An Intermediate Concentration of Calcium with Antioxidant Supplement in Culture Medium Enhances Proliferation and Decreases the Aging of Bone Marrow Mesenchymal Stem Cells.

Human bone marrow stem cells (HBMSCs) are isolated from the bone marrow. Stem cells can self-renew and differentiate into various types of cells. They are able to regenerate kinds of tissue that are potentially used for tissue engineering. To maintain and expand these cells under culture conditions is difficult-they are easily triggered for differentiation or death. In this study, we describe a new culture formula to culture isolated HBMSCs. This new formula was modified from NCDB 153, a medium with low calcium, supplied with 5% FBS, extra growth factor added to it, and supplemented with N-acetyl-L-cysteine and L-ascorbic acid-2-phosphate to maintain the cells in a steady stage. The cells retain these characteristics as primarily isolated HBMSCs. Moreover, our new formula keeps HBMSCs with high proliferation rate and multiple linage differentiation ability, such as osteoblastogenesis, chondrogenesis, and adipogenesis. It also retains HBMSCs with stable chromosome, DNA, telomere length, and telomerase activity, even after long-term culture. Senescence can be minimized under this new formulation and carcinogenesis of stem cells can also be prevented. These modifications greatly enhance the survival rate, growth rate, and basal characteristics of isolated HBMSCs, which will be very helpful in stem cell research.

Effect of Anadenanthera colubrina protease inhibitors as an antiinflamatory mediator.

This work aimed to obtain and characterize protease inhibitors from A. colubrina leaves and evaluate their potential as inflammatory mediator and cell viability. The protein extract was analyzed and characterized by SDS-PAGE, RP-HPLC-PDA, MALDI-TOF/MS and Zeta potential. Bioassays were conducted in order to evaluate the cell viability in RAW 264.7, in vitro (NO and TNF-α production inhibition) and in vivo anti-inflammatory potential, inhibition rate of trypsin and hemagglutination activity from protein extract. The results revealed the presence of bands at 14, 21 and 30 kDa in SDS-PAGE, the RP-HPLC-PDA analysis showed peaks at 12, 13, 28 and 40 minutes and MALDI-TOF/MS showed peaks with 3.4, 4.7, 5.6, 9.4 and 11.2 kDa. The protein extracts presented enzymatic activity inhibition of trypsin (IC50 59.2 µgmL-1), did not show any cytotoxicity to RAW264.7 cells, hemagglutination 8HU and insignificant reduction in NO and TNF-α production and reduced anti-inflammatory potential in vivo compared to dexamethasone.

Dendrosomal nanocurcumin promotes remyelination through induction of oligodendrogenesis in experimental demyelination animal model.

Multiple sclerosis (MS) is an autoimmune disease, associated with central nervous system (CNS) inflammation, demyelination, and axonal loss. Myelin, a multilayer membranous that covers nerve fibers, is essential for rapid impulse conduction. Oligodendrocytes that are generated either from CNS-resident oligodendrocyte progenitor cells (OPCs) or subventricular zone-derived neural stem cells (NSCs) are the myelinating cells of the CNS. The adult CNS maintains a certain endogenous potential to repair myelin damage. However, this process often fails as MS progresses. The origin of this failure is not fully understood, but it is likely to relate to progenitors/stem cells' arrestment in a quiescent state, incapable of generating new oligodendrocyte. Current treatments for MS are immunomodulatory or immunosuppressive medications, with little to no effect on myelin restoration. Recent studies have provided proof-of-principle that CNS remyelination can be promoted either via enhancing endogenous remyelination or by transplanting myelinating cells. Curcumin, a natural polyphenolic compound, has been shown to have therapeutic properties in several neurodegenerative diseases. Here, we investigated the effect of a curcumin nanoformulation, dendrosomal nanoparticles (DNC) on oligodendrogenesis and remyelination, both in vitro and in animal model of demyelination. We indicated that DNC enhanced oligodendrogenesis from NSCs and OPCs, in vitro in dose dependent manner. DNC also induced in vivo remyelination via promotion of oligodendrogenesis. Furthermore, DNC enhanced remyelination capacity of transplanted NSCs through promoting their survival and oligodendrogenesis capacity. Our findings suggest that DNC has significant beneficial effects in demyelinating conditions, either as mono-therapy or as being paired with transplantation approaches.

