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BCR-ABL1/ABL1 p210 measurement by quantitative polymerase chain reaction (qPCR) is used worldwide to monitor the molecular response in chronic myeloid leukemia (CML) patients. Droplet digital polymerase chain reaction (ddPCR) seems to show a greater sensitivity than qPCR, probably due to the high number of replicates analyzed in ddPCR for the comparison. Additionally, in a recently published comparison, ddPCR measurements were not adequately transformed into International Scale (IS). We have analyzed 50 CML patients and ten non-CML donors in parallel by qPCR and ddPCR. To the best of our knowledge, this is the first study comparing both techniques under similar conditions, with BCR-ABL1/ABL1 measurements performed via both techniques transformed into IS. Qualitative and quantitative comparisons showed excellent results. The qualitative correlation showed a Kappa index of 0.94 (95% confidence interval [CI] 0.90–0.98) (P

We evaluated the safety and feasibility of adipose‐derived mesenchymal stem cells to treat endoscopically urinary incontinence after radical prostatectomy in men or female stress urinary. We designed two prospective, nonrandomized phase I‐IIa clinical trials of urinary incontinence involving 9 men (8 treated) and 10 women to test the feasibility and safety of autologous mesenchymal stem cells for this use. Cells were obtained from liposuction containing 150 to 200 g of fat performed on every patient. After 4 to 6 weeks and under sedation, endoscopic intraurethral injection of the cells was performed. On each visit (baseline, 1, 3, 6, and 12 months), clinical parameters were measured, and blood samples, urine culture, and uroflowmetry were performed. Every patient underwent an urethrocystoscopy and urodynamic studies on the first and last visit. Data from pad test, quality‐of‐life and incontinence questionnaires, and pads used per day were collected at every visit. Statistical analysis was done by Wilcoxon signed‐rank test. No adverse effects were observed. Three men (37.5%) and five women (50%) showed an objective improvement of >50% (P, We evaluated the safety and feasibility of adipose‐derived mesenchymal stem cells (MSCs) to treat endoscopically urinary incontinence after radical prostatectomy in men or women stress urinary. Our conclusion is intraurethral application of MSCs are a safe and feasible procedure to treat urinary incontinence. A statistically significant difference was obtained for pad‐test improvement in 3/8 men and 5/10 women.

Objectives. To confirm that patients affected by colorectal cancer have the V2 region of Septin 9 (SEPT9) gene hypermethylated in the circulating free DNA from a peripheral blood sample before surgery and to determine if this hypermethylated DNA disappears from the patients after complete resection of the tumour. Methods. Plasma from 10 patients with colorectal cancer was collected preoperative and three months after surgery. The analysis of the methylation status of the promoter region of the SEPT9 gene was performed using a 7500 Fast Real-Time PCR System. Results. Hypermethylation of SEPT9 gene was detected in 8 out of 10 preoperative samples (one negative result was probed to be a Lynch syndrome) and in 4 out of 10 postoperative samples matching with the cases of recurrence or persistence of disease. This means that, in this sample, the preoperative sensitivity and specificity of the test were 88.9% and 100%, respectively, and there is 100% correlation between the positive results of the SEPT9 test and a recurrence/persistence of the disease in patients after surgical resection. Conclusions. Our study shows that circulating hypermethylated SEPT9 is a specific colorectal cancer biomarker. This hypermethylated SEPT9 DNA disappears around three months after surgery and that circulating hypermethylated SEPT9 may be the first noninvasive marker for postsurgical diagnosis; this conclusion must be confirmed with a more significant number of patients.

The use of mesenchymal stem cells has resulted in a breakthrough for the treatment of complex perianal fistulas in Crohn’s disease. This novel treatment is associated with a minimally invasive surgical technique that can be well defined. However, our previous experience has taught us that neglecting any of the critical steps in the operation can result in frequent treatment failure. We have put together a comprehensive guide, a stepwise algorithm, for our minimally invasive approach to identify common pitfalls and reduce treatment failures. Using data we have collected over the past 15 years from drug development, execution of clinical trials, and enacting an advanced educational training program, we spelt out each stage of the minimally invasive surgical intervention for stem cell delivery for perianal Crohn’s disease. In this article, we provide 21 tips for a correct approach during the five major phases of the surgical procedure. To optimize the efficacy of mesenchymal stem cells for perianal Crohn’s disease, a standardized minimally invasive technique including a reliable and reproducible series of key steps should be utilized.

Aim. To report our experience in a compassionate use program for complex perianal fistula. 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 nonhealing diseases, mostly complex fistula-in-ano, who do not meet criteria to be included in a clinical trial. Candidates had previously undergone multiple surgical interventions that had failed. The intervention consisted of surgery (with closure of the internal opening or a surgical flap performance), followed by stem cell injection. Three types of cells were used for implant: stromal vascular fraction, autologous expanded adipose-derived, or allogenic adipose-derived stem cells. Healing was evaluated at 6th month follow-up. Outcome was classified as partial response or healing. Relapse was evaluated 1 year later. Maximum follow-up period was 48 months. Results. 45 patients (24 male) were included; the mean age was 45 years, which ranged from 24 to 69 years. Since some of them received repeated doses, 52 cases were considered (42 fistula-in-ano, 7 rectovaginal fistulas, 1 urethrorectal fistula, 1 sacral fistula, and 1 hidradenitis suppurativa). Regarding fistula-in-ano, there were 18 Crohn’s-associated and 24 cryptoglandular. 49 cases (94.2%) showed partial response starting 6.5 weeks of follow-up. 24 cases (46.2%) healed in a mean time of 5.5 months. A year later, all patients cured remained healed. No adverse effects related to stem cell therapy were reported. Conclusion. Stem cells are safe and useful for treating anal fistulae. Healing can be achieved in severe cases.

