The Regulatory Approach for Faecal Microbiota Transplantation as Treatment for Clostridioides difficile Infection in Italy.

Faecal microbiota transplantation (FMT) is regarded as an efficacious treatment for recurrent C. difficile infection. Unfortunately, widespread patient access is hindered by regulatory hurdles, which are the primary barriers to incorporating FMT into clinical practice. At the European and International level, there is no uniform perspective on FMT classification, and a coordinated effort is desirable to solve this regulatory puzzle. In this communication, we report the regulatory principles and the implementation approach for FMT application in Italy. Our experience suggests that the EU Tissue and Cell Directives are suited to ensure safe and efficient FMT for C. difficile management, especially through extensive high-quality donor selection and full traceability maintenance.

Intermediate-Intensity Autologous Hematopoietic Stem Cell Transplantation Reduces Serum Neurofilament Light Chains and Brain Atrophy in Aggressive Multiple Sclerosis.

Background: Autologous haematopoietic stem cell transplantation (AHSCT) is highly effective in reducing new inflammatory activity in aggressive multiple sclerosis (MS). A remarkable decrease of serum neurofilament light chains (sNfL) concentration, a marker of axonal damage, was reported in MS following high-intensity regimen AHSCT, but hints for potential neurotoxicity had emerged. sNfL and brain atrophy were therefore analysed in a cohort of patients with aggressive MS treated with intermediate-intensity AHSCT, exploring whether sNfL might be a reliable marker of disability progression independent from new inflammation (i.e. relapses and/or new/gadolinium-enhancing MRI focal lesions). Methods: sNfL concentrations were measured using SIMOA methodology in peripheral blood from relapsing-remitting (RR-) or secondary-progressive (SP-) MS patients undergoing AHSCT (MS AHSCT), collected before transplant and at months 6 and 24 following the procedure. sNfL measured at a single timepoint in SP-MS patients not treated with AHSCT without recent inflammatory activity (SP-MS CTRL) and healthy subjects (HD) were used as controls. The rate of brain volume loss (AR-BVL) was also evaluated by MRI in MS AHSCT cases. Results: Thirty-eight MS AHSCT (28 RR-MS; 10 SP-MS), 22 SP-MS CTRL and 19 HD were included. Baseline median sNfL concentrations were remarkably higher in the MS AHSCT than in the SP-MS CTRL and HD groups (p = 0.005 and <0.0001, respectively), and levels correlated with recent inflammatory activity. After a marginal (not significant) median increase observed at month 6, at month 24 following AHSCT sNfL concentrations decreased compared to baseline by median 42.8 pg/mL (range 2.4-217.3; p = 0.039), reducing by at least 50% in 13 cases, and did not differ from SP-MS CTRL (p = 0.110) but were still higher than in HD (p < 0.0001). Post-AHSCT AR-BVL normalised in 55% of RR-MS and in 30% of SP-MS. The effectiveness and safety of AHSCT were aligned with the literature. Conclusion: sNfL concentrations correlated with recent inflammatory activity and were massively and persistently reduced by intermediate-intensity AHSCT. Association with response to treatment assessed by clinical or MRI outcomes was not observed, suggesting a good sensitivity of sNfL for recent inflammatory activity but low sensitivity in detecting ongoing axonal damage independent from new focal inflammation.

The Italian National Faecal Microbiota Transplantation Program: a coordinated effort against Clostridioides difficile infection.

Clostridioides (previously Clostridium) difficile infection (CDI) is a common cause of antibiotic-associated diarrhea, whose symptoms range from mild diarrhea to life-threatening pseudomembranous colitis. CDI is characterized by significant recurrence rate following initial resolution and recurrent C. difficile infection (rCDI) represents an onerous burden for the healthcare systems. Conventional antibiotic-based approaches are generally used for the treatment of rCDI but the effective therapy remains elusive. Recently, the faecal microbiota transplantation (FMT) has emerged as an alternative therapeutic strategy against rCDI, with high treatment success rate. In 2018, the Italian National FMT Program was launched, with the aim to provide high quality standards in FMT application to adults with rCDI not responding to antibiotic therapy. Here, we sketch out the key characteristics and the progress of the Italian National FMT Program during the COVID-19 pandemic.

