Small Extracellular Vesicles from Human Amniotic Fluid Samples as Promising Theranostics.

Since the first evidence that stem cells can provide pro-resolving effects via paracrine secretion of soluble factors, growing interest has been addressed to define the most ideal cell source for clinical translation. Leftover or clinical waste samples of human amniotic fluid obtained following prenatal screening, clinical intervention, or during scheduled caesarean section (C-section) delivery at term have been recently considered an appealing source of mesenchymal progenitors with peculiar regenerative capacity. Human amniotic fluid stem cells (hAFSC) have been demonstrated to support tissue recovery in several preclinical models of disease by exerting paracrine proliferative, anti-inflammatory and regenerative influence. Small extracellular vesicles (EVs) concentrated from the hAFSC secretome (the total soluble trophic factors secreted in the cell-conditioned medium, hAFSC-CM) recapitulate most of the beneficial cell effects. Independent studies in preclinical models of either adult disorders or severe diseases in newborns have suggested a regenerative role of hAFSC-EVs. EVs can be eventually concentrated from amniotic fluid (hAF) to offer useful prenatal information, as recently suggested. In this review, we focus on the most significant aspects of EVs obtained from either hAFSC and hAF and consider the current challenges for their clinical translation, including isolation, characterization and quantification methods.

Targeting PIK3CA Actionable Mutations in the Circulome: A Proof of Concept in Metastatic Breast Cancer.

The study of circulating cancer-derived components (circulome) is considered the new frontier of liquid biopsy. Despite the recognized role of circulome biomarkers, their comparative molecular profiling is not yet routine. In advanced breast cancer (BC), approximately 40% of hormone-receptor-positive, HER2-negative BC cases harbor druggable PIK3CA mutations suitable for combined alpelisib/fulvestrant treatment. This pilot study investigates PIK3CA mutations in circulating tumor DNA (ctDNA), tumor cells (CTCs), and extracellular vesicles (EVs) with the aim of determining which information on molecular targetable profiling could be recollected in each of them. The in-depth molecular analysis of four BC patients demonstrated, as a proof-of-concept study, that it is possible to retrieve mutational information in the three components. Patient-specific PIK3CA mutations were found in both tissue and ctDNA and in 3/4 cases, as well as in CTCs, in the classical population (large-sized CD45-/EpCAM+/- cells), and/or in the "non-conventional" sub-population (smaller-sized CD44+/EpCAM-/CD45- cells). Consistent mutational profiles of EVs with CTCs suggest that they may have been released by CTCs. This preliminary evidence on the molecular content of the different circulating biomaterials suggests their possible function as a mirror of the intrinsic heterogeneity of BC. Moreover, this study demonstrates, through mutational assessment, the tumor origin of the different CTC sub-populations sustaining the translational value of the circulome for a more comprehensive picture of the disease.

Comprehensive Profiling of Secretome Formulations from Fetal- and Perinatal Human Amniotic Fluid Stem Cells.

We previously reported that c-KIT+ human amniotic-fluid derived stem cells obtained from leftover samples of routine II trimester prenatal diagnosis (fetal hAFS) are endowed with regenerative paracrine potential driving pro-survival, anti-fibrotic and proliferative effects. hAFS may also be isolated from III trimester clinical waste samples during scheduled C-sections (perinatal hAFS), thus offering a more easily accessible alternative when compared to fetal hAFS. Nonetheless, little is known about the paracrine profile of perinatal hAFS. Here we provide a detailed characterization of the hAFS total secretome (i.e., the entirety of soluble paracrine factors released by cells in the conditioned medium, hAFS-CM) and the extracellular vesicles (hAFS-EVs) within it, from II trimester fetal- versus III trimester perinatal cells. Fetal- and perinatal hAFS were characterized and subject to hypoxic preconditioning to enhance their paracrine potential. hAFS-CM and hAFS-EV formulations were analyzed for protein and chemokine/cytokine content, and the EV cargo was further investigated by RNA sequencing. The phenotype of fetal- and perinatal hAFS, along with their corresponding secretome formulations, overlapped; yet, fetal hAFS showed immature oxidative phosphorylation activity when compared to perinatal ones. The profiling of their paracrine cargo revealed some differences according to gestational stage and hypoxic preconditioning. Both cell sources provided formulations enriched with neurotrophic, immunomodulatory, anti-fibrotic and endothelial stimulating factors, and the immature fetal hAFS secretome was defined by a more pronounced pro-vasculogenic, regenerative, pro-resolving and anti-aging profile. Small RNA profiling showed microRNA enrichment in both fetal- and perinatal hAFS-EV cargo, with a stably- expressed pro-resolving core as a reference molecular signature. Here we confirm that hAFS represents an appealing source of regenerative paracrine factors; the selection of either fetal or perinatal hAFS secretome formulations for future paracrine therapy should be evaluated considering the specific clinical scenario.

