Chia-Cheng Chang

Comparative study of human eutopic and ectopic endometrial mesenchymal stem cells and the development of an in vivo endometriotic invasion model

OBJECTIVEnTo elucidate the role of endometrial stem-progenitor cells in the etiology of endometriosis and to develop an animal model to study the invasion ability of endometrial cells.nnnDESIGNnGene expression and cell function studies were designed.nnnSETTINGnKaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan.nnnPATIENT(S)nHuman endometrial mesenchymal stem cells (MSCs) were isolated from 22 different endometrium biopsies after surgery for treatment of endometriosis.nnnINTERVENTION(S)nEndometrial MSCs developed from eutopic and ectopic endometrial tissues.nnnMAIN OUTCOME MEASURE(S)nCharacterization of MSC phenotypes (i.e., differentiation induction and gene expression by flow cytometric analysis); comparative study of cell functions (i.e., cell growth, migration, and invasion assays). The invasion of implants in an animal model was examined by histologic staining.nnnRESULT(S)nWe compared the characteristics of eutopic and ectopic endometrial MSCs from the same endometrial donor. Although both showed similar mesenchymal cell phenotypes, ectopic endometrial MSCs showed distinctly greater ability of cell migration and invasion. Furthermore, in an in vivo cell invasion model using cells grown in scaffold and transplantation in immune-deficient mice, the ectopic endometrial MSCs were found to form many new blood vessels and to invade surrounding tissue.nnnCONCLUSION(S)nThese results indicate unique invasion and angiogenesis characteristics of ectopic endometrial MSCs that may underlie the pathogenesis of ectopic endometriosis. The animal invasion model will be useful for future characterization of endometrial MSCs.

Increasing CD44+/CD24- tumor stem cells, and upregulation of COX-2 and HDAC6, as major functions of HER2 in breast tumorigenesis

BackgroundCancer cells are believed to arise primarily from stem cells. CD44+/CD24- have been identified as markers for human breast cancer stem cells. Although, HER2 is a well known breast cancer oncogene, the mechanisms of action of this gene are not completely understood. Previously, we have derived immortal (M13SV1), weakly tumorigenic (M13SV1R2) and highly tumorigenic (M13SV1R2N1) cell lines from a breast epithelial cell type with stem cell phenotypes after successive SV40 large T-antigen transfection, X-ray irradiation and ectopic expression of HER2/C-erbB2/neu. Recently, we found that M13SV1R2 cells became non-tumorigenic after growing in a growth factor/hormone-deprived medium (R2d cells).ResultsIn this study, we developed M13SV1R2N1 under the same growth factor/hormone-deprived condition (R2N1d cells). This provides an opportunity to analyze HER2 effect on gene expression associated with tumorigenesis by comparative study of R2d and R2N1d cells with homogeneous genetic background except HER2 expression. The results reveal distinct characters of R2N1d cells that can be ascribed to HER2: 1) development of fast-growing tumors; 2) high frequency of CD44+/CD24- cells (~50% for R2N1d vs. ~10% for R2d); 3) enhanced expression of COX-2, HDAC6 mediated, respectively, by MAPK and PI3K/Akt pathways, and many genes associated with inflammation, metastasis, and angiogenesis. Furthermore, HER2 expression can be down regulated in non-adhering R2N1d cells. These cells showed longer latent period and lower rate of tumor development compared with adhering cells.ConclusionsHER2 may induce breast cancer by increasing the frequency of tumor stem cells and upregulating the expression of COX-2 and HDAC6 that play pivotal roles in tumor progression.

Interleukin-1β induces cyclooxygenase-2 expression and promotes the invasive ability of human mesenchymal stem cells derived from ovarian endometrioma

OBJECTIVEnTo elucidate the role of interleukin-1β (IL-1β) on cyclooxygenase-2 (COX-2) expression and invasion of endometrioma-derived ectopic endometrial mesenchymal stem cells (EN-MSCs) and to develop an organoid method to study the invasive ability of endometrial cells.nnnDESIGNnGene expression and cell functions.nnnSETTINGnKaohsiung Medical University, Kaohsiung, Taiwan.nnnPATIENT(S)nHuman eutopic and endometrioma-derived ectopic EN-MSCs were isolated from different endometrium biopsy samples after surgery for treatment of endometriosis.nnnINTERVENTION(S)nChemical treatment of cell culture.nnnMAIN OUTCOME MEASURE(S)nComparative analysis of genomewide messenger RNA (mRNA) expression, cell migration, and invasion abilities in cell culture and organoid culture.nnnRESULT(S)nGene expression profiles revealed that the expression of IL-1β and COX-2 were statistically significantly higher in ectopic EN-MSCs compared with eutopic EN-MSCs. These enhanced expressions coincided with a greater ability for cell migration and invasion in ectopic EN-MSCs and were found to be distinctly regulated by IL-1β which up-regulates COX-2 expression. Furthermore, IL-1β treatment of ectopic EN-MSCs in organoids was found to induce tentacle-like structures that mimicked cell invasion.nnnCONCLUSION(S)nThese results indicate that COX-2 and IL-1β regulate the invasion ability of ectopic EN-MSCs. The information may be useful for developing a new therapeutic strategy for endometriosis. The exxa0vivo invasion model will be useful for characterization of EN-MSCs.

