Preliminary study on non-viral transfection of F9 (factor IX) gene by nucleofection in human adipose-derived mesenchymal stem cells

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

Células progenitoras multipotentes obtenidas del tejido adiposo y su aplicación clínica

La primera vez que se describió la existencia de células progenitoras en el tejido adiposo subcutáneo fue en el año 2001. Se comunicó la exis- tencia de un grupo de células homogéneas con aspecto fibroblástico, obtenidas a partir de la fracción vásculo-estromal de tejido adiposo ex- traido por liposucción, que tenían la capacidad de diferenciarse a osteo- citos, condriocitos y adipocitos. En el año 2004 fueron definidas como ASC (Adipose-derived Stem Cells) y se establecieron los criterios de identificación: 1. Células con morfología fusiforme que se aíslan median- te digestión enzimática y adhesión a los plásticos. 2. Con capacidad de autorrenovación por largos periodos de tiempo. 3. Multipotenciales (se di- ferencian a adipocitos, condrocitos y osteocitos) y 4. Que tienen el si- guiente perfil de expresión: marcaje positivo para CD29, CD44, CD 90 y CD105; y un marcaje negativo para CD34, CD45 y CD133. Se han descrito más de doscientos ciclos de replicación ex vivo de las ASC sin alteraciones genéticas o fenotípicas. En los últimos años se ha observado la expresión de marcadores de células madre embrionarias en la superficie de las ASC (Oct-4, Rex-1 y Sox-2). También se han descrito algunas vías de señalización, que regulan la proliferación y/o la diferen- ciación celular (Wnt, Esfingosilfosforilcolina y FGF-2). También se ha comunicado diferenciación a músculo esquelético y cardiaco, tejido neu- ronal, endotelio vascular, epitelio, tejido hepático y tejido pancreático. Lo que propone a las ASCs como una de las células más prometedoras para su uso en terapia celular

Liquid biopsy by NGS: Differential presence of exons (DPE) is related to metastatic potential in a colon-cancer model in the rat

Differential presence of exons (DPE) is a method of interpretation of exome sequencing, which has been proposed to design a predictive algorithm with clinical value in patients with colorectal cancer (CRC). The goal of the present study was to examine the reproducibility in a rat model of metastatic colon cancer. DHD/K12-TRb cells were injected in syngenic immunocompetent BD-IX rats. Cells were from two stocks with low and normal metastatic potential, and injected into two separate groups of rats. Five to ten weeks after injection, blood samples were taken prior euthanasia and whole exome sequencing performed. Through DPE analysis, we identified a set of exons whose differential presence in plasma allowed us to compare both groups of tumor-bearing animals. A verification test was performed to confirm that the algorithm was able to classify extracted samples into their corresponding groups of origin. The highest mean probability was 0.8954. In conclusion, the DPE analysis in tumor-bearing animals was able to discriminate between different disease status, which fully supports previous results in CRC patients.This studywas funded by two grants from"Instituto de Salud Carlos III", Spain (FIS; refs. PS09/01815 and PI13/01924

MSC therapy ameliorates experimental gouty arthritis hinting an early COX-2 induction

ObjectiveThe specific effect of Adipose-Derived Mesenchymal Stem Cells (Ad-MSC) on acute joint inflammation, where the response mostly depends on innate immunity activation, remains elusive. The pathogenesis of gouty arthritis, characterized by the deposition of monosodium urate (MSU) crystals in the joints, associated to acute flares, has been associated to NLRP3 inflammasome activation and subsequent amplification of the inflammatory response. Our aim was to study the effect of human Ad-MSC administration in the clinical inflammatory response of rabbits after MSU injection, and the molecular mechanisms involved.MethodsAd-MSC were administered by intraarterial route shortly after intraarticular MSU crystal injections. Joint and systemic inflammation was sequentially studied, and the mechanisms involved in NLRP3 inflammasome activation, and the synthesis of inflammatory mediators were assessed in the synovial membranes 72h after insult. Ad-MSC and THP-1-derived macrophages stimulated with MSU were co-cultured in transwell system.ResultsA single systemic dose of Ad-MSC accelerated the resolution of local and systemic inflammatory response. In the synovial membrane, Ad-MSC promoted alternatively M2 macrophage presence, inhibiting NLRP3 inflammasome and inducing the production of anti-inflammatory cytokines, such as IL-10 or TGF-β, and decreasing nuclear factor-κB activity. Ad-MSC induced a net anti-inflammatory balance in MSU-stimulated THP-1 cells, with a higher increase in IL-10 and IDO expression than that observed for IL-1β and TNF.ConclusionOur in vivo and in vitro results showed that a single systemic dose of Ad-MSC decrease the intensity and duration of the inflammatory response by an early local COX-2 upregulation and PGE2 release. Ad-MSCs suppressed NF-kB activity, NLRP3 inflammasome, and promoted the presence of M2 alternative macrophages in the synovium. Therefore, this therapeutic approach could be considered as a pharmacological alternative in patients with comorbidities that preclude conventional treatment

