VEGF overexpressed mesoangioblasts enhance urethral and vaginal recovery following simulated vaginal birth in rats.

Vaginal birth causes pelvic floor injury which may lead to urinary incontinence. Cell therapy has been proposed to assist in functional recovery. We aim to assess if intra-arterial injection of rat mesoangioblasts (MABs) and stable Vascular Endothelial Growth Factor (VEGF)-expressing MABs, improve recovery of urethral and vaginal function following simulated vaginal delivery (SVD). Female rats (n = 86) were assigned to either injection of saline (control), allogeneic-MABs (MABsallo), autologous-MABs (MABsauto) or allogeneic-MABs transduced to stably expressed VEGF (MABsallo-VEGF). One hour after SVD, 0.5 × 106 MABs or saline were injected into the aorta. Primary outcome was urethral (7d and 14d) and vaginal (14d) function; others were bioluminescent imaging for cell tracking (1, 3 and 7d), morphometry (7, 14 and 60d) and mRNAseq (3 and 7d). All MABs injected rats had external urethral sphincter and vaginal function recovery within 14d, as compared to only half of saline controls. Functional recovery was paralleled by improved muscle regeneration and microvascularization. Recovery rate was not different between MABsallo and MABsauto. MABsallo-VEGF accelerated functional recovery and increased GAP-43 expression at 7d. At 3d we detected major transcriptional changes in the urethra of both MABsallo and MABsallo-VEGF-injected animals, with upregulation of Rho/GTPase activity, epigenetic factors and dendrite development. MABSallo also upregulated transcripts that encode proteins involved in myogenesis and downregulated pro-inflammatory processes. MABsallo-VEGF also upregulated transcripts that encode proteins involved in neuron development and downregulated genes involved in hypoxia and oxidative stress. At 7d, urethras of MABsallo-VEGF-injected rats showed downregulation of oxidative and inflammatory response compared to MABSallo. Intra-arterial injection of MABsallo-VEGF enhances neuromuscular regeneration induced by untransduced MABs and accelerates the functional urethral and vaginal recovery after SVD.

Measurement of LAG-3 Expression Across Multiple Staining Platforms With the 17B4 Antibody Clone.

CONTEXT.­: An immunohistochemistry (IHC) assay developed to detect lymphocyte-activation gene 3 (LAG-3), a novel immune checkpoint inhibitor target, has demonstrated high analytic precision and interlaboratory reproducibility using a Leica staining platform, but it has not been investigated on other IHC staining platforms. OBJECTIVE.­: To evaluate the performance of LAG-3 IHC assays using the 17B4 antibody clone across widely used IHC staining platforms: Agilent/Dako Autostainer Link 48 and VENTANA BenchMark ULTRA compared to Leica BOND-RX (BOND-RX). DESIGN.­: Eighty formalin-fixed, paraffin-embedded melanoma tissue blocks were cut into consecutive sections and evaluated using staining platform-specific IHC assays with the 17B4 antibody clone. Duplicate testing was performed on the BOND-RX platform to assess intraplatform agreement. LAG-3 expression using a numeric score was evaluated by a pathologist and with a digital scoring algorithm. LAG-3 positivity was determined from manual scores using a 1% or greater cutoff. RESULTS.­: LAG-3 IHC staining patterns and intensities were visually similar across all 3 staining platforms. Spearman and Pearson correlations were 0.75 or greater for interplatform and BOND-RX intraplatform concordance when LAG-3 expression was evaluated with a numeric score determined by a pathologist. Correlation increased with a numeric score determined with a digital scoring algorithm (Spearman and Pearson correlations ≥0.88 for all comparisons). Overall percentage agreement was 77.5% or greater for interplatform and BOND-RX intraplatform comparisons when LAG-3 positivity was determined using a 1% or greater cutoff. CONCLUSIONS.­: Data presented here demonstrate that LAG-3 expression can be robustly and reproducibly assessed across 3 major commercial IHC staining platforms using the 17B4 antibody clone.

Extracellular vesicle-derived miRNAs improve stem cell-based therapeutic approaches in muscle wasting conditions.