The acidic tumor microenvironment drives a stem-like phenotype in melanoma cells.

Acidosis characterizes the microenvironment of most solid tumors and is considered a new hallmark of cancer. It is mainly caused by both "aerobic" and "anaerobic" glycolysis of differently adapted cancer cells, with the final product lactic acid being responsible of the extracellular acidification. Many evidences underline the role of extracellular acidosis in tumor progression. Among the different findings, we demonstrated that acidosis-exposed cancer cells are characterized by an epithelial-to-mesenchymal transition phenotype with high invasive ability, high resistance to apoptosis, anchorage-independent growth, and drug therapy. Acidic melanoma cells over-express SOX2, which is crucial for the maintenance of their oxidative metabolism, and carbonic anhydrase IX, that correlates with poor prognosis of cancer patients. Considering these evidences, we realized that the profile outlined for acid cancer cells inevitably remind us the stemness profile. Therefore, we wondered whether extracellular acidosis might induce in cancer cells the acquisition of stem-like properties and contribute to the expansion of the cancer stem cell sub-population. We found that a chronic adaptation to acidosis stimulates in cancer cells the expression of stem-related markers, also providing a high in vitro/in vivo clonogenic and trans-differentiating ability. Moreover, we observed that the acidosis-induced stem-like phenotype of melanoma cells was reversible and related to the EMT induction. These findings help to characterize a further aspect of stem cell niche, contributing to the sustainment and expansion of cancer stem cell subpopulation. Thus, the usage of agents controlling tumor extracellular acidosis might acquire great importance in the clinic for the treatment of aggressive solid tumor. KEY MESSAGES: • Extracellular acidosis up-regulates EMT and stem-related markers in melanoma cells • Acidic medium up-regulates in vitro self-renewal capacity of melanoma cells • Chronic acidosis adaptation induces trans-differentiation ability in melanoma cells • Melanoma cells adapted to acidosis show higher tumor-initiating potential than control cells • Extracellular acidosis promotes a stem-like phenotype in prostate and colorectal carcinoma cells.

The TCR Repertoire Reconstitution in Multiple Sclerosis: Comparing One-Shot and Continuous Immunosuppressive Therapies.

Natalizumab (NTZ) and autologous hematopoietic stem cell transplantation (AHSCT) are two successful treatments for relapsing-remitting multiple sclerosis (RRMS), an autoimmune T-cell-driven disorder affecting the central nervous system that is characterized by relapses interspersed with periods of complete or partial recovery. Both RRMS treatments have been documented to impact T-cell subpopulations and the T-cell receptor (TCR) repertoire in terms of clone frequency, but, so far, the link between T-cell naive and memory populations, autoimmunity, and treatment outcome has not yet been established hindering insight into the post-treatment TCR landscape of MS patients. To address this important knowledge gap, we tracked peripheral T-cell subpopulations (naïve and memory CD4+ and CD8+) across 15 RRMS patients before and after two years of continuous treatment (NTZ) and a single treatment course (AHSCT) by high-throughput TCRß sequencing. We found that the two MS treatments left treatment-specific multidimensional traces in patient TCRß repertoire dynamics with respect to clonal expansion, clonal diversity and repertoire architecture. Comparing MS TCR sequences with published datasets suggested that the majority of public TCRs belonged to virus-associated sequences. In summary, applying multi-dimensional computational immunology to a TCRß dataset of treated MS patients, we show that qualitative changes of TCRß repertoires encode treatment-specific information that may be relevant for future clinical trials monitoring and personalized MS follow-up, diagnosis and treatment regimes. Natalizumab (NTZ) and autologous hematopoietic stem cell transplantation (AHSCT) are two successful treatments for relapsing-remitting multiple sclerosis (RRMS), an autoimmune T-cell-driven disorder affecting the central nervous system that is characterized by relapses interspersed with periods of complete or partial recovery. Both RRMS treatments have been documented to impact T-cell subpopulations and the T-cell receptor (TCR) repertoire in terms of clone frequency, but, so far, the link between T-cell naive and memory populations, autoimmunity, and treatment outcome has not yet been established hindering insight into the posttreatment TCR landscape of MS patients. To address this important knowledge gap, we tracked peripheral T-cell subpopulations (naive and memory CD4+ and CD8+) across 15 RRMS patients before and after 2 years of continuous treatment (NTZ) and a single treatment course (AHSCT) by high-throughput TCRß sequencing. We found that the two MS treatments left treatment-specific multidimensional traces in patient TCRß repertoire dynamics with respect to clonal expansion, clonal diversity, and repertoire architecture. Comparing MS TCR sequences with published datasets suggested that the majority of public TCRs belonged to virus-associated sequences. In summary, applying multidimensional computational immunology to a TCRß dataset of treated MS patients, we show that qualitative changes of TCRß repertoires encode treatment-specific information that may be relevant for future clinical trials monitoring and personalized MS follow-up, diagnosis, and treatment regimens.