Conditional knockout of leptin receptor in neural stem cells leads to obesity in mice and affects neuronal differentiation in the hypothalamus early after birth.

Leptin, secreted by peripheral adipocytes, binds the leptin receptor (Lepr) in the hypothalamus, thereby contributing to the regulation of satiety and body weight. Lepr is expressed in the embryonic brain as early as embryonic day 12.5. However, the function of Lepr in neural precursor cells in the brain has not been resolved. To address this issue, we crossed the Leprflox/flox mice with each of Shh-Cre mice (Shh, sonic hedgehog) and Nestin (Nes)-Cre mice. We found that deletion of Lepr specifically in nestin-expressing cells led to extreme obesity, but the conditional null of Lepr in Shh-expressing cells had no obvious phenotype. Moreover, the level of leptin-activated pSTAT3 decreased in the anterior and central subregions of the arcuate hypothalamus of Shh-Cre; Leprflox/flox mice compared with the controls. By contrast, in Nes-Cre; Leprflox/flox mice, the level of leptin-activated pSTAT3 decreased in all subregions including the anterior, central, and posterior arcuate hypothalamus as well as the dorsomedial, ventromedial, and median eminence of the hypothalamus, revealing that the extensive lack of Lepr in the differentiated neurons of the hypothalamus in the conditional null mice. Notably, conditional deletion of Lepr in nestin-expressing cells enhanced the differentiation of neural precursor cells into neurons and oligodendroglia but inhibited differentiation into astrocytes early in postnatal development of hypothalamus. Our results suggest that Lepr expression in neural precursor cells is essential for maintaining normal body weight as well as the differentiation of neural precursor cells to the neural/glial fate in the hypothalamus shortly after birth.

ΔKi67 proliferation index as independent predictive and prognostic factor of outcome in luminal breast cancer: data from neoadjuvant letrozole-based treatment.

A key tool for monitoring breast cancer patients under neoadjuvant treatment is the identification of reliable predictive markers. Ki67 has been identified as a prognostic and predictive marker in ER-positive breast cancer. Ninety ER-positive, HER2 negative locally advanced breast cancer patients received letrozole (2.5 mg daily) and cyclophosphamide (50 mg daily) with/without Sorafenib (400 mg/bid daily) for 6 months before undergoing surgery. Ki67 expression and tumor size measured with caliber were determined at baseline, after 30 days of treatment and at the end of treatment. Patients were assigned to a clinical response category according to Response Evaluation Criteria in Solid Tumors, both at 30 days and before surgery and further classified as high-responder and low-responder according to the median variation of Ki67 values between biopsy and 30 days and between biopsy and surgery time. The predictive role of Ki67 and its changes with regard to clinical response and survival was analyzed. No differences in terms of survival outcomes emerged between the arms of treatment, while we observed a higher percentage of women with progression or stable disease in arm with the combination containing Sorafenib (20.5% vs 7.1%, p = 0.06). Clinical complete responders experienced a greater overall variation in Ki67 when compared with partial responders and patients with progressive/stable disease (66.7% vs 30.7%, p = 0.009). High responders showed a better outcome than low responders in terms of both disease-free survival (p = 0.009) and overall survival (p = 0.002). ΔKi67 score evaluated between basal and residual tumor at definitive surgery showed to be highly predictive of clinical complete response, and a potential parameter to be used for predicting disease-free survival and overall survival in luminal breast cancer treated with neoadjuvant endocrine-based therapy.

Effects of Biotin on survival, ensheathment, and ATP production by oligodendrocyte lineage cells in vitro.

