Probing the influence of strontium doping and annealing temperature on the structure and biocompatibility of hydroxyapatite nanorods.

Among numerous biologically important metal cations, strontium (Sr2+) has received much attention in bone tissue regeneration because of its osteoinductive properties combined with its ability to inhibit osteoclast activity. In this study, strontium-doped hydroxyapatite (Sr-HAp) nanorods with varying molar ratios of Ca : Sr (10 : 0, 9 : 1, 5 : 5, 3 : 7 and 0 : 10) were synthesized using the chemical precipitation technique. The synthesized Sr-HAp nanostructures were characterized using powder X-ray diffraction (XRD), field emission scanning electron microscopy (FESEM), transmission electron microscopy, energy dispersive X-ray spectroscopy, and Raman and Fourier transform infrared (FTIR) spectroscopies to understand their structural and morphological features, and composition. XRD results revealed the formation of HAp nanostructures, whose unit cell volume increased as a function of the dopant level. The reaction process investigation showed the formation of hydroxyapatite (HAp), strontium apatite (SAp) and various Sr-HAp phases. FESEM micrographs displayed the morphological transformation of Sr-HAp from nanorods to nanosheets upon increasing the dopant level. In the FTIR spectra, the bands of the PO43- group shifted towards a lower wavenumber upon increasing the dopant concentration in Sr-HAp that signifies the structural distortion due to the presence of a large amount of strontium ions. The peaks of PO43- and OH- vibrations in the Raman spectra were further analysed to corroborate the structural distortion of Sr-HAp. Selected area electron diffraction patterns obtained using TEM reveal the reduced crystallinity of Sr-HAp due to Sr-doping, which is in line with the XRD results. Finally, the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay showed that the synthesized Sr-HAp has no toxic effect on the survival and growth of mesenchymal stem cells. In summary, the synthesized novel Sr-HAp nanorods exhibit great promise for bone tissue engineering applications.

Th17 cell promotes apoptosis of IL-23R+ neurons in experimental autoimmune encephalomyelitis.

Myelin antigen-reactive Th1 and Th17 cells are critical drivers of central nervous system (CNS) autoimmune inflammation. Transcription factors T-bet and RORγt play a crucial role in the differentiation and function of Th1 and Th17 cells, and impart them a pathogenic role in CNS autoimmune inflammation. Mice deficient in these two factors do not develop experimental autoimmune encephalomyelitis (EAE). While T-bet and RORγt are known to regulate the expression of several cell adhesion and migratory molecules in T cells, their role in supporting Th1 and Th17 trafficking to the CNS is not completely understood. More importantly, once Th1 and Th17 cells reach the CNS, how the function of these transcription factors modulates the local inflammatory response during EAE is unclear. In the present study, we showed that myelin oligodendrocyte glycoprotein 35-55 peptide (MOG35-55)-specific Th1 cells deficient in RORγt could cross the blood-brain barrier (BBB) but failed to induce demyelination, apoptosis of neurons, and EAE. Pathogenic Th17 cell-derived cytokines GM-CSF, TNF-α, IL-17A, and IL-21 significantly increased the surface expression of IL-23R on neuronal cells. Furthermore, we showed that, in EAE, neurons in the brain and spinal cord express IL-23R. IL-23-IL-23R signaling in neuronal cells caused phosphorylation of STAT3 (Ser727 and Tyr705) and induced cleaved caspase 3 and cleaved poly (ADP-ribose) polymerase-1 (PARP-1) molecules in an IL-23R-dependent manner and caused apoptosis. Thus, we provided a mechanism showing that T-bet is required to recruit pathogenic Th17 cells to the CNS and RORγt-mediated inflammatory response to drive the apoptosis of IL-23R+ neurons in the CNS and cause EAE. Understanding detailed molecular mechanisms will help to design better strategies to control neuroinflammation and autoimmunity. ONE SENTENCE SUMMARY: IL-23-IL-23R signaling promotes apoptosis of CNS neurons.

