Telomerase is essential for cardiac differentiation and sustained metabolism of human cardiomyocytes.

Telomeres as the protective ends of linear chromosomes, are synthesized by the enzyme telomerase (TERT). Critically short telomeres essentially contribute to aging-related diseases and are associated with a broad spectrum of disorders known as telomeropathies. In cardiomyocytes, telomere length is strongly correlated with cardiomyopathies but it remains ambiguous whether short telomeres are the cause or the result of the disease. In this study, we employed an inducible CRISPRi human induced pluripotent stem cell (hiPSC) line to silence TERT expression enabling the generation of hiPSCs and hiPSC-derived cardiomyocytes with long and short telomeres. Reduced telomerase activity and shorter telomere lengths of hiPSCs induced global transcriptomic changes associated with cardiac developmental pathways. Consequently, the differentiation potential towards cardiomyocytes was strongly impaired and single cell RNA sequencing revealed a shift towards a more smooth muscle cell like identity in the cells with the shortest telomeres. Poor cardiomyocyte function and increased sensitivity to stress directly correlated with the extent of telomere shortening. Collectively our data demonstrates a TERT dependent cardiomyogenic differentiation defect, highlighting the CRISPRi TERT hiPSCs model as a powerful platform to study the mechanisms and consequences of short telomeres in the heart and also in the context of telomeropathies.

Stepwise combined cell transplantation using mesenchymal stem cells and induced pluripotent stem cell-derived motor neuron progenitor cells in spinal cord injury.

BACKGROUND: Spinal cord injury (SCI) is an intractable neurological disease in which functions cannot be permanently restored due to nerve damage. Stem cell therapy is a promising strategy for neuroregeneration after SCI. However, experimental evidence of its therapeutic effect in SCI is lacking. This study aimed to investigate the efficacy of transplanted cells using stepwise combined cell therapy with human mesenchymal stem cells (hMSC) and induced pluripotent stem cell (iPSC)-derived motor neuron progenitor cells (iMNP) in a rat model of SCI. METHODS: A contusive SCI model was developed in Sprague-Dawley rats using multicenter animal spinal cord injury study (MASCIS) impactor. Three protocols were designed and conducted as follows: (Subtopic 1) chronic SCI + iMNP, (Subtopic 2) acute SCI + multiple hMSC injections, and (Main topic) chronic SCI + stepwise combined cell therapy using multiple preemptive hMSC and iMNP. Neurite outgrowth was induced by coculturing hMSC and iPSC-derived motor neuron (iMN) on both two-dimensional (2D) and three-dimensional (3D) spheroid platforms during mature iMN differentiation in vitro. RESULTS: Stepwise combined cell therapy promoted mature motor neuron differentiation and axonal regeneration at the lesional site. In addition, stepwise combined cell therapy improved behavioral recovery and was more effective than single cell therapy alone. In vitro results showed that hMSC and iMN act synergistically and play a critical role in the induction of neurite outgrowth during iMN differentiation and maturation. CONCLUSIONS: Our findings show that stepwise combined cell therapy can induce alterations in the microenvironment for effective cell therapy in SCI. The in vitro results suggest that co-culturing hMSC and iMN can synergistically promote induction of MN neurite outgrowth.

Endogenous tagging using split mNeonGreen in human iPSCs for live imaging studies.

Endogenous tags have become invaluable tools to visualize and study native proteins in live cells. However, generating human cell lines carrying endogenous tags is difficult due to the low efficiency of homology-directed repair. Recently, an engineered split mNeonGreen protein was used to generate a large-scale endogenous tag library in HEK293 cells. Using split mNeonGreen for large-scale endogenous tagging in human iPSCs would open the door to studying protein function in healthy cells and across differentiated cell types. We engineered an iPS cell line to express the large fragment of the split mNeonGreen protein (mNG21-10) and showed that it enables fast and efficient endogenous tagging of proteins with the short fragment (mNG211). We also demonstrate that neural network-based image restoration enables live imaging studies of highly dynamic cellular processes such as cytokinesis in iPSCs. This work represents the first step towards a genome-wide endogenous tag library in human stem cells.

