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Cancer stem cells (CSCs) drive malignant tumor progression, recurrence, and metastasis with unique characteristics, including self-renewal and resistance to conventional treatments. Conventional differentiation inducers, although promising, have limited cytotoxicity and may inadvertently enhance CSC stemness. To address these challenges, ongoing efforts are dedicated to developing strategies that can effectively combine both cytotoxicity and differentiation-inducing effects. In this study, we introduce oridonin (Ori), a small molecule with dual differentiation-inducing and cytotoxicity properties capable of eliminating tumor CSCs. We isolated CSCs in B16F10 cells using the Hoechst side population method and assessed the differentiation effect of Ori. Ori's differentiation-inducing effect was further evaluated using human acute promyelocytic leukemia. The cytotoxic potential of Ori against MCF-7 and B16F10 cell lines was assessed through various methods. In vivo anti-tumor and anti-CSC efficacy of Ori was investigated using mouse melanoma and CSCs melanoma models. Safety evaluation included zebrafish embryotoxicity and mouse acute toxicity experiments. As a result, Ori effectively dismantles tumorspheres, inhibits proliferation, and reduces the expression of CSC-specific markers. It induces significant differentiation, especially in the case of NB4. Additionally, Ori upregulates TP53 expression, mitigates the hypoxic tumor microenvironment, suppresses stemness, and inhibits PD-L1 expression, prompting a robust anti-cancer immune response. Ori demonstrates pronounced cytotoxicity, inducing notable pro-apoptotic effects on B16F10 and MCF-7 cells, with specific triggering of mitochondrial apoptosis. Importantly, Ori maintains a commendable biosafety record. The dual-action prowess of Ori not only induces the differentiation of CSCs but also dispatches differentiated and residual tumor cells, effectively thwarting the relentless march of tumor progression., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Basophils, rare granulocytes, have long been acknowledged for their roles in type 2 immune responses. However, the mechanisms by which basophils adapt their functions to diverse mammalian microenvironments remain unclear. Recent advancements in specific research tools and single-cell-based technologies have greatly enhanced our understanding of basophils. Several studies have shown that basophils play a role in maintaining homeostasis but can also contribute to pathology in various tissues and organs, including skin, lung, and others. Here, we provide an overview of recent basophil research, including cell development, characteristics, and functions. Based on an increasing understanding of basophil biology, we suggest that the precise targeting of basophil features might be beneficial in alleviating certain pathologies such as asthma, atopic dermatitis (AD), and others., Competing Interests: Declaration of interests The authors declare no conflicts of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Chronic airway inflammation mediated by CD8 + T lymphocytes contributes to the pathogenesis of Chronic obstructive pulmonary disease (COPD). Deciphering the fingerprint of the chronic inflammation orchestrated by CD8 + T cells may allow the development of novel approaches to COPD management. Here, the expression of IL-27 and IFN-γ + CD8 + Tc1 cells were evaluated in patients with COPD and in cigarette smoke-exposed mice. The production of IL-27 by marrow-derived dendritic cells (mDCs) in response to cigarette smoke extract (CSE) was assessed. The role of IL-27 in IFN-γ + CD8 + Tc1 cells was explored. We demonstrated that elevated IL-27 was accompanied by an exaggerated IFN-γ + CD8 + Tc1 response in a smoking mouse model of emphysema. We noted that lung dendritic cells were one of the main sources of IL-27 during chronic cigarette smoke exposure. Moreover, CSE directly induced the production of IL-27 by mDCs in vitro. IL-27 negatively regulated the differentiation of IFN-γ + CD8 + Tc1 cells isolated from cigarette smoke-exposed mice in a STAT1- and STAT3-independent manner. Systemic administration of recombinant IL-27 attenuated IFN-γ + CD8 + Tc1 response in the late phase of cigarette smoke exposure. Our results uncovered that IL-27 negatively regulates IFN-γ + CD8 + Tc1 response in the late stage of chronic cigarette smoke exposure, which may provide a new strategy for the anti-inflammatory treatment of smoking-related COPD/emphysema., (© 2021 Wiley-VCH GmbH.)

Human hepatocyte-like cells (HLCs) derived from human pluripotent stem cells (hPSCs) represent a promising cell source for the assessment of hepatotoxicity and pharmaceutical safety testing. However, the hepatic functionality of HLCs remains significantly inferior to primary human hepatocytes. The bioactive vitamin D (VD), calcitriol, promotes the differentiation of many types of cells, and its deficiency is correlated to the severity of liver diseases. Whether calcitriol contributes to the differentiation of HLCs needs to be explored. Here, we found that the supplementation of calcitriol improved the functionalities of hPSCs-derived HLCs in P450 activities, urea production, and albumin secretion. Moreover, calcitriol also enhanced mitochondrial respiratory function with increased protein expression levels of the subunit of respiratory enzyme complexes in HLCs. Further analyses showed that the mitochondrial biogenesis regulators and mitophagy were increased by calcitriol, thus improving the mitochondrial quality. These improvements in functionality and mitochondrial condition were dependent on vitamin D receptor (VDR) because the improvements were abolished under VDR-deficient conditions. Our finding provides a cost-effective chemical process for HLC maturation to meet the demand for basic research and potential clinic applications., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

During inflammation or tissue injury, pro-inflammatory mediators attract migratory monocytes to inflammatory sites and monocyte-to-macrophage differentiation occurs to activate macrophages. We report here that PAQR11, a member of the progesterone and AdipoQ receptor family, regulates monocyte-to-macrophage differentiation in vitro and in vivo. Paqr11 gene was highly induced during monocyte-to-macrophage differentiation. Knockdown or deletion of Paqr11 inhibited monocyte differentiation but had little effect on macrophage polarization. Mechanistically, PAQR11 promoted cell survival as apoptosis was increased by Paqr11 knockdown or deletion. Activation of the MAPK signalling pathway was involved in the regulatory role of PAQR11 on monocyte differentiation and cell survival. C/EBPβ regulated the expression of Paqr11 at the transcriptional level. In mice, deletion of Paqr11 gene alleviated progression of collagen-induced rheumatoid arthritis. Thus, these results provide strong evidence that PAQR11 has a function in monocyte-to-macrophage differentiation and such function is related to autoimmune disease in vivo., (© 2021 John Wiley & Sons Ltd.)

Objectives: Acute lung injury/acute respiratory distress syndrome (ALI/ARDS) is a severe form of inflammatory lung disease. Its development and progression are regulated by cytokines. The purpose of this study was to determine the effects of HMGB1 involved in the regulation of Treg cells and IL-35., Methods: A cecal ligation and puncture (CLP)-induced ALI model was used to investigate the changes in IL-35, Tregs, and the expression of RAGE and caspase-11 after HMGB1 inhibition (glycyrrhizin was used as an inhibitor of HMGB1). CD4+ naïve T cells sorted from C57BL/6 mice spleens were cultured to explore the role of HMGB1 in the differentiation from CD4+ naïve T cells to Tregs., Results: HMGB1 promoted lung injury and uncontrolled inflammation in the CLP mouse model. HMGB1, NF-κB p65, RAGE, and caspase-11 expression in the lungs of CLP mice decreased significantly after pretreatment with glycyrrhizin. We found that the Treg proportion and IL-35 expression were upregulated in the serum and lung of CLP mice after inhibiting HMGB1. In our in vitro experiments, we found that recombinant HMGB1 significantly suppressed the proportion of CD4+CD25+FOXP3+Tregs differentiated from CD4+ naïve T cells., Conclusions: The inhibition of HMGB1 increased the proportion of Treg and expression of IL-35 and alleviated lung injury in the CLP-induced ALI model. Furthermore, inhibition of HMGB1 reduced caspase-11-dependent pyroptosis in the lungs of the CLP-induced ALI model., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Interleukin (IL)-35, which has an anti-inflammatory role in acute respiratory distress syndrome (ARDS)/acute lung injury (ALI), is relatively promising as a drug target. Studies have shown that curcumin may play a therapeutic role in ALI and enhance the suppressive function of regulatory T cells (Tregs). To illustrate the effect of curcumin on the regulation of Treg cell differentiation and expression of IL-35, we built a cecal ligation and puncture (CLP)-induced acute lung injury mouse mode with curcumin pretreatment. The expression of IL-35 in serum, severity of lung injury, IL-17A in lung tissue, survival rate, Treg-related cytokines levels in serum, nuclear factor-kappa B (NF-κB)'s nuclear translocation in lung tissue, and splenic CD4+CD25+FOXP3+ Tregs were assessed. Furthermore, the proportion of Tregs, STAT5, and IL-35 expression during naïve CD4+ T cell differentiation in vitro was measured. Compared with the CLP group, the increased IL-35 expression in CLP with the curcumin pretreatment (CLP + Cur) group was consistent with the decreased severity of lung injury, IL-17A protein levels in lung tissue, and Treg-related cytokines levels. Pretreatment with curcumin, the survival rate climbed to 50%, while the mortality rate was 100% in the CLP group. In addition, splenic CD4+CD25+FOXP3+ Treg cells increased in the CLP + Cur group. In vitro, CD4+CD25+FOXP3+ Treg cells from naïve CD4+ T cells, STAT5 proportion, and IL-35 expression increased after curcumin treatment. These findings showed that curcumin might regulate IL-35 by activating the differentiation of Treg cells to control the inflammation in acute lung injury.

