Txnip regulates the Oct4-mediated pluripotency circuitry via metabolic changes upon differentiation.

Thioredoxin interacting protein (Txnip) is a stress-responsive factor regulating Trx1 for redox balance and involved in diverse cellular processes including proliferation, differentiation, apoptosis, inflammation, and metabolism. However, the biological role of Txnip function in stem cell pluripotency has yet to be investigated. Here, we reveal the novel functions of mouse Txnip in cellular reprogramming and differentiation onset by involving in glucose-mediated histone acetylation and the regulation of Oct4, which is a fundamental component of the molecular circuitry underlying pluripotency. During reprogramming or PSC differentiation process, cellular metabolic and chromatin remodeling occur in order to change its cellular fate. Txnip knockout promotes induced pluripotency but hinders initial differentiation by activating pluripotency factors and promoting glycolysis. This alteration affects the intracellular levels of acetyl-coA, a final product of enhanced glycolysis, resulting in sustained histone acetylation on active PSC gene regions. Moreover, Txnip directly interacts with Oct4, thereby repressing its activity and consequently deregulating Oct4 target gene transcriptions. Our work suggests that control of Txnip expression is crucial for cell fate transitions by modulating the entry and exit of pluripotency.

Directly reprogrammed natural killer cells for cancer immunotherapy.

Efficacious and accessible sources of natural killer (NK) cells would widen their use as immunotherapeutics, particularly for solid cancers. Here, we show that human somatic cells can be directly reprogrammed into NK cells with a CD56brightCD16bright phenotype using pluripotency transcription factors and an optimized reprogramming medium. The directly reprogrammed NK cells have strong innate-adaptive immunomodulatory activity and are highly potent against a wide range of cancer cells, including difficult-to-treat solid cancers and cancer stem cells. Both directly reprogrammed NK cells bearing a cancer-specific chimeric antigen receptor and reprogrammed NK cells in combination with antibodies competent for antibody-dependent cell-mediated cytotoxicity led to selective anticancer effects with augmented potency. The direct reprogramming of human somatic cells into NK cells is amenable to the production of autologous and allogeneic NK cells, and will facilitate the design and testing of cancer immunotherapies and combination therapies.

Exploration of Alternative Splicing Events in Mesenchymal Stem Cells from Human Induced Pluripotent Stem Cells.

Although comparative genome-wide transcriptomic analysis has provided insight into the biology of human induced pluripotent stem cell-derived mesenchymal stem cells (iMSCs), the distinct alternative splicing (AS) signatures of iMSCs remain elusive. Here, we performed Illumina RNA sequencing analysis to characterize AS events in iMSCs compared with tissue-derived MSCs. A total of 4586 differentially expressed genes (|FC| > 2) were identified between iMSCs and umbilical cord blood-derived MSCs (UCB-MSCs), including 2169 upregulated and 2417 downregulated genes. Of these, 164 differentially spliced events (BF > 20) in 112 genes were identified between iMSCs and UCB-MSCs. The predominant type of AS found in iMSCs was skipped exons (43.3%), followed by retained introns (19.5%), alternative 3' (15.2%) and 5' (12.8%) splice sites, and mutually exclusive exons (9.1%). Functional enrichment analysis showed that the differentially spliced genes (|FC| > 2 and BF > 20) were mainly enriched in functions associated with focal adhesion, extracellular exosomes, extracellular matrix organization, cell adhesion, and actin binding. Splice isoforms of selected genes including TRPT1, CNN2, and AP1G2, identified in sashimi plots, were further validated by RT-PCR analysis. This study provides valuable insight into the biology of iMSCs and the translation of mechanistic understanding of iMSCs into therapeutic applications.

Modeling Sialidosis with Neural Precursor Cells Derived from Patient-Derived Induced Pluripotent Stem Cells.