Mechanisms implicated in disease progression in multiple sclerosis include continued oligodendrocyte (OL)/myelin injury and failure of myelin repair. Underlying causes include metabolic stress with resultant energy deficiency. Biotin is a cofactor for carboxylases involved in ATP production that impact myelin production by promoting fatty acid synthesis. Here, we investigate the effects of high dose Biotin (MD1003) on the functional properties of post-natal rat derived oligodendrocyte progenitor cells (OPCs). A2B5 positive OPCs were assessed using an in vitro injury assay, culturing cells in either DFM (DMEM/F12+N1) or "stress media" (no glucose (NG)-DMEM), with Biotin added over a range from 2.5 to 250 µg/ml, and cell viability determined after 24 hrs. Biotin reduced the increase in OPC cell death in the NG condition. In nanofiber myelination assays, biotin increased the percentage of ensheathing cells, the number of ensheathed segments per cell, and length of ensheathed segments. In dispersed cell culture, Biotin also significantly increased ATP production, assessed using a Seahorse bio-analyzer. For most assays, the positive effects of Biotin were observed at the higher end of the dose-response analysis. We conclude that Biotin, in vitro, protects OL lineage cells from metabolic injury, enhances myelin-like ensheathment, and is associated with increased ATP production.

Enterococcus faecium L-15 Extract Enhances the Self-Renewal and Proliferation of Mouse Skin-Derived Precursor Cells.

Lactic acid bacteria (LAB) in the gastrointestinal tract have beneficial health effects. LAB activate the proliferation of intestinal stem cells and speed the recovery of damaged intestinal cells, but little is known about effect of LAB on other adult stem cells. In this study, a cell-free extract of Enterococcus faecium L-15 (L15) was exposed to mouse skin-derived precursor cells (SKPs), and the changes in characteristics associated with proliferation and self-renewal capacity were investigated. L15 increased the size of the spheres and the proliferation rate of SKPs. Cell cycle analysis revealed that cells in the S-phase increased after treatment with L15. In the L15-treated group, the total number of spheres significantly increased. The expression level of pluripotency marker genes also increased, while the mesenchymal lineage-related differentiation marker genes significantly decreased in the L15-treated group. The PI3K/Akt signaling pathway was activated by L15 in SKPs. These results indicate that L15 enhances proliferation and self-renewal of SKPs and may be used as a supplement for stem cell maintenance or application of stem cell therapy. This is the first report to investigate the functional effects of E. faecium on the proliferation and self-renewal capacity of SKPs.

GPR30-Expressing Gastric Chief Cells Do Not Dedifferentiate But Are Eliminated via PDK-Dependent Cell Competition During Development of Metaplasia.

BACKGROUND & AIMS: Gastric chief cells, a mature cell type that secretes digestive enzymes, have been proposed to be the origin of metaplasia and cancer through dedifferentiation or transdifferentiation. However, studies supporting this claim have had technical limitations, including issues with the specificity of chief cell markers and the toxicity of drugs used. We therefore sought to identify genes expressed specifically in chief cells and establish a model to trace these cells. METHODS: We performed transcriptome analysis of Mist1-CreERT-traced cells, with or without chief cell depletion. Gpr30-rtTA mice were generated and crossed to TetO-Cre mice, and lineage tracing was performed after crosses to R26-TdTomato mice. Additional lineage tracing experiments were performed using Mist1-CreERT, Kitl-CreERT, Tff1-Cre, and Tff2-Cre mice crossed to reporter mice. Mice were given high-dose tamoxifen or DMP-777 or were infected with Helicobacter pylori to induce gastric metaplasia. We studied mice that expressed mutant forms of Ras in gastric cells, using TetO-KrasG12D, LSL-KrasG12D, and LSL-HrasG12V mice. We analyzed stomach tissues from GPR30-knockout mice. Mice were given dichloroacetate to inhibit pyruvate dehydrogenase kinase (PDK)-dependent cell competition. RESULTS: We identified GPR30, the G-protein-coupled form of the estrogen receptor, as a cell-specific marker of chief cells in gastric epithelium of mice. Gpr30-rtTA mice crossed to TetO-Cre;R26-TdTomato mice had specific expression of GPR30 in chief cells, with no expression noted in isthmus stem cells or lineage tracing of glands. Expression of mutant Kras in GPR30+ chief cells did not lead to the development of metaplasia or dysplasia but, instead, led to a reduction in labeled numbers of chief cells and a compensatory expansion of neck lineage, which was derived from upper Kitl+ clones. Administration of high-dose tamoxifen, DMP-777, or H pylori decreased the number of labeled chief cells. Chief cells were eliminated from epithelia via GPR30- and PDK-dependent cell competition after metaplastic stimuli, whereas loss of GRP30 or inhibition of PDK activity preserved chief cell numbers and attenuated neck lineage cell expansion. CONCLUSIONS: In tracing studies of mice, we found that most chief cells are lost during metaplasia and therefore are unlikely to contribute to gastric carcinogenesis. Expansion of cells that coexpress neck and chief lineage markers, known as spasmolytic polypeptide-expressing metaplasia, does not occur via dedifferentiation from chief cells but, rather, through a compensatory response from neck progenitors to replace the eliminated chief cells.

