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Tongue epithelium is one of the most proliferative and regenerative epithelia in our body. However, tongue stem cell research is hampered partly by the lack of optimal animal models to study tongue injury, repair, and regeneration. Here, we establish a novel chemically induced tongue injury-recovery mouse model. Focal application of sodium hydroxide for a limited time led to the denudation of suprabasal layers, leaving the basal layer. Time course study revealed that tongue epithelial cells robustly proliferate over one week after the tongue injury. Importantly, we demonstrated that our novel mouse model could be employed in the lineage tracing of the tongue stem cells under the injury and repair process and further showed that tongue stem cells proliferate faster and generate larger clones in the injury condition than in the steady state condition. Our data indicate the development of a novel chemically induced tongue injury-recovery mouse model for tongue stem cell research, which will significantly facilitate the preclinical study for the pathogenesis and treatment of caustic ingestion.

Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) have been reported to exhibit immature embryonic or fetal cardiomyocyte-like phenotypes. To enhance the maturation of hESC-CMs, we identified a natural steroidal alkaloid, tomatidine, as a new substance that stimulates the maturation of hESC-CMs. Treatment of human embryonic stem cells with tomatidine during cardiomyocyte differentiation stimulated the expression of several cardiomyocyte-specific markers and increased the density of T-tubules. Furthermore, tomatidine treatment augmented the number and size of mitochondria and enhanced the formation of mitochondrial lamellar cristae. Tomatidine treatment stimulated mitochondrial functions, including mitochondrial membrane potential, oxidative phosphorylation, and ATP production, in hESC-CMs. Tomatidine-treated hESC-CMs were more sensitive to doxorubicin-induced cardiotoxicity than the control cells. In conclusion, the present study suggests that tomatidine promotes the differentiation of stem cells to adult cardiomyocytes by accelerating mitochondrial biogenesis and maturation and that tomatidine-treated mature hESC-CMs can be used for cardiotoxicity screening and cardiac disease modeling.

The general bone anabolic effect of photobiomodulation (PBM) is largely accepted. As a result, PBM therapy is expected to be beneficial in the medical fields of dentistry and bone healing. However, most of the previous in vitro studies on PBM and bone metabolism were performed with single-cell cultures of osteoclast-lineage cells or osteoblast-lineage cells. In the present study, the bone-modulating effects of PBM were evaluated in an in vitro osteoblast/osteoclast co-culture system. Mouse bone marrow-derived macrophages (BMMs) and mouse calvarial pre-osteoblasts cells were purified and used as precursor cells for osteoclasts and osteoblasts, respectively. The PBM effects on single-cell culture of osteoclasts or osteoblasts as well as co-culture were examined by 1.2 J/cm2 low-level Ga-Al-As laser (λ = 808 ± 3 nm, 80 mW, and 80 mA; spot size, 1cm2; NDLux, Seoul, Korea) irradiation for 30 s at daily intervals throughout culture period. At the end of culture, the osteoclast differentiation and osteoblast differentiation were assessed by TRAP staining and ALP staining, respectively. The expressions of osteoclastogenic cytokines were evaluated by RT-PCR and Western blot analyses. Under the single-cell culture condition, PBM enhanced osteoblast differentiation but had minor effects on osteoclast differentiation. However, in the co-culture condition, its osteoblastogenic effect was maintained, and osteoclast differentiation was substantially reduced. Subsequent RT-PCR analyses and western blot results revealed marked reduction in receptor activator of NF-κB ligand (RANKL) expression and elevation in osteoprotegerin (OPG) expression by PBM in co-cultured cells. More importantly, these alterations in RANKL/OPG levels were not observed under the single-cell culture conditions. Our results highlight the different effects of PBM on bone cells based on culture conditions. Further, our findings suggest the indirect anti-osteoclastogenic effect of PBM, which is accompanied by a decrease in RANKL expression and an increase in OPG expression.

Vasohibin-1 (VASH1) is a key inhibitor of vascular endothelial growth factor-induced angiogenesis. Although the involvement of VASH1 in various pathological processes has been extensively studied, its role in periodontal disease (PD) remains unclear. We aimed to investigate the role of VASH1 in PD by focusing on osteoclastogenesis regulation. We investigated VASH1 expression in PD by analyzing data from the online Gene Expression Omnibus (GEO) database and using a mouse ligature-induced periodontitis model. The effects of VASH1 on osteoclast differentiation and osteoclastogenesis-supporting cells were assessed in mouse bone marrow-derived macrophages (BMMs) and human gingival fibroblasts (GFs). To identify the stimulant of VASH1, we used culture broth from Porphyromonas gingivalis (Pg), a periopathogen. The GEO database and mouse periodontitis model revealed that VASH1 expression was upregulated in periodontitis-affected gingival tissues, which was further supported by immunohistochemistry and qRT-PCR analyses. VASH1 expression was significantly stimulated in GFs after treatment with the Pg broth. Direct treatment with recombinant VASH1 protein did not stimulate osteoclast differentiation in BMMs but did contribute to osteoclast differentiation by inducing RANKL expression in GFs through a paracrine mechanism. Small interfering RNA-mediated silencing of VASH1 in GFs abrogated RANKL-mediated osteoclast differentiation in BMMs. Additionally, VASH1-activated RANKL expression in GFs was significantly suppressed by MK-2206, a selective inhibitor of AKT. These results suggest that Pg-induced VASH1 may be associated with RANKL expression in GFs in a paracrine manner, contributing to osteoclastogenesis via an AKT-dependent mechanism during PD progression.

The purpose of this study was to evaluate the bone-generating ability of a new bovine-derived xenograft (S1-XB) containing hydrogel. For control purposes, we used Bio-Oss and Bone-XB bovine-derived xenografts. S1-XB was produced by mixing Bone-XB and hydrogel. Cell proliferation and differentiation studies were performed to assess cytotoxicities and cell responses. For in vivo study, 8 mm-sized cranial defects were formed in 16 rats, and then the bone substitutes were transplanted into defect sites in the four study groups, that is, a Bio-Oss group, a Bone-XB group, an S1-XB group, and a control (all n = 4), in the control group defects were left empty. Eight weeks after surgery, new bone formation areas were measured histomorphometrically. In the cell study, extracts of Bio-Oss, Bone-XB, and S1-XB showed good results in terms of the osteogenic differentiation of human mesenchymal stem cells (hMSCs) and no cytotoxic reaction was evident. No significant difference was observed between mean new bone areas in the Bio-Oss (36.93 ± 4.27%), Bone-XB (35.07 ± 3.23%), and S1-XB (30.80 ± 6.41%) groups, but new bone area was significantly smaller in the control group (18.73 ± 5.59%) (p &lt, 0.05). Bovine-derived bone graft material containing hydrogel (S1-XB) had a better cellular response and an osteogenic effect similar to Bio-Oss.