The human body contains around 20,000 different proteins that perform a myriad of essential roles. To understand how these proteins work in healthy individuals and during disease, we need to know their precise locations inside cells and how these locations may change in different situations. Genetic tools known as fluorescent proteins are often used as tags to study the location of specific proteins of interest within cells. When exposed to light, the fluorescent proteins emit specific colours of light that can be observed using microscopes. In a fluorescent protein system known as split mNeonGreen, researchers insert the DNA encoding two fragments of a fluorescent protein (one large, one small) separately into cells. The large fragment can be found throughout the cell, while the small fragment is attached to specific host proteins. When the two fragments meet, they assemble into the full mNeonGreen protein and can fluoresce. Researchers can use split mNeonGreen and other similar systems to generate large libraries of cells where the small fragment of a fluorescent protein is attached to thousands of different host proteins. However, so far these libraries are restricted to a handful of different types of cells. To address this challenge, Husser et al. inserted the DNA encoding the large fragment of mNeonGreen into human cells known as induced pluripotent stem cells, which are able to give rise to any other type of human cell. This then enabled the team to quickly and efficiently generate a library of stem cells that express the small fragment of mNeonGreen attached to different host proteins. Further experiments studied the locations of host proteins in the stem cells just before they divided into two cells. This suggested that there are differences between how induced pluripotent stem cells and other types of cells divide. In the future, the cells and the method developed by Husser et al. may be used by other researchers to create atlases showing where human proteins are located in many other types of cells.

APOE4/4 is linked to damaging lipid droplets in Alzheimer's disease microglia.

Several genetic risk factors for Alzheimer's disease implicate genes involved in lipid metabolism and many of these lipid genes are highly expressed in glial cells1. However, the relationship between lipid metabolism in glia and Alzheimer's disease pathology remains poorly understood. Through single-nucleus RNA sequencing of brain tissue in Alzheimer's disease, we have identified a microglial state defined by the expression of the lipid droplet-associated enzyme ACSL1 with ACSL1-positive microglia being most abundant in patients with Alzheimer's disease having the APOE4/4 genotype. In human induced pluripotent stem cell-derived microglia, fibrillar Aß induces ACSL1 expression, triglyceride synthesis and lipid droplet accumulation in an APOE-dependent manner. Additionally, conditioned media from lipid droplet-containing microglia lead to Tau phosphorylation and neurotoxicity in an APOE-dependent manner. Our findings suggest a link between genetic risk factors for Alzheimer's disease with microglial lipid droplet accumulation and neurotoxic microglia-derived factors, potentially providing therapeutic strategies for Alzheimer's disease.

The role of lipids in genome integrity and pluripotency.

Pluripotent stem cells (PSCs), comprising embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), offer immense potential for regenerative medicine due to their ability to differentiate into all cell types of the adult body. A critical aspect of harnessing this potential is understanding their metabolic requirements during derivation, maintenance, and differentiation in vitro. Traditional culture methods using fetal bovine serum often lead to issues such as heterogeneous cell populations and diminished pluripotency. Although the chemically-defined 2i/LIF medium has provided solutions to some of these challenges, prolonged culturing of these cells, especially female ESCs, raises concerns related to genome integrity. This review discusses the pivotal role of lipids in genome stability and pluripotency of stem cells. Notably, the introduction of lipid-rich albumin, AlbuMAX, into the 2i/LIF culture medium offers a promising avenue for enhancing the genomic stability and pluripotency of cultured ESCs. We further explore the unique characteristics of lipid-induced pluripotent stem cells (LIP-ESCs), emphasizing their potential in regenerative medicine and pluripotency research.

CRISPRi: a way to integrate iPSC-derived neuronal models.

