Ataxia Telangiectasia patient-derived neuronal and brain organoid models reveal mitochondrial dysfunction and oxidative stress.

Ataxia Telangiectasia (AT) is a rare disorder caused by mutations in the ATM gene and results in progressive neurodegeneration for reasons that remain poorly understood. In addition to its central role in nuclear DNA repair, ATM operates outside the nucleus to regulate metabolism, redox homeostasis and mitochondrial function. However, a systematic investigation into how and when loss of ATM affects these parameters in relevant human neuronal models of AT was lacking. We therefore used cortical neurons and brain organoids from AT-patient iPSC and gene corrected isogenic controls to reveal levels of mitochondrial dysfunction, oxidative stress, and senescence that vary with developmental maturity. Transcriptome analyses identified disruptions in regulatory networks related to mitochondrial function and maintenance, including alterations in the PARP/SIRT signalling axis and dysregulation of key mitophagy and mitochondrial fission-fusion processes. We further show that antioxidants reduce ROS and restore neurite branching in AT neuronal cultures, and ameliorate impaired neuronal activity in AT brain organoids. We conclude that progressive mitochondrial dysfunction and aberrant ROS production are important contributors to neurodegeneration in AT and are strongly linked to ATM's role in mitochondrial homeostasis regulation.

Development of Physiologically Relevant Skin Organoids from Human Induced Pluripotent Stem Cells.

The development of skin organs for studying developmental pathways, modeling diseases, or regenerative medicine purposes is a major endeavor in the field. Human induced pluripotent stem cells (hiPSCs) are successfully used to derive skin cells, but the field is still far from meeting the goal of creating skin containing appendages, such as hair follicles and sweat glands. Here, the goal is to generate skin organoids (SKOs) from human skin fibroblast or placental CD34+ cell-derived hiPSCs. With all three hiPSC lines, complex SKOs with stratified skin layers and pigmented hair follicles are generated with different efficacies. In addition, the hiPSC-derived SKOs develop sebaceous glands, touch-receptive Merkel cells, and more importantly eccrine sweat glands. Together, physiologically relevant skin organoids are developed by direct induction of embryoid body formation, along with simultaneous inactivation of transforming growth factor beta signaling, activation of fibroblast growth factor signaling, and inhibition of bone morphogenetic protein signaling pathways. The skin organoids created in this study can be used as valuable platforms for further research into human skin development, disease modeling, or reconstructive surgeries.

SARS-CoV-2 drives NLRP3 inflammasome activation in human microglia through spike protein.

Coronavirus disease-2019 (COVID-19) is primarily a respiratory disease, however, an increasing number of reports indicate that SARS-CoV-2 infection can also cause severe neurological manifestations, including precipitating cases of probable Parkinson's disease. As microglial NLRP3 inflammasome activation is a major driver of neurodegeneration, here we interrogated whether SARS-CoV-2 can promote microglial NLRP3 inflammasome activation. Using SARS-CoV-2 infection of transgenic mice expressing human angiotensin-converting enzyme 2 (hACE2) as a COVID-19 pre-clinical model, we established the presence of virus in the brain together with microglial activation and NLRP3 inflammasome upregulation in comparison to uninfected mice. Next, utilising a model of human monocyte-derived microglia, we identified that SARS-CoV-2 isolates can bind and enter human microglia in the absence of viral replication. This interaction of virus and microglia directly induced robust inflammasome activation, even in the absence of another priming signal. Mechanistically, we demonstrated that purified SARS-CoV-2 spike glycoprotein activated the NLRP3 inflammasome in LPS-primed microglia, in a ACE2-dependent manner. Spike protein also could prime the inflammasome in microglia through NF-κB signalling, allowing for activation through either ATP, nigericin or α-synuclein. Notably, SARS-CoV-2 and spike protein-mediated microglial inflammasome activation was significantly enhanced in the presence of α-synuclein fibrils and was entirely ablated by NLRP3-inhibition. Finally, we demonstrate SARS-CoV-2 infected hACE2 mice treated orally post-infection with the NLRP3 inhibitory drug MCC950, have significantly reduced microglial inflammasome activation, and increased survival in comparison with untreated SARS-CoV-2 infected mice. These results support a possible mechanism of microglial innate immune activation by SARS-CoV-2, which could explain the increased vulnerability to developing neurological symptoms akin to Parkinson's disease in COVID-19 infected individuals, and a potential therapeutic avenue for intervention.

