VEGF-FGF Signaling Activates Quiescent CD63+ Liver Stem Cells to Proliferate and Differentiate.

Understanding the liver stem cells (LSCs) holds great promise for new insights into liver diseases and liver regeneration. However, the heterogenicity and plasticity of liver cells have made it controversial. Here, by employing single-cell RNA-sequencing technology, transcriptome features of Krt19+ bile duct lineage cells isolated from Krt19CreERT; Rosa26R-GFP reporter mouse livers are examined. Distinct biliary epithelial cells which include adult LSCs, as well as their downstream hepatocytes and cholangiocytes are identified. Importantly, a novel cell surface LSCs marker, CD63, as well as CD56, which distinguished active and quiescent LSCs are discovered. Cell expansion and bi-potential differentiation in culture demonstrate the stemness ability of CD63+ cells in vitro. Transplantation and lineage tracing of CD63+ cells confirm their contribution to liver cell mass in vivo upon injury. Moreover, CD63+CD56+ cells are proved to be activated LSCs with vigorous proliferation ability. Further studies confirm that CD63+CD56- quiescent LSCs express VEGFR2 and FGFR1, and they can be activated to proliferation and differentiation through combination of growth factors: VEGF-A and bFGF. These findings define an authentic adult liver stem cells compartment, make a further understanding of fate regulation on LSCs, and highlight its contribution to liver during pathophysiologic processes.

Impaired thymic iNKT cell differentiation at early precursor stage in murine haploidentical bone marrow transplantation with GvHD.

Introduction: Early recovery of donor-derived invariant natural killer T (iNKT) cells are associated with reduced risk of graft-versus-host disease (GvHD) and overall survival. Patients with severe GvHD, however, had much slower iNKT cell reconstitution relative to conventional T cells. Methods: To characterize the delay of iNKT cell reconstitution and explore its possible causes, we used a haploidentical bone marrow transplantation (haplo-BMT) mouse model with GvHD. We found the delayed recovery of thymic and peripheral iNKT cell numbers with markedly decreased thymic NKT1 subset in GvHD mice. The defective generation of thymic iNKT precursors with egress capability contributed to the reduced peripheral iNKT cells in GvHD mice. We further identified intermediate NK1.1- NKT1 precursor subpopulations under steady-state conditions and found that the differentiation of these subpopulations was impaired in the thymi of GvHD mice. Detailed characterization of iNKT precursors and thymic microenvironment showed a close association of elevated TCR/co-stimulatory signaling provided by double positive thymocytes and macrophages with defective down-regulation of proliferation, metabolism, and NKT2 signature in iNKT precursor cells. Correspondingly, NKT2 but not NKT1 differentiation was favored in GvHD mice. Discussion: These data underline the important roles of TCR and co-stimulatory signaling in the differentiation of thymic iNKT subsets under transplantation conditions.

Transcriptional Profiling Reveals Brain Region-Specific Gene Networks Regulated in Exercise in a Mouse Model of Parkinson's Disease.

Background: Exercise plays an essential role in improving motor symptoms in Parkinson's disease (PD), but the underlying mechanism in the central nervous system remains unclear. Methods: Motor ability was observed after 12-week treadmill exercise on a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mouse model of PD. RNA-sequencing on four brain regions (cerebellum, cortex, substantia nigra (SN), and striatum) from control animals, MPTP-induced PD, and MPTP-induced PD model treated with exercise for 12 weeks were performed. Transcriptional networks on the four regions were further identified by an integrative network biology approach. Results: The 12-week treadmill exercise significantly improved the motor ability of an MPTP-induced mouse model of PD. RNA-seq analysis showed SN and striatum were remarkably different among individual region's response to exercise in the PD model. Especially, synaptic regulation pathways about axon guidance, synapse assembly, neurogenesis, synaptogenesis, transmitter transport-related pathway, and synaptic regulation genes, including Neurod2, Rtn4rl2, and Cd5, were upregulated in SN and striatum. Lastly, immunofluorescence staining revealed that exercise rescued the loss of TH+ synapses in the striatal region in PD mice, which validates the key role of synaptic regulation pathways in exercise-induced protective effects in vivo. Conclusion: SN and striatum are important brain regions in which critical transcriptional changes, such as in synaptic regulation pathways, occur after the exercise intervention on the PD model.