The genetic landscape of neurodegenerative diseases encompasses genes affecting multiple cellular pathways which exert effects in an array of neuronal and glial cell-types. Deconvolution of the roles of genes implicated in disease and the effects of disease-associated variants remains a vital step in the understanding of neurodegeneration and the development of therapeutics. Disease modelling using patient induced pluripotent stem cells (iPSCs) has enabled the generation of key cell-types associated with disease whilst maintaining the genomic variants that predispose to neurodegeneration. The use of CRISPR interference (CRISPRi), alongside other CRISPR-perturbations, allows the modelling of the effects of these disease-associated variants or identifying genes which modify disease phenotypes. This review summarises the current applications of CRISPRi in iPSC-derived neuronal models, such as fluorescence-activated cell sorting (FACS)-based screens, and discusses the future opportunities for disease modelling, identification of disease risk modifiers and target/drug discovery in neurodegeneration.

Macrophages derived from human induced pluripotent stem cells (iPSCs) serve as a high-fidelity cellular model for investigating HIV-1, dengue, and influenza viruses.

Macrophages are important target cells for diverse viruses and thus represent a valuable system for studying virus biology. Isolation of primary human macrophages is done by culture of dissociated tissues or from differentiated blood monocytes, but these methods are both time consuming and result in low numbers of recovered macrophages. Here, we explore whether macrophages derived from human induced pluripotent stem cells (iPSCs)-which proliferate indefinitely and potentially provide unlimited starting material-could serve as a faithful model system for studying virus biology. Human iPSC-derived monocytes were differentiated into macrophages and then infected with HIV-1, dengue virus, or influenza virus as model human viruses. We show that iPSC-derived macrophages support the replication of these viruses with kinetics and phenotypes similar to human blood monocyte-derived macrophages. These iPSC-derived macrophages were virtually indistinguishable from human blood monocyte-derived macrophages based on surface marker expression (flow cytometry), transcriptomics (RNA sequencing), and chromatin accessibility profiling. iPSC lines were additionally generated from non-human primate (chimpanzee) fibroblasts. When challenged with dengue virus, human and chimpanzee iPSC-derived macrophages show differential susceptibility to infection, thus providing a valuable resource for studying the species-tropism of viruses. We also show that blood- and iPSC-derived macrophages both restrict influenza virus at a late stage of the virus lifecycle. Collectively, our results substantiate iPSC-derived macrophages as an alternative to blood monocyte-derived macrophages for the study of virus biology. IMPORTANCE: Macrophages have complex relationships with viruses: while macrophages aid in the removal of pathogenic viruses from the body, macrophages are also manipulated by some viruses to serve as vessels for viral replication, dissemination, and long-term persistence. Here, we show that iPSC-derived macrophages are an excellent model that can be exploited in virology.

Generation of two induced pluripotent stem cell lines from healthy patients of African American ancestry.

Stem cells are a resourceful tool for investigating cardiovascular disease in the context of race and gender. Once derived from blood or skin cells, the reprogrammed induced pluripotent stem cells (iPSCs) adopt an embryonic-like pluripotent state, enabling researchers to develop drug screening or disease modeling platforms. Here, we generated two iPSC lines from peripheral blood mononuclear cells (PBMCs) of two healthy African American patients. Both lines display the usual morphology of pluripotent stem cells, demonstrate elevated expression of pluripotent markers, show normal karyotype, and differentiate into all three germ layers in vitro.

Generation of an induced pluripotent stem cell line (UMi043-A) from an African American patient with Alzheimer's disease carrying an ABCA7 deletion (p.Arg578Alafs).