A non-traditional approach to cryopreservation by ultra-rapid cooling for human mesenchymal stem cells.

Cryopreservation is the most common method for long-term cell storage. Successful cryopreservation of cells depends on optimal freezing conditions, freezer storage and a proper thawing technique to minimize the cellular damage that can occur during the cryopreservation process. These factors are especially critical for sensitive stem cells with a consequential and significant impact on viability and functionality. Until now, slow-freezing has been the routine method of cryopreservation but, more recently rapid-cooling techniques have also been proposed. In this study, an ultra-rapid cooling technique [1] was performed for the first time on human mesenchymal stem cells and the effectiveness evaluated in comparison with the conventional slow-freezing procedure. A thin nylon-membrane carrier was used combined with different cryoprotective agents: dimethyl sulfoxide, ethylene glycol and/or trehalose. Various aspects of the low cryoprotective doses and the ultra-rapid cooling procedure of the human mesenchymal stem cells were examined including: the physical properties of the nylon-support, cells encumbrance, viability, proliferation and differentiation. The expression of cell surface markers and apoptosis were also investigated. The study used an ultra-rapid cooling/warming method and showed an overall cell integrity preservation (83-99%), with no significant differences between dimethyl sulfoxide or ethylene glycol treatment (83-87%) and a substantial cell viability of 68% and 51%, respectively. We confirmed a discrepancy also observed by other authors in cell viability and integrity, which implies that caution is necessary when assessing and reporting cell viability data.

Human fetal adrenal cells retain age-related stem- and endocrine-differentiation potential in culture.

The adrenal gland is a multiendocrine organ with a steroidogenic mesenchymal cortex and an inner catecholamine-producing medulla of neuroendocrine origin. After embryonic development, this plastic organ undergoes a functional postnatal remodeling. Elucidating these complex processes is pivotal for understanding the early bases of functional endocrine disorders and tumors affecting the mature gland. We developed an in vitro human adrenal cell model derived from fetal adrenal specimens at different gestational ages, consisting of neuroendocrine and cortical components and expressing the zona and functional markers of the original fetal organ. These cortical and neuroendocrine progenitor cells retain in vitro an intrinsic gestational-age-related differentiation and functional program. In vitro these cells spontaneously form 3-dimensional structure organoids with a structure similar to the fetal gland. The organoids show morphofunctional features and adrenal steroidogenic factor, steroid acute regulatory, cytochrome-P450-17A1, dosage-sensitive, sex-reversal, adrenal hypoplasia-critical region on chromosome X protein , NOTCH1, and nephroblastoma overexpressed/cysteine-rich protein 61/connective tissue growth factor/nephroblastoma overexpressed gene-3; stem (BMI1, nestin); and chromaffin (chromogranin A, tyrosine hydroxylase) markers similar to those of the populations of origin. This in vitro human adrenal system represents a unique but preliminar model for investigating the pathophysiological processes underlying physiologic adrenal remodeling and pathologic alterations involved in organ hypo- and hyperplasia and cancer.-Poli, G., Sarchielli, E., Guasti, D., Benvenuti, S., Ballerini, L., Mazzanti, B., Armignacco, R., Cantini, G., Lulli, M., Chortis, V., Arlt, W., Romagnoli, P., Vannelli, G. B., Mannelli, M., Luconi, M. Human fetal adrenal cells retain age-related stem- and endocrine-differentiation potential in culture.

