Molecular insights on Eltrombopag: potential mitogen stimulants, angiogenesis, and therapeutic radioprotectant through TPO-R activation.

The purpose of this study is to investigate the molecular interactions and potential therapeutic uses of Eltrombopag (EPAG), a small molecule that activates the cMPL receptor. EPAG has been found to be effective in increasing platelet levels and alleviating thrombocytopenia. We utilized computational techniques to predict and confirm the complex formed by the ligand (EPAG) and the Thrombopoietin receptor (TPO-R) cMPL, elucidating the role of RAS, JAK-2, STAT-3, and other essential elements for downstream signaling. Molecular dynamics (MD) simulations were employed to evaluate the stability of the ligand across specific proteins, showing favorable characteristics. For the first time, we examined the presence of TPO-R in human umbilical cord mesenchymal stem cells (hUCMSC) and human gingival mesenchymal stem cells (hGMSC) proliferation. Furthermore, treatment with EPAG demonstrated angiogenesis and vasculature formation of endothelial lineage derived from both MSCs. It also indicated the activation of critical factors such as RUNX-1, GFI-1b, VEGF-A, MYB, GOF-1, and FLI-1. Additional experiments confirmed that EPAG could be an ideal molecule for protecting against UVB radiation damage, as gene expression (JAK-2, ERK-2, MCL-1, NFkB, and STAT-3) and protein CD90/cMPL analysis showed TPO-R activation in both hUCMSC and hGMSC. Overall, EPAG exhibits significant potential in treating radiation damage and mitigating the side effects of radiotherapy, warranting further clinical exploration.

What is the context?● Chemotherapy, radiation treatment, or immunological disorders can cause a decrease in platelet count (thrombocytopenia) or decrease all blood cell types (pancytopenia) in the bone marrow. This can make it challenging to choose the appropriate cancer treatment plan.● Eltrombopag (EPAG) is an oral non-peptide thrombopoietin (TPO) mimetic that activates the cMPL receptor in the body. This activation leads to cell differentiation and proliferation, stimulating platelet production and reducing thrombocytopenia. The cMPL receptor is present in liver cells, megakaryocytes, and hematopoietic cells. However, its effects on stem cell proliferation and differentiation are not entirely understood.What is the new?● This study delves into the molecular interactions and therapeutic applications of EPAG, a small molecule that activates cMPL (TPO-R).● The study offers a comprehensive analysis of the ligand-receptor complex formation, including an examination of downstream signaling elements. Furthermore, molecular dynamics simulations demonstrate the stability of the ligand when interacting with targeted proteins.● The research investigates the presence of TPO-R on stem cell-derived endothelial cells, shedding insight into the ability of EPAG TPO-mimetic to promote angiogenesis and vasculature formation.● The study revealed that EPAG has the potential to protect against UVB-induced radiation damage and stimulate stem cell growth.What is the implications?The study emphasizes the potential of EPAG as a promising option for addressing radiation injury and minimizing the adverse effects of radiotherapy. It could revolutionize treatments not only for thrombocytopenia but also for enhancing the growth of stem cells. Furthermore, the research deepens our understanding of EPAG's molecular mechanisms, providing valuable insights for developing future drugs and therapeutic approaches for cell therapy to treat radiation damage.

Conditioned medium-enriched umbilical cord mesenchymal stem cells: a potential therapeutic strategy for spinal cord injury, unveiling transcriptomic and secretomic insights.

INTRODUCTION: Spinal cord injury (SCI) leads to significant destruction of nerve tissue, causing the degeneration of axons and the formation of cystic cavities. This study aimed to examine the characteristics of human umbilical cord-derived mesenchymal stem cells (HUCMSCs) cultured in a serum-free conditioned medium (CM) and assess their effectiveness in a well-established hemitransection SCI model. MATERIALS AND METHODS: In this study, HUCMSCs cultured medium was collected and characterized by measuring IL-10 and identifying proteomics using mass spectroscopy. This collected serum-free CM was further used in the experiments to culture and characterize the HUMSCs. Later, neuronal cells derived from CM-enriched HUCMSC were tested sequentially using an injectable caffeic acid-bioconjugated gelatin (CBG), which was further transplanted in a hemitransection SCI model. In vitro, characterization of CM-enriched HUCMSCs and differentiated neuronal cells was performed using flow cytometry, immunofluorescence, electron microscopy, and post-transplant analysis using immunohistology analysis, qPCR, in vivo bioluminescence imaging, and behavioral analysis using an infrared actimeter. RESULTS: The cells that were cultured in the conditioned media produced a pro-inflammatory cytokine called IL-10. Upon examining the secretome of the conditioned media, the Kruppel-like family of KRAB and zinc-finger proteins (C2H2 and C4) were found to be activated. Transcriptome analysis also revealed an increased expression of ELK-1, HOXD8, OTX2, YY1, STAT1, ETV7, and PATZ1 in the conditioned media. Furthermore, the expression of Human Stem-101 confirmed proliferation during the first 3 weeks after transplantation, along with the migration of CBG-UCNSC cells within the transplanted area. The gene analysis showed increased expression of Nestin, NeuN, Calb-2, Msi1, and Msi2. The group that received CBG-UCNSC therapy showed a smooth recovery by the end of week 2, with most rats regaining their walking abilities similar to those before the spinal cord injury by week 5. CONCLUSIONS: In conclusion, the CBG-UCNSC method effectively preserved the integrity of the transplanted neuronal-like cells and improved locomotor function. Thus, CM-enriched cells can potentially reduce biosafety risks associated with animal content, making them a promising option for clinical applications in treating spinal cord injuries.

