Hutchinson-Gilford Progeria Syndrome (HGPS), characterised by accelerated ageing, causes cardiovascular defects resembling aspects of cardiovascular ageing. We used human left ventricle cardiomyocytes (CMs) derived from HGPS-induced pluripotent stem cells (iPSCs), and their isogenic-corrected controls, to investigate HGPS-CM dysfunction and identify potential therapies. Our results revealed that HGPS-iPSC-CMs exhibit greater maturity and associated elevated oxidative stress compared to controls, which they could not contend with, leading to cellular senescence. Increased senescence was also observed in cardiac tissue from mouse and human physiologically-aged and HGPS individuals. Functionally, HGPS-iPSC-CMs showed dysregulated mitochondrial respiration and calcium handling. Amongst the six drugs tested, rapamycin and lonafarnib were the most effective against HGPS-cardiac phenotypes. Although lonafarnib raised safety concerns, it partially reverted the cardiac senescent phenotype by inducing cellular autophagy and decreasing progerin expression in progeroid mice. Our study supports the use of HGPS-iPSC-CMs to identify novel biomarkers and therapies for HGPS, and potentially cardiac physiological-ageing. ### Competing Interest Statement The Francis Crick Institute has filed a patent application related to the left ventricle differentiation protocol used in this work (WO 2020/245612), and A.S.B. is listed as an inventor. The Francis Crick Institute has granted an exclusive license to Axol Bioscience to commercialise the protocol for the generation and sale of cardiomyocytes for R&D and the provision of contract research services. N.D., J.C.S., and A.S.B. may benefit from this license. The rest of the authors have no conflict of interest to disclose. FCT, 2022.07615.PTDC, PTDC/BTM-SAL/5174/2020 FEDER, Program COMPETE European projects, RESETaging (ref. 952266), CHAngeing - Connected Hubs in Ageing: Healthy Living to Protect Cerebrovascular Function” (Ref. 101087071), DREAMs - Drug Repurposing with Artificial Intelligence for Muscular Disorders” (Ref: 101080229), PRR project “HfPT” Wellcome Trust, https://ror.org/029chgv08, 210987/Z/18/Z Francis Crick Institute, Cancer Research UK (FC001157), UK Medical Research Council (FC001157), Wellcome Trust (FC001157) Germany

The sympathetic nervous system densely innervates all cardiac chambers and is a key player in cardiac control, yet this relationship has scarcely been investigated using a stem cell-based model. This study investigates the effects that sympathetic neurons (SNs) have on human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) in vitro, and whether they induce any degree of functional or structural maturity in these conventionally immature cells. SNs were isolated from neonatal rat pups, and cocultured with hPSC-CMs for up to 15 days. Structural changes in hPSC-CMs were analysed by microscopy techniques. Fluorescence resonance energy transfer was used to measure second messenger molecule cAMP production and β-adrenergic receptor (βAR) response. Contractile and Ca2+ transient activity was measured using CytoCypher. These cocultures promoted hPSC-CM structural elongation and increased sarcomere organization. Furthermore, the βAR response of cocultured hiPSC-CMs was larger, indicated by increased cAMP production upon neuronal nicotinic stimulation. Faster contraction and ratiometric Ca2+ transient peak height and kinetic parameters strongly indicate increased chronotropic response in coculture. Coculture with SNs also elicited an increase in action potential amplitude and depolarization velocity, further confirming that SNs contribute to hiPSC-CM functional maturation. Overall, we have found that SNs modulate hPSC-CMs in vitro, inducing a more mature functional response. As an in vitro tool, these cocultures could serve as a model of sympathoadrenergic disease, enabling new discovery avenues. KEY POINTS: The sympathetic nervous system controls the involuntary 'fight-or-flight' response, with the heart being one of key target organs. In certain neuro-cardiac diseases, the input from the sympathetic nervous system is hyperregulated, and can lead to increased speed or force of the heart's contraction. Human induced pluripotent stem cells (hiPSCs) represent a rapidly evolving field which allow us to create a cell of interest and model its structural and functional activity in a dish. Here we have created hiPSC-derived cardiomyocytes (hiPSC-CMs) and cocultured them with sympathetic neurons (SNs). We found that SNs are able to modulate structure of the hiPSC-CMs by reducing their circularity and increasing sarcomeric organization, and can significantly increase the speed of contraction and Ca2+ handling. Together, our data provide a platform to investigate the neuro-cardiac relationship in vitro, which could be used for patient-specific disease modelling in future.

