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Dental caries is a highly prevalent chronic disease that leads to dental pulp inflammation. It is treated by removing the damaged tooth structure and applying a material that promotes resolution of pulpal inflammation. Tumor necrosis factor superfamily 14 (TNFSF14) is an immunomodulatory cytokine and a member of the TNF superfamily. This study aimed to evaluate the effect of TNFSF14 on the levels of inflammatory cytokines involved in pulpal inflammation using lipoteichoic acid (LTA)-induced human dental pulp stem cells (hDPSCs). hDPSCs were cultured and induced with LTA, followed by treatment with TNFSF14 at 25 and 50 ng/mL. Cellular viability was evaluated using the Alamar Blue assay. The levels of inflammatory cytokines IL-6, IL-8, IL-10, and TNF-α were quantified using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA). TNFSF14 at 25 and 50 ng/mL significantly reduced the mRNA and protein levels of pro-inflammatory cytokines TNF-α , IL-6 , and IL-8 , and increased the anti-inflammatory cytokine IL-10 . In addition, TNFSF14-treated groups enhanced cell viability. Adding TNFSF14 to LTA-induced hDPSCs regulated the production of inflammatory cytokines by lowering the levels of IL-6, IL-8, and TNF-α and elevating IL-10 levels.

The present study examined the functional activities of the human bone marrow mesenchymal stem cells (hBM-MSCs) under the effects of various concentrations of the inflammatory mediator interleukin 1 beta (IL-1β). The effects of IL-1β on the functional properties of hBM-MSCs were measured using functional assays (adhesion, proliferation, and migration). hBM-MSCs expressions of colony-stimulating factors 1 and 2 (CSF1, CSF2), C-C chemokine receptor type 2 (CCR2), C-X-C chemokine receptor type 1 and 3 (CXCL1, CXCL3), were examined using real-time polymerase chain reaction (RT‒PCR). The pro-inflammatory cytokine IL-1β did not disrupt hBM-MSCs adhesion, but it improved proliferation and migration only up to 50 ng/ml. However, in response to 100 ng/ml IL-1β, cell growth, proliferation, and migration were reduced significantly. The expression of CSF1, CCR2, CXCL3, and IL-1β genes increased with the increase in the concentration of IL-1β. CSF2 and CXCL1 gene expression increased in the 50ng/ml group compared with the 10ng/ml group to be higher than the control group in the 100ng/ml IL-1β group which might facilitate the differentiation, and homing of MSCs to the site of injury and augment their activities in the inflamed microenvironment. The study corroborates the advantages of prior stimulation of mesenchymal stem cells (MSCs) with the cytokine IL-1β, demonstrating an upregulation of key chemokines and cytokines. This upregulation potentially enhances MSCs' ability to differentiate and migrate to injury sites, while also augmenting their functional role within an inflamed microenvironment, thereby amplifying their therapeutic potential.

Background/purpose: Various materials have been used to promote human dental pulp stem cells (hDPSCs) differentiation to produce dentin bridge formation with less-than-optimal results. Polydatin (PD), a naturally present material with osteogenic properties can be a promising material in the pulp regeneration/repair process. The aim of this study was to evaluate the effect of (PD) on the viability and differentiation of human dental pulp stem cells., Materials and Methods: PD effect on hDPSCs in terms of cellular viability, alkaline phosphatase (ALP) production, and messenger RNAs (mRNA) of odontogenic markers production using quantitative reverse transcription polymerase chain reaction (RT-qPCR) were evaluated. In addition, mineral deposits were detected with Alizarin red stain., Results: The viable hDPSCs in the presence of 0.01 μM and 0.1 μM PD were significantly higher than the control on days 3 and 7, respectively. In addition, ALP activity of hDPSCs was significantly increased with 0.01, 0.1, and 1 μM of PD. In addition, increased expression mRNAs of ALP, osteocalcin (OC), osteonectin (ON), osteopontin (OP), Runt-related transcription factor-2 (RUNX-2), dentin sialophosphoprotein (DSPP), and dentin matrix protein-1 (DMP-1) was observed after PD treatment, however, the difference was not statistically significant. Furthermore, increased size of mineral deposits was observed with PD., Conclusion: PD promoted the expression of markers associated with odontogenic differentiation and mineralized tissue deposition in hDPSCs., Competing Interests: The authors have no conflicts of interest relevant to this article., (© 2024 Association for Dental Sciences of the Republic of China. Publishing services by Elsevier B.V.)

