Progeria-based vascular model identifies networks associated with cardiovascular aging and disease.

Hutchinson-Gilford Progeria syndrome (HGPS) is a lethal premature aging disorder caused by a de novo heterozygous mutation that leads to the accumulation of a splicing isoform of Lamin A termed progerin. Progerin expression deregulates the organization of the nuclear lamina and the epigenetic landscape. Progerin has also been observed to accumulate at low levels during normal aging in cardiovascular cells of adults that do not carry genetic mutations linked with HGPS. Therefore, the molecular mechanisms that lead to vascular dysfunction in HGPS may also play a role in vascular aging-associated diseases, such as myocardial infarction and stroke. Here, we show that HGPS patient-derived vascular smooth muscle cells (VSMCs) recapitulate HGPS molecular hallmarks. Transcriptional profiling revealed cardiovascular disease remodeling and reactive oxidative stress response activation in HGPS VSMCs. Proteomic analyses identified abnormal acetylation programs in HGPS VSMC replication fork complexes, resulting in reduced H4K16 acetylation. Analysis of acetylation kinetics revealed both upregulation of K16 deacetylation and downregulation of K16 acetylation. This correlates with abnormal accumulation of error-prone nonhomologous end joining (NHEJ) repair proteins on newly replicated chromatin. The knockdown of the histone acetyltransferase MOF recapitulates preferential engagement of NHEJ repair activity in control VSMCs. Additionally, we find that primary donor-derived coronary artery vascular smooth muscle cells from aged individuals show similar defects to HGPS VSMCs, including loss of H4K16 acetylation. Altogether, we provide insight into the molecular mechanisms underlying vascular complications associated with HGPS patients and normative aging.

Genetic and pharmacological reduction of CDK14 mitigates synucleinopathy.

Parkinson's disease (PD) is a debilitating neurodegenerative disease characterized by the loss of midbrain dopaminergic neurons (DaNs) and the abnormal accumulation of α-Synuclein (α-Syn) protein. Currently, no treatment can slow nor halt the progression of PD. Multiplications and mutations of the α-Syn gene (SNCA) cause PD-associated syndromes and animal models that overexpress α-Syn replicate several features of PD. Decreasing total α-Syn levels, therefore, is an attractive approach to slow down neurodegeneration in patients with synucleinopathy. We previously performed a genetic screen for modifiers of α-Syn levels and identified CDK14, a kinase of largely unknown function as a regulator of α-Syn. To test the potential therapeutic effects of CDK14 reduction in PD, we ablated Cdk14 in the α-Syn preformed fibrils (PFF)-induced PD mouse model. We found that loss of Cdk14 mitigates the grip strength deficit of PFF-treated mice and ameliorates PFF-induced cortical α-Syn pathology, indicated by reduced numbers of pS129 α-Syn-containing cells. In primary neurons, we found that Cdk14 depletion protects against the propagation of toxic α-Syn species. We further validated these findings on pS129 α-Syn levels in PD patient neurons. Finally, we leveraged the recent discovery of a covalent inhibitor of CDK14 to determine whether this target is pharmacologically tractable in vitro and in vivo. We found that CDK14 inhibition decreases total and pathologically aggregated α-Syn in human neurons, in PFF-challenged rat neurons and in the brains of α-Syn-humanized mice. In summary, we suggest that CDK14 represents a novel therapeutic target for PD-associated synucleinopathy.

Tissue-Specific Tumour Suppressor and Oncogenic Activities of the Polycomb-like Protein MTF2.

The Polycomb repressive complex 2 (PRC2) is a conserved chromatin-remodelling complex that catalyses the trimethylation of histone H3 lysine 27 (H3K27me3), a mark associated with gene silencing. PRC2 regulates chromatin structure and gene expression during organismal and tissue development and tissue homeostasis in the adult. PRC2 core subunits are associated with various accessory proteins that modulate its function and recruitment to target genes. The multimeric composition of accessory proteins results in two distinct variant complexes of PRC2, PRC2.1 and PRC2.2. Metal response element-binding transcription factor 2 (MTF2) is one of the Polycomb-like proteins (PCLs) that forms the PRC2.1 complex. MTF2 is highly conserved, and as an accessory subunit of PRC2, it has important roles in embryonic stem cell self-renewal and differentiation, development, and cancer progression. Here, we review the impact of MTF2 in PRC2 complex assembly, catalytic activity, and spatiotemporal function. The emerging paradoxical evidence suggesting that MTF2 has divergent roles as either a tumour suppressor or an oncogene in different tissues merits further investigations. Altogether, our review illuminates the context-dependent roles of MTF2 in Polycomb group (PcG) protein-mediated epigenetic regulation. Its impact on disease paves the way for a deeper understanding of epigenetic regulation and novel therapeutic strategies.

