First clinical application of cord blood mesenchymal stromal cells in children with multi-drug resistant nephrotic syndrome.

BACKGROUND AND OBJECTIVES: Children with multi-drug resistant idiopathic nephrotic syndrome (MDR-INS) usually progress to end-stage kidney disease with a consistent risk of disease recurrence after transplantation. New therapeutic options are needed for these patients. Mesenchymal stromal cells (MSCs) are multipotential non-hematopoietic cells with several immunomodulatory properties and growing clinical applications. Cord blood-derived MSC have peculiar anti-inflammatory and immunosuppressive properties. We aimed at assessing safety and efficacy of cord-blood-derived MSCs (CB-MSCs) in children with MDR-INS. DESIGN, SETTING, PARTICIPANTS: Prospective, open-label, single arm phase I-II pilot study. Pediatric patients with MDR-INS, resistant to at least two lines of therapy, were enrolled. Allogenic CB-MSCs were administered intravenously on days 0, 14, and 21 at a dose of 1.5 × 106 cells/kg. Patients were followed for at least 12 months. The primary outcomes were safety and toxicity. The secondary outcome was remission at 12 months evaluated by urinary protein/urinary creatinine ratio (uPr/uCr). Circulating regulatory T cells (Tregs) were monitored. RESULTS: Eleven pediatric patients with MDR-INS (10 females, median age 13 years) resistant to a median of 3 previous lines of therapy were enrolled. All patients completed the CB-MSC infusion schedule. No patient experienced any infusion-related adverse event or toxicity. Nine patients were assessable for efficacy. At the 12 months follow-up after the treatment, the median uPr/uCr did not change significantly from baseline (8.13 vs. 9.07; p = 0.98), while 3 patients were in partial or complete remission. A lower baseline uPr/uCr was a predictor of remission (2.55 vs. 8.74; p = 0.0238). Tregs count was not associated with CB-MSCs therapy. CONCLUSIONS: CB-MSCs are safe and may have a role in the immunosuppressive therapy of pediatric patients with MDR-INS. This preliminary experience paves the way toward further phase II studies addressing MSC efficacy in immune-mediated kidney diseases.

Human airway organoids and microplastic fibers: A new exposure model for emerging contaminants.

Three-dimensional (3D) structured organoids are the most advanced in vitro models for studying human health effects, but their application to evaluate the biological effects associated with microplastic exposure was neglected until now. Fibers from synthetic clothes and fabrics are a major source of airborne microplastics, and their release from dryer machines is poorly understood. We quantified and characterized the microplastic fibers (MPFs) released in the exhaust filter of a household dryer and tested their effects on airway organoids (1, 10, and 50 µg mL-1) by optical microscopy, scanning electron microscopy (SEM), confocal microscopy and quantitative reverse transcription-polymerase chain reaction (qRT-PCR). While the presence of MPFs did not inhibit organoid growth, we observed a significant reduction of SCGB1A1 gene expression related to club cell functionality and a polarized cell growth along the fibers. The MPFs did not cause relevant inflammation or oxidative stress but were coated with a cellular layer, resulting in the inclusion of fibers in the organoid. This effect could have long-term implications regarding lung epithelial cells undergoing repair. This exposure study using human airway organoids proved suitability of the model for studying the effects of airborne microplastic contamination on humans and could form the basis for further research regarding the toxicological assessment of emerging contaminants such as micro- or nanoplastics.

Validation of an automated cell counting method for cGMP manufacturing of human induced pluripotent stem cells.