Temporal and spatial cellular and molecular pathological alterations with single-cell resolution in the adult spinal cord after injury.

Spinal cord injury (SCI) involves diverse injury responses in different cell types in a temporally and spatially specific manner. Here, using single-cell transcriptomic analyses combined with classic anatomical, behavioral, electrophysiological analyses, we report, with single-cell resolution, temporal molecular and cellular changes in crush-injured adult mouse spinal cord. Data revealed pathological changes of 12 different major cell types, three of which infiltrated into the spinal cord at distinct times post-injury. We discovered novel microglia and astrocyte subtypes in the uninjured spinal cord, and their dynamic conversions into additional stage-specific subtypes/states. Most dynamic changes occur at 3-days post-injury and by day-14 the second wave of microglial activation emerged, accompanied with changes in various cell types including neurons, indicative of the second round of attacks. By day-38, major cell types are still substantially deviated from uninjured states, demonstrating prolonged alterations. This study provides a comprehensive mapping of cellular/molecular pathological changes along the temporal axis after SCI, which may facilitate the development of novel therapeutic strategies, including those targeting microglia.

Transcriptome sequencing reveals Gastrodia elata Blume could increase the cell viability of eNPCs under hypoxic condition by improving DNA damage repair ability.

ETHNOPHARMACOLOGICAL RELEVANCE: Gastrodia elata Blume (GEB), known as Tianma in China, is a traditional medicinal herb that has been reported to have various pharmacological effects and neuroprotection, has long been used for treating dizziness, epilepsy, stroke. However, explanation of its underlying mechanisms remains a great challenge. AIM OF THE STUDY: The neuroprotective mechanism of GEB on hypoxia-induced neuronal injury in cultured mouse embryonic neural progenitor cells (eNPCs) was investigated, with emphasis on the eNPCs proliferation and DNA damage repair. MATERIALS AND METHODS: In this study, hypoxia was focused, which may be caused by stroke or acute cerebral ischemia and is considered as one of the important factors contributing to the Central Nervous System diseases. CoCl2 was adopted to construct a hypoxic/ischemic condition in eNPCs. eNPCs proliferation analysis validated GEB neuroprotective effect under hypoxic/ischemic condition. Transcriptome and weighted gene co-expression network analysis (WGCNA) screened the special gene-network module correlated with what appeared to have significant positive correlation with GEB. Then, Gene ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were performed to explore the biological functions of selected genes in the modules that had high correlation with GEB. RESULTS: GEB has neuroprotective effect and could rescue eNPCs proliferation under hypoxic/ischemic condition induced by CoCl2. Transcriptome and WGCNA unveil the neuroprotective mechanism of GEB on improving DNA damage repair ability by increasing the expression of genes associated with DNA repair and replication. Western blotting and qPCR showed that GEB could improve DNA damage repair ability by increasing the expression of Mcm2, Mcm6, Pold2, Pole, Pole2, Rfc1, Pole4, Dna2 and Rpa2, which were associated with DNA damage and replication. CONCLUSION: Through transcriptome and WGCNA, this study unveiled Gastrodia elata Blume could increase the cell viability of eNPCs under hypoxic condition by improving DNA damage repair ability.

Eradication of specific donor-dependent variations of mesenchymal stem cells in immunomodulation to enhance therapeutic values.

