Cryopreservation and validation of differentiated PDGFRα-positive cells for long term usage in experimentation.

OBJECTIVE: Differentiation of immortalized Mesenchymal Stromal Cells (iMSCs) into PDGFRα-positive cells under controlled growth conditions has several vital implications in functional studies concerned with the pathogenesis of Diabetic Gastroparesis (DGP). A study published previously by our research group demonstrated the importance of these cells as a novel, in-vitro model for investigating the functional role of neuronal nitric oxide synthase. The currently available methods require fresh differentiation of PDGFRα-positive cells for each round of experimentation. This leads to longer delays, higher usage of reagents, and inconsistency in reproducibility of experiments frequently. We thus aimed to establish through validation that cryopreserving and maintaining the iMSC-derived PDGFRα-positive cells for functional investigations help us to overcome these challenges. RESULTS: We demonstrated for the first time that the differentiated PDGFRα-positive cells from iMSCs can be cryopreserved and thawed to be used as per the experimental requirements with prolonged preservation of their characteristics. We assessed the viability of differentiated PDGFRα-positive cells pre- and post-freezing with the subsequent validation of their functional features using flow cytometry, qRT-PCR, and western blotting. We have been successful in demonstrating for the first time that the cryopreservation of previously differentiated PDGFRα-positive cells can be used as a feasible and cost-effective model for experimental reproducibility in functional studies of Diabetes Gastroparesis.

GSK-3α aggravates inflammation, metabolic derangement, and cardiac injury post-ischemia/reperfusion.

Reperfusion after acute myocardial infarction further exaggerates cardiac injury and adverse remodeling. Irrespective of cardiac cell types, loss of specifically the α isoform of the protein kinase GSK-3 is protective in chronic cardiac diseases. However, the role of GSK-3α in clinically relevant ischemia/reperfusion (I/R)-induced cardiac injury is unknown. Here, we challenged cardiomyocyte-specific conditional GSK-3α knockout (cKO) and littermate control mice with I/R injury and investigated the underlying molecular mechanism using an in vitro GSK-3α gain-of-function model in AC16 cardiomyocytes post-hypoxia/reoxygenation (H/R). Analysis revealed a significantly lower percentage of infarct area in the cKO vs. control hearts post-I/R. Consistent with in vivo findings, GSK-3α overexpression promoted AC16 cardiomyocyte death post-H/R which was accompanied by an induction of reactive oxygen species (ROS) generation. Consistently, GSK-3α gain-of-function caused mitochondrial dysfunction by significantly suppressing mitochondrial membrane potential. Transcriptomic analysis of GSK-3α overexpressing cardiomyocytes challenged with hypoxia or H/R revealed that NOD-like receptor (NLR), TNF, NF-κB, IL-17, and mitogen-activated protein kinase (MAPK) signaling pathways were among the most upregulated pathways. Glutathione and fatty acid metabolism were among the top downregulated pathways post-H/R. Together, these observations suggest that loss of cardiomyocyte-GSK-3α attenuates cardiac injury post-I/R potentially through limiting the myocardial inflammation, mitochondrial dysfunction, and metabolic derangement. Therefore, selective inhibition of GSK-3α may provide beneficial effects in I/R-induced cardiac injury and remodeling. KEY MESSAGES: GSK-3α promotes cardiac injury post-ischemia/reperfusion (I/R). GSK-3α regulates inflammatory and metabolic pathways post-hypoxia/reoxygenation (H/R). GSK-3α overexpression upregulates NOD-like receptor (NLR), TNF, NF-kB, IL-17, and MAPK signaling pathways in cardiomyocytes post-H/R. GSK-3α downregulates glutathione and fatty acid metabolic pathways in cardiomyocytes post-H/R.

Biosynthesized Silver Nanoparticles from Cyperus Conglomeratus Root Extract Inhibit Osteogenic Differentiation of Immortalized Mesenchymal Stromal Cells.