Investigation of human hair keratin-based nanofibrous scaffold for skin tissue engineering application.

Keratin-based nanofibers were fabricated using the electrospinning technique, and their potential as scaffolds for tissue engineering was investigated. Keratin, extracted from the human hair, was blended with poly(vinyl alcohol) (PVA) in an aqueous medium. Morphological characterizations of the fabricated PVA-keratin nanofiber (PK-NF) random and aligned scaffolds performed using a scanning electron microscope (SEM) revealed the formation of uniform and randomly oriented nanofibers with an interconnected three-dimensional network structure. The mean diameter of the nanofibers ranged from 100 to 250 nm. Functional groups and structural studies were done by infrared spectroscopy (FTIR) and X-ray diffraction (XRD) analysis. FTIR study suggested that PVA interacted with keratin by hydrogen bonding. Moreover, the in vitro cell culture study could suggest that PK-NF scaffolds were non-cytotoxic by supporting the growth of murine embryonic stem cells (ESCs), human keratinocytes (HaCaT), and dermal fibroblast (NHDF) cell lines. Further, the immunocytochemical characterization revealed the successful infiltration, adhesion, and growth of ESCs, HaCaT, and NHDF cells seeded on PK-NF scaffolds. However, there was no noteworthy difference observed concerning cell growth and viability irrespective of the random and aligned internal fibril arrangement of the PK-NF scaffolds. The infiltration and growth pattern of HaCaT and NHDF cells adjacent to each other in a 3D co-culture study mimicked that of epidermal and dermal skin cells and indeed underscored the potential of PK-NFs as a scaffold for skin tissue engineering.

Fabrication and characterization of ceramic-polymer composite 3D scaffolds and demonstration of osteoinductive propensity with gingival mesenchymal stem cells.

The fabrication of biomaterial 3D scaffolds for bone tissue engineering applications involves the usage of metals, polymers, and ceramics as the base constituents. Notwithstanding, the composite materials facilitating enhanced osteogenic differentiation/regeneration are endorsed as the ideally suited bone grafts for addressing critical-sized bone defects. Here, we report the successful fabrication of 3D composite scaffolds mimicking the ECM of bone tissue by using ∼30 wt% of collagen type I (Col-I) and ∼70 wt% of different crystalline phases of calcium phosphate (CP) nanomaterials [hydroxyapatite (HAp), beta-tricalcium phosphate (ßTCP), biphasic hydroxyapatite (ßTCP-HAp or BCP)], where pH served as the sole variable for obtaining these CP phases. The different Ca/P ratio and CP nanomaterials orientation in these CP/Col-I composite scaffolds not only altered the microstructure, surface area, porosity with randomly oriented interconnected pores (80-450 µm) and mechanical strength similar to trabecular bone but also consecutively influenced the bioactivity, biocompatibility, and osteogenic differentiation potential of gingival-derived mesenchymal stem cells (gMSCs). In fact, BCP/Col-I, as determined from micro-CT analysis, achieved the highest surface area (∼42.6 m2 g-1) and porosity (∼85%), demonstrated improved bioactivity and biocompatibility and promoted maximum osteogenic differentiation of gMSCs among the three. Interestingly, the released Ca2+ ions, as low as 3 mM, from these scaffolds could also facilitate the osteogenic differentiation of gMSCs without even subjecting them to osteoinduction, thereby attesting these CP/Col-I 3D scaffolds as ideally suited bone graft materials.

Elucidating the Anti-Tumorigenic Efficacy of Oltipraz, a Dithiolethione, in Glioblastoma.