CRISPR-Cas9 Direct Fusions for Improved Genome Editing via Enhanced Homologous Recombination.

DNA repair in mammalian cells involves the coordinated action of a range of complex cellular repair machinery. Our understanding of these DNA repair processes has advanced to the extent that they can be leveraged to improve the efficacy and precision of Cas9-assisted genome editing tools. Here, we review how the fusion of CRISPR-Cas9 to functional domains of proteins that directly or indirectly impact the DNA repair process can enhance genome editing. Such studies have allowed the development of diverse technologies that promote efficient gene knock-in for safer genome engineering practices.

Correction of ATM mutations in iPS cells from two ataxia-telangiectasia patients restores DNA damage and oxidative stress responses.

Patients with ataxia-telangiectasia (A-T) lack a functional ATM kinase protein and exhibit defective repair of DNA double-stranded breaks and response to oxidative stress. We show that CRISPR/Cas9-assisted gene correction combined with piggyBac (PB) transposon-mediated excision of the selection cassette enables seamless restoration of functional ATM alleles in induced pluripotent stem cells from an A-T patient carrying compound heterozygous exonic missense/frameshift mutations, and from a patient with a homozygous splicing acceptor mutation of an internal coding exon. We show that the correction of one allele restores expression of ~ 50% of full-length ATM protein and ameliorates DNA damage-induced activation (auto-phosphorylation) of ATM and phosphorylation of its downstream targets, KAP-1 and H2AX. Restoration of ATM function also normalizes radiosensitivity, mitochondrial ROS production and oxidative-stress-induced apoptosis levels in A-T iPSC lines, demonstrating that restoration of a single ATM allele is sufficient to rescue key ATM functions. Our data further show that despite the absence of a functional ATM kinase, homology-directed repair and seamless correction of a pathogenic ATM mutation is possible. The isogenic pairs of A-T and gene-corrected iPSCs described here constitute valuable tools for elucidating the role of ATM in ageing and A-T pathogenesis.

Evaluation of variability in human kidney organoids.

The utility of human pluripotent stem cell-derived kidney organoids relies implicitly on the robustness and transferability of the protocol. Here we analyze the sources of transcriptional variation in a specific kidney organoid protocol. Although individual organoids within a differentiation batch showed strong transcriptional correlation, we noted significant variation between experimental batches, particularly in genes associated with temporal maturation. Single-cell profiling revealed shifts in nephron patterning and proportions of component cells. Distinct induced pluripotent stem cell clones showed congruent transcriptional programs, with interexperimental and interclonal variation also strongly associated with nephron patterning. Epithelial cells isolated from organoids aligned with total organoids at the same day of differentiation, again implicating relative maturation as a confounder. This understanding of experimental variation facilitated an optimized analysis of organoid-based disease modeling, thereby increasing the utility of kidney organoids for personalized medicine and functional genomics.

Kidney organoids from human iPS cells contain multiple lineages and model human nephrogenesis.

The human kidney contains up to 2 million epithelial nephrons responsible for blood filtration. Regenerating the kidney requires the induction of the more than 20 distinct cell types required for excretion and the regulation of pH, and electrolyte and fluid balance. We have previously described the simultaneous induction of progenitors for both collecting duct and nephrons via the directed differentiation of human pluripotent stem cells. Paradoxically, although both are of intermediate mesoderm in origin, collecting duct and nephrons have distinct temporospatial origins. Here we identify the developmental mechanism regulating the preferential induction of collecting duct versus kidney mesenchyme progenitors. Using this knowledge, we have generated kidney organoids that contain nephrons associated with a collecting duct network surrounded by renal interstitium and endothelial cells. Within these organoids, individual nephrons segment into distal and proximal tubules, early loops of Henle, and glomeruli containing podocytes elaborating foot processes and undergoing vascularization. When transcription profiles of kidney organoids were compared to human fetal tissues, they showed highest congruence with first trimester human kidney. Furthermore, the proximal tubules endocytose dextran and differentially apoptose in response to cisplatin, a nephrotoxicant. Such kidney organoids represent powerful models of the human organ for future applications, including nephrotoxicity screening, disease modelling and as a source of cells for therapy.

Use of Human Induced Pluripotent Stem Cells and Kidney Organoids To Develop a Cysteamine/mTOR Inhibition Combination Therapy for Cystinosis.

