NRP1 antagonism as a novel therapeutic target in nasal polyps of patients with chronic rhinosinusitis.

BACKGROUND: Neuropilin-1 (NRP1) is expressed on the surface epithelium of respiratory tract and immune cells, demonstrating its possible function in regulating the immune response in airway disease. However, its role in patient with chronic rhinosinusitis (CRS) remains unknown. This study aimed to elucidate the role of NRP1 in CRS with nasal polyps (CRSwNP). METHODS: Sinonasal biopsy specimens were immunohistochemically stained to investigate NRP1 expression. Double immunofluorescence, immunoblotting, and real-time polymerase chain reaction were performed to evaluate NRP1 in primary human nasal epithelial cells (hNECs). An NRP1 inhibitor was administered to a murine nasal polyp (NP) model. RESULTS: NRP1 was highly expressed in the epithelium in patients with CRSwNP compared to nasal tissue from controls and CRS without NP patients. NRP1 and vascular endothelial growth factor were upregulated in hNECs under hypoxia. Treatment with NRP1 inhibitor (EG00229) reduced the secretion of interleukin (IL)-1ß, IL-6, IL-8, and IL-33 cytokines, as well as inducible nitric oxide synthase, cyclooxygenase-2, and prostaglandin E2 in hNECs. We found that NRP1 was highly expressed in the airway epithelium in the murine NP model. The group treated with the NRP1 inhibitor had significantly fewer nasal polypoid lesions and reduced accumulations of immune cells. CONCLUSIONS: These findings reveal that NRP1 is upregulated in CRS and NP epithelium, and the inhibition of NRP1 may lead to a reduction in NP growth and immune cell infiltration. Our results suggest that NRP1 inhibition could be a novel possibility for treating nasal polyposis.

Ganglioside GD3 Regulates Inflammation and Epithelial-to-Mesenchymal Transition in Human Nasal Epithelial Cells.

Chronic sinusitis with nasal polyps (CRSwNP) is one of the most common chronic inflammatory diseases, and involves tissue remodeling. One of the key mechanisms of tissue remodeling is the epithelial-mesenchymal transition (EMT), which also represents one of the pathophysiological processes of CRS observed in CRSwNP tissues. To date, many transcription factors and forms of extracellular stimulation have been found to regulate the EMT process. However, it is not known whether gangliosides, which are the central molecules of plasma membranes, involved in regulating signal transmission pathways, are involved in the EMT process. Therefore, we aimed to determine the role of gangliosides in the EMT process. First, we confirmed that N-cadherin, which is a known mesenchymal marker, and ganglioside GD3 were specifically expressed in CRSwNP_NP tissues. Subsequently, we investigated whether the administration of TNF-α to human nasal epithelial cells (hNECs) resulted in the upregulation of ganglioside GD3 and its synthesizing enzyme, ST8 alpha-N-acetyl-neuraminide alpha-2,8-sialytransferase 1 (ST8Sia1), and the consequently promoted inflammatory processes. Additionally, the expression of N-cadherin, Zinc finger protein SNAI2 (SLUG), and matrix metallopeptidase 9 (MMP-9) were elevated, but that of E-cadherin, which is known to be epithelial, was reduced. Moreover, the inhibition of ganglioside GD3 expression by the siRNA or exogenous treatment of neuraminidase 3 (NEU 3) led to the suppression of inflammation and EMT. These results suggest that gangliosides may play an important role in prevention and therapy for inflammation and EMT.

Quercetin Induces Mitochondrial Apoptosis and Downregulates Ganglioside GD3 Expression in Melanoma Cells.

Malignant melanoma represents a form of skin cancer characterized by a bleak prognosis and heightened resistance to traditional therapies. Quercetin has demonstrated notable anti-carcinogenic, anti-inflammatory, anti-oxidant, and pharmacological effects across various cancer types. However, the intricate relationship between quercetin's anti-cancer properties and ganglioside expression in melanoma remains incompletely understood. In this study, quercetin manifests specific anti-proliferative, anti-migratory, and cell-cycle arrest effects, inducing mitochondrial dysfunction and apoptosis in two melanoma cancer cell lines. This positions quercetin as a promising candidate for treating malignant melanoma. Moreover, our investigation indicates that quercetin significantly reduces the expression levels of ganglioside GD3 and its synthetic enzyme. Notably, this reduction is achieved through the inhibition of the FAK/paxillin/Akt signaling pathway, which plays a crucial role in cancer development. Taken together, our findings suggest that quercetin may be a potent anti-cancer drug candidate for the treatment of malignant melanoma.

