Research Models to Mimic Necrotizing Enterocolitis and Inflammatory Bowel Diseases: Focus on Extracellular Vesicles Action.

This review focuses on the crucial role of the intestinal epithelium in maintaining intestinal homeostasis and its significance in the pathogenesis of necrotizing enterocolitis (NEC) and inflammatory bowel diseases (IBD). NEC is a devastating neonatal disease, while IBD represents a global healthcare problem with increasing incidence. The breakdown of the intestinal barrier in neonates is considered pivotal in the development and progression of both disorders. This review provides an overview of the current state of in vitro, ex vivo, and animal models to study epithelial injury in NEC and IBD, addressing pertinent questions that engage clinicians and researchers alike. Despite significant advancements in early recognition and aggressive treatment, no single therapy has been conclusively proven effective in reducing the severity of these disorders. Although early interventions have improved clinical outcomes, NEC and IBD continue to impose substantial morbidity, mortality, and economic burdens on affected individuals and society. Consequently, exploring alternative therapeutic options capable of preventing and treating the sequelae of NEC and IBD has become a pressing necessity. In recent decades, extracellular vehicles (EVs) have emerged as a potential solution to modulate the pathogenic mechanism in these multifactorial and complex disorders. Despite the diverse array of proposed models, a comprehensive model to investigate and decelerate the progression of NEC and IBD remains to be established. To bridge the translational gap between preclinical studies and clinical applications, enhancements in the technical development of gut-on-a-chip models and EVs hold considerable promise.

Intratracheal administration of mesenchymal stem cell-derived extracellular vesicles reduces lung injuries in a chronic rat model of bronchopulmonary dysplasia.

Early therapeutic effect of intratracheally (IT)-administered extracellular vesicles secreted by mesenchymal stem cells (MSC-EVs) has been demonstrated in a rat model of bronchopulmonary dysplasia (BPD) involving hyperoxia exposure in the first 2 postnatal weeks. The aim of this study was to evaluate the protective effects of IT-administered MSC-EVs in the long term. EVs were produced from MSCs following GMP standards. At birth, rats were distributed in three groups: (a) animals raised in ambient air for 6 weeks (n = 10); and animals exposed to 60% hyperoxia for 2 weeks and to room air for additional 4 weeks and treated with (b) IT-administered saline solution (n = 10), or (c) MSC-EVs (n = 10) on postnatal days 3, 7, 10, and 21. Hyperoxia exposure produced significant decreases in total number of alveoli, total surface area of alveolar air spaces, and proliferation index, together with increases in mean alveolar volume, mean linear intercept and fibrosis percentage; all these morphometric changes were prevented by MSC-EVs treatment. The medial thickness index for <100 µm vessels was higher for hyperoxia-exposed/sham-treated than for normoxia-exposed rats; MSC-EV treatment significantly reduced this index. There were no significant differences in interstitial/alveolar and perivascular F4/8-positive and CD86-positive macrophages. Conversely, hyperoxia exposure reduced CD163-positive macrophages both in interstitial/alveolar and perivascular populations and MSC-EV prevented these hyperoxia-induced reductions. These findings further support that IT-administered EVs could be an effective approach to prevent/treat BPD, ameliorating the impaired alveolarization and pulmonary artery remodeling also in a long-term model. M2 macrophage polarization could play a role through anti-inflammatory and proliferative mechanisms.

In Utero Transplantation of Expanded Autologous Amniotic Fluid Stem Cells Results in Long-Term Hematopoietic Engraftment.

