Clostridium ventriculi in a cynomolgus monkey with acute gastric dilatation and rupture: A case report.

Acute gastric dilatation (AGD) is one of the most prevalent and life-threatening diseases in nonhuman primates worldwide. However, the etiology of this syndrome has not been determined. Recently, sudden death occurred in a 7-year-old female cynomolgus monkey with a history of fecal microbiota transplantation using diarrheic stools. The monkey had undergone surgery previously. On necropsy, gastric dilatation and rupture demonstrated a tetrad arrangement on histopathologic examination. On 16S rRNA sequencing, a high population of Clostridium ventriculi was identified in the duodenum adjacent to stomach but not in the colon. This paper is the first report of Clostridium ventriculi infection in a cynomolgus macaque with acute gastric dilatation and rupture.

Human umbilical cord blood mesenchymal stem cells engineered to overexpress growth factors accelerate outcomes in hair growth.

Human umbilical cord blood mesenchymal stem cells (hUCB-MSCs) are used in tissue repair and regeneration; however, the mechanisms involved are not well understood. We investigated the hair growth-promoting effects of hUCB-MSCs treatment to determine whether hUCB-MSCs enhance the promotion of hair growth. Furthermore, we attempted to identify the factors responsible for hair growth. The effects of hUCB-MSCs on hair growth were investigated in vivo, and hUCB-MSCs advanced anagen onset and hair follicle neogeneration. We found that hUCB-MSCs co-culture increased the viability and up-regulated hair induction-related proteins of human dermal papilla cells (hDPCs) in vitro. A growth factor antibody array revealed that secretory factors from hUCB-MSCs are related to hair growth. Insulin-like growth factor binding protein-1 (IGFBP-1) and vascular endothelial growth factor (VEGF) were increased in co-culture medium. Finally, we found that IGFBP-1, through the co-localization of an IGF-1 and IGFBP-1, had positive effects on cell viability; VEGF secretion; expression of alkaline phosphatase (ALP), CD133, and ß-catenin; and formation of hDPCs 3D spheroids. Taken together, these data suggest that hUCB-MSCs promote hair growth via a paracrine mechanism.

Cobalt chloride induces neuronal differentiation of human mesenchymal stem cells through upregulation of microRNA-124a.

Human mesenchymal stem cells (hMSCs) are known to have the capacity to differentiate into various cell types, including neurons. To examine our hypothesis that miRNA was involved in neuronal differentiation of hMSCs, CoCl2, a hypoxia-mimicking agent was used to induce neuronal differentiation, which was assessed by determining the expression of neuronal markers such as nestin and Tuj1. Treatment of hMSCs with CoCl2 led to increased expression of miR-124a, a neuron-specific miRNA. HIF-1α silencing and JNK inhibition abolished CoCl2-induced miR-124a expression, suggesting that JNK and HIF-1α signals were required for the miR-124a expression induced by CoCl2 in hMSCs. Overexpression of miR-124a or CoCl2 treatment suppressed the expression of anti-neural proteins such as SCP1 and SOX9. Silencing of both SCP1 and SOX9 induced neuronal differentiation of hMSCs, indicating that suppression of miR-124a targets is important for CoCl2-induced neuronal differentiation of hMSCs. Knockdown of HIF-1α or inhibition of JNK restored the expression of SCP1 and SOX9 in CoCl2-treated cells. Inhibition of miR-124a blocked CoCl2-induced suppression of SCP1 and SOX9 and abolished CoCl2-induced neuronal differentiation of hMSCs. Taken together, we demonstrate that miR-124a is critically regulates CoCl2-induced neuronal differentiation of hMSCs by suppressing the expression of SCP1 and SOX9.

Functional expression of smooth muscle-specific ion channels in TGF-ß(1)-treated human adipose-derived mesenchymal stem cells.