Human induced pluripotent stem cells (hiPSCs) must be manufactured as advanced therapy medicinal products (ATMPs) for innovative tissue replacement clinical applications. Yet, production of hiPSCs under current Good Manufacturing Practice (cGMP) presents many hurdles, such as the large-scale cell expansion needed to reach therapeutically-relevant hiPSC doses. For the monitoring of this phase, a fast and reliable cell counting method should be used. Conventional manual cell counting by the hemocytometer method is dependent on the operator's expertise and is time-consuming. Therefore, automation of sample preparation and analysis is needed to improve precision and rapidity of hiPSC cell counting. We investigated whether an automated cell counting method could be validated for use with hiPSCs, in comparison with a reference cell counting method included in the European Pharmacopeia, 10th edition. The proposed method was the fluorescence imaging-based NucleoCounter NC-100 system, whereas the reference method was manual cell counting using a Bürker hemocytometer. The validation strategy complied with EudraLex cGMP regulations for ATMP manufacturing and ICH Q2(R1) indications for validation of analytical methods. The use of the NucleoCounter NC-100 system for automated cell counting was validated, focusing on accuracy, specificity, intra- and inter-operator reproducibility, range and linearity, showing higher precision than the manual method. The automated method can be used more effectively than the manual one for hiPSC cell counting. Thus, this piece of work paves the way for all cGMP facilities that want to pursue hiPSC manufacturing for clinical use.

A comprehensive report of long-term stability data for a range ATMPs: A need to develop guidelines for safe and harmonized stability studies.

BACKGROUND AIMS: Advanced therapy medicinal products (ATMPs) are novel drugs based on genes, cells or tissues developed to treat many different diseases. Stability studies of each new ATMP need to be performed to define its shelf life and guarantee efficacy and safety upon infusion, and these are presently based on guidelines originally drafted for standard pharmaceutical drugs, which have properties and are stored in conditions quite different from cell products. The aim of this report is to provide evidence-based information for stability studies on ATMPs that will facilitate the interlaboratory harmonization of practices in this area. METHODS: We have collected and analyzed the results of stability studies on 19 different cell-based experimental ATMPs, produced by five authorized cell factories forming the Lombardy "Plagencell network" for use in 36 approved phase I/II clinical trials; most were cryopreserved and stored in liquid nitrogen vapors for 1 to 13 years. RESULTS: The cell attributes collected in stability studies included cell viability, immunophenotype and potency assays, in particular immunosuppression, cytotoxicity, cytokine release and proliferation/differentiation capacity. Microbiological attributes including sterility, endotoxin levels and mycoplasma contamination were also analyzed. All drug products (DPs), cryopreserved in various excipients containing 10% DMSO and in different primary containers, were very stable long term at <-150°C and did not show any tendency for diminished viability or efficacy for up to 13.5 years. CONCLUSIONS: Our data indicate that new guidelines for stability studies, specific for ATMPs and based on risk analyses, should be drafted to harmonize practices, significantly reduce the costs of stability studies without diminishing safety. Some specific suggestions are presented in the discussion.

Process development and validation of expanded regulatory T cells for prospective applications: an example of manufacturing a personalized advanced therapy medicinal product.

BACKGROUND: A growing number of clinical trials have shown that regulatory T (Treg) cell transfer may have a favorable effect on the maintenance of self-tolerance and immune homeostasis in different conditions such as graft-versus-host disease (GvHD), solid organ transplantation, type 1 diabetes, and others. In this context, the availability of a robust manufacturing protocol that is able to produce a sufficient number of functional Treg cells represents a fundamental prerequisite for the success of a cell therapy clinical protocol. However, extended workflow guidelines for nonprofit manufacturers are currently lacking. Despite the fact that different successful manufacturing procedures and cell products with excellent safety profiles have been reported from early clinical trials, the selection and expansion protocols for Treg cells vary a lot. The objective of this study was to validate a Good Manufacturing Practice (GMP)-compliant protocol for the production of Treg cells that approaches the whole process with a risk-management methodology, from process design to completion of final product development. High emphasis was given to the description of the quality control (QC) methodologies used for the in-process and release tests (sterility, endotoxin test, mycoplasma, and immunophenotype). RESULTS: The GMP-compliant protocol defined in this work allows at least 4.11 × 109 Treg cells to be obtained with an average purity of 95.75 ± 4.38% and can be used in different clinical settings to exploit Treg cell immunomodulatory function. CONCLUSIONS: These results could be of great use for facilities implementing GMP-compliant cell therapy protocols of these cells for different conditions aimed at restoring the Treg cell number and function, which may slow the progression of certain diseases.

Age-related changes in the energy of human mesenchymal stem cells.