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.

Objective. The aims of this study are to compare 2 origins of adipose-derived mesenchymal stem cells (MSCs) (omentum and subcutaneous) from 2 pathologies (morbid obesity and cancer) vs healthy donors. Adipose tissue has revealed to be the ideal MSC source. However, in developing adipose-derived stem cells (ASCs) for clinical use, it is important to consider the effects of different fat depots and also the effect of donor variability. Methods. We isolated and characterized the membrane markers and differentiation capacities of ASCs obtained from patients with these diseases and different origin. During the culture period, we further analysed the cells’ proliferation capacity in an in vitro assay as well as their secretome. Results. Adipose-derived stem cells isolated from obese and cancer patients have mesenchymal phenotype and similar cell proliferation as ASCs derived from healthy donors, some higher in cells derived from subcutaneous fat. However, cells from these 2 types of patients do not have the same differentiation potential, especially in cancer patients from omentum, and exhibit distinct secretion of both pro-inflammatory and regulatory cytokines, which could explain the differences in use due to origin as well as pathology associated with the donor. Conclusion. Subcutaneous and omentum ASCs are slightly different; omentum generates fewer cells but with greater anti-inflammatory capacity. Adipose-derived stem cells from patients with either obesity or cancer are slightly altered, which limits their therapeutic properties.

Background aims Successful cell cryopreservation and banking remain a major challenge for the manufacture of cell therapy products, particularly in relation to providing a hermetic, sterile cryovial that ensures optimal viability and stability post-thaw while minimizing exposure to toxic cryoprotective agents, typically dimethyl sulfoxide (Me2SO). Methods In the present study, the authors evaluated the effectiveness and functionality of Limbo technology (Cellulis S.L., Santona, Spain). This system provides a hermetic vial with two compartments (one for adding cells with the cryoprotective agent solution and the other for the diluent solution) and an automated defrosting device. Limbo technology (Cellulis S.L.) allows reduction of the final amount of Me2SO, sidestepping washing and dilution steps and favoring standardization. The study was performed in several Good Manufacturing Practice laboratories manufacturing diverse cell therapy products (human mesenchymal stromal cells, hematopoietic progenitor cells, leukapheresis products, fibroblasts and induced pluripotent stem cells). Laboratories compared Limbo technology (Cellulis S.L.) with their standard cryopreservation procedure, analyzing cell recovery, viability, phenotype and functionality. Results Limbo technology (Cellulis S.L.) maintained the viability and functionality of most of the cell products and preserved sterility while reducing the final concentration of Me2SO. Conclusions Results showed that use of Limbo technology (Cellulis S.L.) offers an overall safe alternative for cell banking and direct infusion of cryopreserved cell products into patients.

The usefulness of local collagenase in therapeutic approaches to solid tumors has been tested recently. In this study, we evaluate the safety and efficacy of intraperitoneal collagenase associated or not to mitomycin for treatment of colorectal peritoneal metastases in an experimental rat model. Using a fixed-dose procedure, we found that a dose of collagenase of 37 IU/mL administered for 15 min with a hyperthermia pump at 37.5 °C, both in isolation or associated to sequential treatment with intraperitoneal mitomycin, led to a macroscopic decrease in tumor volume as evaluated by the modified peritoneal cancer index (mPCI). Concerning the safety of the procedure, the animals showed no physiological or behavioral disorders during 8 weeks of follow-up. Local treatment for peritoneal metastases of colorectal origin with intraperitoneal collagenase has proved safe and effective in an experimental murine model. Therefore, the stroma-first approach by enzymatic breakdown of collagen from the tumor's extracellular matrix provides a new therapeutic target for colorectal peritoneal metastases.

Venous leg ulcers (VLU) represent an uphill economic, health and social burden, aggravated in the elderly. Best-practice care interventions are often insufficient and alternative therapies need to be explored. Herein, we have treated for the first time a chronic VLU in an elderly patient by combining cell therapy and tissue engineering in the context of a compassionate use. The administration of allogeneic adipose-derived mesenchymal stromal cells (MSCs) embedded in a plasma-based bioengineered dermis covering the ulcer bed and also injected into the ulcer margins led to the complete closure of a 10-year recalcitrant VLU in an 85-year-old patient. Regenerative properties of MSCs might be boosted by the use of bioengineered matrices for their delivery.