BACKGROUND: Mutations in CTNS-a gene encoding the cystine transporter cystinosin-cause the rare, autosomal, recessive, lysosomal-storage disease cystinosis. Research has also implicated cystinosin in modulating the mTORC1 pathway, which serves as a core regulator of cellular metabolism, proliferation, survival, and autophagy. In its severest form, cystinosis is characterized by cystine accumulation, renal proximal tubule dysfunction, and kidney failure. Because treatment with the cystine-depleting drug cysteamine only slows disease progression, there is an urgent need for better treatments. METHODS: To address a lack of good human-based cell culture models for studying cystinosis, we generated the first human induced pluripotent stem cell (iPSC) and kidney organoid models of the disorder. We used a variety of techniques to examine hallmarks of cystinosis-including cystine accumulation, lysosome size, the autophagy pathway, and apoptosis-and performed RNA sequencing on isogenic lines to identify differentially expressed genes in the cystinosis models compared with controls. RESULTS: Compared with controls, these cystinosis models exhibit elevated cystine levels, increased apoptosis, and defective basal autophagy. Cysteamine treatment ameliorates this phenotype, except for abnormalities in apoptosis and basal autophagy. We found that treatment with everolimus, an inhibitor of the mTOR pathway, reduces the number of large lysosomes, decreases apoptosis, and activates autophagy, but it does not rescue the defect in cystine loading. However, dual treatment of cystinotic iPSCs or kidney organoids with cysteamine and everolimus corrects all of the observed phenotypic abnormalities. CONCLUSIONS: These observations suggest that combination therapy with a cystine-depleting drug such as cysteamine and an mTOR pathway inhibitor such as everolimus has potential to improve treatment of cystinosis.

Global Proteomic and Methylome Analysis in Human Induced Pluripotent Stem Cells Reveals Overexpression of a Human TLR3 Affecting Proper Innate Immune Response Signaling.

When considering the clinical applications of autologous cell replacement therapy of human induced pluripotent stem cells (iPSC)-derived cells, there is a clear need to better understand what the immune response will be before we embark on extensive clinical trials to treat or model human disease. We performed a detailed assessment comparing human fibroblast cell lines (termed F1) reprogrammed into human iPSC and subsequently differentiated back to fibroblast cells (termed F2) or other human iPSC-derived cells including neural stem cells (NSC) made from either retroviral, episomal, or synthetic mRNA cell reprogramming methods. Global proteomic analysis reveals the main differences in signal transduction and immune cell protein expression between F1 and F2 cells, implicating wild type (WT) toll like receptor protein 3 (TLR3). Furthermore, global methylome analysis identified an isoform of the human TLR3 gene that is not epigenetically reset correctly upon differentiation to F2 cells resulting in a hypomethylated transcription start site in the TLR3 isoform promoter and overexpression in most human iPSC-derived cells not seen in normal human tissue. The human TLR3 isoform in human iPSC-NSC functions to suppress NF-KB p65 signaling pathway in response to virus (Poly IC), suggesting suppressed immunity of iPSC-derived cells to viral infection. The sustained WT TLR3 and TLR3 isoform overexpression is central to understanding the altered immunogenicity of human iPSC-derived cells calling for screening of human iPSC-derived cells for TLR3 expression levels before applications. Stem Cells 2019;37:476-488.

A rat model of ataxia-telangiectasia: evidence for a neurodegenerative phenotype.

Ataxia-telangiectasia (A-T), an autosomal recessive disease caused by mutations in the ATM gene is characterised by cerebellar atrophy and progressive neurodegeneration which has been poorly recapitulated in Atm mutant mice. Consequently, pathways leading to neurodegeneration in A-T are poorly understood. We describe here the generation of an Atm knockout rat model that does not display cerebellar atrophy but instead paralysis and spinal cord atrophy, reminiscent of that seen in older patients and milder forms of the disorder. Loss of Atm in neurons and glia leads to accumulation of cytosolic DNA, increased cytokine production and constitutive activation of microglia consistent with a neuroinflammatory phenotype. Rats lacking ATM had significant loss of motor neurons and microgliosis in the spinal cord, consistent with onset of paralysis. Since short term treatment with steroids has been shown to improve the neurological signs in A-T patients we determined if that was also the case for Atm-deficient rats. Betamethasone treatment extended the lifespan of Atm knockout rats, prevented microglial activation and significantly decreased neuroinflammatory changes and motor neuron loss. These results point to unrepaired damage to DNA leading to significant levels of cytosolic DNA in Atm-deficient neurons and microglia and as a consequence activation of the cGAS-STING pathway and cytokine production. This in turn would increase the inflammatory microenvironment leading to dysfunction and death of neurons. Thus the rat model represents a suitable one for studying neurodegeneration in A-T and adds support for the use of anti-inflammatory drugs for the treatment of neurodegeneration in A-T patients.

