Isolation and Characterization of Stem Cells from the Anal Canal Transition Zone in Pigs.

BACKGROUND: Utilization of autologous stem cells has been proposed for the treatment of anal incontinence despite a lack of understanding of their mechanism of action and of the physiological healing process of anal sphincters after injury. AIMS: We aim to develop a technique allowing isolation and further study of local mesenchymal stem cells, directly from anal canal transition zone in pig. METHODS: Anal canal was resected "en bloc" from two young pigs and further microdissected. The anal canal transition zone was washed and digested with 0.1% type I collagenase for 45 min at 37 °C. The isolated cells were plated on dishes in mesenchymal stem cell medium and trypsinized when confluent. Cells were further used for flow cytometry analysis and differentiation assays. RESULTS: The anal canal transition zone localization was confirmed with H&E staining. Following culture, cells exhibited a typical "fibroblast-like" morphology typical of stem cells. Isolated cells were positive for CD90 and CD44 but negative for CD14, CD34, CD45, CD105, CD106, and SLA-DR. Following incubation with specific differentiation medium, isolated cells differentiated into adipocytes, osteoblasts, and chondrocytes, confirming in vitro multipotency. CONCLUSIONS: Herein, we report for the first time the presence of mesenchymal stem cells in the anal canal transition zone in pigs and the feasibility of their isolation. This preliminary study opens the path to the isolation of human anal canal transition zone mesenchymal stem cells that might be used to study sphincters healing and to treat anal incontinence.

Engineering Islets From Stem Cells: The Optimal Solution for the Treatment of Diabetes?

Diabetes is a metabolic disease characterized by insulin deficiency. Bioengineering of stem cells with the aim to restore insulin production and glucose regulation has the potential to cure diabetic patients. In this review, we focus on the recent developments for bioengineering of induced pluripotent stem cells (iPSCs), mesenchymal stem cells (MSCs), embryonic stem cells (ESCs), and pancreatic progenitor cells in view of generating insulin producing and glucose regulating cells for ß-cell replacement therapies. Recent clinical trials using islet cells derived from stem cells have been initiated for the transplantation into diabetic patients, with crucial bottlenecks of tumorigenesis, post-transplant survival, genetic instability, and immunogenicity that should be further optimized. As a new approach given high expectations, bioengineered islets from stem cells occupies considerable potential for the future clinical application and addressing the treatment dilemma of diabetes.

Cell Therapy for Anal Sphincter Incontinence: Where Do We Stand?

Anal sphincter incontinence is a chronic disease, which dramatically impairs quality of life and induces high costs for the society. Surgery, considered as the best curative option, shows a disappointing success rate. Stem/progenitor cell therapy is pledging, for anal sphincter incontinence, a substitute to surgery with higher efficacy. However, the published literature is disparate. Our aim was to perform a review on the development of cell therapy for anal sphincter incontinence with critical analyses of its pitfalls. Animal models for anal sphincter incontinence were varied and tried to reproduce distinct clinical situations (acute injury or healed injury with or without surgical reconstruction) but were limited by anatomical considerations. Cell preparations used for treatment, originated, in order of frequency, from skeletal muscle, bone marrow or fat tissue. The characterization of these preparations was often incomplete and stemness not always addressed. Despite a lack of understanding of sphincter healing processes and the exact mechanism of action of cell preparations, this treatment was evaluated in 83 incontinent patients, reporting encouraging results. However, further development is necessary to establish the correct indications, to determine the most-suited cell type, to standardize the cell preparation method and to validate the route and number of cell delivery.

Platelet Transforming Growth Factor-ß1 Induces Liver Sinusoidal Endothelial Cells to Secrete Interleukin-6.