Aging is a physiological process that leads to a higher risk for the most devastating diseases. There are a number of theories of human aging proposed, and many of them are directly or indirectly linked to mitochondria. Here, we used mesenchymal stem cells (MSCs) from young and older donors to study age-related changes in mitochondrial metabolism. We have found that aging in MSCs is associated with a decrease in mitochondrial membrane potential and lower NADH levels in mitochondria. Mitochondrial DNA content is higher in aged MSCs, but the overall mitochondrial mass is decreased due to increased rates of mitophagy. Despite the higher level of ATP in aged cells, a higher rate of ATP consumption renders them more vulnerable to energy deprivation compared to younger cells. Changes in mitochondrial metabolism in aged MSCs activate the overproduction of reactive oxygen species in mitochondria which is compensated by a higher level of the endogenous antioxidant glutathione. Thus, energy metabolism and redox state are the drivers for the aging of MSCs/mesenchymal stromal cells.

Identification of the best housekeeping gene for RT-qPCR analysis of human pancreatic organoids.

In the last few years, there has been a considerable increase in the use of organoids, which is a new three-dimensional culture technology applied in scientific research. The main reasons for their extensive use are their plasticity and multiple applications, including in regenerative medicine and the screening of new drugs. The aim of this study was to better understand these structures by focusing on the choice of the best housekeeping gene (HKG) to perform accurate molecular analysis on such a heterogeneous system. This feature should not be underestimated because the inappropriate use of a HKG can lead to misleading data and incorrect results, especially when the subject of the study is innovative and not totally explored like organoids. We focused our attention on the newly described human pancreatic organoids (hPOs) and compared 12 well-known HKGs (ACTB, B2M, EF1α, GAPDH, GUSB, HPRT, PPIA, RNA18S, RPL13A TBP, UBC and YWHAZ). Four different statistical algorithms (NormFinder, geNorm, BestKeeper and ΔCt) were applied to estimate the expression stability of each HKG, and RefFinder was used to identify the most suitable genes for RT-qPCR data normalization. Our results showed that the intragroup and intergroup comparisons could influence the best choice of the HKG, making clear that the identification of a stable reference gene for accurate and reproducible RT-qPCR data normalization remains a critical issue. In summary, this is the first report on HKGs in human organoids, and this work provides a strong basis to pave the way for further gene analysis in hPOs.

Critical Analysis of cGMP Large-Scale Expansion Process in Bioreactors of Human Induced Pluripotent Stem Cells in the Framework of Quality by Design.

Human induced pluripotent stem cells (hiPSCs) are manufactured as advanced therapy medicinal products for tissue replacement applications. With this aim, the feasibility of hiPSC large-scale expansion in existing bioreactor systems under current good manufacturing practices (cGMP) has been tested. Yet, these attempts have lacked a paradigm shift in culture settings and technologies tailored to hiPSCs, which jeopardizes their clinical translation. The best approach for industrial scale-up of high-quality hiPSCs is to design their manufacturing process by following quality-by-design (QbD) principles: a scientific, risk-based framework for process design based on relating product and process attributes to product quality. In this review, we analyzed the hiPSC expansion manufacturing process implementing the QbD approach in the use of bioreactors, stressing the decisive role played by the cell quantity, quality and costs, drawing key QbD concepts directly from the guidelines of the International Council for Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use.

Safety and Effectiveness of Cell Therapy in Neurodegenerative Diseases: Take-Home Messages From a Pilot Feasibility Phase I Study of Progressive Supranuclear Palsy.