Skeletal muscle holds an intrinsic capability of growth and regeneration both in physiological conditions and in case of injury. Chronic muscle illnesses, generally caused by genetic and acquired factors, lead to deconditioning of the skeletal muscle structure and function, and are associated with a significant loss in muscle mass. At the same time, progressive muscle wasting is a hallmark of aging. Given the paracrine properties of myogenic stem cells, extracellular vesicle-derived signals have been studied for their potential implication in both the pathogenesis of degenerative neuromuscular diseases and as a possible therapeutic target. In this study, we screened the content of extracellular vesicles from animal models of muscle hypertrophy and muscle wasting associated with chronic disease and aging. Analysis of the transcriptome, protein cargo, and microRNAs (miRNAs) allowed us to identify a hypertrophic miRNA signature amenable for targeting muscle wasting, consisting of miR-1 and miR-208a. We tested this signature among others in vitro on mesoangioblasts (MABs), vessel-associated adult stem cells, and we observed an increase in the efficiency of myogenic differentiation. Furthermore, injections of miRNA-treated MABs in aged mice resulted in an improvement in skeletal muscle features, such as muscle weight, strength, cross-sectional area, and fibrosis compared to controls. Overall, we provide evidence that the extracellular vesicle-derived miRNA signature we identified enhances the myogenic potential of myogenic stem cells.

Mesoangioblasts at 20: From the embryonic aorta to the patient bed.

In 2002 we published an article describing a population of vessel-associated progenitors that we termed mesoangioblasts (MABs). During the past decade evidence had accumulated that during muscle development and regeneration things may be more complex than a simple sequence of binary choices (e.g., dorsal vs. ventral somite). LacZ expressing fibroblasts could fuse with unlabelled myoblasts but not among themselves or with other cell types. Bone marrow derived, circulating progenitors were able to participate in muscle regeneration, though in very small percentage. Searching for the embryonic origin of these progenitors, we identified them as originating at least in part from the embryonic aorta and, at later stages, from the microvasculature of skeletal muscle. While continuing to investigate origin and fate of MABs, the fact that they could be expanded in vitro (also from human muscle) and cross the vessel wall, suggested a protocol for the cell therapy of muscular dystrophies. We tested this protocol in mice and dogs before proceeding to the first clinical trial on Duchenne Muscular Dystrophy patients that showed safety but minimal efficacy. In the last years, we have worked to overcome the problem of low engraftment and tried to understand their role as auxiliary myogenic progenitors during development and regeneration.

Generation of Human Motor Units with Functional Neuromuscular Junctions in Microfluidic Devices.

Neuromuscular junctions (NMJs) are specialized synapses between the axon of the lower motor neuron and the muscle facilitating the engagement of muscle contraction. In motor neuron disorders, such as amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA), NMJs degenerate, resulting in muscle atrophy and progressive paralysis. The underlying mechanism of NMJ degeneration is unknown, largely due to the lack of translatable research models. This study aimed to create a versatile and reproducible in vitro model of a human motor unit with functional NMJs. Therefore, human induced pluripotent stem cell (hiPSC)-derived motor neurons and human primary mesoangioblast (MAB)-derived myotubes were co-cultured in commercially available microfluidic devices. The use of fluidically isolated micro-compartments allows for the maintenance of cell-specific microenvironments while permitting cell-to-cell contact through microgrooves. By applying a chemotactic and volumetric gradient, the growth of motor neuron-neurites through the microgrooves promoting myotube interaction and the formation of NMJs were stimulated. These NMJs were identified immunocytochemically through co-localization of motor neuron presynaptic marker synaptophysin (SYP) and postsynaptic acetylcholine receptor (AChR) marker α-bungarotoxin (Btx) on myotubes and characterized morphologically using scanning electron microscopy (SEM). The functionality of the NMJs was confirmed by measuring calcium responses in myotubes upon depolarization of the motor neurons. The motor unit generated using standard microfluidic devices and stem cell technology can aid future research focusing on NMJs in health and disease.

Improved functionality and potency of next generation BinMLV viral vectors toward safer gene therapy.

