Neural Stem Cell Line Research Articles

Lipopolysaccharide-induced Autophagy Increases SOX2-positive Astrocytes While Decreasing Neuronal Differentiation in the Adult Hippocampus.

Inflammation alters the neural stem cell (NSC) lineage from neuronal to astrogliogenesis. However, the underlying mechanism is elusive. Autophagy contributes to the decline in adult hippocampal neurogenesis under E. coli lipopolysaccharide (LPS) stimulation. SRY-box transcription Factor 2 (SOX2) is critical for NSC self-renewal and proliferation. In this study, we investigated the role of SOX2 in induced autophagy and hippocampal adult neurogenesis under LPS stimulation. LPS (5 ng•100 g-1•hour-1 for 7 days) was intraperitoneally infused into male Sprague-Dawley rats (8 weeks old) to induce mild systemic inflammation. Beclin 1 and autophagy protein 12 (Atg12) were significantly upregulated concurrent with decreased numbers of Ki67- and doublecortin (DCX)-positive cells in the dentate gyrus. Synchronically, the levels of phospho(p)-mTOR, the p-mTOR/mTOR ratio, p-P85s6k, and the p-P85s6k/P85s6k ratio were suppressed. In contrast, SOX2 expression was increased. The fluorescence micrographs indicated that the colocalization of Beclin 1 and SOX2 was increased in the subgranular zone (SGZ) of the dentate gyrus. Moreover, increased S100β-positive astrocytes were colocalized with SOX2 in the SGZ. Intracerebroventricular infusion of 3-methyladenine (an autophagy inhibitor) effectively prevented the increases in Beclin 1, Atg12, and SOX2. The SOX2+-Beclin 1+ and SOX2+-S100β+ cells were reduced. The levels of p-mTOR and p-P85s6k were enhanced. Most importantly, the number of DCX-positive cells was preserved. Altogether, these data suggest that LPS induced autophagy to inactivate the mTOR/P85s6k pathway, resulting in a decline in neural differentiation. SOX2 was upregulated to facilitate the NSC lineage, while the autophagy milieu could switch the SOX2-induced NSC lineage from neurogenesis to astrogliogenesis.

Establishment and characterization of a cell line from the brain of the Japanese flounder (Paralichthys olivaceus) and its application in the study of viral infection

Fish cell lines are important tools in many biological studies. Here, we established a new Japanese flounder brain (JFB) cell line from the brain tissue of the Japanese flounder (Paralichthys olivaceus), which was successfully sub-cultured for over 120 passages, and consisted predominantly of fibroblast-like cells. Sequencing of the actin gene confirmed that JFB cells were derived from the Japanese flounder, and JFB was also identified as a neural stem cell line on the basis of the mRNA expression of nestin gene. The JFB cells could grow at a temperature ranging from 17 to 29 °C, and 23 °C was the optimum temperature. Karyotype analysis showed that the chromosome mode number was 48, demonstrating a normal diploid chromosome number. The transfection efficiency of pEGFP-N1 in JFB cells was up to 30%. The JFB cells showed susceptibility to Hirame rhabdovirus (HIRRV), Lymphocystis disease virus (LCDV), Bohle virus (BIV) and Chinese giant salamander iridovirus (GSIV), which was demonstrated by varying degrees of cytopathic effects. Viral infection or Poly (I:C) stimulation upregulated the expression of multiple immune-related genes, including IL-1β, IL8, Mx and TNF. These results showed that the newly established JFB cell line is an ideal tool for studying gene manipulation, host-virus interaction and potential vaccines development.

GD2-targeting CAR-T cells enhanced by transgenic IL-15 expression are an effective and clinically feasible therapy for glioblastoma

BackgroundAggressive primary brain tumors such as glioblastoma are uniquely challenging to treat. The intracranial location poses barriers to therapy, and the potential for severe toxicity. Effective treatments for primary brain...