Mesenchymal stromal cells (MSCs) are multipotent cells with anti-inflammatory properties. Here we tested the safety of MSCs in patients with progressive supranuclear palsy (PSP; ClinicalTrials.gov: NCT01824121; Eudract No. 2011-004051-39). Seven patients were treated. To improve the safety, protocol adjustments were made during the performance of the study. The objectives of our work were: (1) to assess the safety of MSCs and (2) to identify critical issues in cell therapies for neurodegenerative diseases. Autologous MSCs from the bone marrow of PSP patients were administered through the internal carotid arteries. 1-year survival and number of severe adverse events were considered as safety endpoints. Clinical rating scales, neuropsychological assessments, gait and posture analysis, single-photon emission computed tomography, positron emission tomography, and brain magnetic resonance (BMR) were performed at different follow-up times. Peripheral blood levels of inflammatory cytokines were measured before and after cell infusion. Six of the seven treated patients were living 1 year after cell infusion. Asymptomatic spotty lesions were observed at BMR after 24 h in six of the seven treated patients. The last patient in the preliminary cohort (Case 5) exhibited transiently symptomatic BMR ischemic alterations. No severe adverse events were recorded in the last two treated patients. Interleukin-8 serum concentrations decreased in three patients (Case 2, 3, and 4). An adaptive study design, appropriate and up-to-date efficacy measures, adequate sample size estimation, and, possibly, the use of a cellular and/or allogeneic cell sources may help in performing phase II trials in the field.

Chondrogenic and BMP-4 primings confer osteogenesis potential to human cord blood mesenchymal stromal cells delivered with biphasic calcium phosphate ceramics.

Bone marrow mesenchymal stem/stromal cells (BMSCs) show great promise for bone repair, however they are isolated by an invasive bone marrow harvest and their regenerative potential decreases with age. Conversely, cord blood can be collected non-invasively after birth and contains MSCs (CBMSCs) that can be stored for future use. However, whether CBMSCs can replace BMSCs targeting bone repair is unknown. This study evaluates the in vitro osteogenic potential of unprimed, osteogenically primed, or chondrogenically primed CBMSCs and BMSCs and their in vivo bone forming capacity following ectopic implantation on biphasic calcium phosphate ceramics in nude mice. In vitro, alkaline phosphatase (intracellular, extracellular, and gene expression), and secretion of osteogenic cytokines (osteoprotegerin and osteocalcin) was significantly higher in BMSCs compared with CBMSCs, while CBMSCs demonstrated superior chondrogenic differentiation and secretion of interleukins IL-6 and IL-8. BMSCs yielded significantly more cell engraftment and ectopic bone formation compared to CBMSCs. However, priming of CBMSCs with either chondrogenic or BMP-4 supplements led to bone formation by CBMSCs. This study is the first direct quantification of the bone forming abilities of BMSCs and CBMSCs in vivo and, while revealing the innate superiority of BMSCs for bone repair, it provides avenues to induce osteogenesis by CBMSCs.

A flow cytometric assay for the quantification of MSC lysis by peripheral blood mononucleated cells.

Mesenchymal stromal cells (MSC) are attractive candidates for the treatment of acute graft versus host disease (aGvHD) or autoimmune disorders. However, mechanisms of MSC recognition remain unclear and there are evidences that MSC are not totally immunoprivileged. Data suggest that MSC undergo apoptosis after infusion in presence of cytotoxic cells and their death could drive immunosuppression. In GvHD patients, that activity was associated with clinical response. It is mandatory to develop an in vitro potency testing predictor of the "in vivo" response to the therapy. We describe a flow cytometric assay based on differential immunostaining of target and effector cells where BM MSC are enumerated with fluorospheres to determine the loss of target cells after co-culture with PB MNC. 6/13 (46%) of BM MSC lots were lysed by PB MNC and the lysis was proportional to the E/T cell ratio. The method overcomes the problems linked to the use of dyes or radioactive, evidencing the limitations linked to the use of a single vital dye and proposing a precise gating strategy based on absolute cell counts where cells are left untouched. The assay is easy and could be used to predict the response of the patients to the therapy.

Blood-derived extracellular vesicles isolated from healthy donors exposed to air pollution modulate in vitro endothelial cells behavior.