Human adipose tissue-derived mesenchymal stem cells (hASCs) have the power to differentiate into various cell types including chondrocytes, osteocytes, adipocytes, neurons, cardiomyocytes, and smooth muscle cells. We characterized the functional expression of ion channels after transforming growth factor-ß1 (TGF-ß1)-induced differentiation of hASCs, providing insights into the differentiation of vascular smooth muscle cells. The treatment of hASCs with TGF-ß1 dramatically increased the contraction of a collagen-gel lattice and the expression levels of specific genes for smooth muscle including α-smooth muscle actin, calponin, smooth mucle-myosin heavy chain, smoothelin-B, myocardin, and h-caldesmon. We observed Ca(2+), big-conductance Ca(2+)-activated K(+) (BKCa), and voltage-dependent K(+) (Kv) currents in TGF-ß1-induced, differentiated hASCs and not in undifferentiated hASCs. The currents share the characteristics of vascular smooth muscle cells (SMCs). RT-PCR and Western blotting revealed that the L-type (Cav1.2) and T-type (Cav3.1, 3.2, and 3.3), known to be expressed in vascular SMCs, dramatically increased along with the Cavß1 and Cavß3 subtypes in TGF-ß1-induced, differentiated hASCs. Although the expression-level changes of the ß-subtype BKCa channels varied, the major α-subtype BKCa channel (KCa1.1) clearly increased in the TGF-ß1-induced, differentiated hASCs. Most of the Kv subtypes, also known to be expressed in vascular SMCs, dramatically increased in the TGF-ß1-induced, differentiated hASCs. Our results suggest that TGF-ß1 induces the increased expression of vascular SMC-like ion channels and the differentiation of hASCs into contractile vascular SMCs.

Mesenchymal stem cells stimulate angiogenesis in a murine xenograft model of A549 human adenocarcinoma through an LPA1 receptor-dependent mechanism.

Carcinoma-associated fibroblasts play a key role in tumorigenesis and metastasis by providing a tumor-supportive microenvironment. In the present study, we demonstrate that conditioned medium from A549 human lung adenocarcinoma cells induces differentiation of human adipose tissue-derived mesenchymal stem cells (hASCs) to carcinoma-associated fibroblasts expressing α-smooth muscle actin, vascular endothelial growth factor, and stromal cell-derived factor-1. A549 conditioned medium-induced differentiation of hASCs to carcinoma-associated fibroblasts was completely abrogated by treatment of hASCs with Ki16425, a lysophosphatidic acid receptor antagonist, or knockdown of lysophosphatidic acid receptor 1 (LPA(1)) expression in hASCs with small interfering RNA or lentiviral short hairpin RNA. Using a murine xenograft transplantation model of A549 cells, we showed that co-transplantation of hASCs with A549 cells stimulated growth of A549 xenograft tumor, angiogenesis, and differentiation of hASCs to carcinoma-associated fibroblasts in vivo. Knockdown of LPA(1) expression in hASCs abrogated hASCs-stimulated growth of A549 xenograft tumor, angiogenesis, and differentiation of hASCs to carcinoma-associated fibroblasts. Moreover, A549 conditioned medium-treated hASCs stimulated tube formation of human umbilical vein endothelial cells by LPA(1)-dependent secretion of vascular endothelial growth factor. These results suggest that A549 cells induce in vivo differentiation of hASCs to carcinoma-associated fibroblasts, which play a key role in tumor angiogenesis within tumor microenvironment, through an LPA-LPA(1)-mediated paracrine mechanism.

Lysophosphatidic acid-induced expression of periostin in stromal cells: Prognoistic relevance of periostin expression in epithelial ovarian cancer.

Lysophosphatidic acid (LPA) is a bioactive lipid crucial for the initiation and progression of ovarian cancer. Identification of LPA-induced biomarkers is necessary for predicting prognosis of ovarian cancer patients. Here we report periostin, an extracellular matrix protein, as an LPA-induced protein in stromal cells and as a prognostic marker in patients with epithelial ovarian cancer (EOC). In human EOC tissues, periostin was mainly expressed in cancer-associated stromal fibroblasts, but not in cancer cells. The expression levels of periostin highly correlated with poor survival and tumor recurrence of ovarian cancer patients. Treatment of human adipose tissue-derived stromal cells with LPA or conditioned media from human ovarian adenocarcinoma cell lines, such as SK-OV-3 and OVCAR-3, induced expression of periostin. The periostin expression induced by cancer-conditioned media was abrogated by silencing of the LPA receptor 1 expression using small hairpin RNA lentivirus. Recombinant periostin stimulated adhesion and invasion of SK-OV-3 human ovarian adenocarcinoma cells and induced expression of matrix metalloprotease-2 in the cancer cells. These results suggest that LPA is associated with the expression of periostin in cancer-associated fibroblasts of EOC.