The general bone anabolic effect of photobiomodulation (PBM) is largely accepted. As a result, PBM therapy is expected to be beneficial in the medical fields of dentistry and bone healing. However, most of the previous in vitro studies on PBM and bone metabolism were performed with single-cell cultures of osteoclast-lineage cells or osteoblast-lineage cells. In the present study, the bone-modulating effects of PBM were evaluated in an in vitro osteoblast/osteoclast co-culture system. Mouse bone marrow-derived macrophages (BMMs) and mouse calvarial pre-osteoblasts cells were purified and used as precursor cells for osteoclasts and osteoblasts, respectively. The PBM effects on single-cell culture of osteoclasts or osteoblasts as well as co-culture were examined by 1.2 J/cm

Periodontitis is a chronic inflammatory disease with alveolar bone resorption and subsequent tooth loss as its ultimate outcomes. Gastrin-releasing peptide (GRP) is a neuropeptide with growth-stimulatory and tumorigenic properties, and neuropeptides have previously been suggested to play a role in the complex cascade of chemical activity associated with periodontal inflammation. In this study, GRP treatment enhanced the differentiation of bone marrow-derived macrophages (BMMs) into osteoclasts, and gastrin-releasing peptide receptor (GRPR) antagonists suppressed the pro-osteoclastogenic effect of GRP. Grpr-siRNA knockdown resulted in a significantly lower number of osteoclasts formed as compared with the control. Interestingly, gene expression analysis indicated downregulation of Grp and Grpr expressions in BMMs during osteoclastogenesis. Moreover, ligature-induced periodontitis model in mice and gingival samples from patients with periodontitis displayed increased immunostaining of GRP in the oral epithelium. Subsequently, stimulation of mouse primary epithelial cells (ECs) and HaCaT cells, human epidermal keratinocytes, with lipopolysaccharides (LPS) of Porphyromonas gingivalis or live P. gingivalis upregulated Grp and Grpr expressions. Finally, coculture of P. gingivalis-stimulated ECs and BMMs using Transwell system revealed that the differentiation of BMMs was induced when subjected to paracrine activation by LPS- as well as live-P. gingivalis stimulated ECs. Taken together, our results demonstrate that the pro-osteoclastogenic properties of BMMs may be modulated by GRP produced by ECs in the periodontal microenvironment.

Cathepsin K (CTSK) is a cysteine protease that is mainly produced from mature osteoclasts and contributes to the destruction of connective tissues and mineralized matrix as a consequence of periodontal disease (PD). However, few studies have reported its regulatory role in osteoclastogenesis-supporting cells in inflammatory conditions. Here, we investigated the role of CTSK in osteoclastogenesis-supporting cells, focusing on the modulation of paracrine function. Microarray data showed that CTSK was upregulated in PD patients compared with healthy individuals, which was further supported by immunohistochemistry and qPCR analyses performed with human gingival tissues. The expression of CTSK in the osteoclastogenesis-supporting cells, including dental pulp stem cells, gingival fibroblasts, and periodontal ligament fibroblasts (PDLFs) was significantly elevated by treatment with inflammatory cytokines such as TNF&alpha, and IL-1&beta, Moreover, TNF&alpha, stimulation potentiated the PDLF-mediated osteoclastogenesis of bone marrow-derived macrophages. Interestingly, small interfering RNA-mediated silencing of CTSK in PDLF noticeably attenuated the TNF&alpha, triggered upregulation of receptor activator of nuclear factor kappa-B ligand (RANKL), macrophage colony-stimulating factor, and RANKL/osteoprotegerin ratio, thereby abrogating the enhanced osteoclastogenesis-supporting activity of PDLF. Collectively, these results suggest a novel role of CTSK in the paracrine function of osteoclastogenesis-supporting cells in periodontal disease.

Multiple mitogenic pathways capable of promoting mammalian cardiomyocyte (CM) proliferation have been identified as potential candidates for functional heart repair following myocardial infarction. However, it is unclear whether the effects of these mitogens are species-specific and how they directly compare in the same cardiac setting. Here, we examined how CM-specific lentiviral expression of various candidate mitogens affects human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and neonatal rat ventricular myocytes (NRVMs) in vitro. In 2D-cultured CMs from both species, and in highly mature 3D-engineered cardiac tissues generated from NRVMs, a constitutively-active mutant form of the human gene Erbb2 (cahErbb2) was the most potent tested mitogen. Persistent expression of cahErbb2 induced CM proliferation, sarcomere loss, and remodeling of tissue structure and function, which were attenuated by small molecule inhibitors of Erk signaling. These results suggest transient activation of Erbb2/Erk axis in cardiomyocytes as a potential strategy for regenerative heart repair.

Letrozole is a reversible nonsteroidal aromatase inhibitor that is widely used in postmenopausal breast cancer patients. It is well established that letrozole decreases bone density owing to estrogen depletion, however, few studies have reported its direct effect on bone cells in vitro. Therefore, we investigated the effect of letrozole on bone metabolism, focusing on osteoclastogenesis. Letrozole did not affect the viability, proliferation, or migration of bone marrow-derived macrophages (BMMs), however, it reduced the multinucleation of immature osteoclasts and subsequent bone resorption in vitro. Overall, letrozole inhibited the expression of dendritic cell-specific transmembrane protein (DC-STAMP), tartrate-resistant acid phosphatase, calcitonin receptor, and cathepsin K. Among them, the reduced expression of DC-STAMP was the most prominent. However, this downregulation of DC-STAMP expression following letrozole treatment was not related to the inhibition of major osteoclastogenesis pathways, such as the nuclear factor-&kappa, B (NF-&kappa, B), c-Fos, and nuclear factor of activated T cell c1 (NFATc1) pathways, but was attributed to the inhibition of p38, which is known to reside upstream of DC-STAMP expression. Notably, the anti-osteoclastogenic effect of letrozole was abolished following treatment with the p38 activator anisomycin. Contrary to our expectations, these results strongly suggest a previously unknown anti-osteoclastogenic activity of letrozole, mediated by the downregulation of the p38/DC-STAMP pathway.