Optimizing proliferation and characterization of multipotent stem cells from porcine adipose tissue

Porcine mesenchymal stem cells have been isolated previously from bone marrow but not from adipose tissue. In the present study a new cell‐culture method, using a low‐calcium medium supplemented with N‐acetyl‐L‐cysteine and L‐ascorbic acid 2‐phosphate (the PM2 medium) was developed to grow pASCs (porcine adipose‐tissue‐derived stem cells). The pASCs developed using the new medium showed a high growth rate and a high proliferation potential, as measured by a cumulative population doubling level (55) that was significantly higher than those reported for ASCs in the literature. These pASCs lacked gap‐junctional intercellular communication and were capable of differentiation into three mesodermal lineages (i.e. adipocytes, osteoblasts and chondrocytes) and an ectodermal lineage (i.e. neural cells). Surprisingly, osteogenic ability, but not adipogenesis, was found to increase dramatically with increasing passages. The high proliferative and differentiation potential of these pASCs should facilitate the development of a large‐animal model to study the use of ASCs in regenerative and reparative medicine.

Bisphenol A at environmentally relevant doses induces cyclooxygenase-2 expression and promotes invasion of human mesenchymal stem cells derived from uterine myoma tissue

OBJECTIVEnUterine myoma is the most common benign reproductive tract tumor in women. Despite its high prevalence, the exact pathogenesis of these benign tumors remains unknown. Toward understanding the pathogenic mechanism of these tumors, we attempted to isolate human uterine myoma mesenchymal stem cells (hUM-MSCs), which may be the target cells for tumorigenesis. Furthermore, we tested the response of these hUM-MSCs to the environmental endocrine disruptor, bisphenol A (BPA), which may mimic the action of estrogen in hormone-sensitive organs such as the uterus.nnnMATERIALS AND METHODSnThe hUM-MSC lines were clonally derived from uterine myoma tissue using the MSU-1 medium supplemented with N-acetyl-l-cysteine and l-ascorbic acid-2-phosphate. These hUM-MSCs were characterized by reverse transcription polymerase chain reaction (RT-PCR) analysis for the expression of mesenchymal stem cell (MSC) surface markers (e.g., CD90 and CD105) and the transcription factor Oct-4. The proliferation potential was measured by the cumulative population doubling level and the colony-forming efficiency.nnnRESULTSnPutative hUM-MSC lines expressed CD90, CD105, and the stem cell marker gene, Oct-4. The cells were capable of differentiating into adipocytes, osteoblasts, and chondrocytes. Bisphenol A treatment of these hUM-MSCs enhanced cell proliferation and colony-forming efficiency in a dose-responsive manner. At an environmentally relevant concentration (10(-8)xa0M), BPA moreover induced cyclooxygenase-2 (COX-2) gene expression and promoted cell migration and invasiveness.nnnCONCLUSIONnThe hUM-MSC cell lines can be isolated from uterine myoma tissues. Bisphenol A could enhance cell proliferation and colony-forming efficiency, induce COX-2 gene expression, and promote migration and invasion of hUM-MSCs. The results imply that BPA has a detrimental effect on female health by promoting uterine tumorigenesis.

Modulation of tumorigenesis and oestrogen receptor‐α expression by cell culture conditions in a stem cell‐derived breast epithelial cell line

Background information. The common phenotypes of cancer and stem cells suggest that cancers arise from stem cells. Oestrogen is one of the few most important determinants of breast cancer, as shown by several lines of convincing evidence. We have previously reported a human breast epithelial cell type (Type 1 HBEC) with stem cell characteristics and ERα (oestrogen receptor α) expression. A tumorigenic cell line, M13SV1R2, was developed from this cell type after SV40 (simian virus 40) large T‐antigen transfection and X‐ray irradiation. The cell line, however, was not responsive to oestrogen for cell growth or tumour development. In the present study, we tested the hypothesis that deprivation of growth factors and hormones may change the tumorigenicity and oestrogen response of this cell line.

Bisphenol A-induced epithelial to mesenchymal transition is mediated by cyclooxygenase-2 up-regulation in human endometrial carcinoma cells.