Sialidosis, caused by a genetic deficiency of the lysosomal sialidase gene (NEU1), is a systemic disease involving various tissues and organs, including the nervous system. Understanding the neurological dysfunction and pathology associated with sialidosis remains a challenge, partially due to the lack of a human model system. In this study, we have generated two types of induced pluripotent stem cells (iPSCs) with sialidosis-specific NEU1G227R and NEU1V275A/R347Q mutations (sialidosis-iPSCs), and further differentiated them into neural precursor cells (iNPCs). Characterization of NEU1G227R- and NEU1V275A/R347Q- mutated iNPCs derived from sialidosis-iPSCs (sialidosis-iNPCs) validated that sialidosis-iNPCs faithfully recapitulate key disease-specific phenotypes, including reduced NEU1 activity and impaired lysosomal and autophagic function. In particular, these cells showed defective differentiation into oligodendrocytes and astrocytes, while their neuronal differentiation was not notably affected. Importantly, we found that the phenotypic defects of sialidosis-iNPCs, such as impaired differentiation capacity, could be effectively rescued by the induction of autophagy with rapamycin. Our results demonstrate the first use of a sialidosis-iNPC model with NEU1G227R- and NEU1V275A/R347Q- mutation(s) to study the neurological defects of sialidosis, particularly those related to a defective autophagy-lysosome pathway, and may help accelerate the development of new drugs and therapeutics to combat sialidosis and other LSDs.

Dysregulation of BRD4 Function Underlies the Functional Abnormalities of MeCP2 Mutant Neurons.

Rett syndrome (RTT), mainly caused by mutations in methyl-CpG binding protein 2 (MeCP2), is one of the most prevalent intellectual disorders without effective therapies. Here, we used 2D and 3D human brain cultures to investigate MeCP2 function. We found that MeCP2 mutations cause severe abnormalities in human interneurons (INs). Surprisingly, treatment with a BET inhibitor, JQ1, rescued the molecular and functional phenotypes of MeCP2 mutant INs. We uncovered that abnormal increases in chromatin binding of BRD4 and enhancer-promoter interactions underlie the abnormal transcription in MeCP2 mutant INs, which were recovered to normal levels by JQ1. We revealed cell-type-specific transcriptome impairment in MeCP2 mutant region-specific human brain organoids that were rescued by JQ1. Finally, JQ1 ameliorated RTT-like phenotypes in mice. These data demonstrate that BRD4 dysregulation is a critical driver for RTT etiology and suggest that targeting BRD4 could be a potential therapeutic opportunity for RTT.

Directly induced human Schwann cell precursors as a valuable source of Schwann cells.

BACKGROUND: Schwann cells (SCs) are primarily responsible for regeneration and repair of the peripheral nervous system (PNS). Renewable and lineage-restricted SC precursors (SCPs) are considered highly desirable and promising cell sources for the production of SCs and for studies of SC lineage development, but SCPs are extremely limited. Here, we present a novel direct conversion strategy for the generation of human SCPs, capable of differentiating into functional SCs. METHODS: Easily accessible human skin fibroblast cells were directly induced into integration-free SCPs using episomal vectors (Oct3/4, Klf4, Sox2, L-Myc, Lin28 and p53 shRNA) under SCP lineage-specific chemically defined medium conditions. Induced SCPs (iSCPs) were further examined for their ability to differentiate into SCs. The identification and functionality of iSCPs and iSCP-differentiated SCs (iSCs) were confirmed according to morphology, lineage-specific markers, neurotropic factor secretion, and/or standard functional assays. RESULTS: Highly pure, Sox 10-positive of iSCPs (more than 95% purity) were generated from human skin fibroblasts within 3 weeks. Established iSCPs could be propagated in vitro while maintaining their SCP identity. Within 1 week, iSCPs could efficiently differentiate into SCs (more than 95% purity). The iSCs were capable of secreting various neurotrophic factors such as GDNF, NGF, BDNF, and NT-3. The in vitro myelinogenic potential of iSCs was assessed by myelinating cocultures using mouse dorsal root ganglion (DRG) neurons or human induced pluripotent stem cell (iPSC)-derived sensory neurons (HSNs). Furthermore, iSC transplantation promoted sciatic nerve repair and improved behavioral recovery in a mouse model of sciatic nerve crush injury in vivo. CONCLUSIONS: We report a robust method for the generation of human iSCPs/iSCs that might serve as a promising cellular source for various regenerative biomedical research and applications, such as cell therapy and drug discovery, especially for the treatment of PNS injury and disorders.

The role of selenium-mediated redox signaling by selenophosphate synthetase 1 (SEPHS1) in hESCs.