In vitro modeling of liver fibrosis with 3D co-culture system using a novel human hepatic stellate cell line.

Hepatic stellate cells (HSCs) play an important role in liver fibrosis; however, owing to the heterogeneity and limited supply of primary HSCs, the development of in vitro liver fibrosis models has been impeded. In this study, we established and characterized a novel human HSC line (LSC-1), and applied it to various types of three-dimensional (3D) co-culture systems with differentiated HepaRG cells. Furthermore, we compared LSC-1 with a commercially available HSC line on conventional monolayer culture. LSC-1 exhibited an overall upregulation of the expression of fibrogenic genes along with increased levels of matrix and adhesion proteins, suggesting a myofibroblast-like or transdifferentiated state. However, activated states reverted to a quiescent-like phenotype when cultured in different 3D culture formats with a relatively soft microenvironment. Additionally, LSC-1 exerted an overall positive effect on co-cultured differentiated HepaRG, which significantly increased hepatic functionality upon long-term cultivation compared with that achieved with other HSC line. In 3D spheroid culture, LSC-1 exhibited enhanced responsiveness to transforming growth factor beta 1 exposure that is caused by a different matrix-related protein expression mechanism. Therefore, the LSC-1 line developed in this study provides a reliable candidate model that can be used to address unmet needs, such as development of antifibrotic therapies.

The Role of CPNE7 (Copine-7) in Colorectal Cancer Prognosis and Metastasis.

Colorectal cancer (CRC) is one of the most common and deadly cancers in the world. However, no effective treatment for the disease has yet been found. For this reason, several studies are being carried out on the treatment of CRC. Currently, there is limited understanding of the role of CPNE7 (copine-7) in CRC progression and metastasis. The results of this study show that CPNE7 exerts an oncogenic effect in CRC. First, CPNE7 was shown to be significantly up-regulated in CRC patient tissues and CRC cell lines compared to normal tissues according to IHC staining, qRT-PCR, and western blotting. Next, this study used both systems of siRNA and shRNA to suppress CPNE7 gene expression to check the CPNE7 mechanism in CRC. The suppressed CPNE7 significantly inhibited the growth of CRC cells in in vitro experiments, including migration, invasion, and semisolid agar colony-forming assay. Moreover, the modified expression of CPNE7 led to a decrease in the levels of genes associated with epithelial-mesenchymal transition (EMT). The epithelial genes E-cadherin (CDH1) and Collagen A1 were upregulated, and the levels of mesenchymal genes such as N-cadherin (CDH2), ZEB1, ZEB2, and SNAIL (SNAL1) were downregulated after CPNE7 inhibition. This study suggests that CPNE7 may serve as a potential diagnostic biomarker for CRC patients.

Circulating Exosomal miR-1290 for Diagnosis of Epithelial Ovarian Cancer.

The aim of the study was to develop a new diagnostic biomarker for identifying serum exosomal miRNAs specific to epithelial ovarian cancer (EOC) and to find out target gene of the miRNA for exploring the molecular mechanisms in EOC. A total of 84 cases of ovarian masses and sera were enrolled, comprising EOC (n = 71), benign ovarian neoplasms (n = 13). We detected expression of candidate miRNAs in the serum and tissue of both benign ovarian neoplasm group and EOC group using real-time polymerase chain reaction. Immunohistochemistry were constructed using formalin fixed paraffin embedded (FFPE) tissue to detect expression level of suppressor of cytokine signaling 4 (SOCS4). In the EOC group, miRNA-1290 was significantly overexpressed in serum exosomes and tissues as compared to benign ovarian neoplasm group (fold change ≥ 2, p < 0.05). We observed area under the receiver operating characteristic curve (AUC) for miR-1290, using a cut-off of 0.73, the exosomal miR-1290 from serum had AUC, sensitivity, and specificity values of 0.794, 69.2 and 87.3, respectively. In immunohistochemical study, expression of SOCS4 in EOC was lower than that in benign ovarian neoplasm. Serum exosomal miR-1290 could be considered as a biomarker for differential diagnosis of EOC from benign ovarian neoplasm and SOCS4 might be potential target gene of miR-1290 in EOC.