Tissue-Engineered Disease Modeling of Lymphangioleiomyomatosis Exposes a Therapeutic Vulnerability to HDAC Inhibition.

Lymphangioleiomyomatosis (LAM) is a rare disease involving cystic lung destruction by invasive LAM cells. These cells harbor loss-of-function mutations in TSC2, conferring hyperactive mTORC1 signaling. Here, tissue engineering tools are employed to model LAM and identify new therapeutic candidates. Biomimetic hydrogel culture of LAM cells is found to recapitulate the molecular and phenotypic characteristics of human disease more faithfully than culture on plastic. A 3D drug screen is conducted, identifying histone deacetylase (HDAC) inhibitors as anti-invasive agents that are also selectively cytotoxic toward TSC2-/- cells. The anti-invasive effects of HDAC inhibitors are independent of genotype, while selective cell death is mTORC1-dependent and mediated by apoptosis. Genotype-selective cytotoxicity is seen exclusively in hydrogel culture due to potentiated differential mTORC1 signaling, a feature that is abrogated in cell culture on plastic. Importantly, HDAC inhibitors block invasion and selectively eradicate LAM cells in vivo in zebrafish xenografts. These findings demonstrate that tissue-engineered disease modeling exposes a physiologically relevant therapeutic vulnerability that would be otherwise missed by conventional culture on plastic. This work substantiates HDAC inhibitors as possible therapeutic candidates for the treatment of patients with LAM and requires further study.

Differentiation and single-cell RNA-seq analyses of human pluripotent-stem-cell-derived renal organoids.

Here, we present a protocol for the maintenance and differentiation of human pluripotent stem cells into renal organoids. We describe steps for using a series of readily made differentiation media, multiplexed sample single-cell RNA-seq analysis, quality control, and validation of organoids using immunofluorescence. This provides a rapid and reproducible model of human kidney development and renal disease modeling. Finally, we detail genome engineering using CRISPR-Cas9 homology-directed repair for the generation of renal disease models. For complete details on the use and execution of this protocol, please refer to Pietrobon et al.1.

Tuberous Sclerosis Complex Kidney Lesion Pathogenesis: A Developmental Perspective.

The phenotypic diversity of tuberous sclerosis complex (TSC) kidney pathology is enigmatic. Despite a well-established monogenic etiology, an incomplete understanding of lesion pathogenesis persists. In this review, we explore the question: How do TSC kidney lesions arise? We appraise literature findings in the context of mutational timing and cell-of-origin. Through a developmental lens, we integrate the critical results from clinical studies, human specimens, and genetic animal models. We also review novel insights gleaned from emerging organoid and single-cell sequencing technologies. We present a new model of pathogenesis which posits a phenotypic continuum, whereby lesions arise by mutagenesis during development from variably timed second-hit events. This model can serve as a conceptual framework for testing hypotheses of TSC lesion pathogenesis, both in the kidney and in other affected tissues.

Erratum: Young Bone Marrow Sca-1 Cells Rejuvenate the Aged Heart by Promoting Epithelial-to-Mesenchymal Transition: Erratum.

[This corrects the article DOI: 10.7150/thno.22788.].

Identification and differential usage of a host metalloproteinase entry pathway by SARS-CoV-2 Delta and Omicron.

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) spike glycoprotein (S) binds to angiotensin-converting enzyme 2 (ACE2) to mediate membrane fusion via two distinct pathways: 1) a surface, serine protease-dependent or 2) an endosomal, cysteine protease-dependent pathway. In this study, we found that SARS-CoV-2 S has a wider protease usage and can also be activated by TMPRSS13 and matrix metalloproteinases (MMPs). We found that MMP-2 and MMP-9 played roles in SARS-CoV-2 S cell-cell fusion and TMPRSS2- and cathepsin-independent viral entry in cells expressing high MMP levels. MMP-dependent viral entry required cleavage at the S1/S2 junction in viral producer cells, and differential processing of variants of concern S dictated its usage; the efficiently processed Delta S preferred metalloproteinase-dependent entry when available, and less processed Omicron S was unable to us metalloproteinases for entry. As MMP-2/9 are released during inflammation, they may play roles in S-mediated cytopathic effects, tropism, and disease outcome.

Renal organoid modeling of tuberous sclerosis complex reveals lesion features arise from diverse developmental processes.

