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(1) Background: Abdominal adhesions are a common disease appearing after any type of abdominal surgery and may prolong surgical time and cause intestinal obstruction, infertility, or chronic pain. We propose the use of intraperitoneal collagenase to perform chemical adhesiolysis based on the pathophysiology and histology of adhesions. (2) Methods: We generated an adhesion model with intraperitoneal polypropylene meshes. Four months later, we evaluated the efficacy of the treatment in blinded form, i.e., 0.05% collagenase vs. placebo at 37 °C for 20 min. Protocol 1: Ten rats with ten mesh fragments, in which an attempt was made to remove the maximum number of meshes in a 5-min period. Protocol 2: Six rats with four mesh fragments in the sides of the abdominal cavity in which adhesiolysis was performed using a device that measures burst pressure. (3) Results: Protocol 1: 42% efficacy in the collagenase group versus 8% in the control group (p < 0.013). Protocol 2: 188.25 mmHg (SD 69.65) in the collagenase group vs. 325.76 mmHg (SD 50.25) in the control group (p < 0.001). (4) Conclusions: Collagenase allows for the safe and effective chemical adhesiolysis in this experimental model of adhesions.

Mesenchymal stromal cells (MSCs) currently constitute the most frequently used cell type in advanced therapies with different purposes, most of which are related with inflammatory processes. Although the therapeutic efficacy of these cells has been clearly demonstrated in different disease animal models and in numerous human phase I/II clinical trials, only very few phase III trials using MSCs have demonstrated the expected potential therapeutic benefit. On the other hand, diverse controversial issues on the biology and clinical applications of MSCs, including their specific phenotype, the requirement of an inflammatory environment to induce immunosuppression, the relevance of the cell dose and their administration schedule, the cell delivery route (intravascular/systemic vs. local cell delivery), and the selected cell product (i.e., use of autologous vs. allogeneic MSCs, freshly cultured vs. frozen and thawed MSCs, MSCs vs. MSC-derived extracellular vesicles, etc.) persist. In the current review article, we have addressed these issues with special emphasis in the new approaches to improve the properties and functional capabilities of MSCs after distinct cell bioengineering strategies.

Mesenchymal stromal cells (MSCs) constitute the cell type more frequently used in many regenerative medicine approaches due to their exclusive immunomodulatory properties, and they have been reported to mediate profound immunomodulatory effects in vivo. Nevertheless, MSCs do not express essential adhesion molecules actively involved in cell migration, a phenotypic feature that hampers their ability to home inflamed tissues following intravenous administration. In this study, we investigated whether modification by fucosylation of murine AdMSCs (mAdMSCs) creates Hematopoietic Cell E-/L-selectin Ligand, the E-selectin-binding CD44 glycoform. This cell surface glycan modification of CD44 has previously shown in preclinical studies to favor trafficking of mAdMSCs to inflamed or injured peripheral tissues. We analyzed the impact that exofucosylation could have in other innate phenotypic and functional properties of MSCs. Compared to unmodified counterparts, fucosylated mAdMSCs demonstrated higher in vitro migration, an altered secretome pattern, including increased expression and secretion of anti-inflammatory molecules, and a higher capacity to inhibit mitogen-stimulated splenocyte proliferation under standard culture conditions. Together, these findings indicate that exofucosylation could represent a suitable cell engineering strategy, not only to facilitate the in vivo MSC colonization of damaged tissues after systemic administration, but also to convert MSCs in a more potent immunomodulatory/anti-inflammatory cell therapy-based product for the treatment of a variety of autoimmune, inflammatory, and degenerative diseases.

Background: To examine the usefulness of green fluorescent protein (GFP) mice for studying the interactions between normal cells and tumor cells in a host, we used a melanoma model in such “green” mice [C57BL/6-Tg(CAG-EGFP)1Osb mice]. Mice were given a subcutaneous injection of B16-F10 cells, and the resultant primary tumors were removed. Then cells from individual tumors were cultured. Results: The proportion of EFGP+ cells was determined by fluorescence-activated cell sorting (FACS) and was 6.8% ± 3.2% (mean ± s.d.) on day 1 of culture, 0.6% ± 0.3% on day 2, and 0.02% ± 0.01% at day 7. In all cases, isolated cells grew at a constant rate, but fluorescence decreased over time and became undetectable on day 14. Cells were tested using PCR for the presence of an EGFP-specific sequence, and results were negative in all cases, thus indicating that the cells did not harbor the host's reporter gene. Cells were also tested for the presence of EGFP mRNA, which was consistently detected for 22 days after the start of culture. The tumorogenicity of the cultured cells was confirmed in GFP mice injected with cells from a selection of cultures. Conclusions: In a melanoma model in GFP mice, the detection of “green” cells in tumors was not equivalent to the detection of host-derived cells. Such “masking” was caused by a transient, but lasting, transfer of EGFP mRNA from the host's normal cells to tumor cells. Thus, an analysis of tumors postmortem by techniques that yield only a single snapshot can lead to incorrect interpretations and erroneous conclusions.How to cite: Garcia-Olmo D, Picazo MG, García-Olmo D. Melanoma transplants in “green” mice: fluorescent cells in tumors are not equivalent to host-derived cells. Electron J Biotechnol 2018;34. https://doi.org/10.1016/j.ejbt.2018.04.007. Keywords: Cancer models, Development of tumors, EGFP, GFP mice, Green-fluorescent cells, Melanoma, Mouse, Oncogenic transformation