Imp and Syp mediated temporal patterning of neural stem cells in the developing Drosophila CNS.

The assembly of complex neural circuits requires that stem cells generate diverse types of neurons in the correct temporal order. Pioneering work in the Drosophila embryonic ventral nerve cord has shown that neural stem cells are temporally patterned by the sequential expression of rapidly changing transcription factors to generate diversity in their progeny. In recent years, a second temporal patterning mechanism, driven by the opposing gradients of the Imp and Syp RNA-binding proteins, has emerged as a powerful way to generate neural diversity. This long-range temporal patterning mechanism is utilized in the extended neural stem cell lineages of the postembryonic fly brain. Here, we review the role played by Imp and Syp gradients in several neural stem cell lineages, focusing on how they specify sequential neural fates through the post-transcriptional regulation of target genes, including the Chinmo and Mamo transcription factors. We further discuss how upstream inputs, including hormonal signals, modify the output of these gradients to couple neurogenesis with the development of the organism. Finally, we review the roles that the Imp and Syp gradients play beyond the generation of diversity, including the regulation of stem cell proliferation, the timing of neural stem cell lineage termination, and the coupling of neuronal birth order to circuit assembly.

Targeting 17β-estradiol biosynthesis in neural stem cells improves stroke outcome.

Dax-1 (dosage-sensitive sex reversal adrenal hypoplasia congenital region on X-chromosome gene 1) blocks 17β-estradiol biosynthesis and its knockdown would be expected to increase 17β-estradiol production. We hypothesized that knockdown of Dax-1 in a conditionally immortalized neural stem cell (NSC) line, MHP36, is a useful approach to increase 17β-estradiol production. Short hairpin (sh) RNA targeted to Dax-1 in NSCs, namely MHP36-Dax1KD cells, resulted in the degradation of Dax-1 RNA and attenuation of Dax-1 protein expression. In vitro, MHP36-Dax1KD cells exhibited overexpression of aromatase and increased 17β-estradiol secretion compared to MHP36 cells. As 17β-estradiol has been shown to promote the efficacy of cell therapy, we interrogated the application of 17β-estradiol-enriched NSCs in a relevant in vivo disease model. We hypothesized that MHP36-Dax1KD cells will enhance functional recovery after transplantation in a stroke model. C57BL/6 male adult mice underwent ischemia/reperfusion by left middle cerebral artery occlusion for 45 min using an intraluminal thread. Two days later male mice randomly received vehicle, MHP36 cells, MHP36-Dax1KD cells, and MHP36 cells suspended in 17β-estradiol (100 nm) or 17β-estradiol alone (100 nm) with serial behavioral testing over 28 days followed by post-mortem histology and blinded analysis. Recovery of sensorimotor function was accelerated and enhanced, and lesion volume was reduced by MHP36-Dax1KD transplants. Regarding mechanisms, immunofluorescence indicated increased synaptic plasticity and neuronal differentiation after MHP36-Dax1KD transplants. In conclusion, knockdown of Dax-1 is a useful target to increase 17β-estradiol biosynthesis in NSCs and improves functional recovery after stroke in vivo, possibly mediated through neuroprotection and improved synaptic plasticity. Therefore, targeting 17β-estradiol biosynthesis in stem cells may be a promising therapeutic strategy for enhancing the efficacy of stem cell-based therapies for stroke.

Neuronal Cells Display Distinct Stability Controls of Alternative Polyadenylation mRNA Isoforms, Long Non-Coding RNAs, and Mitochondrial RNAs.