Circulating angiogenic cells (CACs) have been implicated in the repair of ischemic tissues, and their mobilization from bone marrow is known to be regulated by the activations of chemokine receptors, including CXCR2 and CXCR4. This study was conducted to investigate the role of N-acetylated proline-glycine-proline (Ac-PGP; a collagen-derived chemotactic tripeptide) on CAC mobilization and its therapeutic potential for the treatment of peripheral artery diseases. Ac-PGP was administered daily to a murine hind limb ischemia model, and the effects of Ac-PGP on blood perfusion and CAC mobilization (Sca1+Flk1+ cells) into peripheral blood were assessed. Intramuscular administration of Ac-PGP significantly improved ischemic limb perfusion and increased limb salvage rate by increasing blood vessel formation, whereas Ac-PGP-induced blood perfusion and angiogenesis in ischemic limbs were not observed in CXCR2-knockout mice. In addition, Ac-PGP-induced CAC mobilization was found to occur in wild-type mice but not in CXCR2-knockout mice. Transplantation of bone marrow from green fluorescent protein (GFP) transgenic mice to wild-type mice showed bone marrow-derived cells homed to ischemic limbs after Ac-PGP administration and that GFP-positive cells contributed to the formation of ILB4-positive capillaries and α smooth muscle actin (α-SMA)-positive arteries. These results suggest CXCR2 activation in bone marrow after Ac-PGP administration improves blood perfusion and reduces tissue necrosis by inducing CAC mobilization. These findings suggest a new pharmaceutical basis for the treatment of critical limb ischemia. Stem Cells Translational Medicine 2019;8:236&246

Endothelial progenitor cells (EPCs) promote neovascularization and tissue repair by migrating to vascular injury sites; therefore, factors that enhance EPC homing to damaged tissues are of interest. Here, we provide evidence of the prominent role of the Netrin-4 (NTN4)-Unc-5 Netrin receptor B (UNC5B) axis in EPC-specific promotion of ischemic neovascularization. Our results showed that NTN4 promoted the proliferation, chemotactic migration, and paracrine effects of small EPCs (SEPCs) and significantly increased the incorporation of large EPCs (LEPCs) into tubule networks. Additionally, NTN4 prominently augmented neovascularization in mice with hindlimb ischemia by increasing the homing of exogenously transplanted EPCs to the ischemic limb and incorporating EPCs into vessels. Moreover, silencing of UNC5B, an NTN4 receptor, abrogated the NTN4-induced cellular activities of SEPCs in vitro and blood-flow recovery and neovascularization in vivo in ischemic muscle by reducing EPC homing and incorporation. These findings suggest NTN4 as an EPC-based therapy for treating angiogenesis-dependent diseases.

Increasing evidence suggests that circulating angiogenic cells (CACs) promote repair of ischemic tissues. Activation of formyl peptide receptor 2 (Fpr2) has been reported to stimulate repair of ischemic heart. This study was conducted to investigate the role of Fpr2 on CAC mobilization and cardiac protection in myocardial infarction (MI). WKYMVm, a strong agonist for Fpr2, was administered in a murine model of acute MI, and mobilization of CACs including endothelial progenitor cells (CD34+ Flk1+ or Sca1+ Flk1+ cells) in peripheral blood was monitored. CAC mobilization by daily injection of WKYMVm for the first 4 days after MI was as efficient as granulocyte colony-stimulating factor and provided myocardial protection from apoptosis with increased vascular density and preservation of cardiac function. Transplantation of bone marrow (BM) from green fluorescent protein mice showed that BM-derived cells homed to ischemic heart after WKYMVm treatment and contributed to tissue protection. Transplantation of BM from Fpr2 knockout mice showed that Fpr2 in BM cells is critical in mediation of WKYMVm-stimulated myocardial protection and neovascularization after MI. These results suggest that activation of Fpr2 in BM after WKYMVm treatment provides cardiac protection through mobilization of CACs after MI, which may lead to the development of a new clinical protocol for treating patients with ischemic heart conditions.

Near-infrared (NIR) fluorophores attract increasing attention as a molecular marker (or probe) for in vivo and in vitro biological fluorescence imaging. Three types of new NIR fluorescent conjugated oligoelectrolytes (COEs: Q-FlTBTTFl, Q-FlBBTFl, and Q-FlTBBTTFl) are synthesized with quaternized ammonium ionic groups in their side-chains for water solubility. The emission wavelength is modulated in the range 600-1300 nm, by adjusting the intramolecular charge transfer in the molecular backbone based on the electron-rich fluorene (and/or thiophene) and electron-deficient benzo[2,1,3]thiadiazole (or benzo[1,2-c:4,5-c']bis[1,2,5]thiadiazole) moieties. The COEs show a remarkably larger Stokes shift (147-276 nm) compared to commercial rhodamine and cyanine dyes in water, avoiding self-quenching and interference from the excitation backscattered light. The photoluminescence (PL) quantum efficiency is improved substantially by up to 27.8% in water by fabricating a vesicular complex, COE/v, with a block ionomer, poly[(ethylene oxide)-block-(sodium 2-acrylamido-2-methyl-1-propanesulfonate)]. In vitro cellular uptake images with the COEs are obtained with good biocompatibility by confocal single-photon and two-photon microscopy. The ex vivo and in vivo images of a mouse xenograft model treated with the Q-FlBBTFl/v exhibit a substantially stronger fluorescence signal at the tumor site than at the other organs, highlighting the potential of the COE/v as an NIR fluorescent imaging agent for the diagnosis of cancer.

Objective Human adipose tissue-derived mesenchymal stem cells (ASCs) have been reported to promote angiogenesis and tissue repair. However, poor survival and engraftment efficiency of transplanted ASCs are the major bottlenecks for therapeutic application. The present study aims to improve the therapeutic efficacy of ASCs for peripheral artery diseases. Methods Hydrogen peroxide (H2O2) was used to induce apoptotic cell death in ASCs. To measure apoptosis, we used flow cytometry-based apoptosis analysis and terminal deoxynucleotidyl transferase dUTP nick end labeling staining. A murine hindlimb ischemia model was established to measure the ASC-mediated therapeutic angiogenesis and in vivo survival ability of ASCs. Results We identified that the inhibitor of lamin A-progerin binding, JH4, protects ASCs against H2O2-induced oxidative stress and apoptosis. Co-administration of ASCs with JH4 improved ASC-mediated blood reperfusion recovery and limb salvage compared to that of the control group in a mouse hind limb ischemia model. Immunofluorescence showed that JH4 treatment potentiated ASC-mediated vascular regeneration via reducing ASC apoptosis post transplantation. Conclusion JH4 exerts anti-apoptotic effects in ASCs in conditions of oxidative stress, and contributes to the repair of ischemic hind limb injury by improving cell survival.