Mesenchymal stem cells (MSCs) are one of the most widely clinically trialed stem cells, due to their abilities to differentiate into multiple cell lineages, to secrete regenerative/rejuvenative factors, and to modulate immune functions, among others. In this study, we analyzed human umbilical-cord-derived MSCs from 32 donors and revealed donor-dependent variations in two non-correlated properties, (1) cell proliferation, and (2) immune modulatory functions in vitro and in vivo, which might explain inconsistent clinical efficacies of MSCs. Through unbiased transcriptomic analyses, we discovered that IFN-γ and NF-κB signaling were positively associated with immune modulatory function of MSCs. Activation of these two pathways via IFN-γ and TNF-α treatment eradicated donor-dependent variations. Additional transcriptomic analyses revealed that treatment with these two factors, while having abolished donor-dependent variations in immune modulatory function, did not overall make different donor-derived MSCs the same at whole transcriptomic levels, demonstrating that the cells were still different in many other biological perspectives, and may not perform equally for therapeutic purposes other than immune modulation. Pre-selection or pre-treatment to eradicate MSC variations in a disease-treatment-specific manner would therefore be necessary to ensure clinical efficacies. Together this study provided novel insights into the quality control perspective of using different-donor-derived MSCs to treat inflammation-related clinical conditions and/or autoimmune diseases.

Pten is a key intrinsic factor regulating raphe 5-HT neuronal plasticity and depressive behaviors in mice.

Serotonin (5-HT)-based antidepressants, selective serotonin reuptake inhibitors (SSRIs) aim to enhance serotonergic activity by blocking its reuptake. We propose PTEN as a target for an alternative approach for regulating 5-HT neuron activity in the brain and depressive behaviors. We show that PTEN is elevated in central 5-HT neurons in the raphe nucleus by chronic stress in mice, and selective deletion of Pten in the 5-HT neurons induces its structural plasticity shown by increases of dendritic branching and density of PSD95-positive puncta in the dendrites. 5-HT levels are elevated and electrical stimulation of raphe neurons evokes more 5-HT release in the brain of condition knockout (cKO) mice with Pten-deficient 5-HT neurons. In addition, the 5-HT neurons remain normal electrophysiological properties but have increased excitatory synaptic inputs. Single-cell RNA sequencing revealed gene transcript alterations that may underlay morphological and functional changes in Pten-deficient 5-HT neurons. Finally, Pten cKO mice and wild-type mice treated with systemic application of PTEN inhibitor display reduced depression-like behaviors. Thus, PTEN is an intrinsic regulator of 5-HT neuron activity, representing a novel therapeutic strategy for producing antidepressant action.

Transcriptional heterogeneity of clonal plasma cells and immune evasion in immunoglobulin light chain amyloidosis.

Immunoglobulin light chain amyloidosis (AL amyloidosis) is characterized by the presence of B cells producing amyloidogenic immunoglobulin light chains (LCs). The low frequency of aberrant B cells in AL is often masked by a polyclonal B cell background, making it difficult for treatment. We analyzed the single-cell RNA sequencing data from GEO database to compare the plasma cell (PCs) in four individuals with AL amyloidosis, one AL subject after treatment, and six healthy controls. High interindividual variability in AL-derived PCs in their expression pattern of known overexpressed genes in multiple myeloma and their usage of V regions in LCs was demonstrated. We also found overexpression of MHC class I molecules as one of the common features of clonal PCs in individuals with AL amyloidosis. Significantly reduced frequencies of circulating natural killer (NK) cells were also observed in a small cohort of AL patients when compared to healthy controls. These data demonstrate that aberrant PCs in AL has a highly diverse transcriptome, an upregulation of MHC, and a dampened capability of immunosurveillance by reduction of circulating NK frequencies. The analysis of clonal PCs at single cell level may provide a better approach for precise molecular profiling and diagnosis of AL amyloidosis.

A wave of Foxp3+ regulatory T cell accumulation in the neonatal liver plays unique roles in maintaining self-tolerance.

Newborn animals require tightly regulated local and systemic immune environments to govern the development and maturation of multiple organs/tissues even though the immune system itself is far from mature during the neonatal period. Regulatory T cells (Tregs) are essential for maintaining immune tolerance/homeostasis and modulating inflammatory responses. The features of Tregs in the neonatal liver under steady-state conditions are not well understood. The present study aimed to investigate the phenotype, functions, and significance of neonatal Tregs in the liver. We found a wave of thymus-derived Treg influx into the liver during 1-2 weeks of age. Depletion of these Tregs between days 7 and 11 after birth rapidly resulted in Th1-type liver inflammation and metabolic disorder. More Tregs in the neonatal liver than in the spleen underwent MHC II-dependent activation and proliferation, and the liver Tregs acquired stronger suppressive functions. The transcriptomic profile of these neonatal liver Tregs showed elevated expression of PPARγ and T-bet and features of Tregs that utilize lipid metabolic machinery and are capable of regulating Th1 responses. The accumulation of Tregs with unique features in the neonatal liver is critical to ensure self-tolerance and liver maturation.