Background: We studied the potential of human bone marrow-derived mesenchymal stem cell conditioned media (hBMSC CM) in protecting endothelial cell properties (viability, proliferation, and migrations) from the deleterious effects produced by the inflammatory environment of H 2 O 2 . Additionally, we investigated their impact on the endothelial cells' gene expression of some inflammatory-related genes, namely, TGF- β 1, FOS, ATF3, RAF-1, and SMAD3. Methods: Human umbilical vein endothelial cells (HUVECs) were cultured individually under three conditions: alone, with varying concentrations of H 2 O 2 , or with varying concentrations of H 2 O 2 and hBMSC CM. HUVEC adhesion, proliferation, and migration were evaluated using the xCELLigence system. The HUVECs' gene expressions were evaluated by real-time polymerase chain reaction (RT-PCR). Results: Generally, we observed enhanced HUVEC viability, proliferation, and migration when cultured in media supplemented with H 2 O 2 and hBMSC CM. Furthermore, the CM modulated the expressions of the studied inflammatory-related genes in HUVECs, promoting a more robust cellular response. Conclusion: This study has illuminated the protective role of hBMSC CM in mitigating the damaging effects of H 2 O 2 on endothelial cell function. Our data demonstrate that hBMSC CM enhances the viability, proliferation, and migration of HUVECs even under oxidative stress conditions. Additionally, the conditioned medium was found to modulate the gene expression of pivotal markers related to inflammation, suggesting a favorable influence on cellular response mechanisms., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2024 Abdulmajeed Alqasoumi et al.)

Bone formation is regulated by numerous factors, such as transcription factors, cytokines, and extracellular matrix molecules. Human hormone nuclear receptors (hHNR) are a family of ligand-regulated transcription factors that are activated by steroid hormones, such as estrogen and progesterone, and various lipid-soluble signals, including retinoic acid, oxysterols, and thyroid hormone. We found that an hHNR called NR4A1 was the most highly expressed after human MSC differentiation into osteoblasts by whole-genome microarray. NR4A1 knockout decreased the osteoblastic differentiation of hMSCs in terms of ALPL expression and key marker gene expression. Whole-genome microarray analysis further confirmed the decrease in key pathways when we knocked down NR4A1. Further studies with small molecule activators identified a novel molecule called Elesclomol (STA-4783), which could activate and enhance osteoblast differentiation. Elesclomol activation of hMSCs also induced the gene expression of NR4A1 and rescued the phenotype of NR4A1 KD. In addition, Elesclomol activated the TGF-ß pathway by regulating key marker genes. In conclusion, we first identified the role of NR4A1 in osteoblast differentiation and that Elesclomol is a positive regulator of NR4A1 through activation of the TGF-ß signalling pathway., (© 2023 John Wiley & Sons Ltd.)

Background/purpose: Iloprost has been proposed as a potential biomaterial owing to angiogenic and odontogenic properties. However, the liquid form can limit its use during clinical applications. Mineral trioxide aggregate (MTA) has been used for various dental applications in which cell-material interaction is essential. This study aimed to investigate additive effects of iloprost on the biological properties of MTA on the viability, attachment, migration and differentiation of human mesenchymal stem cells (hMSCs)., Materials and Methods: Standardized human dentin disks were prepared. MTA was prepared by mixing distilled water or iloprost solution, and the lumen of the disks was filled with MTA or MTA-iloprost. hMSCs on disk alone and hMSCs on culture plates were used as controls. Cell viability and attachment were measured after 1, 7 and 14 days using AlamarBlue assay and scanning electron microscopy (SEM). Cell migration in MTA or MTA-iloprost extracts was determined using a wound-healing model.Osteogenic differentiation was evaluated by real-time reverse transcriptase polymerase chain reaction for alkaline phosphatase (ALP), bone sialoprotein (BSP), osteocalcin (OCN), and osteopontin (OSP) gene expressions after 7 and 14 days of osteogenic induction., Results: Cells on MTA-iloprost surface showed similar viability with MTA at 1 and 14 days but enhanced cellular viability and cell spreading compared to MTA at 7 days (p < 0.05). Cell migration was similar by MTA-iloprost and MTA extracts (p > 0.05). MTAiloprost significantly upregulated BSP, OCN and OSP expressions compared to MTA (p < 0.05)., Conclusion: The addition of iloprost to MTA improved the initial cell viability and osteogenic potential of hMSCs., Competing Interests: The authors declare that there is no conflict of interest regarding the publication of this article., (© 2021 Association for Dental Sciences of the Republic of China. Publishing services by Elsevier B.V.)