Selenium (Se) plays a vital role in reactive oxygen species (ROS) homeostasis and redox regulation in intracellular signaling via selenocysteine (Sec), known as the 21st proteinogenic amino acid, but its specific biological functions in development and disease remain undiscovered. In this study, we explored the role of selenophosphate synthetase 1 (SEPHS1) in the pluripotency maintenance and reprogramming. We found that high level of SEPHS1 is retained in undifferentiated embryonic stem cells (ESCs), which is decreased during their differentiation. SEPHS1 knockdown significantly reduced reprogramming efficiency, proving that SEPHS1 is required for acquisition of pluripotency. However, SEPHS1 knockdown did not affect the expression of significant pluripotency genes, suggesting that SEPHS1 may be involved in the survival of pluripotent stem cells rather than in the regulation of pluripotency genes. Transcriptome analysis revealed altered expression of the gene set related to the ROS pathway and apoptosis in SEPHS1-knockdown cells. We also demonstrated the role of SEPHS1 in human ESC clonogenicity, and we found improved single-cell survival of hESCs by selenium treatment in a concentration-dependent manner. Our study implies that hSEPHS1 is a regulator of selenium-mediated redox-signaling in human pluripotent stem cells and plays a role in their survival.

TSPYL5-mediated inhibition of p53 promotes human endothelial cell function.

Testis-specific protein, Y-encoded like (TSPYL) family proteins (TSPYL1-6), which are members of the nucleosome assembly protein superfamily, have been determined to be involved in the regulation of various cellular functions. However, the potential role of TSPYL family proteins in endothelial cells (ECs) has not been determined. Here, we demonstrated that the expression of TSPYL5 is highly enriched in human ECs such as human umbilical vein endothelial cells (HUVECs) and human pluripotent stem cell-differentiated ECs (hPSC-ECs). Importantly, TSPYL5 overexpression was shown to promote EC proliferation and functions, such as migration and tube formation, by downregulating p53 expression. Adriamycin-induced senescence was markedly blocked by TSPYL5 overexpression. In addition, the TSPYL5 depletion-mediated loss of EC functions was blocked by p53 inhibition. Significantly, TSPYL5 overexpression promoted angiogenesis in Matrigel plug and wound repair in a mouse skin wound healing model in vivo. Our results suggest that TSPYL5, a novel angiogenic regulator, plays a key role in maintaining endothelial integrity and function. These findings extend the understanding of TSPYL5-dependent mechanisms underlying the regulation of p53-related functions in ECs.

Effects of the Extracts from Fruit and Stem of Camellia japonica on Induced Pluripotency and Wound Healing.

Small molecules that improve reprogramming, stem cell properties, and regeneration can be widely applied in regenerative medicine. Natural plant extracts represent an abundant and valuable source of bioactive small molecules for drug discovery. Natural products themselves or direct derivatives of them have continued to provide small molecules that have entered clinical trials, such as anticancer and antimicrobial drugs. Here, we tested 3695 extracts from native plants to examine whether they can improve induced pluripotent stem cell (iPSC) generation using genetically homogeneous secondary mouse embryonic fibroblasts (MEFs) harboring doxycycline (dox)-inducible reprograming transgenes. Among the tested extracts, extracts from the fruit and stem of Camellia japonica (CJ) enhanced mouse and human iPSC generation and promoted efficient wound healing in an in vivo mouse wound model. CJ is one of the best-known species of the genus Camellia that belongs to the Theaceae family. Our findings identified the natural plant extracts from the fruit and stem of CJ as novel regulators capable of enhancing cellular reprogramming and wound healing, providing a useful supplement in the development of a more efficient and safer method to produce clinical-grade iPSCs and therapeutics.

Peroxisome Proliferator-Activated Receptor α Agonist and Its Target Nanog Cooperate to Induce Pluripotency.