BACKGROUND: Silver nanoparticles (AgNPs) are a focus of huge interest in biological research, including stem cell research. AgNPs synthesized using Cyperus conglomeratus root extract have been previously reported but their effects on mesenchymal stromal cells have yet to be investigated. OBJECTIVES: The aim of this study is to investigate the effects of C. conglomeratus-derived AgNPs on adipogenesis and osteogenesis of mesenchymal stromal cells. METHODS: AgNPs were synthesized using C. conglomeratus root extract, and the phytochemicals involved in AgNPs synthesis were analyzed using gas chromatography-mass spectrometry (GC-MS). The cytotoxicity of the AgNPs was tested on telomerase-transformed immortalized human bone marrow-derived MSCs-hTERT (iMSC3) and human osteosarcoma cell line (MG-63) using MTT and apoptosis assays. The uptake of AgNPs by both cells was confirmed using inductively coupled plasma-optical emission spectrometry (ICP-OES). Furthermore, the effect of AgNPs on iMSC3 adipogenesis and osteogenesis was analyzed using stain quantification and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). RESULTS: The phytochemicals predominately identified in both the AgNPs and C. conglomeratus root extract were carbohydrates. The AgNP concentrations tested using MTT and apoptosis assays (0.5-64 µg/ml and 1,4 and 32 µg/ml, respectively) showed no significant cytotoxicity on iMSC3 and MG-63. The AgNPs were internalized in a concentration-dependent manner in both cell types. Additionally, the AgNPs exhibited a significant negative effect on osteogenesis but not on adipogenesis. CONCLUSION: C. conglomeratus-derived AgNPs had an impact on the differentiation capacity of iMSC3. Our results indicated that C. conglomeratus AgNPs and the associated phytochemicals could exhibit potential medical applications.

Suppression of endoplasmic reticulum stress reverses hindlimb unloading-induced hepatic cellular processes in mice.

BACKGROUND: The Hindlimb unloaded mouse, an animal model of simulated microgravity demonstrates significant metabolic and hepatic derangements. However, cellular and molecular mechanisms driving liver dysfunction in Hindlimb unloaded mice are poorly characterized. METHODS: We investigated the possible contribution of dysregulated protein homeostasis by endoplasmic reticulum, endoplasmic reticulum stress, to liver dysfunction during HU. C57BL/6j male mice were grouped into ground-based controls or Hindlimb unloaded groups treated daily with vehicle or 4-phenylbutyrate (4-PBA), a potent inhibitor of endoplasmic reticulum stress. Following three weeks of HU, mice were sacrificed, and liver tissues were dissected for further analysis. RESULTS: Hindlimb unloaded was associated with hepatic atrophy and elevated endoplasmic reticulum stress, which was restored by 4-PBA treatment. The Gene Ontology analysis revealed the downregulation of genes primarily involved in liver metabolic and Wingless-related integration site (WNT) signaling pathways, while those related to cytochrome P450, and liver fibrosis were upregulated. The Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed downregulation of several genes involved in metabolic pathways following treatment with 4-PBA, induced by HU. CONCLUSIONS: We report several differential and uniquely expressed genes associated with microgravity-induced elevated ER stress and liver injury. Our data has translational potential in unraveling novel molecular targets for pharmaceutical therapies of liver diseases. GENERAL SIGNIFICANCE: Our novel findings show a pathogenic role for elevated ER stress in liver injury in microgravity conditions.

Disaster-development interface and its impact on emerging vulnerability scenario in Ladakh region of northwestern Himalayas.

The Union Territory of Ladakh, located in the northwestern Himalayan region, is highly vulnerable to natural and anthropogenic hazards like earthquakes, landslides, snow avalanches, flash floods, cloud bursts, and border conflicts. Occurrences of these disasters have significantly influenced the development and vulnerability scenario of Trans-Himalayan Ladakh. Findings reveal that despite suffering losses from natural and human-induced disasters, the region has benefited by grabbing the attention of policymakers at the national level. Consequently, long-term developments were positively impacted, reflecting infrastructural upgradation, improved transportation and communication, profoundly improving the socio-economic well-being of the people. Furthermore, post-disaster developments have managed to showcase the unique physiography and adventurous terrains of Ladakh, promoting tourism as the main economic driver in the region. The exponential growth of tourism and associated sectors have influenced the vulnerability scenario, which was quantified using the multi-criterion-based analytical hierarchical processes (AHP) method, indicating an increase in climate change-related vulnerability, followed by socio-cultural, environmental, and physical vulnerabilities. Specifically, the vulnerabilities with respect to flash floods, landslides, erratic rainfall, haphazard constructions, cultural dilution, water crisis, and changes in land use patterns have been exacerbated across the study area. The study highlights the need for effective management of these emerging vulnerabilities through proper planning to ensure long-term sustainable development goals in this environmentally fragile region.