Inflammatory responses to a pathogenic West Nile virus strain.

BACKGROUND: West Nile virus (WNV) circulates across Australia and was referred to historically as Kunjin virus (WNVKUN). WNVKUN has been considered more benign than other WNV strains circulating globally. In 2011, a more virulent form of the virus emerged during an outbreak of equine arboviral disease in Australia. METHODS: To better understand the emergence of this virulent phenotype and the mechanism by which pathogenicity is manifested in its host, cells were infected with either the virulent strain (NSW2012), or less pathogenic historical isolates, and their innate immune responses compared by digital immune gene expression profiling. Two different cell systems were used: a neuroblastoma cell line (SK-N-SH cells) and neuronal cells derived from induced pluripotent stem cells (iPSCs). RESULTS: Significant innate immune gene induction was observed in both systems. The NSW2012 isolate induced higher gene expression of two genes (IL-8 and CCL2) when compared with cells infected with less pathogenic isolates. Pathway analysis of induced inflammation-associated genes also indicated generally higher activation in infected NSW2012 cells. However, this differential response was not paralleled in the neuronal cultures. CONCLUSION: NSW2012 may have unique genetic characteristics which contributed to the outbreak. The data herein is consistent with the possibility that the virulence of NSW2012 is underpinned by increased induction of inflammatory genes.

Complement C5aR1 Signaling Promotes Polarization and Proliferation of Embryonic Neural Progenitor Cells through PKCζ.

The complement system, typically associated with innate immunity, is emerging as a key controller of nonimmune systems including in development, with recent studies linking complement mutations with neurodevelopmental disease. A key effector of the complement response is the activation fragment C5a, which, through its receptor C5aR1, is a potent driver of inflammation. Surprisingly, C5aR1 is also expressed during early mammalian embryogenesis; however, no clearly defined function is ascribed to C5aR1 in development. Here we demonstrate polarized expression of C5aR1 on the apical surface of mouse embryonic neural progenitor cells in vivo and on human embryonic stem cell-derived neural progenitors. We also show that signaling of endogenous C5a during mouse embryogenesis drives proliferation of neural progenitor cells within the ventricular zone and is required for normal brain histogenesis. C5aR1 signaling in neural progenitors was dependent on atypical protein kinase C ζ, a mediator of stem cell polarity, with C5aR1 inhibition reducing proliferation and symmetric division of apical neural progenitors in human and mouse models. C5aR1 signaling was shown to promote the maintenance of cell polarity, with exogenous C5a increasing the retention of polarized rosette architecture in human neural progenitors after physical or chemical disruption. Transient inhibition of C5aR1 during neurogenesis in developing mice led to behavioral abnormalities in both sexes and MRI-detected brain microstructural alterations, in studied males, demonstrating a requirement of C5aR1 signaling for appropriate brain development. This study thus identifies a functional role for C5a-C5aR1 signaling in mammalian neurogenesis and provides mechanistic insight into recently identified complement gene mutations and brain disorders.SIGNIFICANCE STATEMENT The complement system, traditionally known as a controller of innate immunity, now stands as a multifaceted signaling family with a broad range of physiological actions. These include roles in the brain, where complement activation is associated with diseases, including epilepsy and schizophrenia. This study has explored complement regulation of neurogenesis, identifying a novel relationship between the complement activation peptide C5a and the neural progenitor proliferation underpinning formation of the mammalian brain. C5a was identified as a regulator of cell polarity, with inhibition of C5a receptors during embryogenesis leading to abnormal brain development and behavioral deficits. This work demonstrates mechanisms through which dysregulation of complement causes developmental disease and highlights the potential risk of complement inhibition for therapeutic purposes in pregnancy.

PCDH19 regulation of neural progenitor cell differentiation suggests asynchrony of neurogenesis as a mechanism contributing to PCDH19 Girls Clustering Epilepsy.