Background: Identification of effective treatments in severe cases of COVID-19 requiring mechanical ventilation represents an unmet medical need. Our aim was to determine whether the administration of adipose-tissue derived mesenchymal stromal cells (AT-MSC) is safe and potentially useful in these patients. Methods: Thirteen COVID-19 adult patients under invasive mechanical ventilation who had received previous antiviral and/or anti-inflammatory treatments (including steroids, lopinavir/ritonavir, hydroxychloroquine and/or tocilizumab, among others) were treated with allogeneic AT-MSC. Ten patients received two doses, with the second dose administered a median of 3 days (interquartile range-IQR- 1 day) after the first one. Two patients received a single dose and another patient received 3 doses. Median number of cells per dose was 0.98 × 106 (IQR 0.50 × 106) AT-MSC/kg of recipient's body weight. Potential adverse effects related to cell infusion and clinical outcome were assessed. Additional parameters analyzed included changes in imaging, analytical and inflammatory parameters. Findings: First dose of AT-MSC was administered at a median of 7 days (IQR 12 days) after mechanical ventilation. No adverse events were related to cell therapy. With a median follow-up of 16 days (IQR 9 days) after the first dose, clinical improvement was observed in nine patients (70%). Seven patients were extubated and discharged from ICU while four patients remained intubated (two with an improvement in their ventilatory and radiological parameters and two in stable condition). Two patients died (one due to massive gastrointestinal bleeding unrelated to MSC therapy). Treatment with AT-MSC was followed by a decrease in inflammatory parameters (reduction in C-reactive protein, IL-6, ferritin, LDH and d-dimer) as well as an increase in lymphocytes, particularly in those patients with clinical improvement. Interpretation: Treatment with intravenous administration of AT-MSC in 13 severe COVID-19 pneumonia under mechanical ventilation in a small case series did not induce significant adverse events and was followed by clinical and biological improvement in most subjects. Funding: None. We would like to acknowledge the Instituto de Salud Carlos III (ISCIII) through the project “RD16/0011: Red de Terapia Celular”, from the sub-program RETICS, integrated in the “Plan Estatal de I+D+I 2013-2016” and co-financed by the European Regional Development Fund “A way to make Europe”, groups RD16/0011/0001, -/0002, -/005, -/0013, -/0015, -/0029), the Centro en Red de Medicina Regenerativa y Terapia Celular de Castilla y León, Spain and AvanCell-CM (Red de Investigación de Terapia Celular de la Comunidad de Madrid, Spain), for supporting some personnel and networking activities.

Faecal continence is a complex function involving different organs and systems. Faecal incontinence is a common disorder with different pathogeneses, disabling consequences and high repercussions for quality of life. Current management modalities are not ideal, and the development of new treatments is needed. Since 2008, stem cell therapies have been validated, 36 publications have appeared (29 in preclinical models and seven in clinical settings), and six registered clinical trials are currently ongoing. Some publications have combined stem cells with bioengineering technologies. The aim of this review is to identify and summarise the existing published knowledge of stem cell utilization as a treatment for faecal incontinence. A narrative or descriptive review is presented. Preclinical studies have demonstrated that cellular therapy, mainly in the form of local injections of muscle-derived (muscle derived stem cells or myoblasts derived from them) or mesenchymal (bone-marrow- or adipose-derived) stem cells, is safe. Cellular therapy has also been shown to stimulate the repair of both acute and subacute anal sphincter injuries, and some encouraging functional results have been obtained. Stem cells combined with normal cells on bioengineered scaffolds have achieved the successful creation and implantation of intrinsically-innervated anal sphincter constructs. The clinical evidence, based on adipose-derived stem cells and myoblasts, is extremely limited yet has yielded some promising results, and appears to be safe. Further investigation in both animal models and clinical settings is necessary to drawing conclusions. Nevertheless, if the preliminary results are confirmed, stem cell therapy for faecal incontinence may well become a clinical reality in the near future.

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, Research was supported by grants from the NIH (R01DK108743, R01CA172025, and R01CA207177 to J.M.; R01CA192642 and R01CA218254 to M.T.D.-M.)

Aim To assess KRAS G12D mutation detection by droplet digital PCR (ddPCR) in stool-derived DNA from colorectal cancer (CRC) patients. Methods In this study, tumor tissue and stool samples were collected from 70 patients with stage I-IV CRC diagnosed by preoperative biopsy. KRAS mutational status was determined by pyrosequencing analysis of DNA obtained from formalin-fixed paraffin-embedded (FFPE) tumor tissues. The KRAS G12D mutation was then analyzed by ddPCR in FFPE tumors and stool-derived DNA from patients with this point mutation. Wild-type (WT) tumors, as determined by pyrosequencing, were included as controls; analysis of FFPE tissue and stool-derived DNA by ddPCR was performed for these patients as well. Results Among the total 70 patients included, KRAS mutations were detected by pyrosequencing in 32 (45.71%), whereas 38 (54.29%) had WT tumors. The frequency of KRAS mutations was higher in left-sided tumors (11 located in the right colon, 15 in the left, and 6 in the rectum). The predominant point mutation was KRAS G12D (14.29%, n = 10), which was more frequent in early-stage tumors (I-IIA, n = 7). In agreement with pyrosequencing results, the KRAS G12D mutation was detected by ddPCR in FFPE tumor-derived DNA, and only a residual number of mutated copies was found in WT controls. The KRAS G12D mutation was also detected in stool-derived DNA in 80% of all fecal samples from CRC patients with this point mutation. Conclusion ddPCR is a reliable and sensitive method to analyze KRAS G12D mutation in stool-derived DNA from CRC patients, especially at early stages. This non-invasive approach is potentially applicable to other relevant biomarkers for CRC management.

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)

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.

