Matching Together Living Cells and Prototissues: Will There Be Chemistry?

Scientific advancements in bottom-up synthetic biology have led to the development of numerous models of synthetic cells, or protocells. To date, research has mainly focused on increasing the (bio)chemical complexity of these bioinspired micro-compartmentalized systems, yet the successful integration of protocells with living cells remains one of the major challenges in bottom-up synthetic biology. In this review, we aim to summarize the current state of the art in hybrid protocell/living cell and prototissue/living cell systems. Inspired by recent breakthroughs in tissue engineering, we review the chemical, bio-chemical, and mechano-chemical aspects that hold promise for achieving an effective integration of non-living and living matter. The future production of fully integrated protocell/living cell systems and increasingly complex prototissue/living tissue systems not only has the potential to revolutionize the field of tissue engineering, but also paves the way for new technologies in (bio)sensing, personalized therapy, and drug delivery.

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 colorectal cancer matrix as bioactive microenvironment for in vitro 3D cancer research.

Three-dimensional (3D) cancer models are overlooking the scientific landscape with the primary goal of bridging the gaps between two-dimensional (2D) cell lines, animal models and clinical research. Here, we describe an innovative tissue engineering approach applied to colorectal cancer (CRC) starting from decellularized human biopsies in order to generate an organotypic 3D-bioactive model. This in vitro 3D system recapitulates the ultrastructural environment of native tissue as demonstrated by histology, immunohistochemistry, immunofluorescence and scanning electron microscopy analyses. Mass spectrometry of proteome and secretome confirmed a different stromal composition between decellularized healthy mucosa and CRC in terms of structural and secreted proteins. Importantly, we proved that our 3D acellular matrices retained their biological properties: using CAM assay, we observed a decreased angiogenic potential in decellularized CRC compared with healthy tissue, caused by direct effect of DEFA3. We demonstrated that following a 5 days of recellularization with HT-29 cell line, the 3D tumor matrices induced an over-expression of IL-8, a DEFA3-mediated pathway and a mandatory chemokine in cancer growth and proliferation. Given the biological activity maintained by the scaffolds after decellularization, we believe this approach is a powerful tool for future pre-clinical research and screenings.

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.

Extracellular Matrix and Colorectal Cancer: How Surrounding Microenvironment Affects Cancer Cell Behavior?

Colorectal cancer (CRC) whit more than a million of new cases per year is one of the most common registered cancers worldwide with few treatment options especially for advanced and metastatic patients.The tumor microenvironment is composed by extracellular matrix (ECM), cells, and interstitial fluids. Among all these constituents, in the last years an increased interest around the ECM and its potential role in cancer tumorigenesis is arisen. During cancer progression the ECM structure and composition became disorganized, allowing cellular transformation and metastasis. Up to now, the focus has mainly been on the characterization of CRC microenvironment analyzing separately structural ECM components or cell secretome modifications. A more extensive view that interconnects these aspects should be addressed. In this review, biochemical (secretome) and biomechanical (structure and architecture) changes of tumor microenvironment will be discussed, giving suggestions on how these changes can affect cancer cell behavior. J. Cell. Physiol. 232: 967-975, 2017. © 2016 Wiley Periodicals, Inc.

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.

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.

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.

Three-Dimensional Bioprinting of Naturally Derived Hydrogels for the Production of Biomimetic Living Tissues: Benefits and Challenges.

Three-dimensional bioprinting is the process of manipulating cell-laden bioinks to fabricate living structures. Three-dimensional bioprinting techniques have brought considerable innovation in biomedicine, especially in the field of tissue engineering, allowing the production of 3D organ and tissue models for in vivo transplantation purposes or for in-depth and precise in vitro analyses. Naturally derived hydrogels, especially those obtained from the decellularization of biological tissues, are promising bioinks for 3D printing purposes, as they present the best biocompatibility characteristics. Despite this, many natural hydrogels do not possess the necessary mechanical properties to allow a simple and immediate application in the 3D printing process. In this review, we focus on the bioactive and mechanical characteristics that natural hydrogels may possess to allow efficient production of organs and tissues for biomedical applications, emphasizing the reinforcement techniques to improve their biomechanical properties.

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.

3D in vitro Models of Pathological Skeletal Muscle: Which Cells and Scaffolds to Elect?