Tuberous sclerosis complex (TSC) is a multisystem tumor-forming disorder caused by loss of TSC1 or TSC2. Renal manifestations predominately include cysts and angiomyolipomas. Despite a well-described monogenic etiology, the cellular pathogenesis remains elusive. We report a genetically engineered human renal organoid model that recapitulates pleiotropic features of TSC kidney disease in vitro and upon orthotopic xenotransplantation. Time course single-cell RNA sequencing demonstrates that loss of TSC1 or TSC2 affects multiple developmental processes in the renal epithelial, stromal, and glial compartments. First, TSC1 or TSC2 ablation induces transitional upregulation of stromal-associated genes. Second, epithelial cells in the TSC1-/- and TSC2-/- organoids exhibit a rapamycin-insensitive epithelial-to-mesenchymal transition. Third, a melanocytic population forms exclusively in TSC1-/- and TSC2-/- organoids, branching from MITF+ Schwann cell precursors. Together, these results illustrate the pleiotropic developmental consequences of biallelic inactivation of TSC1 or TSC2 and offer insight into TSC kidney lesion pathogenesis.

Organelle Cooperation in Stem Cell Fate: Lysosomes as Emerging Regulators of Cell Identity.

Regulation of stem cell fate is best understood at the level of gene and protein regulatory networks, though it is now clear that multiple cellular organelles also have critical impacts. A growing appreciation for the functional interconnectedness of organelles suggests that an orchestration of integrated biological networks functions to drive stem cell fate decisions and regulate metabolism. Metabolic signaling itself has emerged as an integral regulator of cell fate including the determination of identity, activation state, survival, and differentiation potential of many developmental, adult, disease, and cancer-associated stem cell populations and their progeny. As the primary adenosine triphosphate-generating organelles, mitochondria are well-known regulators of stem cell fate decisions, yet it is now becoming apparent that additional organelles such as the lysosome are important players in mediating these dynamic decisions. In this review, we will focus on the emerging role of organelles, in particular lysosomes, in the reprogramming of both metabolic networks and stem cell fate decisions, especially those that impact the determination of cell identity. We will discuss the inter-organelle interactions, cell signaling pathways, and transcriptional regulatory mechanisms with which lysosomes engage and how these activities impact metabolic signaling. We will further review recent data that position lysosomes as critical regulators of cell identity determination programs and discuss the known or putative biological mechanisms. Finally, we will briefly highlight the potential impact of elucidating mechanisms by which lysosomes regulate stem cell identity on our understanding of disease pathogenesis, as well as the development of refined regenerative medicine, biomarker, and therapeutic strategies.

Vascular contributions to 16p11.2 deletion autism syndrome modeled in mice.

While the neuronal underpinnings of autism spectrum disorder (ASD) are being unraveled, vascular contributions to ASD remain elusive. Here, we investigated postnatal cerebrovascular development in the 16p11.2df/+ mouse model of 16p11.2 deletion ASD syndrome. We discover that 16p11.2 hemizygosity leads to male-specific, endothelium-dependent structural and functional neurovascular abnormalities. In 16p11.2df/+ mice, endothelial dysfunction results in impaired cerebral angiogenesis at postnatal day 14, and in altered neurovascular coupling and cerebrovascular reactivity at postnatal day 50. Moreover, we show that there is defective angiogenesis in primary 16p11.2df/+ mouse brain endothelial cells and in induced-pluripotent-stem-cell-derived endothelial cells from human carriers of the 16p11.2 deletion. Finally, we find that mice with an endothelium-specific 16p11.2 deletion (16p11.2ΔEC) partially recapitulate some of the behavioral changes seen in 16p11.2 syndrome, specifically hyperactivity and impaired motor learning. By showing that developmental 16p11.2 haploinsufficiency from endothelial cells results in neurovascular and behavioral changes in adults, our results point to a potential role for endothelial impairment in ASD.

Proteome-wide Prediction of Lysine Methylation Leads to Identification of H2BK43 Methylation and Outlines the Potential Methyllysine Proteome.

Protein Lys methylation plays a critical role in numerous cellular processes, but it is challenging to identify Lys methylation in a systematic manner. Here we present an approach combining in silico prediction with targeted mass spectrometry (MS) to identify Lys methylation (Kme) sites at the proteome level. We develop MethylSight, a program that predicts Kme events solely on the physicochemical properties of residues surrounding the putative methylation sites, which then requires validation by targeted MS. Using this approach, we identify 70 new histone Kme marks with a 90% validation rate. H2BK43me2, which undergoes dynamic changes during stem cell differentiation, is found to be a substrate of KDM5b. Furthermore, MethylSight predicts that Lys methylation is a prevalent post-translational modification in the human proteome. Our work provides a useful resource for guiding systematic exploration of the role of Lys methylation in human health and disease.

