Hwan-Wun Liu

Field methylation silencing of the protocadherin 10 gene in cervical carcinogenesis as a potential specific diagnostic test from cervical scrapings

PCDH10 is a member of the protocadherin cell adhesion molecule family, which are frequently downregulated in cancers. This study aimed to characterize the methylation silencing of the PCDH10 gene in the full spectrum of cervical carcinogenesis and to clarify if a field effect of methylation might be a target for a diagnostic test from cervical scrapings. Methylation silencing of PCDH10 was found in four of five cervical cancers and one of two cervical precancerous cell lines, which could be reversed by demethylation treatment. The same methylation was detected in 85.7% (24/28) of invasive cancer tissues, 36.4% (4/11) of high‐grade squamous intraepithelial lesions, 20% (1/5) of low‐grade squamous intraepithelial lesions, and none (0/17) of the normal cervical tissues from non‐cancer subjects. In addition, methylation was also frequently found in histologically ‘normal’ cervical tissues adjacent to cancer lesions (7/13, 53.8%) and, less frequently, in vaginal and endometrial tissues (1/8, 12.5%). Further investigation of cervical scrapings revealed cancer‐specific methylation of PCDH10 with a methylation rate of 71% (22/31) in invasive cancer, 27.9% (12/43) in carcinoma in situ, and none in high‐grade squamous intraepithelial lesions excluding carcinoma in situ (n = 12), low‐grade squamous intraepithelial lesions (n = 27), and normal controls (n = 66) (P < 10−16). Compared to the high‐risk human papilloma virus test, PCDH10 methylation testing of cervical scrapings was more specific (92 vs 60%) but less sensitive (71 vs 96%) in detecting invasive cervical cancer. This study demonstrated field methylation of the PCDH10 gene specifically in the invasion stage of cervical carcinogenesis, which might be used to develop a highly specific diagnostic test for cervical scrapings. (Cancer Sci 2009)

Crosstalk with Cancer-Associated Fibroblasts Increases the Growth and Radiation Survival of Cervical Cancer Cells

Crosstalk between cancer cells and the surrounding cancer associated fibroblasts (CAFs) plays an illusive role in cancer radiotherapy. This study investigated the effect of cancer cell–cancer associated fibroblasts crosstalk on the proliferation and survival of irradiated cervical cancer cells. A pretreatment with conditioned medium from a mixed culture of CAF and HeLa cells (mixCAF) had a stronger effect on enhancing the proliferation and survival of irradiated HeLa cells compared to pretreatment with CAF conditioned medium alone. In addition, pretreatment with a mixed culture of CAF and HeLa cells conditioned medium reduced the levels of two major radiation-induced genes, GADD45 and BTG2, and phosphorylation of p38. Profiling of the growth and survival factors in the conditioned medium revealed PDGF and VEGF, and IGF2, EGF, FGF-4, IGFBPs and GM-CSF to be specifically secreted from HeLa cells and CAFs, respectively. This study demonstrated radiation protective effects of CAF-cancer cell crosstalk, and identified multiple growth factors and radiation response genes that might be involved in these effects.

Human Infrapatellar Fat Pad-Derived Stromal Cells Have More Potent Differentiation Capacity Than Other Mesenchymal Cells and Can Be Enhanced by Hyaluronan.

The microenvironment plays an important role in the homing in and differentiation of stem cells to repair injured tissue. Infrapatellar fat pad stromal cells (IFPSCs) are a promising source of such cells for the repair of articular injury-induced degeneration. This study investigated the chemotaxis of IFPSCs to chondrocytes and the effect of hyaluronan (HA) on the biological and regenerative properties of IFPSCs. The IFPSCs were obtained from patients undergoing arthroscopy and cultured via a standard 2-week culture protocol that yielded more than 10 million cells on passage 3. The results showed that the IFPSCs had a higher capacity for chondrogenic differentiation than mesenchymal cells from body fat, bone marrow, and Whartons jelly of the umbilical cord. The IFPSCs cultured on 25% or 50% HA showed better osteogenic and adipogenic capabilities than those without HA or with 75% HA (p < 0.001). Cultures of the IFPSCs on 25% HA had a fourfold increase in chondrogenic differentiation compared to cultures without HA, which was better than with 50% and 75% HA (p < 0.05). Cell proliferation was not affected by the presence of HA. In conclusion, IFPSCs have a strong potential for chondrogenic regeneration, which can even be augmented in a 25% HA microenvironment.