Thromboxane a(2) induces differentiation of human mesenchymal stem cells to smooth muscle-like cells.

Thromboxane A(2) (TxA(2)) is involved in smooth muscle contraction and atherosclerotic vascular diseases. Accumulating evidence suggests a pivotal role for mesenchymal stem cells (MSCs) in vascular remodeling. In the present study, we demonstrate for the first time that the TxA(2) mimetic U46619 induces differentiation of human adipose tissue-derived MSCs (hADSCs) to smooth muscle-like cells, as demonstrated by increased expression of smooth muscle-specific contractile proteins such as alpha-smooth muscle actin (alpha-SMA), calponin, smoothelin, and smooth muscle-myosin heavy chain. Using an in vitro collagen gel lattice contraction assay, we showed that U46619-induced expression of the contractile proteins was associated with increased contractility of the cells. U46619 increased the intracellular Ca(2+) concentration in hADSCs and pretreatment of the cells with the thromboxane receptor antagonist SQ29548 or the calmodulin (CaM) inhibitor W13 abrogated the U46619-induced alpha-SMA expression and contractility, suggesting a pivotal role of Ca(2+)/CaM in the U46619-stimulated smooth muscle differentiation of hADSCs. In addition, U46619 elicited activation of RhoA in hADSCs, and pretreatment of the cells with the Rho kinase-specific inhibitor Y27632 or overexpression of the dominant-negative mutants of RhoA and Rho kinase blocked U46619-stimulated alpha-SMA expression and contractility. Furthermore, U46619 induced phosphorylation of myosin light chain (MLC) through CaM/MLC kinase- and Rho kinase-dependent pathways, and the MLC kinase inhibitor ML-7 abrogated U46619-induced alpha-SMA expression and contractility. These results suggest that U46619 induces differentiation of hADSCs to contractile smooth muscle-like cells through CaM/MLCK- and RhoA-Rho kinase-dependent actin polymerization.

A Rho kinase/myocardin-related transcription factor-A-dependent mechanism underlies the sphingosylphosphorylcholine-induced differentiation of mesenchymal stem cells into contractile smooth muscle cells.

Sphingosylphosphorylcholine (SPC) induces differentiation of human adipose tissue-derived mesenchymal stem cells (hADSCs) to smooth muscle cells (SMCs). In the present study, we characterized contractile and ion channel properties of SMCs differentiated from hADSCs (hADSC-SMCs) as a result of SPC treatment, and we investigated the molecular mechanisms involved in the SPC-induced differentiation. Using in vitro collagen gel lattice contraction and whole cell patch clamp, we showed that the hADSC-SMCs expressed functional L-type voltage-gated Ca2+ channels and contractile activities in response to KCl, carbachol, and the L-type Ca2+ channel opener Bay K8644, whereas the L-type Ca2+ channel blocker nifedipine abrogated the contractility of hADSC-SMCs. Furthermore, hADSC-SMCs expressed functional big conductance Ca2+-activated K+ (BK(Ca)) channels, and the BK(Ca) channel blocker iberiotoxin potentiated the Bay K8644-stimulated contractility of the hADSC-SMCs, indicating that these cells exhibited SMC-like contractile characteristics. SPC activated RhoA in hADSCs and pretreatment with the Rho kinase inhibitor Y27632 or by overexpression of dominant-negative mutants of RhoA or Rho kinase completely abrogated the SPC-induced differentiation of hADSCs into SMCs. SPC also increased the expression levels of myocardin-related transcription factor (MRTF)-A, a transcription factor involved in smooth muscle differentiation, in hADSCs. Small interference RNA-mediated depletion of endogenous MRTF-A abolished the SPC-induced differentiation of hADSCs into SMCs. Furthermore, SPC promoted nuclear translocation of MRTF-A, and pharmacological inhibition of Rho kinase blocked this effect. These results suggest that SPC induced differentiation of hADSCs into contractile SMCs through a mechanism involving RhoA/Rho kinase-dependent nuclear translocation of MRTF-A.