Diabetes is one of the most common human diseases and 15% of the 200 million diabetics worldwide suffer from diabetic wounds. Development of new therapeutic agents is needed for treatment of diabetic wounds. Wound healing is mediated by multiple steps, including inflammation, epithelialization, neoangiogenesis, and granulation. Formyl peptide receptor 2 has been known to stimulate angiogenesis, which is essential for tissue repair and cutaneous wound healing. In this study, we explored the therapeutic effects of WKYMVm (Trp-Lys-Tyr-Met-Val-D-Met-NH2), a synthetic peptide agonist of formyl peptide receptor 2, on cutaneous wounds in streptozotocin-induced diabetic rats. Topical application of WKYMVm onto cutaneous wounds stimulated formation of von Willebrand factor-positive capillary and α-smooth muscle actin-positive arteriole with a maximal stimulation on day 6, suggesting WKYMVm-stimulated angiogenesis. Infiltration of immune cells could be detected on early phase during wound healing and WKYMVm treatment acutely augmented infiltration of CD68-positive macrophages. In addition, reepithelialization and granulation tissue formation were accelerated by treatment with WKYMVm. These results suggest that WKYMVm has therapeutic effects on diabetic wounds by stimulating angiogenesis and infiltration of immune cells.

A liquid chromatographic-electrospray ionization-time-of-flight/mass spectrometric (LC-ESI-TOF/MS) method was developed and applied for the determination of WKYMVm peptide in rat plasma to support preclinical pharmacokinetics studies. The method consisted of micro-elution solid phase extraction (SPE) for sample preparation and LC-ESI-TOF/MS in the positive ion mode for analysis. Phenanthroline (10mg/mL) was added to rat blood immediately for plasma preparation followed by addition of trace amount of 2M hydrogen chloride (HCl) to plasma before SPE for the stability of WKYMVm peptide. Then, sample preparation using micro-elution SPE was performed with verapamil as an internal standard. A quadratic regression (weighted 1/concentration2), with an equation y=ax2+bx+c, was used to fit calibration curves over the concentration range of 3.02~2200 ng/mL for WKYMVm peptide. The quantification run met the acceptance criteria of ±25 % accuracy and precision values. For quality control samples at 15, 165, and 1820 ng/mL from the quantification experiment, the within-run and the between-run accuracy ranged from 92.5 to 123.4 % with precision values ≤15.1 % for WKYMVm peptide from the nominal values. This novel LC-ESI-TOF/MS method was successfully applied to evaluate the pharmacokinetics of WKYMVm peptide in rat plasma.

Human endothelial progenitor cells (hEPCs) are promising therapeutic resources for wound repair through stimulating neovascularization. However, the hEPCs-based cell therapy has been hampered by poor engraftment of transplanted cells. In this study, we explored the effects of N-acetylated Proline-Glycine-Proline (Ac-PGP), a degradation product of collagen, on hEPC-mediated cutaneous wound healing and neovascularization. Treatment of hEPCs with Ac-PGP increased migration, proliferation, and tube-forming activity of hEPCs in vitro. Knockdown of CXCR2 expression in hEPCs abrogated the stimulatory effects of Ac-PGP on migration and tube formation. In a cutaneous wound healing model of rats and mice, topical application of Ac-PGP accelerated cutaneous wound healing with promotion of neovascularization. The positive effects of Ac-PGP on wound healing and neovascularization were blocked in CXCR2 knockout mice. In nude mice, the individual application of Ac-PGP treatment or hEPC injection accelerated wound healing by increasing neovascularization. Moreover, the combination of Ac-PGP treatment and hEPC injection further stimulated wound healing and neovascularization. Topical administration of Ac-PGP onto wound bed stimulated migration and engraftment of transplanted hEPCs into cutaneous dermal wounds. Therefore, these results suggest novel applications of Ac-PGP in promoting wound healing and augmenting the therapeutic efficacy of hEPCs.

Ischemia is a serious disease, characterized by an inadequate blood supply to an organ or part of the body. In the present study, we evaluated the effects of the anti-microbial peptide SR-0379 on the stem cell-mediated therapy of ischemic diseases. The migratory and tube-forming abilities of human endothelial progenitor cells (EPCs) were enhanced by treatment with SR-0379 in vitro. Intramuscular administration of SR-0379 into a murine ischemic hindlimb significantly enhanced blood perfusion, decreased tissue necrosis, and increased the number of blood vessels in the ischemic muscle. Moreover, co-administration of SR-0379 with EPCs stimulated blood perfusion in an ischemic hindlimb more than intramuscular injection with either SR-0379 or EPCs alone. This enhanced blood perfusion was accompanied by a significant increase in the number of CD31- and α-SMApositive blood vessels in ischemic hindlimb. These results suggest that SR-0379 is a potential drug candidate for potentiating EPC-mediated therapy of ischemic diseases. [BMB Reports 2017; 50(10): 504-509].

Oct4 has been implicated in regulation of pluripotency in embryonic stem cells (ESCs) and reprogramming of somatic cells into induced pluripotent stem cells. However, the molecular mechanisms involved in Oct4-dependent regulation of pluripotency and reprogramming have not been clear. To gain insight into the mechanism of regulation of Oct4-mediated self-renewal of ESCs and reprogramming of somatic cells, we attempted to identify Oct4-binding proteins using affinity purification and mass spectrometry. We identified Reptin, a key component of ATP-dependent chromatin remodeling complexes, as an Oct4-binding protein. Depletion of endogenous Reptin using lentiviral short hairpin RNA (shRNA) led to a decrease in the number and size of alkaline phosphatase-positive colonies of mouse ESCs. In addition, shRNA-mediated silencing of Reptin resulted in decreased expression of pluripotency-specific marker genes, including Oct4, Sox2, Nanog, and SSEA-1. Results of the Oct4 reporter assay showed synergism between Oct4 and Reptin, and depletion of endogenous Reptin abolished Oct4 transcriptional activity. Results of a chromatin immunoprecipitation assay showed the overlapping interaction of Reptin and Oct4 to CR4 in the Oct4 enhancer in ESCs. Knockdown of Reptin using shRNA suppressed the reprogramming of mouse embryonic fibroblasts to induced pluripotent stem cells, whereas overexpression of Reptin resulted in enhanced efficiency of induced pluripotent stem cell generation. These results strongly suggest that Reptin plays a key role in maintaining the pluripotency of ESCs and in establishing the pluripotency during reprogramming of somatic cells by regulation of Oct4-mediated gene regulation. Stem Cells 2014;32:3126–3136