Ganglioside GM3 Up-Regulate Chondrogenic Differentiation by Transform Growth Factor Receptors.

Mesenchymal stem cells, also known as multipotent stromal progenitor cells, can differentiate into cells of mesodermal lineage. Gangliosides are sialic acid-conjugated glycosphingolipids that are believed to regulate cell differentiation and several signaling molecules. These molecules are localized in glycosphingolipid-enriched microdomains on the cell surface and are regulated by glycosphingolipid composition. Transforming growth factor-beta (TGF-ß) signaling plays a critical role in chondrogenic differentiation. However, the role of gangliosides in chondrogenesis is not understood. In this study, the relationship between the ganglioside GM3 and TGF-ß activation, during chondrogenic differentiation, was investigated using an aggregate culture of human synovial membrane-derived mesenchymal stem cells. We showed that the gangliosides GM3 and GD3 were expressed after the chondrogenic differentiation of hSMSC aggregates. To test whether GM3 affected the chondrogenic differentiation of hSMSC aggregates, we used GM3 treatment during chondrogenic differentiation. The results showed that the group treated with 5 µM GM3 had higher expression of chondrogenic specific markers, increased toluidine blue, and safranin O staining, and increased accumulation of glycosaminoglycans compared with the untreated group. Furthermore, GM3 treatment enhanced TGF-ß signaling via SMAD 2/3 during the chondrogenic differentiation of hSMSC aggregates. Taken together, our results suggested that GM3 may be useful in developing therapeutic agents for cell-based articular cartilage regeneration in articular cartilage disease.

Application of Mesenchymal Stem Cells in Inflammatory and Fibrotic Diseases.

Mesenchymal stem cells (MSCs) are multipotent stem cells that can be isolated from various tissues in the adult body. MSCs should be characterized by three criteria for regenerative medicine. MSCs must (1) adhere to plastic surfaces, (2) express specific surface antigens, and (3) differentiate into mesodermal lineages, including chondrocytes, osteoblasts, and adipocytes, in vitro. Interestingly, MSCs have immunomodulatory features and secrete trophic factors and immune receptors that regulate the microenvironment in host tissue. These specific and unique therapeutic properties make MSCs ideal as therapeutic agents in vivo. Specifically, pre-clinical and clinical investigators generated inflammatory and fibrotic diseases models, and then transplantation of MSCs into diseases models for therapeutic effects investigation. In this review, we characterize MSCs from various tissues and describe their applications for treating various inflammation and fibrotic diseases.

Generation of expandable human pluripotent stem cell-derived hepatocyte-like liver organoids.

BACKGROUND & AIMS: The development of hepatic models capable of long-term expansion with competent liver functionality is technically challenging in a personalized setting. Stem cell-based organoid technologies can provide an alternative source of patient-derived primary hepatocytes. However, self-renewing and functionally competent human pluripotent stem cell (PSC)-derived hepatic organoids have not been developed. METHODS: We developed a novel method to efficiently and reproducibly generate functionally mature human hepatic organoids derived from PSCs, including human embryonic stem cells and induced PSCs. The maturity of the organoids was validated by a detailed transcriptome analysis and functional performance assays. The organoids were applied to screening platforms for the prediction of toxicity and the evaluation of drugs that target hepatic steatosis through real-time monitoring of cellular bioenergetics and high-content analyses. RESULTS: Our organoids were morphologically indistinguishable from adult liver tissue-derived epithelial organoids and exhibited self-renewal. With further maturation, their molecular features approximated those of liver tissue, although these features were lacking in 2D differentiated hepatocytes. Our organoids preserved mature liver properties, including serum protein production, drug metabolism and detoxifying functions, active mitochondrial bioenergetics, and regenerative and inflammatory responses. The organoids exhibited significant toxic responses to clinically relevant concentrations of drugs that had been withdrawn from the market due to hepatotoxicity and recapitulated human disease phenotypes such as hepatic steatosis. CONCLUSIONS: Our organoids exhibit self-renewal (expandable and further able to differentiate) while maintaining their mature hepatic characteristics over long-term culture. These organoids may provide a versatile and valuable platform for physiologically and pathologically relevant hepatic models in the context of personalized medicine. LAY SUMMARY: A functionally mature, human cell-based liver model exhibiting human responses in toxicity prediction and drug evaluation is urgently needed for pre-clinical drug development. Here, we develop a novel human pluripotent stem cell-derived hepatocyte-like liver organoid that is critically advanced in terms of its generation method, functional performance, and application technologies. Our organoids can contribute to the better understanding of liver development and regeneration, and provide insights for metabolic studies and disease modeling, as well as toxicity assessments and drug screening for personalized medicine.