Establishment of macaque trophoblast stem cell lines derived from cynomolgus monkey blastocysts.

The placenta forms a maternal-fetal junction that supports many physiological functions such as the supply of nutrition and exchange of gases and wastes. Establishing an in vitro culture model of human and non-human primate trophoblast stem/progenitor cells is important for investigating the process of early placental development and trophoblast differentiation. In this study, we have established five trophoblast stem cell (TSC) lines from cynomolgus monkey blastocysts, named macTSC #1-5. Fibroblast growth factor 4 (FGF4) enhanced proliferation of macTSCs, while other exogenous factors were not required to maintain their undifferentiated state. macTSCs showed a trophoblastic gene expression profile and trophoblast-like DNA methylation status and also exhibited differentiation capacity towards invasive trophoblast cells and multinucleated syncytia. In a xenogeneic chimera assay, these stem cells contributed to trophectoderm (TE) development in the chimeric blastocysts. macTSC are the first primate trophoblast cell lines whose proliferation is promoted by FGF4. These cell lines provide a valuable in vitro culture model to analyze the similarities and differences in placental development between human and non-human primates.

Transcriptomically Guided Mesendoderm Induction of Human Pluripotent Stem Cells Using a Systematically Defined Culture Scheme.

Human pluripotent stem cells (hPSCs) are an essential cell source in tissue engineering, studies of development, and disease modeling. Efficient, broadly amenable protocols for rapid lineage induction of hPSCs are of great interest in the stem cell biology field. We describe a simple, robust method for differentiation of hPSCs into mesendoderm in defined conditions utilizing single-cell seeding (SCS) and BMP4 and Activin A (BA) treatment. BA treatment was readily incorporated into existing protocols for chondrogenic and endothelial progenitor cell differentiation, while fine-tuning of BA conditions facilitated definitive endoderm commitment. After prolonged differentiation in vitro or in vivo, BA pretreatment resulted in higher mesoderm and endoderm levels at the expense of ectoderm formation. These data demonstrate that SCS with BA treatment is a powerful method for induction of mesendoderm that can be adapted for use in mesoderm and endoderm differentiation.

Gene expression profiling of skeletal myogenesis in human embryonic stem cells reveals a potential cascade of transcription factors regulating stages of myogenesis, including quiescent/activated satellite cell-like gene expression.

Human embryonic stem cell (hESC)-derived skeletal muscle progenitors (SMP)-defined as PAX7-expressing cells with myogenic potential-can provide an abundant source of donor material for muscle stem cell therapy. As in vitro myogenesis is decoupled from in vivo timing and 3D-embryo structure, it is important to characterize what stage or type of muscle is modeled in culture. Here, gene expression profiling is analyzed in hESCs over a 50 day skeletal myogenesis protocol and compared to datasets of other hESC-derived skeletal muscle and adult murine satellite cells. Furthermore, day 2 cultures differentiated with high or lower concentrations of CHIR99021, a GSK3A/GSK3B inhibitor, were contrasted. Expression profiling of the 50 day time course identified successively expressed gene subsets involved in mesoderm/paraxial mesoderm induction, somitogenesis, and skeletal muscle commitment/formation which could be regulated by a putative cascade of transcription factors. Initiating differentiation with higher CHIR99021 concentrations significantly increased expression of MSGN1 and TGFB-superfamily genes, notably NODAL, resulting in enhanced paraxial mesoderm and reduced ectoderm/neuronal gene expression. Comparison to adult satellite cells revealed that genes expressed in 50-day cultures correlated better with those expressed by quiescent or early activated satellite cells, which have the greatest therapeutic potential. Day 50 cultures were similar to other hESC-derived skeletal muscle and both expressed known and novel SMP surface proteins. Overall, a putative cascade of transcription factors has been identified which regulates four stages of myogenesis. Subsets of these factors were upregulated by high CHIR99021 or their binding sites were significantly over-represented during SMP activation, ranging from quiescent to late-activated stages. This analysis serves as a resource to further study the progression of in vitro skeletal myogenesis and could be mined to identify novel markers of pluripotent-derived SMPs or regulatory transcription/growth factors. Finally, 50-day hESC-derived SMPs appear similar to quiescent/early activated satellite cells, suggesting they possess therapeutic potential.

Development of a novel screening platform for the identification of small molecule inhibitors of human adenovirus.

