Clonación y caracterización de un gen (APA1) implicado en el control de la expresión de la H+-ATPasa de la membrana plasmática de Saccharomyces cerevisiae

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Bioquímica. Fecha de lectura: 4-7-199

Toxicity study in a pig model of intraperitoneal collagenase as an “enzymatic scalpel” directed to break stroma in order to generate a new perspective for peritoneal carcinomatosis approach: an experimental research

Background: This study aimed to measure the toxicity resulting from collagenase administration to the peritoneal cavity in a pig model as a preliminary step to break down the stroma surrounding tumors. Methods: Eight pigs were treated with 2 different collagenase concentrations previously tested in rats by our group. Time and temperature were controlled using a peritoneal lavage system (PRS System, Combat Medical Ltd.) identical to that used in human surgeries through hyperthermic intraperitoneal chemotherapy (HIPEC); 2 additional pigs were treated with peritoneal lavage only. Samples of blood and peritoneal fluid were collected pre-treatment, immediately after treatment, and 24 h postoperatively. In addition, histological studies and blood collagenase levels were measured. Results: No complications were observed during the surgeries. Intraoperative images evidenced the release of peritoneal tissue during collagenase treatment. After surgery, the animals showed no signs of pain. Diet and mobility were normal at 4 h postoperatively, and there were no significant differences in hematologic or biochemical parameters. Quantification of MMP1 and MMP2 in all samples as measured by absorbance showed no differences in blood collagenase levels between pre-treatment, post-treatment, and 24 h postoperatively. None of the animals treated with collagenase showed peritoneal adhesions during the second surgery. Histologically, peritoneal organs and serous structures did not show any microscopic alterations associated with collagenase treatment in any group. Conclusion: Lavage of the peritoneal cavity with doses of up to 100,000 collagen digestion units/animal for 30 min is safe and removes connective tissue from the peritoneal cavity

Oncological transformation in vitro of hepatic progenitor cell lines isolated from adult mice

Colorectal cancer cells can transfer the oncogene KRAS to distant cells, predisposing them to malignant transformation (Genometastasis Theory). This process could contribute to liver metastasis; besides, hepatic progenitor cells (HPCs) have been found to be involved in liver malignant neoplasms. The objective of this study is to determine if mouse HPCs—Oval cells (OCs)—are susceptible to incorporate Kras GAT (G12D) mutation from mouse colorectal cancer cell line CT26.WT and if OCs with the incorporated mutation behave like malignant cells. To achieve this, three lines of OCs in diferent conditions were exposed to CT26.WT cells through transwell co-culture for a week. The presence of KrasG12D and capacity to form tumors were analyzed in treated samples by droplet digital PCR and colony-forming assays, respectively. The results showed that the KrasG12D mutation was detected in hepatic culture conditions of undiferentiated OCs and these cells were capable of forming tumors in vitro. Therefore, OCs are susceptible to malignant transformation by horizontal transfer of DNA with KrasG12D mutation in an undiferentiated condition associated with the liver microenvironment. This study contributes to a new step in the understanding of the colorectal metastatic processTis study was supported by Conchita Rábago Foundation, Madrid, Spain by a doctoral scholarship to Rocío Olivera-Salazar, Instituto de Salud Carlos III (ISCIII), European Regional Development Fund (ERDF) (PI20/01052), Instituto de Salud Carlos III (ISCIII), and Spanish Network of Cell Terapy (TerCel) (RD16/0011/0013)

Gene and Cell Therapy in Dental Tissue Regeneration

Advanced therapies hold substantial promise for the treatment of periodontal conditions. Gene therapy has the potential to transfer “therapeutic” genes, which express proteins such as bone morphogenetic proteins, osteoprotegerin, and tissue nonspecific alkaline phosphatase, which is deficient in patients with hypophosphatasia, a condition that affects mineralization of teeth and bone. Transferred genes may also express platelet-derived growth factor, which modulates the growth of periodontal tissue and the alveolar bone. As regards cell therapy, several clinical trials have shown that mesenchymal stem cells, when used with different kinds of scaffolds to enable the required three-dimensional environment, possess a bone regeneration potential that is particularly useful in such disorders as osteoporosis and osteonecrosis, or for regenerating alveolar bone (osseointegration) prior to placing a dental implant. However, much work is still required before these new therapies become true alternatives in routine clinical dental practice. Medical advances require investments, which are usually influenced by the priorities of both politicians and society at large. This will contribute to promoting innovation, efficient treatments, medium- and long-term savings, and a higher quality of life