In utero transplantation (IUT) of hematopoietic stem cells (HSCs) has been proposed as a strategy for the prenatal treatment of congenital hematological diseases. However, levels of long-term hematopoietic engraftment achieved in experimental IUT to date are subtherapeutic, likely due to host fetal HSCs outcompeting their bone marrow (BM)-derived donor equivalents for space in the hematopoietic compartment. In the present study, we demonstrate that amniotic fluid stem cells (AFSCs; c-Kit+/Lin-) have hematopoietic characteristics and, thanks to their fetal origin, favorable proliferation kinetics in vitro and in vivo, which are maintained when the cells are expanded. IUT of autologous/congenic freshly isolated or cultured AFSCs resulted in stable multilineage hematopoietic engraftment, far higher to that achieved with BM-HSCs. Intravascular IUT of allogenic AFSCs was not successful as recently reported after intraperitoneal IUT. Herein, we demonstrated that this likely due to a failure of timely homing of donor cells to the host fetal thymus resulted in lack of tolerance induction and rejection. This study reveals that intravascular IUT leads to a remarkable hematopoietic engraftment of AFSCs in the setting of autologous/congenic IUT, and confirms the requirement for induction of central tolerance for allogenic IUT to be successful. Autologous, gene-engineered, and in vitro expanded AFSCs could be used as a stem cell/gene therapy platform for the in utero treatment of inherited disorders of hematopoiesis. Stem Cells 2019;37:1176-1188.

Decellularized Diaphragmatic Muscle Drives a Constructive Angiogenic Response In Vivo.

Skeletal muscle tissue engineering (TE) aims to efficiently repair large congenital and acquired defects. Biological acellular scaffolds are considered a good tool for TE, as decellularization allows structural preservation of tissue extracellular matrix (ECM) and conservation of its unique cytokine reservoir and the ability to support angiogenesis, cell viability, and proliferation. This represents a major advantage compared to synthetic scaffolds, which can acquire these features only after modification and show limited biocompatibility. In this work, we describe the ability of a skeletal muscle acellular scaffold to promote vascularization both ex vivo and in vivo. Specifically, chicken chorioallantoic membrane assay and protein array confirmed the presence of pro-angiogenic molecules in the decellularized tissue such as HGF, VEGF, and SDF-1α. The acellular muscle was implanted in BL6/J mice both subcutaneously and ortotopically. In the first condition, the ECM-derived scaffold appeared vascularized 7 days post-implantation. When the decellularized diaphragm was ortotopically applied, newly formed blood vessels containing CD31⁺, αSMA⁺, and vWF⁺ cells were visible inside the scaffold. Systemic injection of Evans Blue proved function and perfusion of the new vessels, underlying a tissue-regenerative activation. On the contrary, the implantation of a synthetic matrix made of polytetrafluoroethylene used as control was only surrounded by vWF⁺ cells, with no cell migration inside the scaffold and clear foreign body reaction (giant cells were visible). The molecular profile and the analysis of macrophages confirmed the tendency of the synthetic scaffold to enhance inflammation instead of regeneration. In conclusion, we identified the angiogenic potential of a skeletal muscle-derived acellular scaffold and the pro-regenerative environment activated in vivo, showing clear evidence that the decellularized diaphragm is a suitable candidate for skeletal muscle tissue engineering and regeneration.

Functional Human Podocytes Generated in Organoids from Amniotic Fluid Stem Cells.

Generating kidney organoids using human stem cells could offer promising prospects for research and therapeutic purposes. However, no cell-based strategy has generated nephrons displaying an intact three-dimensional epithelial filtering barrier. Here, we generated organoids using murine embryonic kidney cells, and documented that these tissues recapitulated the complex three-dimensional filtering structure of glomerular slits in vivo and accomplished selective glomerular filtration and tubular reabsorption. Exploiting this technology, we mixed human amniotic fluid stem cells with mouse embryonic kidney cells to establish three-dimensional chimeric organoids that engrafted in vivo and grew to form vascularized glomeruli and tubular structures. Human cells contributed to the formation of glomerular structures, differentiated into podocytes with slit diaphragms, and internalized exogenously infused BSA, thus attaining in vivo degrees of specialization and function unprecedented for donor stem cells. In conclusion, human amniotic fluid stem cell chimeric organoids may offer new paths for studying renal development and human podocyte disease, and for facilitating drug discovery and translational research.

Challenges and Strategies for Improving the Regenerative Effects of Mesenchymal Stromal Cell-Based Therapies.