Background: Osteoporosis is associated with a metabolic imbalance between adipogenesis and osteogenesis. We hypothesized that implanting a carrier for differentiated stem cells and signaling molecules inside adipose tissues could be used to enable transdifferentiation between cells, upregulate osteogenesis, and support bone formation, which may regain the balance between osteogenesis and adipogenesis. Methodology: A CL1 human mesenchymal stem cell line was grown in an osteogenic medium to differentiate into osteoblasts, and the differentiated cells were then exposed to an adipogenic medium to stimulate differentiation into adipocytes. Osteogenic and adipogenic differentiation were confirmed by the following assays: alkaline phosphatase staining, Nile red Staining, and quantitative real-time polymerase chain reaction (qPCR). The ratio of adipocytes to osteocytes for both cases was calculated. To evaluate bone induction in vivo , a calcium sulfate/hydroxyapatite cement was prepared in a syringe and then seeded with 10 6 cells/mL of rat bone marrow stromal cells (rMSCs) and covered with 1 mL of tissue culture media containing 0.1 mg of bone morphogenetic protein 7 (BMP-7). The construct was injected into the abdominal fat tissue of 10 male Sprague-Dawley rats. Results: The conversion of osteocytes to adipocytes was 20-fold greater than the reverse conversion, and the area of bone regeneration was 15.7 ± 3.7%, the area of adipose tissue was 65.8 ± 13.1%, and the area of fibrous tissue was 18.3 ± 7.8%. Conclusion: Adipogenic interconversion and associated bone formation demonstrate the potential of a new therapy for balancing osteogenesis and adipogenesis.

Colorectal cancer (CRC) is the fourth most common cancer type globally. Investigating the signaling pathways that maintain cancer cell phenotype can identify new biomarkers for targeted therapy. Aberrant transforming growth factor-β (TGFβ) signaling has been implicated in CRC progression, however, the exact mechanism by which TGFβ exerts its function is still being unraveled. Herein, we investigated TAGLN expression, prognostic value, and its regulation by TGFβ in CRC. While TAGLN was generally found to be downregulated in CRC, elevated expression of TAGLN was associated with advanced CRC stage and predicted poor overall survival (hazard ratio (HR) = 1.8, log-rank test P-value = 0.014) and disease-free survival (HR = 1.6, log-rank test P-value = 0.046), hence implicating TAGLN as poor prognostic factor in CRC. Forced expression of TAGLN was associated with enhanced CRC cell proliferation, clonogenic growth, cell migration and in vivo tumor formation in immunocompromised mice, while targeted depletion of TAGLN exhibited opposing biological effects. Global gene expression profiling of TAGLN-overexpressing or TAGLN-deficient CRC cell lines revealed deregulation of multiple cancer-related genes and signaling pathways. Transmission electron microscopy (TEM) revealed ultrastructural changes due to loss of TAGLN, including disruption of actin cytoskeleton organization and aberrant actin filament distribution. Hierarchical clustering, principle component, and ingenuity pathway analyses revealed distinct molecular profile associated with TAGLN high CRC patients with remarkable activation of a number of mechanistic networks, including SMARCA4, TGFβ1, and P38 MAPK. The P38 MAPK was the top predicted upstream regulator network promoting cell movement through regulation of several intermediate molecules, including TGFβ1. Concordantly, functional categories associated with cellular movement and angiogenesis were also enriched in TAGLN high CRC, supporting a model for the molecular mechanisms linking TGFβ-induced upregulation of TAGLN and CRC tumor progression and suggesting TAGLN as potential prognostic marker associated with advanced CRC pathological stage.