The roles and interactions of platelets and liver sinusoidal endothelial cells in liver regeneration are unclear, and the trigger that initiates hepatocyte proliferation is unknown. We aimed to identify the key factors released by activated platelets that induce liver sinusoidal endothelial cells to produce interleukin-6 (IL-6), a cytokine implicated in the early phase of liver regeneration. We characterized the releasate of activated platelets inducing the in vitro production of IL-6 by mouse liver sinusoidal endothelial cells and observed that the stimulating factor was a thermolabile protein. Following gel filtration, a single fraction of activated platelet releasate induced a maximal IL-6 secretion by liver sinusoidal endothelial cells (90.2 ± 13.9 versus control with buffer, 9.0 ± 0.8 pg/mL, p < 0.05). Mass spectroscopy analysis of this fraction, followed by in silico processing, resulted in a reduced list of 18 candidates. Several proteins from the list were tested, and only recombinant transforming growth factor ß1 (TGF-ß1) resulted in an increased IL-6 production up to 242.7 ± 30.5 pg/mL, which was comparable to non-fractionated platelet releasate effect. Using neutralizing anti-TGF-ß1 antibody or a TGF-ß1 receptor inhibitor, IL-6 production by liver sinusoidal endothelial cells was dramatically reduced. These results support a role of platelet TGF-ß1 ß1 in the priming phase of liver regeneration.

Does Surgical Approach in Pelvic Floor Repair Impact Sexual Function in Women?

INTRODUCTION: Surgical routes used to correct complex pelvic floor disorders (CPFDs) may have a negative impact on women's sexual function. Currently, there is no evidence concerning the impact of a specific surgical procedure on postoperative sexual function in women. AIM: The aim of this study was to compare an abdominal approach with rectopexy and sacrocolpopexy to a perineal procedure with abdominal rectopexy, regarding female sexual function in cases of CPFDs. METHODS: Women who were operated for CPFDs between January 2003 and June 2010 were retrospectively asked to answer the Pelvic Organ Prolapse/Urinary Incontinence Sexual Questionnaire-12, the Miller Score of Incontinence, and a urinary incontinence frequency score. We also questioned them about their sexual function and satisfaction before and after the operation using visual analogic scores. MAIN OUTCOME MEASURE: We compared the Pelvic Organ Prolapse/Urinary Incontinence Sexual Questionnaire-12 before and after the surgery in both groups, and we made an intragroup comparison. RESULTS: There were 334 women identified, but only 51 could be included. Globally, we found no statistically significant differences in terms of sexual function before and after surgery between the 25 groups. Intragroup comparison demonstrated that, within the perineal approach group, patients experienced a decrease in their sexual arousal after the procedure. The choice of surgical route for pelvic floor disorders does not seem to have an impact on the results of postoperative sexual function in women. This study adds to the limited literature on sexual outcomes of surgery for CPFD. It is limited principally due to its retrospective design and the small number of patients included. CONCLUSION: Both surgical routes have very similar outcomes on most sexual questions. A perineal approach combined with abdominal rectopexy did, however, demonstrate a slight decrease in sexual arousal of the patients after the intervention. Zawodnik A, Balaphas A, Buchs NC, et al. Does Surgical Approach in Pelvic Floor Repair Impact Sexual Function in Women? Sex Med 2019;7:522-529.

Platelets and Platelet-Derived Extracellular Vesicles in Liver Physiology and Disease.

Beyond their role in hemostasis, platelets are proposed as key mediators of several physiological and pathophysiological processes of the liver, such as liver regeneration, toxic or viral acute liver injury, liver fibrosis, and carcinogenesis. The effects of platelets on the liver involve interactions with sinusoidal endothelial cells and the release of platelet-contained molecules following platelet activation. Platelets are the major source of circulating extracellular vesicles, which are suggested to play key roles in platelet interactions with endothelial cells in several clinical disorders. In the present review, we discuss the implications of platelet-derived extracellular vesicles in physiological and pathophysiological processes of the liver.

Harnessing Microglia and Macrophages for the Treatment of Glioblastoma.

Glioblastoma multiforme (GBM) is the most malignant form of brain tumors, with a dismal prognosis. During the course of the disease, microglia and macrophages both infiltrate the tumor microenvironment and contribute considerably in glioma development. Thus, tumor-associated microglia and macrophages have recently emerged as potentially key therapeutic targets. Here, we review the physiology of microglia and their responses in brain cancer. We further discuss current treatment options for GBM using radiotherapy, and novel advances in our knowledge of microglia physiology, with emphasis on the recently discovered pathway that controls the baseline motility of microglia processes. We argue that the latter pathway is an interesting therapeutic avenue to pursue for the treatment of glioblastoma.

Evaluation of donor kidneys prior to transplantation: an update of current and emerging methods.