Cell-based therapies have the potential to revolutionize current treatments for diseases with high prevalence and related economic and social burden. Unfortunately, clinical trials have made only modest improvements in restoring normal function to degenerating tissues. This limitation is due, at least in part, to the death of transplanted cells within a few hours after transplant due to a combination of mechanical, cellular, and host factors. In particular, mechanical stress during implantation, extracellular matrix loss upon delivery, nutrient and oxygen deprivation at the recipient site, and host inflammatory response are detrimental factors limiting long-term transplanted cell survival. The beneficial effect of cell therapy for regenerative medicine ultimately depends on the number of administered cells reaching the target tissue, their viability, and their promotion of tissue regeneration. Therefore, strategies aiming at improving viable cell engraftment are crucial for regenerative medicine. Here we review the major factors that hamper successful cell engraftment and the strategies that have been studied to enhance the beneficial effects of cell therapy. Moreover, we provide a perspective on whether mesenchymal stromal cell-derived extracellular vesicle delivery, as a cell-free regenerative approach, may circumvent current cell therapy limitations.

Diverging Concepts and Novel Perspectives in Regenerative Medicine.

Regenerative medicine has rapidly evolved, due to progress in cell and molecular biology allowing the isolation, characterization, expansion, and engineering of cells as therapeutic tools. Despite past limited success in the clinical translation of several promising preclinical results, this novel field is now entering a phase of renewed confidence and productivity, marked by the commercialization of the first cell therapy products. Ongoing issues in the field include the use of pluripotent vs. somatic and of allogenic vs. autologous stem cells. Moreover, the recognition that several of the observed beneficial effects of cell therapy are not due to integration of the transplanted cells, but rather to paracrine signals released by the exogenous cells, is generating new therapeutic perspectives in the field. Somatic stem cells are outperforming embryonic and induced pluripotent stem cells in clinical applications, mainly because of their more favorable safety profile. Presently, both autologous and allogeneic somatic stem cells seem to be equally safe and effective under several different conditions. Recognition that a number of therapeutic effects of transplanted cells are mediated by paracrine signals, and that such signals can be found in extracellular vesicles isolated from culture media, opens novel therapeutic perspectives in the field of regenerative medicine.

Culturing muscle fibres in hanging drop: a novel approach to solve an old problem.

BACKGROUND INFORMATION: The satellite cells (SCs) associated with muscle fibres play a key role in postnatal growth and regeneration of skeletal muscle. Commonly used methods of isolation and in vitro culture of SCs lead to the mixture of their subpopulations that exist within muscle. To solve this problem, we used the well established technique, the hanging drop system, to culture SCs in a three-dimensional environment and thus, to monitor them in their original niche. RESULTS: Using hanging drop technique, we were able to culture SCs associated with the fibre at least for 9 days with one transfer of fibres to the fresh drops. In comparison, in the classical method of myofibres culture, that is, on the dishes coated with Matrigel, SCs leave the fibres within 3 days after the isolation. Cells cultured in both systems differed in expression of Pax7 and MyoD. While almost all cells cultured in adhesion system expressed MyoD before the fifth day of the culture, the majority of SCs cultured in hanging drop still maintained expression of Pax7 and were not characterised by the presence of MyoD. Among the cells cultured with single myofibre for up to 9 days, we identified two different subclones of SCs: low proliferative clone and high proliferative clone, which differed in proliferation rate and membrane potential. CONCLUSIONS: The hanging drop enables the myofibres to be kept in suspension for at least 9 days, and thus, allows SCs and their niche to interact each other for prolonged time. In a consequence, SCs cultured in hanging drop maintain expression of Pax7 while those cultured in a traditional adhesion culture, that is, devoid of signals from the original niche, activate and preferentially undergo differentiation as manifested by expression of MyoD. Thus, the innovative method of SCs culturing in the hanging drop system may serve as a useful tool to study the fate of different subpopulations of these cells in their anatomical location and to determine reciprocal interactions between them and their niche.

The use of human amniotic fluid stem cells as an adjunct to promote pulmonary development in a rabbit model for congenital diaphragmatic hernia.