RNA stability plays an important role in gene expression. Here, using 3′ end sequencing of newly made and pre-existing poly(A)+ RNAs, we compare transcript stability in multiple human cell lines, including HEK293T, HepG2, and SH-SY5Y. We show that while mRNA stability is generally conserved across the cell lines, specific transcripts having a high GC content and possibly more stable secondary RNA structures are relatively more stable in SH-SY5Y cells compared to the other 2 cell lines. These features also differentiate stability levels of alternative polyadenylation (APA) 3′UTR isoforms in a cell type-specific manner. Using differentiation of a neural stem cell line as a model, we show that mRNA stability difference could contribute to gene expression changes in neurogenesis and confirm the neuronal identity of SH-SY5Y cells at both gene expression and APA levels. In addition, compared to transcripts using 3′-most exon cleavage/polyadenylation sites (PASs), those using intronic PASs are generally less stable, especially when the PAS-containing intron is large and has a strong 5′ splice site, suggesting that intronic polyadenylation mostly plays a negative role in gene expression. Interestingly, the differential mRNA stability among APA isoforms appears to buffer PAS choice in these cell lines. Moreover, we found that several other poly(A)+ RNA species, including promoter-associated long noncoding RNAs and transcripts encoded by the mitochondrial genome, are more stable in SH-SY5Y cells than the other 2 cell lines, further highlighting distinct RNA metabolism in neuronal cells. Together, our results indicate that distinct RNA stability control in neuronal cells may contribute to the gene expression and APA programs that define their cell identity.

Glial-secreted Netrins regulate Robo1/Rac1-Cdc42 signaling threshold levels during Drosophila asymmetric neural stem/progenitor cell division

Asymmetric stem cell division (ASCD) is a key mechanism in development, cancer, and stem cell biology. Drosophila neural stem cells, called neuroblasts (NBs), divide asymmetrically through intrinsic mechanisms. Here, we show that the extrinsic axon guidance cues Netrins, secreted by a glial niche surrounding larval brain neural stem cell lineages, regulate NB ASCD. Netrin-Frazzled/DCC signaling modulates, through Abelson kinase, Robo1 signaling threshold levels in Drosophila larval brain neural stem and progenitor cells of NBII lineages. Unbalanced Robo1 signaling levels induce ectopic NBs and progenitor cells due to failures in the ASCD process. Mechanistically, Robo1 signaling directly impinges on the intrinsic ASCD machinery, such as aPKC, Canoe/Afadin, and Numb, through the small GTPases Rac1 and Cdc42, which are required for the localization in mitotic NBs of Par-6, a Cdc42 physical partner and a core component of the Par (Par-6-aPKC-Par3/Bazooka) apical complex.

Egr1 is a 3D matrix-specific mediator of mechanosensitive stem cell lineage commitment.

While extracellular matrix (ECM) mechanics strongly regulate stem cell commitment, the field’s mechanistic understanding of this phenomenon largely derives from simplified two-dimensional (2D) culture substrates. Here, we found a 3D matrix–specific mechanoresponsive mechanism for neural stem cell (NSC) differentiation. NSC lineage commitment in 3D is maximally stiffness sensitive in the range of 0.1 to 1.2 kPa, a narrower and more brain-mimetic range than we had previously identified in 2D (0.75 to 75 kPa). Transcriptomics revealed stiffness-dependent up-regulation of early growth response 1 (Egr1) in 3D but not in 2D. Egr1 knockdown enhanced neurogenesis in stiff ECMs by driving β-catenin nuclear localization and activity in 3D, but not in 2D. Mechanical modeling and experimental studies under osmotic pressure indicate that stiff 3D ECMs are likely to stimulate Egr1 via increases in confining stress during cell volumetric growth. To our knowledge, Egr1 represents the first 3D-specific stem cell mechanoregulatory factor.

Deciphering heterogeneous populations of migrating cells based on the computational assessment of their dynamic properties.

