Rapid and Efficient Direct Conversion of Human Adult Somatic Cells into Neural Stem Cells by HMGA2/let-7b.

A recent study has suggested that fibroblasts can be converted into mouse-induced neural stem cells (miNSCs) through the expression of defined factors. However, successful generation of human iNSCs (hiNSCs) has proven challenging to achieve. Here, using microRNA (miRNA) expression profile analyses, we showed that let-7 microRNA has critical roles for the formation of PAX6/NESTIN-positive colonies from human adult fibroblasts and the proliferation and self-renewal of hiNSCs. HMGA2, a let-7-targeting gene, enables induction of hiNSCs that displayed morphological/molecular features and in vitro/in vivo differentiation potential similar to H9-derived NSCs. Interestingly, HMGA2 facilitated the efficient conversion of senescent somatic cells or blood CD34+ cells into hiNSCs through an interaction with SOX2, whereas other combinations or SOX2 alone showed a limited conversion ability. Taken together, these findings suggest that HMGA2/let-7 facilitates direct reprogramming toward hiNSCs in minimal conditions and maintains hiNSC self-renewal, providing a strategy for the clinical treatment of neurological diseases.

Core-shell nanoparticle controlled hATSCs neurogenesis for neuropathic pain therapy.

A stem cell-based strategy for tissue engineering in regenerative medicine is crucial to produce and effective therapeutic replacement of injured or damaged tissues. This type of therapeutic replacement requires interaction with the cells and tissues via the incorporation of a beneficial physical microenvironment and cellular biochemical signals. Recently, we studied a cell-function modifying factor, core-shell nanoparticles consisting of an SPIO (superparamagnetic iron oxide) core covered with a photonic ZnO shell for human adipose tissue-derived stem cells (hATSCs) that regulate various cellular functions: self-renewal, neurogenesis, and dedifferentiation. We proposed an alternative method of stem cell culture that focuses on the use of Zn++ Finger nanoparticles for stem cell expansion and transdifferentiation modulation in vitro and in in vivo spinal cord injury models. Our study showed that treating hATSC cultures with nanoscale particles could lead to active cell proliferation and self-renewal and could promote nuclear Dicer-regulation of several functional molecules, Oct4 and Glutathione peroxidase 3 (GPx3), and the abundance of specific functional proteins that have been observed using biochemical analysis. These biochemical changes in hATSCs induced the functional development of multiple differentiation potencies such as ß-cells and neural cells; specifically, the ability to differentiation into GABA-secreting cells was significantly improved in in vitro- and in vivo-induced animal lesions with significantly improved therapeutic modality.

MBD6 is a direct target of Oct4 and controls the stemness and differentiation of adipose tissue-derived stem cells.

Argonaute 2 (Ago2) is a pivotal regulator of cell fate in adult stem cells. Its expression is significantly downregulated in late passages of cells, concomitant with a prominent increase in Ago2 cytosolic localization in single cells. Nuclear localization of Ago2 is crucial for the survival, proliferation, and differentiation of hATSCs (human adipose tissue-derived stem cells), mediated by the specific binding of the regulatory regions of functional genes, which positively or negatively altered gene expression. Ago2 targets genes that control stemness, reactive oxygen species scavenging, and microRNA expression, all of which are crucial for hATSC survival and self-renewal. Ago2 promotes cell proliferation and self-renewal by activating the expression of octamer-binding transcription factor 4 (Oct4). We confirmed the direct regulation of Oct4 activity by Ago2, as indicated by the results of the ChIP analysis. Methyl-CpG-binding protein 6 (MBD6) was detected as an Oct4 regulatory gene. As predicted, knockdown of MBD6 expression attenuated cell proliferation and eventually induced cell death. We hypothesized that MBD6 functions downstream of Oct4 in the regulation of stemness-related genes, cell proliferation, self-renewal activity, and survival. MBD6 also promoted cell transdifferentiation into neural and endodermal ß-cells while significantly attenuating differentiation into the mesodermal lineage. We demonstrate that MBD6 is regulated by Ago2 via an interaction with Oct4, which alters self-renewal and gene expression in hATSCs. MBD6 was promoted cell proliferation through a novel set of signal mediators that may influence differentiation by repressing MBD2 and MBD3, which are possibly recruited by germ cell nuclear factor (GCNF).

MicroRNA 486 is a potentially novel target for the treatment of spinal cord injury.