OBJECTIVE: This study aimed to evaluate the potential benefit of intra-tracheal injection of human amniotic fluid stem cells (hAFSC) on pulmonary development combined with TO in a rabbit model for CDH. METHODS: In time-mated pregnant does a left diaphragmatic defect was created at d23 (term = 31). At d28, previously operated fetuses were assigned to either TO and injection with 70 µL of phosphate buffered saline (PBS) or 1.0 × 10(6) c-Kit positive hAFSC expressing LacZ or were left untouched (CDH). Harvesting was done at d31 to obtain their lung-to-body weight ratio (LBWR), airway and vascular lung morphometry, X-gal staining and immunohistochemistry for Ki67 and surfactant protein-B (SP-B). RESULTS: CDH-induced pulmonary hypoplasia is countered by TO + PBS, this reverses LBWR, mean terminal bronchiole density (MTBD) and medial thickness to normal. The additional injection of hAFSC decreases MTBD and results in a non-significant decrease in muscularization of intra-acinary vessels. There were no inflammatory changes and LacZ positive hAFSC were dispersed throughout the lung parenchyma 4 days after injection. CONCLUSION: HAFSC exert an additional effect on TO leading to a decrease in MTBD, a measure of alveolar number surrounding the terminal bronchioles, without signs of toxicity. © 2015 John Wiley & Sons, Ltd.

Amniotic fluid stem cells improve survival and enhance repair of damaged intestine in necrotising enterocolitis via a COX-2 dependent mechanism.

OBJECTIVE: Necrotising enterocolitis (NEC) remains one of the primary causes of morbidity and mortality in neonates and alternative strategies are needed. Stem cells have become a therapeutic option for other intestinal diseases, which share some features with NEC. We tested the hypothesis that amniotic fluid stem (AFS) cells exerted a beneficial effect in a neonatal rat model of NEC. DESIGN: Rats intraperitoneally injected with AFS cells and their controls (bone marrow mesenchymal stem cells, myoblast) were analysed for survival, behaviour, bowel imaging (MRI scan), histology, bowel absorption and motility, immunofluorescence for AFS cell detection, degree of gut inflammation (myeloperoxidase and malondialdehyde), and enterocyte apoptosis and proliferation. RESULTS: AFS cells integrated in the bowel wall and improved rat survival and clinical conditions, decreased NEC incidence and macroscopic gut damage, improved intestinal function, decreased bowel inflammation, increased enterocyte proliferation and reduced apoptosis. The beneficial effect was achieved via modulation of stromal cells expressing cyclooxygenase 2 in the lamina propria, as shown by survival studies using selective and non-selective cyclooxygenase 2 inhibitors. Interestingly, AFS cells differentially expressed genes of the Wnt/ß-catenin pathway, which regulate intestinal epithelial stem cell function and cell migration and growth factors known to maintain gut epithelial integrity and reduce mucosal injury. CONCLUSIONS: We demonstrated here for the first time that AFS cells injected in an established model of NEC improve survival, clinical status, gut structure and function. Understanding the mechanism of this effect may help us to develop new cellular or pharmacological therapies for infants with NEC.

Single-cell PCR analysis of murine embryonic stem cells cultured on different substrates highlights heterogeneous expression of stem cell markers.

BACKGROUND INFORMATION: In the last few years, recent evidence has revealed that inside an apparently homogeneous cell population there indeed appears to be heterogeneity. This is particularly true for embryonic stem (ES) cells where markers of pluripotency are dynamically expressed within the single cells. In this work, we have designed and tested a new set of primers for multiplex PCR detection of pluripotency markers expression, and have applied it to perform a single-cell analysis in murine ES cells cultured on three different substrates that could play an important role in controlling cell behaviour and fate: (i) mouse embryonic fibroblast (MEF) feeder layer, as the standard method for ES cells culture; (ii) Matrigel coating; (iii) micropatterned hydrogel. RESULTS: Compared with population analysis, using a single-cell approach, we were able to evaluate not only the number of cells that maintained the expression of a specific gene but, most importantly, how many cells co-expressed different markers. We found that micropatterned hydrogel seems to represent a good alternative to MEF, as the expression of stemness markers is better preserved than in Matrigel culture. CONCLUSIONS: This single-cell assay allows for the assessment of the stemness maintenance at a single-cell level in terms of gene expression profile, and can be applied in stem cell research to characterise freshly isolated and cultured cells, or to standardise, for instance, the method of culture closely linked to the transcriptional activity and the differentiation potential.

