Teaching innovation experiences in Biomedicine

254 p.La innovación docente en las aulas se ha convertido en un factor esencial en la docencia universitaria y uno de los más claros indicadores de calidad. Sin embargo, la multiplicidad de funciones docentes, de investigación y de gestión a las que tiene que hacer frente en el día a día el profesorado hace que ésta sea una tarea titánica. Con el fin de visibilizar la labor en innovación del profesorado universitario surgió la idea de editar este libro que reúne experiencias e investigaciones docentes en el área de la Biomedicina. La propuesta se hizo a las personas participantes en las IX Jornadas de Docencia en Biología Celular, celebradas en octubre de 2018 en Bilbao, organizadas por la Sociedad Española de Biología Celular (SEBC) en colaboración con la Facultad de Medicina y Enfermería de la Universidad del País Vasco UPV/EHU. Así, el presente libro digital contiene casi una veintena de capítulos que incluyen artículos y revisiones sobre diversos temas. Entre ellos no podía faltar la microscopía; pero el libro incluye también implementación de diversas metodologías docentes, aprendizaje activo, uso de recursos TICs, gamificación, etc

Transscleral Cyclophotocoagulation for the Treatment of Uncontrolled Glaucoma in a Boston Keratoprosthesis Type II Patient

[EN] Postoperative endoscopic cyclophotocoagulation (CPC) for the treatment of glaucoma in patients with Boston keratoprosthesis type II (BKPro II) was first described in 2017 by Poon et al. (Endoscopic cyclophotocoagulation for the treatment of glaucoma in Boston keratoprosthesis type ii patient. J Glaucoma. 2017 Apr;26(4):e146-9). As we do not have this device, we present a case of transscleral CPC (TSCPC), in a BKPro II patient who had graft versus host disease and developed uncontrolled glaucoma. We dissected plane by plane to expose the bare sclera and performed the procedure as traditionally described. We concluded that this is a safe, controlled, and effective option in this patient population where the glaucoma treatment options are very limited. To the best of our knowledge, this is the first case report to describe the surgical technique of TSCPC in a BKPro II patient

Expansion of Human Limbal Epithelial Stem/Progenitor Cells Using Different Human Sera: A Multivariate Statistical Analysis

Transplantation of human cultured limbal epithelial stem/progenitor cells (LESCs) has demonstrated to restore the integrity and functionality of the corneal surface in about 76% of patients with limbal stem cell deficiency. However, there are different protocols for the expansion of LESCs, and many of them use xenogeneic products, being a risk for the patients’ health. We compared the culture of limbal explants on the denuded amniotic membrane in the culture medium—supplemental hormone epithelial medium (SHEM)—supplemented with FBS or two differently produced human sera. Cell morphology, cell size, cell growth rate, and the expression level of differentiation and putative stem cell markers were examined. Several bioactive molecules were quantified in the human sera. In a novel approach, we performed a multivariate statistical analysis of data to investigate the culture factors, such as differently expressed molecules of human sera that specifically influence the cell phenotype. Our results showed that limbal cells cultured with human sera grew faster and contained similar amounts of small-sized cells, higher expression of the protein p63α, and lower of cytokeratin K12 than FBS cultures, thus, maintaining the stem/progenitor phenotype of LESCs. Furthermore, the multivariate analysis provided much data to better understand the obtaining of different cell phenotypes as a consequence of the use of different culture methodologies or different culture components.Research study supported by grants from Mutua Madrileña Foundation (FMM11/02), Basque Foundation for Health Research and Innovation BIOEF (BIO14/TP/002), National Eye Institute (R01EY021797), California Institute for Regenerative Medicine (TR-TR2-01768 and CLIN1-08686) and an unrestricted grant from Research to Prevent Blindness to the Department of Ophthalmology, University of California, Los Angeles. R.H.-M. was supported by fellowships from the University of the Basque Country UPV/EHU and the Jesus de Gangoiti Barrera foundation