A Multicenter Study Investigating Empathy and Burnout Characteristics in Medical Residents with Various Specialties.

We assessed empathy in medical residents, including factors modifying empathy and the relationship between empathy and burnout. Participants (n = 317 residents, response rate = 42%) from 4 university hospitals completed a socio-demographic questionnaire, the Jefferson Scale of Empathy (Health Professional version, Korean edition), and the Maslach Burnout Inventory (MBI). Participants were classified by medical specialty: "people-oriented specialty" (POS group) or "technology-oriented specialty" (TOS group), with more women in the POS than in the TOS group, χ(2) = 14.12, P < 0.001. Being female, married, and having children were factors related to higher empathy (gender, t = -2.129, P = 0.034; marriage, t = -2.078, P = 0.038; children, t = 2.86, P = 0.005). Within specialty group, POS residents showed higher empathy scores in the fourth as compared to the first year, F = 3.166, P = 0.026. Comparing POS and TOS groups by year, fourth year POS residents had significantly higher scores than did fourth year TOS residents, t = 3.349, P = 0.002. There were negative correlations between empathy scores and 2 MBI subscales, emotional exhaustion (EE) and depersonalization (DP). Additionally, first year POS residents had higher DP scores than did first year TOS residents, t = 2.183, P = 0.031. We suggest that factors important for empathy are type of medical specialty, marriage, siblings, and children. Burnout state may be related to decreasing empathy.

Co-culture with human synovium-derived mesenchymal stem cells inhibits inflammatory activity and increases cell proliferation of sodium nitroprusside-stimulated chondrocytes.

Rheumatoid arthritis (RA) and osteoarthritis (OA) are primarily chronic inflammatory diseases. Mesenchymal stem cells (MSCs) have the ability to differentiate into cells of the mesodermal lineage, and to regulate immunomodulatory activity. Specifically, MSCs have been shown to secrete insulin-like growth factor 1 (IGF-1). The purpose of the present study was to examine the inhibitory effects on inflammatory activity from a co-culture of human synovium-derived mesenchymal stem cells (hSDMSCs) and sodium nitroprusside (SNP)-stimulated chondrocytes. First, chondrocytes were treated with SNP to generate an in vitro model of RA or OA. Next, the co-culture of hSDMSCs with SNP-stimulated chondrocytes reduced inflammatory cytokine secretion, inhibited expression of inflammation activity-related genes, generated IGF-1 secretion, and increased the chondrocyte proliferation rate. To evaluate the effect of IGF-1 on inhibition of inflammation, chondrocytes pre-treated with IGF-1 were treated with SNP, and then the production of inflammatory cytokines was analyzed. Treatment with IGF-1 was shown to significantly reduce inflammatory cytokine secretion in SNP-stimulated chondrocytes. Our results suggest that hSDMSCs offer a new strategy to promote cell-based cartilage regeneration in RA or OA.

Overexpression of TGF-ß1 enhances chondrogenic differentiation and proliferation of human synovium-derived stem cells.

Transforming growth factor-beta (TGF-ß) superfamily proteins play a critical role in proliferation, differentiation, and other functions of mesenchymal stem cells (MSCs). During chondrogenic differentiation of MSCs, TGF-ß up-regulates chondrogenic gene expression by enhancing the expression of the transcription factor SRY (sex-determining region Y)-box9 (Sox9). In this study, we investigated the effect of continuous TGF-ß1 overexpression in human synovium-derived MSCs (hSD-MSCs) on immunophenotype, differentiation potential, and proliferation rate. hSD-MSCs were transduced with recombinant retroviruses (rRV) encoding TGF-ß1. The results revealed that continuous overexpression of TGF-ß1 did not affect their phenotype as evidenced by flow cytometry and reverse transcriptase PCR (RT-PCR). In addition, continuous TGF-ß1 overexpression strongly enhanced cell proliferation of hSD-MSCs compared to the control groups. Also, induction of chondrogenesis was more effective in rRV-TGFB-transduced hSD-MSCs as shown by RT-PCR for chondrogenic markers, toluidine blue staining and glycosaminoglycan (GAG)/DNA ratio. Our data suggest that overexpression of TGF-ß1 positively enhances the proliferation and chondrogenic potential of hSD-MSCs.