Bisphosphonate-related osteonecrosis. Application of adipose-derived stem cells in an experimental murine model

Bisphosphonate-related osteonecrosis of the jaw is a pathological condition without effective established treatment and preventive strategies. The aim of this study was to analyse the effect of adipose-derived stem cells (ASC) in an experimental murine model of osteonecrosis. 38 Wistar rats were injected intraperitoneally with zoledronic acid. After treatment, upper jaw molars were extracted. The animals were randomly assigned to one of two groups. In the control group, saline solution was applied over the alveolar sockets after the tooth extractions. In the treatment group, ASCs were applied instead of saline solution. The control and treatment groups were subdivided based on the time of euthanasia. A clinical and histological analysis was performed. The presence of osteonecrosis in alveolar bone was observed in a similar distribution in both groups. In the ASC-treated group, new bone formation was greater than in controls. In this study, application of ASCs showed greater new bone formation in an osteonecrosis-like murine model. Previous inhibited post-extraction bone remodelling could be reactivated, and these findings appeared to be secondary to implantation of ASCs

Optimization of Mesenchymal Stromal Cell (MSC) Manufacturing Processes for a Better Therapeutic Outcome

MSCs products as well as their derived extracellular vesicles, are currently being explored as advanced biologics in cell-based therapies with high expectations for their clinical use in the next few years. In recent years, various strategies designed for improving the therapeutic potential of mesenchymal stromal cells (MSCs), including pre-conditioning for enhanced cytokine production, improved cell homing and strengthening of immunomodulatory properties, have been developed but the manufacture and handling of these cells for their use as advanced therapy medicinal products (ATMPs) remains insufficiently studied, and available data are mainly related to non-industrial processes. In the present article, we will review this topic, analyzing current information on the specific regulations, the selection of living donors as well as MSCs from different sources (bone marrow, adipose tissue, umbilical cord, etc.), in-process quality controls for ensuring cell efficiency and safety during all stages of the manual and automatic (bioreactors) manufacturing process, including cryopreservation, the use of cell banks, handling medicines, transport systems of ATMPs, among other related aspects, according to European and US legislation. Our aim is to provide a guide for a better, homogeneous manufacturing of therapeutic cellular products with special reference to MSCsThis manuscript has been supported by the Instituto de Salud Carlos III (ISCIII) through the project “RD16/0011: Red de Terapia Celular” (Groups: 0001, 0002, 0004, 0005, 0013, 0015, and 0029), fromthe sub-programme RETICS, integrated in the “Plan Estatal de I+D+I 2013-2016” and co-financed by the European Regional Development Fund (ERDF) “A way to make Europe”, and also by the ISCIII through the project RICORS “RD21/0017;TERAV” (Groups: 001, 002, 003, 006, 009 and 010) that is supported by the Next Generation EU program (Plan de Recuperación, Transformación y Resiliencia); and the Regional Government of Madrid (S2017/BMD-3962, Avancell-CM

Stem cell therapy applied for digestive anastomosis: Current state and future perspectives