The Arabian camel is the largest known mammal that can survive in severe hot climatic conditions. We provide the molecular explanation for the thermotolerance of camel granulosa somatic cells after exposure to 45 °C for 2 (acute heat shock) or 20 h (chronic heat shock). The common features of the cellular responses to acute heat stress were the increase of heat shock proteins and DNA repair enzymes expression. Actin polymerization and Rho signaling were critically activated as a cellular defense against heat shock. Cells exposed to chronic heat shock showed altered cell architecture with a decrease in total detected proteins, metabolic enzymes, and cytoskeletal protein expression. Treatment with transforming growth factor beta (TGFβ) pathway inhibitor SB-431542 suppressed the morphological alterations of cells exposed to chronic heat shock. Moreover, during the recovery stage at 38 °C for 24 h, proteomic changes were partially restored with an exponential increase in HSP70 expression, and the cells restored their normal cellular morphology on the 9th day of recovery. Full proteomics data are available via ProteomeXchange with identifier PXD012159. The strategies of cellular defense and tolerance to both thermal conditions reflect the flexible adaptability of camel somatic cells to conserve life under extremely hot conditions., Competing Interests: Author declares that there is no conflicts of interest., (© 2020 THE AUTHORS. Published by Elsevier BV on behalf of Cairo University.)

All-trans retinoic acid (RA) is a metabolite of vitamin A and has pleiotropic actions on many different biological processes, including cell growth and differentiation, and is involved in different aspects of fertility and developmental biology. In the current study, we investigated the effects of RA on camel (Camelus dromedarius) cumulus-oocyte complex in vitro maturation (IVM). IVM medium was supplemented with 0, 10, 20, and 40 µM RA. Application of 20 µM RA significantly reduced the proportion of degenerated oocytes and significantly improved oocyte meiosis and first polar body extrusion compared to the control and other experimental groups. Retinoic acid significantly reduced the mRNA transcript levels of apoptosis-related genes, including BAX and P53, and reduced the BAX/BCL2 ratio. In addition, RA significantly reduced the expression of the Transforming growth factor beta (TGFβ) pathway-related transcripts associated with the actin cytoskeleton, ACTA2 and TAGLN; however, RA increased TGFβ expression in cumulus cells. The small molecule SB-431542 inhibits the TGFβ pathway by inhibiting the activity of activin receptor-like kinases (ALK-4, ALK-5, and ALK-7); however, combined supplementation with RA during IVM compensated for the inhibitory effect of SB-431542 on cumulus expansion, oocyte meiosis I, and first polar body extrusion in activated oocytes. The current study shows the beneficial effects of RA on camel oocyte IVM and provides a model to study the multifunctional mechanisms involved in cumulus expansion and oocyte meiosis, particularly those involved in the TGFβ pathway.

Targeting regulatory signaling pathways that control human bone marrow stromal (skeletal or mesenchymal) stem cell (hBMSC) differentiation and lineage fate determination is gaining momentum in the regenerative medicine field. Therefore, to identify the central regulatory mechanism of osteoblast differentiation of hBMSCs, the molecular phenotypes of two clonal hBMSC lines exhibiting opposite in vivo phenotypes, namely, bone forming (hBMSC +bone ) and non-bone forming (hBMSC -Bone ) cells, were studied. Global transcriptome analysis revealed significant downregulation of several TGFβ responsive genes, namely, TAGLN, TMP1, ACTA2, TGFβ2, SMAD6, SMAD9, BMP2, and BMP4 in hBMSC -Bone cells and upregulation on SERPINB2 and NOG. Transcriptomic data was associated with marked reduction in SMAD2 protein phosphorylation, which thereby implies the inactivation of TGFβ and BMP signaling in those cells. Concordantly, activation of TGFβ signaling in hBMSC -Bone cells using either recombinant TGFβ1 protein or knockdown of SERPINB2 TGFβ-responsive gene partially restored their osteoblastic differentiation potential. Similarly, the activation of BMP signaling using exogenous BMP4 or via siRNA-mediated knockdown of NOG partially restored the differentiation phenotype of hBMSC -Bone cells. Concordantly, recombinant NOG impaired ex vivo osteoblastic differentiation of hBMSC +Bone cells, which was associated with SERBINB2 upregulation. Our data suggests the existence of reciprocal relationship between TGFB and BMP signaling that regulates hBMSC lineage commitment and differentiation, whilst provide a plausible strategy for generating osteoblastic committed cells from hBMSCs for clinical applications.