Stem cells from fetal membranes and amniotic fluid: markers for cell isolation and therapy.

Stem cell therapy is in constant need of new cell sources to conceive regenerative medicine approaches for diseases that are still without therapy. Scientists drew the attention toward amniotic membrane and amniotic fluid stem cells, since these sources possess many advantages: first of all as cells can be extracted from discarded foetal material it is inexpensive, secondly abundant stem cells can be obtained and finally, these stem cell sources are free from ethical considerations. Many studies have demonstrated the differentiation potential in vitro and in vivo toward mesenchymal and non-mesenchymal cell types; in addition the immune-modulatory properties make these cells a good candidate for allo- and xenotransplantation. This review offers an overview on markers characterisation and on the latest findings in pre-clinical or clinical setting of the stem cell populations isolated from these sources.

Extracellular Vesicles From Mesenchymal Umbilical Cord Cells Exert Protection Against Oxidative Stress and Fibrosis in a Rat Model of Bronchopulmonary Dysplasia.

Oxidative stress and fibrosis are important stress responses that characterize bronchopulmonary dysplasia (BPD), a disease for which only a therapy but not a cure has been developed. In this work, we investigated the effects of mesenchymal stromal cells-derived extracellular vesicles (MSC-EVs) on lung and brain compartment in an animal model of hyperoxia-induced BPD. Rat pups were intratracheally injected with MSC-EVs produced by human umbilical cord-derived MSC, following the Good Manufacturing Practice-grade (GMP-grade). After evaluating biodistribution of labelled MSC-EVs in rat pups left in normoxia and hyperoxia, oxidative stress and fibrosis investigation were performed. Oxidative stress protection by MSC-EVs treatment was proved both in lung and in brain. The lung epithelial compartment ameliorated glycosaminoglycan and surfactant protein expression in MSC-EVs-injected rat pups compared to untreated animals. Pups under hyperoxia exhibited a fibrotic phenotype in lungs shown by increased collagen deposition and also expression of profibrotic genes. Both parameters were reduced by treatment with MSC-EVs. We established an in vitro model of fibrosis and another of oxidative stress, and we proved that MSC-EVs suppressed the induction of αSMA, influencing collagen deposition and protecting from the oxidative stress. In conclusion, intratracheal administration of clinical-grade MSC-EVs protect from oxidative stress, improves pulmonary epithelial function, and counteracts the development of fibrosis. In the future, MSC-EVs could represent a new cure to prevent the development of BPD.

Amniotic fluid stem cells restore the muscle cell niche in a HSA-Cre, Smn(F7/F7) mouse model.

Mutations in the survival of motor neuron gene (SMN1) are responsible for spinal muscular atrophy, a fatal neuromuscular disorder. Mice carrying a homozygous deletion of Smn exon 7 directed to skeletal muscle (HSA-Cre, Smn(F7/F7) mice) present clinical features of human muscular dystrophies for which new therapeutic approaches are highly warranted. Herein we demonstrate that tail vein transplantation of mouse amniotic fluid stem (AFS) cells enhances the muscle strength and improves the survival rate of the affected animals. Second, after cardiotoxin injury of the Tibialis Anterior, only AFS-transplanted mice efficiently regenerate. Most importantly, secondary transplants of satellite cells (SCs) derived from treated mice show that AFS cells integrate into the muscle stem cell compartment and have long-term muscle regeneration capacity indistinguishable from that of wild-type-derived SC. This is the first study demonstrating the functional and stable integration of AFS cells into the skeletal muscle, highlighting their value as cell source for the treatment of muscular dystrophies.