Tissue inhibitor of metalloproteinase 1 (TIMP1) has various biological activities including the regulation of cell growth and differentiation. However, its role in bone homeostasis and remodeling remains poorly understood. In this study, we investigate the effects of TIMP1 on osteoblast and osteoclast activity at both cellular and molecular level using siRNA-mediated knockdown technique. Our results show that knockdown of TIMP1 stimulates proliferation and survival, but decreases apoptosis in osteoblastic MC3T3-E1 cells, suggesting that TIMP1 inhibits cell growth. TIMP1 also dampens differentiation of committed osteoblasts, as well as osteoblastogenesis of bone marrow-derived mesenchymal stem cells (BMSCs). We further show that the modulation of TIMP1 on osteoblast activity is independent of its MMP inhibition. Importantly, we uncover that TIMP1 suppresses osteoblast growth and differentiation by targeting the AKT pathway, and this is associated with TIMP1-mediated induction of PTEN via its binding to the cell surface receptor CD44. Therefore, our results highlight a novel TIMP1/CD44/PTEN/AKT signaling nexus that functions as a suppressor of osteoblast activity. Moreover, we show that TIMP1 also inhibits osteoclast differentiation in osteoclast precursor RAW 264.7 cells by targeting the AKT. In conclusion, our results demonstrate that TIMP1 can act as a suppressor of growth and differentiation of osteoblasts and differentiation of osteoclasts through the negative regulation of the AKT pathway. We propose that TIMP1 may serve as a potential target for low bone mass-related skeletal diseases, such as osteoporosis., Competing Interests: Declaration of competing interest The authors declare that there are no competing interests associated with the manuscript., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Oxidative stress (OS) caused by multiple factors occurs after the implantation of bone repair materials. DNA methylation plays an important role in the regulation of osteogenic differentiation. Moreover, recent studies suggest that DNA methyltransferases (Dnmts) are involved in bone formation and resorption. However, the effect and mechanism of DNA methylation changes induced by OS on bone formation after implantation still remain unknown. Three-dimensional (3D) cell culture systems are much closer to the real situation than traditional monolayer cell culture systems in mimicking the in vivo microenvironment. We have developed porous 3D scaffolds composed of mineralized collagen type I, which mimics the composition of the extracellular matrix of human bone. Here, we first established a 3D culture model of human mesenchymal stem cells (hMSCs) seeded in the biomimetic scaffolds using 160 μ M H 2 O 2 to simulate the microenvironment of osteogenesis after implantation. Our results showed that decreased methylation levels of ALP and RUNX2 were induced by H 2 O 2 treatment in hMSCs cultivated in the 3D scaffolds. Furthermore, we found that Dnmt3a was significantly downregulated in a porcine anterior lumbar interbody fusion model and was confirmed to be reduced by H 2 O 2 treatment using the 3D in vitro model. The hypomethylation of ALP and RUNX2 induced by H 2 O 2 treatment was abolished by Dnmt3a overexpression. Moreover, our findings demonstrated that the Dnmt inhibitor 5-AZA can enhance osteogenic differentiation of hMSCs under OS, evidenced by the increased expression of ALP and RUNX2 accompanied by the decreased DNA methylation of ALP and RUNX2. Taken together, these results suggest that Dnmt3a-mediated DNA methylation changes regulate osteogenic differentiation and 5-AZA can enhance osteogenic differentiation via the hypomethylation of ALP and RUNX2 under OS. The biomimetic 3D scaffolds combined with 5-AZA and antioxidants may serve as a promising novel strategy to improve osteogenesis after implantation., Competing Interests: The authors declare that there are no conflicts of interest regarding the publication of this paper. No benefits in any form have been or will be received from a commercial party related directly or indirectly to the subject of this manuscript., (Copyright © 2019 Liangping Li et al.)

Background: iRoot BP Plus is a novel bioceramic endodontic material. Recently, it has been considered as an alternative to MTA which is the most popular scaffold cover during regenerative endodontic therapy. This study aimed to evaluate the effects of iRoot BP Plus on the osteo/odontogenic capacity of bone marrow mesenchymal stem cells (BMMSCs), including the underlying mechanisms., Methods: BMMSCs were collected by a whole marrow method and treated with iRoot BP Plus-conditioned medium (BP-CM). The proliferation ability was evaluated by cell counting kit 8 and flow cytometry. Complete medium was used as a blank control, and 2 mg/ml MTA-conditioned medium was served as a positive control. Alkaline phosphatase (ALP) activity assay, ALP staining, western blot, real-time RT-PCR, Alizarin Red S staining, and immunofluorescence staining were performed to explore the osteo/odontogenic potential and the involvement of MAPK pathways. Besides, autophagy was investigated by western blot, immunofluorescence staining, and transmission electron microscopy.

Sweat gland regeneration is important for patients with an extensive deep burn injury. In previous study, we reported that bone marrow-mesenchymal stem cells (BM-MSCs) could differentiate into sweat gland-like cells (SGLCs), but the underlying molecular mechanism remains unclear. Recently, microRNAs (miRNAs or miRs) are reported to manipulate many biological processes. However, whether the process of MSCs differentiation into sweat gland cells (SGCs) is regulated by miRNAs has not been reported. In this study, BM-MSCs were induced into SGLCs by co-culturing with SGCs. Differential expressions of miRNAs between BM-MSC and SGLCs were determined through miRNAs microarray and 68 miRNAs were found significantly changed in miRNA profile including hsa-miR-138-5p. Bioinformatics analysis showed that hsa-miR-138-5p targeted a group of nuclear factor-κB (NF-κB) related genes which play an important role in skin appendage development. As expected, hsa-miR-138-5p inhibitor transfected into BM-MSCs partly mimicked the effects of co-culture and increased the number of SGLCs by increasing the expression of NF-κB related genes. These results suggest that hsa-miR-138-5p and NF-κB are involved in the regulation of BM-MSCs differentiation into SGLCs. This study may also offer a new approach to yield SGCs for burn patients.