The lack of suitable kidney donor organs has led to rising numbers of patients with end stage renal disease waiting for kidney transplantation. Despite decades of clinical experience and research, no evaluation process that can reliably predict the outcome of an organ has yet been established. This review is an overview of current methods and emerging techniques in the field of donor kidney evaluation prior to transplantation. Established techniques like histological evaluation, clinical scores, and machine perfusion systems offer relatively reliable predictions of delayed graft function but are unable to consistently predict graft survival. Emerging techniques including molecular biomarkers, new imaging technologies, and normothermic machine perfusion offer innovative approaches toward a more global evaluation of an organ with better outcome prediction and possibly even identification of targets for therapeutic interventions prior to transplantation. These techniques should be studied in randomized controlled trials to determine whether they can be safely used in routine clinical practice to ultimately reduce the discard rate and improve graft outcomes.

Beneficial Effects of Human Mesenchymal Stromal Cells on Porcine Hepatocyte Viability and Albumin Secretion.

Porcine hepatocytes transplanted during acute liver failure might support metabolic functions until the diseased liver recovers its function. Here, we isolated high numbers of viable pig hepatocytes and evaluated hepatocyte functionality after encapsulation. We further investigated whether coculture and coencapsulation of hepatocytes with human multipotent mesenchymal stromal cells (MSC) are beneficial on hepatocyte function. Livers from 10 kg pigs (n = 9) were harvested, and hepatocytes were isolated from liver suspensions for microencapsulation using alginate and poly(ethylene-glycol)- (PEG-) grafted alginate hydrogels, either alone or in combination with MSC. Viability, albumin secretion, and diazepam catabolism of hepatocytes were measured for one week. 9.2 ± 3.6 × 109 hepatocytes with 95.2 ± 3.1% viability were obtained after isolation. At day 3, free hepatocytes displayed 99% viability, whereas microencapsulation in alginate and PEG-grafted alginate decreased viability to 62% and 48%, respectively. Albumin secretion and diazepam catabolism occurred in free and microencapsulated hepatocytes. Coencapsulation of hepatocytes with MSC significantly improved viability and albumin secretion at days 4 and 8 (p < 0.05). Coculture with MSC significantly increased and prolonged albumin secretion. In conclusion, we established a protocol for isolation and microencapsulation of high numbers of viable pig hepatocytes and demonstrated that the presence of MSC is beneficial for the viability and function of porcine hepatocytes.

Multipotent mesenchymal stromal cells enhance insulin secretion from human islets via N-cadherin interaction and prolong function of transplanted encapsulated islets in mice.

BACKGROUND: Multipotent mesenchymal stromal cells (MSC) enhance viability and function of islets of Langerhans. We aimed to examine the interactions between human MSC and human islets of Langerhans that influence the function of islets. METHODS: Human MSC and human islets (or pseudoislets, obtained after digestion and reaggregation of islet cells) were cocultured with or without cellular contact and glucose-stimulated insulin secretion assays were performed to assess cell function. The expression of several adhesion molecules, notably ICAM-1 and N-cadherin on islets and MSC, was investigated by qPCR. The role of N-cadherin was analyzed by adding an anti-N-cadherin antibody in islets cultured with or without MSC for 24 h followed by insulin measurements in static incubation assays. Islets and MSC were coencapsulated in new hydrogel microspheres composed of calcium alginate and covalently crosslinked polyethylene glycol. Encapsulated cells were transplanted intraperitoneally in streptozotocin-induced diabetic mice and glycemia was monitored. Islet function was evaluated by the intraperitoneal glucose tolerance test. RESULTS: In vitro, free islets and pseudoislets cocultured in contact with MSC showed a significantly increased insulin secretion when compared to islets or pseudoislets cultured alone or cocultured without cell-to-cell contact with MSC (p < 0.05). The expression of ICAM-1 and N-cadherin was present on islets and MSC. Blocking N-cadherin prevented the enhanced insulin secretion by islets cultured in contact with MSC whereas it did not affect insulin secretion by islets cultured alone. Upon transplantation in diabetic mice, islets microencapsulated together with MSC showed significantly prolonged normoglycemia when compared with islets alone (median 69 and 39 days, respectively, p < 0.01). The intraperitoneal glucose tolerance test revealed an improved glycemic response in mice treated with islets microencapsulated together with MSC compared to mice transplanted with islets alone (p < 0.001). CONCLUSIONS: MSC improve survival and function of islets of Langerhans by cell-to-cell contact mediated by the adhesion molecule N-cadherin.