PCDH19-Girls Clustering Epilepsy (PCDH19-GCE) is a childhood epileptic encephalopathy characterised by a spectrum of neurodevelopmental problems. PCDH19-GCE is caused by heterozygous loss-of-function mutations in the X-chromosome gene, Protocadherin 19 (PCDH19) encoding a cell-cell adhesion molecule. Intriguingly, hemizygous males are generally unaffected. As PCDH19 is subjected to random X-inactivation, heterozygous females are comprised of a mosaic of cells expressing either the normal or mutant allele, which is thought to drive pathology. Despite being the second most prevalent monogeneic cause of epilepsy, little is known about the role of PCDH19 in brain development. In this study we show that PCDH19 is highly expressed in human neural stem and progenitor cells (NSPCs) and investigate its function in vitro in these cells of both mouse and human origin. Transcriptomic analysis of mouse NSPCs lacking Pcdh19 revealed changes to genes involved in regulation of neuronal differentiation, and we subsequently show that loss of Pcdh19 causes increased NSPC neurogenesis. We reprogramed human fibroblast cells harbouring a pathogenic PCDH19 mutation into human induced pluripotent stem cells (hiPSC) and employed neural differentiation of these to extend our studies into human NSPCs. As in mouse, loss of PCDH19 function caused increased neurogenesis, and furthermore, we show this is associated with a loss of human NSPC polarity. Overall our data suggests a conserved role for PCDH19 in regulating mammalian cortical neurogenesis and has implications for the pathogenesis of PCDH19-GCE. We propose that the difference in timing or "heterochrony" of neuronal cell production originating from PCDH19 wildtype and mutant NSPCs within the same individual may lead to downstream asynchronies and abnormalities in neuronal network formation, which in-part predispose the individual to network dysfunction and epileptic activity.

Targeted, Stimuli-Responsive Delivery of Plasmid DNA and miRNAs Using a Facile Self-Assembled Supramolecular Nanoparticle System.

Gene therapy is rapidly regaining traction in terms of research activity and investment across the globe, with clear potential to revolutionize medicine and tissue regeneration. Viral vectors remain the most commonly utilized gene delivery vehicles, due to their high efficiency, however, they are acknowledged to have numerous drawbacks, including limited payload capacity, lack of cell-type specificity, and risk of possible mutations in vivo, hence, patient safety. Synthetic nanoparticle gene delivery systems can offer substantial advantages over viral vectors. They can be utilized as off-the-shelf components to package genetic material, display targeting ligands, and release payloads upon environmental triggers and enable the possibility of programmed cell-specific uptake and transfection. In this study, we have synthesized three functional polymeric building blocks that, in a rapid, facile, tailorable, and stage-wise manner, associate through both electrostatic and noncovalent hydrophobic "host-guest" interactions to form monodisperse self-assembled nanoparticles (SaNP). We show that these SaNPs successfully package significant amounts of microRNA through to plasmid DNA, present desired ligands on their outer surface for targeted receptor-mediated cell-specific uptake and affect efficient translation of packaged plasmids. We confirm that these SaNPs outperform commercially available, gold standard transfection agents in terms of in vitro transfection efficiencies and have very low cytotoxicity. With facile self-assembly and tailorable composition, our SaNP gene delivery system has significant potential in targeted gene therapy applications.

Variability of Gene Expression Identifies Transcriptional Regulators of Early Human Embryonic Development.

An analysis of gene expression variability can provide an insightful window into how regulatory control is distributed across the transcriptome. In a single cell analysis, the inter-cellular variability of gene expression measures the consistency of transcript copy numbers observed between cells in the same population. Application of these ideas to the study of early human embryonic development may reveal important insights into the transcriptional programs controlling this process, based on which components are most tightly regulated. Using a published single cell RNA-seq data set of human embryos collected at four-cell, eight-cell, morula and blastocyst stages, we identified genes with the most stable, invariant expression across all four developmental stages. Stably-expressed genes were found to be enriched for those sharing indispensable features, including essentiality, haploinsufficiency, and ubiquitous expression. The stable genes were less likely to be associated with loss-of-function variant genes or human recessive disease genes affected by a DNA copy number variant deletion, suggesting that stable genes have a functional impact on the regulation of some of the basic cellular processes. Genes with low expression variability at early stages of development are involved in regulation of DNA methylation, responses to hypoxia and telomerase activity, whereas by the blastocyst stage, low-variability genes are enriched for metabolic processes as well as telomerase signaling. Based on changes in expression variability, we identified a putative set of gene expression markers of morulae and blastocyst stages. Experimental validation of a blastocyst-expressed variability marker demonstrated that HDDC2 plays a role in the maintenance of pluripotency in human ES and iPS cells. Collectively our analyses identified new regulators involved in human embryonic development that would have otherwise been missed using methods that focus on assessment of the average expression levels; in doing so, we highlight the value of studying expression variability for single cell RNA-seq data.