Cord blood-derived cytokine-induced killer cells combined with blinatumomab as a therapeutic strategy for CD19+ tumors.

BACKGROUND: Cytokine-induced killer cells (CIKs) are an advanced therapeutic medicinal product (ATMP) that has shown therapeutic activity in clinical trials but needs optimization. We developed a novel strategy using CIKs from banked cryopreserved cord blood units (CBUs) combined with bispecific antibody (BsAb) blinatumomab to treat CD19+ malignancies. METHODS: CB-CIKs were expanded in vitro and fully characterized in comparison with peripheral blood (PB)-derived CIKs. RESULTS: CB-CIKs, like PB-CIKs, were mostly CD3+ T cells with mean 45% CD3+CD56+ and expressing mostly TCR(T cell receptor)αß with a TH1 phenotype. CB-CIK cultures had, however, a larger proportion of CD4+ cells, mostly CD56-, as well as a greater proportion of naïve CCR7+CD45RA+ and a lower percentage of effector memory cells, compared with PB-CIKs. CB-CIKs were very similar to PB-CIKs in their expression of a large panel of co-stimulatory and inhibitory/exhaustion markers, except for higher CD28 expression among CD8+ cells. Like PB-CIKs, CB-CIKs were highly cytotoxic in vitro against natural killer (NK) cell targets and efficiently lysed CD19+ tumor cells in the presence of blinatumomab, with 30-60% lysis of target cells at very low effector:target ratios. Finally, both CB-CIKs and PB-CIKs, combined with blinatumomab, showed significant therapeutic activity in an aggressive PDX Ph+ CD19+ acute lymphoblastic leukemia model in NOD-SCID mice, without sign of toxicity or graft-versus-host disease. The improved expansion protocol was finally validated in good manufacturing practice conditions, showing reproducible expansion of CIKs from cryopreserved cord blood units with a median of 28.8 × 106 CIK/kg. DISCUSSION: We conclude that CB-CIKs, combined with bispecific T-cell-engaging antibodies, offer a novel, effective treatment strategy for leukemia.

Therapeutic Efficacy of Autologous Non-Mobilized Enriched Circulating Endothelial Progenitors in Patients With Critical Limb Ischemia　- The SCELTA Trial.

BACKGROUND: The therapeutic efficacy of bone marrow mononuclear cells (BM-MNC) autotransplantation in critical limb ischemia (CLI) has been reported. Variable proportions of circulating monocytes express low levels of CD34 (CD14+CD34lowcells) and behave in vitro as endothelial progenitor cells (EPCs). The aim of the present randomized clinical trial was to compare the safety and therapeutic effects of enriched circulating EPCs (ECEPCs) with BM-MNC administration.Methods and Results:ECEPCs (obtained from non-mobilized peripheral blood by immunomagnetic selection of CD14+and CD34+cells) or BM-MNC were injected into the gastrocnemius of the affected limb in 23 and 17 patients, respectively. After a mean of 25.2±18.6-month follow-up, both groups showed significant and progressive improvement in muscle perfusion (primary endpoint), rest pain, consumption of analgesics, pain-free walking distance, wound healing, quality of life, ankle-brachial index, toe-brachial index, and transcutaneous PO2. In ECEPC-treated patients, there was a positive correlation between injected CD14+CD34lowcell counts and the increase in muscle perfusion. The safety profile was comparable between the ECEPC and BM-MNC treatment arms. In both groups, the number of deaths and major amputations was lower compared with eligible untreated patients and historical reference patients. CONCLUSIONS: This study supports previous trials showing the efficacy of BM-MNC autotransplantation in CLI patients and demonstrates comparable therapeutic efficacy between BM-MNC and EPEPCs.

The effects of Exendin-4 on bone marrow-derived mesenchymal cells.