Protective effects of human umbilical cord blood­derived mesenchymal stem cells against dexamethasone­induced apoptotic cell death in hair follicles.

Alopecia is a common and distressing condition, and developing new therapeutic agents to prevent hair loss is important. Human umbilical cord blood­derived mesenchymal stem cells (hUCB­MSCs) have been studied intensively in regenerative medicine. However, the therapeutic potential of these cells against hair loss and hair organ damage remains unclear, and the effects of hUCB­MSC transplantation on hair loss require evaluation. The current study aimed to investigate the effects of hUCB­MSCs on hair regression in vivo and restoration of anagen conduction on hair growth in vitro. The effects of hUCB­MSCs were explored in mouse catagen induction models using a topical treatment of 0.1% dexamethasone to induce hair regression. Dexamethasone was also used to simulate a stress environment in vitro. The results demonstrated that hUCB­MSCs significantly prevented hair regression induced by dexamethasone topical stimulation in vivo. Additionally, hUCB­MSCs significantly increased the proliferation of human dermal papilla cells (hDPCs) and HaCaT cells, which are key constituent cells of the hair follicle. Stimulation of vascular endothelial growth factor secretion and decreased expression of DKK­1 by hUCB­MSCs were also observed in hDPCs. Restoration of cell viability by hUCB­MSCs suggested that these cells exerted a protective effect on glucocorticoid stress­associated hair loss. In addition, anti­apoptotic effects and regulation of the autophagic flux recovery were observed in HaCaT cells. The results of the present study indicated that hUCB­MSCs may have the capacity to protect hair follicular dermal papilla cells and keratinocytes, thus preventing hair loss. Additionally, the protective effects of hUCB­MSCs may be resistant to dysregulation of autophagy under harmful stress.

Cancer-derived lysophosphatidic acid stimulates differentiation of human mesenchymal stem cells to myofibroblast-like cells.

Lysophosphatidic acid (LPA) is enriched in ascites of ovarian cancer patients and is involved in growth and invasion of ovarian cancer cells. Accumulating evidence suggests cancer-associated myofibroblasts play a pivotal role in tumorigenesis through secreting stromal cell-derived factor-1 (SDF-1). In the present study, we demonstrate that LPA induces expression of alpha-smooth muscle actin (alpha-SMA), a marker for myofibroblasts, in human adipose tissue-derived mesenchymal stem cells (hADSCs). The LPA-induced expression of alpha-SMA was completely abrogated by pretreatment of the cells with Ki16425, an antagonist of LPA receptors, or by silencing LPA(1) or LPA(2) isoform expression with small interference RNA (siRNA). LPA elicited phosphorylation of Smad2/3, and siRNA-mediated depletion of endogenous Smad2/3 or adenoviral expression of Smad7, an inhibitory Smad, abrogated the LPA induced expression of alpha-SMA and phosphorylation of Smad2/3. LPA-induced secretion of transforming growth factor (TGF)-beta1 in hADSCs, and pretreatment of the cells with SB431542, a TGF-beta type I receptor kinase inhibitor, or anti-TGF-beta1 neutralizing antibody inhibited the LPA-induced expression of alpha-SMA and phosphorylation of Smad2. Furthermore, ascites from ovarian cancer patients or conditioned medium from ovarian cancer cells induced expression of alpha-SMA and phosphorylation of Smad2, and pretreatment of the cells with Ki16425 or SB431542 abrogated the expression of alpha-SMA and phosphorylation of Smad2. In addition, LPA increased the expression of SDF-1 in hADSCs, and pretreatment of the cells with Ki16425 or SB431562 attenuated the LPA-stimulated expression of SDF-1. These results suggest that cancer-derived LPA stimulates differentiation of hADSCs to myofibroblast-like cells and increases SDF-1 expression through activating autocrine TGF-beta1-Smad signaling pathway.

Bradykinin-induced expression of alpha-smooth muscle actin in human mesenchymal stem cells.