Human adenovirus (HAdV) can cause severe disease and death in both immunocompromised and immunocompetent patients. The current standards of treatment are often ineffective, and no approved antiviral therapy against HAdV exists. We report here the design and validation of a fluorescence-based high-content screening platform for the identification of novel anti-HAdV compounds. The screen was conducted using a wildtype-like virus containing the red fluorescent protein (RFP) gene under the regulation of the HAdV major late promoter. Thus, RFP expression allows monitoring of viral late gene expression (a surrogate marker for virus replication), and compounds affecting virus growth can be easily discovered by quantifying RFP intensity. We used our platform to screen ~1200 FDA-approved small molecules, and identified several cardiotonic steroids, corticosteroids and chemotherapeutic agents as anti-HAdV compounds. Our screening platform provides the stringency necessary to detect compounds with varying degrees of antiviral activity, and facilitates drug discovery/repurposing to combat HAdV infections.

The GPI-Linked Protein LY6A Drives AAV-PHP.B Transport across the Blood-Brain Barrier.

Efficient delivery of gene therapy vectors across the blood-brain barrier (BBB) is the holy grail of neurological disease therapies. A variant of the neurotropic vector adeno-associated virus (AAV) serotype 9, called AAV-PHP.B, was shown to very efficiently deliver transgenes across the BBB in C57BL/6J mice. Based on our recent observation that this phenotype is mouse strain dependent, we used whole-exome sequencing-based genetics to map this phenotype to a specific haplotype of lymphocyte antigen 6 complex, locus A (Ly6a) (stem cell antigen-1 [Sca-1]), which encodes a glycosylphosphatidylinositol (GPI)-anchored protein whose function had been thought to be limited to the biology of hematopoiesis. Additional biochemical and genetic studies definitively linked high BBB transport to the binding of AAV-PHP.B with LY6A (SCA-1). These studies identify, for the first time, a ligand for this GPI-anchored protein and suggest a role for it in BBB transport that could be hijacked by viruses in natural infections or by gene therapy vectors to treat neurological diseases.

Rationally Designed 3D Hydrogels Model Invasive Lung Diseases Enabling High-Content Drug Screening.

Cell behavior is highly dependent upon microenvironment. Thus, to identify drugs targeting metastatic cancer, screens need to be performed in tissue mimetic substrates that allow cell invasion and matrix remodeling. A novel biomimetic 3D hydrogel platform that enables quantitative analysis of cell invasion and viability at the individual cell level is developed using automated data acquisition methods with an invasive lung disease (lymphangioleiomyomatosis, LAM) characterized by hyperactive mammalian target of rapamycin complex 1 (mTORC1) signaling as a model. To test the lung-mimetic hydrogel platform, a kinase inhibitor screen is performed using tuberous sclerosis complex 2 (TSC2) hypomorphic cells, identifying Cdk2 inhibition as a putative LAM therapeutic. The 3D hydrogels mimic the native niche, enable multiple modes of invasion, and delineate phenotypic differences between healthy and diseased cells, all of which are critical to effective drug screens of highly invasive diseases including lung cancer.

Targeting the MTF2-MDM2 Axis Sensitizes Refractory Acute Myeloid Leukemia to Chemotherapy.

Deep sequencing has revealed that epigenetic modifiers are the most mutated genes in acute myeloid leukemia (AML). Thus, elucidating epigenetic dysregulation in AML is crucial to understand disease mechanisms. Here, we demonstrate that metal response element binding transcription factor 2/polycomblike 2 (MTF2/PCL2) plays a fundamental role in the polycomb repressive complex 2 (PRC2) and that its loss elicits an altered epigenetic state underlying refractory AML. Unbiased systems analyses identified the loss of MTF2-PRC2 repression of MDM2 as central to, and therefore a biomarker for, refractory AML. Thus, immature MTF2-deficient CD34+CD38- cells overexpress MDM2, thereby inhibiting p53 that leads to chemoresistance due to defects in cell-cycle regulation and apoptosis. Targeting this dysregulated signaling pathway by MTF2 overexpression or MDM2 inhibitors sensitized refractory patient leukemic cells to induction chemotherapeutics and prevented relapse in AML patient-derived xenograft mice. Therefore, we have uncovered a direct epigenetic mechanism by which MTF2 functions as a tumor suppressor required for AML chemotherapeutic sensitivity and identified a potential therapeutic strategy to treat refractory AML.Significance: MTF2 deficiency predicts refractory AML at diagnosis. MTF2 represses MDM2 in hematopoietic cells and its loss in AML results in chemoresistance. Inhibiting p53 degradation by overexpressing MTF2 in vitro or by using MDM2 inhibitors in vivo sensitizes MTF2-deficient refractory AML cells to a standard induction-chemotherapy regimen. Cancer Discov; 8(11); 1376-89. ©2018 AACR. See related commentary by Duy and Melnick, p. 1348 This article is highlighted in the In This Issue feature, p. 1333.