PURPOSE: GLP-1 receptor agonists are antidiabetic drugs currently used in the therapy of type 2 diabetes. Despite several in vitro and in vivo animal studies suggesting a beneficial effect of GLP-1 analogues on bone, in humans their skeletal effects are not clear and clinical studies report conflicting results. METHODS: We differentiated human mesenchymal stromal cells (hMSC) toward the adipogenic and the osteoblastic lineages, analysing the effect of Exendin-4 (EXE) before, during and after specific differentiations. RESULTS: We showed EXE ability to act selectively on a sub-population of hMSC characterised by a more stem potential, shifting them from G1 to S/M phase of cell cycle. We observed that EXE pre-treatment promotes both adipogenic and osteoblastic differentiations, possibly determined by an increased number of uncommitted progenitors. In fully differentiated cells, EXE affects mature adipocytes by increasing lipolysis, otherwise not altering osteoblasts metabolic activity. Moreover, the increased expression of osteoprotegerin, a modulator of the RANK/RANKL system, observed during osteogenic induction in presence of EXE, could negatively modulate osteoclastogenesis. CONCLUSIONS: Our data suggest a complex action of EXE on bone, targeting the proliferation of mesenchymal progenitors, the metabolism of mature adipocytes and the modulation of osteoclastogenesis. Thus, an overall positive effect of this molecule on bone quality might be hypothesised.

Human mesenchymal stromal cell therapy for damaged cochlea repair in nod-scid mice deafened with kanamycin.

BACKGROUND: Kanamycin, mainly used in the treatment of drug-resistant-tuberculosis, is known to cause irreversible hearing loss. Using the xeno-transplant model, we compared both in vitro and in vivo characteristics of human mesenchymal stromal cells (MSCs) derived from adult tissues, bone marrow (BM-MSCs) and adipose tissue (ADSCs). These tissues were selected for their availability, in vitro multipotency and regenerative potential in vivo in kanamycin-deafened nod-scid mice. METHODS: MSCs were isolated from informed donors and expanded ex vivo. We evaluated their proliferation capacity in vitro using the hexosaminidase assay, the phenotypic profile using flow-cytometry of a panel of surface antigens, the osteogenic potential using alkaline phosphatase activity and the adipogenic potential using oil-red-O staining. MSCs were intravenously injected in deafened mice and cochleae, liver, spleen and kidney were sampled 7 and 30 days after transplantation. The dissected organs were analyzed using lectin histochemistry, immunohistochemistry, polymerase chain reaction (PCR) and dual color fluorescence in situ hybridization (DC-FISH). RESULTS: MSCs showed similar in vitro characteristics, but ADSCs appeared to be more efficient after prolonged expansion. Both cell types engrafted in the cochlea of damaged mice, inducing regeneration of the damaged sensory structures. Several hybrid cells were detected in engrafted tissues. DISCUSSION: BM-MSCs and ADSCs showed in vitro characteristics suitable for tissue regeneration and fused with resident cells in engrafted tissues. The data suggest that paracrine effect is the prevalent mechanism inducing tissue recovery. Overall, BM-MSCs and ADSCs appear to be valuable tools in regenerative medicine for hearing loss recovery.

Young Human Cholinergic Neurons Respond to Physiological Regulators and Improve Cognitive Symptoms in an Animal Model of Alzheimer's Disease.

The degeneration of cholinergic neurons of the nucleus basalis of Meynert (NBM) in the basal forebrain (BF) is associated to the cognitive decline of Alzheimer's disease (AD) patients. To date no resolutive therapies exist. Cell-based replacement therapy is a strategy currently under consideration, although the mechanisms underlying the generation of stem cell-derived NBM cholinergic neurons able of functional integration remain to be clarified. Since fetal brain is an optimal source of neuronal cells committed towards a specific phenotype, this study is aimed at isolating cholinergic neurons from the human fetal NBM (hfNBMs) in order to study their phenotypic, maturational and functional properties. Extensive characterization confirmed the cholinergic identity of hfNBMs, including positivity for specific markers (such as choline acetyltransferase) and acetylcholine (Ach) release. Electrophysiological measurements provided the functional validation of hfNBM cells, which exhibited the activation of peculiar sodium (INa) and potassium (IK) currents, as well as the presence of functional cholinergic receptors. Accordingly, hfNBMs express both nicotinic and muscarinic receptors, which were activated by Ach. The hfNBMs cholinergic phenotype was regulated by the nerve growth factor (NGF), through the activation of the high-affinity NGF receptor TrkA, as well as by 17-ß-estradiol through a peculiar recruitment of its own receptors. When intravenously administered in NBM-lesioned rats, hfNBMs determined a significant improvement in memory functions. Histological examination of brain sections showed that hfNBMs (labeled with PKH26 fluorescent dye prior to administration) reached the damaged brain areas. The study provides a useful model to study the ontogenetic mechanisms regulating the development and maintenance of the human brain cholinergic system and to assess new lines of research, including disease modeling, drug discovery and cell-based therapy for AD.