Atomic force microscopy for biomechanical and structural analysis of human dermis: A complementary tool for medical diagnosis and therapy monitoring.

Skin mechanical properties are usually measured considering the entire skin thickness and very little is known about the mechanical behaviour of individual skin layers. We propose atomic force microscopy (AFM) as a tool to quantify nanoscale changes in the biomechanical properties and ultrastructure of human papillary dermis exposed to different mechanical and physical stimuli. Samples from 3 human skin biopsies were studied: one stretched by obesity, one subjected to a high level of sun exposure and normal skin as control. Slices of the papillary dermis layer were harvested at controlled depths from each skin biopsy and 25 µm2 areas of each slice were imaged and D-periodicity of collagen fibres measured by AFM, together with their stiffness. Standard histological analysis was also carried out to correlate biochemical properties and their distribution with stiffness and topography. We obtained similar stiffness values between the sample affected by obesity and the control sample at any depth level into the dermis, while the sun-exposed sample presented a significantly lower stiffness. Additionally, all samples presented an increase in the stiffness at higher depths into the papillary dermis layer. Collagen fibres close to the epidermis of sample affected either by obesity and sun exposure-the former even more than the latter-are thicker and present a larger D-period than those in the control sample. Our results open the possibility to use structural and mechanical analysis based on AFM as a complementary tool for medical diagnosis and therapy monitoring.

Skin Rejuvenation and Volume Enhancement with the Micro Superficial Enhanced Fluid Fat Injection (M-SEFFI) for Skin Aging of the Periocular and Perioral Regions.

BACKGROUND: Adipose-derived stromal and stem cells (ADSC) in autologous fat promises regenerative advantages, and injected into the dermal and subdermal layers, enhances rejuvenation and volume. However, extremely superficial fat injection with current techniques is limited. OBJECTIVES: Efficacy and viability evaluation of fat harvested with extremely small side port (0.3 mm) cannulae without further tissue manipulation for the correction of aging/thin skin in the periocular and perioral regions. METHODS: Micro-superficial enhanced fluid fat injection (M-SEFFI) harvests adipose tissue with a multi-perforated cannula (0.3 mm), and autologous platelet rich plasma (PRP) is added. The tissue is injected into the dermal region of the periocular and perioral zones. Efficacy and viability were evaluated by histological and cell culture analysis. Clinical assessment included retrospective evaluation according to 1 = no effect, 2 = fair effect, 3 = good effect, 4 = excellent effect. RESULTS: Between June 2014 and July 2015, 65 patients (7 men; mean age 49.7 years) were treated with M-SEFFI. No intraoperative complications or visible lumpiness were recorded. Analysis demonstrated mature, viable adipocytes with a strong stromal component. Following PRP addition, there was a greater proliferation noted in the M-SEFFI compared to the SEFFI (0.5 mm). Mean follow-up was 4.1 months. Clinical assessment by surgeons and patients at 1 month was 3.52 and 3.74, and 6 months 3.06 and 2.6 respectively. CONCLUSIONS: M-SEFFI is effective and viable for lump free skin rejuvenation and volume enhancement, through the extraction of smoother ADSC rich, autologous fat tissue that does not require further tissue manipulation, to correct skin aging. LEVEL OF EVIDENCE: 4 Therapeutic.

Chemical and morphological gradient scaffolds to mimic hierarchically complex tissues: From theoretical modeling to their fabrication.

