Randomized CRISPR-Cas Transcriptional Perturbation Screening Reveals Protective Genes against Alpha-Synuclein Toxicity.

The genome-wide perturbation of transcriptional networks with CRISPR-Cas technology has primarily involved systematic and targeted gene modulation. Here, we developed PRISM (Perturbing Regulatory Interactions by Synthetic Modulators), a screening platform that uses randomized CRISPR-Cas transcription factors (crisprTFs) to globally perturb transcriptional networks. By applying PRISM to a yeast model of Parkinson's disease (PD), we identified guide RNAs (gRNAs) that modulate transcriptional networks and protect cells from alpha-synuclein (αSyn) toxicity. One gRNA identified in this screen outperformed the most protective suppressors of αSyn toxicity reported previously, highlighting PRISM's ability to identify modulators of important phenotypes. Gene expression profiling revealed genes differentially modulated by this strong protective gRNA that rescued yeast from αSyn toxicity when overexpressed. Human homologs of top-ranked hits protected against αSyn-induced cell death in a human neuronal PD model. Thus, high-throughput and unbiased perturbation of transcriptional networks via randomized crisprTFs can reveal complex biological phenotypes and effective disease modulators.

Role of the immune system in cardiac tissue damage and repair following myocardial infarction.

INTRODUCTION: The immune system plays a crucial role in the initiation, development, and resolution of inflammation following myocardial infarction (MI). The lack of oxygen and nutrients causes the death of cardiomyocytes and leads to the exposure of danger-associated molecular patterns that are recognized by the immune system to initiate inflammation. RESULTS: At the initial stage of post-MI inflammation, the immune system further damages cardiac tissue to clear cell debris. The excessive production of reactive oxygen species (ROS) by immune cells and the inability of the anti-oxidant system to neutralize ROS cause oxidative stress that further aggravates inflammation. On the other hand, the cells of both innate and adaptive immune system and their secreted factors are critically instrumental in the very dynamic and complex processes of regulating inflammation and mediating cardiac repair. CONCLUSIONS: It is important to decipher the balance between detrimental and beneficial effects of the immune system in MI. This enables us to identify better therapeutic targets for reducing the infarct size, sustaining the cardiac function, and minimizing the likelihood of heart failure. This review discusses the role of both innate and adaptive immune systems in cardiac tissue damage and repair in experimental models of MI.

Human myocardial pericytes: multipotent mesodermal precursors exhibiting cardiac specificity.

Perivascular mesenchymal precursor cells (i.e., pericytes) reside in skeletal muscle where they contribute to myofiber regeneration; however, the existence of similar microvessel-associated regenerative precursor cells in cardiac muscle has not yet been documented. We tested whether microvascular pericytes within human myocardium exhibit phenotypes and multipotency similar to their anatomically and developmentally distinct counterparts. Fetal and adult human heart pericytes (hHPs) express canonical pericyte markers in situ, including CD146, NG2, platelet-derived growth factor receptor (PDGFR) ß, PDGFRα, alpha-smooth muscle actin, and smooth muscle myosin heavy chain, but not CD117, CD133, and desmin, nor endothelial cell (EC) markers. hHPs were prospectively purified to homogeneity from ventricular myocardium by flow cytometry, based on a combination of positive- (CD146) and negative-selection (CD34, CD45, CD56, and CD117) cell lineage markers. Purified hHPs expanded in vitro were phenotypically similar to human skeletal muscle-derived pericytes (hSkMPs). hHPs express mesenchymal stem/stromal cell markers in situ and exhibited osteo-, chondro-, and adipogenic potentials but, importantly, no ability for skeletal myogenesis, diverging from pericytes of all other origins. hHPs supported network formation with/without ECs in Matrigel cultures; hHPs further stimulated angiogenic responses under hypoxia, markedly different from hSkMPs. The cardiomyogenic potential of hHPs was examined following 5-azacytidine treatment and neonatal cardiomyocyte coculture in vitro, and intramyocardial transplantation in vivo. Results indicated cardiomyocytic differentiation in a small fraction of hHPs. In conclusion, human myocardial pericytes share certain phenotypic and developmental similarities with their skeletal muscle homologs, yet exhibit different antigenic, myogenic, and angiogenic properties. This is the first example of an anatomical restriction in the developmental potential of pericytes as native mesenchymal stem cells.