The ATP-binding cassette, subfamily A (ABC1), member 7 (ABCA7) gene is associated with Alzheimer's disease (AD) risk in populations of African, Asian, and European ancestry1-5. Numerous ABCA7 mutations contributing to risk have been identified, including a 44 base pair deletion (rs142076058) specific to individuals of African ancestry and predicted to cause a frameshift mutation (p.Arg578Alafs) (Cukier et al., 2016). The UMi043-A human induced pluripotent stem cell line was derived from an African American individual with AD who is heterozygous for this deletion and is a resource to further investigate ABCA7 and how this African-specific deletion may influence disease pathology.

WNT signalling control by KDM5C during development affects cognition.

Although KDM5C is one of the most frequently mutated genes in X-linked intellectual disability1, the exact mechanisms that lead to cognitive impairment remain unknown. Here we use human patient-derived induced pluripotent stem cells and Kdm5c knockout mice to conduct cellular, transcriptomic, chromatin and behavioural studies. KDM5C is identified as a safeguard to ensure that neurodevelopment occurs at an appropriate timescale, the disruption of which leads to intellectual disability. Specifically, there is a developmental window during which KDM5C directly controls WNT output to regulate the timely transition of primary to intermediate progenitor cells and consequently neurogenesis. Treatment with WNT signalling modulators at specific times reveal that only a transient alteration of the canonical WNT signalling pathway is sufficient to rescue the transcriptomic and chromatin landscapes in patient-derived cells and to induce these changes in wild-type cells. Notably, WNT inhibition during this developmental period also rescues behavioural changes of Kdm5c knockout mice. Conversely, a single injection of WNT3A into the brains of wild-type embryonic mice cause anxiety and memory alterations. Our work identifies KDM5C as a crucial sentinel for neurodevelopment and sheds new light on KDM5C mutation-associated intellectual disability. The results also increase our general understanding of memory and anxiety formation, with the identification of WNT functioning in a transient nature to affect long-lasting cognitive function.

The nuclear factor ID3 endows macrophages with a potent anti-tumour activity.

Macrophage activation is controlled by a balance between activating and inhibitory receptors1-7, which protect normal tissues from excessive damage during infection8,9 but promote tumour growth and metastasis in cancer7,10. Here we report that the Kupffer cell lineage-determining factor ID3 controls this balance and selectively endows Kupffer cells with the ability to phagocytose live tumour cells and orchestrate the recruitment, proliferation and activation of natural killer and CD8 T lymphoid effector cells in the liver to restrict the growth of a variety of tumours. ID3 shifts the macrophage inhibitory/activating receptor balance to promote the phagocytic and lymphoid response, at least in part by buffering the binding of the transcription factors ELK1 and E2A at the SIRPA locus. Furthermore, loss- and gain-of-function experiments demonstrate that ID3 is sufficient to confer this potent anti-tumour activity to mouse bone-marrow-derived macrophages and human induced pluripotent stem-cell-derived macrophages. Expression of ID3 is therefore necessary and sufficient to endow macrophages with the ability to form an efficient anti-tumour niche, which could be harnessed for cell therapy in cancer.

Role of connexin 32 in the directional differentiation of induced pluripotent stem cells into hepatocytes.

This study aimed to explore the role of connexin 32 (Cx32) in the directional differentiation of induced pluripotent stem cells (iPSCs) into hepatocytes. Urine-derived epithelial cells were collected from the fresh urine of a healthy donor and transducted with reprogramming plasmid mixture to generate iPSCs. The iPSCs were then directionally differentiated into hepatocytes. During the differentiation, the upregulated and downregulated groups were treated with vitamin K2 (VK2) and 2-aminoethoxyboronate diphenylester (2-APB) to increase and inhibit Cx32 expression, respectively. The control group was not treated with the regulatory factor. Expression of Cx32 and hepatocyte-specific markers, including AFP, hepatocyte nuclear factor 4α (HNF-4α), albumin (ALB) and cytokeratin 18 (CK18) were detected. It indicated that Cx32 expression was not observed in iPSCs, but gradually increased during the process of hepatic differentiation from iPSCs. Upregulation of Cx32 expression by VK2 treatment promoted hepatocyte maturation and enhanced the expression of the aforementioned hepatic specific markers, whereas downregulation of Cx32 expression by 2-APB treatment had the opposite effects. In conclusion, urine-derived iPSCs could be directionally differentiated into hepatocytes. Up-regulation of Cx32 improves the efficiency and maturity of differentiation of iPSCs into hepatocytes, and Cx32 may be a promoting factor during the process of hepatic differentiation from iPSCs.