Acute respiratory distress syndrome (ARDS) has been the major cause of acute respiratory failure in critical patients and one of the leading causes of death worldwide for several decades. Th17 cells are involved in the occurrence and progression of ARDS. Furthermore, histone acetyltransferase (HAT) p300 is a transcriptional coactivator, and its activity is closely related to cancer and inflammatory diseases. p300 and histone deacetylase 1 (HDAC1) interact with and stabilize the nuclear transcription factor retinoic acid-related orphan receptor gamma t (RORγt) and participate in the regulation of RORγt-mediated IL-17 transcription in T helper 17 (Th17) cell differentiation by acetylation and deacetylation. However, the effect of p300 on RORγt and Th17 cells in ARDS is not well reported. Therefore, we aimed to investigate the clinical features of p300 and its effect on RORγt and Th17 cells in patients with ARDS as well as in lipopolysaccharide-induced acute lung injury (ALI) mouse models. Overexpression of p300 and RORγt mRNA was found in the peripheral blood mononuclear cells from patients with ARDS, especially among non-survivors, compared to that in healthy individuals (P < 0.05). Moreover, the decline of FOXP3 mRNA level correlated with survival and increased RORγt mRNA levels corelated with infection (P < 0.05). Immunohistochemical analysis revealed high p300 and RORγt expression in ALI mouse lung tissues. Inhibitor-mediated knockdown of p300 reduced lung tissue inflammation and lung injury score (P < 0.05). Western blotting and ELISA revealed that p300 inhibitor caused a decrease in the mRNA and protein levels of RORγt as well as interleukin 17 (IL-17) production in ALI mouse lung tissues (P < 0.05). Thus, our findings suggest that p300 may play a key role in ARDS by positively regulating RORγt transcription and is a potential new immunotherapy target for ARDS., (Copyright © 2018. Published by Elsevier B.V.)

The human GLI3 protein has a dual function as a transcriptional activator or repressor of hedgehog signaling, depending on the proteolytic processing forms of GLI3. In this study, we established a compound heterozygous GLI3 mutant human embryonic stem cell line (WAe001-A-20) through CRISPR/Cas9 editing. The WAe001-A-20 cells carried two deletions on two different alleles of exon 2 of GLI3, respectively, which resulted in a frame shift and early termination in the translation of GLI3. Moreover, WAe001-A-20 maintains a normal karyotype, parental cell morphology, pluripotent phenotype and the ability to differentiate into three germ layers. Resource table., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Interstitial fibrosis serves a causal role in the development of heart failure following acute and chronic myocardial infarction, and anti‑fibrotic therapy represents a promising strategy to mitigate this pathological process. Oxymatrine (OMT) exerts a number of pharmacological effects on the cardiovascular system, but its anti‑cardiovascular disease mechanisms remain unclear. The purpose of the present study was to investigate the effect of OMT administration on transforming growth factor (TGF)‑β1‑induced cardiac fibroblast (CFB) proliferation and abnormal differentiation, and to elucidate the underlying mechanisms. Primary CFBs were isolated from neonatal rats and used for experimental treatments. TGF‑β1 stimulation in CFBs resulted in increased proliferation, increased α‑smooth muscle actin (SMA) and type I and type III collagen expression, and increased p38 mitogen‑activated protein kinase (MAPK) and extracellular signal‑regulated kinase (ERK)1/2 phosphorylation. Treatment with OMT and SB431542 (a TGF‑β1 receptor inhibitor) attenuated the proliferation and abnormal differentiation of CFBs induced by TGF‑β1, and decreased p38MAPK and ERK1/2 phosphorylation. In addition, treatment with SB203580 (a p38MAPK inhibitor) or PD98059 (an ERK1/2 inhibitor), but not by SP600125 (a c‑jun N‑terminal kinase1/2/3 inhibitor), inhibited the TGF‑β1 stimulated CFB proliferation, as well as the elevation of α‑SMA and the deposition of type I and type III collagen, suggesting that ERK1/2 and p38MAPK signaling may be important in the in the process of myocardial fibrosis. In conclusion, the present study revealed that OMT treatment inhibited CFB proliferation and the CFB‑myofibroblast transition induced by TGF‑β1, at least in part through inhibition of ERK1/2 and p38MAPK signaling.

BACKGROUND Interleukin-17A (IL-17A) is not only an important modulator of inflammatory reactions, but also affects bone metabolism, which is involved in osteogenic differentiation of stem cells. However, the role and mechanism of IL-17A in osteogenic differentiation of bone mesenchymal stem cells (BMSCs) are not fully understood. In this study, we investigated the role and mechanism of IL-17A in osteogenic differentiation of BMSCs. MATERIAL AND METHODS The osteogenic differentiation of BMSCs was induced by osteoblast-induction medium with IL-17A or without IL-17A. The osteogenic differentiation of BMSCs was confirmed by the alkaline phosphatase and alizarin red staining. The lentiviral plasmid was used to construct the sFRP1-shRNA expression vector. The associated osteogenic differentiation marks (RUNX2, ALP, OPN), Wnt signaling pathway inhibitor (sFRP1), and modulators of Wnt signaling pathway (Wnt3, Wnt6) were detected by qRT-PCR and Western blot method. RESULTS The results showed that the addition of IL-17A inhibited osteogenic differentiation of BMSCs. IL-17A induced up-regulated expression of sFRP1 and down-regulated expression of Wnt3 and Wnt6 in BMSCs. In addition, sFRP1-shRNA abolished the inhibition effect of IL-17A in osteogenic differentiation of BMSCs and induced up-regulated expression of Wnt3 and Wnt6 in the Wnt signaling pathway in BMSCs. CONCLUSIONS Our findings show that IL-17A inhibits osteogenic differentiation of bone mesenchymal stem cells via the Wnt signaling pathway.

Human induced pluripotent stem cells (hiPSCs) may provide potential resource for regenerative medicine research, including generation of insulin-producing cells for diabetes research and insulin production. Testosterone (T) is an androgen hormone which promotes protein synthesis and improves the management of type 2 diabetes in clinical studies. Concurrently, co-existed hyperandrogenism and hyperinsulinism is frequently observed in polycystic ovary syndrome, congenital adrenal hyperplasia and some of Wermer's syndrome. However, the relationship among androgens, insulin and the differentiation of pancreatic β cells is still not fully clear. Here we find that T improves the differentiation efficiency of insulin-producing cells from hiPSCs. The addition of T into routine differentiation formula for pancreatic β cells increases the differentiation efficiency from 12% to 35%. The administration of T promotes the expression of key genes associated with β cells differentiation including NGN3, NEUROD1 and INS. This finding benefits the ongoing process to optimize the differentiation protocol of pancreatic β cells from hiPSCs, and provides some degree of understanding the clinical management of T for type 2 diabetes.

Objectives: Induced pluripotent stem cell (iPSC)-derived cardiomyocytes are a promising source of cells for regenerative heart disease therapies, but progress towards their use has been limited by their low differentiation efficiency and high cellular heterogeneity. Previous studies have demonstrated expression of adrenergic receptors (ARs) in stem cells after differentiation; however, roles of ARs in fate specification of stem cells, particularly in cardiomyocyte differentiation and development, have not been characterized., Materials and Methods: Murine-induced pluripotent stem cells (miPSCs) were cultured in hanging drops to form embryoid bodies, cells of which were then differentiated into cardiomyocytes. To determine whether ARs regulated miPSC differentiation into cardiac lineages, effects of the AR agonist, epinephrine (EPI), on miPSC differentiation and underlying signalling mechanisms, were evaluated., Results: Treatment with EPI, robustly enhanced miPSC cardiac differentiation, as indicated by increased expression levels of cardiac-specific markers, GATA4, Nkx2.5 and Tnnt2. Although β-AR signalling is the foremost signalling pathway in cardiomyocytes, EPI-enhanced cardiac differentiation depended more on α-AR signalling than β-AR signalling. In addition, selective activation of α 1 -AR signalling with specific agonists induced vigorous cardiomyocyte differentiation, whereas selective activation of α 2 - or β-AR signalling induced no or less differentiation, respectively. EPI- and α 1 -AR-dependent cardiomyocyte differentiation from miPSCs occurred through specific promotion of CPC proliferation via the MEK-ERK1/2 pathway and regulation of miPS cell-cycle progression., Conclusions: These results demonstrate that activation of ARs, particularly of α 1 -ARs, promoted miPSC differentiation into cardiac lineages via MEK-ERK1/2 signalling., (© 2016 John Wiley & Sons Ltd.)