Glioblastoma multiforme (GBM), the most aggressive primary brain tumor, displays a highly infiltrative growth pattern and remains refractory to chemotherapy. Phytochemicals carrying specificity and low cytotoxicity may serve as potent and safer alternatives to conventional chemotherapy for treating GBM. We have evaluated the anticancer effects of Oltipraz (Olt), a synthetic dithiolethione found in many vegetables, including crucifers. While Olt exposure was non-toxic to the HEK-293 cell line, it impaired the cell growth in three GBM cell lines (LN18, LN229, and U-87 MG), arresting those at the G2/M phase. Olt-exposed GBM cells induced the generation of reactive oxygen species (ROS), mitochondrial depolarization, caspase 3/7-mediated apoptosis, nuclear condensation, and DNA fragmentation, and decreased glutathione, a natural ROS scavenger, as well as vimentin and ß-catenin, the EMT-associated markers. Its effect on a subpopulation of GBM cells exhibiting glioblastoma stem cell (GSCs)-like characteristics revealed a reduced expression of Oct4, Sox2, CD133, CD44, and a decrease in ALDH+, Nestin+ and CD44+ cells. In contrast, there was an increase in the expression of GFAP and GFAP+ cells. The Olt also significantly suppressed the oncosphere-forming ability of cells. Its efficacy was further validated in vivo, wherein oral administration of Olt could suppress the ectopically established GBM tumor growth in SCID mice. However, there was no alteration in body weight, organ ratio, and biochemical parameters, reflecting the absence of any toxicity otherwise. Together, our findings could demonstrate the promising chemotherapeutic efficacy of Olt with potential implications in treating GBM.

Leishmania intercepts IFN-γR signaling at multiple levels in macrophages.

IFN-γ, a type 2 interferon and a cytokine, is critical for both innate and adaptive immunity. IFN-γ binds to the IFN-γRs on the cell membrane of macrophages, signals through JAK1-STAT-1 pathway and induces IFN-γ-stimulated genes (ISGs). As Leishmania amastigotes reside and replicate within macrophages, IFN-γ mediated macrophage activation eventuate in Leishmania elimination. As befits the principle of parasitism, the impaired IFN-γ responsiveness in macrophages ensures Leishmania survival. IFN-γ responsiveness is a function of integrated molecular events at multiple levels in the cells that express IFN-γ receptors. In Leishmania-infected macrophages, reduced IFN-γRα expression, impaired IFN-γRα and IFN-γRß hetero-dimerization due to altered membrane lipid composition, reduced JAK-1 and STAT-1 phosphorylation but increased STAT-1 degradation and impaired ISGs induction collectively determine the IFN-γ responsiveness and the efficacy of IFN-γ induced antileishmanial function of macrophages. Therefore, parasite load is not only decided by the levels of IFN-γ produced but also by the IFN-γ responsiveness. Indeed, in Leishmania-infected patients, IFN-γ is produced but IFN-γ signalling is downregulated. However, the molecular mechanisms of IFN-γ responsiveness remain unclear. Therefore, we review the current understanding of IFN-γ responsiveness of Leishmania-infected macrophages.

Cancer stem cells and tumor heterogeneity: Deciphering the role in tumor progression and metastasis.

Tumor heterogeneity, in principle, reflects the variation among different cancer cell populations. It can be termed inter- or intra-tumoral heterogeneity, respectively, based on its occurrence in various tissues from diverse patients or within a single tumor. The intra-tumoral heterogeneity is one of the leading causes of cancer progression and treatment failure, with the cancer stem cells (CSCs) contributing immensely to the same. These niche cells, similar to normal stem cells, possess the characteristics of self-renewal and differentiation into multiple cell types. Moreover, CSCs contribute to tumor growth and surveillance by promoting recurrence, metastasis, and therapeutic resistance. Diverse factors, including intracellular signalling pathways and tumor microenvironment (TME), play a vital role in regulating these CSCs. Although a panel of markers is considered to identify the CSC pool in various cancers, further research is needed to discriminate cancer-specific CSC markers in those. CSCs have also been found to be promising therapeutic targets for cancer therapy. Several small molecules, natural compounds, antibodies, chimeric antigen receptor T (CAR-T) cells, and CAR-natural killer (CAR-NK) cells have emerged as therapeutic tools for specific targeting of CSCs. Interestingly, many of these are in clinical trials too. Despite being a much-explored avenue of research for years, and we have come to understand its nitty-gritty, there is still a tremendous gap in our knowledge concerning its precise genesis and regulation. Hence, a concrete understanding is needed to assess the CSC-TME link and how to target different cancer-specific CSCs by designing newer tools. In this review, we have summarized CSC, its causative, different pathways and factors regulating its growth, association with tumor heterogeneity, and last but not least, discussed many of the promising CSC-targeted therapies for combating cancer metastasis.