Perlecan (HSPG2), a heparan sulfate proteoglycan similar to agrin, is key for extracellular matrix (ECM) maturation and stabilization. Although crucial for cardiac development, its role remains elusive. We show that perlecan expression increases as cardiomyocytes mature in vivo and during human pluripotent stem cell differentiation to cardiomyocytes (hPSC-CMs). Perlecan-haploinsuffient hPSCs (HSPG2(+/-)) differentiate efficiently, but late-stage CMs have structural, contractile, metabolic, and ECM gene dysregulation. In keeping with this, late -stage HSPG2(+/-) hPSC-CMs have immature features, including reduced alpha-actinin expression and increased glycolytic metabolism and proliferation. Moreover, perlecan-haploinsuffient engineered heart tissues have reduced tissue thickness and force generation. Conversely, hPSC-CMs grown on a perlecanpeptide substrate are enlarged and display increased nucleation, typical of hypertrophic growth. Together, perlecan appears to play the opposite role of agrin, promoting cellular maturation rather than hyperplasia and proliferation. Perlecan signaling is likely mediated via its binding to the dystroglycan complex. Targeting perlecan-dependent signaling may help reverse the phenotypic switch common to heart failure.

During embryonic development, mutually antagonistic signaling cascades determine gonadal fate toward a testicular or ovarian identity. Errors in this process result in disorders of sex development (DSDs), characterized by discordance between chromosomal, gonadal, and anatomical sex. The absence of an appropriate, accessible in vitro system is a major obstacle in understanding mechanisms of sex-determination/DSDs. Here, we describe protocols for differentiation of mouse and human pluripotent cells toward gonadal progenitors. Transcriptomic analysis reveals that the in vitro–derived murine gonadal cells are equivalent to embryonic day 11.5 in vivo progenitors. Using similar conditions, Sertoli-like cells derived from 46,XY human induced pluripotent stem cells (hiPSCs) exhibit sustained expression of testis-specific genes, secrete anti-Müllerian hormone, migrate, and form tubular structures. Cells derived from 46,XY DSD female hiPSCs, carrying an NR5A1 variant, show aberrant gene expression and absence of tubule formation. CRISPR-Cas9–mediated variant correction rescued the phenotype. This is a robust tool to understand mechanisms of sex determination and model DSDs.

Skeletal muscle is a complex tissue composed of multinucleated myofibers responsible for force generation that are supported by multiple cell types. Many severe and lethal disorders affect skeletal muscle; therefore, engineering models to reproduce such cellular complexity and function are instrumental for investigating muscle pathophysiology and developing therapies. Here, we detail the modular 3D bioengineering of multilineage skeletal muscles from human induced pluripotent stem cells, which are first differentiated into myogenic, neural and vascular progenitor cells and then combined within 3D hydrogels under tension to generate an aligned myofiber scaffold containing vascular networks and motor neurons. 3D bioengineered muscles recapitulate morphological and functional features of human skeletal muscle, including establishment of a pool of cells expressing muscle stem cell markers. Importantly, bioengineered muscles provide a high-fidelity platform to study muscle pathology, such as emergence of dysmorphic nuclei in muscular dystrophies caused by mutant lamins. The protocol is easy to follow for operators with cell culture experience and takes between 9 and 30 d, depending on the number of cell lineages in the construct. We also provide examples of applications of this advanced platform for testing gene and cell therapies in vitro, as well as for in vivo studies, providing proof of principle of its potential as a tool to develop next-generation neuromuscular or musculoskeletal therapies. The authors present a protocol for the modular 3D bioengineering of multilineage skeletal muscles from human induced pluripotent stem cells, along with assays to characterize morphological and functional features of the artificial muscle constructs.