Accumulating evidence has indicated that microRNAs (miRNAs) and endoplasmic reticulum (ER) stress play critical roles in myoblast differentiation. However, the regulation roles of miRNAs and ER stress in myogenic differentiation have not been fully revealed and need to be further studied. Here, we discovered that the expression levels of miR-181a-5p were strongly upregulated during C2C12 cell differentiation. miR-181a-5p overexpression promoted ER stress and differentiation of C2C12 cells, which was accompanied by increasing expression levels of marker genes related to ER stress-mediated apoptosis and myogenic differentiation. Opposite results were observed after inhibition of the miR-181a-5p expression. The gain- and loss-of-function experiments on C2C12 cells showed that miR-181a-5p affected the development of muscle fiber type, but had no significant influence on C2C12 cell proliferation. In the ER-stressed C2C12 cells induced by thapsigargin (Tg), the expression levels of both miR-181a-5p and marker genes related to ER stress and myogenesis were upregulated. In the ER-stressed C2C12 cells and porcine muscle fibroblast (PMF) cells pretreated with Tg, we found that miR-181a-5p targeted glucose-regulated protein, 78kDa/binding immunoglobulin protein (GRP78/BIP), and influenced cell apoptosis. In conclusion, these results indicate that miR-181a-5p and ER stress have positive synergistic effects on myogenic differentiation by increasing the expression levels of myogenic differentiation key genes and activating the ER stress-mediated apoptosis signaling pathway., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Gingival-derived mesenchymal stem cells (GMSCs) have recently been harvested; however, the use of GMSCs in periodontal tissue engineering requires further study. The present study established an indirect co‑culture system between rat apical tooth germ‑conditioned medium (APTG‑CM) and GMSCs, in order to determine the effects on periodontal tissue differentiation in vitro and in vivo. Using the limiting dilution technique, single‑colony derived human GMSCs and periodontal ligament stem cells (PDLSCs) were isolated and expanded to obtain homogeneous populations. PDLSCs were used as a positive control group. Cell cycle distribution, alkaline phosphatase (ALP) activity, mineralization behavior, expression of genes associated with a cementoblast phenotype (osteocalcin, bone sialoprotein, ALP, type I collagen, cementum‑derived protein 23), and in vivo differentiation capacities of GMSCs/PDLSCs co‑cultured with APTG‑CM were evaluated. Flow cytometry indicated that GMSCs and PDLSCs were positive for STRO‑1 and CD105, whereas CD45 expression was negative. The cell types were capable of forming colonies, and of osteogenic and adipogenic differentiation in response to appropriate stimuli. The induced GMSCs and PDLSCs exhibited numerous characteristics associated with cementoblast lineages, as indicated by increased proliferation and ALP activity, and upregulated expression of cementum‑associated genes in vitro. In vivo, cementum/periodontal ligament‑like structures were shown to form along the dentin surface and ceramic bovine bone in GMSCs and PDLSCs induced by APTG‑CM group. Conversely, vertical fibers could not insert in the control group, which was not co‑cultured with APTG‑CM. In conclusion, GMSCs are likely to have a role in periodontal tissue regeneration. In addition, APTG‑CM was able to provide a cementogenic microenvironment and promote differentiation of GMSCs along the cementoblastic lineage.

The enhancer of zeste 2 polycomb repressive complex 2 subunit (Ezh2) is a histone-lysine Nmethyltransferase enzyme that participates in DNA methylation. Ezh2 has also been reported to play crucial roles in stem cell proliferation and differentiation. However, the detailed expression profile of Ezh2 during mouse cochlear development has not been investigated. Here, we examined the spatiotemporal expression of Ezh2 in the cochlea during embryonic and postnatal development. Ezh2 expression began to be observed in the whole otocyst nuclei at embryonic day 9.5 (E9.5). At E12.5, Ezh2 was expressed in the nuclei of the cochlear prosensory epithelium. At E13.5 and E15.5, Ezh2 was expressed from the apical to the basal turns in the nuclei of the differentiating cochlear epithelium. At postnatal day (P) 0 and 7, the Ezh2 expression was located in the nuclei of the cochlear epithelium in all three turns and could be clearly seen in outer and inner hair cells, supporting cells, the stria vascularis, and spiral ganglion cells. Ezh2 continued to be expressed in the cochlear epithelium of adult mice. Our results provide the basic Ezh2 expression pattern and might be useful for further investigating the detailed role of Ezh2 during cochlear development.

Lgr5+ supporting cells (SCs) are enriched hair cell (HC) progenitors in the cochlea, and several studies have shown a difference in the proliferation and HC regeneration ability of SCs between the apical and basal turns. However, the detailed differences between the transcriptomes of the apical and basal Lgr5+ SCs have not yet been investigated. We found that when isolated by FACS, Lgr5+ cells from the apex generated significantly more HCs and had significantly higher proliferation and mitotic HC regeneration ability compared to those from the base. Next, we used microarray analysis to determine the transcriptome expression profiles of Lgr5+ progenitors from the apex and the base. We first analyzed the genes that were enriched and differentially expressed in Lgr5+ progenitors from the apex and the base. Then we analyzed the cell cycle genes and the transcription factors that might regulate the proliferation and differentiation of Lgr5+ progenitors. Lastly, to further analyze the role of differentially expressed genes and to gain an overall view of the gene network in cochlear HC regeneration, we created a protein-protein interaction network. Our datasets suggest the possible genes that might regulate the proliferation and HC regeneration ability of Lgr5+ progenitors, and these genes might provide new therapeutic targets for HC regeneration in the future., Competing Interests: The authors declare that they have no competing interests.

Physical exercise is able to improve skeletal health. However, the mechanisms are poorly known. Irisin, a novel exercise-induced myokine, secreted by skeletal muscle in response to exercise, have been shown to mediate beneficial effects of exercise in many disorders. In the current study, we demonstrated that irisin promotes osteoblast proliferation, and increases the expression of osteoblastic transcription regulators, such as Runt-related transcription factor-2, osterix/sp7; and osteoblast differentiation markers, including alkaline phosphatase, collagen type 1 alpha-1, osteocalcin, and osteopontin in vitro. Irisin also increase ALP activity and calcium deposition in cultured osteoblast. These osteogenic effects were mediated by activating the p38 mitogen-activated protein kinase (p-p38 MAPK) and extracellular signal-regulated kinase (ERK). Inhibition of p38 MAPK by SB023580 or pERK by U0126 abolished the proliferation and up-regulatory effects of irisin on Runx2 expression and ALP activity. Together our observation suggest that irisin directly targets osteoblast, promoting osteoblast proliferation and differentiation via activating P38/ERK MAP kinase signaling cascades in vitro. Whether irisin can be utilized as the therapeutic agents for osteopenia and osteoporosis is worth to be further pursued.

Background: Pulmonary adventitial fibroblasts (PAFs) are activated under stress stimuli leading to their differentiation into myofibroblasts, which is involved in vessel remodeling. 15-HETE is known as an important factor in vessel remodeling under hypoxia; however, the role of 15-HETE in PAF phenotypic alteration is not clear., Results: The effect of 15-HETE on PAF phenotypic alterations was investigated in the present study. PAFs were treated with 15-HETE (0.5 μM) for 24 h, and the myofibroblast marker α-smooth muscle actin (α-SMA) was analyzed. The 15-HETE induced α-SMA expression and cell morphology. 15-HETE upregulated FGF-2 levels in PAFs, and knockdown FGF-2 by siRNAs blocked the enhanced α-SMA expression induced by 15-HETE. p38 kinase was activated, and blocked depressed 15-HETE-induced FGF-2 expression. The downstream of p38 pathway, Egr-1 activation, was also raised by 15-HETE treatment, and silenced Egr-1 suppressed the 15-HETE-induced upregulation of FGF-2. TGF-β1 was upregulated with FGF-2 treatment, and α-SMA expression induced by FGF-2 was inhibited after the cell was transferred with TGF-β1 siRNA. Meanwhile, FGF-2 increased α-SMA expression and improved proliferation, which was associated with p27(kip1) and cyclin E variation., Conclusions: The above results suggest that p38/Egr-1 pathway-mediated FGF-2 is involved in 15-HETE-induced differentiation of PAFs into myofibroblasts and cell proliferation.

