Inhibition of DNA methyltransferases and histone deacetylases induces astrocytic differentiation of neural progenitors

AbstractUnderstanding how to specify rapid differentiation of human neural progenitor towards enriched non-transformed human astrocyte progenitors will provide a critical cell source to further our understanding of how astrocytes play a pivotal role in neural function and development. Human neural progenitors derived from pluripotent embryonic stem cells and propagated in adherent serum-free cultures provide a fate restricted renewable source for quick production of neural cells; however, such cells are highly refractive to astrocytogenesis and show a strong neurogenic bias, similar to neural progenitors from the early embryonic central nervous system (CNS). We found that several astrocytic genes are hypermethylated in such progenitors potentially preventing generation of astrocytes and leading to the proneuronal fate of these progenitors. However, epigenetic modification by Azacytidine (Aza-C) and Trichostatin A (TSA), with concomitant signaling from BMP2 and LIF in neural progenitor cultures shifts this bias, leading to expression of astrocytic markers as early as 5days of differentiation, with near complete suppression of neuronal differentiation. The resultant cells express major astrocytic markers, are amenable to co-culture with neurons, can be propagated as astrocyte progenitors and are cryopreservable. Although previous reports have generated astrocytes from pluripotent cells, the differentiation required extensive culture or selection based on cell surface antigens. The development of a label free and rapid differentiation process will expedite future derivation of astrocytes from various sources pluripotent cells including, but not limited to, human astrocytes associated with various neurological diseases

Porcine induced pluripotent stem cells produce chimeric offspring

Ethical and moral issues rule out the use of human induced pluripotent stem cells (iPSCs) in chimera studies that would determine the full extent of their reprogrammed state, instead relying on less rigorous assays such as teratoma formation and differentiated cell types. To date, only mouse iPSC lines are known to be truly pluripotent. However, initial mouse iPSC lines failed to form chimeric offspring, but did generate teratomas and differentiated embryoid bodies, and thus these specific iPSC lines were not completely reprogrammed or truly pluripotent. Therefore, there is a need to address whether the reprogramming factors and process used eventually to generate chimeric mice are universal and sufficient to generate reprogrammed iPSC that contribute to chimeric offspring in additional species. Here we show that porcine mesenchymal stem cells transduced with 6 human reprogramming factors (POU5F1, SOX2, NANOG, KLF4, LIN28, and C-MYC) injected into preimplantation-stage embryos contributed to multiple tissue types spanning all 3 germ layers in 8 of 10 fetuses. The chimerism rate was high, 85.3% or 29 of 34 live offspring were chimeras based on skin and tail biopsies harvested from 2-to 5-day-old pigs. The creation of pluripotent porcine iPSCs capable of generating chimeric offspring introduces numerous opportunities to study the facets significantly affecting cell therapies, genetic engineering, and other aspects of stem cell and developmental biology

Integrated analysis of long-noncoding RNA and circular RNA expression in Peste-des-Petits-Ruminants Virus (PPRV) infected marmoset B lymphocyte (B95a) cells

Not AvailablePeste-des-Petits-Ruminants (PPR) or goat plague is an important viral disease of sheep and goats caused by the small ruminant morbilli virus or PPR virus (PPRV). Long non coding RNAs (lncRNA) and circular RNAs (circRNA) play a pivotal role in several biological processes including regulation of virus-host interactions. The present study explored the expression of lncRNA, circRNA and their functions in PPRV infected B-lymphocyte (B95a) cells. The results revealed a total of 4531 lncRNA and 2348 circRNA expression in both mock and PPRV infected samples. Analysis of differentially expressed (DE) RNA identified 123 DE-lncRNA and 39 DE-circRNA as significantly dysregulated. Functional analysis of cis-target genes of DE-lncRNA indicated activation of TCF dependent WNT signaling and PKN1 stimulated transcription process. Interactions (sponging) of microRNA (miRNA) revealed 344 DE-lncRNA-miRNA and 93 DE-circRNA-miRNA pairs. The competing endogenous RNA (ceRNA) network of lncRNA/circRNA-miRNA-mRNA in PPRV infected B95a cells was represented by 69 ceRNA pairs. We validated the DE-circRNA by targeted amplification and sequencing of back spliced junctions (BSJs). The present study revealed a profile of lncRNA, circRNA and their potential ceRNA network in PPRV infection. The results provide insight for better understanding of PPRV-host interactions.DBT/Wellcome Trust India Alliance Grant no: IA/E/17/1/503689 awarded to Basavaraj Sajjanar