Synthesis Strategies to Extend the Variety of Alginate-Based Hybrid Hydrogels for Cell Microencapsulation.

The production of hydrogel microspheres (MS) for cell immobilization, maintaining the favorable properties of alginate gels but presenting enhanced performance in terms of in vivo durability and physical properties, is desirable to extend the therapeutic potential of cell transplantation. A novel type of hydrogel MS was produced by straightforward functionalization of sodium alginate (Na-alg) with heterotelechelic poly(ethylene glycol) (PEG) derivatives equipped with either end thiol or 1,2-dithiolane moieties. Activation of the hydroxyl moieties of the alginate backbone in the form of imidazolide intermediate allowed for fast conjugation to PEG oligomers through a covalent carbamate linkage. Evaluation of the modified alginates for the preparation of MS combining fast ionic gelation ability of the alginate carboxylate groups and slow covalent cross-linking provided by the PEG-end functionalities highlighted the influence of the chemical composition of the PEG-grafting units on the physical characteristics of the MS. The mechanical properties of the MS (resistance and shape recovery) and durability of PEG-grafted alginates in physiological environment can be adjusted by varying the nature of the end functionalities and the length of the PEG chains. In vitro cell microencapsulation studies and preliminary in vivo assessment suggested the potential of these hydrogels for cell transplantation applications.

Testing of microencapsulated porcine hepatocytes in a new model of fulminant liver failure in baboons.

BACKGROUND: There is no standard therapy for acute liver failure. Hepatocyte transplantation has been proposed for temporary liver function support, while the injured liver regenerates or while waiting for transplantation. We have previously shown such efficacy for microencapsulated porcine hepatocytes in mice with fulminant liver failure. We aimed to establish a large animal model for fulminant liver failure to assess the efficacy of microencapsulated porcine hepatocytes in temporary liver function support. METHODS: The model was developed in baboons; for testing microencapsulated hepatocytes, the best condition was 75% hepatectomy and 60 min warm ischemia time. Fulminant liver failure was characterized by steep increases in liver biochemical parameters, severe steatosis, and massive hepatocyte necrosis during the first 10 days. Hepatocytes from miniature swine were microencapsulated in alginate-poly-l-lysine microspheres, and transplanted intraperitoneally immediately after hepatectomy and warm ischemia (80-120 mL packed hepatocytes in 200-350 mL microspheres, about 30%-50% of the baboon's native liver volume). RESULTS: In the control group, three of five animals were sacrificed after 6-10 days because of fulminant liver failure, and two of five animals recovered normal liver function and survived until elective euthanasia (28 days). In the treatment group of four animals, one animal developed liver failure but survived to 21 days, and three animals recovered completely with normal liver function. CONCLUSIONS: The results indicate that microencapsulated porcine hepatocytes provide temporary liver function support in baboons with fulminant liver failure. These data support development of this cell therapy product toward clinical trials in patients with acute liver failure.

Microencapsulation of Hepatocytes and Mesenchymal Stem Cells for Therapeutic Applications.

Encapsulated hepatocyte transplantation and encapsulated mesenchymal stem cell transplantation are newly developed potential treatments for acute and chronic liver diseases, respectively. Cells are microencapsulated in biocompatible semipermeable alginate-based hydrogels. Microspheres protect cells against antibodies and immune cells, while allowing nutrients, small/medium size proteins and drugs to diffuse inside and outside the polymer matrix. Microencapsulated cells are assessed in vitro and designed for experimental transplantation and for future clinical applications.Here, we describe the protocol for microencapsulation of hepatocytes and mesenchymal stem cells within hybrid poly(ethylene glycol)-alginate hydrogels.

[Cystic lesions of the pancreas from the general practitioner's point of view]. / Les lésions kystiques du pancréas sous l'angle du médecin de premier recours.

The detection rate of cystic lesions of the pancreas has increased following the widespread use of high-resolution imaging technologies. CT-scan, MRI and echo-endoscopy are diagnostic modalities. Pseudocyst is the most common lesion. It is benign and can be managed with endoscopic treatment. Mucinous cystic neoplasia and Intraductal Papillary Mucinous Neoplasia (IPMN) carry a risk for malignant transformation. The surgical treatment of these lesions has to be discussed by a multidisciplinary board. Serous cystic neoplasia and pseudopapillar and solid neoplasia are two rare types of lesion. The aim of this article is to present the diagnostic pathway and the management of these lesions from the general practitioner point of view.

