Self-renewal Markers Research Articles

A novel undifferentiated spermatogonia-specific surface protein 1 (USSP1) in neonatal mice

Mammalian spermatogenesis is maintained by a rare population of spermatogonial stem cells (SSCs), which are important for male fertility. SSCs remain a subset of undifferentiated spermatogonia, which can be isolated by a combination of surface markers. Specific markers to identify and isolate undifferentiated spermatogonia are lacking. Ussp1, a transcript previously annotated as long noncoding RNA (RIKEN cDNA 4933427D06, Gene ID: 232217), virtually encodes a membrane protein, USSP1, in a highly testis-specific manner in mouse. We demonstrate its expression on the membrane of undifferentiated spermatogonia by a homemade polyclonal rabbit antibody against the protein. In vivo, USSP1+ clusters consist mainly of As, Apr (GFRα1+) and Aal (PLZF+) cells. USSP1+ cells exhibit enrichment of undifferentiated spermatogonia, as shown by increased expression of SSC self-renewal molecular markers and the potential to form SSC clones in vitro and in vivo. However, Ussp1 knockout did not affect the number of SSCs or spermatogenesis in mice. Thy1+ cells from Ussp1 null mice did not show any defect in the SSC colony formation capacity, indicating that USSP1 is not essential for SSC self-renewal. Our data demonstrate that Ussp1 is specifically expressed in undifferentiated murine spermatogonia, indicating the potential to sort undifferentiated spermatogonia with USSP1 antibodies. Ussp1 might be a good maker for SSC enrichment in neonatal mice.

Specific Transcription Factors Distinguish Umbilical Cord Mesenchymal Stem Cells From Fibroblasts

Now at the forefront of scientific research, stem cells play a crucial role in regenerative medicine and cell therapy. Although noted for their great promise in understanding organismal development and potential as a treatment for various diseases, stem cell research and applications have been limited by ethical and political concerns. However, recent research regarding induced pluripotent stem cells (iPSCs) has revolutionized the principle of stem cell-based treatment, especially since certain ethical controversies are no longer applicable. In 2009, scientists discovered that differentiated somatic cells could be induced to their stem-cell state by expressing transcription factors specific to self-renewal and potency. Since iPSCs are derived from one’s own somatic cells, they bypass ethical and political concerns. Our objective is to further investigate whether specific protein markers, inherent to stem cells and their properties of self-renewal and potency, can be used to identify umbilical cord mesenchymal stem cells (UC-MSCs). We compared fibroblasts as a control cell type because of their similar physical structure to stem cells and their lack of self-renewal and potency-specific markers. We cultured both cell lines and measured protein levels of four selected factors (β-actin, Klf4, Nanog, and Sox2) using western blot techniques. Our results revealed that these selected proteins were expressed exclusively by UC-MSCs and not by fibroblasts, successfully demonstrating that specific protein markers can be used to distinguish UC-MSCs.

Novel role of O-glycosyltransferases GALNT3 and B3GNT3 in the self-renewal of pancreatic cancer stem cells

BackgroundGlycosylation plays a critical role in the aggressiveness of pancreatic cancer (PC). Emerging evidences indicate significant involvement of cancer stem cells (CSCs) in PC aggressiveness. However, the importance of glycosylation in pancreatic cancer stem cells (PCSCs) is yet to be addressed. Hence, we evaluated the potential role of glycosylation in maintenance of stemness of PCSCs.MethodsEffect of glycosylation specific inhibitors on growth and PCSCs of PC cells was assessed by MTT assay and Side Population (SP) analysis. Isolated PCSCs/SP were characterized using molecular and functional assays. Expression of tumor-associated carbohydrate antigens (TACAs) was analyzed in PCSCs by western blotting. Effect of tunicamycin on PCSCs was analyzed by tumorsphere, clonogenicity, migration assay and immunoblotting for CSCs markers. The differential expression of glycogenes in PCSCs compared to non-CSCs were determined by RT-qPCR, immunoblotting and immunofluorescence. Co-expression of GALNT3 and B3GNT3 with CD44v6 was assessed in progression stages of KrasG12D; Pdx-1-Cre (KC) and KrasG12D; p53R172H; Pdx-1-Cre (KPC) tumors by immunofluorescence. Transient and CRISPR/Cas9 silencing of GALNT3 and B3GNT3 was performed to examine their effect on CSCs maintenance.ResultsInhibition of glycosylation decreased growth and CSCs/SP in PC cells. PCSCs overexpressed CSC markers (CD44v6, ESA, SOX2, SOX9 and ABCG2), exhibited global expressional variation of TACAs and showed higher self-renewal potential. Specifically, N-glycosylation inhibition, significantly decreased tumorsphere formation, migration, and clonogenicity of PCSCs, as well as hypo-glycosylated CD44v6 and ESA. Of note, glycosyltransferases (GFs), GALNT3 and B3GNT3, were significantly overexpressed in PCSCs and co-expressed with CD44v6 at advanced PDAC stages in KC and KPC tumors. Further, GALNT3 and B3GNT3 knockdown led to a decrease in the expression of cell surface markers (CD44v6 and ESA) and self-renewal markers (SOX2 and OCT3/4) in PCSCs. Interestingly, CD44v6 was modified with sialyl Lewis a in PCSCs. Finally, CRISPR/Cas9-mediated GALNT3 KO significantly decreased self-renewal, clonogenicity, and migratory capacity in PCSCs.ConclusionsTaken together, for the first time, our study showed the importance of glycosylation in mediating growth, stemness, and maintenance of PCSCs. These results indicate that elevated GALNT3 and B3GNT3 expression in PCSCs regulate stemness through modulating CSC markers.