Fully automated, clinical-grade bone marrow processing: a single-centre experience.

BACKGROUND: Clinical grade processing of harvested bone marrow is required in various clinical situations, particularly in the management of ABO mismatching in allogeneic haematopoietic stem cell transplantation (HSCT) and in regenerative medicine. MATERIAL AND METHODS: We report a single-centre experience using a fully automated, clinical grade, closed system (Sepax, Biosafe, Switzerland). From 2003 to 2015, 125 procedures were performed in our laboratory, including buffy-coat production for HSCT (n=58), regenerative medicine in an orthopaedic setting (n=54) and density-gradient separation in a trial for treatment of critical limb ischaemia (n=13). RESULTS: Buffy coat separation resulted in a median volume reduction of 85% (range, 75-87%), providing satisfactory red blood cell depletion (69%, range 30-88%) and a median recovery of CD34 cells of 96% (range, 81-134%) in the setting of allogeneic HSCT. Significantly greater volume reduction (90%; range, 90-92%) and red blood cell depletion (88%; range, 80-93%) were achieved by the new SmartRedux software released for Sepax2, validated in the last eight allogeneic HSCT. The density gradient separation programme resulted in complete red blood cell depletion associated with high CD34 recovery (69%; range, 36-124%). No reactions related to the quality of the product were reported. Time to engraftment following allogeneic HSCT was in the normal range. No cases of microbiological contamination related to the manipulation were reported. DISCUSSION: Clinical grade, automated bone marrow manipulation with Sepax was shown to be effective, giving operator-independent results and could be used for a broad range of clinical applications.

Tumor Necrosis Factor-α Impairs Kisspeptin Signaling in Human Gonadotropin-Releasing Hormone Primary Neurons.

Context: Inflammatory pathways may impair central regulatory networks involving gonadotropin-releasing hormone (GnRH) neuron activity. Studies in humans are limited by the lack of human GnRH neuron cell lines. Objective: To establish an in vitro model of human GnRH neurons and analyze the effects of proinflammatory cytokines. Design: The primary human fetal hypothalamic cells (hfHypo) were isolated from 12-week-old fetuses. Responsiveness to kisspeptin, the main GnRH neurons' physiological regulator, was evaluated for biological characterization. Tumor necrosis factor alpha (TNF-α) was used as a proinflammatory stimulus. Main Outcome Measures: Expression of specific GnRH neuron markers by quantitative reverse transcription-polymerase chain reaction, flow cytometry, and immunocytochemistry analyses; and GnRH-releasing ability and electrophysiological changes in response to kisspeptin. Results: The primary hfHypo showed a high percentage of GnRH-positive cells (80%), expressing a functional kisspeptin receptor (KISS1R) and able to release GnRH in response to kisspeptin. TNF-α exposure determined a specific inflammatory intracellular signaling and reduced GnRH secretion, KISS1R expression, and kisspeptin-induced depolarizing effect. Moreover, hfHypo possessed a primary cilium, whose assembly was inhibited by TNF-α. Conclusion: The hfHypo cells represent a novel tool for investigations on human GnRH neuron biology. TNF-α directly affects GnRH neuron function by interfering with KISS1R expression and ciliogenesis, thereby impairing kisspeptin signaling.

Triazole RGD antagonist reverts TGFß1-induced endothelial-to-mesenchymal transition in endothelial precursor cells.