Phenotypic expression of alpha-smooth muscle actin (alpha-SMA), a smooth muscle marker, has been implicated in vascular diseases, fibrosis, wound healing, and tissue remodeling. Bradykinin (BK), a vasoactive peptide produced during tissue injury, plays a key role in inflammatory and vascular responses associated with tissue injury. In the present study, we demonstrated for the first time that BK treatment increased alpha-SMA expression in human adipose tissue-derived mesenchymal stem cells (hADSCs). This BK-induced alpha-SMA expression was abrogated by small interfering RNA (siRNA)-mediated depletion of endogenous myocardin, a transcription factor involved in smooth muscle differentiation. BK also increased the intracellular calcium concentration ([Ca(2+)](i)), a response that was completely blocked by treatment with a BK B2 receptor-specific antagonist (HOE 140), suggesting that the BK B2 receptor was participating in BK-induced cellular responses. In addition, BK induced the secretion of transforming growth factor-beta1 (TGF-beta1) and autocrine activation of Smad2. Pretreatment with a TGF-beta type I receptor kinase inhibitor (SB-431542), small interfering RNA-mediated depletion of endogenous Smad2, or adenoviral expression of Smad7 (an inhibitory Smad isoform) all blocked BK-induced alpha-SMA expression and Smad2 phosphorylation. Furthermore, a MEK-specific inhibitor (U0126) abrogated BK-induced TGF-beta1 secretion, Smad2 phosphorylation, and alpha-SMA expression. These results suggest that BK induced expression of alpha-SMA in hADSCs through ERK-dependent activation of the autocrine TGF-beta1-Smad2 crosstalk pathway.

Angiotensin II-induced differentiation of adipose tissue-derived mesenchymal stem cells to smooth muscle-like cells.

Angiotensin II (Ang II) is involved in the development of cardiovascular disease and vascular remodeling. In this study, we demonstrate that treatment of human adipose tissue-derived mesenchymal stem cells (hADSCs) with Ang II increased the expression of smooth muscle-specific genes, including alpha-smooth muscle actin (alpha-SMA), calponin, h-caldesmon, and smooth muscle myosin heavy chain (SM-MHC), and also elicited the secretion of transforming growth factor-beta1 (TGF-beta1) and delayed phosphorylation of Smad2. The Ang II-induced expression of alpha-SMA and delayed phosphorylation of Smad2 were blocked by pretreatment of the cells with a TGF-beta type I receptor kinase inhibitor, SB-431542, small interference RNA-mediated depletion of endogenous Smad2, and adenoviral expression of Smad7. Furthermore, the Ang II-induced TGF-beta1 secretion, alpha-SMA expression, and delayed phosphorylation of Smad2 in hADSCs were abrogated by the MEK inhibitor U0126, suggesting a pivotal role of MEK/ERK pathway in the Ang II-induced activation of TGF-beta1-Smad2 signaling pathway. The smooth muscle-like cells which were differentiated from hADSCs by Ang II treatment exhibited contraction in response to 60mM KCl. These results suggest that Ang II induces differentiation of hADSCs to contractile smooth muscle-like cells through ERK-dependent activation of the autocrine TGF-beta1-Smad2 crosstalk pathway.

Lysophosphatidic acid in malignant ascites stimulates migration of human mesenchymal stem cells.

Lysophosphatidic acid (LPA) is elevated in ascites of ovarian cancer patients and is involved in growth and invasion of ovarian cancer cells. Accumulating evidence suggests a pivotal role of mesenchymal stem cells (MSCs) or stromal cells in tumorigenesis. In the present study, we demonstrated that ascites from ovarian cancer patients and LPA increased migration of human MSCs. The migration of MSCs induced by LPA and malignant ascites was completely abrogated by pretreatment with Ki16425, an antagonist of LPA receptors, and by silencing of endogenous LPA(1), but not LPA(2), with small interference RNA, suggesting a key role of LPA played in the malignant ascites-induced migration. LPA induced activation of ERK through pertussis toxin-sensitive manner, and pretreatment of MSCs with U0126, a MEK inhibitor, or pertussis toxin attenuated the LPA-induced migration. Moreover, LPA induced activation of RhoA in MSCs, and pretreatment of the cells with Y27632, a Rho kinase inhibitor, markedly inhibited the LPA-induced migration. In addition, LPA and malignant ascites increased intracellular concentration of calcium in MSCs, and Ki16425 completely inhibited the elevation of intracellular calcium. These results suggest that LPA is a crucial component of the malignant ascites which induce the migration of MSCs and elevation of intracellular calcium.

