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.

miRNome and Proteome Profiling of Human Keratinocytes and Adipose Derived Stem Cells Proposed miRNA-Mediated Regulations of Epidermal Growth Factor and Interleukin 1-Alpha.

Wound healing is regulated by complex crosstalk between keratinocytes and other cell types, including stem cells. In this study, a 7-day direct co-culture model of human keratinocytes and adipose-derived stem cells (ADSCs) was proposed to study the interaction between the two cell types, in order to identify regulators of ADSCs differentiation toward the epidermal lineage. As major mediators of cell communication, miRNome and proteome profiles in cell lysates of cultured human keratinocytes and ADSCs were explored through experimental and computational analyses. GeneChip® miRNA microarray, identified 378 differentially expressed miRNAs; of these, 114 miRNAs were upregulated and 264 miRNAs were downregulated in keratinocytes. According to miRNA target prediction databases and the Expression Atlas database, 109 skin-related genes were obtained. Pathway enrichment analysis revealed 14 pathways including vesicle-mediated transport, signaling by interleukin, and others. Proteome profiling showed a significant upregulation of the epidermal growth factor (EGF) and Interleukin 1-alpha (IL-1α) compared to ADSCs. Integrated analysis through cross-matching the differentially expressed miRNA and proteins suggested two potential pathways for regulations of epidermal differentiation; the first is EGF-based through the downregulation of miR-485-5p and miR-6765-5p and/or the upregulation of miR-4459. The second is mediated by IL-1α overexpression through four isomers of miR-30-5p and miR-181a-5p.

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.

Xeno-free workflow exhibits comparable efficiency and quality of keratinocytes isolated from human skin biopsies.

INTRODUCTION: Regenerative solutions of the skin represent a hope for burn victims with extensive skin loss and chronic wound patients. The development of xeno-free workflow is crucial for clinical application in compliance with the directives of the European Medicines Agency. This study aimed at evaluating the outcome of the xeno-free isolation workflow of keratinocytes from human skin biopsy. METHODS: Skin biopsies were obtained from volunteers. The epidermis was digested with TrypLE™ Select, which was deactivated by dilution or with trypsin, deactivated by media with fetal bovine serum. Freshly isolated cells were compared for total cell number, viability, activity of caspase 3, gene expression and the presence of the keratinocyte surface markers cytokeratin 14. The cells were cultured in xeno-free conditions for one week and characterized regarding the number and viability as well as the metalloproteinase secretion. RESULTS: The number of obtained cells was similar in both workflows. The cell viability was less in the TrypLE group, with slight reduction of the cell surface marker cytokeratin 14. Caspase 3 activity was comparable as well as the gene expression of the apoptotic markers BAX, BCL2 and SLUG, as well as the keratinocyte markers cytokeratin 14, stratifin and filaggrin. Upon culture, the number of keratinocytes, their viability and secretion of matrix metalloproteinases 1 and 10 were equal in both groups. CONCLUSION: This study reports the possibility of isolating functioning and viable keratinocytes through a xeno-free workflow for clinical application.

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.

Involvement of ß-catenin in Androgen-induced Mesenchymal Transition of Breast MDA-MB-453 Cancer Cells.

Purpose The cellular and molecular dynamics of DHT-induced EMT in MDA-MB-453 cells were investigated.Methods:PCR arrays were used to examine the expression of EMT-regulatory genes. Immunoblotting was used to detect protein levels and confirm protein-protein interaction following immunoprecipitation. Immunofluorescence was used to observe rearrangement of the actin cytoskeleton and cell morphology. Cell migration was assessed by transwell assayResults: Change of cell morphology was concomitant with increased cell migration after treating cells with DHT. Exposure of cells to DHT for one hour was sufficient to induce changes in cell morphology and actin cytoskeleton after 72 hours indicating altered gene expression. A long-term lasting nuclear translocation of AR was observed after a short exposure of cells to DHT. Investigating the expression of 84 EMT-related genes revealed down-expression of ß-catenin, N-cadherin, and TCF-4 and increased expression of Slug, all of which were confirmed at the protein level. Yet, not only early interaction of AR and ß-catenin was observed following AR activation, inhibition of ß-catenin blocked DHT-induced mesenchymal transition and migration. Wnt signaling was found to be partially important in DHT-induced morphological alteration. The mesenchymal transition of cells could be induced by treating cells with an inhibitor of glycogen synthase kinase-3ß, an enzyme that inhibits ß-catenin; this morphological transition could be reversed by antagonizing AR suggesting that AR functions downstream of ß-catenin.Conclusions: These results suggest that MDA-MB-453 cells undergo partial EMT induced by DHT, ß-catenin is critical for this phenotypic change, and AR probably reciprocally mediates the mesenchymal transition of these cells upon activation of GSK-3 ß.