Endochondral ossification is the process by which long bones are formed; the process of long bone formation is regulated by numerous factors such as transcription factors, cytokines, and extracellular matrix molecules. Human hormone Nuclear receptors (hHNR) are a family of ligand-regulated transcription factors that are activated by steroid hormones, such as estrogen and progesterone, and various lipid-soluble signals, including retinoic acid, oxysterols, and thyroid hormone. Whole genome microarray data from our previous study revealed that most hHNR's are up-regulated during osteoblast differentiation in hMSCS. NR2F1 was among the highest expressed hHNR during osteogenesis, NR2F1 belongs to the steroid/thyroid hormone nuclear receptor superfamily. NR2F1 is designated as an orphan nuclear receptor because its ligands are unknown. NR2F1 plays a wide range of roles, including cell differentiation, cancer progression, and central and peripheral neurogenesis. Identifying signaling networks involved in osteoblast differentiation is important in orchestrating new therapeutic and clinical applications in bone biology. This study aimed to identify alterations in signaling networks mediated by NR2F1 in osteoblast differentiation. siRNA-mediated down-regulation of NR2F1 leads to impairment in the differentiation of hBMSC-TERT to osteoblast; gene-expression results confirmed the down-regulation of osteoblast markers such as RUNX2, ALPL, OSC, and BSP. Global whole gene expression analysis revealed that most down-regulated genes were associated with osteoblast differentiation (DDIT3, BMP2). Pathway analysis revealed prominent signaling pathways that were down-regulated, including the TGFβ pathway and MAPK pathway. Functional studies on NR2F1 transfected cells, during osteoblast differentiation in combination with TGFβ1 and BMP-2, showed that TGFβ1 does not recover osteoblast differentiation, whereas BMP-2 rescues osteoblast differentiation in NR2F1 siRNA transfected cells. Thus, our results showed that BMP-2 could intervene in NR2F1 down-regulated signaling pathways to recover osteoblast differentiation., (Copyright © 2018 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.)

The mammalian ovary is a highly dynamic organ, in which proliferation and differentiation occur constantly during the entire life span, particularly in camels that are characterized by a follicular wave pattern and induced ovulation. Granulosa cells are the main cells of mature follicles. Two distinct cell types, namely, the mural and cumulus granulosa cells are distinguished on the basis of antral fluid increase. The multipotency of follicular fluid and the luteinizing cell were recently demonstrated. However, reports regarding the plasticity of cumulus cells are lacking. We obtained cumulus cells from cumulus-oocyte complexes and showed that camel cumulus cells expressed stem cell mRNA transcripts (POU5A1, KLF4, SOX2, and MYC) and were able to differentiate into other non-ovarian follicular cell types in vitro, such as neurons, osteoblasts, and adipocytes. In contrast, removal of the ooplasm (oocytectemy) showed no effect on cumulus cell proliferation and differentiation. This is the first report to identify an invaluable source of multipotent stem cells, which is routinely discarded during in vitro embryo production. The plasticity and transdifferentiation capability of camel cumulus cells definitely requires attention as it provides a cheap biological experimental model for basic research in stem cells and for understanding ovarian differentiation, both of which are relevant for use in regenerative medicine and tissue engineering in humans and animals., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The dromedary camel (Camel dromedarius) is physiologically well adapted to life in hot, dry and barren land. In the present study, we report the tolerance of camel oocytes and cumulus cells to acute and chronic heat shock. Camel oocytes and cumulus cells were exposed to acute (45 °C for 2 h) and chronic (45 °C for 20 h) heat shock. Our results demonstrated that acute and chronic heat shock altered malondialdehyde concentration, which is a marker for oxidative stress. Furthermore, the heat shock reduced glutathione levels during in vitro oocyte maturation. The expression of two well-known heat shock proteins HSP70 and HSP90 were increased similarly in oocytes and cumulus cells after acute heat shock. Oocytes were less tolerant to the short acute heat shock, and showed decreased maturation, which leads to reduction in ooplasmic diameter and an increase in chromosomal count abnormalities. Furthermore, the pro-apoptotic genes P53 and BAX had increased expression levels, whereas for the anti-apoptotic gene such as BCL2 expression levels was decreased. On the other hand, the cumulus cells tolerated acute and chronic heat shock, as evident by the increase in HSP70 and HSP90 expression and steady expression levels of P53, BAX, and BCL2 after acute hyperthermia. Cumulus cells regained their vitality and ability to proliferate after chronic hyperthermia and showed wound healing capabilities after 9 days of chronic hyperthermia. Collectively, these results indicate the adaptive tolerance of camel somatic cells to acute and chronic heat shock, which is lethal to cells in many other mammals., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

