Transplanting Human Neural Stem Cells with ≈50% Reduction of SOX9 Gene Dosage Promotes Tissue Repair and Functional Recovery from Severe Spinal Cord Injury.

Neural stem cells (NSCs) derived from human pluripotent stem cells (hPSCs) are considered a major cell source for reconstructing damaged neural circuitry and enabling axonal regeneration. However, the microenvironment at the site of spinal cord injury (SCI) and inadequate intrinsic factors limit the therapeutic potential of transplanted NSCs. Here, it is shown that half dose of SOX9 in hPSCs-derived NSCs (hNSCs) results in robust neuronal differentiation bias toward motor neuron lineage. The enhanced neurogenic potency is partly attributed to the reduction of glycolysis. These neurogenic and metabolic properties retain after transplantation of hNSCs with reduced SOX9 expression in a contusive SCI rat model without the need for growth factor-enriched matrices. Importantly, the grafts exhibit excellent integration properties, predominantly differentiate into motor neurons, reduce glial scar matrix accumulation to facilitate long-distance axon growth and neuronal connectivity with the host as well as dramatically improve locomotor and somatosensory function in recipient animals. These results demonstrate that hNSCs with half SOX9 gene dosage can overcome extrinsic and intrinsic barriers, representing a powerful therapeutic potential for transplantation treatments for SCI.

Cytoprotection of baicalein against oxidative stress-induced cardiomyocytes injury through the Nrf2/Keap1 pathway.

Baicalein is one of the major flavonoids found in the root of Scutellaria baicalensis Georgi. Previous studies suggest that baicalein displays protective effect on experimental cardiac models in vitro and in vivo. However, the mode of action remains unclear. Here, we showed that baicalein conferred cardioprotective effect against oxidative stress-induced cell injury in H9c2 cells and human embryonic stem cells-derived cardiomyocytes. Immunoprecipitation with anti-NF-E2-related factor 2 (Nrf2) antibody in baicalein-treated cells demonstrated that baicalein effectively disrupted the association between Nrf2 and Kelch-like epichlorohydrin-associated protein 1 (Keap1). In addition, the unbounded Nrf2 translocated from cytoplasm to nucleus and increased Nrf2/heme oxygenase-1 (HO-1) content in a time-dependent manner. Moreover, antioxidant response element transcriptional activity was enhanced by baicalein treatment, and the Nrf2 siRNA transfection could block the cytoprotective effect of baicalein. Taken together, these results demonstrate that baicalein protected cardiomyocytes against oxidative stress-induced cell injury through the Nrf2/Keap1 pathway.

Excess of O-linked N-acetylglucosamine modifies human pluripotent stem cell differentiation.

O-linked-N-acetylglucosamine (O-GlcNAc), a post translational modification, has emerged as an important cue in controlling key cell mechanisms. Here, we investigate O-GlcNAc's role in the maintenance and differentiation of human pluripotent stem cells (hPSC). We reveal that protein expression of O-GlcNAc transferase and hydrolase both decreases during hPSC differentiation. Upregulating O-GlcNAc with O-GlcNAc hydrolase inhibitors has no significant effect on either the maintenance of pluripotency in hPSC culture, or the loss of pluripotency in differentiating hPSC. However, in spontaneously differentiating hPSC, excess O-GlcNAc alters the expression of specific lineage markers: decrease of ectoderm markers (PAX6 by 53-88%, MSX1 by 26-49%) and increase of adipose-related mesoderm markers (PPARγ by 28-100%, C/EBPα by 46-135%). All other lineage markers tested (cardiac, visceral-endoderm, trophectoderm) remain minimally affected by upregulated O-GlcNAc. Interestingly, we also show that excess O-GlcNAc triggers a feedback mechanism that increases O-GlcNAc hydrolase expression by 29-91%. To the best of our knowledge, this is the first report demonstrating that excess O-GlcNAc does not affect hPSC pluripotency in undifferentiated maintenance cultures; instead, it restricts the hPSC differentiation towards specific cell lineages. These data will be useful for developing targeted differentiation protocols and aid in understanding the effects of O-GlcNAc on hPSC differentiation.

Enhanced production of neuroprogenitors, dopaminergic neurons, and identification of target genes by overexpression of sonic hedgehog in human embryonic stem cells.