MicroRNAs have been shown to effectively regulate gene expression at the translational level. Recently, we identified novel microRNAs that were upregulated in a mouse model of spinal cord injury. Among those, we have focused on microRNA 486, which directly represses NeuroD6 expression through a conserved sequence in its untranslated region. We correlated the overexpression of microRNA 486 in motor neurons with a poor outcome due to progressive neurodegeneration and a pathophysiology that is mediated by reactive oxygen species. The expression of microRNA 486 was induced by reactive oxygen species that were produced by inflammatory factors, and reactive oxygen species were accumulated in response to the knockdown of NeuroD6, which enhances the downregulation of glutathione peroxidase 3 and thioredoxin-like 1 after traumatic spinal cord injury. NeuroD6 directly bound to regulatory regions of thioredoxin-like 1 and glutathione peroxidase 3 in motor neurons and activated their expression, which promoted reactive oxygen species scavenging. Moreover, knocking down microRNA 486 induced the expression of NeuroD6, which effectively ameliorated the spinal cord injury and allowed the mice to recover motor function. The infusion of exogenic NeuroD6 in spinal cord injury lesions effectively blocked apoptosis by reactivating thioredoxin-like 1 and glutathione peroxidase 3, which was accompanied by a recovery of motor function. Collectively, these findings have identified a novel microRNA in spinal cord injury lesions called microRNA 486, demonstrating a new role for NeuroD6 in neuroprotection, and suggest a potential therapeutic target for spinal cord injuries.

Novel GSK-3ß inhibitors and CBM-1078 guide hATSCs' deaging via Oct4 and ß-catenin activation.

UNLABELLED: Abstract Aims: The fate decision of adult stem cells is determined by the activation of specific intracellular signaling pathways after exposure to specific stimuli. In this study, we demonstrated specific functions of a novel small molecule, CBM-1078, that induced cell self-renewal via Oct4- and canonical Wnt/ß-catenin-mediated deaging in cultured human adipose tissue-derived stem cells (hATSCs). RESULTS: As a potential glycogen synthase kinase-3ß (GSK-3ß) inhibitor, CBM-1078 primarily activated ß-catenin and Oct4 expression after inhibition of GSK-3ß. Treatment of hATSCs with CBM-1078 led to transdifferentiation toward a neural precursor cell fate after transient self-renewal, and the cells were capable of differentiation into gamma-Aminobutyric acid (GABA)-secreting neuronal cells with pain-modulating functions in an animal model of neuropathic pain. During cell self-renewal, CBM-1078 directs the translocalization of ß-catenin and Oct4 into the nucleus, an event that is crucial for the cooperative activation of hATSC neurogenesis via Oct4 and Wnt/ß-catenin. Nuclear-localized ß-catenin and Oct4 act together to regulate the expression of Oct4, Nanog, Sox2, ß-catenin, c-Myc, and STAT3 after binding to the regulatory regions of these genes. Nuclear Oct4 and Wnt3a/ß-catenin also control cell growth by binding to the promoters of STAT3, Gli3, and c-Myc after complex formation and direct interaction. CBM-1078 actively enhanced the DNA-binding affinity of Oct4 and ß-catenin to functional genes and activated the Wnt/ß-catenin pathway to promote hATSC reprogramming. INNOVATION AND CONCLUSION: This study revealed the value of a single small molecule, CBM-1078, showing a definitive cell reprogramming mechanism. Finally, we confirmed the therapeutic potential of GABA-hATSCs for treatment of neuropathic pain, which could be used for therapeutic purposes in humans. Antioxid. Redox Signal. 00, 000-000.

miR23b ameliorates neuropathic pain in spinal cord by silencing NADPH oxidase 4.

AIMS: Neuropathic pain is a well-known type of chronic pain caused by damage to the nervous system. Until recently, researchers have been primarily focused on identifying the cellular or chemical sources of neuropathic pain or have approached neuropathic pain via the basis of biological study. We investigated whether mmu-mir-23b (miR23b) infusion can alleviate pain by compensating for the abnormally downregulated miR23b by reducing the expression of its target gene, NADPH oxidase 4 (NOX4), a reactive oxygen species (ROS) family member overexpressed in neuropathic pain. RESULTS: Ectopic miR23b expression effectively downregulated NOX4 and was normalized to GAD65/67 expression. Moreover, the animals with neuropathic pain showed significant improvements in the paw withdrawal thresholds following miR23b infusion. Normalizing miR23b expression in tissue lesions caused by neuropathic pain induction reduced inflammatory mediator expression and increased the level of several ROS scavengers. Moreover, GABAergic neurons coexpressed suboptimal levels of miR23b and elevated NOX4/ROS after pain induction at the cellular level. MiR23b protects GABAergic neurons against ROS/p38/JNK-mediated apoptotic death. By evaluating the functional behavior of the mice receiving pain/miR23b, normal/anti-miR23b, or anti-miR23b/si-NOX4, the positive role of miR23b and the negative role of NOX4 in neuropathic pain were confirmed. INNOVATION AND CONCLUSION: Based on this study, we conclude that miR23b plays a crucial role in the amelioration of neuropathic pain in the injured spinal cord by inactivating its target gene, NOX4, and protecting GABAergic neurons from cell death. We finally suggest that miR23b may provide attractive diagnostic and therapeutic resources for effective pain modulation in neuropathic pain.