The pharmaceutical compounds that modulate pluripotent stem cell (PSC) identity and function are increasingly adopted to generate qualified PSCs and their derivatives, which have promising potential in regenerative medicine, in pursuit of more accuracy and safety and less cost. Here, we demonstrate the peroxisome proliferator-activated receptor α (PPARα) agonist as a novel enhancer of pluripotency acquisition and induced pluripotent stem cell (iPSC) generation. We found that PPARα agonist, examined and selected Food and Drug Administration (FDA) -approved compound libraries, increase the expression of pluripotency-associated genes, such as Nanog, Nr5A2, Oct4, and Rex1, during the reprogramming process and facilitate iPSC generation by enhancing their reprogramming efficiency. A reprogramming-promoting effect of PPARα occurred via the upregulation of Nanog, which is essential for the induction and maintenance of pluripotency. Through bioinformatic analysis, we identified putative peroxisome proliferator responsive elements (PPREs) located within the promoter region of the Nanog gene. We also determined that PPARα can activate Nanog transcription by specific binding to putative PPREs. Taken together, our findings suggest that PPARα is an important regulator of PSC pluripotency and reprogramming, and PPARα agonists can be used to improve PSC technology and regenerative medicine.

Pharmacological Regulation of Oxidative Stress in Stem Cells.

Oxidative stress results from an imbalance between reactive oxygen species (ROS) production and antioxidant defense mechanisms. The regulation of stem cell self-renewal and differentiation is crucial for early development and tissue homeostasis. Recent reports have suggested that the balance between self-renewal and differentiation is regulated by the cellular oxidation-reduction (redox) state; therefore, the study of ROS regulation in regenerative medicine has emerged to develop protocols for regulating appropriate stem cell differentiation and maintenance for clinical applications. In this review, we introduce the defined roles of oxidative stress in pluripotent stem cells (PSCs) and hematopoietic stem cells (HSCs) and discuss the potential applications of pharmacological approaches for regulating oxidative stress in regenerative medicine.

DSG2 Is a Functional Cell Surface Marker for Identification and Isolation of Human Pluripotent Stem Cells.

Pluripotent stem cells (PSCs) represent the most promising clinical source for regenerative medicine. However, given the cellular heterogeneity within cultivation and safety concerns, the development of specific and efficient tools to isolate a pure population and eliminate all residual undifferentiated PSCs from differentiated derivatives is a prerequisite for clinical applications. In this study, we raised a monoclonal antibody and identified its target antigen as desmoglein-2 (DSG2). DSG2 co-localized with human PSC (hPSC)-specific cell surface markers, and its expression was rapidly downregulated upon differentiation. The depletion of DSG2 markedly decreased hPSC proliferation and pluripotency marker expression. In addition, DSG2-negative population in hPSCs exhibited a notable suppression in embryonic body and teratoma formation. The actions of DSG2 in regulating the self-renewal and pluripotency of hPSCs were predominantly exerted through the regulation of ß-catenin/Slug-mediated epithelial-to-mesenchymal transition. Our results demonstrate that DSG2 is a valuable PSC surface marker that is essential for the maintenance of PSC self-renewal.

Generation and Fusion of Human Cortical and Medial Ganglionic Eminence Brain Organoids.

Three-dimensional (3D) brain organoid culture has become an essential tool for investigating human brain development and modeling neurological disorders during the past few years. Given the specific regionalization during brain development, it is important to produce distinct brain organoids that reproduce different brain regions and their interaction. The authors' laboratory recently established the platform to generate brain organoids resembling the medial ganglionic eminence (MGE), a specific brain region responsible for interneurogenesis, and found when fusing with organoid resembling the cortex, the fused organoids enabled modeling of interneuron migration in the brain. This unit describes four basic protocols that have been successfully applied in the authors' laboratory, covering the generation of embryonic body (EB) with neuroectodermal fate, the production of MGE organoids (hMGEOs) and cortical organoids (hCOs), and the fusion of the two organoids.

Clump-passaging-based efficient 3D culture of human pluripotent stem cells under chemically defined conditions.

Large-scale production of human pluripotent stem cells (hPSCs) in an efficient and safe manner is crucial to the successful application of hPSCs in biomedical research and regenerative medicine. Three-dimensional culture methods for hPSCs have been extensively studied using single-cell passaging approaches; however, these techniques have been challenged by the induction of massive cell death and accumulation of genomic abnormalities. In this work, we developed and optimized a novel, simple clump-passaging method for in vitro hPSCs 3-dimensional (3D) culture that can be exploited for large-scale production. Fully grown hPSC spheroids were dissociated into smaller-sized spheroid clumps by simple treatment with enzyme-free dissociation buffer, and clumped hPSCs were inoculated and maintained for 3D suspension culture. Our clump-passaging method effectively increased the hPSCs survival rate after subculture and supported scalable hPSCs 3D expansion. We also tested and selected chemically defined media formulations that are suitable for 3D culture and commercially available. Overall, our clump-passaging and expansion method demonstrated high survival and expansion rates for hPSC spheroids compared with conventional methods and may also have the advantage of maintaining genomic stability.