Recurrent anal fistulae: Limited surgery supported by stem cells

AIM: To study the results of stem-cell therapy under a Compassionate-use Program for patients with recurrent anal fistulae. METHODS: Under controlled circumstances, and approved by European and Spanish laws, a Compassionate-use Program allows the use of stem-cell therapy for patients with very complex anal fistulae. Candidates had previously undergone multiple surgical interventions that had failed to resolve the fistulae, and presented symptomatic recurrence. The intervention consisted of limited surgery (with closure of the internal opening), followed by local implant of stem cells in the fistula- tract wall. Autologous expanded adipose-derived stem cells were the main cell type selected for implant. The first evaluation was performed on the 8th postoperative week; outcome was classified as response or partial response. Evaluation one year after the intervention confirmed if complete healing of the fistula was achieved. RESULTS: Ten patients (8 male) with highly recurrent and complex fistulae were treated (mean age: 49 years, range: 28-76 years). Seven cases were non- Crohn’s fistulae, and three were Crohn’s-associated fistulae. Previous surgical attempts ranged from 3 to 12. Two patients presented with preoperative incontinence (Wexner scores of 12 and 13 points). After the intervention, six patients showed clinical response on the 8th postoperative week, with a complete cessation of suppuration from the fistula. Three patients presented a partial response, with an evident decrease in suppuration. A year later, six patients (60%) remained healed, with complete reepithelization of the external opening. Postoperative Wexner Scores were 0 in six cases. The two patients with previous incontinence improved their scores from 12 to 8 points and from 13 to 5 points. No adverse reactions or complications related to stem-cell therapy were reported during the study period. CONCLUSION: Stem cells are safe and useful for treating anal fistulae. Healing can be achieved in severe cases, sparing fecal incontinence risk, and improving previous scorin

The role of mucin cell-free DNA detection as a new marker for the study of acellular pseudomyxoma peritonei of appendicular origin by liquid biopsy

Background: Acellular pseudomyxoma peritonei (aPMP) is a rare peritoneal malignancy characterized by the accumulation of large amounts of mucin (lacking tumor cells) in the peritoneum. Many cases account for several kilograms of mucin to be screened by the pathologist. This is a comprehensive study of three patients with aPMP, whose tumors showed KRAS mutation, allowing for the tracking of this marker by liquid biopsy. Methods: Pre and post-surgery plasma, and mucin removed during cytoreductive surgery were collected from the patients. KRAS mutations were analyzed using droplet digital polymerase chain reaction (ddPCR). Mucin was injected in mice. KRAS and cytokine levels were measured in plasma of the mice using ddPCR and a magnetic bead-based assay. Mucin microbiome was analyzed by 16S rRNA sequencing. Results: KRAS mutations were detected in mucin cell-free DNA (cfDNA) from the three patients but not in the pre or post-surgery plasma. Electron microscopy detected microparticles (diameter <0.4 µm) in mucin. Mucin from one patient grew up inside the peritoneal cavity of mice and human KRAS was identified in mucin cfDNA, but not in plasma. All mucins showed the same bacterial profile. Cytokine levels were slightly altered in mice. Conclusions: The three aPMP patients included in this study shared some common aspects: the absence of tumor cells in mucin, the presence of KRAS mutated cfDNA in mucin, and the absence of this tumor-derived mutation in the bloodstream, providing additional information to the routine pathological examinations and suggesting that mucin cfDNA could potentially play a role in aPMP recurrence and prognosis.The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was funded by a grant from the ‘FIS-ISCIII-FEDER’ [Fondo de Investigaciones Sanitarias-Instituto de Salud Carlos III-Fondo Europeo de Desarrollo Regional (European Regional Development Fund)], Ministry of Health, Spain (grant number PI17/01233)