Transdifferentiation of human gingival mesenchymal stem cells into functional keratinocytes by Acalypha indica in three-dimensional microenvironment.

Gingival tissue is reportedly a promising, easily accessible, abundant resource of mesenchymal stem cells (MSC) for use in various tissue engineering strategies. Human gingival MSC (HGMSCs) were successfully isolated from gingival tissue and characterized. To analyze in a two-dimensional form, HGMSCs were cultured with basal medium and induced with 25 µg/ml of Acalypha indica. Quantitative real-time polymerase chain reaction (qPCR) and western blot analysis showed the presence of keratinocyte-specific markers, including cytokeratin-5 and involucrin. To further assess its capability for stratification akin to human keratinocytes, HGMSCs were encapsulated in a HyStem® -HP Cell Culture Scaffold Kit and cultured in the presence of A. indica. Calcein AM staining indicated that the HyStem® -HP Scaffold Kit has excellent biocompatibility. Immunofluorescence and qPCR analysis revealed the presence of keratinocyte-specific markers. The study concluded that the three-dimensional microenvironment is a novel method for inducing epidermal differentiation of HGMSCs to engineer epidermal substitutes with the help of A. indica, which provides an alternative strategy for skin tissue engineering.

4PBA strongly attenuates endoplasmic reticulum stress, fibrosis, and mitochondrial apoptosis markers in cyclosporine treated human gingival fibroblasts.

Cyclosporine induces overgrowth of human gingiva. Previously we have shown (i) cyclosporine-inducing ER stress in human gingival fibroblasts (HGF), (ii) increased matrix protein expression, and (iii) interference with mitochondrial pro- and anti-apoptotic factors. This study was undertaken to assess the effects of melatonin (an antioxidant), 4PBA (an ER stress inhibitor), and simvastatin on the expression of ER Stress markers as well as on matrix and mitochondrial markers. HGF incubated with cyclosporine, or without melatonin/4PBA/statin. After 24 hr of incubation, mRNA expression of ER stress markers (GRP78, CHOP, XBP1, and XBPs) and matrix protein markers (like α-SMA, VEGF, TGF-ß, CTGF), and mitochondrial apoptosis markers estimated and compared with housekeeping gene GAPDH. Compared to the control cyclosporine significantly augmented ER Stress and matrix proteins, which decreased significantly with the use of melatonin, 4PBA, and simvastatin. The mitochondrial proapoptotic molecule cyclophilin D, as well as Bcl2 expression also decreased after PBA treatment, paralleling an increase in cytochrome c expression. The effect of 4PBA was much more pronounced than the influence of other two. In conclusion, 4PBA could be a viable therapeutic option for drug-induced gingival overgrowth.

Gingival spheroids possess multilineage differentiation potential.

Recently studies have demonstrated HGMSCs as ideal candidates for regenerative study. Interestingly we found that HGMSCs derived spheroids are more potent and maintain the properties of stemness convincingly compared to conventional culture methods. During the culture, GMSCs instinctively accumulated into spheroids and display multipotent STRO-1 and Vimentin-positive cells. Reduced phenotypic expression of CD73, CD105, and elevated expression STRO-1 and CD-34. Pluripotent nature of S-GMSCs putatively shown the expression of OCT4A, NANOG, SOX-2, SSEA4, TRA-1-60, and TRA-181. Also, levels of protein are much higher in spheroid than dissociated culture. On endothelial induction, spheroid differentiated and developed a vascular structure with positive expression of CD31 and on neuronal induction showed positivity for TUJ1 and E-Cadherin. Importantly, undifferentiated state of S-GMSCs exhibited significant upregulation of aforementioned pluripotent genes and lack of pro-inflammatory cytokines IL-6 and amplified ARF signal confirming that the spheroids are not teratoma formation. However, higher of CAP1, CP, TGFß, OPN, PPARÉ£, TUJ1, and NESTIN expression observed in spheroids, and minimal expression of the same markers were observed in adherent GMSCs respectively. Ahead of dissociated gingival culture, spheroid provides enhanced viable, pluripotent, and multilineage ability. This study suggested that S-GMSCs increased the chances of therapeutic efficacy in the regenerative applications.

Increased Advanced Oxidation Protein Products Generation by Cyclosporine-A and Angiotensin II in Human Gingival Fibroblasts - Ex-vivo Study.