Fibrosis is the dramatic consequence of a dysregulated reparative process in which activated fibroblasts (myofibroblasts) and Transforming Growth Factor ß1 (TGFß1) play a central role. When exposed to TGFß1, fibroblast and epithelial cells differentiate in myofibroblasts; in addition, endothelial cells may undergo endothelial-to-mesenchymal transition (EndoMT) and actively participate to the progression of fibrosis. Recently, the role of αv integrins, which recognize the Arg-Gly-Asp (RGD) tripeptide, in the release and signal transduction activation of TGFß1 became evident. In this study, we present a class of triazole-derived RGD antagonists that interact with αvß3 integrin. Above different compounds, the RGD-2 specifically interferes with integrin-dependent TGFß1 EndoMT in Endothelial Colony-Forming Cells (ECPCs) derived from circulating Endothelial Precursor Cells (ECPCs). The RGD-2 decreases the amount of membrane-associated TGFß1, and reduces both ALK5/TGFß1 type I receptor expression and Smad2 phosphorylation in ECPCs. We found that RGD-2 antagonist reverts EndoMT, reducing α-smooth muscle actin (α-SMA) and vimentin expression in differentiated ECPCs. Our results outline the critical role of integrin in fibrosis progression and account for the opportunity of using integrins as target for anti-fibrotic therapeutic treatment.

Local injection of bone marrow progenitor cells for the treatment of anal sphincter injury: in-vitro expanded versus minimally-manipulated cells.

BACKGROUND: Anal incontinence is a disabling condition that adversely affects the quality of life of a large number of patients, mainly with anal sphincter lesions. In a previous experimental work, in-vitro expanded bone marrow (BM)-derived mesenchymal stem cells (MSC) were demonstrated to enhance sphincter healing after injury and primary repair in a rat preclinical model. In the present article we investigated whether unexpanded BM mononuclear cells (MNC) may also be effective. METHODS: Thirty-two rats, divided into groups, underwent sphincterotomy and repair (SR) with primary suture of anal sphincters plus intrasphincteric injection of saline (CTR), or of in-vitro expanded MSC, or of minimally manipulated MNC; moreover, the fourth group underwent sham operation. At day 30, histologic, morphometric, in-vitro contractility, and functional analysis were performed. RESULTS: Treatment with both MSC and MNC improved muscle regeneration and increased contractile function of anal sphincters after SR compared with CTR (p < 0.05). No significant difference was observed between the two BM stem cell types used. GFP-positive cells (MSC and MNC) remained in the proximity of the lesion site up to 30 days post injection. CONCLUSIONS: In the present study we demonstrated in a preclinical model that minimally manipulated BM-MNC were as effective as in-vitro expanded MSC for the recovery of anal sphincter injury followed by primary sphincter repair. These results may serve as a basis for improving clinical applications of stem cell therapy in human anal incontinence treatment.

Searching for Classical Brown Fat in Humans: Development of a Novel Human Fetal Brown Stem Cell Model.

The potential therapeutic applications of targeting brown adipose tissue open new clinical avenues in fighting against metabolic pathologies. However, due to the limited extension in adult humans of brown depots, which are dramatically reduced after birth, solid cell models to study human brown adipogenesis and its regulatory factors in pathophysiology are urgently needed. Here, we generated a novel human model of brown adipose stem cells, hfB-ASC, derived for the first time from fetal interscapular brown fat depots. Besides the characterization of their stem and classical brown adipose properties, we demonstrated that these cells retain a specific intrinsic differentiation program to functional brown adipocytes, even spontaneously generating organoid structures with brown features. Moreover, for the first time, we investigated the thermogenic and electrophysiological activity of the in vitro-derived fetal brown adipocytes compared to their undifferentiated precursors hfB-ASC, in basal and norepinephrine-induced conditions. In conclusion, from interscapular brown fat of the human fetus we developed and functionally characterized a novel physiological brown adipose stem cell model early programmed to brown differentiation, which may represent a unique opportunity for further studies on brown adipogenesis processes in humans as well as the most suitable target to study novel therapeutic approaches for stimulating brown activity in metabolic pathologies. Stem Cells 2016;34:1679-1691.