Expansion and preservation of the functional activity of adult hematopoietic stem cells cultured ex vivo with a histone deacetylase inhibitor.

Attempts to expand ex vivo the numbers of human hematopoietic stem cells (HSCs) without compromising their marrow repopulating capacity and their ability to establish multilineage hematopoiesis has been the subject of intense investigation. Although most such efforts have focused on cord blood HSCs, few have been applied to adult HSCs, a more clinically relevant HSC source for gene modification. To date, the strategies that have been used to expand adult HSCs have resulted in modest effects or HSCs with lineage bias and a limited ability to generate T cells in vivo. We previously reported that culturing umbilical cord blood CD34+ cells in serum-free media supplemented with valproic acid (VPA), a histone deacetylase inhibitor, and a combination of cytokines led to the expansion of the numbers of fully functional HSCs. In the present study, we used this same approach to expand the numbers of adult human CD34+ cells isolated from mobilized peripheral blood and bone marrow. This approach resulted in a significant increase in the numbers of phenotypically defined HSCs (CD34+CD45RA-CD90+D49f+). Cells incubated with VPA also exhibited increased aldehyde dehydrogenase activity and decreased mitochondrial membrane potential, each functional markers of HSCs. Grafts harvested from VPA-treated cultures were able to engraft in immune-deficient mice and, importantly, to generate cellular progeny belonging to each hematopoietic lineage in similar proportion to that observed with unmanipulated CD34+ cells. These data support the utility of VPA-mediated ex vivo HSC expansion for gene modification of adult HSCs.

Overview of Molecular Mechanisms Involved in Herbal Compounds for Inhibiting Osteoclastogenesis from Macrophage Linage RAW264.7.

Differentiation from RAW264.7 cells to osteoclasts rely on many signaling pathways, such as NF-κB, MAPK, Akt and others. However, the specific underlying mechanisms are not clear. Recently, much works have focused on the inhibitory effects of plant derived compounds in the differentiation from RAW264.7 to osteoclasts. However, the specific mechanisms remain unclear. In this paper, we summarize a lot of plant derived compounds which exert blocking effect on the progression of differentiation via signaling pathways.

Vitamin D increases remyelination by promoting oligodendrocyte lineage differentiation.