NADH improves AIF dimerization and inhibits apoptosis in iPSCs-derived neurons from patients with auditory neuropathy spectrum disorder.

Auditory neuropathy spectrum disorder (ANSD) is a hearing impairment involving disruptions to inner hair cells (IHCs), ribbon synapses, spiral ganglion neurons (SGNs), and/or the auditory nerve itself. The outcomes of cochlear implants (CI) for ANSD are variable and dependent on the location of lesion sites. Discovering a potential therapeutic agent for ANSD remains an urgent requirement. Here, 293T stable transfection cell lines and patient induced pluripotent stem cells (iPSCs)-derived auditory neurons carrying the apoptosis inducing factor (AIF) p.R422Q variant were used to pursue a therapeutic regent for ANSD. Nicotinamide adenine dinucleotide (NADH) is a main electron donor in the electron transport chain (ETC). In 293T stable transfection cells with the p.R422Q variant, NADH treatment improved AIF dimerization, rescued mitochondrial dysfunctions, and decreased cell apoptosis. The effects of NADH were further confirmed in patient iPSCs-derived neurons. The relative level of AIF dimers was increased to 150.7 % (P = 0.026) from 59.2 % in patient-neurons upon NADH treatment. Such increased AIF dimerization promoted the mitochondrial import of coiled-coil-helix-coiled-coil-helix domain-containing protein 4 (CHCHD4), which further restored mitochondrial functions. Similarly, the content of mitochondrial calcium (mCa2+) was downregulated from 136.7 % to 102.3 % (P = 0.0024) in patient-neurons upon NADH treatment. Such decreased mCa2+ levels inhibited calpain activity, ultimately reducing the percentage of apoptotic cells from 30.5 % to 21.1 % (P = 0.021). We also compared the therapeutic effects of gene correction and NADH treatment on hereditary ANSD. NADH treatment had comparable restorative effects on functions of ANSD patient-specific cells to that of gene correction. Our findings offer evidence of the molecular mechanisms of ANSD and introduce NADH as a potential therapeutic agent for ANSD therapy.

Modelling post-implantation human development to yolk sac blood emergence.

Implantation of the human embryo begins a critical developmental stage that comprises profound events including axis formation, gastrulation and the emergence of haematopoietic system1,2. Our mechanistic knowledge of this window of human life remains limited due to restricted access to in vivo samples for both technical and ethical reasons3-5. Stem cell models of human embryo have emerged to help unlock the mysteries of this stage6-16. Here we present a genetically inducible stem cell-derived embryoid model of early post-implantation human embryogenesis that captures the reciprocal codevelopment of embryonic tissue and the extra-embryonic endoderm and mesoderm niche with early haematopoiesis. This model is produced from induced pluripotent stem cells and shows unanticipated self-organizing cellular programmes similar to those that occur in embryogenesis, including the formation of amniotic cavity and bilaminar disc morphologies as well as the generation of an anterior hypoblast pole and posterior domain. The extra-embryonic layer in these embryoids lacks trophoblast and shows advanced multilineage yolk sac tissue-like morphogenesis that harbours a process similar to distinct waves of haematopoiesis, including the emergence of erythroid-, megakaryocyte-, myeloid- and lymphoid-like cells. This model presents an easy-to-use, high-throughput, reproducible and scalable platform to probe multifaceted aspects of human development and blood formation at the early post-implantation stage. It will provide a tractable human-based model for drug testing and disease modelling.

iPS-cell-derived microglia promote brain organoid maturation via cholesterol transfer.