© The Author(s) 2019., Colorectal cancer (CRC) with CpG island methylator phenotype (CIMP) is recognized as a subgroup of CRC that shows association with particular genetic defects and patient outcomes. We analyzed CIMP status of 229 individuals with CRC using an eight-marker panel (CACNA1G, CDKN2A, CRABP1, IGF2, MLH1, NEUROG1, RUNX3 and SOCS1); CIMP-(+) tumors were defined as having ≥ 5 methylated markers. Patients were divided into individuals who developed a “unique” CRC, which were subclassified into early-onset CRC (EOCRC) and late-onset CRC (LOCRC), and patients with multiple primary CRCs subclassified into synchronous CRC (SCRC) and metachronous CRC (MCRC). We found 9 (15.2%) CIMP-(+) EOCRC patients related with the proximal colon (p = 0.008), and 19 (26.8%) CIMP-(+) LOCRC patients associated with tumor differentiation (p = 0.045), MSI status (p = 0.021) and BRAF mutation (p = 0.001). Thirty-five (64.8%) SCRC patients had at least one CIMP-(+) tumor and 20 (44.4%) MCRC patients presented their first tumor as CIMP-(+). Thirty-nine (72.2%) SCRC patients showed concordant CIMP status in their simultaneous tumors. The differences in CIMP-(+) frequency between groups may reflect the importance of taking into account several criteria for the development of multiple primary neoplasms. Additionally, the concordance between synchronous tumors suggests CIMP status is generally maintained in SCRC patients., This work was funded by Projects PI10/00683 and PI16/01650 to J.P, and PI16/01920 to R.G.S, from the Spanish Ministry of Health and Consumer Afairs and FEDER, and by Project 2012-0036 from the Mutua Madrileña Foundation. This work was supported by R01 (CA72851, CA181572, CA184792, CA202797) and U01 (CA187956, CA214254) grants from the National Cancer Institute, NIH; RP140784 from the Cancer Prevention Research Institute of Texas; grants from the Baylor Foundation and Baylor Scott & White Research Institute, Dallas, TX, USA to AG.

The Collaborative Working Group “Noma Project Team”., Cell therapy is a progressively growing field that is rapidly moving from preclinical model development to clinical application. Outcomes obtained from clinical trials reveal the therapeutic potential of stem cell-based therapy to deal with unmet medical treatment needs for several disorders with no therapeutic options. Among adult stem cells, mesenchymal stem cells (MSCs) are the leading cell type used in advanced therapies for the treatment of autoimmune, inflammatory and vascular diseases. To date, the safety and feasibility of autologous MSC-based therapy has been established; however, their indiscriminate use has resulted in mixed outcomes in preclinical and clinical studies. While MSCs derived from diverse tissues share common properties depending on the type of clinical application, they markedly differ within clinical trials in terms of efficacy, resulting in many unanswered questions regarding the application of MSCs. Additionally, our experience in clinical trials related to critical limb ischemia pathology (CLI) shows that the therapeutic efficacy of these cells in different animal models has only been partially reproduced in humans through clinical trials. Therefore, it is crucial to develop new research to identify pitfalls, to optimize procedures and to clarify the repair mechanisms used by these cells, as well as to be able to offer a next generation of stem cell that can be routinely used in a cost-effective and safe manner in stem cell-based therapies targeting CLI., The authors are supported by the Fundación Progreso y Salud, Consejería de Salud, Junta de Andalucía; FEDER co-funded grants from Instituto de Salud Carlos III (Red TerCel-Grant RD16/0011/0034 and RD16/0011/0013; PIC18/00010; PI16/00259, PI17/02104, and PI-0272-2017), Fundación Andaluza de I+D and Al-Andalus Biopharma Project (FAID-2018-1); and Juvenile Diabetes Research Foundation, JDRF 2-SRA-2019-837-S-B I to (BS). VC-G is a recipient of a Sara Borrell contract (CD16/00118) from FEDER co-funded grants from Instituto de Salud Carlos III.

Our aim was to characterize and validate that the location and age of onset of the tumor are both important criteria to classify colorectal cancer (CRC). We analyzed clinical and molecular characteristics of early-onset CRC (EOCRC) and late-onset CRC (LOCRC), and we compared each tumor location between both ages-of-onset. In right-sided colon tumors, early-onset cases showed extensive Lynch syndrome (LS) features, with a relatively low frequency of chromosomal instability (CIN), but a high CpG island methylation phenotype. Nevertheless, late-onset cases showed predominantly sporadic features and microsatellite instability cases due to BRAF mutations. In left colon cancers, the most reliable clinical features were the tendency to develop polyps as well as multiple primary CRC associated with the late-onset subset. Apart from the higher degree of CIN in left-sided early-onset cancers, differential copy number alterations were also observed. Differences among rectal cancers showed that early-onset rectal cancers were diagnosed at later stages, had less association with polyps, and more than half of them were associated with a familial LS component. Stratifying CRC according to both location and age-of-onset criteria is meaningful, not only because it correlates the resulting categories with certain molecular bases, but with the confirmation across larger studies, new therapeutical algorithms could be defined according to this subclassification., This work was funded by Project PI10/0683 and PI13/0127 and PI16/01650 to J.P, and PI16/01920 to R.G.S, and PI17/01233 to D.G-O from the Spanish Ministry of Health and Consumer Affairs, and co-funded by the European Regional Development Fund (FEDER); and supported by grants R01 CA72851, CA18172, CA184792, and U01 CA187956 from the National Cancer Institute, National Institutes of Health to Ajay Goel

Introduction The ability of mesenchymal stromal cells (MSC) to suppress T-cell function has prompted their therapeutic use for graft-versus-host disease (GVHD) control. However, as MSC also modulate the activity of NK cells, which play an important role in graft-versus-leukemia (GVL) reaction, their administration could hamper this beneficial effect of allogeneic hematopoietic stem cell transplantation. MSC can be expanded from several sources, especially bone marrow and fat, but it is not well established if the cell source makes a difference in their immunoregulatory capacity. Objective The aim of this study was to compare the immunomodulatory effect of MSC derived from bone marrow (BM-CSM) or adipose tissue (AT-MSC) on NK cells, to determine whether the use of MSC from one or the other origin could be more favorable to preserve NK cell activity and, therefore, GVL. Methods Human NK cells were stimulated with IL-15 in the presence of BM-MSC or AT-MSC. The effect of both MSC populations on NK cell proliferation, cell cycle progression, and CD56 expression was analyzed by flow cytometry. Cytokine secretion was measured by ELISA, and cytotoxic activity was assessed by calcein release assays. Results Although both BM-MSC and AT-MSC induced a similar inhibition of NK cell proliferation, only BM-MSC decreased significantly NK cell cytotoxic activity and showed a trend for a higher reduction of IFN-γ secretion. Conclusion These results suggest that, in the context of GVHD inhibition, the use of AT-MSC rather than BM-MSC could further preserve NK cell activity and, thus, favor GVL.