INTRODUCTION: Several experimental studies have suggested the potential remyelinating effects of vitamin D (VitD) supplements regardless of the presence of VitD deficiency. This study aims to analyze neurogenesis in a model of toxic demyelination in order to evaluate the effects of VitD on demyelination and remyelination. MATERIAL AND METHODS: We used 24 male Wistar rats that had received surgical lesions to the corpus callosum and were injected with lysolecithin. Rats were divided into three groups: Group 1 included eight rats with lesions to the corpus callosum but not lysolecithin injections (sham group), group 2 included eight rats with lesions to the corpus callosum that were injected with lysolecithin (lysolecithin group), and group 3 included eight rats with lesions that were injected with lysolecithin and received VitD (VitD group). We analyzed neurogenesis both in the subventricular zone and at the lesion site. RESULTS: Administration of VitD promotes the proliferation and differentiation of neural stem cells in the subventricular zone and the migration of these cells to the lesion site in the corpus callosum; these cells subsequently differentiate into oligodendrocyte lineage cells and produce myelin basic protein. This phenomenon was not caused by microglial activation, which was less marked in rats receiving VitD. Megalin expression did not increase at the lesion site, which suggests that VitD is internalized by other mechanisms. CONCLUSION: Our results support the hypothesis that regardless of the presence of VitD deficiency, treatment with VitD may contribute to remyelination by promoting the proliferation of oligodendrocyte precursor cells.

Intra-pancreatic tissue-derived mesenchymal stromal cells: a promising therapeutic potential with anti-inflammatory and pro-angiogenic profiles.

BACKGROUND: Human pancreata contain many types of cells, such as endocrine islets, acinar, ductal, fat, and mesenchymal stromal cells (MSCs). MSCs are important and shown to have a promising therapeutic potential to treat various disease conditions. METHODS: We investigated intra-pancreatic tissue-derived (IPTD) MSCs isolated from tissue fractions that are routinely discarded during pancreatic islet isolation of human cadaveric donors. Furthermore, whether pro-angiogenic and anti-inflammatory properties of these cells could be enhanced was investigated. RESULTS: IPTD-MSCs were expanded in GMP-compatible CMRL-1066 medium supplemented with 5% human platelet lysate (hPL). IPTD-MSCs were found to be highly pure, with > 95% positive for CD90, CD105, and CD73, and negative for CD45, CD34, CD14, and HLA-DR. Immunofluorescence staining of pancreas tissue demonstrated the presence of CD105+ cells in the vicinity of islets. IPTD-MSCs were capable of differentiation into adipocytes, chondrocytes, and osteoblasts in vitro, underscoring their multipotent features. When these cells were cultured in the presence of a low dose of TNF-α, gene expression of tumor necrosis factor alpha-stimulated gene-6 (TSG-6) was significantly increased, compared to control. In contrast, treating cells with dimethyloxallyl glycine (DMOG) (a prolyl 4-hydroxylase inhibitor) enhanced mRNA levels of nuclear factor erythroid 2-related factor 2 (NRF2) and vascular endothelial growth factor (VEGF). Interestingly, a combination of TNF-α and DMOG stimulated the optimal expression of all three genes in IPTD-MSCs. Conditioned medium of IPTD-MSCs treated with a combination of DMOG and TNF-α contained higher levels of pro-angiogenic (VEGF, IL-6, and IL-8) compared to controls, promoting angiogenesis of human endothelial cells in vitro. In contrast, levels of MCP-1, a pro-inflammatory cytokine, were reduced in the conditioned medium of IPTD-MSCs treated with a combination of DMOG and TNF-α. CONCLUSIONS: The results demonstrate that IPTD-MSCs reside within the pancreas and can be separated as part of a standard islet-isolation protocol. These IPTD-MSCs can be expanded and potentiated ex vivo to enhance their anti-inflammatory and pro-angiogenic profiles. The fact that IPTD-MSCs are generated in a GMP-compatible procedure implicates a direct clinical application.

Omega-3 Docosahexaenoic Acid Is a Mediator of Fate-Decision of Adult Neural Stem Cells.