The unique spliceosome signature of human pluripotent stem cells is mediated by SNRPA1, SNRPD1, and PNN.

Spliceosomes are the core host of pre-mRNA splicing, allowing multiple protein isoforms to be produced from a single gene. Herein, we reveal that spliceosomes are more abundant in human pluripotent stem cells (hPSs), including human embryonic stem cells (hESs) and human induced pluripotent stem cells (hiPSs), than non-hPSs, and their presence is associated with high transcriptional activity. Supportively, spliceosomal components involved in the catalytically active pre-mRNA splicing step were mainly co-localized with hPS spliceosomes. By profiling the gene expression of 342 selected splicing factors, we found that 71 genes were significantly altered during the reprogramming of human somatic cells into hiPSs. Among them, SNRPA1, SNRPD1, and PNN were significantly up-regulated during the early stage of reprogramming, identified as hub genes by interaction network and cluster analysis. SNRPA1, SNRPD1, or PNN depletion led to a pronounced loss of pluripotency and significantly blocked hiPS generation. SNRPA1, SNRPD1, and PNN co-localized with the hPS spliceosomes, physically interacted with each other, and positively influenced the appearance of hPS spliceosomes. Our data suggest that SNRPA1, SNRPD1, and PNN are key players in the regulation of pluripotency-specific spliceosome assembly and the acquisition and maintenance of pluripotency.

Upregulation of mitochondrial NAD+ levels impairs the clonogenicity of SSEA1+ glioblastoma tumor-initiating cells.

Emerging evidence has emphasized the importance of cancer therapies targeting an abnormal metabolic state of tumor-initiating cells (TICs) in which they retain stem cell-like phenotypes and nicotinamide adenine dinucleotide (NAD+) metabolism. However, the functional role of NAD+ metabolism in regulating the characteristics of TICs is not known. In this study, we provide evidence that the mitochondrial NAD+ levels affect the characteristics of glioma-driven SSEA1+ TICs, including clonogenic growth potential. An increase in the mitochondrial NAD+ levels by the overexpression of the mitochondrial enzyme nicotinamide nucleotide transhydrogenase (NNT) significantly suppressed the sphere-forming ability and induced differentiation of TICs, suggesting a loss of the characteristics of TICs. In addition, increased SIRT3 activity and reduced lactate production, which are mainly observed in healthy and young cells, appeared following NNT-overexpressed TICs. Moreover, in vivo tumorigenic potential was substantially abolished by NNT overexpression. Conversely, the short interfering RNA-mediated knockdown of NNT facilitated the maintenance of TIC characteristics, as evidenced by the increased numbers of large tumor spheres and in vivo tumorigenic potential. Our results demonstrated that targeting the maintenance of healthy mitochondria with increased mitochondrial NAD+ levels and SIRT3 activity could be a promising strategy for abolishing the development of TICs as a new therapeutic approach to treating aging-associated tumors.

Schwann Cell Precursors from Human Pluripotent Stem Cells as a Potential Therapeutic Target for Myelin Repair.

Schwann cells play a crucial role in successful nerve repair and regeneration by supporting both axonal growth and myelination. However, the sources of human Schwann cells are limited both for studies of Schwann cell development and biology and for the development of treatments for Schwann cell-associated diseases. Here, we provide a rapid and scalable method to produce self-renewing Schwann cell precursors (SCPs) from human pluripotent stem cells (hPSCs), using combined sequential treatment with inhibitors of the TGF-ß and GSK-3 signaling pathways, and with neuregulin-1 for 18 days under chemically defined conditions. Within 1 week, hPSC-derived SCPs could be differentiated into immature Schwann cells that were functionally confirmed by their secretion of neurotrophic factors and their myelination capacity in vitro and in vivo. We propose that hPSC-derived SCPs are a promising, unlimited source of functional Schwann cells for treating demyelination disorders and injuries to the peripheral nervous system.