Molecular and cellular signaling pathways are involved in the process of neural differentiation from human embryonic stem cells (hESC) to terminally differentiated neurons. The Sonic hedgehog (SHH) morphogen is required to direct the differentiation of hESC to several neural subtypes, for example, dopaminergic (DA) or motor neurons. However, the roles of SHH signaling and the pathway target genes that regulate the diversity of cellular responses arising from SHH activation during neurogenesis of hESC have yet to be elucidated. In this study, we report that overexpression of SHH in hESC promotes the derivation of neuroprogenitors (NP), increases proliferation of NP, and subsequently increases the yield of DA neurons. Next, gene expression changes resulting from the overexpression of SHH in hESC-derived NP were examined by genome-wide transcriptional profiling. Categorizing the differentially expressed genes according to the Gene Ontology biological processes showed that they are involved in numerous cellular processes, including neural development, NP proliferation, and neural specification. In silico GLI-binding sites analysis of the differentially expressed genes also identified a set of putative novel direct target genes of SHH in hESC-derived NP, which are involved in nervous system development. Electrophoretic mobility shift assays and promoter-luciferase assays confirmed that GLI1 binds to the promoter region and activates transcription of HEY2, a NOTCH signaling target gene. Taken together, our data provide evidence for the first time that there is cross-talk between the NOTCH and SHH signaling pathways in hESC-derived NP and also provide significant new insights into transcriptional targets in SHH-mediated neural differentiation of hESC.

Integrated transcriptome and binding sites analysis implicates E2F in the regulation of self-renewal in human pluripotent stem cells.

Rapid cellular growth and multiplication, limited replicative senescence, calibrated sensitivity to apoptosis, and a capacity to differentiate into almost any cell type are major properties that underline the self-renewal capabilities of human pluripotent stem cells (hPSCs). We developed an integrated bioinformatics pipeline to understand the gene regulation and functions involved in maintaining such self-renewal properties of hPSCs compared to matched fibroblasts. An initial genome-wide screening of transcription factor activity using in silico binding-site and gene expression microarray data newly identified E2F as one of major candidate factors, revealing their significant regulation of the transcriptome. This is underscored by an elevated level of its transcription factor activity and expression in all tested pluripotent stem cell lines. Subsequent analysis of functional gene groups demonstrated the importance of the TFs to self-renewal in the pluripotency-coupled context; E2F directly targets the global signaling (e.g. self-renewal associated WNT and FGF pathways) and metabolic network (e.g. energy generation pathways, molecular transports and fatty acid metabolism) to promote its canonical functions that are driving the self-renewal of hPSCs. In addition, we proposed a core self-renewal module of regulatory interplay between E2F and, WNT and FGF pathways in these cells. Thus, we conclude that E2F plays a significant role in influencing the self-renewal capabilities of hPSCs.

Technical advances to genetically engineering human embryonic stem cells.

Human embryonic stem cells (hESC) are important to basic scientific research as an in vitro model system for the study of human development and to clinical research as an invaluable cell source for regenerative medicine. The ability to genetically engineer hESC is a critical resource as it facilitates many fundamental studies to understand gene regulation and cell development. These techniques include (1) unidirectional or reversible; (2) non-, pseudo- or completely site-specific; and (3) endogenous and/or pre-engineered DNA sequences modification; where each has its own strengths and limitations. This article reviews the various methodologies to genetically engineer hESC to achieve a stable gene insertion or deletion. We discuss the existing challenges of the well-established methodologies (lentivirus and Cre/loxP system), and further examine recent advances in this field, such as the latest genetic modifying tools (phiC31 integrase, PiggyBac transposase and zinc finger nucleases). We also propose new opportunities for future developments to aid genetic modifications of hESC, and new applications for future basic and therapeutic research in hESC.

Role of Sonic hedgehog signaling and the expression of its components in human embryonic stem cells.