The mammalian brain is enriched with lipids that serve as energy catalyzers or secondary messengers of essential signaling pathways. Docosahexaenoic acid (DHA) is an omega-3 fatty acid synthesized de novo at low levels in humans, an endogenous supply from its precursors, and is mainly incorporated from nutrition, an exogeneous supply. Decreased levels of DHA have been reported in the brains of patients with neurodegenerative diseases. Preventing this decrease or supplementing the brain with DHA has been considered as a therapy for the DHA brain deficiency that could be linked with neuronal death or neurodegeneration. The mammalian brain has, however, a mechanism of compensation for loss of neurons in the brain: neurogenesis, the birth of neurons from neural stem cells. In adulthood, neurogenesis is still present, although at a slower rate and with low efficiency, where most of the newly born neurons die. Neural stem/progenitor cells (NSPCs) have been shown to require lipids for proper metabolism for proliferation maintenance and neurogenesis induction. Recent studies have focused on the effects of these essential lipids on the neurobiology of NSPCs. This review aimed to introduce the possible use of DHA to impact NSPC fate-decision as a therapy for neurodegenerative diseases.

Mitochondrial Targeting of Antioxidants Alters Pancreatic Acinar Cell Bioenergetics and Determines Cell Fate.

Mitochondrial dysfunction is a core feature of acute pancreatitis, a severe disease in which oxidative stress is elevated. Mitochondrial targeting of antioxidants is a potential therapeutic strategy for this and other diseases, although thus far mixed results have been reported. We investigated the effects of mitochondrial targeting with the antioxidant MitoQ on pancreatic acinar cell bioenergetics, adenosine triphosphate (ATP) production and cell fate, in comparison with the non-antioxidant control decyltriphenylphosphonium bromide (DecylTPP) and general antioxidant N-acetylcysteine (NAC). MitoQ (µM range) and NAC (mM range) caused sustained elevations of basal respiration and the inhibition of spare respiratory capacity, which was attributable to an antioxidant action since these effects were minimal with DecylTPP. Although MitoQ but not DecylTPP decreased cellular NADH levels, mitochondrial ATP turnover capacity and cellular ATP concentrations were markedly reduced by both MitoQ and DecylTPP, indicating a non-specific effect of mitochondrial targeting. All three compounds were associated with a compensatory elevation of glycolysis and concentration-dependent increases in acinar cell apoptosis and necrosis. These data suggest that reactive oxygen species (ROS) contribute a significant negative feedback control of basal cellular metabolism. Mitochondrial targeting using positively charged molecules that insert into the inner mitochondrial member appears to be deleterious in pancreatic acinar cells, as does an antioxidant strategy for the treatment of acute pancreatitis.

Comparative study of different anticoagulants and coagulants in the evaluation of clinical application of platelet-rich plasma (PRP) standardization.

To investigate the impact of different anticoagulants and coagulants with autologous platelet-rich plasma (PRP) in order to evaluate the clinical application of PRP standardization. Bone marrow stem cells (BMSCs) were seeded into autologous PRP gel scaffolds with different anticoagulants (EDTA, heparin sodium HS, and sodium citrate SC) as well as control group (the whole blood group). Quality of PRP was evaluated and flow cytometric assay was used to detect the activity of the platelet (CD62p, PAC-1). BMSCs were also seeded into PRP with different coagulants (Thrombin, Collagen-I, ADP) as well as PRP un-activated (negative group) and L-DMEM complete culture without PRP (control group). The effects of different coagulants with PRP on proliferation, osteogenic differentiation of BMSCs were analyzed by methyl thiazolyl tetrazolium assay (MTT), ALP staining, Von Kossa staining, Confocal microscopic observation, RT-PCR and Western Blot at the morphological, cellular and molecular levels. Different anticoagulants (EDTA, HS, and SC) could affect the quality of PRP. EDTA group revealed the best quality and activity (CD62p, PAC-1). With different coagulants (Thrombin, Collagen-I and ADP) in the proliferation of BMSCs, the MTT assay showed that the proliferation of BMSCs was increased in all groups with time. On the sixth day of culture, the cell number of each PRP group was significantly higher than that in the control group (P < 0.05), while the most rapidly increasing was found in Collagen-I group. The cumulative release of growth factor (TGF-ß1, PDGF) at each time point in the PRP gel of the four groups was higher than that in the control group (P < 0.05). Collagen-I was considered as the best PRP coagulant. When thrombin was used as a platelet coagulant, the release of growth factor in PRP was rapid and direct, while the release of growth factor in Collagen-I-activated PRP was sustained and slow, and the total release of ADP-activated PRP growth factors was the lowest. The study demonstrated the similar outcome in osteogenic differentiation. In terms of gene expression and western bolt, the PCR results showed that the expression levels of OCN gene and RUNX2 protein in each PRP group were higher than that in the control group (P < 0.05). Different anticoagulants caused different degrees of lysis and spontaneous activation of platelets, which lead to different quality of PRP. Compared with HS and SC, EDTA could maintain the structural integrity of platelets, reduce their spontaneous activation, and increase the release of PRP growth factors for a longer period of time, thus ensuring the biomass of PRP. In addition, different coagulants also showed different results in the proliferation as well as osteogenic differentiation of BMSCs. Compared with Thrombin and ADP, Collagen-I may be a better choice.