Transplantation of IGF-1-induced BMSC-derived NPCs promotes tissue repair and motor recovery in a rat spinal cord injury model.

Bone marrow-derived mesenchymal stem cells (BMSCs) have therapeutic potential for spinal cord injury (SCI). We have shown that insulin-like growth factor 1 (IGF-1) enhances the cellular proliferation and survivability of BMSCs-derived neural progenitor cells (NPCs) by downregulating miR-22-3p. However, the functional application of BMSCs-derived NPCs has not been investigated fully. In this study, we demonstrate that knockdown of endogenous miR-22-3p in BMSCs-derived NPCs upregulates Akt1 expression, leading to enhanced cellular proliferation. RNASeq analysis reveals 3,513 differentially expressed genes in NPCs. The upregulated genes in NPCs enrich the gene ontology term associated with nervous system development. Terminally differentiated NPCs generate cells with neuronal-like morphology and phenotypes. Transplantation of NPCs in the SCI rat model results in better recovery in locomotor and sensory functions 4 weeks after transplantation. Altogether, the result of this study demonstrate that NPCs derived with IGF-1 supplementation could be differentiated into functional neural lineage cells and are optimal for stem cell therapy in SCI.

Mitigating sarcoplasmic reticulum stress limits disuse-induced muscle loss in hindlimb unloaded mice.

Muscle disuse in the hindlimb unloaded (HU) mice causes significant atrophy and weakness. However, the cellular and molecular mechanisms driving disuse-muscle atrophy remain elusive. We investigated the potential contribution of proteins dysregulation by sarcoplasmic reticulum (SR), a condition called SR stress, to muscle loss during HU. Male, c57BL/6j mice were assigned to ground-based controls or HU groups treated with vehicle or 4-phenylbutyrate (4-PBA), a potent inhibitor of SR stress, once a day for three weeks. We report that the 4-PBA reduced the SR stress and partly reversed the muscle atrophy and weakness in the HU mice. Transcriptome analysis revealed that several genes were switched on (n = 3688) or differentially expressed (n = 1184) due to HU. GO, and KEGG term analysis revealed alterations in pathways associated with the assembly of cilia and microtubules, extracellular matrix proteins regulation, calcium homeostasis, and immune modulation during HU. The muscle restoration with 4-PBA partly reversed these changes along with differential and unique expression of several genes. The analysis of genes among the two comparisons (HU-v vs. control and HU-t vs. HU-v.) shows 841 genes were overlapped between the two comparisons and they may be regulated by 4-PBA. Altogether, our findings suggest that the pharmacological suppression of SR stress may be an effective strategy to prevent disuse-induced muscle weakness and atrophy.

Increased strength in the Col-Tgel induces apoptosis in the human dental pulp stem cells: 3D culturing of human dental pulp stem cells at different strengths of collagen.