Tissue-Restricted Stem Cells as Starting Cell Source for Efficient Generation of Pluripotent Stem Cells: An Overview.

Induced pluripotent stem cells (iPSCs) have vast biomedical potential concerning disease modeling, drug screening and discovery, cell therapy, tissue engineering, and understanding organismal development. In the year 2006, a groundbreaking study reported the generation of iPSCs from mouse embryonic fibroblasts by viral transduction of four transcription factors, namely, Oct4, Sox2, Klf4, and c-Myc. Subsequently, human iPSCs were generated by reprogramming fibroblasts as a starting cell source using two reprogramming factor cocktails [(i) OCT4, SOX2, KLF4, and c-MYC, and (ii) OCT4, SOX2, NANOG, and LIN28]. The wide range of applications of these human iPSCs in research, therapeutics, and personalized medicine has driven the scientific community to optimize and understand this reprogramming process to achieve quality iPSCs with higher efficiency and faster kinetics. One of the essential criteria to address this is by identifying an ideal cell source in which pluripotency can be induced efficiently to give rise to high-quality iPSCs. Therefore, various cell types have been studied for their ability to generate iPSCs efficiently. Cell sources that can be easily reverted to a pluripotent state are tissue-restricted stem cells present in the fetus and adult tissues. Tissue-restricted stem cells can be isolated from fetal, cord blood, bone marrow, and other adult tissues or can be obtained by differentiation of embryonic stem cells or trans-differentiation of other tissue-restricted stem cells. Since these cells are undifferentiated cells with self-renewal potential, they are much easier to reprogram due to the inherent characteristic of having an endogenous expression of few pluripotency-inducing factors. This review presents an overview of promising tissue-restricted stem cells that can be isolated from different sources, namely, neural stem cells, hematopoietic stem cells, mesenchymal stem cells, limbal epithelial stem cells, and spermatogonial stem cells, and their reprogramming efficacy. This insight will pave the way for developing safe and efficient reprogramming strategies and generating patient-specific iPSCs from tissue-restricted stem cells derived from various fetal and adult tissues.

An Overview on Promising Somatic Cell Sources Utilized for the Efficient Generation of Induced Pluripotent Stem Cells.

Human induced Pluripotent Stem Cells (iPSCs) have enormous potential in understanding developmental biology, disease modeling, drug discovery, and regenerative medicine. The initial human iPSC studies used fibroblasts as a starting cell source to reprogram them; however, it has been identified to be a less appealing somatic cell source by numerous studies due to various reasons. One of the important criteria to achieve efficient reprogramming is determining an appropriate starting somatic cell type to induce pluripotency since the cellular source has a major influence on the reprogramming efficiency, kinetics, and quality of iPSCs. Therefore, numerous groups have explored various somatic cell sources to identify the promising sources for reprogramming into iPSCs with different reprogramming factor combinations. This review provides an overview of promising easily accessible somatic cell sources isolated in non-invasive or minimally invasive manner such as keratinocytes, urine cells, and peripheral blood mononuclear cells used for the generation of human iPSCs derived from healthy and diseased subjects. Notably, iPSCs generated from one of these cell types derived from the patient will offer ethical and clinical advantages. In addition, these promising somatic cell sources have the potential to efficiently generate bona fide iPSCs with improved reprogramming efficiency and faster kinetics. This knowledge will help in establishing strategies for safe and efficient reprogramming and the generation of patient-specific iPSCs from these cell types.