Silencing of miR20a is crucial for Ngn1-mediated neuroprotection in injured spinal cord.

MicroRNAs (miRNAs) compose a relatively new discipline in biomedical research, and many physiological processes in disease have been associated with changes in miRNA expression. Several studies report that miRNAs participate in biological processes such as the control of secondary injury in several disease models. Recently, we identified novel miRNAs that were abnormally up-regulated in a traumatic spinal cord injury (SCI). In the current study, we focused on miR20a, which causes continuing motor neuron degeneration when overexpressed in SCI lesions. Blocking miR20a in SCI animals led to neural cell survival and eventual neurogenesis with rescued expression of the key target gene, neurogenin 1 (Ngn1). Infusion of siNgn1 resulted in functional deficit in the hindlimbs caused by aggressive secondary injury and actively enhanced the inflammation involved in secondary injury progression. The events involving miR20a underlie motor neuron and myelin destruction and pathophysiology and ultimately block regeneration in injured spinal cords. Inhibition of miR20a expression effectively induced definitive motor neuron survival and neurogenesis, and SCI animals showed improved functional deficit. In this study, we showed that abnormal expression of miR20a induces secondary injury, which suggests that miR20a could be a potential target for therapeutic intervention following SCI.

Human umbilical cord blood-derived mesenchymal stem cells protect against neuronal cell death and ameliorate motor deficits in Niemann Pick type C1 mice.

Niemann Pick disease type C1 (NPC) is an autosomal recessive disease characterized by progressive neurological deterioration leading to premature death. In this study, we hypothesized that human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) have the multifunctional abilities to ameliorate NPC symptoms in the brain. To test this hypothesis, hUCB-MSCs were transplanted into the hippocampus of NPC mice in the early asymptomatic stage. This transplantation resulted in the recovery of motor function in the Rota Rod test and impaired cholesterol homeostasis leading to increased levels of cholesterol efflux-related genes such as LXRα, ABCA1, and ABCG5 while decreased levels of 3-hydroxy-3-methylglutaryl coenzyme A reductase were observed in NPC mice. In the cerebrum, hUCB-MSCs enhanced neuronal cell survival and proliferation, where they directly differentiated into electrically active MAP2-positive neurons as demonstrated by whole-cell patch clamping. In addition, we observed that hUCB-MSCs reduced Purkinje neuronal loss by suppression of inflammatory and apoptotic signaling in the cerebellum as shown by immunohistochemistry. We further investigated how hUCB-MSCs enhance cellular survival and inhibit apoptosis in NPC mice. Neuronal cell survival was associated with increased PI3K/AKT and JAK2/STAT3 signaling; moreover, hUCB-MSCs modulated the levels of GABA/glutamate transporters such as GAT1, EAAT2, EAAT3, and GAD6 in NPC mice as assessed by Western blot analysis. Taken together, our findings suggest that hUCB-MSCs might play multifunctional roles in neuronal cell survival and ameliorating motor deficits of NPC mice.

Vascular endothelial growth factor/kinase insult domain receptor (KDR)/fetal liver kinase 1 (FLK1)-mediated skin-epithelial progenitor cells reprogramming.

Vascular endothelial growth factor (VEGF) is an angiogenic protein that has effects on damaged neurons. We investigated VEGF function and found that it effectively induces the transition of skin-derived epithelial progenitor cells (EPCs) to more primitive stem cells. This change was accompanied by the epigenetic reprogramming of many genes. Among these genes are several stem-cell-associated transcription factors, such as Rex1, Oct4, Nanog, and Sox2. The VEGF-induced reprogramming of EPCs occurred through the FLK1 receptor and Janus kinase (JAK)/signal transducer- and activator of transcription 3 (STAT3) phosphorylation. When we engrafted VEGF-sensitized EPCs into sites of brain trauma, both engrafted VEGF/EPCs and endogenous cells showed functionally active neurogenesis and potent immunomodulatory function. These results indicate that VEGF actively induces the reprogramming of EPCs to become more primitive stem cells that display active cell growth, neurogenesis, migration, and survival behaviors, which may lead to a novel therapeutic strategy for central nervous system disorders.

DHP-derivative and low oxygen tension effectively induces human adipose stromal cell reprogramming.

