generation-human-motor-units-with-functional-neuromuscular-junctions

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 ...

KU Leuven - University of Leuven

Despite advances in cardiovascular biology and medical therapy, heart disorders are the leading cause of death worldwide. Cell-based regenerative therapies become a promising treatment for patients affected by heart failure, but also underline the need for reproducible results in preclinical and clinical studies for safety and efficacy. Enthusiasm has been tempered by poor engraftment, survival and differentiation of the injected adult stem cells. The crucial challenge is identification and selection of the most suitable stem cell type for cardiac regenerative medicine. Human pluripotent stem cells (PSCs) have emerged as attractive cell source to obtain cardiomyocytes (CMs), with potential applications, including drug discovery and toxicity screening, disease modelling and innovative cell therapies. Lessons from embryology offered important insights into the development of stem cell-derived CMs. However, the generation of a CM population, uniform in cardiac subtype, adult maturation and functional properties, is highly recommended. Moreover, hurdles regarding tumorigenesis, graft cell death, immune rejection and arrhythmogenesis need to be overcome in clinical practice. Here we highlight the recent progression in PSC technologies for the regeneration of injured heart. We review novel strategies that might overcome current obstacles in heart regenerative medicine, aiming at improving cell survival and functional integration after cell transplantation.

Stem Cell Technology in Cardiac Regeneration: A Pluripotent Stem Cell Promise.

Non-coding RNAs (ncRNAs) are emerging players in muscle regulation. Based on their length and differences in molecular structure, ncRNAs are subdivided into several categories including small interfering RNAs, stable non-coding RNAs, microRNAs (miRs), long non-coding RNAs (lncRNAs), and circular RNAs. miRs and lncRNAs are able to post-transcriptionally regulate many genes and bring into play several traits simultaneously due to a myriad of different targets. Recent studies have emphasized their importance in cardiac regeneration and repair. As their altered expression affects cardiac function, miRs and lncRNAs could be potential targets for therapeutic intervention. In this context, miR- and lncRNA-based gene therapies are an interesting field for harnessing the complexity of ncRNA-based therapeutic approaches in cardiac diseases. In this review we will focus on lncRNA- and miR-driven regulations of cardiac development and repair. Finally, we will summarize miRs and lncRNAs as promising candidates for the treatment of heart diseases.

Interactions between microRNAs and long non-coding RNAs in cardiac development and repair.

The molecular mechanisms underlying tissue regeneration and wound healing are still poorly understood despite their importance. In this paper we develop a bioinformatics approach, combining biology and network theory to drive experiments for better understanding the genetic underpinnings of wound healing mechanisms and for selecting potential drug targets. We start by selecting literature-relevant genes in murine wound healing, and inferring from them a Protein-Protein Interaction (PPI) network. Then, we analyze the network to rank wound healing-related genes according to their topological properties. Lastly, we perform a procedure for in-silico simulation of a treatment action in a biological pathway. The findings obtained by applying the developed pipeline, including gene expression analysis, confirms how a network-based bioinformatics method is able to prioritize candidate genes for in vitro analysis, thus speeding up the understanding of molecular mechanisms and supporting the discovery of potential drug targets.

Exploring Wound-Healing Genomic Machinery with a Network-Based Approach.

Dental pulp represents an easily accessible autologous source of adult stem cells. A subset of these cells, named dental pulp pluripotent-like stem cells (DPPSC), shows high plasticity and can undergo multiple population doublings, making DPPSC an appealing tool for tissue repair or maintenance.

Human dental pulp pluripotent-like stem cells promote wound healing and muscle regeneration.

Neural tube defects (NTDs) are severe congenital abnormalities, caused by failed closure of neural tube during early embryonic development. Periconceptional folic acid (FA) supplementation greatly reduces the risk of NTDs. However, the molecular mechanisms behind NTDs and the preventive role of FA remain unclear. Here, we use human induced pluripotent stem cells (iPSCs) derived from fetuses with spina bifida aperta (SBA) to study the pathophysiology of NTDs and explore the effects of FA exposure. We report that FA exposure in SBA model is necessary for the proper formation and maturation of neural tube structures and robust differentiation of mesodermal derivatives. Additionally, we show that the folate antagonist methotrexate dramatically affects the formation of neural tube structures and FA partially reverts this aberrant phenotype. In conclusion, we present a novel model for human NTDs and provide evidence that it is a powerful tool to investigate the molecular mechanisms underlying NTDs, test drugs for therapeutic approaches.