Polymorphisms in the lectin pathway of complement activation influence the incidence of acute rejection and graft outcome after kidney transplantation.

There are conflicting data on the role of the lectin pathway of complement activation and its recognition molecules in acute rejection and outcome after transplantation. To help resolve this we analyzed polymorphisms and serum levels of lectin pathway components in 710 consecutive kidney transplant recipients enrolled in the nationwide Swiss Transplant Cohort Study, together with all biopsy-proven rejection episodes and 1-year graft and patient survival. Functional mannose-binding lectin (MBL) levels were determined in serum samples, and previously described MBL2, ficolin 2, and MBL-associated serine protease 2 polymorphisms were genotyped. Low MBL serum levels and deficient MBL2 diplotypes were associated with a higher incidence of acute cellular rejection during the first year, in particular in recipients of deceased-donor kidneys. This association remained significant (hazard ratio 1.75, 95% confidence interval 1.18-2.60) in a Cox regression model after adjustment for relevant covariates. In contrast, there was no significant association with rates of antibody-mediated rejection, patient death, early graft dysfunction or loss. Thus, results in a prospective multicenter contemporary cohort suggest that MBL2 polymorphisms result in low MBL serum levels and are associated with acute cellular rejection after kidney transplantation. Since MBL deficiency is a relatively frequent trait in the normal population, our findings may lead to individual risk stratification and customized immunosuppression.

Current status of hepatocyte xenotransplantation.

The treatment of acute liver failure, a condition with high mortality, comprises optimal clinical care, and in severe cases liver transplantation. However, there are limitations in availability of organ donors. Hepatocyte transplantation is a promising alternative that could fill the medical need, in particular as the bridge to liver transplantation. Encapsulated porcine hepatocytes represent an unlimited source that could function as a bioreactor requiring minimal immunosuppression. Besides patients with acute liver failure, patients with alcoholic hepatitis who are unresponsive to a short course of corticosteroids are a target for hepatocyte transplantation. In this review we present an overview of the innate immune barriers in hepatocyte xenotransplantation, including the role of complement and natural antibodies; the role of phagocytic cells and ligands like CD47 in the regulation of phagocytic cells; and the role of Natural Killer cells. We present also some illustrations of physiological species incompatibilities in hepatocyte xenotransplantation, such as incompatibilities in the coagulation system. An overview of the methodology for cell microencapsulation is presented, followed by proof-of-concept studies in rodent and nonhuman primate models of fulminant liver failure: these studies document the efficacy of microencapsulated porcine hepatocytes which warrants progress towards clinical application. Lastly, we present an outline of a provisional clinical trial, that upon completion of preclinical work could start within the upcoming 2-3 years.

[Robotic-assisted hepatic surgery]. / La chirurgie hépatique robot-assistée.

Open surgery is currently the gold standard for most liver resection. Laparoscopic hepatic surgery is currently gaining significance, but technical challenges remain. Surgical robotics has been developed to overcome these technical limitations and to enable more difficult minimally invasive procedures. At our institution, 16 robotic hepatic resections have been performed since 2010. Shorter length of stay on intermediate care unit and shorter overall hospitalization has been observed with the robotic patients when compared to open hepatic resection. Overall, the literature shows promising data with demonstration of general feasibility of robotic liver surgery. However, more systematic research is needed to precisely determine the potential advantages of robotics over alternative approaches and its overall role for hepatic resections.

Integrated analysis of mRNA and miRNA expression in response to interleukin-6 in hepatocytes.

The expression of plasma proteins changes dramatically as a result of cytokine induction, particularly interleukin-6, and their levels are used as clinical markers of inflammation. miRNAs are important regulators of gene expression and play significant roles in many inflammatory diseases and processes. The interactions between miRNAs and the genes that they regulate during the acute phase response have not been investigated. We examined the effects of IL-6 stimulation on the transcriptome and miRNome of human and mouse primary hepatocytes and the HepG2 cell line. Using an integrated analysis, we identified differentially expressed miRNAs whose seed sequences are significantly enriched in the 3' untranslated regions of differentially expressed genes, many of which are involved in inflammation-related pathways. Our finding that certain miRNAs may de-repress critical acute phase proteins within acute timeframes has important biological and clinical implications.