TGF β is a potent regulator of several biological functions in many cell types, but its role in the differentiation of human bone marrow-derived skeletal stem cells (hMSCs) is currently poorly understood. In the present study, we demonstrate that a single dose of TGF β 1 prior to induction of osteogenic or adipogenic differentiation results in increased mineralized matrix or increased numbers of lipid-filled mature adipocytes, respectively. To identify the mechanisms underlying this TGF β -mediated enhancement of lineage commitment, we compared the gene expression profiles of TGF β 1-treated hMSC cultures using DNA microarrays. In total, 1932 genes were upregulated, and 1298 genes were downregulated. Bioinformatics analysis revealed that TGF β l treatment was associated with an enrichment of genes in the skeletal and extracellular matrix categories and the regulation of the actin cytoskeleton. To investigate further, we examined the actin cytoskeleton following treatment with TGF β 1 and/or cytochalasin D. Interestingly, cytochalasin D treatment of hMSCs enhanced adipogenic differentiation but inhibited osteogenic differentiation. Global gene expression profiling revealed a significant enrichment of pathways related to osteogenesis and adipogenesis and of genes regulated by both TGF β 1 and cytochalasin D. Our study demonstrates that TGF β 1 enhances hMSC commitment to either the osteogenic or adipogenic lineages by reorganizing the actin cytoskeleton.

TGF-β1, a multifunctional regulator of cell growth and differentiation, is the most abundant bone matrix growth factor. During differentiation of human bone stromal cells (hBMSCs), which constitute bone marrow osteoblast (OS) and adipocyte (AD) progenitor cells, continuous TGF-β1 (10 ng/ml) treatment enhanced OS differentiation as evidenced by increased mineralised matrix production. Conversely, pulsed TGF-β1 administration during the commitment phase increased mature lipid-filled adipocyte numbers. Global gene expression analysis using DNA microarrays in hBMSCs treated with TGF-β1 identified 1587 up- and 1716 down-regulated genes in OS-induced, TGF-β1-treated compared to OS-induced hBMSCs (2.0 fold change (FC), p < 0.05). Gene ontology (GO) analysis revealed enrichment in 'osteoblast differentiation' and 'skeletal system development-associated' genes and up-regulation of several genes involved in 'osteoblastic-differentiation related signalling pathways'. In AD-induced, TGF-β1-treated compared to AD-induced hBMSCs, we identified 323 up- and 369 down-regulated genes (2.0 FC, p < 0.05) associated with 'fat cell differentiation', 'fatty acid derivative biosynthesis process', 'fatty acid derivative metabolic process', and 'inositol lipid-mediated'. Serpin peptidase inhibitor, clade B (ovalbumin), member 2 (SERPINB2) was down-regulated 3-fold in TGF-β1-treated hBMSCs. siRNA-mediated SERPINB2 inhibition enhanced OS and AD differentiation. Thus, TGF-β signalling is important for hBMSC OS and AD differentiation and SERPINB2 is a TGF-β-responsive gene that plays a negative regulatory role in hBMSC differentiation.