Dysfunctional mitochondria accumulate in a skeletal muscle knockout model of Smn1, the causal gene of spinal muscular atrophy.

The approved gene therapies for spinal muscular atrophy (SMA), caused by loss of survival motor neuron 1 (SMN1), greatly ameliorate SMA natural history but are not curative. These therapies primarily target motor neurons, but SMN1 loss has detrimental effects beyond motor neurons and especially in muscle. Here we show that SMN loss in mouse skeletal muscle leads to accumulation of dysfunctional mitochondria. Expression profiling of single myofibers from a muscle specific Smn1 knockout mouse model revealed down-regulation of mitochondrial and lysosomal genes. Albeit levels of proteins that mark mitochondria for mitophagy were increased, morphologically deranged mitochondria with impaired complex I and IV activity and respiration and that produced excess reactive oxygen species accumulated in Smn1 knockout muscles, because of the lysosomal dysfunction highlighted by the transcriptional profiling. Amniotic fluid stem cells transplantation that corrects the SMN knockout mouse myopathic phenotype restored mitochondrial morphology and expression of mitochondrial genes. Thus, targeting muscle mitochondrial dysfunction in SMA may complement the current gene therapy.

A perfusion-based three-dimensional cell culture system to model alveolar rhabdomyosarcoma pathological features.

Although a rare disease, rhabdomyosarcoma (RMS) is one of the most common cancers in children the more aggressive and metastatic subtype is the alveolar RMS (ARMS). Survival outcomes with metastatic disease remain dismal and the need for new models that recapitulate key pathological features, including cell-extracellular matrix (ECM) interactions, is warranted. Here, we report an organotypic model that captures cellular and molecular determinants of invasive ARMS. We cultured the ARMS cell line RH30 on a collagen sponge in a perfusion-based bioreactor (U-CUP), obtaining after 7 days a 3D construct with homogeneous cell distribution. Compared to static culture, perfusion flow induced higher cell proliferation rates (20% vs. 5%), enhanced secretion of active MMP-2, and upregulation of the Rho pathway, associated with cancer cell dissemination. Consistently, the ECM genes LAMA1 and LAMA2, the antiapoptotic gene HSP90, identified in patient databases as hallmarks of invasive ARMS, were higher under perfusion flow at mRNA and protein level. Our advanced ARMS organotypic model mimics (1) the interactions cells-ECM, (2) the cell growth maintenance, and (3) the expression of proteins that characterize tumor expansion and aggressiveness. In the future, the perfusion-based model could be used with primary patient-derived cell subtypes to create a personalized ARMS chemotherapy screening system.

Expression of Basement Membrane Molecules by Wharton Jelly Stem Cells (WJSC) in Full-Term Human Umbilical Cords, Cell Cultures and Microtissues.

Wharton's jelly stem cells (WJSC) from the human umbilical cord (UC) are one of the most promising mesenchymal stem cells (MSC) in tissue engineering (TE) and advanced therapies. The cell niche is a key element for both, MSC and fully differentiated tissues, to preserve their unique features. The basement membrane (BM) is an essential structure during embryonic development and in adult tissues. Epithelial BMs are well-known, but similar structures are present in other histological structures, such as in peripheral nerve fibers, myocytes or chondrocytes. Previous studies suggest the expression of some BM molecules within the Wharton's Jelly (WJ) of UC, but the distribution pattern and full expression profile of these molecules have not been yet elucidated. In this sense, the aim of this histological study was to evaluate the expression of main BM molecules within the WJ, cultured WJSC and during WJSC microtissue (WJSC-MT) formation process. Results confirmed the presence of a pericellular matrix composed by the main BM molecules-collagens (IV, VII), HSPG2, agrin, laminin and nidogen-around the WJSC within UC. Additionally, ex vivo studies demonstrated the synthesis of these BM molecules, except agrin, especially during WJSC-MT formation process. The WJSC capability to synthesize main BM molecules could offer new alternatives for the generation of biomimetic-engineered substitutes where these molecules are particularly needed.