Human dental pulp stem cells (HDPSCs) have great potential to be used in regenerative medicine. To use these stem cells effectively for this purpose, they should be grown in a 3D cell culture that mimics their natural niches instead of a 2D conventional cell culture. The aim of this study was to grow the HDPSCs in the 3D cell culture created by Transglutaminase-crosslinked collagen hydrogels (Col-Tgel) in two different strengths to find a suitable 3D cell culture environment for these stem cells. Two stiffness of the 3D Col-Tgel were used to grow the HDPSCs: soft and medium matrix with strength of 0.9-1.5 kPa and 14-20 kPa, respectively. HDPSCs express markers similar to MSCs, therefore seven such markers were analyzed in the HDPSCs during their growth in the 2D and in the 3D soft and medium Col-Tgel. The CD105 and CD90 markers were significantly (p < 0.05) downregulated in HDPSCs cultured in both 3D cell culture conditions compared with HDPSCs in 2D cell culture. Furthermore, CD34 marker, a negative marker, expressed by a few cells in HDPSCs culture was upregulated (p < 0.05) in HDPSCs cultured in medium 3D Col-Tgel, indicating cells that expressing the marker grow better in medium 3D Col-Tgel. The apoptosis results revealed that HDPSCs in medium 3D Col-Tgel had the least number of live cells and a significantly (p < 0.05) higher early apoptosis rate compared to HDPSCs in 2D and 3D Col-Tgel medium. MTT analysis also showed a significant difference among the three cell culture conditions. We conclude that HDPSCs cultured on 3D soft Col-Tgel showed better proliferation than cells cultured in 3D medium gel. These results demonstrate that the ideal environment to grow HDPSCs in 3D is the soft Col-Tgel not medium Col-Tgel.

Transcriptomic profiling of the telomerase transformed Mesenchymal stromal cells derived adipocytes in response to rosiglitazone.

BACKGROUND: Differentiation of Immortalized Human Bone Marrow Mesenchymal Stromal Cells - hTERT (iMSC3) into adipocytes is in vitro model of obesity. In our earlier study, rosiglitazone enhanced adipogenesis particularly the brown adipogenesis of iMSC3. In this study, the transcriptomic profiles of iMSC3 derived adipocytes with and without rosiglitazone were analyzed through mRNA sequencing. RESULTS: A total of 1508 genes were differentially expressed between iMSC3 and the derived adipocytes without rosiglitazone treatment. GO and KEGG enrichment analyses revealed that rosiglitazone regulates PPAR and PI3K-Akt pathways. The constant rosiglitazone treatment enhanced the expression of Fatty Acid Binding Protein 4 (FABP4) which enriched GO terms such as fatty acid binding, lipid droplet, as well as white and brown fat cell differentiation. Moreover, the constant treatment upregulated several lipid droplets (LDs) associated proteins such as PLIN1. Rosiglitazone also activated the receptor complex PTK2B that has essential roles in beige adipocytes thermogenic program. Several uniquely expressed novel regulators of brown adipogenesis were also expressed in adipocytes derived with rosiglitazone: PRDM16, ZBTB16, HOXA4, and KLF15 in addition to other uniquely expressed genes. CONCLUSIONS: Rosiglitazone regulated several differentially regulated genes and non-coding RNAs that warrant further investigation about their roles in adipogenesis particularly brown adipogenesis.

Significant transcriptomic changes are associated with the inhibitory effects of 5-aza-2-deoxycytidine during adipogenic differentiation of MG-63 cells.

Our previous study revealed that the treatment of 5-aza-2-deoxycytidine (5-aza) inhibited while treatment of suberoylanilide hydroxamic acid (SAHA) enhanced the adipogenic differentiation of MG-63 cells. In this study, we examined the transcriptomic profiles of the derived adipocyte-like cells from MG-63 cells in the presence of 5-aza (Treatment 1) and SAHA (Treatment 2). Genome wide expression analysis showed high within sample variability for the adipocytes derived with 5-aza versus vehicle. Additionally, the expression profile of 5-aza derived cells was separated from the other sample groups. Differential analysis on the pairwise comparison of 5-aza versus control and SAHA versus 5-aza identified 1290 and 1086 differentially expressed (DE) genes, respectively. Furthermore, some overlap was observed between the up and down-regulated DE genes of 5-aza versus control and SAHA versus control (jaccard score 0.3) as well as between the differentially regulated genes of 5-aza versus control and 5-aza versus SAHA (jaccard score 0.29). A total of 73 transcription factors (TFs) were differentially expressed across all the pair wise comparisons with some overlap between the under and over expressed TFs of 5-aza versus control and 5-aza versus SAHA (jaccard score 0.29). Unsupervised clustering of TFs showed that the samples within the group are consistent in expression and the samples cluster in accordance with the group. Several GO terms related to enhanced adipogenesis such as neutral lipid biosynthetic process, lipid metabolic processes, cellular amide metabolic processes and cellular carbohydrate metabolic processes were enriched in the down regulated genes of 5-aza derived adipocytes versus control, indicating 5-aza inhibit the adipogenic differentiation of MG-63 cells. GSEA analysis on selected gene sets of MAPK and PI3K signaling pathway in MSigDB identified the pathways were up-regulated in 5-aza versus control. This study revealed that inhibition of MG-63 adipogenesis due to 5-aza treatment is associated with large transcriptomics changes and further research is needed to unravel the roles of these genes in the adipogenesis.