Enhancement of hydrophilicity, biocompatibility and biodegradability of poly(ε-caprolactone) electrospun nanofiber scaffolds using poly(ethylene glycol) and poly(L-lactide-co-ε-caprolactone-co-glycolide) as additives for soft tissue engineering.

In this study, poly(ε-caprolactone) (PCL) has been blended with a more hydrophilic poly(ethylene glycol) (PEG) and with a biocompatible block-co-polymer: poly(L-lactide-co-ε-caprolactone-co-glycolide) (PLCG) in order to improve hydrophilicity, biocompatibility and biodegradability of PCL. PCL and the blend solutions were subjected to electrospinning to produce nanofiber scaffolds by the addition of only 1 wt% of PEG and PLCG either singly or in combination in PCL to retain the mechanical properties of the scaffolds. PCL-PEG-PLCG ternary and two binary (PCL-PEG and PCL-PLCG) blend nanofiber scaffolds have been prepared for comparison. The resulting nanofibers showed a smooth and flaw-free surface and the diameter of the nanofibers displayed a normal distribution. The PCL-PEG nanofiber scaffold showed improved hydrophilicity [water contact angle (WCA) ∼84°] over pristine PCL (WCA ∼127°); while PCL-PLCG and PCL-PEG-PLCG scaffolds exhibited absolute wetting by water, likely due to high porosity. In vitro biocompatibility studies using gingival mesenchymal stem cells (gMSCs) suggested that, both the PCL and the blend scaffolds were biocompatible supporting cell-viability and growth of gMSCs following their seeding on these scaffolds. Biodegradation studies in phosphate buffer solution showed that the addition of PEG and PLCG in PCL increased the weight loss of scaffolds with time, indicating higher extent of biodegradation in the blend scaffolds and the weight loss followed the power law curve with time.

Glycogen synthase kinase 3ß inhibitor- CHIR 99021 augments the differentiation potential of mesenchymal stem cells.

AIM: Mesenchymal stem cells (MSCs) are immunomodulatory, non-teratogenic and multipotent alternatives to embryonic or induced pluripotent stem cells (ESCs or iPSCs). However, the potency of MSCs is not equivalent to the pluripotency of ESCs or iPSCs. We used CHIR 99021 to improve current protocols and methods of differentiation for the enhanced transdifferentiation potency of MSCs. MAIN METHODS: We used Flurescence activated cell sorter (FACS) for MSC immunophenotyping and biochemical assay for demonstrating the trilineage potential of MSCs. We used real-time polymerase chain reaction, immunocytochemistry and Western blotting assay for analyzing the expression of lineage-specific markers. KEY FINDINGS: CHIR 99021 treatment of MSCs resulted in enhanced transdifferentiation into neurological, hepatogenic and cardiomyocyte lineages with standardized protocols of differentiation. CHIR 99021-treated MSCs showed increased nuclear localization of ß-catenin. These MSCs showed a significantly increased deposition of active histone marks (H3K4Me3, H3K36Me3), whereas no change was observed in repressive marks (H3K9Me3, H3K27Me3). Differential methylation profiling showed demethylation of the transcription factor OCT4 promoter region with subsequent analysis revealing increased gene expression and protein content. The HLA-DR antigen was absent in CHIR 99021-treated MSCs and their differentiated cell types, indicating their immune-privileged status. Karyotyping analysis showed that CHIR 99021-treated MSCs were genomically stable. Teratoma analysis of nude mice injected with CHIR 99021-treated MSCs showed the increased presence of cell types of mesodermal origin at the site of injection. SIGNIFICANCE: MSCs pretreated with CHIR 99021 can be potent, abundant alternative sources of stem cells with enhanced differentiation capabilities that are well suited to cell-based regenerative therapy.

Deciphering the Epitranscriptomic Signatures in Cell Fate Determination and Development.