To analyze the possible clonal origin of a part of Synchronous colorectal cancer (SCRC), we studied 104 paired‐SCRCs from 52 consecutive patients without hereditary forms of CRC. We used a Single‐Nucleotide Polymorphism array to characterize the genomic profiles, and subsequently used a statistical application to define them according to clonality within the same individual. We categorized the ensuing groups according to colonic location to identify differential phenotypes. The SCRC Monoclonal group (M) (19 cases) was divided into Monosegmental (MM) and Pancolonic (MP) groups. The SCRC Polyclonal group (P) (33 cases) was also divided into Monosegmental (PM) and Pancolonic (PP), the first exhibiting preference for left colon. The MM group showed a high rate of mucinous tumors, the lowest mean‐number of tumors and associated‐polyps, and the worst prognosis. The MP group included the largest mean‐number of associated‐polyps, best prognosis and familial cancer component. The PM group seemed to be a “frontier” group. Finally, the PP group also exhibited a mucin component, the highest mean‐number of tumors (4.6) compared with the mean‐number of polyps (7.7), poor prognosis and sporadic cases. Most relevant differential genomic regions within M groups were gains on 1q24 and 8q24, and deletions on 1p21 and 1p23 for MM, while within P were the gains on 7q36 and deletions on 1p36 for PM. The statistical application employed seems to define clonality more accurately in SCRC ‐more likely to be polyclonal in origin‐, and together with the tumor locations, helped us to configure a classification with prognostic and clinical value., This work was funded by Projects PI10/00683 and PI16/01650 to J.P, and PI16/01920 to R.G.S, from the Spanish Ministry of Health and Consumer Affairs and FEDER, by Project 2012–0036 from the Mutua Madrileña Foundation, and supported by the CA72851, CA181572, CA184792, CA187956 and CA202797 grants from the National Cancer Institute, National Institute of Health; RP140784 from the Cancer Prevention Research Institute of Texas; grants from the Sammons Cancer Center and Baylor Foundation, as well as funds from the Baylor Scott & White Research Institute, Dallas, TX, USA.

Anal fistula is a challenging condition both for surgeons and patients. Recurrent fistula, Crohn's disease, or autoimmune disorders add further complexity to this situation. Numerous clinical trials have now demonstrated that cell-based therapy appears to be a good complement to fistulous surgery. As in any new treatment, especially that involving living cells, appropriate application is paramount to achieve optimal outcomes. As stem cell-based treatments are gaining a strong foothold in fistula management worldwide, we herein aim to share our mesenchymal stem cell surgical protocol. With the goal of optimizing results of this emerging therapy, we have improved and refined our protocol over the past 17 years of working with stem cells in clinical trials. The protocol consists of nine reproducible steps for mesenchymal stem cell application inside the fistulous tract, and has proven to be safe and effective in several studies, including international phase III clinical trials.

Background aims In this report, we describe the successful bronchoscopic management of bronchopleural fistula in two patients, using autologous adipose-derived stromal cells. Cell therapy was considered for 2 cases of bronchopleural fistula refractory to conventional surgical treatment after control of the primary disease was confirmed and active pleural infection was ruled out. Briefly, adipose-derived stem cells were first isolated from lipoaspirate and used without cell expansion. In 24 months, we have not received more patients with bronchopleural fistula in our hospital and we have not been able to include more patients. Methods Briefly, adipose-derived stem cells were first isolated from lipo-aspirate and used without cell expansion. A bronchopleural fistula was identified through bronchoscopy, and the mucosa surrounding the fistula was ablated with an argon plasma coagulator. Isolated stem cells were then endoscopically injected into the de-epithelialized area and fistulous tract. If an open thoracostomy was present at the time of the intervention, the same procedure was performed on the pleural side. Bronchoscopic follow-up was scheduled weekly during the first month, monthly during the first year, and then yearly. The underlying etiologies were left pneumonectomy and right lower video-assisted lobectomy for non–small-cell lung cancer. The sizes of the fistulas were 6 mm and 3 mm in diameter, respectively. Results Both patients were discharged on the first postoperative day. The 3-year follow-up revealed a successful and maintained fistula closure, no treatment-related adverse reactions, nonlocal malignant recurrence and improved quality of life. Conclusions This preliminary study showed that bronchoscopic application of autologous adipose-derived stem cells is a feasible, safe and effective procedure for treating bronchopleural fistula.