The release of Extracellular Vesicles (EVs) into the bloodstream is positively associated with Particulate Matter (PM) exposure, which is involved in endothelial dysfunction and related to increased risk of cardiovascular disease. Obesity modifies the effects of PM exposure on heart rate variability and markers of inflammation, oxidative stress, and acute phase response. We isolated and characterized plasmatic EVs from six healthy donors and confirmed a positive association with PM exposure. We stratified for Body Mass Index (BMI) and observed an increased release of CD61+ (platelets) and CD105+ (endothelium) derived-EVs after high PM level exposure in Normal Weight subjects (NW) and no significant variations in Overweight subjects (OW). We then investigated the ability to activate endothelial primary cells by plasmatic EVs after both high and low PM exposure. NW-high-PM EVs showed an increased endothelial activation, measured as CD105+/CD62e+ (activated endothelium) EVs ratio. On the contrary, cells treated with OW-high-PM EVs showed reduced endothelial activation. These results suggest the ability of NW plasmatic EVs to communicate to endothelial cells and promote the crosstalk between activated endothelium and peripheral cells. However, this capacity was lost in OW subjects. Our findings contribute to elucidate the role of EVs in endothelial activation after PM exposure.

Mesenchymal stromal cells and their secreted extracellular vesicles as therapeutic tools for COVID-19 pneumonia?

The COVID-19 epidemic represents an unprecedented global health emergency, further aggravated by the lack of effective therapies. For this reason, several clinical trials are testing different off-label drugs, already approved for other pathologies. Mesenchymal stem/stromal cells (MSCs) have been tested during the last two decades for the treatment of various pathologic conditions, including acute and chronic lung diseases, both in animal models and in patients. In particular, promising results have been obtained in the experimental therapy of acute respiratory distress syndrome, which represents the most threatening complication of COVID-19 infection. Furthermore, more recently, great interest has been devoted to the possible clinical applications of extracellular vesicles secreted by MSCs, nanoparticles that convey much of the biological effects and of the therapeutic efficacy of their cells of origin. This review summarizes the experimental evidence underlying the possible use of MSCs and of MSC-EVs in severe COVID-19 infection and underlines the need to evaluate the possible efficacy of these therapeutic approaches through controlled studies under the supervision of the Regulatory Authorities.

A circular RNA map for human induced pluripotent stem cells of foetal origin.

BACKGROUND: Adult skin fibroblasts represent the most common starting cell type used to generate human induced pluripotent stem cells (F-hiPSC) for clinical studies. Yet, a foetal source would offer unique advantages, primarily the absence of accumulated somatic mutations. Herein, we generated hiPSC from cord blood multipotent mesenchymal stromal cells (MSC-hiPSC) and compared them with F-hiPSC. Assessment of the full activation of the pluripotency gene regulatory network (PGRN) focused on circular RNA (circRNA), recently proposed to participate in the control of pluripotency. METHODS: Reprogramming was achieved by a footprint-free strategy. Self-renewal and pluripotency of cord blood MSC-hiPSC were investigated in vitro and in vivo, compared to parental MSC, to embryonic stem cells and to F-hiPSC. High-throughput array-based approaches and bioinformatics analyses were applied to address the PGRN. FINDINGS: Cord blood MSC-hiPSC successfully acquired a complete pluripotent identity. Functional comparison with F-hiPSC showed no differences in terms of i) generation of mesenchymal-like derivatives, ii) their subsequent adipogenic, osteogenic and chondrogenic commitment, and iii) their hematopoietic support ability. At the transcriptional level, specific subsets of mRNA, miRNA and circRNA (n = 4,429) were evidenced, casting a further layer of complexity on the PGRN regulatory crosstalk. INTERPRETATION: A circRNA map of transcripts associated to naïve and primed pluripotency is provided for hiPSC of clinical-grade foetal origin, offering insights on still unreported regulatory circuits of the PGRN to consider for the optimization and development of efficient differentiation protocols for clinical translation. FUNDING: This research was funded by Ricerca Corrente 2012-2018 by the Italian Ministry of Health.

Standardized GMP-compliant scalable production of human pancreas organoids.