Porous multiphase scaffolds have been proposed in different tissue engineering applications because of their potential to artificially recreate the heterogeneous structure of hierarchically complex tissues. Recently, graded scaffolds have been also realized, offering a continuum at the interface among different phases for an enhanced structural stability of the scaffold. However, their internal architecture is often obtained empirically and the architectural parameters rarely predetermined. The aim of this work is to offer a theoretical model as tool for the design and fabrication of functional and structural complex graded scaffolds with predicted morphological and chemical features, to overcome the time-consuming trial and error experimental method. This developed mathematical model uses laws of motions, Stokes equations, and viscosity laws to describe the dependence between centrifugation speed and fiber/particles sedimentation velocity over time, which finally affects the fiber packing, and thus the total porosity of the 3D scaffolds. The efficacy of the theoretical model was tested by realizing engineered graded grafts for osteochondral tissue engineering applications. The procedure, based on combined centrifugation and freeze-drying technique, was applied on both polycaprolactone (PCL) and collagen-type-I (COL) to test the versatility of the entire process. A functional gradient was combined to the morphological one by adding hydroxyapatite (HA) powders, to mimic the bone mineral phase. Results show that 3D bioactive morphologically and chemically graded grafts can be properly designed and realized in agreement with the theoretical model. Biotechnol. Bioeng. 2016;113: 2286-2297. © 2016 Wiley Periodicals, Inc.

Interaction Between Breast Cancer Cells and Adipose Tissue Cells Derived from Fat Grafting.

BACKGROUND: Adipose tissue transplantation has the benefit of providing both regenerative and aesthetic outcomes in breast cancer treatment. However, the transplanted tissue can stimulate the growth of residual cancer cells. OBJECTIVES: The aim of this study is to identify the interactions between adipose tissue cell subpopulations and human cancer cell lines. METHODS: Intact adipose tissue from lipofilling procedures as well as fibroblasts derived from adipose tissue, were cocultured in the presence of MDA-MB-231, MCF-7 e ZR-75-1 breast cancer cell lines. The influence on cancer cell lines of fibroblasts, induced to differentiate into specific adipocytes, was also assayed. RESULTS: All cancer cell lines displayed a significant increase in proliferation rate when cocultured in the presence of either intact adipose tissue or induced adipocytes. To a lesser extent, uninduced fibroblasts stimulate breast cancer cell proliferation. CONCLUSIONS: Recent studies have shown that the microenvironment surrounding breast cancer cells may stimulate growth and promote progression of residual cancer cells when surgery is performed on the main tumor mass. Accordingly, the graft of adipose tissue could potentially promote or accelerate the development of a subclinical tumor or support its locoregional recurrence. Our data suggest that adipocytes have a remarkable influence on the proliferation of cancer cell lines. The oncological safety of the lipofilling procedure outcome is still debated; thus, further studies and consistent follow-up examination are needed.

Superficial Enhanced Fluid Fat Injection (SEFFI) to Correct Volume Defects and Skin Aging of the Face and Periocular Region.

BACKGROUND: Although recent research on micro fat has shown the potential advantages of superficial implantation and high stem cell content, clinical applications thus far have been limited. OBJECTIVES: The authors report their experience with superficial enhanced fluid fat injection (SEFFI) for the correction of volume loss and skin aging of the face in general and in the periocular region. METHODS: The finer SEFFI preparation (0.5 mL) was injected into the orbicularis in the periorbital and perioral areas, and the 0.8-mL preparation was injected subdermally elsewhere in the face. RESULTS: The records of 98 consecutive patients were reviewed. Average follow-up time was 6 months, and average volume of implanted fat was 20 mL and 51.4 mL for the 0.5-mL and 0.8-mL preparations, respectively. Good or excellent results were achieved for volume restoration and skin improvement in all patients. Complications were minor and included an oil cyst in 3 patients. The smaller SEFFI quantity (0.5 mL) was well suited to correct volume loss in the eyelids, especially the deep upper sulcus and tear trough, whereas the larger SEFFI content was effective for larger volume deficits in other areas of the face, including the brow, temporal fossa, zygomatic-malar region, nasolabial folds, marionette lines, chin, and lips. CONCLUSIONS: The fat administered by SEFFI is easily harvested via small side-port cannulae, yielding micro fat that is rich in viable adipocytes and stem cells. Both volumes of fat (0.5 mL and 0.8 mL) were effective for treating age-related lipoatrophy, reducing facial rhytids, and improving skin quality. LEVEL OF EVIDENCE: 4 Therapeutic.

Chronic lymphocytic leukemia nurse-like cells express hepatocyte growth factor receptor (c-MET) and indoleamine 2,3-dioxygenase and display features of immunosuppressive type 2 skewed macrophages.