Pericyte regulation of vascular remodeling through the CXC receptor 3.

OBJECTIVE: To understand the role, if any, played by pericytes in the regulation of newly formed vessels during angiogenesis. In this study, we investigate whether pericytes regulate the number of nascent endothelial tubes. APPROACH AND RESULTS: Using an in vitro angiogenesis assay (Matrigel assay), we demonstrate that pericytes can inhibit vessel formation and induce vessel dissociation via CXCR3-induced involution of the endothelial cells. In a coculture Matrigel assay for cord formation, pericytes prevented endothelial cord formation of human dermal microvascular endothelial cells but not umbilical vein endothelial cells. Blockade of endothelial CXCR3 function or expression inhibited the repressing effect of the pericytes. We further show that pericytes are also able to induce regression of newly formed microvascular cords through CXCR3 activation of calpain. When CXCR3 function was inhibited by a neutralizing antibody or downregulated by siRNA, cord regression mediated by pericytes was abolished. CONCLUSIONS: We show for the first time that pericytes regulate angiogenic vessel formation, and that this is mediated through CXCR3 expressed on endothelial cells. This suggests a role for pericytes in the pruning of immature vessels overproduced during wound repair.

Beneficial effect of mechanical stimulation on the regenerative potential of muscle-derived stem cells is lost by inhibiting vascular endothelial growth factor.

OBJECTIVE: We previously reported that mechanical stimulation increased the effectiveness of muscle-derived stem cells (MDSCs) for tissue repair. The objective of this study was to determine the importance of vascular endothelial growth factor (VEGF) on mechanically stimulated MDSCs in a murine model of muscle regeneration. APPROACH AND RESULTS: MDSCs were transduced with retroviral vectors encoding the LacZ reporter gene (lacZ-MDSCs), the soluble VEGF receptor Flt1 (sFlt1-MDSCs), or a short hairpin RNA (shRNA) targeting messenger RNA of VEGF (shRNA_VEGF MDSCs). Cells were subjected to 24 hours of mechanical cyclic strain and immediately transplanted into the gastrocnemius muscles of mdx/scid mice. Two weeks after transplantation, angiogenesis, fibrosis, and regeneration were analyzed. There was an increase in angiogenesis in the muscles transplanted with mechanically stimulated lacZ-MDSCs compared with nonstimulated lacZ-MDSCs, sFlt1-MDSCs, and shRNA _VEGF MDSCs. Dystrophin-positive myofiber regeneration was significantly lower in the shRNA_VEGF-MDSC group compared with the lacZ-MDSC and sFlt1-MDSC groups. In vitro proliferation of MDSCs was not decreased by inhibition of VEGF; however, differentiation into myotubes and adhesion to collagen were significantly lower in the shRNA_VEGF-MDSC group compared with the lacZ-MDSC and sFlt1-MDSC groups. CONCLUSIONS: The beneficial effects of mechanical stimulation on MDSC-mediated muscle repair are lost by inhibiting VEGF.

Human skeletal muscle cells with a slow adhesion rate after isolation and an enhanced stress resistance improve function of ischemic hearts.

Identification of cells that are endowed with maximum potency could be critical for the clinical success of cell-based therapies. We investigated whether cells with an enhanced efficacy for cardiac cell therapy could be enriched from adult human skeletal muscle on the basis of their adhesion properties to tissue culture flasks following tissue dissociation. Cells that adhered slowly displayed greater myogenic purity and more readily differentiated into myotubes in vitro than rapidly adhering cells (RACs). The slowly adhering cell (SAC) population also survived better than the RAC population in kinetic in vitro assays that simulate conditions of oxidative and inflammatory stress. When evaluated for the treatment of a myocardial infarction (MI), intramyocardial injection of the SACs more effectively improved echocardiographic indexes of left ventricular (LV) remodeling and contractility than the transplantation of the RACs. Immunohistological analysis revealed that hearts injected with SACs displayed a reduction in myocardial fibrosis and an increase in infarct vascularization, donor cell proliferation, and endogenous cardiomyocyte survival and proliferation in comparison with the RAC-treated hearts. In conclusion, these results suggest that adult human skeletal muscle-derived cells are inherently heterogeneous with regard to their efficacy for enhancing cardiac function after cardiac implantation, with SACs outperforming RACs.