Decreased left ventricle (LV) function caused by genetic mutations or injury often leads to debilitating and fatal cardiovascular disease. LV cardiomyocytes are, therefore, a potentially valuable therapeutical target. Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) are neither homogeneous nor functionally mature, which reduces their utility. Here, we exploit cardiac development knowledge to instruct differentia-tion of hPSCs specifically toward LV cardiomyocytes. Correct mesoderm patterning and retinoic acid pathway blocking are essential to generate near-homogenous LV-specific hPSC-CMs (hPSC-LV-CMs). These cells transit via first heart field progenitors and display typical ventricular action potentials. Impor-tantly, hPSC-LV-CMs exhibit increased metabolism, reduced proliferation, and improved cytoarchitecture and functional maturity compared with age-matched cardiomyocytes generated using the standard WNT-ON/WNT-OFF protocol. Similarly, engineered heart tissues made from hPSC-LV-CMs are better organized, produce higher force, and beat more slowly but can be paced to physiological levels. Together, we show that functionally matured hPSC-LV-CMs can be obtained rapidly without exposure to current maturation regimes.

ABSTRACT The spinal cord emerges from a niche of neuromesodermal progenitors (NMPs) formed and maintained by Wnt/FGF signals at the posterior end of the embryo. NMPs can be generated from human pluripotent stem cells and hold promise for spinal cord replacement therapies. However, NMPs are transient, which complicates the full range production of rostrocaudal spinal cord identities in vitro . Here we report the generation of NMP-derived pre-neural progenitors (PNPs) with stem cell-like self-renewal capacity. PNPs maintain pre-spinal cord identity by co-expressing the transcription factors SOX2 and CDX2, and lose mesodermal potential by downregulating TBXT. For 7 to 10 passages PNPs divide to self-renew and to make trunk neural crest (NC), while gradually adopting a more posterior identity by activating colinear HOX gene expression. This HOX clock can be halted at the thoracic level for up to 30 passages by blocking the trunk-to-tail transition through GDF11-mediated signal inhibition.

Abstract During embryonic development, mutually antagonistic signaling cascades determine the fate of the bipotential gonad towards a testicular or ovarian identity. Errors in this process result in human Disorders of Sex Development (DSDs), where there is discordance between chromosomal, gonadal, and anatomical sex. The absence of an appropriate, accessible in-vitro system is a major obstacle in understanding mechanisms of sex-determination/DSDs. Here, we describe protocols for differentiation of mouse and human pluripotent cells towards gonadal progenitors. Transcriptomic analysis reveals that the in-vitro-derived murine gonadal cells are equivalent to E11.5 in-vivo progenitors. Using similar conditions, Sertoli-like cells derived from 46,XY human induced pluripotent stem cells (hiPSCs) exhibit sustained expression of testis-specific genes, secrete AMH, migrate and form 3D tubular structures on a specially designed microfluidic device. The cells derived from a 46,XY DSD female hiPSCs, carrying a NR5A1 variant, show aberrant gene expression and absence of tubule formation. CRISPR/Cas9-mediated correction of the variant rescued the phenotype. This is a robust tool to understand mechanisms of sex-determination and model DSDs. Presentation: Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

The therapeutic benefit of stimulating the cGMP pathway as a form of treatment to combat heart failure, as well as other fibrotic pathologies, has become well established. However, the development and signal compartmentation of this crucial pathway has so far been overlooked. We studied how the three main cGMP pathways, namely, nitric oxide (NO)-cGMP, natriuretic peptide (NP)-cGMP, and β3-adrenoreceptor (AR)-cGMP, mature over time in culture during cardiomyocyte differentiation from human pluripotent stem cells (hPSC-CMs). After introducing a cGMP sensor for Förster Resonance Energy Transfer (FRET) microscopy, we used selective phosphodiesterase (PDE) inhibition to reveal cGMP signal compartmentation in hPSC-CMs at various times of culture. Methyl-β-cyclodextrin was employed to remove cholesterol and thus to destroy caveolae in these cells, where physical cGMP signaling compartmentalization is known to occur in adult cardiomyocytes. We identified PDE3 as regulator of both the NO-cGMP and NP-cGMP pathway in the early stages of culture. At the late stage, the role of the NO-cGMP pathway diminished, and it was predominantly regulated by PDE1, PDE2, and PDE5. The NP-cGMP pathway shows unrestricted locally and unregulated cGMP signaling. Lastly, we observed that maturation of the β3-AR-cGMP pathway in prolonged cultures of hPSC-CMs depends on the accumulation of caveolae. Overall, this study highlighted the importance of structural development for the necessary compartmentation of the cGMP pathway in maturing hPSC-CMs.