Effect of Qinghuang Powder () Combined with Bupi Yishen Decoction () in Treating Patients with Refractory Cytopenia with Multilineage Dysplasia through Regulating DNA Methylation.

OBJECTIVE: To explore the effect of Qinghuang Powder (QHP,()combined with Bupi Yishen Decoction (BPYS, ) on myelodysplastic syndromes (MDS) patients with refractory cytopenia with multilineage dysplasia (RCMD) and determine the change of DNA methylation in MDS-RCMD patients after the treatment of Chinese medicine formula. METHODS: All 308 MDS-RCMD patients were treated with QHP combined with BPYS for 2 months at least, absolute neutrophil count (ANC), hemoglobin (Hb), platelets (PLT), primitive bone marrow cells and chromosome karyotype were chosen as the main evaluation indexes to analyze the treatment effect according to criteria from the MDS International Working Group. Then 43 bone marrow samples from 15 MDS-RCMD patients and 28 healthy donors were obtained for the examination of DNA methylation. Gene Ontology (GO) and Pathway analysis were applied to analyze the methylation data. RESULTS: The overall MDS response rate to QHP was 61.68% (190/360) including hematologic improvement-neutrophil (HI-N) or hematologic improvement-erythroid (HI-E) or hematologic improvement-platelet (HI-P). Patients with anemia had a better response rate than patients with neutropenia or thrombocypenia (55.88% vs 31.54% or 55.88% vs. 36.9%). The DNA methylation microarray analysis disclosed that 4,257 hypermethylated genes were demethylated upon the treatment with QHP and BPYS. GO analysis and Pathway analysis showed that these demethylated genes were involved in a lot of tumor-related pathways and functions. CONCLUSIONS: QHP combined with BPYS could effectively treat MDS-RCMD patients through hematologic improvement (HI-N, HI-P or HI-E) and PLT and RBC transfusion independence due to the demethylation, thereby providing another choice for the treatment of patients with MDS-RCMD.

Transcription factor induced conversion of human fibroblasts towards the hair cell lineage.

Hearing loss is the most common sensorineural disorder, affecting over 5% of the population worldwide. Its most frequent cause is the loss of hair cells (HCs), the mechanosensory receptors of the cochlea. HCs transduce incoming sounds into electrical signals that activate auditory neurons, which in turn send this information to the brain. Although some spontaneous HC regeneration has been observed in neonatal mammals, the very small pool of putative progenitor cells that have been identified in the adult mammalian cochlea is not able to replace the damaged HCs, making any hearing impairment permanent. To date, guided differentiation of human cells to HC-like cells has only been achieved using either embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs). However, use of such cell types suffers from a number of important disadvantages, such as the risk of tumourigenicity if transplanted into the host´s tissue. We have obtained cells expressing hair cell markers from cultures of human fibroblasts by overexpression of GFI1, Pou4f3 and ATOH1 (GPA), three genes that are known to play a critical role in the development of HCs. Immunocytochemical, qPCR and RNAseq analyses demonstrate the expression of genes typically expressed by HCs in the transdifferentiated cells. Our protocol represents a much faster approach than the methods applied to ESCs and iPSCs and validates the combination of GPA as a set of genes whose activation leads to the direct conversion of human somatic cells towards the hair cell lineage. Our observations are expected to contribute to the development of future therapies aimed at the regeneration of the auditory organ and the restoration of hearing.