Estrogen induces oligodendrocyte remyelination in response to demyelination in the central nervous system. Our objective was to determine the effects of 17 β -estradiol (E2) on Schwann cell function and peripheral nerve remyelination after injury. Adult male C57BL/6J mice were used to prepare the sciatic nerve transection injury model and were randomly categorized into control and E2 groups. To study myelination in vitro, dorsal root ganglion (DRG) explant culture was prepared using 13.5-day-old mouse embryos. Primary Schwann cells were isolated from the sciatic nerves of 1- to 3-day-old Sprague-Dawley rats. Immunostaining for myelin basic protein (MBP) expression and toluidine blue staining for myelin sheaths demonstrated that E2 treatment accelerates early remyelination in the "nerve bridge" region between the proximal and distal stumps of the transection injury site in the mouse sciatic nerve. The 5-bromo-2'-deoxyuridine incorporation assay revealed that E2 promotes Schwann cell proliferation in the bridge region and in the primary culture, which is blocked using AKT inhibitor MK2206. The in vitro myelination in the DRG explant culture determined showed that the MBP expression in the E2-treated group is higher than that in the control group. These results show that E2 promotes Schwann cell proliferation and myelination depending on AKT activation.

Unlabelled: The cell fate determinant Numb is aberrantly expressed in cancer. Numb is alternatively spliced, with one isoform containing a long proline-rich region (PRR(L) ) compared to the other with a short PRR (PRR(S) ). Recently, PRR(L) was reported to enhance proliferation of breast and lung cancer cells. However, the importance of Numb alternative splicing in hepatocellular carcinoma (HCC) remains unexplored. We report here that Numb PRR(L) expression is increased in HCC and associated with early recurrence and reduced overall survival after surgery. In a panel of HCC cell lines, PRR(L) generally promotes and PRR(S) suppresses proliferation, migration, invasion, and colony formation. Knockdown of PRR(S) leads to increased Akt phosphorylation and c-Myc expression, and Akt inhibition or c-Myc silencing dampens the proliferative impact of Numb PRR(S) knockdown. In the cell models explored in this study, alternative splicing of Numb PRR isoforms is coordinately regulated by the splicing factor RNA-binding Fox domain containing 2 (RbFox2) and the kinase serine/arginine protein-specific kinase 2 (SRPK2). Knockdown of the former causes accumulation of PRR(L) , while SRPK2 knockdown causes accumulation of PRR(S) . The subcellular location of SRPK2 is regulated by the molecular chaperone heat shock protein 90, and heat shock protein 90 inhibition or knockdown phenocopies SRPK2 knockdown in promoting accumulation of Numb PRR(S) . Finally, HCC cell lines that predominantly express PRR(L) are differentially sensitive to heat shock protein 90 inhibition., Conclusion: Alternative splicing of Numb may provide a useful prognostic biomarker in HCC and is pharmacologically tractable., (© 2015 by the American Association for the Study of Liver Diseases.)

Background: Environmental factors are important for stem cell lineage specification, and increasing evidence indicates that the nanoscale geometry/topography of the extracellular matrix (ECM) directs stem cell fate. Recently, many three-dimensional (3D) biomimetic nanofibrous scaffolds resembling many characteristics of the native ECM have been used in stem cell-based myocardial tissue engineering. However, the biophysical role and underlying mechanism of 3D nanofibrous scaffolds in cardiomyocyte differentiation of induced pluripotent stem cells (iPSCs) remain unclear., Results: Here, we fabricated a 3D poly-(ε-caprolactone) (PCL) nanofibrous scaffold using the electrospinning method and verified its nanotopography and porous structure by scanning electron microscopy. We seeded murine iPSCs (miPSCs) directly on the 3D PCL nanofibrous scaffold and initiated non-directed, spontaneous differentiation using the monolayer method. After the 3D PCL nanofibrous scaffold was gelatin coated, it was suitable for monolayer miPSC cultivation and cardiomyocyte differentiation. At day 15 of differentiation, miPSCs differentiated into functional cardiomyocytes on the 3D PCL nanofibrous scaffold as evidenced by positive immunostaining of cardiac-specific proteins including cardiac troponin T (cTnT) and myosin light chain 2a (MLC2a). In addition, flow cytometric analysis of cTnT-positive cells and cardiac-specific gene and protein expression of cTnT and sarcomeric alpha actinin (α-actinin) demonstrated that the cardiomyocyte differentiation of miPSCs was more efficient on the 3D PCL nanofibrous scaffold than on normal tissue culture plates (TCPs). Furthermore, early inhibition of Wnt/β-catenin signaling by the selective antagonist Dickkopf-1 significantly reduced the activity of Wnt/β-catenin signaling and decreased the cardiomyocyte differentiation of miPSCs cultured on the 3D PCL nanofibrous scaffold, while the early activation of Wnt/β-catenin signaling by CHIR99021 further increased the cardiomyocyte differentiation of miPSCs., Conclusion: These results indicated that the electrospun 3D PCL nanofibrous scaffolds directly promoted the cardiomyocyte differentiation of miPSCs, which was mediated by the activation of the Wnt/β-catenin signaling during the early period of differentiation. These findings highlighted the biophysical role of 3D nanofibrous scaffolds during the cardiomyocyte differentiation of miPSCs and revealed its underlying mechanism involving Wnt/β-catenin signaling, which will be helpful in guiding future stem cell- and scaffold-based myocardium bioengineering.

Nucleophosmin (NPM1) is a multifunctional protein that functions as a molecular chaperone, shuttling between the nucleolus and the cytoplasm. In up to one third of patients with acute myeloid leukemia, mutation of NPM1 results in the aberrant cytoplasmic accumulation of mutant protein and is thought to be responsible for leukemogenesis. Deguelin, a rotenoid isolated from several plant species, has been shown to be a strong anti-tumor agent. Human leukemia cell lines were used for in vitro studies. Drug efficacy was evaluated by apoptosis and differentiation assays, and associated molecular events were assessed by Western blot. Gene silencing was performed using small interfering RNA (siRNA). Deguelin exhibited strong cytotoxic activity in the cell line of OCI-AML3 and selectively down-regulated the NPM1 mutant protein, which was accompanied by up-regulation of the activity of caspase-6 and caspase-8 in high concentrations. Deguelin induced differentiation of OCI-AML3 cells at a nontoxic concentration which was associated with a decrease in expression of activated caspase-8, p53, p21, and the 30-kD form of CCAAT/enhancer binding protein α (C/EBPα), whereas no effects were found in OCIM2 cells expressing NPM-wt. Moreover, treatment with siRNA in the NPM mutant cell line OCI-AML3 decreased expression of p53, p21, pro-caspase-8, and the 30-kD form of C/EBPα, and it inhibited proliferation and induced differentiation of the OCI-AML3 cells. In conclusion, deguelin is a potent in vitro inhibitor of the mutant form of NPM1, which provides the molecular basis for its anti-leukemia activities in NPM1 mutant acute myeloid leukemia cells.

Background: Primary culture of rat osteoblast cells serves as a model in in vitro research of bone metabolism. But most of studies about osteoblast use fetal or newborn rats for cell isolation, including researches about the post-menopausal osteoporosis. This will affect the authenticity and reliability of the results. These differences have been a concern in recent years. So far, little research has focused on adult female rat osteoblasts., Methods: In this study, a systematic study was done regarding the proliferation, differentiation, and mineralization of adult female rat osteoblasts., Results: The study showed that the age of the host may affect the growth and the mineralization ability of osteoblast., Conclusions: The result of the study suggests that in vitro studies related to osteoblasts should select the right host and appropriate observation points according to the cell growth characteristics in order to ensure the consistency of the actual changes.

Neural stem cells (NSCs) are important pluripotent stem cells, which have potential applications in cell replacement therapy. Brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) have been demonstrated to exert a marked impact on the proliferation and differentiation of NSCs. The effects of NGF, BDNF, and BDNF combined with NGF on NSC neuronal differentiation and the possible mechanisms for these effects were investigated in this study. An adherent monolayer culture was employed to obtain highly homogeneous NSCs. The cells were divided into four groups: Control, NGF, BDNF and combination (BDNF + NGF) groups. Neuron differentiation was examined using immunocytochemistry and phospho-extracellular signal-regulated kinase (p‑ERK) levels were analyzed using western blotting. Reverse transcription polymerase chain reaction was used to measure the mRNA expression levels of the HES1, HES5, MASH1, NGN1 and NeuroD transcription factors at different time intervals following neurotrophin-induced differentiation. NGF and BDNF were observed to induce NSC neuronal differentiation, and β-tubulin III-positive cells and p-ERK expression levels were highest in the NGF + BDNF combination group at all time points. The proportion of β-tubulin Ⅲ-positive neurons in each group was associated with the expression levels of MASH1, NGN1 and NeuroD in the group. In conclusion, BDNF combined with NGF significantly improved NSC neuronal differentiation, which may provide support for the practical application of NSCs in neurodegenerative diseases.