Aim To establish a rat model of anal sphincter injury and test different systems to provide stem cells to injured area. Methods Adipose-derived stem cells (ASCs) were isolated from BDIX rats and were transfected with green fluorescent protein (GFP) for cell tracking. Biosutures (sutures covered with ASCs) were prepared with 1.5 x 106 GFP-ASCs, and solutions of 106 GFP-ASCs in normal saline were prepared for injection. Anorectal normal anatomy was studied on Wistar and BDIX female rats. Then, we designed an anal sphincter injury model consisting of a 1-cm extra-mucosal miotomy beginning at the anal verge in the anterior middle line. The sphincter lesion was confirmed with conventional histology (hematoxylin and eosin) and immunofluorescence with 4', 6-diamidino-2-phenylindole (commonly known as DAPI), GFP and α-actin. Functional effect was assessed with basal anal manometry, prior to and after injury. After sphincter damage, 36 BDIX rats were randomized to three groups for: (1) Cell injection without repair; (2) biosuture repair; and (3) conventional suture repair and cell injection. Functional and safety studies were conducted on all the animals. Rats were sacrificed after 1, 4 or 7 d. Then, histological and immunofluorescence studies were performed on the surgical area. Results With the described protocol, biosutures had been covered with at least 820000-860000 ASCs, with 100% viability. Our studies demonstrated that some ASCs remained adhered after suture passage through the muscle. Morphological assessment showed that the rat anal anatomy is comparable with human anatomy; two sphincters are present, but the external sphincter is poorly developed. Anal sphincter pressure data showed spontaneous, consistent, rhythmic anal contractions, taking the form of "plateaus" with multiple twitches (peaks) in each pressure wave. These basal contractions were very heterogeneous; their frequency was 0.91-4.17 per min (mean 1.6980, SD 0.57698), their mean duration was 26.67 s and mean number of peaks was 12.53. Our morphological assessment revealed that with the aforementioned surgical procedure, both sphincters were completely sectioned. In manometry, the described activity disappeared and was replaced by a gentle oscillation of basal line, without a recognizable pattern. Surprisingly, these findings appeared irrespective of injury repair or not. ASCs survived in this potentially septic area for 7 d, at least. We were able to identify them in 84% of animals, mainly in the muscular section area or in the tissue between the muscular endings. ASCs formed a kind of "conglomerate" in rats treated with injections, while in the biosuture group, they wrapped the suture. ASCs were also able to migrate to the damaged zone. No relevant adverse events or mortality could be related to the stem cells in our study. We also did not find unexpected tissue growths. Conclusion The proposed procedure produces a consistent sphincter lesion. Biosutures and injections are suitable for cell delivery. ASCs survive and are completely safe in this clinical setting.

[Background]: Since there is a predilection of some clinical and molecular features for a given tumor location, we assessed whether this can be confirmed in late-onset colorectal cancer (LOCRC). [Results]: Right colon cancers showed features associated with sporadic Microsatellite Instability: predominance of female cases and BRAF mutations, and an important mucinous component. Left colon cancers developed a higher number of polyps and multiple primary CRCs, showed the strongest familial component, and had better prognosis. Rectal cancers showed a predominantly sporadic phenotype, with worse prognosis and a CpG Island Methylator Phenotype (CIMP)-High. No copy number alterations (CNAs) greater than or equal to 50% were observed in this LOCRC group, and the most recurrent alterations were losses at 5q13 and 14q11, and gains at 7q11, 7q21-q22, 19p13-p12, 19q13 and 20p11-q11. KRAS and PIK3CA were the only mutated genes showing differences according to the tumor location, mainly for right colon cancers. [Materials and Methods]: We analyzed clinical and molecular characteristics of LOCRC at different tumor locations in order to determine if there are differential phenotypes related with the location in the colon. [Conclusions]: Categorizing LOCRC according to tumor location appears to be an adequate first step to resolving the heterogeneity of this subset of CRC., This work was funded by Projects PI10/0683, PI13/01741, PI13/0127 and PI14/00459 from the Spanish Ministry of Health and Consumer Affairs and FEDER, and was approved by the Ethics Committee of our Institution.

Idiopathic pulmonary fibrosis (IPF) is a progressive interstitial lung disease with poor prognosis. Adipose-derived stem cells (ADSC) have demonstrated regenerative properties in several tissues. The hypothesis of this study was that airway transplantation of ADSC could protect against bleomycin (BLM)-induced pulmonary fibrosis (PF). Fifty-eight lungs from 29 male Sprague-Dawley rats were analyzed. Animals were randomly divided into five groups: a) control (n=3); b) sham (n=6); c) BLM (n=6); d) BLM+ADSC-2d (n=6); and e) BLM+ADSC-14d (n=8). Animals received 500 µL saline (sham), 2.5 UI/kg BLM in 500 µL saline (BLM), and 2×106 ADSC in 100 µL saline intratracheally at 2 (BLM+ADSC-2d) and 14 days (BLM+ADSC-14d) after BLM. Animals were sacrificed at 28 days. Blinded Ashcroft score was used to determine pulmonary fibrosis extent on histology. Hsp27, Vegf, Nfkβ, IL-1, IL-6, Col4, and Tgfβ1 mRNA gene expression were determined using real-time quantitative-PCR. Ashcroft index was: control=0; sham=0.37±0.07; BLM=6.55±0.34 vs sham (P=0.006). BLM vs BLM+ADSC-2d=4.63±0.38 (P=0.005) and BLM+ADSC-14d=3.77±0.46 (P=0.005). BLM vs sham significantly increased Hsp27 (P=0.018), Nfkβ (P=0.009), Col4 (P=0.004), Tgfβ1 (P=0.006) and decreased IL-1 (P=0.006). BLM+ADSC-2d vs BLM significantly decreased Hsp27 (P=0.009) and increased Vegf (P=0.006), Nfkβ (P=0.009). BLM+ADSC-14d vs BLM significantly decreased Hsp27 (P=0.028), IL-6 (P=0.013), Col4 (P=0.002), and increased Nfkβ (P=0.040) and Tgfβ1 (P=0.002). Airway transplantation of ADSC significantly decreased the fibrosis rate in both early and established pulmonary fibrosis, modulating the expression of Hsp27, Vegfa, Nfkβ, IL-6, Col4, and Tgfβ1. From a translational perspective, this technique could become a new adjuvant treatment for patients with IPF.