Cardiomyocytes rely on specialised metabolism to meet the high energy demand of the heart. During heart development, metabolism matures and shifts from the predominant utilisation of glycolysis and glutamine oxidation towards lactate and fatty acid oxidation. Iron deficiency (ID) leads to cellular metabolism perturbations. However, the exact alterations in substrate metabolism during ID are poorly defined. Using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM), the present study investigated changes in major metabolic substrate utilisation in the context of ID or upon transferrin rescue. Typically, during hiPSC-CM differentiation, the greatest increase in total metabolic output and rate was seen in fatty acid metabolism. When ID was induced, hiPSC-CMs displayed increased reliance on glycolytic metabolism, and six TCA cycle, five amino acid, and four fatty acid substrates were significantly impaired. Transferrin rescue was able to improve TCA cycle substrate metabolism, but the amino acid and fatty acid metabolism remained perturbed. Replenishing iron stores partially reverses the adverse metabolic changes that occur during ID. Understanding the changes in metabolic substrate utilisation and their modification may provide potential for discovery of new biomarkers and therapeutic targets in cardiovascular diseases.

3 Patricia R. Pitrez, Luís Estronca, Luís Monteiro, Guillem Colell, Helena Vazão, Deolinda 4 Santinha, Karim Harhouri, Daniel Thornton, Claire Navarro, Anne-Laure Egesipe, Tânia 5 Carvalho, Rodrigo L. dos Santos, Nicolas Lévy, James C. Smith, João Pedro de Magalhães, 6 Alessandro Ori, Andreia Bernardo, Annachiara De Sandre-Giovannoli, Xavier Nissan, Anna 7 Rosell, Lino Ferreira* 8 9 Affiliations: 10 Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal 11 Faculty of Medicine, University of Coimbra, 3000-548, Coimbra, Portugal 12 Neurovascular Research Laboratory. Vall d’Hebron Research institute. Passeig Vall d’Hebron 11913 129, 08035, Barcelona, Spain 14 Molecular Genetics Laboratory, Department of Medical Genetics, La Timone Children’s hospital, 15 Marseille, France 16 Integrative Genomics of Ageing Group, Institute of Ageing and Chronic Disease, University of 17 Liverpool, Liverpool L7 8TX, United Kingdom 18 Aix Marseille Univ, INSERM, GMGF, Marseille, France 19 CECS, I-STEM, AFM, Institute for Stem Cell Therapy and Exploration of Monogenic diseases, 20 Evry cedex, France 21 IMM, Instituto de Medicina Molecular, Universidade de Lisboa, Lisbon, Portugal 22 Cambridge Science Park, Mogrify Ltd, 25 Milton Road, Cambridge, CB4 0FW, UK 23 Developmental Biology Laboratory, The Francis Crick Institute, London, NW1 1AT UK 24 Leibniz Institute on Aging-Fritz Lipmann Institute, 07745 Jena, Germany 25

Hutchinson-Gilford Progeria Syndrome (HGPS) is a premature aging disease in children that leads to early death. Smooth muscle cells (SMCs) are the most affected cells in HGPS individuals, although the reason for such vulnerability remains poorly understood. In this work, we develop a microfluidic chip formed by HGPS-SMCs generated from induced pluripotent stem cells (iPSCs), to study their vulnerability to flow shear stress. HGPS-iPSC SMCs cultured under arterial flow conditions detach from the chip after a few days of culture; this process is mediated by the upregulation of metalloprotease 13 (MMP13). Importantly, double-mutant Lmna G609G/G609G Mmp13 −/− mice or Lmna G609G/G609G Mmp13 +/+ mice treated with a MMP inhibitor show lower SMC loss in the aortic arch than controls. MMP13 upregulation appears to be mediated, at least in part, by the upregulation of glycocalyx. Our HGPS-SMCs chip represents a platform for developing treatments for HGPS individuals that may complement previous pre-clinical and clinical treatments.

