quickly during the past decades in North America and Europe. In this regard, a better understanding of the pluripotent stem cell niche where these malignancies arise will provide further insights into the origin of TGCTs and the mechanisms underlying their growth and invasion of adjacent and distant tissues.

Embryonic stem cell transplantation into seminiferous tubules: a model for the study of invasive germ cell tumors of the testis.

Over the last 15 years, cell transplantation into seminiferous tubules has become a valuable tool to study germinal cell biology and related matters. This is particularly so, because the blood-testis permeability barrier establishes a sealed compartment which protect against certain influences such as immunological rejection. In the light of the functional and genetic similarities between carcinoma in situ (CIS) of the testis and embryonic stem (ES) cells, our laboratory has developed a tumor assay to study cancer invasion processes in testicular germ cell tumors (TGCT) based on the transplantation of ES cells into the seminiferous tubules. Here, we describe this new tumor assay and provide additional information regarding the transplantation techniques used and their application for the study of TGCTs. Finally, we discuss the practical implications of our experimental approach and its potential application for the understanding of TGCT invasive processes and the development of new antineoplastic strategies.

Reprogramming of melanoma cells by embryonic microenvironments.

In recent years, the reversion of the cancer phenotype of human melanoma cells in developing zebrafish and chick embryos has been reported. The aim of this review is to revise these and other related contributions regarding the regulation of embryonic cancer and to provide a framework with which to understand results from our laboratory on the interactions of human melanoma cells with post-implanted mouse embryos cultured in vitro. To this end, we used the A375 human melanoma cell line transfected with the green fluorescent protein (GFP) gene. Labeled cells were transplanted onto the surface of the developing visceral endoderm of 7.5 dpc mouse embryos. Subsequently, we cultured the transplanted embryos for three days and monitored the movements of GFP labeled human melanoma cells by confocal microscopy. Our results show that ectopic melanoma cells internalize and migrate inside the embryo body in a way reminiscent of neural crest cells. The absence of localized tumor growth after 72 hours of in vitro embryo co-culture suggests that malignant phenotype inhibiting factors are active at the gastrulating stage and during early organogenesis. These results complement previous reports of growth regulation of B16 mouse melanoma cells by 10 dpc mouse embryonic skin (Gerschenson et al., 1986). Further research is required to elucidate the final fate of melanoma cells in mammalian embryos and the details of the signaling pathways underlying tumor growth regulation. Understanding regulation of melanoma cells by young embryos could represent a starting point for a developmental theory of the pathogenesis of melanoma, and for future developments of more physiologically-based anticancer therapies for this and indeed, other types of aggressive tumor.

Hypoxia and pluripotency in embryonic and embryonal carcinoma stem cell biology.

Low oxygen availability (hypoxia) is a hallmark of rapidly proliferating tumors and has been suggested to be a characteristic of the embryonic and adult stem cell niche. The idea of relating cancer to stem cells is increasingly popular due to the identification of specific cancer stem cells sharing the typical plasticity and motility of pluripotent stem cells. Hypoxia plays a critical role in early embryonic development and in tumor progression, participating in processes such as angiogenesis, apoptosis, cell migration, invasion and metastasis. Some of the molecular pathways that have been shown to mediate these hypoxia-induced responses, such as the hypoxia inducible factor (HIF)-1alpha and Notch signaling, appear to be active in both embryonic and neoplastic pluripotent stem cells. Nevertheless, the mechanisms underlying these regulatory processes are not yet fully understood. In this review, we attempt to shed some light on the mechanisms involved in hypoxia-dependent processes related to stem cell features and tumor progression, such as the maintenance of the undifferentiated state, cell proliferation, tumor neovascularization, extra-cellular matrix degradation and motility factor up-regulation. With this purpose in mind, we summarize recent observations in embryonic, adult and cancer stem cells that demonstrate the parallelism existing in their hypoxia responses. Finally, based on the observations of our own laboratory and others, we suggest that the comparative analysis of the response to low oxygen levels of embryonic stem cells and cancer stem cells (such as embryonal carcinoma cells), may throw fresh light on our understanding of the mechanisms underlying hypoxia-induced invasiveness and the resistance to anticancer treatments, thereby stimulating the development of novel therapeutic strategies.

Plasma membrane and nuclear envelope integrity during the blebbing stage of apoptosis: a time-lapse study.

BACKGROUND INFORMATION: The execution phase of apoptosis is characterized by extensive blebbing of the plasma membrane, which usually results in secondary lysis in vitro. To analyse the permeability of cellular membranes during this process, we induced apoptosis in human melanoma A375 cells that had been transfected with fluorescently tagged proteins which were targeted to different subcellular locations. RESULTS: The dual treatment of resveratrol and butyrate produced a synergistic induction of apoptosis by blocking different phases of the cell cycle. Changes in the plasma membrane, nuclear envelope and nucleoli were monitored by time-lapse confocal microscopy. Fluorescently labelled proteins were not mis-localized from their original locations in any of the cells undergoing blebbing for several hours. Thus the maintenance of karyophilic and nucleolar proteins within the nucleus during the blebbing stage and the accessibility of vital selective chromatin dyes confirmed a functional preservation of the nuclear compartment until the final necrotic blister. The translocation of phosphatidylserine to the outer leaflet of the plasma membrane was not detected during the blebbing period. CONCLUSION: These results show that the functional integrity of the nuclear envelope and plasma membrane may be conserved until the end of the execution phase of apoptosis.