Cobalt Chloride Enhances the Anti-Inflammatory Potency of Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells through the ERK-HIF-1α-MicroRNA-146a-Mediated Signaling Pathway.

Human mesenchymal stem cells (hMSCs), including human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs), which have high proliferation capacity and immunomodulatory properties, are considered to be a good candidate for cell-based therapies. hMSCs show enhanced therapeutic effects via paracrine secretion or cell-to-cell contact that modulates inflammatory or immune reactions. Here, treatment with cobalt chloride (CoCl2) was more effective than naïve hUCB-MSCs in suppressing inflammatory responses in a coculture system with phytohemagglutinin- (PHA-) activated human peripheral blood mononuclear cells (hPBMCs). Furthermore, the effect of CoCl2 is exerted by promoting the expression of anti-inflammatory mediators (e.g., PGE2) and inhibiting that of inflammatory cytokines (e.g., TNF-α and IFN-γ). Treatment of hUCB-MSCs with CoCl2 leads to increased expression of microRNA- (miR-) 146a, which was reported to modulate anti-inflammatory responses. Hypoxia-inducible factor- (HIF-) 1α silencing and ERK inhibition abolished CoCl2-induced miR-146a expression, suggesting that ERK and HIF-1α signals are required for CoCl2-induced miR-146a expression in hUCB-MSCs. These data suggest that treatment with CoCl2 enhances the immunosuppressive capacity of hUCB-MSCs through the ERK-HIF-1α-miR-146a-mediated signaling pathway. Furthermore, pretreatment of transplanted MSCs with CoCl2 can suppress lung inflammation more than naïve MSCs can in a mouse model of asthma. These findings suggest that CoCl2 may improve the therapeutic effects of hUCB-MSCs for the treatment of inflammatory diseases.

Sphingosylphosphorylcholine stimulates expression of fibronectin through TGF-beta1-Smad-dependent mechanism in human mesenchymal stem cells.

Sphingosylphosphorylcholine (SPC) has been reported to stimulate the expression of fibronectin (FN), which plays a key role in cell recruitment and adhesion during wound healing. In a previous study, we reported that SPC induces differentiation of human adipose tissue-derived mesenchymal stem cells (hATSCs) to smooth muscle-like cell types through ERK-dependent autocrine secretion of TGF-beta1 and delayed activation of the TGF-beta1-Smad pathway. In the present study, we demonstrated that SPC dose- and time-dependently increased the expression of FN in hATSCs. Pretreatment of the cells with U0126, an MEK inhibitor, markedly attenuated the SPC-induced expression of FN and delayed phosphorylation of Smad2, suggesting that ERK is involved in the SPC induction of FN expression through activation of Smad2. In addition, the SPC-induced expression of FN and delayed activation of Smad2 were abrogated by SB-431542, a TGF-beta type I receptor kinase inhibitor, or anti-TGF-beta1 neutralizing antibody. Furthermore, the SPC-induced expression of FN was abrogated by adenoviral expression of Smad7, an inhibitory Smad, or short interference RNA (siRNA)-mediated depletion of endogenous Smad2 expression, suggesting that SPC induces the expression of FN through ERK-dependent activation of the TGF-beta1-Smad2 crosstalk pathway. Adhesion of U937 monocytic cells to hATSCs was enhanced by pretreatment of hATSCs with SPC or TGF-beta1 for 4 days, and the peptide GRGDSP (an antagonist of fibronectin receptors) blocked the adhesion of U937 cells to the hATSCs. These results led us to suggest that SPC-induced FN expression plays a pivotal role in the wound healing by stimulating adhesion and recruitment of leukocytes.

Oncostatin M decreases adiponectin expression and induces dedifferentiation of adipocytes by JAK3- and MEK-dependent pathways.