SummaryNeuronal migration is a highly dynamic process, and multiple cell movement metrics can be extracted from time-lapse imaging datasets. However, these parameters alone are often insufficient to evaluate the heterogeneity of neuroblast populations. We developed an analytical pipeline based on reducing the dimensions of the dataset by principal component analysis (PCA) and determining sub-populations using k-means, supported by the elbow criterion method and validated by a decision tree algorithm. We showed that neuroblasts derived from the same adult neural stem cell (NSC) lineage as well as across different lineages are heterogeneous and can be sub-divided into different clusters based on their dynamic properties. Interestingly, we also observed overlapping clusters for neuroblasts derived from different NSC lineages. We further showed that genetic perturbations or environmental stimuli affect the migratory properties of neuroblasts in a sub-cluster-specific manner. Our data thus provide a framework for assessing the heterogeneity of migrating neuroblasts.

Characterisation of ReNcells CX and VM stimulated with interleukin-1β and lipopolysaccharide

Abnormal and dysregulated neuroinflammation has been linked to many neurological disorders and neurodegenerative diseases. Understanding the mechanisms of neuroinflammation, their impact on neurodevelopment and how neuroinflammation might be modulated, are currently considered to be critical to improving neurological treatment. ReNcell CX (originating from the cortical region) and VM (originating from the ventral mesencephalon) are human immortalised neural stem cell lines, that have the potential to be used as experimental models for investigating neuroinflammation in vitro. However, the information on the inflammation response of these cells is limited. This is especially more so for undifferentiated ReNcells. In this report we demonstrate using ELISA that cultured, undifferentiated ReNcell CX and VM produce significant amounts of IL-6 in response to IL-1β treatment, but not to LPS treatment. Additionally, conventional RT-PCR showed that ReNcell CX cells expressed TNFR1 and NF-κB, whereas ReNcell VM expressed only NF-κB. Our results encourage further investigation into the relationship between 1L-1β and IL-6 in both ReNcell CX and VM. Moreover, TNF-α treatment might potentially affect neuroinflammation in ReNcell CX, while activation of the NF-κB pathway could also play a critical part in neuroinflammation.

179 Clinical Outcomes From a Single-Site Phase I Study of Neural Stem Cell Transplantation for Chronic Spinal Cord Injury

INTRODUCTION: We previously reported safe implantation and improvement in motor and sensory scores using neural stem cell line (NSC) NSI-566 in chronic thoracic complete spinal cord injury (SCI) patients. METHODS: A total of four subjects received NSI-566 spinal cord implantation. All subjects tolerated the procedure well with no serious adverse events in the immediate post-procedure period. Prospective data have been collected including ISNCSCI scores, functional and pain surveys, SCIM scores, electromyography (EMG), Brain Motor Control Assessment (BMCA), and serial MRI. RESULTS: Two patients experienced improvements in neurological level of injury (NLI), motor score, and sensory score. BMCA showed newly developed muscle responsiveness in lower limbs to reinforcement maneuvers in one patient. Three subjects experienced new EMG activity in lower muscle groups. Two patients had overall decreased pain scores post-operatively, which included both discomfort and allodynia in the transition zone, surgical site pain, and neuropathic and nociceptive pain elsewhere. MR imaging showed no radiographical evidence of immediate or delayed complications after the stem cell injections. CONCLUSION: Our current clinical data demonstrate that spinal grafting of the human NSI-566 cell line in chronic spinal trauma patients is safe with no detectable side effects identified at 60 months after cell delivery. Despite the small sample size, the key strengths of the study are the extensive follow-up period and the timeline of treatment. Like other Phase I studies, this study was intended to provide proof of safety and tolerability and proof of concept data that will justify the next cohort.