Precise regulation of transcriptome modulates several vital aspects in an organism that includes gene expression, cellular activities and development, and its perturbation ensuing pathological conditions. Around 150 post-transcriptional modifications of RNA have been identified till date, which are evolutionarily conserved and likewise prevalent across RNA classes including messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), and detected less frequently in small nuclear RNA (snRNA) and microRNAs (miRNA). Among the RNA modifications documented, N6-methyladenosine (m6A) is the best characterised till date. Also, N1-methyladenosine (m1A), 5-methylcytosine (m5C) and pseudouridine (Ψ) are some of the other prominent modifications detected in coding and non-coding RNAs. "Epitranscriptome", ensemble of these post-transcriptional RNA modifications, precisely coordinates gene expression and biological processes. Current literatures suggest the critical involvement of epitranscriptomics in several organisms during early development, contributing to cell fate specification and physiology. Indeed, epitranscriptomics similar to DNA epigenetics involves combinatorial dynamics provided by modified RNA molecules and associated protein complexes, which function as "writers", "erasers" and "readers" of these modifications. A novel code orchestrating gene expression during cell fate determination is generated by the coordinated effects of different classes of modified RNAs and its regulator proteins. In this review, we summarize the current knowhow on N6-methyladenosine (m6A), 5-methylcytosine (m5C) and pseudouridine (ψ) modifications in RNA, the associated regulator proteins and enumerate how the epitranscriptomic regulations are involved in cell fate determination.

Effect of Silver-Containing Titania Layers for Bioactivity, Antibacterial Activity, and Osteogenic Differentiation of Human Mesenchymal Stem Cells on Ti Metal.

Biomaterials with better osteogenic capacity, rapid osteo-integration, and higher mechanical strength are undoubtedly preferred for successful bone implant development. A porous sodium hydrogen titanate layer was formed on Ti metal by NaOH treatment, and the Na+ ions were replaced by Ag+ ions by subsequent AgNO3 treatment that formed silver-containing hydrogen titanate. Heat treatment at 600 °C transformed sodium hydrogen titanate into sodium titanate with sheet-like morphology, whereas silver-containing hydrogen titanate was converted to anatase TiO2 with an elongated rod-like structure. Further increment in temperature lead to the formation of rutile TiO2 with distracted network morphology. Between these two, the anatase TiO2 was ascertained to be bioactive by being capable of forming bonelike apatite in simulated body fluid within a period of 12 h. The concentration of silver on Ti metal was further optimized for better antibacterial activity against S. aureus and biocompatibility toward bone cells. A detailed investigation of thus optimized silver-containing Ti metal on the proliferation and differentiation of multipotent human mesenchymal stem cells further proved their biocompatibility nature and facilitation of osteogenic differentiation, thereby conferring those as ideally suited materials for bioimplant development in bone tissue engineering.

Influence of doping ion, capping agent and pH on the fluorescence properties of zinc sulfide quantum dots: Sensing of Cu2+ and Hg2+ ions and their biocompatibility with cancer and fungal cells.

Herein, a facile one-pot synthetic method was explored for the fabrication of glutathione capped Mn2+ doped­zinc sulphide quantum dots (GSH-Mn2+-ZnS QDs) for both fluorescent detection of Cu2+ and Hg2+ ions and for fluorescence imaging of two cancer (RIN5F and MDAMB231) and fungal (Rhizopus oryzae) cells. Particularly, doping of Mn2+ into ZnS QDs nanocrystal structure resulted a great improvement in the fluorescence properties of ZnS QDs. The emission peak of undoped ZnS QDs was found at 447 nm, which is due to the large number of surface defects in the ZnS QDs nanostructures. Under identical conditions, there is a good linear relationship between the quenching of fluorescence intensity and analytes (Cu2+ and Hg2+ ions) concentration in the range of 0.005 to 0.2 mM and of 0.025 to 0.4 mM for Cu2+ and Hg2+ ions, respectively. The GSH-Mn2+-ZnS QDs exhibit least cytotoxicity against RIN5F and MDAMB231 cells, demonstrating the multifunctional applications in sensing of metal ions and biocompatibility towards cancer (RIN5F and MDAMB231) and fungal (Rhizopus oryzae) cells.