Single cell transcriptomic analysis of human mesenchymal stem cells reveals limited heterogeneity.

Mesenchymal stem cells (MSCs) are a population of multipotent cells with a superior ability to promote tissue repair by regulating regeneration and inflammation. Effective application of MSCs in disease treatment relies on the production of relatively homogeneous cell population. However, the cellular heterogeneity and the differentiation trajectories of in vitro expanded MSCs remain largely unclear. We profiled the transcriptomes of 361 single MSCs derived from two umbilical cords (UC-MSCs). These UC-MSCs were harvested at different passages and stimulated with or without inflammatory cytokines. Weighted gene correlation network analysis revealed that UC-MSCs surprisingly possess only limited heterogeneity, regardless of donors, and passages. We also found that upon pretreatment with inflammatory cytokines (IFNγ and TNFα), a classical strategy that can improve the efficiency of MSC-based therapy, MSCs exhibited uniformed changes in gene expression. Cell cycle-based principal component analysis showed that the limited heterogeneity identified in these UC-MSCs was strongly associated with their entrance into the G2/M phase. This was further proven by the observation that one featured gene, CD168, was expressed in a cell cycle-dependent manner. When CD168high UC-MSCs were sorted and cultured in vitro, they again showed similar CD168 expression patterns. Our results demonstrated that in vitro expanded UC-MSCs are a well-organized population with limited heterogeneity dominated by cell cycle status. Thus, our studies provided information for standardization of MSCs for disease treatment.

Hemojuvelin is a novel suppressor for Duchenne muscular dystrophy and age-related muscle wasting.

BACKGROUND: Muscle wasting occurs in response to various physiological and pathological conditions, including ageing and Duchenne muscular dystrophy (DMD). Transforming growth factor-ß1 (TGF-ß1) contributes to muscle pathogenesis in elderly people and DMD patients; inhibition of TGF-ß1 signalling is a promising therapeutic strategy for muscle-wasting disorders. Hemojuvelin (HJV or Hjv as the murine homologue) is a membrane-bound protein that is highly expressed in skeletal muscle, heart, and liver. In hepatic cells, Hjv acts as a coreceptor for bone morphogenetic protein, a TGF-ß subfamily member. The aim of this study was to investigate whether Hjv plays an essential role in muscle physiological and pathophysiological processes by acting as a coreceptor for TGF-ß1 signalling. METHODS: Conventional and conditional Hjv knockout mice as well as mdx and aged mice transfected with Hjv overexpression vector were used to study the role of Hjv in muscle physiology and pathophysiology. qRT-PCR, western blotting, and immunohistochemistry examinations were conducted to evaluate gene, protein, and structural changes in vivo and in vitro. Exercise endurance was determined using treadmill running test, and muscle force was detected by an isometric transducer. RNA interference, immunoprecipitation, and dual-luciferase reporter assays were utilized to explore the mechanism by which Hjv regulates TGF-ß1 signalling in skeletal muscle. RESULTS: Conventional and conditional Hjv knockout mice displayed muscle atrophy, fibrosis, reduced running endurance, and muscle force. HJV was significantly down-regulated in the muscles of DMD patients (n = 3, mean age: 11.7 ± 5.7 years) and mdx mice as well as in those of aged humans (n = 10, 20% women, mean age: 75.1 ± 9.5 years) and mice. Overexpression of Hjv rescued dystrophic and age-related muscle wasting. Unlike its function in hepatic cells, the bone morphogenetic protein downstream phosphorylated p-Smad1/5/8 signalling pathway was unchanged, but TGF-ß1, TGF-ß receptor II (TßRII), and p-Smad2/3 expression were increased in Hjv-deficient muscles. Mechanistically, loss of Hjv promoted activation of Smad3 signalling induced by TGF-ß1, whereas Hjv overexpression inhibited TGF-ß1/Smad3 signalling by directly interacting with TßRII on the muscle membrane. CONCLUSIONS: Our findings identify an unrecognized role of HJV in skeletal muscle by regulating TGF-ß1/Smad3 signalling as a coreceptor for TßRII. Unlike the TGF-ß1/Smad3 pathway, HJV could be a reliable drug target as its expression is not widespread. Novel therapeutic strategies could potentially be devised to interfere only with the muscle function of HJV to treat DMD and age-related muscle wasting.