Stem cell therapy for heart failure in the clinics: new perspectives in the era of precision medicine and artificial intelligence.

Stem/progenitor cells have been widely evaluated as a promising therapeutic option for heart failure (HF). Numerous clinical trials with stem/progenitor cell-based therapy (SCT) for HF have demonstrated encouraging results, but not without limitations or discrepancies. Recent technological advancements in multiomics, bioinformatics, precision medicine, artificial intelligence (AI), and machine learning (ML) provide new approaches and insights for stem cell research and therapeutic development. Integration of these new technologies into stem/progenitor cell therapy for HF may help address: 1) the technical challenges to obtain reliable and high-quality therapeutic precursor cells, 2) the discrepancies between preclinical and clinical studies, and 3) the personalized selection of optimal therapeutic cell types/populations for individual patients in the context of precision medicine. This review summarizes the current status of SCT for HF in clinics and provides new perspectives on the development of computation-aided SCT in the era of precision medicine and AI/ML.

Perivascular cells for regenerative medicine.

Mesenchymal stem/stromal cells (MSC) are currently the best candidate therapeutic cells for regenerative medicine related to osteoarticular, muscular, vascular and inflammatory diseases, although these cells remain heterogeneous and necessitate a better biological characterization. We and others recently described that MSC originate from two types of perivascular cells, namely pericytes and adventitial cells and contain the in situ counterpart of MSC in developing and adult human organs, which can be prospectively purified using well defined cell surface markers. Pericytes encircle endothelial cells of capillaries and microvessels and express the adhesion molecule CD146 and the PDGFRß, but lack endothelial and haematopoietic markers such as CD34, CD31, vWF (von Willebrand factor), the ligand for Ulex europaeus 1 (UEA1) and CD45 respectively. The proteoglycan NG2 is a pericyte marker exclusively associated with the arterial system. Besides its expression in smooth muscle cells, smooth muscle actin (αSMA) is also detected in subsets of pericytes. Adventitial cells surround the largest vessels and, opposite to pericytes, are not closely associated to endothelial cells. Adventitial cells express CD34 and lack αSMA and all endothelial and haematopoietic cell markers, as for pericytes. Altogether, pericytes and adventitial perivascular cells express in situ and in culture markers of MSC and display capacities to differentiate towards osteogenic, adipogenic and chondrogenic cell lineages. Importantly, adventitial cells can differentiate into pericyte-like cells under inductive conditions in vitro. Altogether, using purified perivascular cells instead of MSC may bring higher benefits to regenerative medicine, including the possibility, for the first time, to use these cells uncultured.

A synthetic transcription platform for programmable gene expression in mammalian cells.

Precise, scalable, and sustainable control of genetic and cellular activities in mammalian cells is key to developing precision therapeutics and smart biomanufacturing. Here we create a highly tunable, modular, versatile CRISPR-based synthetic transcription system for the programmable control of gene expression and cellular phenotypes in mammalian cells. Genetic circuits consisting of well-characterized libraries of guide RNAs, binding motifs of synthetic operators, transcriptional activators, and additional genetic regulatory elements express mammalian genes in a highly predictable and tunable manner. We demonstrate the programmable control of reporter genes episomally and chromosomally, with up to 25-fold more activity than seen with the EF1α promoter, in multiple cell types. We use these circuits to program the secretion of human monoclonal antibodies and to control T-cell effector function marked by interferon-γ production. Antibody titers and interferon-γ concentrations significantly correlate with synthetic promoter strengths, providing a platform for programming gene expression and cellular function in diverse applications.

Transplantation of Purified Human Pericytes for Myocardial Repair in SCID-hu Mice.

The physiological, pathological, and regenerative roles of pericytes as microvascular mural cells and multipotent precursors have gained significant attention. The capacity to prospectively purify pericytes from multiple organs enables the investigation of their tissue-specific regenerative capabilities. Here, we describe the application of purified human pericytes for cardiac regeneration post-infarct in an immunodeficient mouse model. This protocol includes experimental details of pericyte isolation from both human skeletal and cardiac muscle, an immunodeficient mouse model of acute myocardial infarction, and xenogeneic pericyte transplantation.