To develop safer retroviral murine leukemia virus (MLV)-based vectors, we previously mutated and re-engineered the MLV integrase: the W390A mutation abolished the interaction with its cellular tethering factors, BET proteins, and a retargeting peptide (the chromodomain of the CBX1 protein) was fused C-terminally. The resulting BET-independent MLVW390A-CBX was shown to integrate efficiently and more randomly, away from typical retroviral markers. In this study, we assessed the functionality and stability of expression of the redistributed MLVW390A-CBX vector in more depth, and evaluated safety using a clinically more relevant vector design encompassing a self-inactivated (SIN) LTR and a weak internal elongation factor 1α short (EFS) promoter. MLVW390A-CBX-EFS produced like MLVWT and efficiently transduced laboratory cells and primary human CD34+ hematopoetic stem cells (HSC) without transgene silencing over time, while displaying a more preferred, redistributed, and safer integration pattern. In a human mesoangioblast (MAB) stem cell model, the myogenic fusion capacity was hindered following MLVWT transduction, while this remained unaffected when applying MLVW390A-CBX. Likewise, smooth muscle cell differentiation of MABs was unaltered by MLVW390A-CBX-EFS. Taken together, our results underscore the potential of MLVW390A-CBX-EFS as a clinically relevant viral vector for ex-vivo gene therapy, combining efficient production with a preferable integration site distribution profile and stable expression over time.

Human motor units in microfluidic devices are impaired by FUS mutations and improved by HDAC6 inhibition.

Neuromuscular junctions (NMJs) ensure communication between motor neurons (MNs) and muscle; however, in MN disorders, such as amyotrophic lateral sclerosis (ALS), NMJs degenerate resulting in muscle atrophy. The aim of this study was to establish a versatile and reproducible in vitro model of a human motor unit to investigate the effects of ALS-causing mutations. Therefore, we generated a co-culture of human induced pluripotent stem cell (iPSC)-derived MNs and human primary mesoangioblast-derived myotubes in microfluidic devices. A chemotactic and volumetric gradient facilitated the growth of MN neurites through microgrooves resulting in the interaction with myotubes and the formation of NMJs. We observed that ALS-causing FUS mutations resulted in reduced neurite outgrowth as well as an impaired neurite regrowth upon axotomy. NMJ numbers were likewise reduced in the FUS-ALS model. Interestingly, the selective HDAC6 inhibitor, Tubastatin A, improved the neurite outgrowth, regrowth, and NMJ morphology, prompting HDAC6 inhibition as a potential therapeutic strategy for ALS.

Isolation of Mammalian Mesoangioblasts: A Subset of Pericytes with Myogenic Potential.

Mesoangioblasts (MABs) are vessel-associated stem cells that express pericyte markers and are originally isolated from the embryonic dorsal aorta. From postnatal small vessels of skeletal muscle and heart, it is possible to isolate cells with similar characteristics to embryonic MABs. Adult MABs have the capacity to self-renew and to differentiate into cell types of mesodermal lineages upon proper culture conditions. To date, the origin of MABs and the relationship with other muscle stem cells are still debated. Recently, in a phase I-II clinical trial, intra-arterial HLA-matched MABs were proved to be relatively safe. Novel information on MAB pure populations is desirable, and implementation of their therapeutic potential is mandatory to approach efficacy in MAB-based treatments. This chapter provides an overview of the current techniques for isolation and characterization of rodent, canine, human, and equine adult MABs.

Zeb2 Regulates Myogenic Differentiation in Pluripotent Stem Cells.

Skeletal muscle differentiation is triggered by a unique family of myogenic basic helix-loop-helix transcription factors, including MyoD, MRF-4, Myf-5, and Myogenin. These transcription factors bind promoters and distant regulatory regions, including E-box elements, of genes whose expression is restricted to muscle cells. Other E-box binding zinc finger proteins target the same DNA response elements, however, their function in muscle development and regeneration is still unknown. Here, we show that the transcription factor zinc finger E-box-binding homeobox 2 (Zeb2, Sip-1, Zfhx1b) is present in skeletal muscle tissues. We investigate the role of Zeb2 in skeletal muscle differentiation using genetic tools and transgenic mouse embryonic stem cells, together with single-cell RNA-sequencing and in vivo muscle engraftment capability. We show that Zeb2 over-expression has a positive impact on skeletal muscle differentiation in pluripotent stem cells and adult myogenic progenitors. We therefore propose that Zeb2 is a novel myogenic regulator and a possible target for improving skeletal muscle regeneration. The non-neural roles of Zeb2 are poorly understood.

(Epi)genetic Modifications in Myogenic Stem Cells: From Novel Insights to Therapeutic Perspectives.