Intravitreal gene therapy restores the autophagy-lysosomal pathway and attenuates retinal degeneration in cathepsin D-deficient mice

Loss of vision due to progressive retinal degeneration is a hallmark of neuronal ceroid lipofuscinoses (NCL), a group of fatal neurodegenerative lysosomal storage diseases. Enzyme substitution therapies represent promising treatment options for NCLs caused by dysfunctions of soluble lysosomal enzymes. Here, we compared the efficacy of a cell-based enzyme substitution strategy and a gene therapy approach to attenuate the retinal pathology in cathepsin D- (CTSD) deficient mice, an animal model of CLN10 disease. Levels of enzymatically active CTSD in mutant retinas were significantly higher after an adeno-associated virus vector-mediated CTSD transfer to retinal glial cells and retinal pigment epithelial cells than after intravitreal transplantations of a CTSD overexpressing clonal neural stem cell line. In line with this finding, the gene therapy treatment restored the disrupted autophagy-lysosomal pathway more effectively than the cell-based approach, as indicated by a complete clearance of storage, significant attenuation of lysosomal hypertrophy, and normalized levels of the autophagy marker sequestosome 1/p62 and microtubule-associated protein 1 light chain 3-II. While the cell-based treatment did not prevent the rapidly progressing loss of various retinal cell types, the gene therapy approach markedly attenuated retinal degeneration as demonstrated by a pronounced rescue of photoreceptor cells and rod bipolar cells.

ADAM10 facilitates rapid neural stem cell cycling and proper positioning within the subventricular zone niche via JAMC/RAP1Gap signaling.

The mechanisms that regulate neural stem cell (NSC) lineage progression and maintain NSCs within different domains of the adult neural stem cell niche, the subventricular zone are not well defined. Quiescent NSCs are arranged at the apical ventricular wall, while mitotically activated NSCs are found in the basal, vascular region of the subventricular zone. Here, we found that ADAM10 (a disintegrin and metalloproteinase 10) is essential in NSC association with the ventricular wall, and via this adhesion to the apical domain, ADAM10 regulates the switch from quiescent and undifferentiated NSC to an actively proliferative and differentiating cell state. Processing of JAMC (junctional adhesion molecule C) by ADAM10 increases Rap1GAP activity. This molecular machinery promotes NSC transit from the apical to the basal compartment and subsequent lineage progression. Understanding the molecular mechanisms responsible for regulating the proper positioning of NSCs within the subventricular zone niche and lineage progression of NSCs could provide new targets for drug development to enhance the regenerative properties of neural tissue.

N-Cadherin adhesive ligation regulates mechanosensitive neural stem cell lineage commitment in 3D matrices.

During differentiation, neural stem cells (NSCs) encounter diverse cues from their niche, including not only biophysical cues from the extracellular matrix (ECM) but also cell-cell communication. However, it is still poorly understood how these cues cumulatively regulate mechanosensitive NSC fate commitment, especially in 3D matrices that better mimic in vivo systems. Here, we develop a click chemistry-based 3D hydrogel material system to fully decouple cell-cell and cell-ECM interactions by functionalizing small peptides: the HAVDI motif from N-cadherin and RGD motif from fibronectin. The hydrogel is engineered to range in stiffness from 75 Pa to 600 Pa. Interestingly, HAVDI-mediated interaction shows increased neurogenesis, except for the softest gel (75 Pa). Moreover, the HAVDI ligation attenuates the mechanosensing state of NSCs, exhibiting restricted cytoskeletal formation and RhoA signaling. Given that mechanosensitive neurogenesis has been reported to be regulated by cytoskeletal formation, our finding suggests that the enhanced neurogenesis in the HAVDI-modified gel may be highly associated with the HAVDI interaction-mediated attenuation of mechanosensing. Furthermore, NSCs in the HAVDI gel shows higher β-catenin activity, which has been known to promote neurogenesis. Our findings provide critical insights into how mechanosensitive NSC fate commitment is regulated as a consequence of diverse interactions in 3D microenvironments.

Regulation of Neural Stem Cell Competency and Commitment during Indirect Neurogenesis.