Hepatocyte growth factor, produced by stromal and follicular dendritic cells, and present at high concentrations in the sera of patients with chronic lymphocytic leukemia, prolongs the survival of leukemic B cells by interacting with their receptor, c-MET. It is, however, unknown whether hepatocyte growth factor influences microenvironmental cells, such as nurse-like cells, which deliver survival signals to the leukemic clone. We evaluated the expression of c-MET on nurse-like cells and monocytes from patients with chronic lymphocytic leukemia and searched for phenotypic/functional features supposed to be influenced by the hepatocyte growth factor/c-MET interaction. c-MET is expressed at high levels on nurse-like cells and at significantly higher levels than normal on monocytes from patients. Moreover, the hepatocyte growth factor/c-MET interaction activates STAT3(TYR705) phosphorylation in nurse-like cells. Indoleamine 2,3-dioxygenase, an enzyme modulating T-cell proliferation and induced on normal monocytes after hepatocyte growth factor treatment, was detected together with interleukin-10 on nurse-like cells, and on freshly-prepared patients' monocytes. Immunohistochemical/immunostaining analyses demonstrated the presence of c-MET(+) and indoleamine 2,3-dioxygenase(+) cells in lymph node biopsies, co-expressed with CD68 and vimentin. Furthermore nurse-like cells and chronic lymphocytic monocytes significantly inhibited T-cell proliferation, prevented by anti-transforming growth factor beta and interleukin-10 antibodies and indoleamine 2,3-dioxygenase inhibitors, and supported CD4(+)CD25(high+)/FOXP3(+) T regulatory cell expansion. We suggest that nurse-like cells display features of immunosuppressive type 2 macrophages: higher hepatocyte growth factor levels, produced by leukemic or other microenvironmental surrounding cells, may cooperate to induce M2 polarization. Hepatocyte growth factor may thus have a dual pathophysiological role: directly through enhancement of survival of the leukemic clone and indirectly by favoring T-cell immunosuppression.

Defining an optimal stromal derived factor-1 presentation for effective recruitment of mesenchymal stem cells in 3D.

In "situ" tissue engineering is a promising approach in regenerative medicine, envisaging to potentiate the physiological tissue repair processes by recruiting the host's own cellular progenitors at the lesion site by means of bioactive materials. Despite numerous works focused the attention in characterizing novel chemoattractant molecules, only few studied the optimal way to present signal in the microenvironment, in order to recruit cells more effectively. In this work, we have analyzed the effects of gradients of stromal derived factor-1 (SDF-1) on the migratory behavior of human mesenchymal stem cells (MSCs). We have characterized the expression of the chemokine-associated receptor, CXCR4, using cytofluorimetric and real-time PCR analyses. Gradients of SDF-1 were created in 3D collagen gels in a chemotaxis chamber. Migration parameters were evaluated using different chemoattractant concentrations. Our results show that cell motion is strongly affected by the spatio-temporal features of SDF-1 gradients. In particular, we demonstrated that the presence of SDF-1 not only influences cell motility but alters the cell state in terms of SDF-1 receptor expression and productions, thus modifying the way cells perceive the signal itself. Our observations highlight the importance of a correct stimulation of MSCs by means of SDF-1 in order to implement on effective cell recruitment. Our results could be useful for the creation of a "cell instructive material" that is capable to communicate with the cells and control and direct tissue regeneration. Biotechnol. Bioeng. 2014;111: 2303-2316. © 2014 Wiley Periodicals, Inc.

A novel scaffold geometry for chondral applications: theoretical model and in vivo validation.

A theoretical model of the 3D scaffold internal architecture has been implemented with the aim to predict the effects of some geometrical parameters on total porosity, Young modulus, buckling resistance and permeability of the graft. This model has been adopted to produce porous poly-caprolacton based grafts for chondral tissue engineering applications, best tuning mechanical and functional features of the scaffolds. Material prototypes were produced with an internal geometry with parallel oriented cylindrical pores of 200 µm of radius (r) and an interpore distance/pores radius (d/r) ratio of 1. The scaffolds have been then extensively characterized; progenitor cells were then used to test their capability to support cartilaginous matrix deposition in an ectopic model. Scaffold prototypes fulfill both the chemical-physical requirements, in terms of Young's modulus and permeability, and the functional needs, such as surface area per volume and total porosity, for an enhanced cellular colonization and matrix deposition. Moreover, the grafts showed interesting chondrogenic potential in vivo, besides offering adequate mechanical performances in vitro, thus becoming a promising candidate for chondral tissues repair. Finally, a very good agreement was found between the prediction of the theoretical model and the experimental data. Many assumption of this theoretical model, hereby applied to cartilage, may be transposed to other tissue engineering applications, such as bone substitutes.