Phytochemical profile and antiproliferative activities of acetone extracts of Asplenium polypodioides Blume. and A. dalhousiae Hook. in MDA-MB-231 breast cancer cells.

The plants extracts are widely used in traditional medicines and hence considered a potential source for drug discovery. In this study, we assessed the phytochemical composition of Asplenium dalhousiae and Asplenium polypodioides in acetone extracts and checked its antiproliferative potential in MDA-MB-231 cells. We found that both plants are rich in phenolic and flavonoid compounds and are efficient in antioxidant activities. The total phenolic compounds in A. dalhousiae were 44.15 ± 1.38 µg/mg whereas in A. polypodioides were 27.73 ± 1.35 µg/mg. Total flavonoids in A. dalhousiae were 105.39 ± 2.92 µg/mg whereas in A. polypodioides were 101.56 ± 1.75 µg/mg. The ferric reducing power assay indicates 66.38 ± 2.6% reduction by A. dalhousiae whereas 78.43 ± 0.47% reduction by A. polypodioides. Similarly, the total antioxidant capacity of A. dalhousiae was found to be 59.95 ± 1.13 whereas for A. polypodioides the recorded value was 33.03 ± 1.67%. Using GCMS analysis, we identified 25 compounds in A. dalhousiae whereas 26 in A. polypodioides. Four of these compounds are common in both plants. The morphological study and MTT assay revealed that both plants have antiproliferative potential as both plants exerted significant effects on the shape of the MDA-MB-231 cells and inhibited cellular proliferation in time and dose dependent manner. We conclude that both Asplenium plants have potential anticancer compounds.

Comparative anticancer activities of Ficus carica and Ficus salicifolia latex in MDA-MB-231 cells.

Ficus latex is rich in polyphenolic compounds and hence considered as an antioxidant and anti-proliferative. Many studies are available on Ficus carica (common fig) whereas Ficus salicifolia is less studied. F. salicifolia grows in a harsh dry environment, therefore its latex was selected in the current study along with the F. carica for their comparative anti-cancer potential and the involved molecular mechanism. Triple-negative breast cancer (TNBC) derived MDA-MB-231 cells were used in the study. MTT and morphological studies indicated that the latex of both plants has anti-proliferative effects. To know their anti-metastatic effects, a wound-healing assay was performed. Both species were able to maintain the wound size compared to the untreated cells indicating their anti-metastatic effects. Using a DNA damage assay kit, we found that both fig species have genotoxic and cytotoxic effects in MDA-MB-231 cells compared to the untreated control. To know the potential molecular mechanism involved, we used a human kinase array kit. We found that ERK2, CREB, and AKT2 were downregulated after treatment the MDA-Mb-231 cells with the latex of F. carica. We assumed that F. salicifolia will also affect the same pathways, however after confirmation through real-time (RT)-PCR, downregulations of the above mentioned pathways were confirmed in cells treated with F. carica latex, however, in cells treated with F. salicifolia the selected genes were upregulated at the transcriptional level. We conclude that latex of both species of ficus have anti-cancer effects in MDA-MB-231 cells, however differ in their level of toxicity and the mechanism of action at the molecular level.