Skeletal muscle is a fundamental tissue of the human body with great plasticity and adaptation to diseases and injuries. Recreating this tissue in vitro helps not only to deepen its functionality, but also to simulate pathophysiological processes. In this review we discuss the generation of human skeletal muscle three-dimensional (3D) models obtained through tissue engineering approaches. First, we present an overview of the most severe myopathies and the two key players involved: the variety of cells composing skeletal muscle tissue and the different components of its extracellular matrix. Then, we discuss the peculiar characteristics among diverse in vitro models with a specific focus on cell sources, scaffold composition and formulations, and fabrication techniques. To conclude, we highlight the efficacy of 3D models in mimicking patient-specific myopathies, deepening muscle disease mechanisms or investigating possible therapeutic effects.

Customized bioreactor enables the production of 3D diaphragmatic constructs influencing matrix remodeling and fibroblast overgrowth.

The production of skeletal muscle constructs useful for replacing large defects in vivo, such as in congenital diaphragmatic hernia (CDH), is still considered a challenge. The standard application of prosthetic material presents major limitations, such as hernia recurrences in a remarkable number of CDH patients. With this work, we developed a tissue engineering approach based on decellularized diaphragmatic muscle and human cells for the in vitro generation of diaphragmatic-like tissues as a proof-of-concept of a new option for the surgical treatment of large diaphragm defects. A customized bioreactor for diaphragmatic muscle was designed to control mechanical stimulation and promote radial stretching during the construct engineering. In vitro tests demonstrated that both ECM remodeling and fibroblast overgrowth were positively influenced by the bioreactor culture. Mechanically stimulated constructs also increased tissue maturation, with the formation of new oriented and aligned muscle fibers. Moreover, after in vivo orthotopic implantation in a surgical CDH mouse model, mechanically stimulated muscles maintained the presence of human cells within myofibers and hernia recurrence did not occur, suggesting the value of this approach for treating diaphragm defects.

Porcine Decellularized Diaphragm Hydrogel: A New Option for Skeletal Muscle Malformations.

Hydrogels are biomaterials that, thanks to their unique hydrophilic and biomimetic characteristics, are used to support cell growth and attachment and promote tissue regeneration. The use of decellularized extracellular matrix (dECM) from different tissues or organs significantly demonstrated to be far superior to other types of hydrogel since it recapitulates the native tissue's ECM composition and bioactivity. Different muscle injuries and malformations require the application of patches or fillers to replenish the defect and boost tissue regeneration. Herein, we develop, produce, and characterize a porcine diaphragmatic dECM-derived hydrogel for diaphragmatic applications. We obtain a tissue-specific biomaterial able to mimic the complex structure of skeletal muscle ECM; we characterize hydrogel properties in terms of biomechanical properties, biocompatibility, and adaptability for in vivo applications. Lastly, we demonstrate that dECM-derived hydrogel obtained from porcine diaphragms can represent a useful biological product for diaphragmatic muscle defect repair when used as relevant acellular stand-alone patch.

Extracellular Vesicles Secreted by Mesenchymal Stromal Cells Exert Opposite Effects to Their Cells of Origin in Murine Sodium Dextran Sulfate-Induced Colitis.

Several reports have described a beneficial effect of Mesenchymal Stromal Cells (MSCs) and of their secreted extracellular vesicles (EVs) in mice with experimental colitis. However, the effects of the two treatments have not been thoroughly compared in this model. Here, we compared the effects of MSCs and of MSC-EV administration in mice with colitis induced by dextran sulfate sodium (DSS). Since cytokine conditioning was reported to enhance the immune modulatory activity of MSCs, the cells were kept either under standard culture conditions (naïve, nMSCs) or primed with a cocktail of pro-inflammatory cytokines, including IL1ß, IL6 and TNFα (induced, iMSCs). In our experimental conditions, nMSCs and iMSCs administration resulted in both clinical and histological worsening and was associated with pro-inflammatory polarization of intestinal macrophages. However, mice treated with iEVs showed clinico-pathological improvement, decreased intestinal fibrosis and angiogenesis and a striking increase in intestinal expression of Mucin 5ac, suggesting improved epithelial function. Moreover, treatment with iEVs resulted in the polarization of intestinal macrophages towards and anti-inflammatory phenotype and in an increased Treg/Teff ratio at the level of the intestinal lymph node. Collectively, these data confirm that MSCs can behave either as anti- or as pro-inflammatory agents depending on the host environment. In contrast, EVs showed a beneficial effect, suggesting a more predictable behavior, a safer therapeutic profile and a higher therapeutic efficacy with respect to their cells of origin.