Standardized Method to Functionalize Plasma-Extracellular Vesicles via Copper-Free Click Chemistry for Targeted Drug Delivery Strategies.

Extracellular vesicles (EVs) have emerged as potential vehicles for targeted drug delivery and diagnostic applications. However, achieving consistent and reliable functionalization of EV membranes remains a challenge. Copper-catalyzed click chemistry, commonly used for EV surface modification, poses limitations due to cytotoxicity and interference with biological systems. To overcome these limitations, we developed a standardized method for functionalizing an EV membrane via copper-free click chemistry. EVs derived from plasma hold immense potential as diagnostic and therapeutic agents. However, the isolation and functionalization of EVs from such a complex biofluid represent considerable challenges. We compared three different EV isolation methods to obtain an EV suspension with an optimal purity/yield ratio, and we identified sucrose cushion ultracentrifugation (sUC) as the ideal protocol. We then optimized the reaction conditions to successfully functionalize the plasma-EV surface through a copper-free click chemistry strategy with a fluorescently labeled azide, used as a proof-of-principle molecule. Click-EVs maintained their identity, size, and, more importantly, capacity to be efficiently taken up by responder tumor cells. Moreover, once internalized, click EVs partially followed the endosomal recycling route. The optimized reaction conditions and characterization techniques presented in this study offer a foundation for future investigations and applications of functionalized EVs in drug delivery, diagnostics, and therapeutics.

Pilot study investigating BP-180 in extracellular vesicles derived from blister fluid of bullous pemphigoid patients.

Bullous pemphigoid (BP) is an autoimmune blistering disease that targets the haemidesmosomal proteins, mainly BP180. Extracellular vesicles (EVs) have been demonstrated to carry tissue-specific autoantigens in the setting of autoimmune diseases and transplant organ rejection; this phenomenon was demonstrated to have pathogenic implications in autoimmune diseases and to correlate with transplant rejection severity. The purpose of this study was to identify the presence of BP targeted autoantigens in blister fluid derived EVs. We isolated, by size exclusion chromatography, EVs derived from blisters of BP-patients and from suction blisters of healthy donors. EV characterization was performed by flow cytometry and nanoparticle tracking analysis. Western blot analysis was used to investigate the presence of autoantigens. A suspension enriched in EVs was efficiently obtained from blister fluid from patients and healthy donors. EV-enriched fractions were enriched in particles with a size distribution characterizing small-EVs (main peak was present at 94.5 nm). BP180 was found, by western blot analysis, in EVs derived from blister fluid of 3 out 6 BP patients and in none of EVs isolated from suction blister fluid of healthy donors. BP230 and Dsg1 were not detectable in EVs of any of the samples. No specific clinical characteristics seemed to correlate to the presence of BP180 in EVs. The discovery of BP180 in EVs derived from blister fluid might help understanding BP pathogenesis.

In vivo lamellar bone formation in fibre coated MgCHA-PCL-composite scaffolds.

Bio-inspired materials with controlled topography have gained increasing interest in regenerative medicine, because of their ability to reproduce the physical features of natural extracellular matrix, thus amplifying certain biological responses both in vitro and in vivo, such as contact guidance and differentiation. However, information on the ability to adapt this high cell potential to 3D scaffolds, effective to be implanted in clinical bone defect, is still missing. Here, we examine the pattern of bone tissue generated within the implant in an ectopic model, seeding bone marrow progenitor cells onto PCL-MgCHA scaffolds. This composite material presented a porous structure with micro/nanostructured surfaces obtained by combining phase inversion/salt leaching and electrospinning techniques. Histological analysis of grafts harvested after 1-2-6 months from implantation highlights an extent of lamellar bone tissue within interconnected pores of fibre coated PCL-MgCHA composites, whereas uncoated scaffolds displayed sparse deposition of bone. Pure PCL scaffolds did not reveal any trace of bone for the overall 6 months of observation. In conclusion, we show that a structural modification in scaffold design is able to enhance bone regeneration possibly mimicking some physiological cues of the natural tissue.

The influence of plasma technology coupled to chemical grafting on the cell growth compliance of 3D hydroxyapatite scaffolds.