Biodistribution of Intratracheal, Intranasal, and Intravenous Injections of Human Mesenchymal Stromal Cell-Derived Extracellular Vesicles in a Mouse Model for Drug Delivery Studies.

Mesenchymal stromal cell-derived extracellular vesicles (MSC-EVs) are extensively studied as therapeutic tools. Evaluation of their biodistribution is fundamental to understanding MSC-EVs' impact on target organs. In our work, MSC-EVs were initially labeled with DiR, a fluorescent lipophilic dye, and administered to BALB/c mice (2.00 × 1010 EV/mice) through the following routes: intravenous (IV), intratracheal (IT) and intranasal (IN). DiR-labeled MSC-EVs were monitored immediately after injection, and after 3 and 24 hours (h). Whole-body analysis, 3 h after IV injection, showed an accumulation of MSC-EVs in the mice abdominal region, compared to IT and IN, where EVs mainly localized at the levels of the chest and brain region, respectively. After 24 h, EV-injected mice retained a stronger positivity in the same regions identified after 3 h from injection. The analyses of isolated organs confirmed the accumulation of EVs in the spleen and liver after IV administration. Twenty-four hours after the IT injection of MSC-EVs, a stronger positivity was detected selectively in the isolated lungs, while for IN, the signal was confined to the brain. In conclusion, these results show that local administration of EVs can increase their concentration in selective organs, limiting their systemic biodistribution and possibly the extra-organ effects. Biodistribution studies can help in the selection of the most appropriate way of administration of MSC-EVs for the treatment of different diseases.

Recommendations from the COST action CA17116 (SPRINT) for the standardization of perinatal derivative preparation and in vitro testing.

Many preclinical studies have shown that birth-associated tissues, cells and their secreted factors, otherwise known as perinatal derivatives (PnD), possess various biological properties that make them suitable therapeutic candidates for the treatment of numerous pathological conditions. Nevertheless, in the field of PnD research, there is a lack of critical evaluation of the PnD standardization process: from preparation to in vitro testing, an issue that may ultimately delay clinical translation. In this paper, we present the PnD e-questionnaire developed to assess the current state of the art of methods used in the published literature for the procurement, isolation, culturing preservation and characterization of PnD in vitro. Furthermore, we also propose a consensus for the scientific community on the minimal criteria that should be reported to facilitate standardization, reproducibility and transparency of data in PnD research. Lastly, based on the data from the PnD e-questionnaire, we recommend to provide adequate information on the characterization of the PnD. The PnD e-questionnaire is now freely available to the scientific community in order to guide researchers on the minimal criteria that should be clearly reported in their manuscripts. This review is a collaborative effort from the COST SPRINT action (CA17116), which aims to guide future research to facilitate the translation of basic research findings on PnD into clinical practice.

In vivo tissue engineering of functional skeletal muscle by freshly isolated satellite cells embedded in a photopolymerizable hydrogel.

The success of skeletal muscle reconstruction depends on finding the most effective, clinically suitable strategy to engineer myogenic cells and biocompatible scaffolds. Satellite cells (SCs), freshly isolated or transplanted within their niche, are presently considered the best source for muscle regeneration. Here, we designed and developed the delivery of either SCs or muscle progenitor cells (MPCs) via an in situ photo-cross-linkable hyaluronan-based hydrogel, hyaluronic acid-photoinitiator (HA-PI) complex. Partially ablated tibialis anterior (TA) of C57BL/6J mice engrafted with freshly isolated satellite cells embedded in hydrogel showed a major improvement in muscle structure and number of new myofibers, compared to muscles receiving hydrogel + MPCs or hydrogel alone. Notably, SCs embedded in HA-PI also promoted functional recovery, as assessed by contractile force measurements. Tissue reconstruction was associated with the formation of both neural and vascular networks and the reconstitution of a functional SC niche. This innovative approach could overcome previous limitations in skeletal muscle tissue engineering.