A new model to study neurodegeneration in ataxia oculomotor apraxia type 2.

Ataxia oculomotor apraxia type 2 (AOA2) is a rare autosomal recessive cerebellar ataxia. Recent evidence suggests that the protein defective in this syndrome, senataxin (SETX), functions in RNA processing to protect the integrity of the genome. To date, only patient-derived lymphoblastoid cells, fibroblasts and SETX knockdown cells were available to investigate AOA2. Recent disruption of the Setx gene in mice did not lead to neurobehavioral defects or neurodegeneration, making it difficult to study the etiology of AOA2. To develop a more relevant neuronal model to study neurodegeneration in AOA2, we derived neural progenitors from a patient with AOA2 and a control by induced pluripotent stem cell (iPSC) reprogramming of fibroblasts. AOA2 iPSC and neural progenitors exhibit increased levels of oxidative damage, DNA double-strand breaks, increased DNA damage-induced cell death and R-loop accumulation. Genome-wide expression and weighted gene co-expression network analysis in these neural progenitors identified both previously reported and novel affected genes and cellular pathways associated with senataxin dysfunction and the pathophysiology of AOA2, providing further insight into the role of senataxin in regulating gene expression on a genome-wide scale. These data show that iPSCs can be generated from patients with the autosomal recessive ataxia, AOA2, differentiated into neurons, and that both cell types recapitulate the AOA2 cellular phenotype. This represents a novel and appropriate model system to investigate neurodegeneration in this syndrome.

ATM-dependent phosphorylation of MRE11 controls extent of resection during homology directed repair by signalling through Exonuclease 1.

The MRE11/RAD50/NBS1 (MRN) complex plays a central role as a sensor of DNA double strand breaks (DSB) and is responsible for the efficient activation of ataxia-telangiectasia mutated (ATM) kinase. Once activated ATM in turn phosphorylates RAD50 and NBS1, important for cell cycle control, DNA repair and cell survival. We report here that MRE11 is also phosphorylated by ATM at S676 and S678 in response to agents that induce DNA DSB, is dependent on the presence of NBS1, and does not affect the association of members of the complex or ATM activation. A phosphosite mutant (MRE11S676AS678A) cell line showed decreased cell survival and increased chromosomal aberrations after radiation exposure indicating a defect in DNA repair. Use of GFP-based DNA repair reporter substrates in MRE11S676AS678A cells revealed a defect in homology directed repair (HDR) but single strand annealing was not affected. More detailed investigation revealed that MRE11S676AS678A cells resected DNA ends to a greater extent at sites undergoing HDR. Furthermore, while ATM-dependent phosphorylation of Kap1 and SMC1 was normal in MRE11S676AS678A cells, there was no phosphorylation of Exonuclease 1 consistent with the defect in HDR. These results describe a novel role for ATM-dependent phosphorylation of MRE11 in limiting the extent of resection mediated through Exonuclease 1.

Brief report: complement C5a promotes human embryonic stem cell pluripotency in the absence of FGF2.

The complement activation product, C5a, is a pivotal member of the innate immune response; however, a diverse number of nonimmune functions are now being ascribed to C5a signaling, including roles during embryonic development. Here, we identify the expression of the C5a precursor protein, C5, as well as the C5a receptors, C5aR and C5L2, in both human embryonic stem cells and human-induced pluripotent stem cells. We show that administration of a physiologically relevant dose of purified human C5a (1 nM) stimulates activation of ERK1/2 and AKT signaling pathways, and is able to promote maintenance of the pluripotent state in the absence of FGF2. C5a also reduced cell loss following dissociation of human pluripotent stem cells. Our results reveal that complement C5a signaling supports human stem cell pluripotency and survival, and thus may play a key role in shaping early human embryonic development.