Adiponectin, an adipokine secreted from adipocytes, plays a crucial role in the regulation of glucose and lipid metabolism. In the present study, we examine the role of the IL-6 family of cytokines in the expression of adiponectin in human adipocytes derived from human adipose tissue-derived stromal cells. Oncostatin M (OSM), but not IL-6, attenuated the expression level of adiponectin dose- and time-dependently, and the inhibitory effect of OSM on adiponectin expression was as potent as that of TNF-alpha. The OSM-induced down-regulation of adiponectin expression was correlated with the down-regulation of PPARgamma2 and lipoprotein lipase, markers for adipogenic differentiation, and depletion of intracellular lipid droplets, suggesting dedifferentiation of adipocytes in response to OSM. OSM induced phosphorylation of STAT1, and treatment of adipocytes with JAK3 inhibitor WHI-P131 or MEK inhibitor U0126, but not with JAK2 inhibitor AG490, prevented the activation of STAT1. Furthermore, the OSM-induced suppression of adiponectin expression and dedifferentiation of adipocytes were ameliorated by WHI-P131 or U0126, but not by AG490. These results suggest that OSM inhibits adiponectin expression by inducing dedifferentiation of adipocytes through signaling pathways involving JAK3 and MEK, but not JAK2.

Optimization of culture conditions for rapid clinical-scale expansion of human umbilical cord blood-derived mesenchymal stem cells.

BACKGROUND: Mesenchymal stem cells (MSCs) have broad-spectrum therapeutic effects in various diseases, and thus have many clinical applications. However, it is difficult to produce sufficient numbers of MSCs for clinical use, and improved culture systems are required. Here, we report the effects of calcium (Ca2+) and hypoxia on the proliferation of human umbilical cord blood-derived MSCs (hUCB-MSCs). In addition, we determined the optimal conditions of these two factors for the large-scale culture of hUCB-MSCs. METHODS: hUCB-MSCs were maintained under hypoxic conditions (3% O2) with 1.8 mM Ca2+ during long-term culture, and their proliferation was evaluated. To characterize the underlying mechanisms, the effects on hypoxia-inducible factor (HIF)-1α and the extracellular signal-regulated kinase (ERK) signaling pathways were investigated. The therapeutic effects in a mouse emphysema model were analyzed and compared with those of naive MSCs. RESULTS: The proliferation of Ca2+/hypoxia-treated hUCB-MSCs was increased compared with that observed using either calcium or hypoxia culture alone, without loss of stem cell marker expression or differentiation ability. The enhancement of the proliferation capacity of hUCB-MSCs by the synergistic effects of Ca2+ and hypoxia was dependent on the expression of HIF-1α and the ERK signaling pathway. The proliferation of Ca2+/hypoxia-treated hUCB-MSCs resulted in a delayed senescence phenotype and increased the expression levels of stemness genes such as Oct4 and Nanog compared to those observed in conventional culture conditions. In addition, Ca2+/hypoxia-treated MSCs transplantation in the mouse emphysema model showed the same therapeutic effects as observed with naive MSCs. CONCLUSIONS: These findings suggest that a Ca2+/hypoxia-based expansion system has applications for the large-scale production of MSCs for therapeutic purposes.

Role of MEK-ERK pathway in sphingosylphosphorylcholine-induced cell death in human adipose tissue-derived mesenchymal stem cells.