Exploiting the power of LINE-1 retrotransposon mutagenesis for identification of genes involved in embryonic stem cell differentiation.

Identifying the genes or epigenetic factors that control the self-renewal and differentiation of stem cells is critical to understanding the molecular basis of cell commitment. Although a number of insertional mutagenesis vectors have been developed for identifying gene functions in animal models, the L1 retrotransposition system offers additional advantages as a tool to disrupt genes in embryonic stem cells in order to identify their functions and the phenotypes associated with them. Recent advances in producing synthetic versions of L1 retrotransposon vector system and the optimization of techniques to accurately identify retrotransposon integration sites have increased their utility for gene discovery applications. We have developed a novel episomal, nonviral L1 retrotransposon vector using scaffold/matrix attachment regions that provides stable, sustained levels of retrotransposition in cell cultures without being affected by epigenetic silencing or from some of the common problems of vector integration. This modified vector contains a GFP marker whose expression occurs only after successful gene disruption events and thus the cells with disrupted genes can be easily picked for functional analysis. Here we present a method to disrupt gene function in embryonic stem cells that aid in the identification of genes involved in stem cell differentiation processes. The methods presented here can be easily adapted to the study of other types of cancer stem cells or induced pluripotent stem cells using the L1 retrotransposon as an insertional mutagen.

Notch exhibits ligand bias and maneuvers stage-specific steering of neural differentiation in embryonic stem cells.

Notch dictates multiple developmental events, including stem cell maintenance and differentiation, through intercellular communication. However, its temporal influence during early development and, of particular interest, its regulation of binary fate decision at different stages during neurogenesis are among the least explored. Here, using an embryonic stem cell (ESC) model, we have deciphered Notch ligand preference during ESC commitment to different germ layers and determined the stage-specific temporal effect of Notch during neural differentiation. ESCs during maintenance remain impervious to Notch inhibition. However, Notch activation promotes differentiation even in the presence of leukemia inhibitory factor (LIF), displaying ligand preference-associated lineage discrimination, where Jagged-1 favors neural commitment and Delta-like-4 favors the mesoderm. This differential ligand action involves a combination of Notch receptors influencing specific downstream target gene expression. Though Notch activation during early neural differentiation specifically promotes neural stem cells or early neural progenitors and delays their maturation, its inhibition promotes late neural progenitors and expedites neurogenesis, with a preference for neurons over glia. However, gliogenesis is promoted upon Notch activation only when executed in combination with ciliary neurotrophic factor. Thus, our investigation underscores a multifaceted role of Notch, demonstrating the interdependency of ligand usage and lineage specification and Notch acting as a master switch, displaying stage-specific influence on neurogenesis.

Temporal and contextual orchestration of cardiac fate by WNT-BMP synergy and threshold.

Cardiomyogenic development proceeds with a cascade of intricate signalling events that operate in a temporo-spatial fashion to specify cardiac cell fate during early embryogenesis. In fact, conflicting reports exist regarding the role of Wnt/ß-catenin signalling during cardiomyogenesis. Here, we describe a dose-dependent and temporal effect of Wnt/ß-catenin signalling on in vitro cardiomyogenesis using embryonic stem cells (ESCs) as a model system. We could demonstrate that canonical Wnt activation during early stage of differentiation either through ligand or by GSK3ß inhibition helped in maintaining Oct4 and Nanog expressions, and in parallel, it promoted mesoderm and endoderm inductions. In contrast, it led to attenuation in cardiomyogenesis that was reversed by moderate concentration of DKK1, but not soluble Fz8. However, higher DKK1 could also block cardiomyogenesis, suggesting thereby governance of a particular signalling threshold underlying this developmental event. Interestingly, Wnt signalling activation at early stage modulated BMP4 expression in a stage-specific manner. Wnt activation, synchronized with BMP4 and brachyury up-regulation at early stage, correlated well with mesoderm induction. Conversely, Wnt activation led to BMP4 and Wnt5a down-regulation at late stage culminating in cardiomyogenic attenuation. Our findings suggested the existence of precise regulatory machinery with context-dependent role of Wnt for fine tuning mesoderm induction and its derivatives, through establishment of Wnt gradient during ESCs' differentiation. Moreover, contrary to mere activation/inhibition, a specific threshold of Wnt and BMP and their synergy seemed necessary for providing the guiding cues in orchestrating mesoderm induction and subsequent cardiomyogenesis.