Tadalafil reduces visceral adipose tissue accumulation by promoting preadipocytes differentiation towards a metabolically healthy phenotype: Studies in rabbits.

Development of metabolically healthy adipocytes within dysfunctional adipose tissue may represent an attractive way to counteract metabolic syndrome (MetS). In an experimental animal model of high fat diet (HFD)-induced MetS, in vivo, long- and short-term tadalafil treatments were able to reduce visceral adipose tissue (VAT) accumulation and hypertriglyceridemia, and to induce the expression in VAT of the brown fat-specific marker, uncoupling protein 1 (UCP1). VAT preadipocytes (PAD), isolated from the tadalafil-treated HFD rabbits, showed: i) a multilocular morphology; ii) an increased expression of brown fat-specific genes (such as UCP1 and CIDEA); iii) improved mitochondrial structure and dynamic and reduced superoxide production; iv) improved insulin sensitivity. Similar effects were obtained after in vitro tadalafil treatment in HFD rPAD. In conclusion, tadalafil counteracted HFD-associated VAT alterations, by restoring insulin-sensitivity and prompting preadipocytes differentiation towards a metabolically healthy phenotype.

MSCs seeded on bioengineered scaffolds improve skin wound healing in rats.

Growing evidence has shown the promise of mesenchymal stromal cells (MSCs) for the treatment of cutaneous wound healing. We have previously demonstrated that MSCs seeded on an artificial dermal matrix, Integra (Integra Lifesciences Corp., Plainsboro, NJ) enriched with platelet-rich plasma (Ematrix) have enhanced proliferative potential in vitro as compared with those cultured on the scaffold alone. In this study, we extended the experimentation by evaluating the efficacy of the MSCs seeded scaffolds in the healing of skin wounds in an animal model in vivo. It was found that the presence of MSCs within the scaffolds greatly ameliorated the quality of regenerated skin, reduced collagen deposition, enhanced reepithelization, increased neo-angiogenesis, and promoted a greater return of hair follicles and sebaceous glands. The mechanisms involved in these beneficial effects were likely related to the ability of MSCs to release paracrine factors modulating the wound healing response. MSC-seeded scaffolds, in fact, up-regulated matrix metalloproteinase 9 expression in the extracellular matrix and enhanced the recruitment of endogenous progenitors during tissue repair. In conclusion, the results of this study provide evidence that the treatment with MSC-seeded scaffolds of cutaneous wounds contributes to the recreation of a suitable microenvironment for promoting tissue repair/regeneration at the implantation sites.

Donor-Specific Anti-HLA Antibodies in Huntington's Disease Recipients of Human Fetal Striatal Grafts.

Fetal grafting in a human diseased brain was thought to be less immunogenic than other solid organ transplants, hence the minor impact on the efficacy of the transplant. How much prophylactic immune protection is required for neural allotransplantation is also debated. High-sensitive anti-HLA antibody screening in this field has never been reported. Sixteen patients with Huntington's disease underwent human fetal striatal transplantation in the frame of an open-label observational trial, which is being carried out at Florence University. All patients had both brain hemispheres grafted in two separate robotic-stereotactic procedures. The trial started in February 2006 with the first graft to the first patient (R1). R16 was given his second graft on March 2011. All patients received triple immunosuppressive treatment. Pre- and posttransplant sera were analyzed for the presence of anti-HLA antibodies using the multiplexed microsphere-based suspension array Luminex xMAP technology. Median follow-up was 38.5 months (range 13-85). Six patients developed anti-HLA antibodies, which turned out to be donor specific. Alloimmunization occurred in a time window of 0-49 months after the first neurosurgical procedure. The immunogenic determinants were non-self-epitopes from mismatched HLA antigens. These determinants were both public epitopes shared by two or more HLA molecules and private epitopes unique to individual HLA molecules. One patient had non-donor-specific anti-HLA antibodies in her pretransplant serum sample, possibly due to previous sensitization events. Although the clinical significance of donor-specific antibodies is far from being established, particularly in the setting of neuronal transplantation, these findings underline the need of careful pre- and posttransplant immunogenetic evaluation of patients with intracerebral grafts.