Exercise Reverses Dysregulation of T-Cell-Related Function in Blood Leukocytes of Patients With Parkinson's Disease.

Parkinson's disease (PD) is a common neurodegenerative disease with movement and balance impairments. Although studies have reported improvement of motor symptoms with physical exercise, the mechanisms by which exercise is beneficial remains poorly understood. Our study addresses the exercise-induced changes to peripheral immune cells by interrogating the transcriptome of blood-derived leukocytes in PD patients before and after exercise. Patients attended 1 h exercise classes twice a week for 12 weeks. Leukocytes were collected at the beginning and end of the study for gene expression analysis by RNA-seq or quantitative real-time PCR. We correlated differentially expressed genes after exercise with clinical measures and analyzed the potential functions of gene changes with Kyoto Encyclopedia of Genes and Genomes pathway and Gene Ontology analysis. Exercise improved measures of movement and balance when compared with scores before the exercise program. Among the gene changes, Kyoto Encyclopedia of Genes and Genomes and Gene Ontology analysis suggests that T-cell receptor signaling, T-cell activation, and T-cell migration pathways were downregulated, while the T-cell receptor signaling pathway was the most significantly correlated with clinical measures. To further investigate T-cell-related changes in PD leukocytes, we reanalyzed the differentially expressed genes from publicly available microarray data and found that genes in the T-cell activation, differentiation, and migration pathways were upregulated in PD samples compared to controls in a time-dependent manner. Together, our findings suggest that exercise rehabilitation may improve movement and balance in PD patients by reversing the upregulated T-cell activation pathways associated with PD. This study was registered with the Chinese Clinical Trial Registry under ChiCTR-TRC-14004707. Registered on May 27, 2014.

Systematic Analysis of RNA Regulatory Network in Rat Brain after Ischemic Stroke.

Although extensive studies have identified large number of microRNAs (miRNAs) and long noncoding RNAs (lncRNAs) in ischemic stroke, the RNA regulation network response to focal ischemia remains poorly understood. In this study, we simultaneously interrogate the expression profiles of lncRNAs, miRNAs, and mRNAs changes during focal ischemia induced by transient middle cerebral artery occlusion. A set of 1924 novel lncRNAs were identified and may involve brain injury and DNA repair as revealed by coexpression network analysis. Furthermore, many short interspersed elements (SINE) mediated lncRNA:mRNA duplexes were identified, implying that lncRNAs mediate Staufen1-mediated mRNA decay (SMD) which may play a role during focal ischemia. Moreover, based on the competitive endogenous RNA (ceRNA) hypothesis, a stroke regulatory ceRNA network which reveals functional lncRNA:miRNA:mRNA interactions was revealed in ischemic stroke. In brief, this work reports a large number of novel lncRNAs responding to focal ischemia and constructs a systematic RNA regulation network which highlighted the role of ncRNAs in ischemic stroke.

Single-cell transcriptomics reveals gene signatures and alterations associated with aging in distinct neural stem/progenitor cell subpopulations.