BACKGROUND: Organoids are three-dimensional in vitro-grown cell clusters that recapitulate key features of native organs. In regenerative medicine, organoid technology represents a promising approach for the replacement of severely damaged organs, such as the pancreas in patients with type 1 diabetes. Isolation human pancreas organoids (hPOs) in chemically defined serum-free culture media would be a major milestone for this approach. METHODS: Starting from discarded pancreatic tissues, we developed a large-scale process for obtaining clinically relevant quantities of undifferentiated organoids, obviating enzymatic digestion and operator-dependent pancreatic ducts picking steps. hPO identity was characterized by molecular and flow cytometry analysis. RESULTS: This work demonstrates that it is possible to obtain a large-scale production of organoids. We introduced some innovations in the isolation, expansion, and freezing of hPOs from five donors. First of all, the choice of the starting material (islet-depleted pancreas) that allows obtaining a high quantity of hPOs at low passages. On the other hand, we introduced mechanical dissociation and we eliminated the picking step to exclude the operator-depending steps, without affecting the success of the culture (100% success rate). Another important improvement was to replace R-spondin-1 (Rspo1) conditioned medium with Rspo1 recombinant molecule to obtain a well-defined composition of the expansion medium. Finally, we implemented a GMP-compliant freezing protocol. hPOs showed exponential growth with diameter and area that increased three- and eight-fold in 7 days, respectively. Immunophenotypic profile and gene expression analysis revealed that hPOs were composed of ductal (82.33 ± 8.37%), acinar (2.80 ± 1.25%) cells, and pancreatic progenitors (5.81 ± 2.65%). CONCLUSION: This work represents a milestone for a GMP-compliance hPO production and, ultimately, their clinical application as a type 1 diabetes therapy.

NG2 as an Identity and Quality Marker of Mesenchymal Stem Cell Extracellular Vesicles.

The therapeutic potential of mesenchymal stem cell (MSC) extracellular vesicles (EV) is currently under investigation in many pathological contexts. Both adult and perinatal MSC are being considered as sources of EV. Herein, we address antigen expression of cord blood and bone marrow MSC and released EV to define an identity and quality parameter of MSC EV as a medicinal product in the context of clinical applications. The research focuses on EV-shuttled neural/glial antigen 2 (NG2), which has previously been detected as a promising surface marker to distinguish perinatal versus adult MSC. Indeed, NG2 was significantly more abundant in cord blood than bone marrow MSC and MSC EV. Ultracentrifuge-isolated EV were then challenged for their pro-angiogenic properties on an xCELLigence system as quality control. NG2+ cord blood MSC EV, but not bone marrow MSC EV, promote bFGF and PDGF-AA proliferative effect on endothelial cells. Likewise, they successfully rescue angiostatin-induced endothelial cell growth arrest. In both cases, the effects are NG2-dependent. These results point at NG2 as an identity and quality parameter for cord blood MSC EV, paving the way for their clinical translation.

Central metabolism of functionally heterogeneous mesenchymal stromal cells.

Metabolism and mitochondrial biology have gained a prominent role as determinants of stem cell fate and function. In the context of regenerative medicine, innovative parameters predictive of therapeutic efficacy could be drawn from the association of metabolic or mitochondrial parameters to different degrees of stemness and differentiation potentials. Herein, this possibility was addressed in human mesenchymal stromal/stem cells (hMSC) previously shown to differ in lifespan and telomere length. First, these hMSC were shown to possess significantly distinct proliferation rate, senescence status and differentiation capacity. More potential hMSC were associated to higher mitochondrial (mt) DNA copy number and lower mtDNA methylation. In addition, they showed higher expression levels of oxidative phosphorylation subunits. Consistently, they exhibited higher coupled oxygen consumption rate and lower transcription of glycolysis-related genes, glucose consumption and lactate production. All these data pointed at oxidative phosphorylation-based central metabolism as a feature of higher stemness-associated hMSC phenotypes. Consistently, reduction of mitochondrial activity by complex I and III inhibitors in higher stemness-associated hMSC triggered senescence. Finally, functionally higher stemness-associated hMSC showed metabolic plasticity when challenged by glucose or glutamine shortage, which mimic bioenergetics switches that hMSC must undergo after transplantation or during self-renewal and differentiation. Altogether, these results hint at metabolic and mitochondrial parameters that could be implemented to identify stem cells endowed with superior growth and differentiation potential.

Generation of a Functioning and Self-Renewing Diaphragmatic Muscle Construct.