Biomarker Discovery by Modeling Behçet's Disease with Patient-Specific Human Induced Pluripotent Stem Cells.

Behçet's disease (BD) is a chronic inflammatory and multisystemic autoimmune disease of unknown etiology. Due to the lack of a specific test for BD, its diagnosis is very difficult and therapeutic options are limited. Induced pluripotent stem cell (iPSC) technology, which provides inaccessible disease-relevant cell types, opens a new era for disease treatment. In this study, we generated BD iPSCs from patient somatic cells and differentiated them into hematopoietic precursor cells (BD iPSC-HPCs) as BD model cells. Based on comparative transcriptome analysis using our BD model cells, we identified eight novel BD-specific genes, AGTR2, CA9, CD44, CXCL1, HTN3, IL-2, PTGER4, and TSLP, which were differentially expressed in BD patients compared with healthy controls or patients with other immune diseases. The use of CXCL1 as a BD biomarker was further validated at the protein level using both a BD iPSC-HPC-based assay system and BD patient serum samples. Furthermore, we show that our BD iPSC-HPC-based drug screening system is highly effective for testing CXCL1 BD biomarkers, as determined by monitoring the efficacy of existing anti-inflammatory drugs. Our results shed new light on the usefulness of patient-specific iPSC technology in the development of a benchmarking platform for disease-specific biomarkers, phenotype- or target-driven drug discovery, and patient-tailored therapies.

Pro-fibrotic effects of PFKFB4-mediated glycolytic reprogramming in fibrous dysplasia.

Fibrous dysplasia (FD) caused by a mosaic somatic mutation of GNAS is characterized by replacement of the affected bone with abnormal fibrous tissue. Herein, we present novel disease models for FD developed with pairs of isogenic wild-type and GNAS(R201H)-mutated induced pluripotent stem cells (iPSCs) and their derivative mesenchymal stem cells (MSCs). Both 2D and 3D MSC culture models for FD successfully reflect FD's typical molecular characteristics, such as enhanced cAMP level, PKA activity, CREB1 phosphorylation and the pathologic fibrotic phenotype. The fibrotic features of GNAS(R201H) FD model cells were closely linked to augmented glycolysis and depended on glycolytic PFKFB4 and the activation of pro-fibrotic TGFß signalling. Either depletion of PFKFB4 or inhibition of glycolysis or TGFß signalling potentially blocked fibrosis progression in GNAS(R201H) FD model cells. Our FD models could facilitate a better mechanistic understanding of FD and help develop effective therapeutics for FD and other fibrosis diseases.

Restoration of Mitochondrial NAD+ Levels Delays Stem Cell Senescence and Facilitates Reprogramming of Aged Somatic Cells.

The fundamental tenet that aging is irreversible has been challenged by the development of reprogramming technology that can restore molecular and cellular age by reversing the progression of aging. The use of cells from aged individuals as sources for reprogramming or transplantation creates a major barrier in stem cell therapy with respect to cell quality and quantity. Here, we investigated the molecular features underlying senescence and rejuvenation during aged cell reprogramming and identified novel factors that can overcome age-associated barriers. Enzymes, such as nicotinamide nucleotide transhydrogenase (NNT) and nicotinamide mononucleotide adenylyltransferase 3 (NMNAT3), that control mitochondrial NAD+ levels appear to be susceptible to aging. In aged cells, mitochondrial NAD+ levels decrease, accompanied by reduced SIRT3 activity; these changes severely impede cell fate transition. However, in cells collected from aged p16 knockout mice, which exhibit delayed cellular senescence, no changes in NNT or NMNAT3 expression were found. Importantly, restoring mitochondrial NAD+ levels by overexpressing NNT and NMNAT3 enhanced reprogramming efficiency of aged somatic cells and extended the lifespan of human mesenchymal stem cells by delaying replicative senescence. These results demonstrate that maintenance of mitochondrial NAD+ levels is critical for reversing the mechanisms of aging and ensuring that cells collected from aged individuals are of high quality. Stem Cells 2016;34:2840-2851.