Neural induction from ES cells portrays default commitment but instructive maturation.

The neural induction has remained a debatable issue pertaining to whether it is a mere default process or it involves precise instructive cues. We have chosen the embryonic stem (ES) cell model to address this issue. In a devised monoculture strategy, the cell-cell interaction availed through optimum cell plating density could define the niche for the attainment of efficient in vitro neurogenesis from the ES cells. The medium plating density was found ideal in generating optimum number of progenitors and also yielded about 80% mature neurons in a serum free culture set up barring any exogenous inducers. We could also demarcate and quantify the neural stem cells/progenitors among the heterogeneous cell population of differentiating ES cells using nestin intron II driven EGFP expression as a tool. The one week post-plating was determined to be the critical time window for optimum neural progenitor generation from ES cells that helped us further in purifying these cells and in demonstrating their proliferation and multipotent differentiation potential. Seeding cells at varying densities, we could decipher an interesting paradoxical scenario that interlinked both commitment and maturation with the initial plating density having a vital influence on neuronal maturation but not specification and the secretory factors were apparently playing a key role during this process. Thus it was comprehended that, the neural specification was a default process independent of exogenous factors and cellular interaction. Conversely, a defined number of cells at the specification stage itself seemed critical to provide an auto-/paracrine means of signaling threshold for the maturation process to materialize.

Derivation and characterization of neural cells from embryonic stem cells using nestin enhancer.

The embryonic stem (ES) cells derived from the inner cell mass of the blastula stage embryo bear the complete repertoire of the complex organizational blueprint of an organism. These fascinating cells are bestowed with pluripotent characteristics and can be directed to differentiate into various lineages in vitro. Hence, these cells are being explored as an ideal in vitro model in gaining insight into early developmental events. Using the ES cell system, we have tried to investigate the early neurogenic proceedings. We have taken advantage of nestin enhancer-mediated cell trapping using the live reporter-based system. We monitored live the ES cell differentiation into neural lineage by following the enhanced green fluorescent protein expression profile in a number of stable ES cell clones generated by us in which the enhanced green fluorescent protein expression was regulated by the nestin enhancer. This strategy has helped us in both qualitative and quantitative detection and characterization of neural progenitor population and the differentiated progenies.

Differential subunit composition of the G protein-activated inward-rectifier potassium channel during cardiac development.

Parasympathetic slowing of the heart rate is predominantly mediated by acetylcholine-dependent activation of the G protein-gated potassium (K+) channel (IK,ACh). This channel is composed of 2 inward-rectifier K+ (Kir) channel subunits, Kir3.1 and Kir3.4, that display distinct functional properties. Here we show that subunit composition of IK,ACh changes during embryonic development. At early stages, IK,ACh is primarily formed by Kir3.1, while in late embryonic and adult cells, Kir3.4 is the predominant subunit. This change in subunit composition results in reduced rectification of IK,ACh, allowing for marked K+ currents over the whole physiological voltage range. As a consequence, IK,ACh is able to generate the membrane hyperpolarization that underlies the strong negative chronotropy occurring in late- but not early-stage atrial cardiomyocytes upon application of muscarinic agonists. Both strong negative chronotropy and membrane hyperpolarization can be induced in early-stage cardiomyocytes by viral overexpression of the mildly rectifying Kir3.4 subunit. Thus, a switch in subunit composition is used to adopt IK,ACh to its functional role in adult cardiomyocytes.