The development of advanced materials with biomimetic features in order to elicit desired biological responses and to guarantee tissue biocompatibility is recently gaining attention for tissue engineering applications. Bioceramics, such as hydroxyapatite-based biomaterials are now used in a number of different applications throughout the body, covering all areas of the skeleton, due to their biological and chemical similarity to the inorganic phases of bones. When bioactive sintered hydroxyapatite (HA) is desired, biomolecular modification of these materials is needed. In the present work, we investigated the influence of plasma surface modification coupled to chemical grafting on the cell growth compliance of HA 3D scaffolds.

A Two-Step Approach to Tune the Micro and Nanoscale Morphology of Porous Niobium Oxide to Promote Osteointegration.

We present a two-step surface modification process to tailor the micro and nano morphology of niobium oxide layers. Niobium was firstly anodized in spark regime in a Ca- and P-containing solution and subsequently treated by acid etching. The effects of anodizing time and applied potential on the surface morphology is investigated with SEM and AFM, complemented by XPS compositional analysis. Anodizing with a limiting potential of 250 V results in the fast growth of oxide layers with a homogeneous distribution of micro-sized pores. Cracks are, however, observed on 250 V grown layers. Limiting the anodizing potential to 200 V slows down the oxide growth, increasing the anodizing time needed to achieve a uniform pore coverage but produces fracture-free oxide layers. The surface nano morphology is further tuned by a subsequent acid etching process that leads to the formation of nano-sized pits on the anodically grown oxide surface. In vitro tests show that the etching-induced nanostructure effectively promotes cell adhesion and spreading onto the niobium oxide surface.

Circulating miRNAs in Breast Cancer Diagnosis and Prognosis.

Great improvement has been made in the diagnosis and therapy of breast cancer patients. However, the identification of biomarkers for early diagnosis, prognosis, therapy assessment and monitoring, including drug resistance and the early detection of micro-metastases, is still lacking. Recently, circulating microRNAs (miRNAs), circulating freely in the blood stream or entrapped in extracellular vesicles (EVs), have been shown to have a potential diagnostic, prognostic or predictive power. In this review, recent findings are summarized, both at a preclinical and clinical level, related to miRNA applicability in the context of breast cancer. Different aspects, including clinical and technical challenges, are discussed, describing the potentialities of miRNA use in breast cancer. Even though more methodological standardized studies conducted in larger and selected patient cohorts are needed to support the effective clinical utility of miRNA as biomarkers, they could represent novel and accessible tools to be transferred into clinical practice.

Preconditioned Mesenchymal Stromal Cell-Derived Extracellular Vesicles (EVs) Counteract Inflammaging.

Inflammaging is one of the evolutionarily conserved mechanisms underlying aging and is defined as the long-term consequence of the chronic stimulation of the innate immune system. As macrophages are intimately involved in initiating and regulating the inflammatory process, their dysregulation plays major roles in inflammaging. The paracrine factors, and in particular extracellular vesicles (EVs), released by mesenchymal stromal cells (MSCs) retain immunoregulatory effects on innate and adaptive immune responses. In this paper, we demonstrate that EVs derived from MSCs preconditioned with hypoxia inflammatory cytokines exerted an anti-inflammatory role in the context of inflammaging. In this study, macrophages isolated from aged mice presented elevated pro-inflammatory factor levels already in basal conditions compared to the young counterpart, and this pre-activation status increased when cells were challenged with IFN-γ. EVs were able to attenuate the age-associated inflammation, inducing a decrease in the expression of TNF-α, iNOS, and the NADase CD38. Moreover, we demonstrate that EVs counteracted the mitochondrial dysfunction that affected the macrophages, reducing lipid peroxidation and hindering the age-associated impairment of mitochondrial complex I activity, oxygen consumption, and ATP synthesis. These results indicate that preconditioned MSC-derived EVs might be exploited as new anti-aging therapies in a variety of age-related diseases.

Investigating the Paracrine Role of Perinatal Derivatives: Human Amniotic Fluid Stem Cell-Extracellular Vesicles Show Promising Transient Potential for Cardiomyocyte Renewal.