Arylated gold nanoparticles have no effect on the adipogenic differentiation of MG-63 cells nor regulate any key signaling pathway during the differentiation.

OBJECTIVE: MG-63 cells that have osteoblastic and adipogenic differentiation potential were evaluated for internalization, and adipogenic differentiation in the presence and absence of the covalently functionalized aryl gold nanoparticles (AuNPs-C6H4-4-COOH). RESULTS: Inductively coupled plasma, flow cytometry and confocal microscopy analyses confirmed that gold nanoparticles were easily internalized by MG-63 cells. The MG-63 cells were differentiated into adipocytes without gold-aryl nanoparticles and with the gold-aryl nanoparticles at 5 µM concentration in both induction and maintenance media. The lipid content assay and the relative expressions of PPAR-γ, ADR1, GLUT1 and GLUT4 genes showed no significant variation with and without the gold nanoparticles treatment. Differential phosphorylation levels of 43 kinases phosphorylation sites were evaluated using the human phospho-kinase array to assess the effect of the gold nanoparticles on the signaling pathways during the differentiation. No kinase phosphorylation site was differentially phosphorylated with two or more folds after the nanoparticles treatment after the first day as well as at the end of MG-63 cells differentiation. The gold-aryl nanoparticles do not affect MG-63 cells differentiation into adipocytes neither do they affect any key signaling pathway. These properties make these gold nanoparticles suitable for future drug delivery and medical applications.

Differentiated PDGFRα-Positive Cells: A Novel In-Vitro Model for Functional Studies of Neuronal Nitric Oxide Synthase.

It is evident that depletion of interstitial cells and dysfunction of nitric oxide (NO) pathways are key players in development of several gastrointestinal (GI) motility disorders such as diabetic gastroparesis (DGP). One of the main limitations of DGP research is the lack of isolation methods that are specific to interstitial cells, and therefore conducting functional studies is not feasible. The present study aims (i) to differentiate telomerase transformed mesenchymal stromal cells (iMSCs) into platelet-derived growth factor receptor-α-positive cells (PDGFRα-positive cells) using connective tissue growth factor (CTGF) and L-ascorbic acids; (ii) to investigate the effects of NO donor and inhibitor on the survival rate of differentiated PDGFRα-positive cells; and (iii) to evaluate the impact of increased glucose concentrations, mimicking diabetic hyperglycemia, on the gene expression of neuronal nitric oxide synthase (nNOS). A fibroblastic differentiation-induction medium supplemented with connective tissue growth factor was used to differentiate iMSCs into PDGFRα-positive cells. The medium was changed every day for 21 days to maintain the biological activity of the growth factors. Gene and protein expression, scanning electron and confocal microscopy, and flow cytometry analysis of several markers were conducted to confirm the differentiation process. Methyl tetrazolium cell viability, nitrite measurement assays, and immunostaining were used to investigate the effects of NO on PDGFRα-positive cells. The present study, for the first time, demonstrated the differentiation of iMSCs into PDGFRα-positive cells. The outcomes of the functional studies showed that SNAP (NO donor) increased the survival rate of differentiated PDGFRα-positive cells whereas LNNA (NO inhibitor) attenuated these effects. Further experimentations revealed that hyperglycemia produced a significant increase in expression of nNOS in PDGFRα-positive cells. Differentiation of iMSCs into PDGFRα-positive cells is a novel model to conduct functional studies and to investigate the involvement of NO pathways. This will help in identifying new therapeutic targets for treatment of DGP.

Anticancer activities of selected Emirati Date (Phoenix dactylifera L.) varieties pits in human triple negative breast cancer MDA-MB-231 cells.