Cardiomyocyte cell division and replication in mammals proceed through embryonic development and abruptly decline soon after birth. The process governing cardiomyocyte cell cycle arrest is poorly understood. Here we carry out whole-exome sequencing in an infant with evidence of persistent postnatal cardiomyocyte replication to determine the genetic risk factors. We identify compound heterozygous ALMS1 mutations in the proband, and confirm their presence in her affected sibling, one copy inherited from each heterozygous parent. Next, we recognize homozygous or compound heterozygous truncating mutations in ALMS1 in four other children with high levels of postnatal cardiomyocyte proliferation. Alms1 mRNA knockdown increases multiple markers of proliferation in cardiomyocytes, the percentage of cardiomyocytes in G2/M phases, and the number of cardiomyocytes by 10% in cultured cells. Homozygous Alms1-mutant mice have increased cardiomyocyte proliferation at 2 weeks postnatal compared with wild-type littermates. We conclude that deficiency of Alström protein impairs postnatal cardiomyocyte cell cycle arrest.

Wogonoside is the main flavonoid component derived from the root of Scutellaria baicalensis Georgi. It is a popular Chinese herbal medicine with the potential to treat hematologic malignancies. In this study, we investigated the anticancer effects of wogonoside in acute myeloid leukemia (AML) cell lines and primary patient-derived AML cells. Wogonoside exerted antiproliferative properties both in vitro and in vivo. Furthermore, it efficiently inhibited the proliferation of U937 and HL-60 cells through the induction of G1 phase arrest and the promotion of differentiation. We also demonstrated that wogonoside significantly increased the transcription of phospholipid scramblase 1 (PLSCR1) due to its influence on the expression of cell cycle- and differentiation-related genes, including the upregulation of p21waf1/cip1 and downregulation of the oncogenic protein c-Myc. Wogonoside also promoted PLSCR1 trafficking into the nucleus and facilitated its binding to the inositol 1,4,5-trisphosphate receptor 1 (IP3R1) promoter, thus increasing the expression of IP3R1. Finally, inhibition of PLSCR1 expression with small interfering RNA partially blocked wogonoside-induced cell cycle arrest and differentiation and disturbed the wogonoside-associated molecular events. The results of this study therefore suggest that wogonoside may represent a therapeutic candidate for the treatment of AML.

Smad7 is an intracellular inhibitory protein that antagonizes the signaling of TGF-β family members. Deletion of Smad7 in the mouse leads to an abnormality in heart development. However, whether Smad7 has a functional role in the development of other organs has been elusive. Here we present evidence that Smad7 imparts a role to eye development in the mouse. Smad7 is expressed in both the lens and retina in the developing embryonic eye. Depletion of Smad7 caused various degrees of coloboma and microphthalmia with alterations in cell apoptosis and proliferation in eyes. Smad7 was implicated in lens differentiation but was not required for the induction of the lens placode. The development of the periocular mesenchyme was retarded with the down-regulation of Bmp7 and Pitx2 in mutant mice. Retinal spatial patterning was affected by Smad7 deletion and was accompanied by altered bone morphogenetic protein (BMP) signaling. At late gestation stages, TGF-β signaling was up-regulated in the differentiating retina. Smad7 mutant mice displayed an expanded optic disc with increasing of sonic hedgehog (SHH) signaling. Furthermore, loss of Smad7 led to a temporal change in retinal neurogenesis. In conclusion, our study suggests that Smad7 is essential for eye development. In addition, our data indicate that alterations in the signaling of BMP, TGF-β, and SHH likely underlie the defects in eye development caused by Smad7 deletion.

Objective: To investigate the involvement of transcription factor Foxa2 in cardiac differentiation in P19 embryonal carcinoma cells and its molecular mechanism., Methods: P19 cells were induced to differentiate into cardiomyocytes by adding dimethyl sulfoxide (DMSO) into the culture medium of their embryoid bodies (EBs). The mRNA levels of pluripotency markers of embryonic pluripotent stem cells, cardiac differentiation related genes, and Foxa2 in the cell samples at different time points of cardiac differentiation were detected by reverse transcription PCR (RT-PCR). Differentiated and mature cardiomyocytes were identified by immunofluorescence. Eukaryotic expression plasmid pCMV-rFoxa2 (rat Foxa2) was transfected into P19 cells, and clonal populations of P19 cells that stably expressed green fluorescence protein (GFP)-rFoxa2 were isolated to enhance the expression levels of Foxa2 in P19 cells. The mRNA and protein levels of pluripotency markers and cardiac differentiation related genes in the above cell samples were detected by RT-PCR and Western blot. The mRNA levels of cardiac differentiation related genes in EBs differentiation system were also examined., Results: P19 cells differentiated into cardiomyocytes in the presence of DMSO, accompanied by stimulated expression of Foxa2. Transfection of pCMV-rFoxa2 plasmids into P19 cells upregulated rFoxa2 expression transiently and activated the transcription of its downstream cardiac inducer Cerberus1 (Cer1). The expression of pluripotency marker Nanog was suppressed and the expression of cardiac inducer Sonic Hedgehog (Shh) was elevated in GFP-rFoxa2 P19 cells. The expression of Cer1 and cardiac muscle marker actin, alpha cardiac muscle 1 (Actc1) was upregulated in EBs of GFP-rFoxa2 P19 cells., Conclusion: Foxa2 participates in cardiac differentiation in P19 embryonal carcinoma cells. Foxa2 may inhibit Nanog expression and stimulate the expression of Cer1 and Shh directly during cardiac differentiation in P19 cells in the presence of DMSO.

The easily accessible mesenchymal stem cells in the Wharton's jelly of human umbilical cord tissue (hUCMSCs) have excellent proliferation and differentiation potential, but it remains unclear whether hUCMSCs can differentiate into odontoblasts. In this study, mesenchymal stem cells were isolated from the Wharton's jelly of human umbilical cord tissue using the simple method of tissue blocks culture attachment. UCMSC surface marker expression was then evaluated for the isolated cells using flow cytometry. The third-passage hUCMSCs induced by conditioned medium from developing tooth germ cells (TGC-CM) displayed high alkaline phosphatase (ALP) levels (P < 0.001), an enhanced ability to proliferate (P < 0.05), and the presence of mineralized nodules. These effects were not observed in cells treated with regular medium. After induction of hUCMSCs, the results of reverse transcriptional polymerase chain reaction (PCR) indicated that the dentin sialophosphoprotein (DSPP) and dentin matrix protein 1 (DMP1) genes were significantly tested. Additionally, dentin sialoprotein (DSP) and DMP1 demonstrated significant levels of staining in an immunofluorescence analysis. In contrast, the control cells failed to display the characteristics of odontoblasts. Taken together, these results suggest that hUCMSCs can be induced to differentiate into odontoblast-like cells with TGC-CM and provide a novel strategy for tooth regeneration research.

Aims: To examine whether transplantation of adipose-derived stem cells (ADSCs) induces neural differentiation and improves neural function in a rat intracerebral hemorrhage (ICH) model., Methods: Adipose-derived stem cells cells were isolated from inguinal fat pad of rat. ICH was induced by injection of collagenase type IV into the right basal ganglia of rat. Forty-eight hours after ICH, ADSCs cells (10 μL of 2-4 × 10(7) cells/mL) were injected into the right lateral cerebral ventricle. The differentiation of ADSCs was detected in vitro and in vivo. The neural function was evaluated with Zea Longa 5-grade scale at day 1, 3, 7, 14, or 28., Results: Our data demonstrated that ADSCs differentiated into cells that shared the similarities of neurons or astrocytes in vitro. Transplantation of ADSCs decreased cell apoptosis and the transplanted ADSCs were able to differentiate into neuron-like and astrocyte-like cells around the hematoma, accompanied with upregulation of vascular endothelial growth factor expression and improvement of neural function., Conclusions: Our data suggest that transplantation of ADSCs could be a therapeutic approach for ICH stroke., (© 2012 Blackwell Publishing Ltd.)