We isolated and characterized trophoblast from in vivo-derived camel embryos and compared with embryonic stem-like cells. Camel embryos were flushed on day 8 post-insemination and used to derive trophectoderm and embryonic stem-like cells under feeder-free culture conditions using a basement membrane matrix. Embryos were evaluated for the expression of POU5F1, MYC, KLF4, SOX2, CDX2, and KRT8 mRNA transcripts by relative quantitative polymerase chain reaction. Camel embryos grew and expanded to ∼4.5 mm and maintained their vesicular shape in vitro for 21 days post-insemination. Trophoblast and embryonic stem-like cell lines grew under feeder-free culture conditions and showed distinct morphological criteria and normal chromosomal counts. Embryonic stem-like cells showed positive staining in the alkaline phosphatase reaction. Trophoblast cells showed a significant increase in CDX2, KRT8, KLF4, and SOX2 expression compared with embryonic stem-like cells and whole embryos. Embryonic stem-like cells showed a significant decrease in CDX2 expression and increase in SOX2 and KRT8 expression compared to embryonic expression. POU5F1 and MYC expression showed no difference between embryos and both cell lines. We characterized embryo survival in vitro, particularly the derivation of trophectoderm and embryonic stem-like cells, providing a foundation for further analysis of early embryonic development and placentation in camels., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Understanding the regulatory networks underlying lineage differentiation and fate determination of human bone marrow stromal cells (hBMSC) is a prerequisite for their therapeutic use. The goal of the current study was to unravel the novel role of the low-density lipoprotein receptor-related protein 3 (LRP3) in regulating the osteogenic and adipogenic differentiation of immortalized hBMSCs. Gene expression profiling revealed significantly higher LRP3 levels in the highly osteogenic hBMSC clone imCL1 than in the less osteogenic clone imCL2, as well as a significant upregulation of LRP3 during the osteogenic induction of the imCL1 clone. Data from functional and gene expression assays demonstrated the role of LRP3 as a molecular switch promoting hBMSC lineage differentiation into osteoblasts and inhibiting differentiation into adipocytes. Interestingly, microRNA (miRNA) expression profiling identified miR-4739 as the most under-represented miRNA (-36.11 fold) in imCL1 compared to imCL2. The TargetScan prediction algorithm, combined with functional and biochemical assays, identified LRP3 mRNA as a novel target of miR-4739, with a single potential binding site for miR-4739 located in the LRP3 3' UTR. Regulation of LRP3 expression by miR-4739 was subsequently confirmed by qRT-PCR, western blotting, and luciferase assays. Over-expression of miR-4739 mimicked the effects of LRP3 knockdown on promoting adipogenic and suppressing osteogenic differentiation of hBMSCs. Hence, we report for the first time a novel biological role for the LRP3/hsa-miR-4739 axis in balancing osteogenic and adipocytic differentiation of hBMSCs. Our data support the potential utilization of miRNA-based therapies in regenerative medicine., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

Human bone marrow-derived stromal stem cells (hBMSC) exhibit multiple functions, including differentiation into skeletal cells (progenitor function), hematopoiesis support, and immune regulation (nonprogenitor function). We have previously demonstrated the presence of morphological and functional heterogeneity of hBMSC cultures. In the present study, we characterized in detail two hTERT-BMSC clonal cell populations termed here CL1 and CL2 that represent an opposing phenotype with respect to morphology, markers expression: alkaline phosphatase (ALP) and CD146, and ex vivo differentiation potential. CL1 differentiated readily to osteoblasts, adipocytes, and chondrocytes as shown by expression of lineage specific genes and proteins. Whole genome transcriptome profiling of CL1 versus CL2 revealed enrichment in CL1 of bone-, mineralization-, and skeletal muscle-related genes, for example, ALP, POSTN, IGFBP5 BMP4, and CXCL12. On the other hand, CL2 transcriptome was enriched in immune modulatory genes, for example, CD14, CD99, NOTCH3, CXCL6, CFB, and CFI. Furthermore, gene expression microarray analysis of osteoblast differentiated CL1 versus CL2 showed significant upregulation in CL1 of bone development and osteoblast differentiation genes which included several homeobox genes: TBX15, HOXA2 and HOXA10, and IGF1, FGFR3, BMP6, MCAM, ITGA10, IGFBP5, and ALP. siRNA-based downregulation of the ALP gene in CL1 impaired osteoblastic and adipocytic differentiation. Our studies demonstrate the existence of molecular and functional heterogeneity in cultured hBMSC. ALP can be employed to identify osteoblastic and adipocytic progenitor cells in the heterogeneous hBMSC cultures.