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.This research study was supported by grants from the Department of Heath of the Basque Government (RIS3, 2019222049), the University of the Basque Country UPV/EHU-Instituto Clinico Quirurgico de Oftalmologia ICQO (US19/18), and MCI/AEI/FEDER, UE (grant number RTI2018-097100-B-C22). C.R.-V. was supported by a fellowship from the University of the Basque Country UPV/EHU

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, p63 alpha, ABCG2, Ki67, Integrin beta 4, 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 p63 alpha 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.This research study has been funded by grants from the Department of Heath of the Basque Government (RIS3, 2019222049) and the University of the Basque Country UPV/EHU-Instituto Clinico Quirurgico de Oftalmologia ICQO (US19/18). C. R-V. has received fellowship support from the University of the Basque Country UPV/EH

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.This work was fully supported by a collaborative fundamental research grant from the Basque Government Elkartek program under grant nº. B4H KK-2019-0006-BC

Pirin is a prognostic marker of human melanoma that dampens the proliferation of malignant cells by downregulating JARID1B/KDM5B expression

Originally considered to act as a transcriptional co‑factor, Pirin has recently been reported to play a role in tumorigenesis and the malignant progression of many tumors. Here, we have analyzed the diagnostic and prognostic value of Pirin expression in the early stages of melanoma, and its role in the biology of melanocytic cells. Pirin expression was analyzed in a total of 314 melanoma biopsies, correlating this feature with the patient’s clinical course. Moreover, PIR downregulated primary melanocytes were analyzed by RNA sequencing, and the data obtained were validated in human melanoma cell lines overexpressing PIR by functional assays. The immunohistochemistry multivariate analysis revealed that early melanomas with stronger Pirin expression were more than twice as likely to develop metastases during the follow‑up. Transcriptome analysis of PIR downregulated melanocytes showed a dampening of genes involved in the G1/S transition, cell proliferation, and cell migration. In addition, an in silico approach predicted that JARID1B as a potential transcriptional regulator that lies between PIR and its downstream modulated genes, which was corroborated by co‑transfection experiments and functional analysis. Together, the data obtained indicated that Pirin could be a useful marker for the metastatic progression of melanoma and that it participates in the proliferation of melanoma cells by regulating the slow‑cycling JARID1B gene.This project was supported by grants from the Basque Government (KK2017-041 and KK2020-00069 to M.D.B.), the UPV/EHU (GIU17/066 to M.D.B.), H2020-ESCEL JTI (15/01 to M.D.B.) and MINECO (PCIN-2015-241 to M.D.B.). CP holds a predoctoral fellowship from the Basque Government. Part of this project is under European patent No. EP3051291 (EP14796149.4): “Method for diagnosis and prognosis of cutaneous melanoma”, Univer- sity of the Basque Country (UPV/EHU). The authors acknowledge the technical support SGIker resources at the UPV/EHU for the computational calculations, which were carried out in the Arina Informatics Cluster. The authors are grateful to the Basque Biobank for providing the biopsy samples and in particular, to María Jesús Fernández and Arantza Perez Dobaran for their technical support with the immunohistochemistry

Teaching innovation experiences in Biomedicine

254 p.La innovación docente en las aulas se ha convertido en un factor esencial en la docencia universitaria y uno de los más claros indicadores de calidad. Sin embargo, la multiplicidad de funciones docentes, de investigación y de gestión a las que tiene que hacer frente en el día a día el profesorado hace que ésta sea una tarea titánica. Con el fin de visibilizar la labor en innovación del profesorado universitario surgió la idea de editar este libro que reúne experiencias e investigaciones docentes en el área de la Biomedicina. La propuesta se hizo a las personas participantes en las IX Jornadas de Docencia en Biología Celular, celebradas en octubre de 2018 en Bilbao, organizadas por la Sociedad Española de Biología Celular (SEBC) en colaboración con la Facultad de Medicina y Enfermería de la Universidad del País Vasco UPV/EHU. Así, el presente libro digital contiene casi una veintena de capítulos que incluyen artículos y revisiones sobre diversos temas. Entre ellos no podía faltar la microscopía; pero el libro incluye también implementación de diversas metodologías docentes, aprendizaje activo, uso de recursos TICs, gamificación, etc