Flavonoid is an important group of natural products which exerted anticancer effects against various cancers. III-10 is a newly synthesized flavonoid with a pyrrolidinyl and a benzyl group substitution. It shares a same carbon skeleton with flavonoid which indicates that it may also have potential anticancer activity. To investigate whether III-10 could express anticancer effect on human U937 leukemia cells through differentiation induction, a series of experiments were processed. MTT and trypan-blue excluding assays showed that III-10 possessed growth and viability inhibition effects on U937 cells. Giemsa staining was implemented to observe the morphologic changes of U937 cells after III-10 treatment. Nitroblue tetrazolium (NBT) reduction assay and cell surface antigen flow cytometry were carried out to figure out the way III-10 induced U937 cells to differentiate. These experiments revealed that III-10 could induce U937 cells to differentiate into monocyte-like cells. Western blots and immunofluorescence were performed to inspect the possible underlying mechanism. The results revealed that differentiation-related proteins phospholipids scramblase 1 (PLSCR1) and promyelocytic leukemia protein (PML) were up-regulated after III-10 treatment. And III-10 stimulated PLSCR1 and PML probably through activation of protein kinase Cδ (PKCδ). All these results suggested that III-10 was a prospective anticancer compound and was requisite to be proceeded further investigation., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Purpose: To compare the corneal epitheliotrophic capacity of different human blood-derived preparations, including cord blood serum (CBS), peripheral blood serum (PBS), and fresh frozen plasma (FFP) on bovine corneal epithelial cells., Materials and Methods: The concentrations of epithelial growth factor, transforming growth factor β1, insulin-like growth factor 1, hyaluronic acid, fibronectin, albumin, glucose, and calcium in different human blood derivatives were evaluated using enzyme-linked immunosorbent assay or biochemical methods. Cultivated bovine corneal epithelial cells were incubated with various blood derivatives, and cell proliferation, migration, and differentiation were evaluated by colorimetric assay, Boyden chamber chemotaxis assay, wounding assay, scanning electron microscopy, and transepithelial electric resistance measurements. Wound closure was assessed using a scratch-induced directional wounding assay., Results: Of the 3 human blood derivatives evaluated, CBS had the highest concentrations of epithelial growth factor, transforming growth factor β1, and hyaluronic acid (P < 0.05). FFP had the lowest concentration of calcium and the highest concentration of glucose (P < 0.05). CBS demonstrated the highest ability to promote cellular proliferation, followed by PBS and FFP (P < 0.05). CBS was also the best in promoting cellular differentiation because scanning electron microscopy demonstrated coherent monolayers of flattened and polygonal-shaped cells with evenly distributed microvilli. Transepithelial electric resistance was noted to be the highest for cells incubated in CBS, indicating formation of well-differentiated cells with functional tight junction (P < 0.05). Cells cultivated with FFP were the least capable of promoting proliferation, migration, and differentiation., Conclusions: Different human blood derivatives may have different concentrations of epitheliotrophic factors. CBS is generally superior to PBS in promoting corneal epithelial proliferation and differentiation.

Transcription factor FoxA1 plays a critical role during embryonic development and is activated during retinoic acid (RA)-induced neural differentiation of pluripotent P19 embryonal carcinoma cells at the early stage, which is marked by decreased expression of Nanog and increased expression of neural stem cell marker Nestin. To further understand how FoxA1 mediates neural differentiation, we have overexpressed FoxA1 through an adenovirus vector in P19 cells and identified that early neurogenesis-related sonic hedgehog (Shh) gene is activated directly by FoxA1. Knockdown of FoxA1 expression during P19 cell neural differentiation results in prevention of Shh and Nestin induction. FoxA1 binds to Shh promoter at -486 to -462 bp region and activates the promoter in cotransfection assays. Furthermore, overexpression of FoxA1 alone in P19 cells stimulates expression of Nestin and results in decreased protein levels of Nanog. During RA-induced P19 cell differentiation, elevated levels of FoxA1 increase the population of neurons, evidenced by stimulated expression of neuron-specific Neurofilament-1 and Tubulin betaIII. Together, our results suggest a critical involvement of FoxA1 in stimulating neural differentiation of pluripotent stem cells at early stages.

Skin-derived precursors (SKPs) are multipotent dermal precursors that share similarities with neural crest stem cells and that can give rise to peripheral neural and some mesodermal cell types, such as adipocytes. Here, we have asked whether rodent or human SKPs can generate other mesenchymally derived cell types, with a particular focus on osteocytes and chondrocytes. In culture, rodent and human foreskin-derived SKPs differentiated into alkaline-positive, collagen type-1-positive, mineralizing osteocytes, and into collagen type-II-positive chondrocytes that secreted chondrocyte-specific proteoglycans. Clonal analysis demonstrated that SKPs efficiently generated these skeletogenic cell types, and that they were multipotent with regard to the osteogenic and chondrogenic lineages. To ask if SKPs could generate these same lineages in vivo, genetically tagged, undifferentiated rat SKPs were transplanted into a tibial bone fracture model. Over the ensuing 6 weeks, many of the transplanted cells survived within the bone callus, where they were morphologically and phenotypically similar to the endogenous mesenchymal/osteogenic cells. Moreover, some transplanted cells adopted a mature osteocyte phenotype and integrated into the newly formed bone. Some transplanted cells also differentiated into chondrocytes and into smooth muscle cells and/or pericytes that were associated with blood vessels. Thus, both rodent and human SKPs generate skeletogenic cell types in culture, and the injured bone environment is sufficient to instruct SKPs to differentiate down an osteogenic lineage, in a fashion similar to the endogenous mesenchymal precursors.

Aim: aTo study the effect of human nuclear distribution C èhNUDCé on human megakaryocyte proliferation and differentiation from cord blood CD34(+) cells in vitro., Methods: aHuman CD34(+) cells were isolated using the Dynal CD34 Progenitor Cell Selection System from umbilical cord blood. The CD34(+) cells were then cultured in serum free methylcellulose semi-solid media, the morphologic aspects and number of small, medium and large CFU-MK colonies were observed and scored on the day12 by microscopy analysis. The CD34(+) cells were cultured in serum free liquid media, cells were removed on day 10 and formation of CD41(+) in human megakaryocyte and its DNA polyploidization of nuclear were analyzed on a FACsort flowcytometer., Results: ahNUDC supported the formation of small and medium CFU-MK colony in serum free semi-solid media. Furthermore, hNUDC induced a remarkable increase in expression of the megakaryocyte cell surface marker CD41(+) and stimulated the CD41(+) DNA polyploidization more effectively than TPO., Conclusion: hNUDC may play an important role in megakaryocyte proliferation and differentiation.

Aim: To investigate the role of histone deacetylases (HDACs) inhibitor FK228 in the IL-3 mediated proliferation and differentiation of human erythroid progenitor cells., Methods: CD34(+) cells were separated from the granulocyte colony-stimulating factor(G-CSF)-mobilized peripheral blood of cancer patients and cultured for 7 days with FK228 of different concentrations, stem cell factor(SCF) and interleukin 3 (IL-3) in serum-free medium. Flow cytometry was performed to analyze the expression of CD14, GPA, CD15 and CD36 on the cells and the colony assay of erythroid cells was conducted. Then the CD36(+)GPA(-) cells were enriched and cultured with with IL-3 and 0.5 microg/L FK228 supplemented serum-free semisolid cultures and the formation test of erythroid colony was conducted., Results: FK228 enhanced the generation of CD36(+) cells in a dose-dependent manner, in spite of that it displayed no influence on the generation of CD14(+) and CD15(+) cells. 0.5 g/L FK228 induced a remarkable increase in the cell number of CD36(+) cells cultured with IL-3; FK228 also significantly increased the number and size of CD36(+) cells colony cultured with IL-3 containing semisolid cultures. Moreover, FK228 augmented the formation of CD36(+) cells colony from CD36(+)GPA(-) cells cultured with IL-3., Conclusion: FK228 can promote the IL-3 mediated proliferation and differentiation of human erythroid progenitor cells.