High-throughput 5' UTR engineering for enhanced protein production in non-viral gene therapies.

Despite significant clinical progress in cell and gene therapies, maximizing protein expression in order to enhance potency remains a major technical challenge. Here, we develop a high-throughput strategy to design, screen, and optimize 5' UTRs that enhance protein expression from a strong human cytomegalovirus (CMV) promoter. We first identify naturally occurring 5' UTRs with high translation efficiencies and use this information with in silico genetic algorithms to generate synthetic 5' UTRs. A total of ~12,000 5' UTRs are then screened using a recombinase-mediated integration strategy that greatly enhances the sensitivity of high-throughput screens by eliminating copy number and position effects that limit lentiviral approaches. Using this approach, we identify three synthetic 5' UTRs that outperform commonly used non-viral gene therapy plasmids in expressing protein payloads. In summary, we demonstrate that high-throughput screening of 5' UTR libraries with recombinase-mediated integration can identify genetic elements that enhance protein expression, which should have numerous applications for engineered cell and gene therapies.

Synthetic biology: at the crossroads of genetic engineering and human therapeutics-a Keystone Symposia report.

Synthetic biology has the potential to transform cell- and gene-based therapies for a variety of diseases. Sophisticated tools are now available for both eukaryotic and prokaryotic cells to engineer cells to selectively achieve therapeutic effects in response to one or more disease-related signals, thus sparing healthy tissue from potentially cytotoxic effects. This report summarizes the Keystone eSymposium "Synthetic Biology: At the Crossroads of Genetic Engineering and Human Therapeutics," which took place on May 3 and 4, 2021. Given that several therapies engineered using synthetic biology have entered clinical trials, there was a clear need for a synthetic biology symposium that emphasizes the therapeutic applications of synthetic biology as opposed to the technical aspects. Presenters discussed the use of synthetic biology to improve T cell, gene, and viral therapies, to engineer probiotics, and to expand upon existing modalities and functions of cell-based therapies.

Decellularized neonatal cardiac extracellular matrix prevents widespread ventricular remodeling in adult mammals after myocardial infarction.

Heart disease remains a leading killer in western society and irreversibly impacts the lives of millions of patients annually. While adult mammals do not possess the ability to regenerate functional cardiac tissue, neonatal mammals are capable of robust cardiomyocyte proliferation and regeneration within a week of birth. Given this change in regenerative function through development, the extracellular matrix (ECM) from adult tissues may not be conducive to promoting cardiac regeneration, although conventional ECM therapies rely exclusively on adult-derived tissues. Therefore the potential of ECM derived from neonatal mouse hearts (nmECM) to prevent adverse ventricular remodeling in adults was investigated using an in vivo model of acute myocardial infarction (MI). Following a single administration of nmECM, we observed a significant improvement in heart function while adult heart-derived ECM (amECM) did not improve these parameters. Treatment with nmECM limits scar expansion in the left ventricle and promotes revascularization of the injured region. Furthermore, nmECM induced expression of the ErbB2 receptor, simulating a neonatal-like environment and promoting neuregulin-1 associated cardiac function. Inhibition of the ErbB2 receptor effectively prevents these actions, suggesting its role in the context of nmECM as a therapy. This study shows the potential of a neonatal-derived biological material in vivo, diverting from the conventional use of adult-derived ECM therapies in research and the clinic. STATEMENT OF SIGNIFICANCE: The of use extracellular matrix biomaterials to aid tissue repair has been previously reported in many forms of injury. The majority of ECM studies to date utilized ECM derived from adult tissues that are not able to fully regenerate functional tissue. In contrast, this study tests the ability of ECM derived from a regenerative organ, the neonatal heart, to stimulate functional cardiac repair after MI. This study is the first to test its potential in vivo. Our results indicate that extracellular factors present in the neonatal environment can be used to alter the healing response in adults, and we have identified the role of ErbB2 in neonatal ECM-based cardiac repair.

A controlled release system for simultaneous delivery of three human perivascular stem cell-derived factors for tissue repair and regeneration.