BACKGROUND AND METHODS: In this study, we utilized a combination of low oxygen tension and a novel anti-oxidant, 4-(3,4-dihydroxy-phenyl)-derivative (DHP-d) to directly induce adipose tissue stromal cells (ATSC) to de-differentiate into more primitive stem cells. De-differentiated ATSCs was overexpress stemness genes, Rex-1, Oct-4, Sox-2, and Nanog. Additionally, demethylation of the regulatory regions of Rex-1, stemnesses, and HIF1alpha and scavenging of reactive oxygen species were finally resulted in an improved stem cell behavior of de-differentiate ATSC (de-ATSC). Proliferation activity of ATSCs after dedifferentiation was induced by REX1, Oct4, and JAK/STAT3 directly or indirectly. De-ATSCs showed increased migration activity that mediated by P38/JUNK and ERK phosphorylation. Moreover, regenerative efficacy of de-ATSC engrafted spinal cord-injured rats and chemical-induced diabetes animals were significantly restored their functions. CONCLUSIONS/SIGNIFICANCE: Our stem cell remodeling system may provide a good model which would provide insight into the molecular mechanisms underlying ATSC proliferation and transdifferentiation. Also, these multipotent stem cells can be harvested may provide us with a valuable reservoir of primitive and autologous stem cells for use in a broad spectrum of regenerative cell-based disease therapy.

Regulation of adipose tissue stromal cells behaviors by endogenic Oct4 expression control.

BACKGROUND: To clarify the role of the POU domain transcription factor Oct4 in Adipose Tissue Stromal Cells (ATSCs), we investigated the regulation of Oct4 expression and other embryonic genes in fully differentiated cells, in addition to identifying expression at the gene and protein levels. The ATSCs and several immature cells were routinely expressing Oct4 protein before and after differentiating into specific lineages. METHODOLOGY/PRINCIPAL FINDINGS AND CONCLUSIONS: Here, we demonstrated the role of Oct4 in ATSCs on cell proliferation and differentiation. Exogenous Oct4 improves adult ATSCs cell proliferation and differentiation potencies through epigenetic reprogramming of stemness genes such as Oct4, Nanog, Sox2, and Rex1. Oct4 directly or indirectly induces ATSCs reprogramming along with the activation of JAK/STAT3 and ERK1/2. Exogenic Oct4 introduced a transdifferentiation priority into the neural lineage than mesodermal lineage. Global gene expression analysis results showed that Oct4 regulated target genes which could be characterized as differentially regulated genes such as pluripotency markers NANOG, SOX2, and KLF4 and markers of undifferentiated stem cells FOXD1, CDC2, and EPHB1. The negatively regulated genes included FAS, TNFR, COL6A1, JAM2, FOXQ1, FOXO1, NESTIN, SMAD3, SLIT3, DKK1, WNT5A, BMP1, and GLIS3 which are implicated in differentiation processes as well as a number of novel genes. Finally we have demonstrated the therapeutic utility of Oct4/ATSCs were introduced into the mouse traumatic brain, engrafted cells was more effectively induces regeneration activity with high therapeutic modality than that of control ATSCs. Engrafted Oct4/ATSCs efficiently migrated and transdifferentiated into action potential carrying, functionally neurons in the hippocampus and promoting the amelioration of lesion cavities.

IFATS collection: Selenium induces improvement of stem cell behaviors in human adipose-tissue stromal cells via SAPK/JNK and stemness acting signals.

In the present study, the potential of selenium to enhance stem cell behavior through improvement of human adipose tissue-derived stromal cells (ATSCs) and the associated molecular mechanism was evaluated. Selenium-induced improvement in stem cell behavior of human ATSCs caused expression of several genes, indicating downregulated mature cell marker proteins coupled with increased cell growth and telomerase activities after the overexpression of Rex1, Nanog, OCT4, SOX2, KLF4, and c-Myc. Also, selenium-treated ATSCs significantly downregulated p53 and p21 tumor suppressor gene products. Selenium induced active growth and growth enhanced by the activation of signal proteins in ATSCs via the inhibition of reactive oxygen species-mediated phospho-stress-activated protein kinase/c-Jun N-terminal protein kinase activation. The selenium-induced activation of extracellular regulated kinases 1/2 and Akt in ATSCs resulted in a subsequent induction of the expression of stemness transcription factors, particularly Rex1, Nanog, and Oct4, along with definitive demethylation on regulatory regions of Rex-1, Nanog, and Oct4. The results of our small interfering RNA knockdown experiment showed that Rex1 plays a major role in the proliferation of selenium-induced ATSCs. Selenium-treated ATSCs also exhibited more profound differentiation into mesodermal and neural lineages. We performed a direct comparison of gene expression profiles in control ATSCs and selenium-treated ATSCs and delineated specific members of important growth factor, signaling, cell adhesion, and transcription factor families. The observations of improved life span and multipotency of selenium-treated ATSCs clearly indicate that selenium-treated ATSCs represent an extraordinarily useful candidate cell source for tissue regeneration. Disclosure of potential conflicts of interest is found at the end of this article.