Summary: Most colorectal cancer (CRC)-related deaths are due to liver metastases. PKCζ is a tumor suppressor in CRC with reduced expression in metastasis. Given the importance of microRNAs (miRNAs) in regulating cellular plasticity, we performed an unbiased screening and identified the miR-200 family as the most relevant miRNAs downregulated by PKCζ deficiency. The regulation of the intracellular levels of miR-200 by PKCζ is post-transcriptional and involves their secretion in extracellular vesicles. Here, we identified ADAR2 as a direct substrate of PKCζ in CRC cells. Phosphorylation of ADAR2 regulates its editing activity, which is required to maintain miR-200 steady-state levels, suggesting that the PKCζ/ADAR2 axis regulates miR-200 secretion through RNA editing. Loss of this axis results in epithelial-to-mesenchymal transition (EMT) and increased liver metastases, which can be inhibited in vivo by blocking miR-200 release. Therefore, the PKCζ/ADAR2 axis is a critical regulator of CRC metastases through modulation of miR-200 levels. : Shelton et al. demonstrate that the loss of the tumor suppressor PKCζ in colorectal cancer cells results in the downregulation of miR-200, leading to increased epithelial-to-mesenchymal transition, cell invasion, and liver metastasis. This is mediated by an increase in miR-200 secretion through the inactivation of the RNA editing enzyme ADAR2, identifying a key vulnerability in liver metastasis. Keywords: PRKCZ, atypical PKC, ADAR2, colorectal cancer, metastasis, EMT, mir-200, extracellular vesicles, tumor suppressors, RNA editing

Myocardial infarction has been carefully studied in numerous experimental models. Most of these models are based on electrophysiological and functional data, and pay less attention to histological discoveries. During the last decade, treatment using advanced therapies, mainly cell therapy, has prevailed from among all the options to be studied for treating myocardial infarction. In our study we wanted to show the fundamental histological parameters to be evaluated during the development of an infarction on an experimental model as well as treatment with mesenchymal stem cells derived from adipose tissue applied intra-lesionally. The fundamental parameters to study in infarcted tissue at the histological level are the cells involved in the inflammatory process (lymphocytes, macrophages and M2, neutrophils, mast cells and plasma cells), neovascularization processes (capillaries and arterioles) and cardiac cells (cardiomyocytes and Purkinje fibers). In our study, we used intramyocardial injection of mesenchymal stem cells into the myocardial infarction area 1 hour after arterial occlusion and allowed 1 month of evolution before analyzing the modifications on the normal tissue inflammatory infiltrate. Acute inflammation was shortened, leading to chronic inflammation with abundant plasma cells and mast cells and complete disappearance of neutrophils. Another benefit was an increase in the number of vessels formed. Cardiomyocytes and Purkinje fibers were better conserved, both from a structural and metabolic point of view, possibly leading to reduced morbidity in the long term. With this study we present the main histological aspects to be evaluated in future assays, complementing or explaining the electrophysiological and functional findings.

Background Diabetes is one of the major risk factors for peripheral arterial disease. In patients in whom surgery cannot be performed, cell therapy may be an alternative treatment. Since time is crucial for these patients, we propose the use of allogenic mesenchymal cells. Methods We obtained mesenchymal cells derived from the fat tissue of a healthy Sprague-Dawley rat. Previous diabetic induction with streptozotocin in 40 male Sprague-Dawley rats, ligation plus left iliac and femoral artery sections were performed as a previously described model of ischemia. After 10 days of follow-up, macroscopic and histo-pathological analysis was performed to evaluate angiogenic and inflammatory parameters in the repair of the injured limb. All samples were evaluated by the same blind researcher. Statistical analysis was performed using the SPSS v.11.5 program (P

Background. Hemophilia is a rare recessive X-linked disease characterized by a deficiency of coagulation factor VIII or factor IX. Its current treatment is merely palliative. Advanced therapies are likely to become the treatment of choice for the disease as they could provide a curative treatment. Methods. The present study looks into the use of a safe non-viral transfection method based on nucleofection to express and secrete human clotting factor IX (hFIX) where human adipose tissue derived mesenchymal stem cells were used as target cells in vitro studies and NOD. Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice were used to analyze factor IX expression in vivo studies. Previously, acute liver injury was induced by an injected intraperitoneal dose of 500 mg/kg body weight of acetaminophen. Results. Nucleofection showed a percentage of positive cells ranging between 30.7% and 41.9% and a cell viability rate of 29.8%, and cells were shown to secrete amounts of hFIX between 36.8 and 71.9 ng/mL. hFIX levels in the blood of NSG mice injected with ASCs transfected with this vector, were 2.7 ng/mL 48 h after injection. Expression and secretion of hFIX were achieved both in vitro cell culture media and in vivo in the plasma of mice treated with the transfected ASCs. Such cells are capable of eventually migrating to a previously damaged target tissue (the liver) where they secrete hFIX, releasing it to the bloodstream over a period of at least five days from administration. Conclusions. The results obtained in the present study may form a preliminary basis for the establishment of a future ex vivo non-viral gene/cellular safe therapy protocol that may eventually contribute to advancing the treatment of hemophilia.