Transplanted stem/progenitor cells improve tissue healing and regeneration anatomically and functionally, mostly due to their secreted trophic factors. However, harsh conditions at the site of injury, including hypoxia, oxidative and inflammatory stress, increased fibrosis and insufficient angiogenesis, and in some cases immunological response or incompatibility, are detrimental to stem cell survival. To overcome the complexity and deficiencies of stem cell therapy, the coacervate delivery platform is deemed promising because it offers controlled and sustained release using heparin to recapitulate the binding and stabilization of extracellular proteins by heparan sulphates in native tissues. Here we show that recombinant alternatives of three key factors [vascular endothelial growth factor (VEGF), monocyte chemoattractant protein-1 (MCP-1) and interleukin-6 (IL-6)], commonly produced by perivascular stem cells under various stress conditions, can be successfully incorporated into a heparin-based coacervate. We characterized the release profile of the triply incorporated factors from the complex coacervate. The coacervate-released factors were able to exert their desired biological activities in vitro: VEGF stimulated human umbilical vein endothelial cell proliferation, MCP-1 elevated macrophage migration and IL-6 increased IgM production by IL-6-dependent cell line. Thus, a controlled release system can be used for simultaneous delivery of three stem cell-derived factors and could be useful for tissue repair and regenerative medicine.

Skeletal and cardiac muscle pericytes: Functions and therapeutic potential.

Pericytes are periendothelial mesenchymal cells residing within the microvasculature. Skeletal muscle and cardiac pericytes are now recognized to fulfill an increasing number of functions in normal tissue homeostasis, including contributing to microvascular function by maintaining vessel stability and regulating capillary flow. In the setting of muscle injury, pericytes contribute to a regenerative microenvironment through release of trophic factors and by modulating local immune responses. In skeletal muscle, pericytes also directly enhance tissue healing by differentiating into myofibers. Conversely, pericytes have also been implicated in the development of disease states, including fibrosis, heterotopic ossication and calcification, atherosclerosis, and tumor angiogenesis. Despite increased recognition of pericyte heterogeneity, it is not yet clear whether specific subsets of pericytes are responsible for individual functions in skeletal and cardiac muscle homeostasis and disease.

Preconditioning of Human Mesenchymal Stem Cells to Enhance Their Regulation of the Immune Response.

Mesenchymal stem cells (MSCs) have attracted the attention of researchers and clinicians for their ability to differentiate into a number of cell types, participate in tissue regeneration, and repair the damaged tissues by producing various growth factors and cytokines, as well as their unique immunoprivilege in alloreactive hosts. The immunomodulatory functions of exogenous MSCs have been widely investigated in immune-mediated inflammatory diseases and transplantation research. However, a harsh environment at the site of tissue injury/inflammation with insufficient oxygen supply, abundance of reactive oxygen species, and presence of other harmful molecules that damage the adoptively transferred cells collectively lead to low survival and engraftment of the transferred cells. Preconditioning of MSCs ex vivo by hypoxia, inflammatory stimulus, or other factors/conditions prior to their use in therapy is an adaptive strategy that prepares MSCs to survive in the harsh environment and to enhance their regulatory function of the local immune responses. This review focuses on a number of approaches in preconditioning human MSCs with the goal of augmenting their capacity to regulate both innate and adaptive immune responses.

Decellularized zebrafish cardiac extracellular matrix induces mammalian heart regeneration.

Heart attack is a global health problem that leads to significant morbidity, mortality, and health care burden. Adult human hearts have very limited regenerative capability after injury. However, evolutionarily primitive species generally have higher regenerative capacity than mammals. The extracellular matrix (ECM) may contribute to this difference. Mammalian cardiac ECM may not be optimally inductive for cardiac regeneration because of the fibrotic, instead of regenerative, responses in injured adult mammalian hearts. Given the high regenerative capacity of adult zebrafish hearts, we hypothesize that decellularized zebrafish cardiac ECM (zECM) made from normal or healing hearts can induce mammalian heart regeneration. Using zebrafish and mice as representative species of lower vertebrates and mammals, we show that a single administration of zECM, particularly the healing variety, enables cardiac functional recovery and regeneration of adult mouse heart tissues after acute myocardial infarction. zECM-treated groups exhibit proliferation of the remaining cardiomyocytes and multiple cardiac precursor cell populations and reactivation of ErbB2 expression in cardiomyocytes. Furthermore, zECM exhibits pro-proliferative and chemotactic effects on human cardiac precursor cell populations in vitro. These contribute to the structural preservation and correlate with significantly higher cardiac contractile function, notably less left ventricular dilatation, and substantially more elastic myocardium in zECM-treated hearts than control animals treated with saline or decellularized adult mouse cardiac ECM. Inhibition of ErbB2 activity abrogates beneficial effects of zECM administration, indicating the possible involvement of ErbB2 signaling in zECM-mediated regeneration. This study departs from conventional focuses on mammalian ECM and introduces a new approach for cardiac tissue regeneration.