Microglia are specialized brain-resident macrophages that arise from primitive macrophages colonizing the embryonic brain1. Microglia contribute to multiple aspects of brain development, but their precise roles in the early human brain remain poorly understood owing to limited access to relevant tissues2-6. The generation of brain organoids from human induced pluripotent stem cells recapitulates some key features of human embryonic brain development7-10. However, current approaches do not incorporate microglia or address their role in organoid maturation11-21. Here we generated microglia-sufficient brain organoids by coculturing brain organoids with primitive-like macrophages generated from the same human induced pluripotent stem cells (iMac)22. In organoid cocultures, iMac differentiated into cells with microglia-like phenotypes and functions (iMicro) and modulated neuronal progenitor cell (NPC) differentiation, limiting NPC proliferation and promoting axonogenesis. Mechanistically, iMicro contained high levels of PLIN2+ lipid droplets that exported cholesterol and its esters, which were taken up by NPCs in the organoids. We also detected PLIN2+ lipid droplet-loaded microglia in mouse and human embryonic brains. Overall, our approach substantially advances current human brain organoid approaches by incorporating microglial cells, as illustrated by the discovery of a key pathway of lipid-mediated crosstalk between microglia and NPCs that leads to improved neurogenesis.

Neutralizing antibodies with neurotropic factor treatment maintain neurodevelopmental gene expression upon exposure to human cytomegalovirus.

IMPORTANCE: Human cytomegalovirus (HCMV) infection is the leading cause of non-heritable birth defects worldwide. HCMV readily infects the early progenitor cell population of the developing brain, and we have found that infection leads to significantly downregulated expression of key neurodevelopmental transcripts. Currently, there are no approved therapies to prevent or mitigate the effects of congenital HCMV infection. Therefore, we used human-induced pluripotent stem cell-derived organoids and neural progenitor cells to elucidate the glycoproteins and receptors used in the viral entry process and whether antibody neutralization was sufficient to block viral entry and prevent disruption of neurodevelopmental gene expression. We found that blocking viral entry alone was insufficient to maintain the expression of key neurodevelopmental genes, but neutralization combined with neurotrophic factor treatment provided robust protection. Together, these studies offer novel insight into mechanisms of HCMV infection in neural tissues, which may aid future therapeutic development.

Laminin fragments conjugated with perlecan's growth factor-binding domain differentiate human induced pluripotent stem cells into skin-derived precursor cells.

Deriving stem cells to regenerate full-thickness human skin is important for treating skin disorders without invasive surgical procedures. Our previous protocol to differentiate human induced pluripotent stem cells (iPSCs) into skin-derived precursor cells (SKPs) as a source of dermal stem cells employs mouse fibroblasts as feeder cells and is therefore unsuitable for clinical use. Herein, we report a feeder-free method for differentiating iPSCs into SKPs by customising culture substrates. We immunohistochemically screened for laminins expressed in dermal papillae (DP) and explored the conditions for inducing the differentiation of iPSCs into SKPs on recombinant laminin E8 (LM-E8) fragments with or without conjugation to domain I of perlecan (PDI), which binds to growth factors through heparan sulphate chains. Several LM-E8 fragments, including those of LM111, 121, 332, 421, 511, and 521, supported iPSC differentiation into SKPs without PDI conjugation. However, the SKP yield was significantly enhanced on PDI-conjugated LM-E8 fragments. SKPs induced on PDI-conjugated LM111-E8 fragments retained the gene expression patterns characteristic of SKPs, as well as the ability to differentiate into adipocytes, osteocytes, and Schwann cells. Thus, PDI-conjugated LM-E8 fragments are promising agents for inducing iPSC differentiation into SKPs in clinical settings.

Transient naive reprogramming corrects hiPS cells functionally and epigenetically.