Plant-derived mAbs have effective anti-cancer activities by increasing ganglioside expression in colon cancers.

An epithelial cell adhesion molecule (EpCAM) was selectively expressed in human colorectal carcinoma. Treatment with plant-derived anti-EpCAM mAb (mAbP CO17-1A) and RAW264.7 cells inhibited cell growth in the human colorectal cancer cell line SW620. In SW620 treated with mAbP CO17-1A and RAW264.7 cells, expression of p53 and p21 increased, whereas the expression of G1 phase-related proteins, cyclin D1, CDK4, cyclin E, and CDK2, decreased, similar to mammalian-derived mAb (mAbM) CO17-1A. Similar to mAbM CO17-1A, treatment with mAbP CO17-1A and RAW264.7 cell decreased the expression of anti-apoptotic protein, Bcl-2, but the expression of pro-apoptotic proteins Bax, TNF-α, caspase-3, caspase-6, caspase-8 and caspase-9, increased. Cells treated with mAbP CO17-1A and RAW264.7 cells expressed metastasis-related gangliosides, GM1 and GD1a, similar to mAbM CO17-1A. These results suggest that mAbP CO17-1A is as effective on anti-cancer activity as mAbM CO17-1A.

c-Src inhibitor PP2 inhibits head and neck cancer progression through regulation of the epithelial-mesenchymal transition.

Head and neck squamous cell carcinoma (HNSCC) is one of the most common cancer, causing considerable mortality and morbidity worldwide. Although HNSCC management has been extensively studied, the treatment outcomes have not improved - the 5-year survival rate of patients with HNSCC is 40%. Recent studies on the development of a novel HNSCC treatment have highlighted proto-oncogene tyrosine-protein kinase Src (c-Src) as one of the major therapeutic targets. However, the clinical efficacy of c-Src inhibitors against HNSCC was not comparable to that obtained in vitro. Furthermore, the molecular mechanisms underlying the efficacy of c-Src inhibitors remain elusive. In this study, we assessed the efficacy of 4-amino-5-(4-chlorophenyl)-7-(dimethylethyl)pyrazolo[3,4-d] pyrimidine (PP2), a selective c-Src inhibitor on HSNCC. Nine HNSCC cell lines (SNU1041, Fraud, SNU46, SNU1076, SNU899, SCC1483, YD15, YD9, and YD10-) were screened, and the effects of PP2 were evaluated using wound healing, apoptosis, and invasion assays. Western blot analysis of downstream markers was conducted to assess the specific mechanism of action of PP2 in HNSCC. The therapeutic efficacy of PP2 was further evaluated in xenograft mice. PP2 reduced tumor cell growth both in vitro and in vivo. Furthermore, it enhanced tumor cell apoptosis in cell lines and prevented metastasis in mice. PP2 also regulated the epithelial-mesenchymal transition pathway downstream of c-Src. More specifically, in SCC1483 and YD15PP2 HNSCC cell lines, PP2 exposure downregulated Erk, Akt/Slug, and Snail but upregulated E-cadherin. These results suggest that PP2 inhibits cell growth and progression in HNSCC by regulating the epithelial-mesenchymal transition pathway.

7,8-Dihydroxyflavone induces mitochondrial apoptosis and down-regulates the expression of ganglioside GD3 in malignant melanoma cells.

Malignant melanoma is a skin cancer with poor prognosis and high resistance to conventional treatment. 7,8-dihydroxyflavone (7,8-DHF) has shown anti-carcinogenic, anti-inflammatory, anti-oxidant, and pharmacological effects in several types of cancer. However, the relationship between ganglioside expression and the anti-cancer effects of 7,8-DHF in melanoma is not fully understood. In the present study, 7,8-DHF exhibits specific anti-proliferation, anti-migration, and G2/M phase cell-cycle arrest effects on both melanoma cancer cell lines, and induces mitochondrial dysfunction and apoptosis, making it a potent candidate for anti-melanoma treatment. Furthermore, we confirmed that 7,8-DHF significantly reduces the expression levels of ganglioside GD3 and its synthase, which are known to be closely involved in carcinogenesis. Taken together, our findings suggest that 7,8-DHF may be a potent anti-cancer drug candidate for the treatment of malignant melanoma.