Aging associated cognitive decline has been linked to dampened neural stem/progenitor cells (NSC/NPCs) activities manifested by decreased proliferation, reduced propensity to produce neurons, and increased differentiation into astrocytes. While gene transcription changes objectively reveal molecular alterations of cells undergoing various biological processes, the search for molecular mechanisms underlying aging of NSC/NPCs has been confronted by the enormous heterogeneity in cellular compositions of the brain and the complex cellular microenvironment where NSC/NPCs reside. Moreover, brain NSC/NPCs themselves are not a homogenous population, making it even more difficult to uncover NSC/NPC sub-type specific aging mechanisms. Here, using both population-based and single cell transcriptome analyses of young and aged mouse forebrain ependymal and subependymal regions and comprehensive "big-data" processing, we report that NSC/NPCs reside in a rather inflammatory environment in aged brain, which likely contributes to the differentiation bias towards astrocytes versus neurons. Moreover, single cell transcriptome analyses revealed that different aged NSC/NPC subpopulations, while all have reduced cell proliferation, use different gene transcription programs to regulate age-dependent decline in cell cycle. Interestingly, changes in cell proliferation capacity are not influenced by inflammatory cytokines, but likely result from cell intrinsic mechanisms. The Erk/Mapk pathway appears to be critically involved in regulating age-dependent changes in the capacity for NSC/NPCs to undergo clonal expansion. Together this study is the first example of using population and single cell based transcriptome analyses to unveil the molecular interplay between different NSC/NPCs and their microenvironment in the context of the aging brain.

Telomere heterogeneity linked to metabolism and pluripotency state revealed by simultaneous analysis of telomere length and RNA-seq in the same human embryonic stem cell.

BACKGROUND: Telomere length heterogeneity has been detected in various cell types, including stem cells and cancer cells. Cell heterogeneity in pluripotent stem cells, such as embryonic stem cells (ESCs), is of particular interest; however, the implication and mechanisms underlying the heterogeneity remain to be understood. Single-cell analysis technology has recently been developed and effectively employed to investigate cell heterogeneity. Yet, methods that can simultaneously measure telomere length and analyze the global transcriptome in the same cell have not been available until now. RESULTS: We have established a robust method that can simultaneously measure telomere length coupled with RNA-sequencing analysis (scT&R-seq) in the same human ESC (hESC). Using this method, we show that telomere length varies with pluripotency state. Compared to those with long telomere, hESCs with short telomeres exhibit the lowest expressions of TERF1/TRF1, and ZFP42/REX1, PRDM14 and NANOG markers for pluripotency, suggesting that these hESCs are prone to exit from the pluripotent state. Interestingly, hESCs ubiquitously express NOP10 and DKC1, stabilizing components of telomerase complexes. Moreover, new candidate genes, such as MELK, MSH6, and UBQLN1, are highly expressed in the cluster of cells with long telomeres and higher expression of known pluripotency markers. Notably, short telomere hESCs exhibit higher oxidative phosphorylation primed for lineage differentiation, whereas long telomere hESCs show elevated glycolysis, another key feature for pluripotency. CONCLUSIONS: Telomere length is a marker of the metabolic activity and pluripotency state of individual hESCs. Single cell analysis of telomeres and RNA-sequencing can be exploited to further understand the molecular mechanisms of telomere heterogeneity.

Modeling Rett Syndrome Using TALEN-Edited MECP2 Mutant Cynomolgus Monkeys.

Gene-editing technologies have made it feasible to create nonhuman primate models for human genetic disorders. Here, we report detailed genotypes and phenotypes of TALEN-edited MECP2 mutant cynomolgus monkeys serving as a model for a neurodevelopmental disorder, Rett syndrome (RTT), which is caused by loss-of-function mutations in the human MECP2 gene. Male mutant monkeys were embryonic lethal, reiterating that RTT is a disease of females. Through a battery of behavioral analyses, including primate-unique eye-tracking tests, in combination with brain imaging via MRI, we found a series of physiological, behavioral, and structural abnormalities resembling clinical manifestations of RTT. Moreover, blood transcriptome profiling revealed that mutant monkeys resembled RTT patients in immune gene dysregulation. Taken together, the stark similarity in phenotype and/or endophenotype between monkeys and patients suggested that gene-edited RTT founder monkeys would be of value for disease mechanistic studies as well as development of potential therapeutic interventions for RTT.