The skeletal muscle is considered to be an ideal target for stem cell therapy as it has an inherent regenerative capacity. Upon injury, the satellite cells, muscle stem cells that reside under the basal lamina of the myofibres, start to differentiate in order to reconstitute the myofibres while maintaining the initial stem cell pool. In recent years, it has become more and more evident that epigenetic mechanisms such as histon modifications, DNA methylations and microRNA modulations play a pivatol role in this differentiation process. By understanding the mechanisms behind myogenesis, researchers are able to use this knowledge to enhance the differentiation and engraftment potential of different muscle stem cells. Besides manipulation on an epigenetic level, recent advances in the field of genome-engineering allow site-specific modifications in the genome of these stem cells. Combining epigenetic control of the stem cell fate with the ability to site-specifically correct mutations or add genes for further cell control, can increase the use of stem cells as treatment of muscular dystrophies drastically. In this review, we will discuss the advances that have been made in genome-engineering and the epigenetic regulation of muscle stem cells and how this knowledge can help to get stem cell therapy to its full potential.

Fate of mesoangioblasts in a vaginal birth injury model: influence of the route of administration.

Currently cell therapy is considered as an experimental strategy to assist the healing process following simulated vaginal birth injury in rats, boosting the functional and morphologic recovery of pelvic floor muscles and nerves. However, the optimal administration route and dose still need to be determined. Mesangioblasts theoretically have the advantage that they can differentiate in skeletal and smooth muscle. We investigated the fate of mesoangioblasts transduced with luciferase and green fluorescent protein reporter genes (rMABseGFP/fLUC) using bioluminescence, immunofluorescence and RT-PCR in rats undergoing simulated birth injury. rMABseGFP/fLUC were injected locally, intravenously and intra-arterially (common iliacs and aorta). Intra-arterial delivery resulted in the highest amount of rMABseGFP/fLUC in the pelvic organs region and in a more homogeneous distribution over all relevant pelvic organs. Sham controls showed that the presence of the injury is important for recruitment of intra-arterially injected rMABseGFP/fLUC. Injection through the aorta or bilaterally in the common iliac arteries resulted in comparable numbers of rMABseGFP/fLUC in the pelvic organs, yet aortic injection was faster and gave less complications.

Aging affects the in vivo regenerative potential of human mesoangioblasts.

Sarcopenia is the age-related loss of muscle mass, strength, and function. Although the role of human satellite cells (SCs) as adult skeletal muscle stem cells has been deeply investigated, little is known about the impact of aging on muscle interstitial stem cells. Here, we isolated the non-SC CD56- fraction from human muscle biopsies of young and elderly subjects. The elderly interstitial cell population contained a higher number of CD15+ and PDGFRα+ cells when compared to young samples. In addition, we found that the CD56- /ALP+ cells were well represented as a multipotent stem cell population inside the CD56- fraction. CD56- /ALP+ /CD15- cells were clonogenic, and since they were myogenic and expressed NG2, α-SMA and PDGFRß can be considered mesoangioblasts (MABs). Interestingly, elderly MABs displayed a dramatic impairment in the myogenic differentiation ability in vitro and when transplanted in dystrophic immunodeficient Sgcb-null Rag2-null γc-null mice. In addition, elderly MABs proliferated less, but yet retained other multilineage capabilities. Overall, our results indicate that aging negatively impacted on the regenerative potential of MABs and this should be carefully considered for potential therapeutic applications of MABs.

Morphological and functional analyses of skeletal muscles from an immunodeficient animal model of limb-girdle muscular dystrophy type 2E.

INTRODUCTION: Limb-girdle muscular dystrophy type 2E (LGMD2E) is caused by mutations in the ß-sarcoglycan gene, which is expressed in skeletal, cardiac, and smooth muscles. ß-Sarcoglycan-deficient (Sgcb-null) mice develop severe muscular dystrophy and cardiomyopathy with focal areas of necrosis. METHODS: In this study we performed morphological (histological and cellular characterization) and functional (isometric tetanic force and fatigue) analyses in dystrophic mice. Comparison studies were carried out in 1-month-old (clinical onset of the disease) and 7-month-old control mice (C57Bl/6J, Rag2/γc-null) and immunocompetent and immunodeficient dystrophic mice (Sgcb-null and Sgcb/Rag2/γc-null, respectively). RESULTS: We found that the lack of an immunological system resulted in an increase of calcification in striated muscles without impairing extensor digitorum longus muscle performance. Sgcb/Rag2/γc-null muscles showed a significant reduction of alkaline phosphate-positive mesoangioblasts. DISCUSSION: The immunological system counteracts skeletal muscle degeneration in the murine model of LGMD2E. Muscle Nerve, 2018.