Generation of An Induced Pluripotent Stem Cell Line from Human Liver Fibroblasts from A Patient with Combined Hepatocellular-Cholangiocarcinoma.

Objective: Combined hepatocellular-cholangiocarcinoma (cHCC-CC) is a rare type of primary liver cancer with characteristics of both hepatocellular carcinoma (HCC) and cholangiocarcinoma (CC). The pathogenesis of cHCCCC is poorly understood due to a shortage of suitable in vitro models. Due to scarce availability of human liver tissue, induced pluripotent stem cells (iPSCs) are a useful alternative source to produce renewable liver cells. For use in the development of liver pathology models, here we successfully developed and evaluated iPSCs from liver fibroblasts of a patient with cHCC-CC. Materials and Methods: In this experimental study, human liver fibroblasts (HLFs) were obtained from the liver biopsy of a 69-year-old male patient with cHCC-CC and transduced with a retroviral cocktail that included four factors - OCT4, SOX2, KLF4, and c-MYC (OSKM). Pluripotency of the iPSCs was determined by alkaline phosphatase (AP) staining, quantitative real-time polymerase chain reaction (PCR), and immunofluorescence. We induced in vitro embryoid body (EB) formation and performed an in vivo teratoma assay to confirm their differentiation capacity into the three germ layers. Results: HLF iPSCs derived from the cHCC-CC patient displayed typical iPSC-like morphology and pluripotency marker expression. The proficiency of the iPSCs to differentiate into three germ layers was assessed both in vitro and in vivo. Compared to normal control iPSCs, differentiated HLF iPSCs showed increased expressions of HCC markers alpha-fetoprotein (AFP) and Dickkopf-1 (DKK1) and the CC marker cytokeratin 7 (CK7), and a decreased expression of the CC tumour suppressor SRY-related HMG-box 17 (SOX17). Conclusion: We established HLF iPSCs using liver fibroblasts from a patient with cHCC-CC for the first time. The HLF iPSCs maintained marker expression in the patient when differentiated into EBs. Therefore, HLF iPSCs may be a sustainable cell source for modelling cHCC-CC and beneficial for understanding liver cancer pathology and developing therapies for cHCC-CC treatment.

Tobacco Smoking Could Accentuate Epithelial-Mesenchymal Transition and Th2-Type Response in Patients With Chronic Rhinosinusitis With Nasal Polyps.

Tobacco smoking (TS) has been known as one of the most potent risk factors for airway inflammatory diseases. However, there has been a paucity of information regarding the immunologic alteration mediated by TS in patients with chronic rhinosinusitis with nasal polyps (CRSwNP). To identify the effect of TS, we harvested human tissue samples (never smoker: n=41, current smoker: n=22, quitter: n=23) and analyzed the expression of epithelial-derived cytokines (EDCs) such as IL-25, IL-33, and thymic stromal lymphopoietin. The expressions of Th2 cytokines and total serum IgE showed a type-2 inflammatory alteration by TS. In addition, the epithelial marker E-cadherin and epithelial-mesenchymal transition (EMT)-associated markers (N-cadherin, α-SMA, and vimentin) were evaluated. Histological analysis showed that EDC expressions were upregulated in the current smoker group and downregulated in the quitter group. These expression patterns were consistent with mRNA and protein expression levels. We also found that the local Th2 cytokine expression and IgE class switching, as well as serum IgE levels, were elevated in the current smoker group and showed normal levels in the quitter group. Furthermore, the expressions of E-cadherin decreased while those of N-cadherin, α-SMA, and vimentin increased in the current smoker group compared those in the never smoker group. Taken together, these results indicate that TS contributes to the deterioration of pathogenesis by releasing local EDCs and Th2 cytokines, resulting in EMT in patients with CRSwNP. We verified that alterations of immunological response by TS in sinonasal epithelium can play a vital role in leading to CRSwNP.

Inhibition of ganglioside GD1a synthesis suppresses the differentiation of human mesenchymal stem cells into osteoblasts.