Indirect neurogenesis, during which neural stem cells generate neurons through intermediate progenitors, drives the evolution of lissencephalic brains to gyrencephalic brains. The mechanisms that specify intermediate progenitor identity and that regulate stem cell competency to generate intermediate progenitors remain poorly understood despite their roles in indirect neurogenesis. Well-characterized lineage hierarchy and available powerful genetic tools for manipulating gene functions make fruit fly neural stem cell (neuroblast) lineages an excellent in vivo paradigm for investigating the mechanisms that regulate neurogenesis. Type II neuroblasts in fly larval brains repeatedly undergo asymmetric divisions to generate intermediate neural progenitors (INPs) that undergo limited proliferation to increase the number of neurons generated per stem cell division. Here, we review key regulatory genes and the mechanisms by which they promote the specification and generation of INPs, safeguarding the indirect generation of neurons during fly larval brain neurogenesis. Homologs of these regulators of INPs have been shown to play important roles in regulating brain development in vertebrates. Insight into the precise regulation of intermediate progenitors will likely improve our understanding of the control of indirect neurogenesis during brain development and brain evolution.

Physical therapy exerts sub-additive and suppressive effects on intracerebral neural stem cell implantation in a rat model of stroke

Intracerebral cell therapy (CT) is emerging as a new therapeutic paradigm for stroke. However, the impact of physical therapy (PT) on implanted cells and their ability to promote recovery remains poorly understood. To address this translational issue, a clinical-grade neural stem cell (NSC) line was implanted into peri-infarct tissue using MRI-defined injection sites, two weeks after stroke. PT in the form of aerobic exercise (AE) was administered 5 × per week post-implantation using a paradigm commonly applied in patients with stroke. A combined AE and CT exerted sub-additive therapeutic effects on sensory neglect, whereas AE suppressed CT effects on motor integration and grip strength. Behavioral testing emerged as a potentially major component for task integration. It is expected that this study will guide and inform the incorporation of PT in the design of clinical trials evaluating intraparenchymal NSCs implantation for stroke.

Pilot RNAi Screen in Drosophila Neural Stem Cell Lineages to Identify Novel Tumor Suppressor Genes Involved in Asymmetric Cell Division.

A connection between compromised asymmetric cell division (ACD) and tumorigenesis was proven some years ago using Drosophila larval brain neural stem cells, called neuroblasts (NBs), as a model system. Since then, we have learned that compromised ACD does not always promote tumorigenesis, as ACD is an extremely well-regulated process in which redundancy substantially overcomes potential ACD failures. Considering this, we have performed a pilot RNAi screen in Drosophila larval brain NB lineages using RasV12 scribble (scrib) mutant clones as a sensitized genetic background, in which ACD is affected but does not cause tumoral growth. First, as a proof of concept, we have tested known ACD regulators in this sensitized background, such as lethal (2) giant larvae and warts. Although the downregulation of these ACD modulators in NB clones does not induce tumorigenesis, their downregulation along with RasV12 scrib does cause tumor-like overgrowth. Based on these results, we have randomly screened 79 RNAi lines detecting 15 potential novel ACD regulators/tumor suppressor genes. We conclude that RasV12 scrib is a good sensitized genetic background in which to identify tumor suppressor genes involved in NB ACD, whose function could otherwise be masked by the high redundancy of the ACD process.

Culturing and Expansion of "Clinical Grade" Neural Stem Cells from the Fetal Human Central Nervous System.

NSCs have been demonstrated to be very useful in grafts into the mammalian central nervous system to investigate the exploitation of NSC for the therapy of neurodegenerative disorders in animal models of neurodegenerative diseases. To push cell therapy in CNS on stage of clinical application, it is necessary to establish a continuous and standardized, clinical grade (i.e., produced following the good manufacturing practice guidelines) human neural stem cell lines.In this chapter we will illustrate some of the protocols for the production and characterization routinely used into our GMP "cell factory" for the production of "clinical grade" human neural stem cell lines already in use in clinical trials on neurodegenerative diseases, particularly amyotrophic lateral sclerosis (ALS- Clinicaltrials.gov number NCT01640067) and secondary progressive multiple sclerosis (SPMS- Clinicaltrials.gov number NCT03282760).