Endothelial colony-forming cells (ECFCs) are recruited to the sites of ischemic injury in order to contribute to neovascularization and repair of injured tissues. However, therapeutic potential of ECFCs is limited due to low homing and engraftment efficiency of transplanted ECFCs. The G-protein-coupled formyl peptide receptor (FPR) 2 has been implicated in regulation of inflammation and angiogenesis, while the role of FPR2 in homing and engraftment of ECFCs and neovascularization in ischemic tissues has not been fully defined. This study was undertaken to investigate the effects of WKYMVm, a selective FPR2 agonist isolated by screening synthetic peptide libraries, on homing ability of ECFCs and vascular regeneration of ischemic tissues. WKYMVm stimulated chemotactic migration, angiogenesis, and proliferation ability of human ECFCs in vitro. Small interfering RNA-mediated silencing of FPR2, but not FPR3, abrogated WKYMVm-induced migration and angiogenesis of ECFCs. Intramuscular injection of WKYMVm resulted in attenuation of severe hind limb ischemia and promoted neovascularization in ischemic limb. ECFCs transplanted via tail vein into nude mice were incorporated into capillary vessels in the ischemic hind limb, resulting in augmented neovascularization and improved ischemic limb salvage. Intramuscular injection of WKYMVm promoted homing of exogenously administered ECFCs to the ischemic limb and ECFC-mediated vascular regeneration. Silencing of FPR2 expression in ECFCs resulted in abrogation of WKYMVm-induced in vivo homing of exogenously transplanted ECFCs to the ischemic limb, neovascularization, and ischemic limb salvage. These results suggest that WKYMVm promotes repair of ischemic tissues by stimulating homing of ECFCs and neovascularization via a FPR2-dependent mechanism. Stem Cells 2014;32:779–790

Background: Transcriptional co-activator with PDZ-binding motif (TAZ), a downstream effector of the Hippo pathway, has been reported to regulate organ size, tissue homeostasis, and tumorigenesis by acting as a transcriptional co-activator. Lysophosphatidic acid (LPA) is a bioactive lipid implicated in tumorigenesis and metastasis of ovarian cancer through activation of G protein-coupled receptors. However, the involvement of TAZ in LPA-induced tumorigenesis of ovarian cancer has not been elucidated. Methods: In order to demonstrate the role of TAZ in LPA-stimulated tumorigenesis, the effects of LPA on TAZ expression and cell migration were determined by Western blotting and chemotaxis analyses in R182 human epithelial ovarian cancer cells. Results and Conclusion: Treatment of R182 cells with the LPA receptor inhibitor Ki16425 blocked LPA-induced cell migration. In addition, transfection of R182 cells with small interfering RNA specific for LPA receptor 1 resulted in abrogation of LPA-stimulated cell migration. LPA induced phosphorylation of ERK and p38 MAP kinase in R182 cells and pretreatment of cells with the MEK-ERK pathway inhibitor U0126, but not the p38 MAPK inhibitor SB202190, resulted in abrogation of LPA-induced cell migration. Pretreatment of R182 cells with U0126 attenuated LPA-induced mRNA levels of TAZ and its transcriptional target genes, such as CTGF and CYR61, without affecting phosphorylation level of YAP. These results suggest that MEK-ERK pathway plays a key role in LPA-induced cell migration and mRNA expression of TAZ in R182 cells, without affecting stability of TAZ protein. In addition, small interfering RNA-mediated silencing of TAZ expression attenuated LPA-stimulated migration of R182 cells. These results suggest that TAZ plays a key role in LPA-stimulated migration of epithelial ovarian cancer cells. Copyright (c) 2013 S. Karger AG, Basel.

Objective— Mesenchymal stem cells are useful for vascular regeneration of injured tissues. Macrophages are involved in acute or chronic inflammatory diseases, and interleukin-1β (IL-1β), a proinflammatory cytokine, plays a key role in the activation of macrophages within injured tissues. To explore the role of macrophages on mesenchymal stem cell–mediated vascular regeneration, we examined the effects of IL-1β–activated macrophages on differentiation of human adipose tissue–derived mesenchymal stem cells (hASCs) to smooth muscle cells (SMCs) and the vascular regenerative capacity of the differentiated SMCs in a hindlimb ischemia animal model. Methods and Results— We demonstrate that IL-1β–conditioned medium from RAW 264.7 macrophages induces differentiation of human adipose tissue–derived mesenchymal stem cells to α-smooth muscle actin–positive SMCs, and the differentiated SMCs exhibited increased contractility in response to KCl and carbachol treatment. Transplantation of the differentiated SMCs attenuated severe hindlimb ischemia and promoted vascular regeneration. IL-1β treatment stimulated secretion of prostaglandin F 2α from RAW 264.7 cells. Small interfering RNA–mediated silencing of the prostaglandin F 2α receptor completely abrogated IL-1β conditioned medium–stimulated α-smooth muscle actin expression. Moreover, prostaglandin F 2α treatment stimulated expression of α-smooth muscle actin in human adipose tissue–derived mesenchymal stem cells. Conclusion— These results suggest that IL-1β–activated macrophages promote differentiation of human adipose tissue–derived mesenchymal stem cells to SMCs through a prostaglandin F 2α –mediated paracrine mechanism.

Mesenchymal stem cells stimulate tumor growth in vivo through a lysophosphatidic acid (LPA)-dependent mechanism. However, the molecular mechanism by which mesenchymal stem cells stimulate tumorigenesis is largely elusive. In the present study, we demonstrate that conditioned medium from A549 human lung adenocarcinoma cells (A549 CM) induces expression of periostin, an extracellular matrix protein, in human adipose tissue-derived mesenchymal stem cells (hASCs). A549 CM-stimulated periostin expression was abrogated by pretreatment of hASCs with the LPA receptor 1 (LPA1) inhibitor Ki16425 or short hairpin RNA-mediated silencing of LPA1, suggesting a key role of the LPA–LPA1 signaling axis in A549 CM-stimulated periostin expression. Using a xenograft transplantation model of A549 cells, we demonstrated that co-injection of hASCs potentiated tumor growth of A549 cells in vivo and that co-transplanted hASCs expressed not only periostin but also α-smooth muscle actin (α-SMA), a marker of carcinoma-associated fibroblasts. Small interfering RNA- or short hairpin RNA-mediated silencing of periostin resulted in blockade of LPA-induced α-SMA expression in hASCs. In addition, silencing of periostin resulted in blockade of hASC-stimulated growth of A549 xenograft tumors and in vivo differentiation of transplanted hASCs to α-SMA-positive carcinoma-associated fibroblasts. Conditioned medium derived from LPA-treated hASCs (LPA CM) potentiated proliferation and adhesion of A549 cells and short interfering RNA-mediated silencing or immunodepletion of periostin from LPA CM abrogated proliferation and adhesion of A549 cells. These results suggest a pivotal role for hASC-secreted periostin in growth of A549 xenograft tumors within the tumor microenvironment.