Cardiomyocyte renewal represents an unmet clinical need for cardiac regeneration. Stem cell paracrine therapy has attracted increasing attention to resurge rescue mechanisms within the heart. We previously characterized the paracrine effects that human amniotic fluid-derived stem cells (hAFSC) can exert to provide cardioprotection and enhance cardiac repair in preclinical models of myocardial ischemia and cardiotoxicity. Here, we analyze whether hAFSC secretome formulations, namely, hAFSC conditioned medium (hAFSC-CM) over extracellular vesicles (hAFSC-EVs) separated from it, can induce cardiomyocyte renewal. c-KIT+ hAFSC were obtained by leftover samples of II trimester prenatal amniocentesis (fetal hAFSC) and from clinical waste III trimester amniotic fluid during scheduled C-section procedures (perinatal hAFSC). hAFSC were primed under 1% O2 to enrich hAFSC-CM and EVs with cardioactive factors. Neonatal mouse ventricular cardiomyocytes (mNVCM) were isolated from cardiac tissue of R26pFUCCI2 mice with cell cycle fluorescent tagging by mutually exclusive nuclear signal. mNVCM were stimulated by fetal versus perinatal hAFSC-CM and hAFSC-EVs to identify the most promising formulation for in vivo assessment in a R26pFUCCI2 neonatal mouse model of myocardial infarction (MI) via intraperitoneal delivery. While the perinatal hAFSC secretome did not provide any significant cardiogenic effect, fetal hAFSC-EVs significantly sustained mNVCM transition from S to M phase by 2-fold, while triggering cytokinesis by 4.5-fold over vehicle-treated cells. Treated mNVCM showed disorganized expression of cardiac alpha-actinin, suggesting cytoskeletal re-arrangements prior to cell renewal, with a 40% significant downregulation of Cofilin-2 and a positive trend of polymerized F-Actin. Fetal hAFSC-EVs increased cardiomyocyte cell cycle progression by 1.8-fold in the 4-day-old neonatal left ventricle myocardium short term after MI; however, such effect was lost at the later stage. Fetal hAFSC-EVs were enriched with a short isoform of Agrin, a mediator of neonatal heart regeneration acting by YAP-related signaling; yet in vitro application of YAP inhibitor verteporfin partially affected EV paracrine stimulation on mNVCM. EVs secreted by developmentally juvenile fetal hAFSC can support cardiomyocyte renewal to some extension, via intercellular conveyance of candidates possibly involving Agrin in combination with other factors. These perinatal derivative promising cardiogenic effects need further investigation to define their specific mechanism of action and enhance their potential translation into therapeutic opportunity.

An interaction between hepatocyte growth factor and its receptor (c-MET) prolongs the survival of chronic lymphocytic leukemic cells through STAT3 phosphorylation: a potential role of mesenchymal cells in the disease.

BACKGROUND: Chronic lymphocytic leukemia cells are characterized by an apparent longevity in vivo which is lost when they are cultured in vitro. Cellular interactions and factors provided by the microenvironment appear essential to cell survival and may protect leukemic cells from the cytotoxicity of conventional therapies. Understanding the cross-talk between leukemic cells and stroma is of interest for identifying signals supporting disease progression and for developing novel therapeutic strategies. DESIGN AND METHODS: Different cell types, sharing a common mesenchymal origin and representative of various bone marrow components, were used to challenge the viability of leukemic cells in co-cultures and in contact-free culture systems. Using a bioinformatic approach we searched for genes shared by lineages prolonging leukemic cell survival and further analyzed their biological role in signal transduction experiments. RESULTS: Human bone marrow stromal cells, fibroblasts, trabecular bone-derived cells and an osteoblast-like cell line strongly enhanced survival of leukemic cells, while endothelial cells and chondrocytes did not. Gene expression profile analysis indicated two soluble factors, hepatocyte growth factor and CXCL12, as potentially involved. We demonstrated that hepatocyte growth factor and CXCL12 are produced only by mesenchymal lineages that sustain the survival of leukemic cells. Indeed chronic lymphocytic leukemic cells express a functional hepatocyte growth factor receptor (c-MET) and hepatocyte growth factor enhanced the viability of these cells through STAT3 phosphorylation, which was blocked by a c-MET tyrosine kinase inhibitor. The role of hepatocyte growth factor was confirmed by its short interfering RNA-mediated knock-down in mesenchymal cells. CONCLUSIONS: The finding that hepatocyte growth factor prolongs the survival of chronic lymphocytic leukemic cells is novel and we suggest that the interaction between hepatocyte growth factor-producing mesenchymal and neoplastic cells contributes to maintenance of the leukemic clone.