Background Genomic imprinting, resulting in parent-of-origin specific gene expression, plays a critical role in mammalian development. Here, we apply allele-specific RNA-seq on isogenic B6D2F1 mice to assay imprinted genes in tissues from early embryonic tissues between E3.5 and E7.25 and in pluripotent cell lines to evaluate maintenance of imprinted gene expression. For the cell lines, we include embryonic stem cells (ESCs) and epiblast stem cells (EpiSCs) derived from fertilized embryos and from embryos obtained after nuclear transfer (NT) or parthenogenetic activation (PGA). Results As homozygous genomic regions of PGA-derived cells are not compatible with allele-specific RNA-seq, we developed an RNA-seq-based genotyping strategy allowing identification of informative heterozygous regions. Global analysis shows that proper imprinted gene expression as observed in embryonic tissues is largely lost in the ESC lines included in this study, which mainly consisted of female ESCs. Differentiation of ESC lines to embryoid bodies or NPCs does not restore monoallelic expression of imprinted genes, neither did reprogramming of the serum-cultured ESCs to the pluripotent ground state by the use of 2 kinase inhibitors. Fertilized EpiSC and EpiSC-NT lines largely maintain imprinted gene expression, as did EpiSC-PGA lines that show known paternally expressed genes being silent and known maternally expressed genes consistently showing doubled expression. Notably, two EpiSC-NT lines show aberrant silencing of Rian and Meg3, two critically imprinted genes in mouse iPSCs. With respect to female EpiSC, most of the lines displayed completely skewed X inactivation suggesting a (near) clonal origin. Conclusions Altogether, our analysis provides a comprehensive overview of imprinted gene expression in pluripotency and provides a benchmark to allow identification of cell lines that faithfully maintain imprinted gene expression and therefore retain full developmental potential.

During early mammalian development, transient pools of pluripotent cells emerge that can be immortalised upon stem cell derivation. The pluripotent state, 'naïve' or 'primed', depends on the embryonic stage and derivation conditions used. Here we analyse the temporal gene expression patterns of mouse, cattle and porcine embryos at stages that harbour different types of pluripotent cells. We document conserved and divergent traits in gene expression, and identify predictor genes shared across the species that are associated with pluripotent states in vivo and in vitro Amongst these are the pluripotency-linked genes Klf4 and Lin28b The novel genes discovered include naïve- (Spic, Scpep1 and Gjb5) and primed-associated (Sema6a and Jakmip2) genes as well as naïve to primed transition genes (Dusp6 and Trip6). Both Gjb5 and Dusp6 play a role in pluripotency since their knockdown results in differentiation and downregulation of key pluripotency genes. Our interspecies comparison revealed new insights of pluripotency, pluripotent stem cell identity and a new molecular criterion for distinguishing between pluripotent states in various species, including human.

Wnt signaling is a key regulator of vertebrate heart development; however, specific roles for human cardiomyocyte development remain uncertain. Here we use human embryonic stem cells (hESCs) to analyze systematically in human cardiomyocyte development the expression of endogenous Wnt signaling components, monitor pathway activity, and dissect stage-specific requirements for canonical and noncanonical Wnt signaling mechanisms using small-molecule inhibitors. Our analysis suggests that WNT3 and WNT8A, via FZD7 and canonical signaling, regulate BRACHYURY expression and mesoderm induction; that WNT5A/5B, via ROR2 and noncanonical signaling, regulate MESP1 expression and cardiovascular development; and that later in development WNT2, WNT5A/5B, and WNT11, via FZD4 and FZD6, regulate functional cardiomyocyte differentiation via noncanonical Wnt signaling. Our findings confirm in human development previously proposed roles for canonical Wnt signaling in sequential stages of vertebrate cardiomyogenesis, and identify more precise roles for noncanonical signaling and for individual Wnt signal and Wnt receptor genes in human cardiomyocyte development.