Isolation of Perivascular Multipotent Precursor Cell Populations from Human Cardiac Tissue.

Multipotent mesenchymal stem/stromal cells (MSC) were conventionally isolated, through their plastic adherence, from primary tissue digests whilst their anatomical tissue location remained unclear. The recent discovery of defined perivascular and MSC cell marker expression by perivascular cells in multiple tissues by our group and other researchers has provided an opportunity to prospectively isolate and purify specific homogenous subpopulations of multipotent perivascular precursor cells. We have previously demonstrated the use of fluorescent activated cell sorting (FACS) to purify microvascular CD146+CD34- pericytes and vascular CD34+CD146- adventitial cells from human skeletal muscle. Herein we describe a method to simultaneously isolate these two perivascular cell subsets from human myocardium by FACS, based on the expression of a defined set of cell surface markers for positive and negative selections. This method thus makes available two specific subpopulations of multipotent cardiac MSC-like precursor cells for use in basic research and/or therapeutic investigations.

Regenerative Translation of Human Blood-Vessel-Derived MSC Precursors.

Mesenchymal stem/stromal cells (MSCs) represent a promising adult progenitor cell source for tissue repair and regeneration. Their mysterious identity in situ has gradually been unveiled by the accumulating evidence indicating an association between adult multipotent stem/progenitor cells and vascular/perivascular niches. Using immunohistochemistry and fluorescence-activated cell sorting, we and other groups have prospectively identified and purified subpopulations of multipotent precursor cells associated with the blood vessels within multiple human organs. The three precursor subsets, myogenic endothelial cells (MECs), pericytes (PCs), and adventitial cells (ACs), are located, respectively, in the three structural tiers of typical blood vessels: intima, media, and adventitia. MECs, PCs, and ACs have been extensively characterized in prior studies and are currently under investigation for their therapeutic potentials in preclinical animal models. In this review, we will briefly discuss the identification, isolation, and characterization of these human blood-vessel-derived stem cells (hBVSCs) and summarize the current status of regenerative applications of hBVSC subsets.

Controlled dual delivery of fibroblast growth factor-2 and Interleukin-10 by heparin-based coacervate synergistically enhances ischemic heart repair.

Myocardial infarction (MI) causes myocardial necrosis, triggers chronic inflammatory responses, and leads to pathological remodeling. Controlled delivery of a combination of angiogenic and immunoregulatory proteins may be a promising therapeutic approach for MI. We investigated the bioactivity and therapeutic potential of an injectable, heparin-based coacervate co-delivering an angiogenic factor, fibroblast growth factor-2 (FGF2), and an anti-inflammatory cytokine, Interleukin-10 (IL-10) in a spatially and temporally controlled manner. Coacervate delivery of FGF2 and IL-10 preserved their bioactivities on cardiac stromal cell proliferation in vitro. Upon intramyocardial injection into a mouse MI model, echocardiography revealed that FGF2/IL-10 coacervate treated groups showed significantly improved long-term LV contractile function and ameliorated LV dilatation. FGF2/IL-10 coacervate substantially augmented LV myocardial elasticity. Additionally, FGF2/IL-10 coacervate notably enhanced long-term revascularization, especially at the infarct area. In addition, coacervate loaded with 500 ng FGF2 and 500 ng IL-10 significantly reduced LV fibrosis, considerably preserved infarct wall thickness, and markedly inhibited chronic inflammation at the infarct area. These results indicate that FGF2/IL-10 coacervate has notably greater therapeutic potential than coacervate containing only FGF2. Overall, our data suggest therapeutically synergistic effects of FGF-2/IL-10 coacervate, particularly coacervate with FGF2 and 500 ng IL-10, for the treatment of ischemic heart disease.