Sphingosylphosphorylcholine (SPC) is a bioactive lipid molecule involved in a variety of cellular responses. In the present study, we demonstrated that treatment of human adipose tissue-derived mesenchymal stem cells (hATSCs) with D-erythro-SPC resulted in apoptosis-like cell death, as demonstrated by decreased cell viability, DNA strand breaks, the increase of sub-G1 fraction, cytochrome c release into cytosol, and activation of caspase-3. In contrast, the exposure of hATSCs to L-threo-SPC did not induce the cell death, suggesting that the SPC-induced cell death was selective for the D-erythro-stereoisomer of SPC. The D-erythro-SPC-induced cell death was prevented by DEVD-CHO, a caspase-3 specific inhibitor, and Z-VAD-FMK, a general caspase inhibitor, suggesting that the SPC-induced cell death of hATSCs occurs through the cytochrome c- and caspase-3-dependent pathways. In addition, D-erythro-SPC treatment stimulated the activation of mitogen-activated protein kinases, such as ERK and c-Jun NH2-terminal protein kinase (JNK), and the D-erythro-SPC-induced cell death was completely prevented by pretreatment with the MEK inhibitor, U0126, but not by pretreatment with the JNK inhibitor, SP600125, and the p38 MAPK inhibitor, SB202190, suggesting a specific involvement of ERK in the D-erythro-SPC-induced cell death. Pretreatment with U0126 attenuated the D-erythro-SPC-induced release of cytochrome c. From these results, we suggest that ERK is involved in the SPC-induced cell death of hATSC through stimulation of the cytochrome c/caspase-3-dependent pathway.

Lysophosphatidylcholine suppresses apoptosis and induces neurite outgrowth in PC12 cells through activation of phospholipase D2.

Lysophosphatidylcholine (LPC) is a bioactive lipid generated by phospholipase A2-mediated hydrolysis of phosphatidylcholine. In the present study, we demonstrate that LPC stimulates phospholipase D2 (PLD2) activity in rat pheochromocytoma PC12 cells. Serum deprivation induced cell death of PC12 cells, as demonstrated by decreased viability, DNA fragmentation, and increased sub-G1 fraction of cell cycle. LPC treatment protected PC12 cells partially from the cell death and induced neurite outgrowth of the cells. Overexpression of PLD2 drastically enhanced the LPC-induced inhibition of apoptosis and neuritogenesis. Pretreatment of the cells with 1-butanol, a PLD inhibitor, completely abrogated the LPC-induced inhibition of apoptosis and neurite outgrowth in PC12 cells overexpressing PLD2. These results indicate that LPC possesses the neurotrophic effects, such as anti-apoptosis and neurite outgrowth, through activation of PLD2.

Sphingosylphosphorylcholine generates reactive oxygen species through calcium-, protein kinase Cdelta- and phospholipase D-dependent pathways.

Sphingosylphosphorylcholine (SPC) is a bioactive lipid molecule involved in numerous biological processes. Treatment of MS1 pancreatic islet endothelial cells with SPC increased phospholipase D (PLD) activity in a time- and dose-dependent manner. In addition, treatment of the MS1 cells with 10 microM SPC induced stimulation of phospholipase C (PLC) activity and transient elevation of intracellular Ca2+. The SPC-induced PLD activation was prevented by pretreatment of the MS1 cells with a PLC inhibitor, U73122, and an intracellular Ca2+-chelating agent, BAPTA-AM. This suggests that PLC-dependent elevation of intracellular Ca2+ is involved in the SPC-induced activation of PLD. The SPC-dependent PLD activity was also almost completely prevented by pretreatment with pan-specific PKC inhibitors, GF109203X and RO-31-8220, and with a PKCdelta-specific inhibitor, rottlerin, but not by pretreatment with GO6976, a conventional PKC isozymes-specific inhibitor. Adenoviral overexpression of a kinase-deficient mutant of PKCdelta attenuated the SPC-induced PLD activity. These results suggest that PKCdelta plays a crucial role for the SPC-induced PLD activation. The SPC-induced PLD activation was preferentially potentiated in COS-7 cells transfected with PLD2 but not with PLD1, suggesting a specific implication of PLD2 in the SPC-induced PLD activation. SPC treatment induced phosphorylation of PLD2 in COS-7 cells, and overexpression of the kinase-deficient mutant of PKCdelta prevented the SPC-induced phosphorylation of PLD2. Furthermore, SPC treatment generated reactive oxygen species (ROS) in MS1 cells and the SPC induced production of ROS was inhibited by pretreatment with U73122, BAPTA-AM, and rottlerin. In addition, pretreatment with a PLD inhibitor 1-butanol and overexpression of a lipase-inactive mutant of PLD2 but not PLD1 attenuated the SPC-induced generation of ROS. These results suggest that PLC-, Ca2+-, PKCdelta-, and PLD2-dependent pathways are essentially required for the SPC induced ROS generation.