Folic Acid Exposure Rescues Spina Bifida Aperta Phenotypes in Human Induced Pluripotent Stem Cell Model.

Progressive muscle degeneration followed by dilated cardiomyopathy is a hallmark of muscular dystrophy. Stem cell therapy is suggested to replace diseased myofibers by healthy myofibers, although so far, we are faced by low efficiencies of migration and engraftment of stem cells. Chemokines are signalling proteins guiding cell migration and have been shown to tightly regulate muscle tissue repair. We sought to determine which chemokines are expressed in dystrophic muscles undergoing tissue remodelling. Therefore, we analysed the expression of chemokines and chemokine receptors in skeletal and cardiac muscles from Sarcoglycan-α null, Sarcoglycan-β null and immunodeficient Sgcβ-null mice. We found that several chemokines are dysregulated in dystrophic muscles. We further show that one of these, platelet-derived growth factor-B, promotes interstitial stem cell migration. This finding provides perspective to an approachable mechanism for improving stem cell homing towards dystrophic muscles.

Growth Factor Screening in Dystrophic Muscles Reveals PDGFB/PDGFRB-Mediated Migration of Interstitial Stem Cells.

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.

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

Defective cell migration causes delayed wound healing (WH) and chronic skin lesions. Autologous micrograft (AMG) therapies have recently emerged as a new effective and affordable treatment able to improve wound healing capacity. However, the precise molecular mechanism through which AMG exhibits its beneficial effects remains unrevealed. Herein we show that AMG improves skin re-epithelialization by accelerating the migration of fibroblasts and keratinocytes. More specifically, AMG-treated wounds showed improvement of indispensable events associated with successful wound healing such as granulation tissue formation, organized collagen content, and newly formed blood vessels. We demonstrate that AMG is enriched with a pool of WH-associated growth factors that may provide the starting signal for a faster endogenous wound healing response. This work links the increased cell migration rate to the activation of the extracellular signal-regulated kinase (ERK) signaling pathway, which is followed by an increase in matrix metalloproteinase expression and their extracellular enzymatic activity. Overall we reveal the AMG-mediated wound healing transcriptional signature and shed light on the AMG molecular mechanism supporting its potential to trigger a highly improved wound healing process. In this way, we present a framework for future improvements in AMG therapy for skin tissue regeneration applications.

Autologous micrograft accelerates endogenous wound healing response through ERK-induced cell migration.

Cardiac development in the human embryo is characterized by the interactions of several transcription and growth factors leading the heart from a primordial linear tube into a synchronous contractile four-chamber organ. Studies on cardiogenesis showed that cell proliferation, differentiation, fate specification and morphogenesis are spatiotemporally coordinated by cell-cell interactions and intracellular signalling cross-talks. In recent years, research has focused on a class of inter- and intra-cellular modulators called non-coding RNAs (ncRNAs), transcribed from the noncoding portion of the DNA and involved in the proper formation of the heart. In this chapter, we will summarize the current state of the art on the roles of three major forms of ncRNAs [microRNAs (miRNAs), long ncRNAs (lncRNAs) and circular RNAs (circRNAs)] in orchestrating the four sequential phases of cardiac organogenesis.

Comprehensive Overview of Non-coding RNAs in Cardiac Development.

Fibrosis and fat replacement in skeletal muscle are major complications that lead to a loss of mobility in chronic muscle disorders, such as muscular dystrophy. However, the in vivo properties of adipogenic stem and precursor cells remain unclear, mainly due to the high cell heterogeneity in skeletal muscles. Here, we use single-cell RNA sequencing to decomplexify interstitial cell populations in healthy and dystrophic skeletal muscles. We identify an interstitial CD142-positive cell population in mice and humans that is responsible for the inhibition of adipogenesis through GDF10 secretion. Furthermore, we show that the interstitial cell composition is completely altered in muscular dystrophy, with a near absence of CD142-positive cells. The identification of these adipo-regulatory cells in the skeletal muscle aids our understanding of the aberrant fat deposition in muscular dystrophy, paving the way for treatments that could counteract degeneration in patients with muscular dystrophy.

Interstitial Cell Remodeling Promotes Aberrant Adipogenesis in Dystrophic Muscles.