Surgical repair of large muscular defects requires the use of autologous graft transfer or prosthetic material. Naturally derived matrices are biocompatible materials obtained by tissue decellularization and are commonly used in clinical practice. Despite promising applications described in the literature, the use of acellular matrices to repair large defects has been only partially successful, highlighting the need for more efficient constructs. Scaffold recellularization by means of tissue engineering may improve not only the structure of the matrix, but also its ability to functionally interact with the host. The development of such a complex construct is challenging, due to the complexity of the native organ architecture and the difficulties in recreating the cellular niche with both proliferative and differentiating potential during growth or after damage. In this study, we tested a mouse decellularized diaphragmatic extracellular matrix (ECM) previously described by our group, for the generation of a cellular skeletal muscle construct with functional features. The decellularized matrix was stored using different conditions to mimic the off-the-shelf clinical need. Pediatric human muscle precursors were seeded into the decellularized scaffold, demonstrating proliferation and differentiation capability, giving rise to a functioning three-dimensional skeletal muscle structure. Furthermore, we exposed the engineered construct to cardiotoxin injury and demonstrated its ability to activate a regenerative response in vitro promoting cell self-renewal and a positive ECM remodeling. Functional reconstruction of an engineered skeletal muscle with maintenance of a stem cell pool makes this a promising tool toward future clinical applications in diaphragmatic regeneration. Stem Cells Translational Medicine 2019;8:858&869.

FOXP1 circular RNA sustains mesenchymal stem cell identity via microRNA inhibition.

Stem cell identity and plasticity are controlled by master regulatory genes and complex circuits also involving non-coding RNAs. Circular RNAs (circRNAs) are a class of RNAs generated from protein-coding genes by backsplicing, resulting in stable RNA structures devoid of free 5' and 3' ends. Little is known of the mechanisms of action of circRNAs, let alone in stem cell biology. In this study, for the first time, we determined that a circRNA controls mesenchymal stem cell (MSC) identity and differentiation. High-throughput MSC expression profiling from different tissues revealed a large number of expressed circRNAs. Among those, circFOXP1 was enriched in MSCs compared to differentiated mesodermal derivatives. Silencing of circFOXP1 dramatically impaired MSC differentiation in culture and in vivo. Furthermore, we demonstrated a direct interaction between circFOXP1 and miR-17-3p/miR-127-5p, which results in the modulation of non-canonical Wnt and EGFR pathways. Finally, we addressed the interplay between canonical and non-canonical Wnt pathways. Reprogramming to pluripotency of MSCs reduced circFOXP1 and non-canonical Wnt, whereas canonical Wnt was boosted. The opposing effect was observed during generation of MSCs from human pluripotent stem cells. Our results provide unprecedented evidence for a regulatory role for circFOXP1 as a gatekeeper of pivotal stem cell molecular networks.

FGF23 and Fetuin-A Interaction and Mesenchymal Osteogenic Transformation.

Recently, we found a strict bone association between Fibroblast growth factor 23 (FGF23) and Fetuin-A, both involved in cardiovascular and mineral bone disorders. In this study, an uninvestigated bone marrow positivity for both was found. Though the role of exogenous FGF23 on mesenchymal cells (MSCs) was reported, no information is as yet available on the possible production of this hormone by MSCs. To further analyze these uninvestigated aspects, we studied human primary cells and mouse and human cell lines by means of immunostaining, qRT-PCR, enzyme linked immunosorbent assays, chromatin immunoprecipitation, transfection, and a streamlined approach for the FGF23⁻Fetuin-A interaction called Duolink proximity ligation assay. Mesenchymal cells produce but do not secrete FGF23 and its expression increases during osteo-differentiation. Fibroblast growth factor 23 is also involved in the regulation of Fetuin-A by binding directly to the Fetuin-A promoter and then activating its transcription. Both FGF23 overexpression and addition induced an upregulation of Fetuin-A in the absence of osteo-inducer factors. Fibroblast growth factor 23 and Fetuin-A promoter were increased by osteo-inducer factors with this effect being abolished after FGF23 silencing. In conclusion, both FGF23 and Fetuin-A are present and strictly linked to each other in MSCs with FGF23 driving Fetuin-A production. This mechanism suggests a role for these two proteins in the osteoblast differentiation.