Characterization of HLA-G and Related Immunosuppressive Effects in Human Umbilical Cord Stroma-Derived Stem Cells:

Mesenchymal stem cells (MSCs) and especially those derived from fetal tissues exert a potent immunosuppressive effect that can be enhanced under inflammatory conditions. This study aimed to explore the immunosuppressive properties of human umbilical cord mesenchymal stem cells (HUCMSCs). We found that HLA-G, the nonclassical HLA allele with strong immune-inhibitory properties, was much more expressed on the HUCMSCs than on MSCs of other origins. Flow cytometry revealed that 90.8% of the HUCMSCs expressed HLA-G. RT-PCR revealed expression of HLA-G1, HLA-G5, and HLA-G7 in all of four HUCMSC lines. In a mixed lymphocyte reaction assay, the HUCMSCs inhibited the proliferation of lymphocytes by 35 ± 3% and could be reversed by treatment with an HLA-G blocking antibody. Upon coculture with the HUCMSCs, peripheral blood mononuclear cells expressed lower levels of proinflammatory mediators such as IL-6, TNF-α, and VEGF-α. This immunosuppressive effect was enhanced when the HUCMSCs were pretreated with IFN-γ, such that the expression of HLA-G was highly activated and HLA-DR diminished. The same phenomenon was not observed in MSCs derived from bone marrow or the placenta. In a xenograft rejection assay, the HUCMSCs survived in immunocompetent mice, whereas primary fibroblasts did not survive. This study confirms the HLA-G-related immunosuppressive property of HUCMSCs, which is more potent than MSCs of other origin. A good tolerance of this mesenchymal stem cell in allogeneic transplantation can thus be anticipated.

Long-term persistence of human papillomavirus in environments.

OBJECTIVE The possibility of its indirect transmission of human papillomavirus (HPV) via formites has been widely raised but with no biological proof. This study explored the durability of HPV16 pseudoviruses and native viruses in different environmental contamination scenarios. METHODS Pseudoviruses were mixed with PBS, cervico-vaginal secretion (CVS), or serum to simulate contamination by genital warts, vaginal discharge or menstruation, respectively, and subjected to in-vitro cell infection assay. The integrity of native HPV16 from CVS of infected women was detected by conformation-specific antibody. RESULTS In viruses exposed to PBS, a persistent infectivity of 30% was noted for at least 7 days. A similar persistence but lower (18%) infectivity was noted in those exposed to CVS. In serum-containing medium, the infection ratio rose initially, remained stable for three more days then rapidly decreased thereafter. Upon desiccation, infectivity was persistently low (10%). Finally, intact native HPV was detectable after 5 days of environmental exposure. CONCLUSION This study demonstrated the high environmental survivability of HPV. However, survivability was lower in viruses exposed to CVS or desiccation.

Mesenchymal Stem Cells and Their Clinical Applications in Osteoarthritis.

Osteoarthritis is a chronic degenerative joint disorder characterized by articular cartilage destruction and osteophyte formation. Chondrocytes in the matrix have a relatively slow turnover rate, and the tissue itself lacks a blood supply to support repair and remodeling. Researchers have evaluated the effectiveness of stem cell therapy and tissue engineering for treating osteoarthritis. All sources of stem cells, including embryonic, induced pluripotent, fetal, and adult stem cells, have potential use in stem cell therapy, which provides a permanent biological solution. Mesenchymal stem cells (MSCs) isolated from bone marrow, adipose tissue, and umbilical cord show considerable promise for use in cartilage repair. MSCs can be sourced from any or all joint tissues and can modulate the immune response. Additionally, MSCs can directly differentiate into chondrocytes under appropriate signal transduction. They also have immunosuppressive and anti-inflammatory paracrine effects. This article reviews the current clinical applications of MSCs and future directions of research in osteoarthritis.

Enhanced differentiation and clonogenicity of human endometrial polyp stem cells

Endometrial polyps arise from endometrial overgrowth and may cause intermenstrual bleeding, irregular bleeding, and menorrhagia. In this study, endometrial polyps were harvested from hysterectomized specimens from 6 female patients not on hormone therapy. Endometrial polyp mesenchymal stem cells (EPMSCs) were isolated and characterized. Selected cells were spindle-shaped, and expressed surface markers CD90 and CD146. The EPMSCs proliferated actively in vitro. A colony-forming study demonstrates that EPMSCs had a colony-generating capacity. When cultured in a defined medium, EPMSCs can differentiate to osteoblast-, adipocyte-, and neuron-like cells. No telomerase reverse transcriptase (TERT) expression was noted. Experimental results demonstrate that EPMSCs are a population of mesenchymal progenitor cells existing in human endometrial polyps that are capable of proliferation, differentiation, and colonogenicity exceeding that of bone marrow stem cells and endometrial stromal cells. These EPMSCs may be an alternative resource of adult stem cells for future regenerative therapy.