Targeted mass spectrometry for monitoring of neural differentiation.

ABSTRACTHuman multipotent neural stem cells could effectively be used for the treatment of a variety of neurological disorders. However, a defining signature of neural stem cell lines that would be expandable, non-tumorigenic, and differentiate into desirable neuronal/glial phenotype after in vivo grafting is not yet defined. Employing a mass spectrometry approach, based on selected reaction monitoring, we tested a panel of well-described culture conditions, and measured levels of protein markers routinely used to probe neural differentiation, i.e. POU5F1 (OCT4), SOX2, NES, DCX, TUBB3, MAP2, S100B, GFAP, GALC, and OLIG1. Our multiplexed assay enabled us to simultaneously identify the presence of pluripotent, multipotent, and lineage-committed neural cells, thus representing a powerful tool to optimize novel and highly specific propagation and differentiation protocols. The multiplexing capacity of this method permits the addition of other newly identified cell type-specific markers to further increase the specificity and quantitative accuracy in detecting targeted cell populations. Such an expandable assay may gain the advantage over traditional antibody-based assays, and represents a method of choice for quality control of neural stem cell lines intended for clinical use.

HiPSC Modeling of Lineage-Specific Smooth Muscle Cell Defects Caused by TGFBR1A230T Variant, and Its Therapeutic Implications for Loeys-Dietz Syndrome.

Loeys-Dietz syndrome (LDS) is an inherited disorder predisposing individuals to thoracic aortic aneurysm and dissection. Currently, there are no medical treatments except surgical resection. Although the genetic basis of LDS is well-understood, molecular mechanisms underlying the disease remain elusive, impeding the development of a therapeutic strategy. In addition, aortic smooth muscle cells (SMCs) have heterogenous embryonic origins, depending on their spatial location, and lineage-specific effects of pathogenic variants on SMC function, likely causing regionally constrained LDS manifestations, have been unexplored. We identified an LDS family with a dominant pathogenic variant in the TGFBR1 gene (TGFBR1A230T) causing aortic root aneurysm and dissection. To accurately model the molecular defects caused by this mutation, we used human induced pluripotent stem cells from a subject with normal aorta to generate human induced pluripotent stem cells carrying TGFBR1A230T, and corrected the mutation in patient-derived human induced pluripotent stem cells using CRISPR-Cas9 gene editing. After their lineage-specific SMC differentiation through cardiovascular progenitor cell (CPC) and neural crest stem cell lineages, we used conventional molecular techniques and single-cell RNA sequencing to characterize the molecular defects. The resulting data led to subsequent molecular and functional rescue experiments using activin A and rapamycin. Our results indicate the TGFBR1A230T mutation impairs contractile transcript and protein levels, and function in CPC-SMC, but not in neural crest stem cell-SMC. Single-cell RNA sequencing results implicate defective differentiation even in TGFBR1A230T/+ CPC-SMC including disruption of SMC contraction and extracellular matrix formation. Comparison of patient-derived and mutation-corrected cells supported the contractile phenotype observed in the mutant CPC-SMC. TGFBR1A230T selectively disrupted SMAD3 (SMAD family member 3) and AKT (AKT serine/threonine kinase) activation in CPC-SMC, and led to increased cell proliferation. Consistently, single-cell RNA sequencing revealed molecular similarities between a loss-of-function SMAD3 mutation (SMAD3c.652delA/+) and TGFBR1A230T/+. Last, combination treatment with activin A and rapamycin during or after SMC differentiation significantly improved the mutant CPC-SMC contractile gene expression and function, and rescued the mechanical properties of mutant CPC-SMC tissue constructs. This study reveals that a pathogenic TGFBR1 variant causes lineage-specific SMC defects informing the etiology of LDS-associated aortic root aneurysm. As a potential pharmacological strategy, our results highlight a combination treatment with activin A and rapamycin that can rescue the SMC defects caused by the variant.