Inflammatory Chemokines MIP-1δ and MIP-3α Are Involved in the Migration of Multipotent Mesenchymal Stromal Cells Induced by Hepatoma Cells.

In vivo, bone marrow-derived multipotent mesenchymal stromal cells (MSC) have been identified at sites of tumors, suggesting that specific signals mobilize and activate MSC to migrate to areas surrounding tumors. The signals and migratory mechanisms that guide MSC are not well understood. Here, we investigated the migration of human MSC induced by conditioned medium of Huh-7 hepatoma cells (Huh-7 CM). Using a transwell migration system, we showed that human MSC migration was increased in the presence of Huh-7 CM. Using a human cytokine antibody array, we detected increased levels of MIP-1δ and MIP-3α in Huh-7 CM. Recombinant chemokines MIP-1δ and MIP-3α induced MSC migration. Anti-MIP-1δ and anti-MIP-3α antibodies added to Huh-7 CM decreased MSC migration, further suggesting that MIP-1δ and MIP-3α were implicated in the Huh-7 CM-induced MSC migration. By real-time polymerase chain reaction, we observed an absence of chemokine receptors CCR2 and CXCR2 and low expression of CCR1, CCR5, and CCR6 in MSC. Expression of these chemokine receptors was not regulated by Huh-7 CM. Furthermore, matrix metalloproteinase 1 (MMP-1) expression was strongly increased in MSC after incubation with Huh-7 CM, suggesting that MSC migration depends on MMP-1 activity. The signaling pathway MAPK/ERK was activated by Huh-7 CM but its inhibition by PD98059 did not impair Huh-7 CM-induced MSC migration. Further, long-term incubation of MSC with MIP-1δ increased α-smooth muscle actin expression, suggesting its implication in the Huh-7 CM-induced evolvement of MSC into myofibroblasts. In conclusion, we report that two inflammatory cytokines, MIP-1δ and MIP-3α, are able to increase MSC migration in vitro. These cytokines might be responsible for migration and evolvement of MSC into myofibroblasts around tumors.

Microencapsulated human mesenchymal stem cells decrease liver fibrosis in mice.

BACKGROUND & AIMS: Mesenchymal stem cell (MSC) transplantation was shown to be effective for the treatment of liver fibrosis, but the mechanisms of action are not yet fully understood. We transplanted encapsulated human MSCs in two mouse models of liver fibrosis to determine the mechanisms behind the protective effect. METHODS: Human bone marrow-derived MSCs were microencapsulated in novel alginate-polyethylene glycol microspheres. In vitro, we analyzed the effect of MSC-conditioned medium on the activation of hepatic stellate cells and the viability, proliferation, cytokine secretion, and differentiation capacity of encapsulated MSCs. The level of fibrosis induced by bile duct ligation (BDL) or carbon tetrachloride (CCl4) was assessed after intraperitoneal transplantation of encapsulated MSCs, encapsulated human fibroblasts, and empty microspheres. RESULTS: MSC-conditioned medium inhibited hepatic stellate cell activation and release of MSC secreted anti-apoptotic (IL-6, IGFBP-2) and anti-inflammatory (IL-1Ra) cytokines. Viability, proliferation, and cytokine secretion of microencapsulated MSCs were similar to those of non-encapsulated MSCs. Within the microspheres, MSCs maintained their capacity to differentiate into adipocytes, chondrocytes, and osteocytes. 23% (5/22) of the MSC clones were able to produce anti-inflammatory IL-1Ra in vitro. Microencapsulated MSCs significantly delayed the development of BDL- and CCl4-induced liver fibrosis. Fibroblasts had an intermediate effect against CCl4-induced fibrosis. Mice transplanted with encapsulated MSCs showed lower mRNA levels of collagen type I, whereas levels of matrix metalloproteinase 9 were significantly higher. Human IL-1Ra was detected in the serum of 36% (4/11) of the mice transplanted with microencapsulated MSCs. CONCLUSIONS: MSC-derived soluble molecules are responsible for an anti-fibrotic effect in experimental liver fibrosis.