Interleukin-6 from Ovarian Mesenchymal Stem Cells Promotes Proliferation, Sphere and Colony Formation and Tumorigenesis of an Ovarian Cancer Cell Line SKOV3

The origin of the majority of epithelial ovarian cancers (EOC) is regarded as extraovarian, with the ovary being the secondary site. The aim of this study was to explore the possible role of ovarian mesenchymal stem cells (OvMSCs) and secreted IL-6 in the development of EOC. OvMSCs were derived from normal ovarian stroma. Cell surface markers and differentiation capability were determined. The effects of IL-6 and conditioned medium of OvMSCs on the malignant phenotype of SKOV3 ovarian cancer cells were tested, and the status of STAT3 and ERK phosphorylation was investigated. OvMSCs had similar surface marker profiles as bone marrow mesenchymal stem cells, i.e., CD44 (+), CD90 (+) and CD45 (-), and was readily inducible to osteogenic, adipogenic and chondrogenic differentiation. OvMSCs secreted an extremely high level (>2500 pg/ml) of IL-6. Treatment of SKOV3 cells with conditioned media from OvMSCs increased cell proliferation, tumor sphere formation and anchorage independent growth, and resulted in activation of STAT3 but not ERK. Coinjection of OvMSCs with SKOV3 cell enhanced tumorigenesis in NOD-SCID mice. All of these behaviors were blocked by IL-6 receptor blocking antibody administered in vitro or in vivo. The OvMSCs alone injected into mice had no tumor growth after 3 months. By secreting high levels of IL-6, OvMSCs enhance the proliferation, sphere and colony formation and tumorigenesis of SKOV3 cells.

Robotic single-site supracervical hysterectomy with manual morcellation: Preliminary experience

AIM To evaluate the feasibility, safety and peri- and postoperative outcomes of robotic single-site supracervical hysterectomy (RSSSH) for benign gynecologic disease. METHODS We report 3 patients who received RSSSH for adenomyosis of the uterus from November 2015 to April 2016. We evaluated the feasibility, safety and outcomes among these patients. RESULTS The mean surgical time was 244 min and the estimated blood loss was 216 mL, with no blood transfusion necessitated. The docking time was shortened gradually from 30 to 10 min. We spent 148 min on console operation. Manual morcellation time was also short, ranging from 5 to 10 min. The mean hospital stay was 5 d. Lower VAS pain score was also noted. There is no complication during or after surgery. CONCLUSION RSSSH is feasible and safe, incurs less postoperative pain and gives good cosmetic appearance. The technique of in-bag, manual morcellation can avoid tumor dissemination.

Transplanting human umbilical cord mesenchymal stem cells and hyaluronate hydrogel repairs cartilage of osteoarthritis in the minipig model

Objectives: Osteoarthritis (OA) is a chronic disease of degenerative joints. Mesenchymal stem cells (MSCs) have been used for cartilage regeneration in OA. We investigated the therapeutic potential of human umbilical cord-derived MSCs (HUCMSCs) with hyaluronic acid (HA) hydrogel transplanted into a porcine OA preclinical model. Materials and Methods: The HUCMSCs were characterized with respect to morphology, surface markers, and differentiation capabilities. Quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) was used to examine gene expressions in a HUCMSC–HA coculture. Two healthy female minipigs weighing 30–40 kg and aged approximately 4 months were used in this large animal study. A full-thickness chondral injury was created in the trochlear groove of each of the pigs rear knees. After 3 weeks, a second osteochondral defect was created. Then, 1.5 mL of a HUCMSC (5 × 106 cells) and HA composite (4%) was transplanted into the chondral-injured area in the right knee of each pig. Using the same surgical process, an osteochondral defect (untreated) was created in the left knee as a control. The pigs were sacrificed 12 weeks after transplantation. Macroscopic and microscopic histologies, qRT-PCR, and immunostaining evaluated the degree of chondral degradation. Results: The HUCMSCs exhibited typical MSC characteristics, including spindle morphology, expression of surface markers (positive for CD29, CD4, CD73, CD90, and human leukocyte antigen [HLA]-ABC; negative for CD34, CD45, and HLA-DR), and multipotent differentiation (adipogenesis, osteogenesis, and chondrogenesis). More extensive proliferation of HUCMSCs was noted with 4% and 25% of HA than without HA. Expression of COL2A1 and aggrecan in the HUCMSC-derived chondrocytes was increased when HA was included. The treated knees showed significant gross and histological improvements in hyaline cartilage regeneration when compared to the control knees. The International Cartilage Repair Society histological score was higher for the treated knees than the control knees. Conclusion: Our findings suggest that cartilage regeneration using a mixture of HUCMSCs and HA in a large animal model may be an effective treatment for OA, and this study is a stepping stone toward the future clinical trials.