Endothelial progenitor cells (EPCs) can be isolated from human bone marrow or peripheral blood and reportedly contribute to neovascularization. Aptamers are 40-120-mer nucleotides that bind to a specific target molecule, as antibodies do. To utilize apatmers for isolation of EPCs, in the present study, we successfully generated aptamers that recognize human CD31, an endothelial cell marker. CD31 aptamers bound to human umbilical cord blood-derived EPCs and showed specific interaction with human CD31, but not with mouse CD31. However, CD31 aptamers showed non-specific interaction with CD31-negative 293FT cells and addition of polyanionic competitor dextran sulfate eliminated non-specific interaction without affecting cell viability. From the mixture of EPCs and 293FT cells, CD31 aptamers successfully isolated EPCs with 97.6% purity and 94.2% yield, comparable to those from antibody isolation. In addition, isolated EPCs were decoupled from CD31 aptamers with a brief treatment of high concentration dextran sulfate. EPCs isolated with CD31 aptamers and subsequently decoupled from CD31 aptamers were functional and enhanced the restoration of blood flow when transplanted into a murine hindlimb ischemia model. In this study, we demonstrated isolation of foreign material-free EPCs, which can be utilized as a universal protocol in preparation of cells for therapeutic transplantation.

Diabetes is one of the most common human diseases and 15% of the 200 million diabetics worldwide suffer from diabetic wounds. Development of new therapeutic agents is needed for treatment of diabetic wounds. Wound healing is mediated by multiple steps, including inflammation, epithelialization, neoangiogenesis, and granulation. Formyl peptide receptor 2 has been known to stimulate angiogenesis, which is essential for tissue repair and cutaneous wound healing. In this study, we explored the therapeutic effects of WKYMVm (Trp-Lys-Tyr-Met-Val-D-Met-NH2), a synthetic peptide agonist of formyl peptide receptor 2, on cutaneous wounds in streptozotocin-induced diabetic rats. Topical application of WKYMVm onto cutaneous wounds stimulated formation of von Willebrand factor-positive capillary and α-smooth muscle actin-positive arteriole with a maximal stimulation on day 6, suggesting WKYMVm-stimulated angiogenesis. Infiltration of immune cells could be detected on early phase during wound healing and WKYMVm treatment acutely augmented infiltration of CD68-positive macrophages. In addition, reepithelialization and granulation tissue formation were accelerated by treatment with WKYMVm. These results suggest that WKYMVm has therapeutic effects on diabetic wounds by stimulating angiogenesis and infiltration of immune cells.

Embryonic stem (ES) cells are pluripotent cells characterized by self-renewability and differentiation potential. Induced pluripotent stem (iPS) cells are ES cell-equivalent cells derived from somatic cells by the introduction of core reprogramming factors. ES and iPS cells are important sources for understanding basic biology and for generating therapeutic cells for clinical applications. Tribbles homolog 2 (Trib2) functions as a scaffold in signaling pathways. However, the relevance of Trib2 to the pluripotency of ES and iPS cells is unknown. In the present study, we elucidated the importance of Trib2 in maintaining pluripotency in mouse ES cells and in generating iPS cells from somatic cells through the reprogramming process. Trib2 expression decreased as ES cells differentiated, and Trib2 knockdown in ES cells changed their colony morphology while reducing the activity of alkaline phosphatase and the expression of the pluripotency marker genes Oct4, Sox2, Nanog and Klf4. Trib2 directly interacted with Oct4 and elevated Oct4 promoter activity. During the generation of iPS cells, Trib2 knockdown decreased the reprogramming efficiency of mouse embryonic fibroblasts, whereas Trib2 overexpression significantly increased their reprogramming efficiency. In summary, our results suggest that Trib2 is important for maintaining self-renewal in ES cells and for pluripotency induction during the reprogramming process.

Endothelial progenitor cells (EPCs) hold a great promise as a therapeutic mediator in treatment of ischemic disease conditions. The discovery of EPCs in adult blood has been a cause of significant enthusiasm in the field of endothelial cell research and numerous clinical trials have been expedited. After more than a decade of research in basic science and clinical applications, limitations and new strategies of EPC therapeutics have emerged. With various phenotypes, vague definitions, and uncertain distinction from hematopoietic cells, understanding EPC biology remains challenging. However, EPCs, still hold great hope for treatment of critical ischemic injury as low concern regarding safety can accelerate the clinical applications from basic findings. This review provides an introduction to EPC as cellular therapeutics, which highlights a recent finding that EPC homing was promoted through FPR2 signaling.

Prostate cancer is the most frequently diagnosed malignancy and the second leading cause of cancer mortality among men in the United States. Accumulating evidence suggests that lysophosphatidic acid (LPA) serves as an autocrine/paracrine mediator to affect initiation, progression and metastasis of prostate cancer. In the current study, we demonstrate that LPA stimulates migration and proliferation of highly metastatic human prostate cancer, PC-3M-luc-C6 cells. LPA-induced migration of PC-3M-luc-C6 cells was abrogated by pretreatment of PC-3M-luc-C6 cells with the LPA receptor 1/3 inhibitor Ki16425 or small interfering RNA (siRNA)-mediated silencing of endogenous LPA receptor 1, implicating a key role of the LPA-LPA receptor 1 signaling axis in migration of PC-3M-luc-C6 cells. In addition, LPA treatment resulted in augmented expression levels of Kruppel-like factor 4 (KLF4), and siRNA or short-hairpin RNA (shRNA)-mediated silencing of KLF4 expression resulted in the abolishment of LPA-stimulated migration and proliferation of PC-3M-luc-C6 cells. shRNA-mediated silencing of KLF4 expression resulted in the inhibition of in vivo growth of PC-3M-luc-C6 cells in a xenograft transplantation animal model. Taken together, these results suggest a key role of LPA-induced KLF4 expression in cell migration and proliferation of prostate cancer cells in vitro and in vivo.