The fine structures of mouse embryonic stem cells (mESCs) grown as colonies and differentiated in three-dimensional (3D) culture as embryoid bodies (EBs) were analyzed by transmission electron microscopy. Undifferentiated mESCs expressed markers that proved their pluripotency. Differentiated EBs expressed different differentiation marker proteins from the three germ layers. The ultrastructure of mESCs revealed the presence of microvilli on the cell surfaces, large and deep infolded nuclei, low cytoplasm-to-nuclear ratios, frequent lipid droplets, nonprominent Golgi apparatus, and smooth endoplasmic reticulum. In addition, we found prominent juvenile mitochondria and free ribosomes-rich cytoplasm in mESCs. Ultrastructure of the differentiated mESCs as EBs showed different cell arrangements, which indicate the different stages of EB development and differentiation. The morphologies of BALB/c and 129 W9.5 EBs were very similar at day 4, whereas C57BL/6 EBs were distinct from the others at day 4. This finding suggested that differentiation of EBs from different cell lines occurs in the same pattern but not at the same rate. Conversely, the ultrastructure results of BALB/c and 129 W9.5 ESCs revealed differentiating features, such as the dilated profile of a rough endoplasmic reticulum. In addition, we found low expression levels of undifferentiated markers on the outer cells of BALB/c and 129 W9.5 mESC colonies, which suggests a faster differentiation potential.

Background: Embryonic stem cells (ESCs) have the potential to form teratomas when implanted into immunodeficient mice, but data in immunocompetent mice are limited. We therefore investigated teratoma formation after implantation of three different mouse ESC (mESC) lines into immunocompetent mice., Materials and Methods: BALB/c mice were injected with three highly germline competent mESCs (129Sv, BALB/c and C57BL/6) subcutaneously or under the kidney capsule. After 4 weeks, mice were euthanized and examined histologically for teratoma development. The incidence, size and composition of teratomas were compared using Pearson Chi-square, t-test for dependent variables, one-way analysis of variance and the nonparametric Kruskal- Wallis analysis of variance and median test., Results: Teratomas developed from all three cell lines. The incidence of formation was significantly higher under the kidney capsule compared to subcutaneous site and occurred in both allogeneic and syngeneic mice. Overall, the size of teratoma was largest with the 129Sv cell line and under the kidney capsule. Diverse embryonic stem cell-derived tissues, belonging to the three embryonic germ layers, were encountered, reflecting the pluripotency of embryonic stem cells. Most commonly represented tissues were nervous tissue, keratinizing stratified squamous epithelium (ectoderm), smooth muscle, striated muscle, cartilage, bone (mesoderm), and glandular tissue in the form of gut- and respiratory-like epithelia (endoderm)., Conclusions: ESCs can form teratomas in immunocompetent mice and, therefore, removal of undifferentiated ESC is a pre-requisite for a safe use of ESC in cell-based therapies. In addition the genetic relationship of the origin of the cell lines to the ability to transplant plays a major role.

Derivation of bone forming cells (osteoblasts) from human embryonic stem cells (hESCs) is a prerequisite for their use in clinical applications. However, there is no standard protocol for differentiating hESCs into osteoblastic cells. The aim of this study was to identify the emergence of a human stromal (mesenchymal and skeletal) stem cell (hMSC)-like population, known to be osteoblastic cell precursors and to test their osteoblastic differentiation capacity in ex vivo cultures and in vivo. We cultured hESCs in a feeder-free environment using serum replacement and as suspension aggregates (embryoid bodies; hEBs). Over a 20 day developmental period, the hEBs demonstrated increasing enrichment for cells expressing hMSC markers: CD29, CD44, CD63, CD56, CD71, CD73, CD105, CD106, and CD166 as revealed by immunohistochemical staining and flow cytometry (fluorescence-activated cell sorting) analysis. Ex vivo differentiation of hEBs using bone morphogenic protein 2 (BMP2) combined with standard osteoblast induction medium led to weak osteoblastic induction. Conversely, subcutaneous in vivo implantation of day 20 hEBs in immune deficient mice, mixed with hydroxyapatite/tricalcium phosphate (HA/TCP) as an osteoconductive scaffold, revealed bone and cartilage, and fibrous tissue elements after 8 weeks. These tissues were of human origin and there was no evidence of differentiation to nonmesodermal tissues. hEBs implanted in the absence of HA/TCP formed vacuolated tissue containing glandular, fibrous and muscle-like tissue elements. Conversely, implantation of undifferentiated hESCs resulted in the formation of a teratoma containing a mixture of endodermal, mesodermal, and ectodermal tissues. Our study demonstrates that hMSC-like cells can be obtained from hESCs and they can be induced to form skeletal tissues in vivo when combined with HA/TCP. These findings are relevant for tissue engineering and suggest that differentiated hEBs can provide an unlimited source for functional osteogenic cells.