Stem Cell Reports最新文献

Pax3-induced pluripotent stem cell-derived myogenic progenitors display an embryonic molecular signature but become postnatal upon transplantation. Because this correlates with upregulation of Notch signaling, here we probed whether NOTCH1 is required for in vivo maturation by performing gain- and loss-of-function studies in inducible Pax3 (iPax3) myogenic progenitors. Transplantation studies revealed that Notch1 signaling did not change the number of donor-derived fibers; however, the NOTCH1 overexpression cohorts showed enhanced satellite cell engraftment and more mature fibers, as indicated by fewer fibers expressing the embryonic myosin heavy-chain isoform and more type IIX fibers. While donor-derived Pax7+ cells were detected in all transplants, in the absence of Notch1, secondary grafts exhibited a high fraction of these cells in the interstitial space, indicating that NOTCH1 is required for proper satellite cell homing. Transcriptional profiling of NOTCH1-modified donor-derived satellite cells suggests that this may be due to changes in the extracellular matrix organization, cell cycle, and metabolism.

Adult hippocampal neurogenesis (AHN), the process of generating new neurons from adult neural stem/progenitor cells (NSPCs), is crucial for cognitive functions and is influenced by numerous factors, including metabolic processes. Pyruvate kinase M2 (PKM2), a key rate-limiting enzyme in glycolysis, catalyzes the production of pyruvate, which undergoes either oxidative phosphorylation or anaerobic oxidation. We observed that PKM2 is highly expressed in NSPCs, but its significance remains unclear for AHN and cognition. Using knockdown or knockout strategies, we discovered that PKM2 deficiency led to reduced AHN and impaired cognitive functions. Furthermore, we observed that knockout of PKM2 resulted in lower L-lactate levels, and supplementing L-lactate in PKM2 knockout mice improved AHN and cognitive functions. Mechanistically, L-lactate restored neurogenesis via monocarboxylate transporter 2 (MCT2), but not hydroxycarboxylic acid receptor 1. In summary, our findings demonstrate that PKM2 is essential for AHN, and lactate supplementation can restore neurogenesis in an MCT2-dependent manner.

Embryonic signaling pathways exert stage-specific effects during cardiac development, yet the precise signals for proliferation or maturation remain elusive. To uncover the cues for proliferation, we performed a combinatory cell-cycle screen for insulin and glycogen synthase kinase-3 (GSK3) inhibition in spontaneously beating human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Our analysis for proliferation, and subsequential downstream sarcomere development, gene expression analysis, and molecular interventions identified a temporal interplay between insulin/Akt/FOXO and CHIR99021/Wnt/GSK3/TCF signaling. Combined pathway activation led to proliferation of immature hiPSC-CMs with low sarcomere and mitochondria content, while, in the absence of pathway activators, cardiomyocytes rapidly exited the cell cycle and fetched higher organization of sarcomeres and mitochondria. Our data demonstrate two important pathways, which enhance proliferation and inhibit maturation, and provide molecular mechanistic understanding of these cell fate decisions in immature hiPSC-CMs.

Heparan sulfate (HS) is an anionic polysaccharide generated by all animal cells, but our understanding of its roles in human pluripotent stem cell (hPSC) self-renewal and differentiation is limited. We derived HS-deficient hPSCs by disrupting the EXT1 glycosyltransferase. These EXT1^-/- hPSCs maintain self-renewal and pluripotency under standard culture conditions that contain high levels of basic fibroblast growth factor(bFGF), a requirement for sufficient bFGF signaling in the engineered cells. Intriguingly, Activin/Nodal signaling is also compromised in EXT1^-/- hPSCs, highlighting HS's previously unexplored involvement in this pathway. As a result, EXT1^-/- hPSCs fail to differentiate into mesoderm or endoderm lineages. Unexpectedly, HS is dispensable for early ectodermal differentiation of hPSCs but still critical in generating motor neurons. Those derived from HS-deficient hPSCs lack proper neuronal projections and show alterations in axonogenesis gene expression. Thus, our study uncovers expected and unexpected mechanistic roles of HS in hPSC fate decisions.