Digestive tract resections are usually followed by an anastomosis. Anastomotic leakage, normally due to failed healing, is the most feared complication in digestive surgery because it is associated with high morbidity and mortality. Despite technical and technological advances and focused research, its rates have remained almost unchanged the last decades. In the last two decades, stem cells (SCs) have been shown to enhance healing in animal and human studies; hence, SCs have emerged since 2008 as an alternative to improve anastomoses outcomes. Aim: To summarise the published knowledge of SC utilisation as a preventative tool for hollow digestive viscera anastomotic or suture leaks. Methods: PubMed, Science Direct, Scopus and Cochrane searches were performed using the key words "anastomosis", "colorectal/colonic anastomoses", "anastomotic leak", "stem cells", "progenitor cells", "cellular therapy" and "cell therapy" in order to identify relevant articles published in English and Spanish during the years of 2000 to 2021. Studies employing SCs, performing digestive anastomoses in hollow viscera or digestive perforation sutures and monitoring healing were finally included. Reference lists from the selected articles were reviewed to identify additional pertinent articles.Given the great variability in the study designs, anastomotic models, interventions (SCs, doses and vehicles) and outcome measures, performing a reliable meta-analysis was considered impossible, so we present the studies, their results and limitations. Results: Eighteen preclinical studies and three review papers were identified; no clinical studies have been published and there are no registered clinical trials. Experimental studies, mainly in rat and porcine models and occasionally in very adverse conditions such as ischaemia or colitis, have been demonstrated SCs as safe and have shown some encouraging morphological, functional and even clinical results. Mesenchymal SCs are mostly employed, and delivery routes are mainly local injections and cell sheets followed by biosutures (sutures coated by SCs) or purely topical. As potential weaknesses, animal models need to be improved to make them more comparable and equivalent to clinical practice, and the SC isolation processes need to be standardised. There is notable heterogeneity in the studies, making them difficult to compare. Further investigations are needed to establish the indications, the administration system, potential adjuvants, the final efficacy and to confirm safety and exclude definitively oncological concerns. Conclusion: The future role of SC therapy to induce healing processes in digestive anastomoses/sutures still needs to be determined and seems to be currently far from clinical us

Enhanced anti-inflammatory effects of mesenchymal stromal cells mediated by the transient ectopic expression of CXCR4 and IL10

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliationsBackground Mesenchymal stromal cells (MSCs) constitute one of the cell types most frequently used in cell therapy. Although several studies have shown the efficacy of these cells to modulate inflammation in different animal models, the results obtained in human clinical trials have been more modest. Here, we aimed at improving the therapeutic properties of MSCs by inducing a transient expression of two molecules that could enhance two different properties of these cells. With the purpose of improving MSC migration towards inflamed sites, we induced a transient expression of the C-X-C chemokine receptor type 4 (CXCR4). Additionally, to augment the anti-inflammatory properties of MSCs, a transient expression of the anti-inflammatory cytokine, interleukin 10 (IL10), was also induced. Methods Human adipose tissue-derived MSCs were transfected with messenger RNAs carrying the codon-optimized versions of CXCR4 and/or IL10. mRNA-transfected MSCs were then studied, first to evaluate whether the characteristic phenotype of MSCs was modified. Additionally, in vitro and also in vivo studies in an LPS-induced inflamed pad model were conducted to evaluate the impact associated to the transient expression of CXCR4 and/or IL10 in MSCs. Results Transfection of MSCs with CXCR4 and/or IL10 mRNAs induced a transient expression of these molecules without modifying the characteristic phenotype of MSCs. In vitro studies then revealed that the ectopic expression of CXCR4 significantly enhanced the migration of MSCs towards SDF-1, while an increased immunosuppression was associated with the ectopic expression of IL10. Finally, in vivo experiments showed that the co-expression of CXCR4 and IL10 increased the homing of MSCs into inflamed pads and induced an enhanced anti-inflammatory effect, compared to wild-type MSCs. Conclusions Our results demonstrate that the transient co-expression of CXCR4 and IL10 enhances the therapeutic potential of MSCs in a local inflammation mouse model, suggesting that these mRNA-modified cells may constitute a new step in the development of more efficient cell therapies for the treatment of inflammatory diseasesThis work was supported by the following public grants: Fondo de Investigaciones Sanitarias, Instituto de Salud Carlos III Ministerio de Ciencia, Innovación y Universidades and Fondo Europeo de Desarrollo Regional (FEDER) ((RETICS-RD16/0011/0011, PIE15/00048, PI18-01379), and Dirección General de Investigación de la Comunidad de Madrid (AvanCell-CM; Ref S2017/BMD-3692