Periostin, an extracellular matrix protein, is expressed in injured tissues, such as the heart with myocardial infarction, and promotes angiogenesis and tissue repair. However, the molecular mechanism associated with periostin-stimulated angiogenesis and tissue repair is still unclear. In order to clarify the role of periostin in neovascularization, we examined the effect of periostin in angiogenic potentials of human endothelial colony forming cells (ECFCs) in vitro and in an ischemic limb animal model. Recombinant periostin protein stimulated the migration and tube formation of ECFCs. To identify the functional domains of periostin implicated in angiogenesis, five fragments of periostin, including four repeating FAS-1 domains and a carboxyl terminal domain, were expressed in Escherichia coli and purified to homogeneity. Of the five different domains, the first FAS-1 domain stimulated the migration and tube formation of human ECFCs as potent as the whole periostin. Chemotactic migration of ECFCs induced by the full length and the first FAS-1 domain of periostin was abrogated by blocking antibodies against β3 and β5 integrins. Intramuscular injection of the full length and the first FAS-1 domain of periostin into the ischemic hindlimb of mice attenuated severe limb loss and promoted blood perfusion and homing of intravenously administered ECFCs to the ischemic limb. These results suggest that the first FAS-1 domain is responsible for periostin-induced migration and angiogenesis and it can be used as a therapeutic tool for treatment of peripheral artery occlusive disease by stimulating homing of ECFCs.

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-β1 (TGF-β1)-induced differentiation of hASCs, providing insights into the differentiation of vascular smooth muscle cells. The treatment of hASCs with TGF-β1 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 Ca2+, big-conductance Ca2+-activated K+ (BKCa), and voltage-dependent K+ (Kv) currents in TGF-β1-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 Cavβ1 and Cavβ3 subtypes in TGF-β1-induced, differentiated hASCs. Although the expression-level changes of the β-subtype BKCa channels varied, the major α-subtype BKCa channel (KCa1.1) clearly increased in the TGF-β1-induced, differentiated hASCs. Most of the Kv subtypes, also known to be expressed in vascular SMCs, dramatically increased in the TGF-β1-induced, differentiated hASCs. Our results suggest that TGF-β1 induces the increased expression of vascular SMC-like ion channels and the differentiation of hASCs into contractile vascular SMCs.

Human dental pulp cells (hDPCs) are a valuable source for the generation of patient-specific human induced pluripotent stem cells (hiPSCs). An advanced strategy for the safe and efficient reprogramming of hDPCs and subsequent lineage-specific differentiation is a critical step toward clinical application. In present research, we successfully generated hDPC-iPSCs using only two non-oncogenic factors: Oct4 and Sox2 (2F hDPC-hiPSCs) and evaluated the feasibility of hDPC-iPSCs as substrates for endothelial progenitor cells (EPCs), contributing to EPC-based therapies. Under conventional differentiation conditions, 2F hDPC-hiPSCs showed higher differentiation efficiency, compared to hiPSCs from other cell types, into multipotent CD34+ EPCs (2F-hEPCs) capable to differentiate into functional endothelial and smooth muscle cells. The angiogenic and neovasculogenic activities of 2F-hEPCs were confirmed using a Matrigel plug assay in mice. In addition, the therapeutic effects of 2F-hEPC transplantation were confirmed in mouse models of hind-limb ischemia and myocardial infarction. Importantly, 2F-EPCs effectively integrated into newly formed vascular structures and enhanced neovascularization via likely both direct and indirect paracrine mechanisms. 2F hDPC-hiPSCs have a robust capability for the generation of angiogenic and vasculogenic EPCs, representing a strategy for patient-specific EPC therapies and disease modeling, particularly for ischemic vascular diseases.

Oncostatin M, a member of the interleukin-6 family of cytokines, has been implicated in tumorigenesis of human prostate cancer. In the current study, we demonstrate that oncostatin M promotes human adipose tissue-derived mesenchymal stem cell-stimulated tumor growth in an in vivo xenograft transplantation model of the human prostate cancer cell line PC-3M-luc-C6, a PC3M cell line expressing the luciferase gene. Conditioned medium derived from oncostatin M-treated mesenchymal stem cells stimulated adhesion of PC-3M-luc-C6 cells. We identified TGFBI and periostin, extracellular matrix proteins implicated in tumorigenesis and metastasis, as oncostatin M-induced secreted proteins in mesenchymal stem cells. Treatment with oncostatin M stimulated secretion of periostin and TGFBI from mesenchymal stem cells in a time-dependent manner. Immunodepletion of TGFBI and periostin from conditioned medium derived from oncostatin M-treated mesenchymal stem cells resulted in abrogation of adhesion of PC-3M-luc-C6 cells stimulated by oncostatin M-conditioned medium. In addition, small interfering RNA-mediated silencing of TGFBI and periostin resulted in abrogation of cell adhesion stimulated by oncostatin M-conditioned medium. These results suggest that mesenchymal stem cell-derived TGFBI and periostin play a key role in tumorigenesis by stimulating adhesion of prostate cancer cells.

Reprogramming of somatic cells to pluripotent cells requires the introduction of factors driving fate switches. Viral delivery has been the most efficient method for generation of induced pluripotent stem cells. Transfection, which precedes virus production, is a commonly-used process for delivery of nucleic acids into cells. The aim of this study is to evaluate the efficiency of PLGA/ bPEI nanoparticles in transfection and virus production. Using a modified method of producing PLGA nanoparticles, PLGA/bPEI-DNA nanoparticles were examined for transfection efficiency and virus production yield in comparison with PLGA-DNA, bPEI-DNA nanoparticles or liposome-DNA complexes. After testing various ratios of PLGA, bPEI, and DNA, the ratio of 6: 3: 1 (PLGA: bPEI: DNA, w/w/w) was determined to be optimal, with acceptable cellular toxicity. PLGA/bPEI-DNA (6: 3: 1) nanoparticles showed superior transfection efficiency, especially in multiple gene transfection, and viral yield when compared with liposome-DNA complexes. The culture supernatants of HEK293FT cells transfected with PLGA/bPEI-DNA of viral constructs containing reprogramming factors (Oct4, Sox2, Klf4, or c-Myc) successfully and more efficiently generated induced pluripotent stem cell colonies from mouse embryonic fibroblasts. These results strongly suggest that PLGA/bPEI-DNA nanoparticles can provide significant advantages in studying the effect of multiple factor delivery such as in reprogramming or direct conversion of cell fate.