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 Results Seventy percent of the rats treated with streptozotocin met the criteria for diabetes. Macroscopically, cell-treated rats presented better general and lower ischemic clinical status, and histologically, a better trend towards angiogenesis, greater infiltration of type 2 macrophages and a shortening of the inflammatory process. However, only the inflammatory variables were statistically significant. No immunological reaction was observed with the use of allogeneic cells. Discussion The application of allogeneic ASCs in a hind limb ischemic model in diabetic animals shows no rejection reactions and a reduction in inflammatory parameters in favor of better repair of damaged tissue. These results are consistent with other lines of research in allogeneic cell therapy. This approach might be a safe, effective treatment option that makes it feasible to avoid the time involved in the process of isolation, expansion and production of the use of autologous cells.

The clinical management of cancer has evolved in recent years towards more personalized strategies that require a comprehensive knowledge of the complex molecular features involved in tumor growth and evolution, and the development of drug resistance mechanisms leading to disease progression. Droplet digital PCR (ddPCR) has become one of the most accurate and reliable tools for the examination of genetic alterations in a wide variety of cancers because of its high sensitivity and specificity. ddPCR is currently being applied for absolute allele quantification, rare mutation detection, analysis of copy number variations, DNA methylation, and gene rearrangements in different kinds of clinical samples. This methodology has proven useful for the evaluation of archival tumor tissues, where poor DNA quality and limited sample availability are major obstacles for standard methods, providing less subjective and more automated quantitative results. However, most applications of ddPCR in cancer are focused on liquid biopsies (including cell-free DNA as well as circulating tumor cells) because these represent non-invasive alternatives to tissue biopsies that can more accurately reflect intratumoral heterogeneity and track the dynamic changes in tumor burden that occur in response to treatment at different times during follow-up. A broad spectrum of molecular markers have been interrogated in blood using ddPCR for diagnostic, predictive, and monitoring purposes in various malignancies. Emerging alternative approaches using other body fluids such as cerebrospinal fluid and urine are also currently being developed. This article aims to give a complete overview of ddPCR applications for molecular screening in oncology.

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, This study has been funded by Instituto de Salud Carlos III (ISCIII) through the Spanish Net of Cell Therapy (TerCel), RETICS subprogram of the I+D+I 2013-2016 [RD16/0011/0013 funded by ISCIII and co-founded by European Regional Development Fund (ERDF)]

Background. A DNA-RNA-lipoprotein complex, termed a virtosome, is released spontaneously from healthy human, other mammalian, avian, amphibian and plant cells in a regulated and energy-dependent manner. Studies on human and mouse lymphocytes, hepatocytes, NIH 3T3 cells and mouse tumour cell lines have shown that virtosomes may be acting as inter-cellular messengers. In particular, virtosomes from non-dividing cells blocked 3H-thymidine incorporation into DNA in tumour cell lines. Objective. Study of the effect of virtosomes on tumors "in vivo" and "in vitro" Methods and Results. The present study shows in vitro effects of virtosomes isolated from rat liver, essentially non-dividing cell populations, on cultures of healthy smooth muscle cells (SMC), human umbilical vein endothelial cells (HUVEC), human fibroblasts (h-fibroblasts) and mouse embryonic fibroblasts (NIH-3T3) together with two tumour cell lines, human Duke’s type B colorectal adenocarcinoma cells (SW480) and human connective tissue fibrosarcoma cells (HT1080). Multiplication of all cell lines was inhibited by the liver virtosomal preparation even with various dilutions of the extract (100 - 0.5%). In an in vivo study, tumours were initiated by subcutaneous injection of 1.0 x 106 DHD/K12-PROb cells in 6 weeks old male BIDX rats. Visible tumours (

KRAS mutations are responsible for resistance to anti-epidermal growth factor receptor (EGFR) therapy in colorectal cancer patients. These mutations sometimes appear once treatment has started. Detection of KRAS mutations in circulating cell-free DNA in plasma (“liquid biopsy”) by droplet digital PCR (ddPCR) has emerged as a very sensitive and promising alternative to serial biopsies for disease monitoring. In this study, KRAS G12V mutation was analyzed by ddPCR in plasma DNA from 10 colorectal cancer patients and compared to six healthy donors. The percentage of KRAS G12V mutation relative to wild-type sequences in tumor-derived DNA was also determined. KRAS G12V mutation circulating in plasma was detected in 9 of 10 colorectal cancer patients whose tumors were also mutated. Colorectal cancer patients had 35.62 copies of mutated KRAS/mL plasma, whereas in healthy controls only residual copies were found (0.62 copies/mL, p = 0.0066). Interestingly, patients with metastatic disease showed a significantly higher number of mutant copies than M0 patients (126.25 versus 9.37 copies/mL, p = 0.0286). Wild-type KRAS was also significantly elevated in colorectal cancer patients compared to healthy controls (7718.8 versus 481.25 copies/mL, p = 0.0002). In conclusion, KRAS G12V mutation is detectable in plasma of colorectal cancer patients by ddPCR and could be used as a non-invasive biomarker., This study was funded by a grant from “Fondo de Investigaciones Sanitarias-FEDER”, Ministry of Health, Spain (FIS; PI13/01924) and the Spanish Ministry of Health and Consumer Affairs (via a cooperative network-FEDER [TerCel RD12- 0019-0035])

In vitro studies of partially purified virtosomes from rat liver showed inhibition of cell multiplication in four normal and two tumour cell lines. In vivo, the liver virtosomes slowed tumour growth and limited metastases in rats bearing DHD/K12-PROb cell initiated tumours.