Chronic arsenic exposure in humans is associated with cancers of the skin, lung, bladder and other tissues. There is evidence that folate deficiency may increase susceptibility to arsenic effects, including skin lesions. K6/ODC mice develop skin tumors when exposed to 10ppm sodium arsenite for 5 months. In the current study, K6/ODC mice maintained on either a folate deficient or folate sufficient diet were exposed to 0, 1, or 10ppm sodium arsenite in the drinking water for 30 days. Total RNA was isolated from skin samples and gene expression analyzed using Affymetrix Mouse 430 2.0 GeneChips. Data from 24 samples, with 4 mice in each of the 6 treatment groups, were RMA normalized and analyzed by two-way ANOVA using GeneSpring. Top gene ontology (GO) categories for genes responding significantly to both arsenic treatment and folate deficiency include nucleotide metabolism and cell organization and biogenesis. For many of these genes, folate deficiency magnifies the response to arsenic treatment. In particular, expression of markers of epidermal differentiation, e.g., loricrin, small proline rich proteins and involucrin, was significantly reduced by arsenic in the folate sufficient animals, and reduced further or at a lower arsenic dose in the folate deficient animals. In addition, expression of a number of epidermal cell growth/proliferation genes and cellular movement genes was altered. These results indicate that arsenic disrupts the normal balance of cell proliferation and differentiation, and that folate deficiency exacerbates these effects, consistent with the view that folate deficiency is a nutritional susceptibility factor for arsenic-induced skin tumorigenesis.

Produced by dietary fiber, butyrate is a potential chemopreventive agent against colon cancer. It stimulates proliferation of normal colonic epithelial cells but induces growth inhibition, differentiation, apoptosis, or a combination of effects in colon carcinoma cells. In this study, we used cDNA membrane arrays and real-time reverse transcriptase-polymerase chain reaction to identify stress genes that were differentially regulated by sodium butyrate (NaB) in HT 29 human colon carcinoma cells. The results indicated that a group of heat shock protein (hsp) genes were upregulated by 3 mM NaB within the first 24 hours of exposure. Because the transcription of hsp genes is under the control of heat shock factors (HSFs), we measured the effects of overexpressed HSF-1 on the responses of HT 29 cells to NaB. Overexpression of HSF-1 inhibited NaB-induced differentiation as measured by alkaline phosphatase activity and carcinoembryonic antigen expression. These results suggest that increased expression of HSFs and Hsps might render colon carcinoma cells resistant to the chemopreventive effects of butyrate.

Transforming growth factor-beta1 (TGF-beta1), an immunosuppressive cytokine, inhibits cytotoxic T cell (CTL) immune responses. In contrast, 4-1BB (CD137), a costimulatory molecule in the tumor necrosis factor (TNF) receptor family, amplifies CTL-mediated antitumor immune responses. We investigated whether TGF-beta1 responses could be reversed by 4-1BB costimulation during in vitro differentiation of naive CD8+ T cells into effector CTL cells. TGF-beta1 potently suppressed CTL differentiation of human cord blood naive CD8+ T cells as determined by reduced induction of characteristic phenotypes of effector cells and cytotoxic activity. TGF-beta1-mediated suppression of CTL differentiation was abrogated by 4-1BB costimulation but not by CD28 or another member in the TNF receptor family, CD30. 4-1BB costimulation suppressed Smad2 phosphorylation induced by TGF-beta1, suggesting that 4-1BB effects were at the level of TGF-beta1 signaling. 4-1BB effects on the TGF-beta1-mediated suppression were enhanced by interleukin 12 (IL-12) but counteracted by IL-4; 4-1BB expression was up- or down-regulated, respectively, by IL-12 and IL-4. IL-4 was more dominant than IL-12 when both cytokines were present during 4-1BB costimulation in the presence of TGF-beta1. This indicates critical roles for IL-4 and IL-12 in regulating 4-1BB effects on TGF-beta1-mediated suppression.

Myostatin is a recently discovered member of transforming growth factor beta (TGFbeta) superfamily and shares similar structure features with other members of TGFbeta superfamily. For a better understanding of molecular mechanism of myostatin function, the production of C-terminal truncated form of recombinant myostatin protein (rMSTN) in E. coli was previously reported. Herein, the functional role of the recombinant myostatin in regulating myogenesis in a chicken embryonic myoblasts (CEMs) system was determined. By using flow cytometric analysis, the myostatin was found to inhibit cell cycle transition from G1 to S phase and result in a cell cycle arrest at G1. In addition, myostatin blocked the multi-nucleus myotube formation and caused a decreased expression of the muscle cell differentiation markers (myogenin and MHC) in CEMs. In this study, a rabbit polyclonal antibody against myostatin was produced and high affinity and specificity of this anti-myostatin antibody to recombinant and endogenous myostatin were assayed by Western blot analysis. Further studies showed that the antibody could also recognize the tissue endogenous myostatin of human, mouse and rat. A specific 40 kD band was detected in chicken muscle, which suggested that chicken myostatin might have different splicing pattern. Immunofluorescence assay indicated that myostatin predominantly existed in the cytosol in C2C12 cells. Taken together, the results show that myostatin inhibits chicken muscle cells proliferation and differentiation and down-regulates expression of two differentiation marker gene in CEMs. Remarkably, production of functional recombinant myostatin protein and its specific antibody provides important reagents for unraveling molecular mechanisms underlying myostatin action during myogenesis.

The tight junction protein claudin‐7 is essential for tight junction function and intestinal homeostasis. Cldn7 deletion in mice leads to an inflammatory bowel disease‐like phenotype exhibiting severe intestinal epithelial damage, weight loss, inflammation, mucosal ulcerations, and epithelial hyperplasia. Claudin‐7 has also been shown to be involved in cancer metastasis and invasion. Here, we test our hypothesis that claudin‐7 plays an important role in regulating colonic intestinal stem cell function. Conditional knockout of Cldn7 in the colon led to impaired epithelial cell differentiation, hyperproliferative epithelium, a decrease in active stem cells, and dramatically altered gene expression profiles. In 3D colonoid culture, claudin‐7–deficient crypts were unable to survive and form spheroids, emphasizing the importance of claudin‐7 in stem cell survival. Inhibition of the Hippo pathway or activation of Notch signaling partially rescued the defective stem cell behavior. Concurrent Notch activation and Hippo inhibition resulted in restored colonoid survival, growth, and differentiation to the level comparable to those of wild‐type derived crypts. In this study, we highlight the essential role of claudin‐7 in regulating Notch and Hippo signaling–dependent colonic stem cell functions, including survival, self‐renewal, and differentiation. These new findings may shed light on potential avenues to explore for drug development in colorectal cancer. [ABSTRACT FROM AUTHOR]

Atoh1 overexpression is essential for hair cell (HC) regeneration in the sensory epithelium of mammalian auditory and vestibular organs. However, Atoh1 overexpression alone cannot induce fully mature and functional HCs in the mammalian inner ear. In the current study, we investigated the effect of Atoh1 constitutive overexpression in native HCs by manipulating Atoh1 expression at different developmental stages. We demonstrated that constitutive overexpression of Atoh1 in native vestibular HCs did not affect cell survival but did impair vestibular function by interfering with the subtype differentiation of HCs and hair bundle development. In contrast, Atoh1 overexpression in cochlear HCs impeded their maturation, eventually leading to gradual HC loss in the cochlea and hearing dysfunction. Our study suggests that time-restricted Atoh1 expression is essential for the differentiation and survival of HCs in the inner ear, and this is pivotal for both hearing and vestibular function re-establishment through Atoh1 overexpression-induced HC regeneration strategies. [ABSTRACT FROM AUTHOR]