Mesenchymal stem cells are useful for vascular regeneration of injured tissues. Macrophages are involved in acute or chronic inflammatory diseases, and interleukin-1β (IL-1β), a proinflammatory cytokine, plays a key role in the activation of macrophages within injured tissues. To explore the role of macrophages on mesenchymal stem cell-mediated vascular regeneration, we examined the effects of IL-1β-activated macrophages on differentiation of human adipose tissue-derived mesenchymal stem cells (hASCs) to smooth muscle cells (SMCs) and the vascular regenerative capacity of the differentiated SMCs in a hindlimb ischemia animal model.We demonstrate that IL-1β-conditioned medium from RAW 264.7 macrophages induces differentiation of human adipose tissue-derived mesenchymal stem cells to α-smooth muscle actin-positive SMCs, and the differentiated SMCs exhibited increased contractility in response to KCl and carbachol treatment. Transplantation of the differentiated SMCs attenuated severe hindlimb ischemia and promoted vascular regeneration. IL-1β treatment stimulated secretion of prostaglandin F(2α) from RAW 264.7 cells. Small interfering RNA-mediated silencing of the prostaglandin F(2α) receptor completely abrogated IL-1β conditioned medium-stimulated α-smooth muscle actin expression. Moreover, prostaglandin F(2α) treatment stimulated expression of α-smooth muscle actin in human adipose tissue-derived mesenchymal stem cells.These results suggest that IL-1β-activated macrophages promote differentiation of human adipose tissue-derived mesenchymal stem cells to SMCs through a prostaglandin F(2α)-mediated paracrine mechanism.

Background: Transforming growth factor-beta 1 (TGF-beta 1) induces the differentiation of human adipose tissue-derived mesenchymal stem cells (hASCs) into smooth muscle cells. Lipid rafts are cholesterol-rich microdomains in cell membranes that reportedly play a key role in receptor-mediated signal transduction and cellular responses. In order to clarify whether lipid rafts are involved in TGF-beta 1-induced differentiation of hASCs into smooth muscle cells, we analyzed the lipid raft proteome of hASCs. Methods and Results: Pretreatment of hASCs with the lipid raft disruptor methyl-beta-cyclodextrin abrogated TGF-beta 1-induced expression of alpha-smooth muscle actin, a smooth muscle cell marker, suggesting a pivotal role of lipid rafts in TGF-beta 1-induced differentiation of hASCs to smooth muscle cells. Sucrose density gradient centrifugation along with a shotgun proteomic strategy using liquid chromatography-tandem mass spectrometry identified 1002 individual proteins as the lipid raft proteome, and 242 of these were induced by TGF-beta 1 treatment. ADAM12, a disintegrin and metalloproteases family member, was identified as the most highly up-regulated protein in response to TGF-beta 1 treatment. TGF-beta 1 treatment of hASCs stimulated the production of both ADAM12 protein and mRNA. Silencing of endogenous ADAM12 expression using lentiviral small hairpin RNA or small interfering RNA abrogated the TGF-beta 1-induced differentiation of hASCs into smooth muscle cells. Conclusions: These results suggest a pivotal role for lipid raft-associated ADAM12 in the TGF-beta 1-induced differentiation of hASCs into smooth muscle cells.

Lysophosphatidic acid (LPA) stimulates growth and invasion of ovarian cancer cells and tumor angiogenesis. Cancer-derived LPA induces differentiation of human adipose tissue-derived mesenchymal stem cells (hASCs) to alpha-smooth muscle actin (alpha-SMA)-positive cancer-associated fibroblasts. Presently, we explored whether cancer-derived LPA regulates secretion of pro-angiogenic factors from hASCs. Conditioned medium (CM) from the OVCAR-3 and SKOV3 ovarian cancer cell lines stimulated secretion angiogenic factors such as stromal-derived factor-1 alpha (SDF-1 alpha) and VEGF from hASCs. Pretreatment with the LPA receptor inhibitor Ki16425 or short hairpin RNA lentiviral silencing of the LPA((1)) receptor abrogated the cancer CM-stimulated expression of alpha-SMA, SDF-1, and VEGF from hASCs. LPA induced expression of myocardin and myocardin-related transcription factor-A, transcription factors involved in smooth muscle differentiation, in hASCs. siRNA-mediated depletion of endogenous myocardin and MRTF-A abrogated the expression of alpha-SMA, but not SDF-1 and VEGF. LPA activated RhoA in hASCs and pretreatment with the Rho kinase inhibitor Y27632 completely abrogated the LPA-induced expression of alpha-SMA, SDF-1, and VEGF in hASCs. Moreover, LPA-induced alpha-SMA expression was abrogated by treatment with the ERK inhibitor U0126 or the phosphoinositide-3-kinase inhibitor LY294002, but not the PLC inhibitor U73122. LPA-induced VEGF secretion was inhibited by LY294002, whereas LPA-induced SDF-1 secretion was markedly attenuated by U0126, U73122, and LY294002. These results suggest that cancer-secreted LPA induces differentiation of hASCs to cancer-associated fibroblasts through multiple signaling pathways involving Rho kinase, ERK, PLC, and phosphoinositide-3-kinase.

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 (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.

Background Periostin, an extracellular matrix protein, is expressed in bone, more specifically, the periosteum and periodontal ligaments, and plays a key role in formation and metabolism of bone tissues. Human adipose tissue-derived mesenchymal stem cells (hASCs) have been reported to differentiate into osteoblasts and stimulate bone repair. However, the role of periostin in hASC-mediated bone healing has not been clarified. In the current study, we examined the effect of periostin on bone healing capacity of hASCs in a critical size calvarial defect model. Methods and Results Recombinant periostin protein stimulated migration, adhesion, and proliferation of hASCs in vitro. Implantation of either hASCs or periostin resulted in slight, but not significant, stimulation of bone healing, whereas co-implantation of hASCs together with periostin further potentiated bone healing. In addition, the number of Ki67-positive proliferating cells was significantly increased in calvarial defects by co-implantation of both hASCs and periostin. Consistently, proliferation of administered hASCs was stimulated by co-implantation with periostin in vivo. In addition, co-delivery of hASCs with periostin resulted in markedly increased numbers of CD31-positive endothelial cells and α-SMA-positive arterioles in calvarial defects. Conclusions These results suggest that recombinant periostin potentiates hASC-mediated bone healing by stimulating proliferation of transplanted hASCs and angiogenesis in calvarial defects.