By Targeting Atg7 MicroRNA-143 Mediates Oxidative Stress-Induced Autophagy of c-Kit+ Mouse Cardiac Progenitor Cells.

Therapeutic efficiency of cardiac progenitor cells (CPCs) transplantation is limited by its low survival and retention in infarcted myocardium. Autophagy plays a critical role in regulating cell death and apoptosis, but the role of microRNAs (miRNAs) in oxidative stress-induced autophagy of CPCs remains unclear. This study aimed to explore if miRNAs mediate autophagy of c-kit+ CPCs. We found that the silencing of miR-143 promoted the autophagy of c-kit+ CPCs in response to H2O2, and the protective effect of miR-143 inhibitor was abrogated by autophagy inhibitor 3-methyladenine (3-MA). Furthermore, autophagy-related gene 7 (Atg7) was identified as the target gene of miR-143 by dual luciferase reporter assays. In vivo, after transfection with miR-143 inhibitor, c-kit+ CPCs from green fluorescent protein transgenic mice were more observed in infarcted mouse hearts. Moreover, transplantation of c-kit+ CPCs with miR-143 inhibitor improved cardiac function after myocardial infarction. Take together, our study demonstrated that miR-143 mediates oxidative stress-induced autophagy to enhance the survival of c-kit+ CPCs by targeting Atg7, which will provide a complementary approach for improving CPC-based heart repair.

miR-219 Cooperates with miR-338 in Myelination and Promotes Myelin Repair in the CNS.

A lack of sufficient oligodendrocyte myelination contributes to remyelination failure in demyelinating disorders. miRNAs have been implicated in oligodendrogenesis; however, their functions in myelin regeneration remained elusive. Through developmentally regulated targeted mutagenesis, we demonstrate that miR-219 alleles are critical for CNS myelination and remyelination after injury. Further deletion of miR-338 exacerbates the miR-219 mutant hypomyelination phenotype. Conversely, miR-219 overexpression promotes precocious oligodendrocyte maturation and regeneration processes in transgenic mice. Integrated transcriptome profiling and biotin-affinity miRNA pull-down approaches reveal stage-specific miR-219 targets in oligodendrocytes and further uncover a novel network for miR-219 targeting of differentiation inhibitors including Lingo1 and Etv5. Inhibition of Lingo1 and Etv5 partially rescues differentiation defects of miR-219-deficient oligodendrocyte precursors. Furthermore, miR-219 mimics enhance myelin restoration following lysolecithin-induced demyelination as well as experimental autoimmune encephalomyelitis, principal animal models of multiple sclerosis. Together, our findings identify context-specific miRNA-regulated checkpoints that control myelinogenesis and a therapeutic role for miR-219 in CNS myelin repair.

Osteogenic differentiation of human gingival mesenchymal stem cells by Aristolochia bracteolata supplementation through enhanced Runx2 expression.

The phenotypic characteristics of human gingival derived mesenchymal stem cells (HGMSCs) on induction with total methanol extract of Aristolochia bracteolata have been evaluated. HGMSCs were cultured in control and two different induction medium: Control medium (basal medium), OM1 (Standard induction medium), and OM2 (100 µg/ml of A. bracteolata). Osteogenic differentiation of the cultured cells was assessed by studying the calcium deposition and osteoblastic gene expression. OM2 medium showed an enhanced osteogenic differentiation potential than OM1 as measured by increased calcium deposition and elevated expression of Runx2, osteopontin, osteonectin, osteocalcin, Collagen type I, and ALP levels in comparison with OM1 differentiated cells. We conclude that at 100 µg/ml A. bracteolata has induced HGMSC differentiation into osteogenic lineage consequent to enhanced Runx2 expression and related osteogenic genes.