The aim of this clinical trial was to determine the safety and feasibility of expanded allogeneic adipose-derived stem cells to treat Crohn’s-related rectovaginal fistula (CRRVF). We designed a phase I–II clinical trial (https://ClinicalTrials.gov, NCT00999115) to treat 10 patients with CRRVF. Patients receiving biological therapy during follow-up were excluded. Curettage was performed, and a vaginal or rectal flap was added if the surgeon considered it necessary. The therapeutic protocol included intralesional injection of 20 million stem cells in the vaginal walls (submucosal area) and fistula tract. Healing was evaluated 12 weeks later. If the fistula had not healed, a second dose of 40 million stem cells was administered. Patient follow-up was 52 weeks from last cell injection. Healing was defined as re-epithelialization of both vaginal and rectal sides and absence of vaginal drainage. Cytokines and immunological blood tests were monitored. Serious adverse events or rejection issues were not observed. Five patients were excluded because biologic drugs were required to treat a Crohn's disease flare-up during follow-up. Cytokine profiles and immunotoxicity assays showed no statistically significant alterations. Sixty percent of the nonexcluded patients achieved a complete healing. Expanded allogeneic adipose-derived stem-cell injection is a safe and feasible therapy for treating CRRVF, and the healing success rate seems promising (60%). The results of this trial encourage further exploration into this therapy. Significance This may be the first publication in which allogeneic stem cells to treat rectovaginal fistula in Crohn’s disease seem to be a feasible and safe treatment. Additional studies are necessary to confirm the efficacy profile of the allogeneic stem cells strategy in a controlled design.

Background & Aims: Therapies for perianal fistulas in patients with Crohn's disease are often ineffective in producing long-term healing. We performed a randomized placebo-controlled trial to determine the long-term efficacy and safety of a single local administration of allogeneic expanded adipose-derived stem cells (Cx601) in patients with Crohn's disease and perianal fistulas. Methods: We performed a double-blind study at 49 hospitals in Europe and Israel, comprising 212 patients with Crohn's disease and treatment-refractory, draining, complex perianal fistulas. Patients were randomly assigned (1:1) to groups given a single local injection of 120 million Cx601 cells or placebo (control), in addition to the standard of care. Efficacy endpoints evaluated in the modified intention-to-treat population (randomly assigned, treated, and with 1 or more post-baseline efficacy assessment) at week 52 included combined remission (closure of all treated external openings draining at baseline with absence of collections >2 cm, confirmed by magnetic resonance imaging) and clinical remission (absence of draining fistulas). Results: The study's primary endpoint, at week 24, was previously reported (combined remission in 51.5% of patients given Cx601 vs 35.6% of controls, for a difference of 15.8 percentage points; 97.5% confidence interval [CI] 0.5–31.2; P =.021). At week 52, a significantly greater proportion of patients given Cx601 achieved combined remission (56.3%) vs controls (38.6%) (a difference of 17.7 percentage points; 95% CI 4.2–31.2; P =.010), and clinical remission (59.2% vs 41.6% of controls, for a difference of 17.6 percentage points; 95% CI 4.1–31.1; P =.013). Safety was maintained throughout week 52; adverse events occurred in 76.7% of patients in the Cx601 group and 72.5% of patients in the control group. Conclusion: In a phase 3 trial of patients with Crohn's disease and treatment-refractory complex perianal fistulas, we found Cx601 to be safe and effective in closing external openings, compared with placebo, after 1 year. ClinicalTrials.gov no: NCT01541579.

Upper limb critical ischemia is rarely due to arteriosclerosis alone. Cases with no surgical option are even less frequent [1]. A growing body of evidence suggests possible benefits of regenerative medicine in this condition using stem cells and growth factors, especially in cases with no other options [2,3]. The use of adipose tissue–derived progenitors as a therapeutic has grown substantially in the past decade. Multiple laboratories have established that stromal cells similar to those identified in bone marrow can be isolated in a reproducible manner from adipose tissue that is either resected as intact tissue or aspirated using tumescent liposuction.The minced adipose tissue is then digested by one or more of the following: collagenase, dispase, trypsin or related enzymes. After neutralization of the enzymes, the released elements, defined as the stromal vascular fraction (SVF), are separated from the mature adipocytes by differential centrifugation.The SVF consists of a heterogeneous mesenchymal population of cells that includes not only adipose stromal and hematopoietic stem and progenitor cells but also endothelial cells, erythrocytes, fibroblasts, lymphocytes, monocyte/ macrophages and pericytes, among others.When SVF cells are seeded into culture, a subset of elongated cells begins to adhere to the tissue culture plastic ware (Figure 1A).These cells can be purified further using a combination of washing steps and culture expansion with media similar to those used for bone marrow mesenchymal stromal cells to deplete most of the hematopoietic cell population from the SVF cells. This process allows the emergence of an adherent cell population termed adipose tissue–derived stromal cells (ASC). Although ASC are less heterogeneous than SVF cells, they are by no means homogeneous. ASC include multipotent cells with the ability to differentiate into adipocytes, chondrocytes and osteoblasts, among other lineage pathways [4]. One main difference between SVF cell and ASC suspensions is the high level of CD45+ cells in the SVF cells and the low or undetectable level in ASC. The International Society for Cellular Therapy has proposed adaptation of the characterization strategy for MSC for the phenotyping of the SVF cells using multicolor analysis [4]. We report the case of a multifactorial dominanthand ischemia in which all surgical and endovascular options had been performed with no result before successful treatment with this new line of treatment, avoiding major amputation.