<i><span style="font-size:11.0pt;font-family: "Times New Roman";mso-fareast-font-family:"Times New Roman";mso-bidi-font-family: Mangal;mso-ansi-language:EN-GB;mso-fareast-language:EN-US;mso-bidi-language: HI" lang="EN-GB">In vitro</span></i><span style="font-size:11.0pt;font-family: "Times New Roman";mso-fareast-font-family:"Times New Roman";mso-bidi-font-family: Mangal;mso-ansi-language:EN-GB;mso-fareast-language:EN-US;mso-bidi-language: HI" lang="EN-GB"> cloning of canine parvovirus NS1 gene and reporter gene GFP in eukaryotic expression vector pVIVO2-mcs and characterization of the double gene construct in mammalian cells</span>

41-46The use of chemotherapy and/or radiotherapy for treatment of cancer is limited due to genotoxic side effects on healthy cells, involvement of anti-apoptotic signal transduction pathways that prevent cell death, and requirement of functional p53 for induction of apoptosis in cancerous cells. Efforts are beings made worldwide to develop new anticancer therapies as an alternative to chemotherapy. And viral gene therapy is one of the most potent therapeutics that is being ventured worldwide. Canine parvovirus-2 (CPV-2) is one of those viruses that have an inherent oncolytic property. The non-structural protein-1 (NS1 protein) of CPV-2 plays a major role in parvoviral cytotoxicity and pathogenicity in permissive cells. The oncolytic potential of CPV2-NS1 has been established in vitro. Prior to taking up the in vivo studies, the present study was undertaken to clone Canine Parvovirus NS1 gene and reporter gene GFP in eukaryotic expression vector pVIVO2-mcs, and to characterize the double construct in mammalian cells. The genes were successfully cloned in pVIVO2-mcs and characterized for their expression as demonstrated by fluorescence microscopy and immunofluorescence staining. This characterized double gene construct will further be used to evaluate the oncolytic potential of CPV-2 NS1 in experimentally induced in vivo tumour model

Genome-wide expression analysis reveals different heat shock responses in indigenous (Bos indicus) and crossbred (Bos indicus X Bos taurus) cattle

Abstract Environmental heat stress in dairy cattle leads to poor health, reduced milk production and decreased reproductive efficiency. Multiple genes interact and coordinate the response to overcome the impact of heat stress. The present study identified heat shock regulated genes in the peripheral blood mononuclear cells (PBMC). Genome-wide expression patterns for cellular stress response were compared between two genetically distinct groups of cattle viz., Hariana (B. indicus) and Vrindavani (B. indicus X B. taurus). In addition to major heat shock response genes, oxidative stress and immune response genes were also found to be affected by heat stress. Heat shock proteins such as HSPH1, HSPB8, FKB4, DNAJ4 and SERPINH1 were up-regulated at higher fold change in Vrindavani compared to Hariana cattle. The oxidative stress response genes (HMOX1, BNIP3, RHOB and VEGFA) and immune response genes (FSOB, GADD45B and JUN) were up-regulated in Vrindavani whereas the same were down-regulated in Hariana cattle. The enrichment analysis of dysregulated genes revealed the biological functions and signaling pathways that were affected by heat stress. Overall, these results show distinct cellular responses to heat stress in two different genetic groups of cattle. This also highlight the long-term adaptation of B. indicus (Hariana) to tropical climate as compared to the crossbred (Vrindavani) with mixed genetic makeup (B. indicus X B. taurus)