The transcription factor brachyury (T, BRA) is one of the first markers of gastrulation and lineage specification in vertebrates. Despite its wide use and importance in stem cell and developmental biology, its functional genomic targets in human cells are largely unknown. Here, we use differentiating human embryonic stem cells to study the role of BRA in activin A-induced endoderm and BMP4-induced mesoderm progenitors. We show that BRA has distinct genome-wide binding landscapes in these two cell populations, and that BRA interacts and collaborates with SMAD1 or SMAD2/3 signalling to regulate the expression of its target genes in a cell-specific manner. Importantly, by manipulating the levels of BRA in cells exposed to different signalling environments, we demonstrate that BRA is essential for mesoderm but not for endoderm formation. Together, our data illuminate the function of BRA in the context of human embryonic development and show that the regulatory role of BRA is context dependent. Our study reinforces the importance of analysing the functions of a transcription factor in different cellular and signalling environments.

It is imperative to unveil the full range of differentiated cell types into which human pluripotent stem cells (hPSCs) can develop. The need is twofold: it will delimit the therapeutic utility of these stem cells and is necessary to place their position accurately in the developmental hierarchy of lineage potential. Accumulated evidence suggested that hPSC could develop in vitro into an extraembryonic lineage (trophoblast (TB)) that is typically inaccessible to pluripotent embryonic cells during embryogenesis. However, whether these differentiated cells are truly authentic TB has been challenged. In this debate, we present a case for and a case against TB differentiation from hPSCs. By analogy to other differentiation systems, our debate is broadly applicable, as it articulates higher and more challenging standards for judging whether a given cell type has been genuinely produced from hPSC differentiation.

Vascular smooth muscle cells (SMCs) arise from diverse developmental origins. Regional distribution of vascular diseases may, in part, be attributed to this inherent heterogeneity in SMC lineage. Therefore, systems for generating human SMC subtypes of distinct embryonic origins would represent useful platforms for studying the influence of SMC lineage on the spatial specificity of vascular disease. Here we describe how human pluripotent stem cells can be differentiated into distinct populations of SMC subtypes under chemically defined conditions. The initial stage (days 0–5 or 0–7) begins with the induction of three intermediate lineages: neuroectoderm, lateral plate mesoderm and paraxial mesoderm. Subsequently, these precursor lineages are differentiated into contractile SMCs (days 5–19+). At key stages, the emergence of lineage-specific markers confirms recapitulation of embryonic developmental pathways and generation of functionally distinct SMC subtypes. The ability to derive an unlimited supply of human SMCs will accelerate applications in regenerative medicine and disease modeling.

Embryonic Stem Cells (ESCs) and Epiblast Stem Cells (EpiSCs) are the in vitro representatives of naïve and primed pluripotency, respectively. It is currently unclear how their epigenomes underpin the phenotypic and molecular characteristics of these distinct pluripotent states. Here, we performed a genome-wide comparison of DNA methylation between ESCs and EpiSCs by MethylCap-Seq. We observe that promoters are preferential targets for methylation in EpiSC compared to ESCs, in particular high CpG island promoters. This is in line with upregulation of the de novo methyltransferases Dnmt3a1 and Dnmt3b in EpiSC, and downregulation of the demethylases Tet1 and Tet2. Remarkably, the observed DNA methylation signature is specific to EpiSCs and differs from that of their in vivo counterpart, the postimplantation epiblast. Using a subset of promoters that are differentially methylated, we show that DNA methylation is established within a few days during in vitro outgrowth of the epiblast, and also occurs when ESCs are converted to EpiSCs in vitro. Once established, this methylation is stable, as ES-like cells obtained by in vitro reversion of EpiSCs display an epigenetic memory that only extensive passaging and sub-cloning are able to almost completely erase.