Cells undergo a major epigenome reconfiguration when reprogrammed to human induced pluripotent stem cells (hiPS cells). However, the epigenomes of hiPS cells and human embryonic stem (hES) cells differ significantly, which affects hiPS cell function1-8. These differences include epigenetic memory and aberrations that emerge during reprogramming, for which the mechanisms remain unknown. Here we characterized the persistence and emergence of these epigenetic differences by performing genome-wide DNA methylation profiling throughout primed and naive reprogramming of human somatic cells to hiPS cells. We found that reprogramming-induced epigenetic aberrations emerge midway through primed reprogramming, whereas DNA demethylation begins early in naive reprogramming. Using this knowledge, we developed a transient-naive-treatment (TNT) reprogramming strategy that emulates the embryonic epigenetic reset. We show that the epigenetic memory in hiPS cells is concentrated in cell of origin-dependent repressive chromatin marked by H3K9me3, lamin-B1 and aberrant CpH methylation. TNT reprogramming reconfigures these domains to a hES cell-like state and does not disrupt genomic imprinting. Using an isogenic system, we demonstrate that TNT reprogramming can correct the transposable element overexpression and differential gene expression seen in conventional hiPS cells, and that TNT-reprogrammed hiPS and hES cells show similar differentiation efficiencies. Moreover, TNT reprogramming enhances the differentiation of hiPS cells derived from multiple cell types. Thus, TNT reprogramming corrects epigenetic memory and aberrations, producing hiPS cells that are molecularly and functionally more similar to hES cells than conventional hiPS cells. We foresee TNT reprogramming becoming a new standard for biomedical and therapeutic applications and providing a novel system for studying epigenetic memory.

Co-transplantation of autologous Treg cells in a cell therapy for Parkinson's disease.

The specific loss of midbrain dopamine neurons (mDANs) causes major motor dysfunction in Parkinson's disease, which makes cell replacement a promising therapeutic approach1-4. However, poor survival of grafted mDANs remains an obstacle to successful clinical outcomes5-8. Here we show that the surgical procedure itself (referred to here as 'needle trauma') triggers a profound host response that is characterized by acute neuroinflammation, robust infiltration of peripheral immune cells and brain cell death. When midbrain dopamine (mDA) cells derived from human induced pluripotent stem (iPS) cells were transplanted into the rodent striatum, less than 10% of implanted tyrosine hydroxylase (TH)+ mDANs survived at two weeks after transplantation. By contrast, TH- grafted cells mostly survived. Notably, transplantation of autologous regulatory T (Treg) cells greatly modified the response to needle trauma, suppressing acute neuroinflammation and immune cell infiltration. Furthermore, intra-striatal co-transplantation of Treg cells and human-iPS-cell-derived mDA cells significantly protected grafted mDANs from needle-trauma-associated death and improved therapeutic outcomes in rodent models of Parkinson's disease with 6-hydroxydopamine lesions. Co-transplantation with Treg cells also suppressed the undesirable proliferation of TH- grafted cells, resulting in more compact grafts with a higher proportion and higher absolute numbers of TH+ neurons. Together, these data emphasize the importance of the initial inflammatory response to surgical injury in the differential survival of cellular components of the graft, and suggest that co-transplanting autologous Treg cells effectively reduces the needle-trauma-induced death of mDANs, providing a potential strategy to achieve better clinical outcomes for cell therapy in Parkinson's disease.

Generation of a human induced pluripotent stem cell line (UQi001-A-1) edited with the CRISPR-Cas9 system to carry the heterozygous TARDBP c.1144G > A (p.A382T) missense mutation.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease in which the TDP-43 protein is believed to play a central role in disease pathophysiology. Using the CRISPR-Cas9 system, we introduced the heterozygous c.1144G > A (p.A382T) missense mutation in exon 6 of the TARDBP gene into an iPSC line derived from a healthy individual. These edited iPSCs displayed normal cellular morphology, expressed major pluripotency markers, were capable of tri-lineage differentiation, and possessed a normal karyotype.