In this study, we investigated the regulatory role of ganglioside GD1a in the differentiation of osteoblasts from human mesenchymal stem cells (hMSCs) by using lentivirus-containing short hairpin (sh)RNA to knockdown ST3 ß-galactoside α-2, 3-sialyltransferase 2 (ST3Gal II) mRNA expression. After hMSCs were infected for 72 h with the lentivirus constructed with ST3Gal II shRNAs, the puromycin-resistant cells were selected and subcultured to produce hMSCs with ST3Gal II mRNA knockdown. The hMSCs established from human dental papilla abundantly expressed CD44 and CD105, but not CD45 and CD117. Osteoblasts that differentiated from normal hMSCs showed a significant increase in alkaline phosphatase (ALP) activity and ganglioside GD1a expression level compared with those in hMSCs. Lentiviral infection of hMSCs successfully induced a marked inhibition of ST3Gal II mRNA expression and caused a significant decrease in ALP activity and ganglioside GD1a expression. During osteoblastic differentiation, the increased ALP activity remarkably reduced by suppression of ganglioside GD1a expression by ST3Gal II shRNA. Ganglioside GD1a and ALP were mainly expressed in the cell body of hMSCs and osteoblasts with colocalization. The phosphorylation of extracellular signal-regulated kinases (ERK) 1/2 mitogen-activated protein (MAP) kinase and epidermal growth factor receptor (EGFR) was significantly reduced in the osteoblasts that had differentiated from the hMSCs with ST3Gal II mRNA knockdown. These results suggest that ganglioside GD1a plays an important role in the regulation of osteoblastic differentiation of hMSCs through the activation of ERK 1/2 MAP kinase and EGFR.

Long-Term Expansion of Functional Human Pluripotent Stem Cell-Derived Hepatic Organoids.

A human cell-based liver model capable of long-term expansion and mature hepatic function is a fundamental requirement for pre-clinical drug development. We previously established self-renewing and functionally mature human pluripotent stem cell-derived liver organoids as an alternate to primary human hepatocytes. In this study, we tested long-term prolonged culture of organoids to increase their maturity. Organoid growing at the edge of Matrigel started to deteriorate two weeks after culturing, and the expression levels of the functional mature hepatocyte marker ALB were decreased at four weeks of culture. Replating the organoids weekly at a 1：2 ratio in fresh Matrigel, resulted in healthier morphology with a thicker layer compared to organoids maintained on the same Matrigel and significantly increased ALB expression until three weeks, although, it decreased sharply at four weeks. The levels of the fetal hepatocyte marker AFP were considerably increased in long-term cultures of organoids. Therefore, we performed serial passaging of organoids, whereby they were mechanically split weekly at a 1：3∼1：5 ratio in fresh Matrigel. The organoids expanded so far over passage 55, or 1 year, without growth retardation and maintained a normal karyotype after long-term cryopreservation. Differentiation potentials were maintained or increased after long-term passaging, while AFP expression considerably decreased after passaging. Therefore, these data demonstrate that organoids can be exponentially expanded by serial passaging, while maintaining long-term functional maturation potential. Thus, hepatic organoids can be a practical and renewable cell source for human cell-based and personalized 3D liver models.

In Vivo and In Vitro Anticancer Activity of Doxorubicin-loaded DNA-AuNP Nanocarrier for the Ovarian Cancer Treatment.

In this study, we have determined the anticancer activity of doxorubicin (Dox)-loaded DNA/gold nanoparticle (AuNP) nanocarrier (Dox-DNA-AuNP) for the treatment of ovarian cancer. The anticancer effect of Dox-DNA-AuNP was evaluated in vitro using the EZ-Cytox cell viability assay on three human ovarian cancer cell lines, SK-OV-3, HEY A8, and A2780. Dox-DNA-AuNP exhibited outstanding activity with good IC50 values of 4.8, 7.4, and 7.6 nM for SK-OV-3, HEY A8, and A2780, respectively. In vivo evaluation further demonstrated the superior anticancer effects of Dox-DNA-AuNP by inhibiting tumor growth compared to free Dox in an established SK-OV-3 xenograft mice model. Dox-DNA-AuNP showed about a 2.5 times higher tumor growth inhibition rate than free Dox. Furthermore, the immunohistochemical analysis of Ki67 antigen expression showed the lowest number of proliferative cells in the ovarian tumor tissue treated with Dox-DNA-AuNP. These results suggest Dox-DNA-AuNP might be a potential effective agent in ovarian cancer chemotherapy.