The date palm (Phoenix dactylifera L.) is an important fruit crop with significant pharmaceutical potential. Little data are available on comparative pharmaceutical importance of the date pits. We designed this study to assess the antitumorigenic effects of date palm pits extracts from different Emiratis varieties. We used MDA-MB-231 cells derived from triple negative breasts cancer tissues as a model. We found that out of the 17 date pits extracts from 6 Emiratis varieties, three (Khalas extract in water + acetone (1:1), Abu-Maan extract in MeOH + Chloroform (1:1) and Mabroom extract in water + acetone (1:1)) were found effectively cytotoxic and changed morphology of cells in dose and time dependent manner. We found the maximum effect at 2.5 mg/mL concentration at 72 h. We calculated IC50 values for these varieties at 24 h. IC50 values for Khalas, Abu-Maan and Mabroom were 0.982 mg/mL, 1.149 mg/mL and 2.213 mg/mL respectively. We treated the cells with IC50 values of extracts and observed changes in protein profile using human kinase array kit. After analyzing the results, we suggest that EGFR/ERK/FAK pathway, eNOS and src family proteins are targets of these extracts. We conclude that date pits extracts can be a possible therapeutic agent against cancer and we suggest further studies.

Significant transcriptomic changes are associated with differentiation of bone marrow-derived mesenchymal stem cells into neural progenitor-like cells in the presence of bFGF and EGF.

INTRODUCTION: Mesenchymal stem cells (MSCs) isolated from bone marrow have different developmental origins, including neural crest. MSCs can differentiate into neural progenitor-like cells (NPCs) under the influence of bFGF and EGF. NPCs can terminally differentiate into neurons that express beta-III-tubulin and elicit action potential. The main aim of the study was to identify key genetic markers involved in differentiation of MSCs into NPCs through transcriptomic analysis. METHOD: Total RNA was isolated from MSCs and MSCs-derived NPCs followed by cDNA library construction for transcriptomic analysis. Sample libraries that passed the quality and quantity assessments were subjected to high throughput mRNA sequencing using NextSeq®500. Differential gene expression analysis was performed using the DESeq2 R package with MSC samples being a reference group. The expression of eight differentially regulated genes was counter validated using real-time PCR. RESULTS: In total, of the 3,252 differentially regulated genes between MSCs and NPCs with two or more folds, 1,771 were upregulated genes, whereas 1,481 were downregulated in NPCs. Amongst these differential genes, 104 transcription factors were upregulated, and 45 were downregulated in NPCs. Neurogenesis related genes were upregulated in NPCs and the main non-redundant gene ontology (GO) terms enriched in NPCs were the autonomic nervous system, cell surface receptor signalling pathways), extracellular structure organisation, and programmed cell death. The main non-redundant GO terms enriched in MSCs included cytoskeleton organisation cytoskeleton structural constituent, mitotic cell cycle), and the mitotic cell cycle process Gene set enrichment analysis also confirmed cell cycle regulated pathways as well as Biocarta integrin pathway were upregulated in MSCs. Transcription factors enrichment analysis by ChEA3 revealed Foxs1 and HEYL, amongst the top five transcription factors, inhibits and enhances, respectively, the NPCs differentiation of MSCs. CONCLUSIONS: The vast differences in the transcriptomic profiles between NPCs and MSCs revealed a set of markers that can identify the differentiation stage of NPCs as well as provide new targets to enhance MSCs differentiation into NPCs.

Exploring the effect of epigenetic modifiers on developing insulin-secreting cells.

Diabetes is a worldwide health problem with increasing incidence. The current management modalities did not succeed to decrease comorbidities. This study aimed at enhancing the regenerative solution for diabetes by improving the differentiation of mesenchymal stromal cells (MSC) into glucose-sensitive, insulin-secreting cells through an epigenetic modification approach. A 3-day treatment protocol with the epigenetic modifiers, either decitabine (5-aza-2'-deoxycytidine; Aza); a DNA methylation inhibitor or Vorinostat (suberoylanilide hydroxamic acid; SAHA); a histone deacetylase inhibitor was added to two different human stem cell lines. The cells followed a multi-step differentiation protocol that provided the critical triggers in a temporal approach. Aza-pretreated group showed higher intracellular expression of insulin and the transcription factor 'PDX-1'. The cells responded to the high glucose challenge by secreting insulin in the media, as shown by ELISA. Gene expression showed induction of the genes for insulin, the glucose transporter 2, glucokinase, as well as the transcription factors MafA and NKX6.1. Although SAHA showed upregulation of insulin secretion, in comparison to control, the cells could not respond to the high glucose challenge. Interestingly, Aza-treated cells showed a significant decrease in the global DNA methylation level at the end of the culture. In conclusion, this additional step with Aza could enhance the response of MSC to the classical differentiation protocol for insulin-secreting cells and may help in establishing a regenerative solution for patients with diabetes.