Downregulation of the Long Non-Coding RNA Meg3 Promotes Angiogenesis After Ischemic Brain Injury by Activating Notch Signaling.

Angiogenesis after ischemic brain injury contributes to the restoration of blood supply in the ischemic zone. Strategies to improve angiogenesis may facilitate the function recovery after stroke. Recent researches have demonstrated that dysfunction of long non-coding RNAs are associated with angiogenesis. We have previously reported that long non-coding RNAs (lncRNAs) are aberrantly expressed in ischemic stroke. However, little is known about long non-coding RNAs and theirs role in angiogenesis after stroke. In this study, we identified a rat lncRNAs, Meg3, and found that Meg3 was significantly decreased after ischemic stroke. Overexpression of Meg3 suppressed functional recovery and decreased capillary density after ischemic stroke. Downregulation of Meg3 ameliorated brain lesion and increased angiogenesis after ischemic stroke. Silencing of Meg3 resulted in a proangiogenic effect evidenced by increased endothelial cell migration, proliferation, sprouting, and tube formation. Mechanistically, we showed that Meg3 negatively regulated notch pathway both in vivo and in vitro. Inhibition of notch signaling in endothelial cells reversed the proangiogenic effect induced by Meg3 downregulation. This study revealed the function of Meg3 in ischemic stroke and elucidated its mechanism in angiogenesis after ischemic stroke.

Targeted Differentiation of Regional Ventral Neuroprogenitors and Related Neuronal Subtypes from Human Pluripotent Stem Cells.

Embryoid body (EB) formation and adherent culture (AD) paradigms are equivalently thought to be applicable for neural specification of human pluripotent stem cells. Here, we report that sonic hedgehog-induced ventral neuroprogenitors under EB conditions are fated to medial ganglionic eminence (MGE), while the AD cells mostly adopt a floor-plate (FP) fate. The EB-MGE later on differentiates into GABA and cholinergic neurons, while the AD-FP favors dopaminergic neuron specification. Distinct developmental, metabolic, and adhesion traits in AD and EB cells may potentially account for their differential patterning potency. Gene targeting combined with small-molecule screening experiments identified that concomitant inhibition of Wnts, STAT3, and p38 pathways (3i) could largely convert FP to MGE under AD conditions. Thus, differentiation paradigms and signaling regulators can be integrated together to specify distinct neuronal subtypes for studying and treating related neurological diseases, such as epilepsy, Alzheimer's disease, and Parkinson's disease.

Aberrant transcriptional networks in step-wise neurogenesis of paroxysmal kinesigenic dyskinesia-induced pluripotent stem cells.

Paroxysmal kinesigenic dyskinesia (PKD) is an episodic movement disorder with autosomal-dominant inheritance and marked variability in clinical manifestations.Proline-rich transmembrane protein 2 (PRRT2) has been identified as a causative gene of PKD, but the molecular mechanism underlying the pathogenesis of PKD still remains a mystery. The phenotypes and transcriptional patterns of the PKD disease need further clarification. Here, we report the generation and neural differentiation of iPSC lines from two familial PKD patients with c.487C>T (p. Gln163X) and c.573dupT (p. Gly192Trpfs*8) PRRT2 mutations, respectively. Notably, an extremely lower efficiency in neural conversion from PKD-iPSCs than control-iPSCs is observed by a step-wise neural differentiation method of dual inhibition of SMAD signaling. Moreover, we show the high expression level of PRRT2 throughout the human brain and the expression pattern of PRRT2 in other human tissues for the first time. To gain molecular insight into the development of the disease, we conduct global gene expression profiling of PKD cells at four different stages of neural induction and identify altered gene expression patterns, which peculiarly reflect dysregulated neural transcriptome signatures and a differentiation tendency to mesodermal development, in comparison to control-iPSCs. Additionally, functional and signaling pathway analyses indicate significantly different cell fate determination between PKD-iPSCs and control-iPSCs. Together, the establishment of PKD-specific in vitro models and the illustration of transcriptome features in PKD cells would certainly help us with better understanding of the defects in neural conversion as well as further investigations in the pathogenesis of the PKD disease.