Multiple sclerosis (MS) is an autoimmune inflammatory demyelinating disease that results in motor, sensory, cognitive, and affective deficits. Hippocampal demyelination, a common occurrence in MS, is linked to impaired cognitive function and mood. Despite this, the precise mechanisms underlying cognitive impairments in MS remain elusive. Pleiotrophin (PTN), secreted by neural stem cells and astrocytes, plays a crucial role in regulating cognition. This study investigates the role of astrocyte-derived PTN. We found that genetic deletion of astrocyte-derived PTN hinders hippocampal neurogenesis. Additionally, conditional ablation of PTN in astrocytes exacerbates neurogenic deficits in the demyelinated hippocampus. Importantly, overexpression of PTN in astrocytes reverses neurogenic and cognitive impairments caused by demyelination, underscoring PTN's protective role in MS. PTN cooperates with protein tyrosine phosphatase receptor type Z1 (PTPRZ1) or anaplastic lymphoma kinase (ALK) receptors to activate the AKT signaling pathway, thereby enhancing hippocampal neurogenesis and cognition in demyelinated mice. These findings illuminate novel effects of astrocyte-derived PTN on hippocampal neurogenesis and cognition.

Complement factor H (CFH) common genetic variants have been associated with age-related macular degeneration (AMD). While most previous in vitro RPE studies focused on the common p.His402Tyr CFH variant, we characterized rare CFH variants that are highly penetrant for AMD using induced pluripotent stem-cell-derived retinal pigment epithelium (iPSC-RPE). Our results show that lower factor H (FH) levels are detected in AMD RPE, which potentially disrupt canonical and non-canonical roles of FH. Specifically, AMD RPE displays higher inflammation rate and a reduced set of differentially expressed genes compared to control RPE upon N-retinylidene-N-retinylethanolamine (A2E) and blue light challenge. Additionally, cholesterol efflux and photoreceptor outer segment (POS) phagocytosis defects, dysregulated complement levels, larger sub-RPE deposits, and increased mitochondrial stress were observed in AMD RPE. Thus, our study reveals new non-canonical roles for FH in regulating important RPE functions.

This study introduces a novel method for rapidly and efficiently inducing human spinal lower motor neurons (LMNs) from induced pluripotent stem cells (iPSCs) to eventually elucidate the pathomechanisms of amyotrophic lateral sclerosis (ALS) and facilitate drug screening. Previous methods were limited by low induction efficiency, poor LMN purity, or labor-intensive induction and evaluation processes. Our protocol overcomes these challenges, achieving around 80% induction efficiency within just two weeks by combining a small molecule-based approach with transcription factor transduction. Moreover, to exclude non-LMN cells from the analysis, we utilized time-lapse microscopy and machine learning to analyze the morphology and viability of iPSC-derived LMNs on a single-cell basis, establishing an effective pathophysiological evaluation system. This rapid, efficient, and streamlined protocol, along with our single-cell-based evaluation method, enables large-scale analysis and drug screening using iPSC-derived motor neurons.

It is widely recognized that the glycocalyx has significant implications in regulating the self-renewal and differentiation of adult stem cells; however, its composition remains poorly understood. Here, we show that the fucose-binding Aleuria aurantia lectin (AAL) binds differentially to basal cells in the stratified epithelium of the human limbus, hair follicle epithelium, and meibomian gland duct. Using fluorescence-activated cell sorting in combination with single-cell transcriptomics, we find that most epithelial progenitor cells and melanocytes in the limbus display low AAL staining (AAL^low) on their cell surface, an attribute that is gradually lost in epithelial cells as they differentiate into mature corneal cells. AAL^low epithelial cells were enriched in putative limbal stem cell markers and displayed high clonogenic capacity. Further analyses revealed that AAL^low epithelial cells had reduced expression of GDP-mannose-4,6-dehydratase, an enzyme catalyzing the first and limiting step in the de novo biosynthesis of GDP-fucose, and that inhibition of fucosylation using a small-molecule fucose analog stimulated the proliferative potential of limbal epithelial cells ex vivo. These results provide crucial insights into the distinctive composition of the glycocalyx in adult stem cells and underscore the significance of fucose modulation in the therapeutic regeneration of the human limbal stem cell niche.

Gene editing strategies to safely and robustly modify the Alzheimer's disease-associated APOE4 isoform are still lacking. Prime editing (PE) enables the precise introduction of genetic variants with minimal unintended editing and without donor templates. However, it requires optimization for each target site and has not yet been applied to APOE4 gene editing. Here, we screened PE guide RNA (pegRNA) parameters and PE systems for introducing the APOE4 variant and applied the optimized PE strategy to generate disease-relevant human induced pluripotent stem cell models. We show that introducing a single-nucleotide difference required for APOE4 correction inhibits PE activity. To advance efficient and robust genome engineering of precise genetic variants, we further present a reliable PE enrichment strategy based on diphtheria toxin co-selection. Our work provides an optimized and reproducible genome engineering pipeline to generate APOE4 disease models and outlines novel strategies to accelerate genome editing in cellular disease model generation.