Many studies have highlighted the correlation between the increase of bisphenol A (BPA) level in the environment and the incidence of tumor in humans. In human carcinogenesis, the overexpression of cyclooxygenase-2 (COX-2) and epithelial-mesenchymal transition (EMT) are closely related with tumor development. In this study, human endometrial carcinoma cells line (RL95-2) was used to investigate whether BPA can induce EMT and COX-2 expression. The results show that BPA increased growth rate and colony-forming efficiency in a dose-dependent manner, induced EMT and COX-2 gene expression and promoted the migration and invasion ability of RL95-2 cells. Furthermore, our study showed that the expression of COX-2 was essential for BPA-induced cell migration and invasion. The results of this study provide new insights into the mechanism of endometrial cancer cell growth and invasion and potential therapeutic strategy.

Promotion of epithelial–mesenchymal transition and tumor growth by 17β‐estradiol in an ER+/HER2+ cell line derived from human breast epithelial stem cells

A tumorigenic cell line with estrogen receptor and HER2 expression (ER/HER2+), R2N1d, was developed from a human breast epithelial cell type with stem cell characteristics in a growth factor/hormone‐deprived cell culture condition. This study was undertaken to test whether tumor growth and other biological effects could be induced by estrogen in this cell line. The results clearly show that estrogen treatment greatly promoted the tumor growth of R2N1d cells in immune‐deficient mice. Estrogen treatment of R2N1d cells in vitro was also found to induce other phenotypic changes related to breast carcinogenesis, that is, 1) the induction of epithelial–mesenchymal transition (EMT) shown by molecular and functional marker changes; 2) a significant increase of the CD44high/CD24−/low stem cell population; 3) the enhancement of cell growth rate and colony‐forming ability; and 4) the acquisition of metastatic ability, that is, increased cell migration and invasiveness. From these results, we conclude that 1) estrogen could induce EMT and cancer stem cells and promote tumor growth in ER+/HER2+ cells known to be derived from human breast epithelial stem cells, and 2) normal stem cells could give rise to cancer stem cells.

Upregulation of Nanog and Sox‐2 genes following ectopic expression of Oct‐4 in amniotic fluid mesenchymal stem cells

Octamer‐binding transcription factor 4 (Oct‐4), an important gene regulating stem cell pluripotency, is well‐known for its ability to reprogram somatic cells in vitro, either alone or in concert with other factors. The aim of this study was to assess the effect of ectopic expression of Oct human amniotic fluid stem cells. We developed a novel method for isolation of putative human amniotic fluid‐derived multipotent stem cells. These cells showing mesenchymal stem cell phenotypes (human amniotic fluid‐derived mesenchymal stem cells, hAFMSCs) were transfected with a plasmid carrying genes for Oct‐4 and the green fluorescent protein (GFP). The stably transfected cells, hAFMSCs‐Oct4/GFP, were selected by using G418 and found to express the GFP reporter gene under the control of Oct‐4 promoter. We found that hAFMSCs developed by our method possess very high self‐renewal ability (about 78 cumulative population doublings) and multilineage differentiation potency. Significantly, the hAFMSCs‐Oct4/GFP cells showed enhanced expression of the three major pluripotency genes Oct‐4, Nanog, and Sox‐2, and increased colony‐forming ability and growth rate compared with the parental hAFMSCs. We demonstrated that the ectopic expression of Oct‐4 gene in hAFMSCs with high self‐renewal ability could upregulate Nanog and Sox‐2 gene expression and enhance cell growth rate and colony‐forming efficiency. Therefore, the ectopic expression of Oct‐4 could be a strategy to develop pluripotency in hAFMSCs for clinical applications.

Lifespan Extension and Sustained Expression of Stem Cell Phenotype of Human Breast Epithelial Stem Cells in a Medium with Antioxidants.

We have previously reported the isolation and culture of a human breast epithelial cell type with stem cell characteristics (Type I HBEC) from reduction mammoplasty using the MSU-1 medium. Subsequently, we have developed several different normal human adult stem cell types from different tissues using the K-NAC medium. In this study, we determined whether this low calcium K-NAC medium with antioxidants (N-acetyl-L-cysteine and L-ascorbic acid-2-phosphate) is a better medium to grow human breast epithelial cells. The results clearly show that the K-NAC medium is a superior medium for prolonged growth (cumulative population doubling levels ranged from 30 to 40) of normal breast epithelial cells that expressed stem cell phenotypes. The characteristics of these mammary stem cells include deficiency in gap junctional intercellular communication, expression of Oct-4, and the ability to differentiate into basal epithelial cells and to form organoid showing mammary ductal and terminal end bud-like structures. Thus, this new method of growing Type I HBECs will be very useful in future studies of mammary development, breast carcinogenesis, chemoprevention, and cancer therapy.