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.

Protein-Coated Aryl Modified Gold Nanoparticles for Cellular Uptake Study by Osteosarcoma Cancer Cells.

Gold nanoparticles coated with proteins have shown extraordinary biocompatibility which advanced to several nanomedicine engineering applications. We synthesized protein-coated gold nanoparticles using green and chemical reduction routes for cellular uptake study. In the current work, we coated gold-aryl nanoparticles of the type AuNPs-C6H4-4-COOH with bovine serum albumin (BSA), collagen, zein, and lysozyme proteins. Both routes were carried out without phase-transfer catalysts or extraneous stabilizing agents. High crystallinity of the AuNPs synthesized by the green route can be seen in transmission electron microscopy images. Osteosarcoma cancer cells are malignant bone tumors with abnormal cellular functions. Studies using MG-63 cells will provide mechanistic suggestions on the details of the amplification in tumors. We studied the cellular uptake of the bioconjugates by MG-63 osteosarcoma cells using laser confocal fluorescence microscopy (LCFM) and flow cytometry. In the LCFM study, BSA-AuNPs were uptaken most efficiently of all protein-coated gold nanoparticles synthesized by the green route. Lysozyme-AuNPs synthesized by the chemical reduction method were mostly efficiently internalized by MG-63 cells among all AuNPs. Zein- and lysozyme-coated AuNPs, though of relatively small size, prepared by the green method were not efficiently uptaken by MG-63. The two nanoparticles are negatively charged, and zein is also a hydrophobic coat. The difference in hydrophobicity and charge might have affected the internalization. All of those coated nanoparticles that were efficiently uptaken can potentially be used as diagnostic and therapeutic agents for osteosarcoma.

A Novel Nonsense Mutation (c.414G>A; p.Trp138*) in CLDN14 Causes Hearing Loss in Yemeni Families: A Case Report.

Non-syndromic hearing loss (NSHL) is a hereditary disorder that affects many populations. Many genes are involved in NSHL and the mutational load of these genes often differs among ethnic groups. Claudin-14 (CLDN14), a tight junction protein, is known to be associated with NSHL in many populations. In this study, we aimed to identify the responsible variants in 3 different Yemeni families affected with NSHL. Firstly, clinical exome sequencing (CES) performed for 3 affected patients from these different families identified a new nonsense variant (c.414G > A) in CLDN14. This variant was then confirmed by Sanger sequencing and PCR-RFLP. Subsequently, four microsatellite markers were used to genotype these families, which revealed a founder effect for this variant. Overall, this study illustrates the implication of the CLDN14 gene in the Yemeni population with NSHL and identifies a new founder variant.

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.

Paradoxical effects of the epigenetic modifiers 5-aza-deoxycytidine and suberoylanilide hydroxamic acid on adipogenesis.

Adipogenesis is an important biological process that is linked to obesity and metabolic disorders. On the other hand, fat regeneration is crucial as a restorative approach following mastectomy or severe burn injury. Furthermore, optimizing an in-vitro model of adipogenesis, which would help in understanding the possible effects and/or side effects of fat-soluble drugs and anti-obesity remedies, in addition to the developmental studies. Epigenetic is an important factor that is involved in cellular differentiation and commitment. This study aimed at investigating the effect of DNA methylation and histone deactylases inhibitors, 5-Aza-deoxycytidine (5-Aza-dC) and Suberoylanilide hydroxamic acid (SAHA), on the adipogenic differentiation process. The two modifiers were applied according to our previously published protocol, followed by three cycles of a classical, two-step adipogenesis protocol. The cells pretreated with SAHA showed enhanced expression of the many adipogenic genes, including peroxisome proliferator-activated receptor-γ as well as the accumulation of intracytoplasmic fat as shown by oil red and Nile red staining and the secretion of adipokines, such as MCP-1 and IP-10. On contrary, 5-Aza-dC inhibited all these markers. In conclusion, adding the reported step with SAHA to the differentiation protocols could have an impact on the progress of the in-vitro fat regenerative approach. The possible role of 5-Aza-dC in the inhibition of adipogenesis can be of clinical interest and will need further characterization in the future.