Muscle functional recovery is driven by extracellular vesicles combined with muscle extracellular matrix in a volumetric muscle loss murine model.

Biological scaffolds derived from decellularized tissues are being investigated as a promising approach to repair volumetric muscle losses (VML). Indeed, extracellular matrix (ECM) from decellularized tissues is highly biocompatible and mimics the original tissue. However, the development of fibrosis and the muscle stiffness still represents a major problem. Intercellular signals mediating tissue repair are conveyed via extracellular vesicles (EVs), biologically active nanoparticles secreted by the cells. This work aimed at using muscle ECM and human EVs derived from Wharton Jelly mesenchymal stromal cells (MSC EVs) to boost tissue regeneration in a VML murine model. Mice transplanted with muscle ECM and treated with PBS or MSC EVs were analyzed after 7 and 30 days. Flow cytometry, tissue analysis, qRT-PCR and physiology test were performed. We demonstrated that angiogenesis and myogenesis were enhanced while fibrosis was reduced after EV treatment. Moreover, the inflammation was directed toward tissue repair. M2-like, pro-regenerative macrophages were significantly increased in the MSC EVs treated group compared to control. Strikingly, the histological improvements were associated with enhanced functional recovery. These results suggest that human MSC EVs can be a naturally-derived boost able to ameliorate the efficacy of tissue-specific ECM in muscle regeneration up to the restored tissue function.

Young at Heart: Combining Strategies to Rejuvenate Endogenous Mechanisms of Cardiac Repair.

True cardiac regeneration of the injured heart has been broadly described in lower vertebrates by active replacement of lost cardiomyocytes to functionally and structurally restore the myocardial tissue. On the contrary, following severe injury (i.e., myocardial infarction) the adult mammalian heart is endowed with an impaired reparative response by means of meager wound healing program and detrimental remodeling, which can lead over time to cardiomyopathy and heart failure. Lately, a growing body of basic, translational and clinical studies have supported the therapeutic use of stem cells to provide myocardial regeneration, with the working hypothesis that stem cells delivered to the cardiac tissue could result into new cardiovascular cells to replenish the lost ones. Nevertheless, multiple independent evidences have demonstrated that injected stem cells are more likely to modulate the cardiac tissue via beneficial paracrine effects, which can enhance cardiac repair and reinstate the embryonic program and cell cycle activity of endogenous cardiac stromal cells and resident cardiomyocytes. Therefore, increasing interest has been addressed to the therapeutic profiling of the stem cell-derived secretome (namely the total of cell-secreted soluble factors), with specific attention to cell-released extracellular vesicles, including exosomes, carrying cardioprotective and regenerative RNA molecules. In addition, the use of cardiac decellularized extracellular matrix has been recently suggested as promising biomaterial to develop novel therapeutic strategies for myocardial repair, as either source of molecular cues for regeneration, biological scaffold for cardiac tissue engineering or biomaterial platform for the functional release of factors. In this review, we will specifically address the translational relevance of these two approaches with ad hoc interest in their feasibility to rejuvenate endogenous mechanisms of cardiac repair up to functional regeneration.

Extracellular Matrix-Derived Hydrogels as Biomaterial for Different Skeletal Muscle Tissue Replacements.

Recently, skeletal muscle represents a complex and challenging tissue to be generated in vitro for tissue engineering purposes. Several attempts have been pursued to develop hydrogels with different formulations resembling in vitro the characteristics of skeletal muscle tissue in vivo. This review article describes how different types of cell-laden hydrogels recapitulate the multiple interactions occurring between extracellular matrix (ECM) and muscle cells. A special attention is focused on the biochemical cues that affect myocytes morphology, adhesion, proliferation, and phenotype maintenance, underlining the importance of topographical cues exerted on the hydrogels to guide cellular orientation and facilitate myogenic differentiation and maturation. Moreover, we highlight the crucial role of 3D printing and bioreactors as useful platforms to finely control spatial deposition of cells into ECM based hydrogels and provide the skeletal muscle native-like tissue microenvironment, respectively.