Rosiglitazone Enhances Browning Adipocytes in Association with MAPK and PI3-K Pathways During the Differentiation of Telomerase-Transformed Mesenchymal Stromal Cells into Adipocytes.

Obesity is a major risk for diabetes. Brown adipose tissue (BAT) mediates production of heat while white adipose tissue (WAT) function in the storage of fat. Roles of BAT in the treatment of obesity and related disorders warrants more investigation. Peroxisome proliferator activator receptor gamma (PPAR-γ) is the master regulator of both BAT and WAT adipogenesis and has roles in glucose and fatty acid metabolism. Adipose tissue is the major expression site for PPAR-γ. In this study, the effects of rosiglitazone on the brown adipogenesis and the association of MAPK and PI3K pathways was investigated during the in vitro adipogenic differentiation of telomerase transformed mesenchymal stromal cells (iMSCs). Our data indicate that 2 µM rosiglitazone enhanced adipogenesis by over-expression of PPAR-γ and C/EBP-α. More specifically, brown adipogenesis was enhanced by the upregulation of EBF2 and UCP-1 and evidenced by multilocular fatty droplets morphology of the differentiated adipocytes. We also found that rosiglitazone significantly activated MAPK and PI3K pathways at the maturation stage of differentiation. Overall, the results indicate that rosiglitazone induced overexpression of PPAR-γ that in turn enhanced adipogenesis, particularly browning adipogenesis. This study reports the browning effects of rosiglitazone during the differentiation of iMSCs into adipocytes in association with the activation of MAPK and PI3K signaling pathways.

Comparison of Classifier Architectures for Online Neural Spike Sorting.

High-density, intracranial recordings from micro-electrode arrays need to undergo Spike Sorting in order to associate the recorded neuronal spikes to particular neurons. This involves spike detection, feature extraction, and classification. To reduce the data transmission and power requirements, on-chip real-time processing is becoming very popular. However, high computational resources are required for classifiers in on-chip spike-sorters, making scalability a great challenge. In this review paper, we analyze several popular classifiers to propose five new hardware architectures using the off-chip training with on-chip classification approach. These include support vector classification, fuzzy C-means classification, self-organizing maps classification, moving-centroid K-means classification, and Cosine distance classification. The performance of these architectures is analyzed in terms of accuracy and resource requirement. We establish that the neural networks based Self-Organizing Maps classifier offers the most viable solution. A spike sorter based on the Self-Organizing Maps classifier, requires only 7.83% of computational resources of the best-reported spike sorter, hierarchical adaptive means, while offering a 3% better accuracy at 7 dB SNR.

MicroRNA expression profile of bone marrow mesenchymal stem cell-derived neural progenitor by microarray under the influence of EGF, bFGF and IGF-1.

Recently there has been growing interest in the differentiation of mesenchymal stem cells (MSCs) into neural lineages. Research suggests that MSCs can be differentiated into neural progenitor-like cells (NPCs) under the specific influence of paracrine factors particularly epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). Our recent research has found that the addition of insulin-like growth factor 1 (IGF-1) with the combination of the EGF and bFGF could significantly improve the growth and survivability of MSC-derived NPCs. To unravel the molecular mechanism of the improved differentiation we compared the microRNA expression profiles of the differentiation under various combinations of growth factors. MSCs were differentiated into neural lineage in 3 groups; Group A (EGF + bFGF), Group B (EGF + bFGF + IGF-1), and Group C (without growth factor). Regulated microRNAs during the early differentiation were identified by detailed microRNA profiling using Affymetrix GeneChip version 2.0 at three time intervals (day 1, day 3 and day 5). The data were deposited in the Gene Expression Omnibus, series GSE60060.