Contractile myofiber units are mainly composed of thick myosin and thin actin (F-actin) filaments. F-Actin interacts with Microtubule Associated Monooxygenase, Calponin And LIM Domain Containing 2 (MICAL2). Indeed, MICAL2 modifies actin subunits and promotes actin filament turnover by severing them and preventing repolymerization. In this study, we found that MICAL2 increases during myogenic differentiation of adult and pluripotent stem cells (PSCs) towards skeletal, smooth and cardiac muscle cells and localizes in the nucleus of acute and chronic regenerating muscle fibers. In vivo delivery of Cas9-Mical2 guide RNA complexes results in muscle actin defects and demonstrates that MICAL2 is essential for skeletal muscle homeostasis and functionality. Conversely, MICAL2 upregulation shows a positive impact on skeletal and cardiac muscle commitments. Taken together these data demonstrate that modulations of MICAL2 have an impact on muscle filament dynamics and its fine-tuned balance is essential for the regeneration of muscle tissues.

MICAL2 is essential for myogenic lineage commitment.

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.

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

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.

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

Muscular dystrophies are debilitating neuromuscular disorders for which no cure exists. As this disorder affects both cardiac and skeletal muscle, patients would benefit from a cellular therapy that can simultaneously regenerate both tissues. The current protocol to derive bipotent mesodermal progenitors which can differentiate into cardiac and skeletal muscle relies on the spontaneous formation of embryoid bodies, thereby hampering further clinical translation. Additionally, as skeletal muscle is the largest organ in the human body, a high myogenic potential is necessary for successful regeneration. Here, we have optimized a protocol to generate chemically defined human induced pluripotent stem cell-derived mesodermal progenitors (cdMiPs). We demonstrate that these cells contribute to myotube formation and differentiate into cardiomyocytes, both in vitro and in vivo. Furthermore, the addition of valproic acid, a clinically approved small molecule, increases the potential of the cdMiPs to contribute to myotube formation that can be prevented by NOTCH signaling inhibitors. Moreover, valproic acid pre-treated cdMiPs injected in dystrophic muscles increase physical strength and ameliorate the functional performances of transplanted mice. Taken together, these results constitute a novel approach to generate mesodermal progenitors with enhanced myogenic potential using clinically approved reagents.

Article	Year
Stem Cell Technology in Cardiac Regeneration: A Pluripotent Stem Cell Promise. EBioMedicine\| PubMed ID: 28169191	2017
Interactions between microRNAs and long non-coding RNAs in cardiac development and repair. Pharmacological research\| PubMed ID: 28629929	2018
Exploring Wound-Healing Genomic Machinery with a Network-Based Approach. Pharmaceuticals (Basel, Switzerland)\| PubMed ID: 28635674	2017
Human dental pulp pluripotent-like stem cells promote wound healing and muscle regeneration. Stem cell research & therapy\| PubMed ID: 28750661	2017
Folic Acid Exposure Rescues Spina Bifida Aperta Phenotypes in Human Induced Pluripotent Stem Cell Model. Scientific reports\| PubMed ID: 29440666	2018
Growth Factor Screening in Dystrophic Muscles Reveals PDGFB/PDGFRB-Mediated Migration of Interstitial Stem Cells. International journal of molecular sciences\| PubMed ID: 30841538	2019
(Epi)genetic Modifications in Myogenic Stem Cells: From Novel Insights to Therapeutic Perspectives. Cells\| PubMed ID: 31075875	2019
Autologous micrograft accelerates endogenous wound healing response through ERK-induced cell migration. Cell death and differentiation\| PubMed ID: 31654035	2020
Comprehensive Overview of Non-coding RNAs in Cardiac Development. Advances in experimental medicine and biology\| PubMed ID: 32285413	2020
Interstitial Cell Remodeling Promotes Aberrant Adipogenesis in Dystrophic Muscles. Cell reports\| PubMed ID: 32375047	2020
MICAL2 is essential for myogenic lineage commitment. Cell death & disease\| PubMed ID: 32811811	2020
Isolation of Mammalian Mesoangioblasts: A Subset of Pericytes with Myogenic Potential. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 33576976	2021
Human motor units in microfluidic devices are impaired by FUS mutations and improved by HDAC6 inhibition. Stem cell reports\| PubMed ID: 33891869	2021
Valproic acid stimulates myogenesis in pluripotent stem cell-derived mesodermal progenitors in a NOTCH-dependent manner. Cell death & disease\| PubMed ID: 34226515	2021

Maurilio Sampaolesi

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