Human embryonic stem cells (hESC) are characterized by their ability to self-renew and differentiate into all cell types of the body, making them a valuable resource for regenerative medicine. Yet, the molecular mechanisms by which hESC retain their capacity for self-renewal and differentiation remain unclear. The Hedgehog signaling pathway plays a pivotal role in organogenesis and differentiation during development, and is also involved in the proliferation and cell-fate specification of neural stem cells and neural crest stem cells. As there has been no detailed study of the Sonic hedgehog (SHH) signaling pathway in hESC, this study examines the expression and functional role of SHH during hESC self-renewal and differentiation. Here, we show the gene and protein expression of key components of the SHH signaling pathway in hESC and differentiated embryoid bodies. Despite the presence of functioning pathway components, SHH plays a minimal role in maintaining pluripotency and regulating proliferation of undifferentiated hESC. However, during differentiation with retinoic acid, a GLI-responsive luciferase assay and target genes PTCH1 and GLI1 expression reveal that the SHH signaling pathway is highly activated. Besides, addition of exogenous SHH to hESC differentiated as embryoid bodies increases the expression of neuroectodermal markers Nestin, SOX1, MAP2, MSI1, and MSX1, suggesting that SHH signaling is important during hESC differentiation toward the neuroectodermal lineage. Our findings provide a new insight in understanding the SHH signaling in hESC and the further development of hESC differentiation for regenerative medicine.

KLF4 and PBX1 directly regulate NANOG expression in human embryonic stem cells.

Insight into the regulation of core transcription factors is important for a better understanding of the molecular mechanisms that control self-renewal and pluripotency of human ESCs (hESCs). However, the transcriptional regulation of NANOG itself in hESCs has largely been elusive. We established a NANOG promoter luciferase reporter assay as a fast read-out for indicating the pluripotent status of hESCs. From the functional cDNA screens and NANOG promoter characterization, we successfully identified a zinc finger transcription factor KLF4 and a homeodomain transcription factor PBX1 as two novel transcriptional regulators that maintain the pluripotent and undifferentiated state of hESCs. We showed that both KLF4 and PBX1 mRNA and protein expression were downregulated during hESC differentiation. In addition, overexpression of KLF4 and PBX1 upregulated NANOG promoter activity and also the endogenous NANOG protein expression in hESCs. Direct binding of KLF4 on NANOG proximal promoter and PBX1 on a new upstream enhancer and proximal promoter were confirmed by chromatin immunoprecipitation and electrophoretic mobility shift assay. Knockdown of KLF4/PBX1 or mutation of KLF4/PBX1 binding motifs significantly downregulated NANOG promoter activity. We also showed that specific members of the SP/KLF and PBX family are functionally redundant at the NANOG promoter and that KLF4 and PBX1 cooperated with OCT4 and SOX2, and transactivated synergistically the NANOG promoter activity. Our results show two novel upstream transcription activators of NANOG that are functionally important for the self-renewal of hESC and provide new insights into the expanded regulatory circuitry that maintains hESC pluripotency.

Generation of high-level stable transgene expressing human embryonic stem cell lines using Chinese hamster elongation factor-1 alpha promoter system.

The utilization of human embryonic stem cells (hESC) in regenerative medicine largely depends on the development of technologies that will allow efficient genetic manipulation of the cells in vitro. Although a few studies have described the transfection of hESC for generation of reporter lines stably expressing specific transgenes driven by different promoters, the optimal choice of promoter system for driving transgene in hESC has yet to be elucidated. We show for the first time that Chinese hamster elongation factor-1 alpha (CHEF1) promoter robustly drove reporter gene expression higher than the human elongation factor 1 alpha (hEF1 alpha), other constitutive Chinese hamster promoters, human cytomegalovirus (CMV) immediate early enhancer/promoter and SV40 promoters in hESC by quantitative analysis. We also successfully generated stably transfected hESC lines using this CHEF1 promoter system and demonstrated that they continued to express enhanced green fluorescent protein (EGFP) during prolonged undifferentiated proliferation, in differentiated embryoid bodies (EBs), and in teratomas without transgene silencing. By immunofluorescence staining and D ow cytometry analysis, the pluripotent markers, OCT-4, SSEA-4, and TRA-1-60, continued to be expressed in undifferentiated CHEF1-EGFP expressing hESC lines. When the stably transfected hESC were directed to differentiate into neural precursors in vitro, high-level EGFP expression was maintained and co-expression of neural markers, Nestin, and beta-tubulin III was observed. The morphology, karyotype, and telomerase activity of CHEF1-EGFP expressing hESC were normal after >50 continuous passages, and the cells retained the ability to differentiate into derivatives of the three germ layers in vitro as confirmed by RT-PCR analysis and immunocytochemical staining and in vivo teratoma formation. Therefore, stable CHEF1-EGFP hESC lines retained the capability for self-renewal and pluripotency. The novel CHEF1 promoter system described here enables high-level transgene expression in the stably transfected hESC. It may have signi, cant implication for uses in bioprocess development and future development of gene-modified hESC in tissue regeneration and transplantation applications.