INTRODUCTION: Cyclosporin-A (CsA), an immunosuppressant, induces renal fibrosis and Renin Angiotensin System (RAS) is known to play a major role. CsA has the potential to increase the oxidative stress; specifically through the Advanced Oxidation Protein Products (AOPP) which could possibly stimulate fibrosis. A similar type of pathology occurs even in the gingiva known as CsA Induced Gingival Overgrowth (CIGO). AIM: This study was undertaken to estimate the AOPP generation by Human Gingival Fibroblasts (HGF) under the influence of CsA and Angiotensin II (Ang II). MATERIALS AND METHODS: Six healthy gingival tissue samples were obtained during crown lengthening procedure and primary HGF were cultured using enzymatic digestion method. The ideal non-cytotoxic concentrations of CsA and Ang II were identified using cytotoxicity assay. Later, HGF were incubated with CsA and Ang II for 12 hours and AOPP assay was performed at zero and one hour interval. RESULTS: There was a statistically significant increase in AOPP production in both the CsA and Ang II when compared to the control group with a p value<0.05. CONCLUSION: CsA can induce oxidative stress and preventing/controlling it may be necessary to prevent untoward effect of the drug.

Cyclosporine-A induces endoplasmic reticulum stress and influences pro-apoptotic factors in human gingival fibroblasts.

Cyclosporine-A (CsA) induces gingival overgrowth. Cyclosporine's anti-apoptotic activity in human gingival fibroblast is due to desensitization of mitochondrial permeability transition pore (MPTP) and augmentation of anti-apoptotic, Bcl2. Alternative mechanisms of apoptosis exist involving enzymes like calcium-dependent Calpain and signaling events related to apoptosis, like Glycogen synthase kinase 3ß (GSK3ß) and protein kinase A (PKA). Cyclosporine-A in renal tubular cells induces endoplasmic reticulum stress (ER stress) which has not been explored in gingival overgrowth. Hence, this study was carried out to assess the influence of Cyclosporine-on ER stress and on the alternate anti-apoptotic mechanisms. Human gingival fibroblasts were treated with CsA, and expression of ER stress markers, such as binding immunoglobulin protein and CCAAT/enhancer-binding protein homologous protein (CHOP), MPTP, and expression of Calpain & GSK3ß /PKA were estimated. The results showed CsA-added fibroblast significantly increasing the expression of Endoplasmic reticulum stress markers. Contrary to usual ER stress outcome of apoptosis, we observed Cyclosporine's anti-apoptotic action in spite of augmented ER stress markers. We conclude that CsA's independent action on different organelles may alter the conventional outcome of ER stress.

Comparision of Gingival and Umbilical Cord Stem Cells Based on Its Modulus and Neuronal Differentiation.

The availability of Human Umbilical Cord-derived Mesenchymal Stem Cells (HUCMSCs) from a single sex being a major limitation for the utilization of a potential stem cell, it is highly desirable to utilize, an autogenous pluripotent cell with desirable biological and mechanical properties in clinical situations. Comparison of Human Gingival Mesenchymal Stem Cells (HGMSCs) with HUCMSCs demonstrates; MSCs derived from gingiva have higher proliferation rate and higher population doubling time than Umbilical Cord. Unlike HUCMSCs, immunofluorescence studies showed the presence of pluripotency markers OCT-4 and NANOG predominantly in the cytoplasm of HGMSCs which was confirmed by Western blot. The mechanical property, such as modulus of elasticity of HGMSCs, is on par with HUCMSCs, but the surface roughness found to be lesser in HGMSCs, which may suggest HGMSCs greater adhesive property to the extracellular matrix. There is a marginal difference in the neuronal differentiation rate between HGMSCs and HUCMSCs; both the cells expressed positivity for several neuronal lineage markers. Hence, HGMSCs represent an autogenous source of mesenchymal stem cells, which are easy to procure with least morbidity, multipotent in nature with desirable biological, and mechanical properties, probably an ideal candidate for clinical applications. J. Cell. Biochem. 118: 2000-2008, 2017. © 2017 Wiley Periodicals, Inc.

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

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

Differentiation of human gingival mesenchymal stem cells into neuronal lineages in 3D bioconjugated injectable protein hydrogel construct for the management of neuronal disorder

The success of regeneration attempt is based on an ideal combination of stem cells, scaffolding and growth factors. Tissue constructs help to maintain stem cells in a required area for a desired time. There is a need for easily obtainable cells, potentially autologous stem cells and a biologically acceptable scaffold for use in humans in different difficult situations. This study aims to address these issues utilizing a unique combination of stem cells from gingiva and a hydrogel scaffold, based on a natural product for regenerative application. Human gingival mesenchymal stem cells (HGMSCs) were, with due induction, differentiated to neuronal lineages to overcome the problems associated with birth tissue-related stem cells. The differentiation potential of neuronal lineages was confirmed with suitable specific markers. The properties of mesenchymal stem cells in encapsulated form were observed to be similar to free cells. The encapsulated cells (3D) were then subjected to differentiation into neuronal lineages with suitable inducers, and the morphology and gene expression of transient cells were analyzed. HGMSCs was differentiated into neuronal lineages as both free and encapsulated forms without any significant differences. The presence of Nissl bodies and the neurite outgrowth confirm the differentiation. The advantages of this new combination appear to make it a promising tissue construct for translational application.