MicroRNAs promote skeletal muscle differentiation of mesodermal iPSC-derived progenitors.

Muscular dystrophies (MDs) are often characterized by impairment of both skeletal and cardiac muscle. Regenerative strategies for both compartments therefore constitute a therapeutic avenue. Mesodermal iPSC-derived progenitors (MiPs) can regenerate both striated muscle types simultaneously in mice. Importantly, MiP myogenic propensity is influenced by somatic lineage retention. However, it is still unknown whether human MiPs have in vivo potential. Furthermore, methods to enhance the intrinsic myogenic properties of MiPs are likely needed, given the scope and need to correct large amounts of muscle in the MDs. Here, we document that human MiPs can successfully engraft into the skeletal muscle and hearts of dystrophic mice. Utilizing non-invasive live imaging and selectively induced apoptosis, we report evidence of striated muscle regeneration in vivo in mice by human MiPs. Finally, combining RNA-seq and miRNA-seq data, we define miRNA cocktails that promote the myogenic potential of human MiPs.

Equine-Induced Pluripotent Stem Cells Retain Lineage Commitment Toward Myogenic and Chondrogenic Fates.

Induced pluripotent stem cells (iPSCs) hold great potential not only for human but also for veterinary purposes. The equine industry must often deal with health issues concerning muscle and cartilage, where comprehensive regenerative strategies are still missing. In this regard, a still open question is whether equine iPSCs differentiate toward muscle and cartilage, and whether donor cell type influences their differentiation potential. We addressed these questions through an isogenic system of equine iPSCs obtained from myogenic mesoangioblasts (MAB-iPSCs) and chondrogenic mesenchymal stem cells (MSC-iPSCs). Despite similar levels of pluripotency characteristics, the myogenic differentiation appeared enhanced in MAB-iPSCs. Conversely, the chondrogenic differentiation was augmented in MSC-iPSCs through both teratoma and in vitro differentiation assays. Thus, our data suggest that equine iPSCs can differentiate toward the myogenic and chondrogenic lineages, and can present a skewed differentiation potential in favor of the source cell lineage.

Unconventional Players on the Striated Muscle Field: microRNAs, Signaling Pathways and Epigenetic Regulators.

Striated muscle regeneration holds an intrinsic complexity governed by many orchestrated events. When the fine balance of regulatory machineries is under strain, the homeostatic conditions are lost and degeneration starts to occur. This is the case for inherited and acquired diseases of both cardiac and skeletal muscles. A wide range of factors are currently under scrutiny for better understanding the details underlying de-/re-generation processes, of both genetic and non-genetic nature. This review focuses on three classes of non-genetic factors regulating striated muscle regeneration, i.e. microRNAs, signaling pathways and epigenetic regulators.

Mesodermal iPSC-derived progenitor cells functionally regenerate cardiac and skeletal muscle.

Conditions such as muscular dystrophies (MDs) that affect both cardiac and skeletal muscles would benefit from therapeutic strategies that enable regeneration of both of these striated muscle types. Protocols have been developed to promote induced pluripotent stem cells (iPSCs) to differentiate toward cardiac or skeletal muscle; however, there are currently no strategies to simultaneously target both muscle types. Tissues exhibit specific epigenetic alterations; therefore, source-related lineage biases have the potential to improve iPSC-driven multilineage differentiation. Here, we determined that differential myogenic propensity influences the commitment of isogenic iPSCs and a specifically isolated pool of mesodermal iPSC-derived progenitors (MiPs) toward the striated muscle lineages. Differential myogenic propensity did not influence pluripotency, but did selectively enhance chimerism of MiP-derived tissue in both fetal and adult skeletal muscle. When injected into dystrophic mice, MiPs engrafted and repaired both skeletal and cardiac muscle, reducing functional defects. Similarly, engraftment into dystrophic mice of canine MiPs from dystrophic dogs that had undergone TALEN-mediated correction of the MD-associated mutation also resulted in functional striatal muscle regeneration. Moreover, human MiPs exhibited the same capacity for the dual differentiation observed in murine and canine MiPs. The findings of this study suggest that MiPs should be further explored for combined therapy of cardiac and skeletal muscles.