MiR-19b-3p integrates Jak-Stat signaling pathway through Plzf to regulate self-renewal in dairy goat male germline stem cells.

Jak-Stat pathway is the first pathway identified to stimulate spermatogonial stem cells (SSCs) self-renewal and maintenance activity. Recent studies have showed that stat3 a crucial gene implicated in this pathway can regulate self-renewal in male germline stem cell. In our previous study, we demonstrated that miR-19b-3p induces cell proliferation and reduces heterochromatin through Plzf which also regulates the balance between cell self-renewal and differentiation. Because miRNA can target several genes and to understand more about Plzf, a crucial transcription factor of SSCs, we performed microarray and found that miR-19b-3p integrate Jak-Stat through Plzf to regulate cell self-renewal. Our results demonstrated that miR-19b-3p induces Jak-Stat when Plzf is downregulated; overexpression of Plzf reversed the trend and shown an existence of feedback (-/+) between Plzf and GHR. The cell self-renewal markers CD49f, GFRα1, Oct4 and cKIT analyzed in the both groups miR-19b-3p and Plzf-overexpressing compared to their respective control confirm miR-19b-3p regulates cell pluripotency and self-renewal in goat male germline stem cells through Plzf. Together our finding revels that miR-19b-3p control Jak-Stat signaling through Plzf.

IRAK1 Augments Cancer Stemness and Drug Resistance via the AP-1/AKR1B10 Signaling Cascade in Hepatocellular Carcinoma

Frequent relapse and drug resistance in patients with hepatocellular carcinoma (HCC) can be attributed to the existence of tumor-initiating cells (TIC) within the tumor bulk. Therefore, targeting liver TICs may improve the prognosis of these patients. From transcriptome sequencing of 16 pairs of clinical HCC samples, we report that interleukin-1 receptor-associated kinase 1 (IRAK1) in the TLR/IRAK pathway is significantly upregulated in HCC. IRAK1 overexpression in HCC was further confirmed at the mRNA and protein levels and correlated with advanced tumor stages and poor patient survival. Interestingly, IRAK4, an upstream regulator of IRAK1, was also consistently upregulated. IRAK1 regulated liver TIC properties, including self-renewal, tumorigenicity, and liver TIC marker expression. IRAK1 inhibition sensitized HCC cells to doxorubicin and sorafenib treatment in vitro via suppression of the apoptotic cascade. Pharmacological inhibition of IRAK1 with a specific IRAK1/4 kinase inhibitor consistently suppressed liver TIC populations. We identified aldo-keto reductase family 1 member 10 (AKR1B10) as a novel downstream target of IRAK1, which was found to be overexpressed in HCC and significantly correlated with IRAK1 expression. Knockdown of AKR1B10 negated IRAK1-induced TIC functions via modulation of the AP-1 complex. Inhibition of IRAK1/4 inhibitor in combination with sorafenib synergistically suppressed tumor growth in an HCC xenograft model. In conclusion, targeting the IRAK4/IRAK1/AP-1/AKR1B10 signaling pathway may be a potential therapeutic strategy against HCC.Significance: IRAK4/IRAK1/AP-1/AKR1B10 signaling pathway regulates cancer stemness and drug resistance and may be a novel therapeutic target in HCC. Cancer Res; 78(9); 2332-42. ©2018 AACR.

Transglutaminase 2 Regulates Self-renewal and Stem Cell Marker of Human Colorectal Cancer Stem Cells.

The aim of this study was to investigate the role of transglutaminase 2 (TGM2) in colorectal cancer stem cells (CCSCs). We used the TU12 cell line possessing CD133-expressing CCSCs. After isolating CD133 (-) and CD133 (+) CCSCs, we overexpressed and knocked-down TGM2 to investigate its role in human CCSCs. The expression level of TGM2 was 25-fold higher in tumorigenic cells than non-tumorigenic cells. We found that knockdown of TGM2 by specific RNA interference markedly inhibited cell growth and caused down-regulation of the stemness markers, CD133, SOX2, and β-catenin. We further demonstrated that knockdown of TGM2 inhibited cell metastatic abilities by down-regulating N-cadherin and vimentin and up-regulating E-cadherin. These findings revealed that TGM2 expression is markedly increased in human colorectal cancer and that down-regulation of TGM2 in tumors may serve as a treatment for colorectal cancer patients. Therefore, this study indicate that TGM2 affects the metastatic potential and stemness of CCSCs by regulating EMT- and stemness-related proteins. The metastatic potential of CSCs arises from highly expressed TGM2.

Combined mild hypoxia and bone marrow mesenchymal stem cells improve expansion and HOXB4 gene expression of human cord blood CD34+ stem cells

Cord blood (CB) is a rich source of hematopoietic stem cells (HSC). It has been used successfully to treat a variety of hematological and non-hematological disorders. Beside the advantages of CB, its main disadvantages are the limited number of stem cells available for transplantation and delayed engraftment. Identifying strategies to enhance expansion and self-renewal of HSCs can improve transplantation efficiency. The aim of this study was to examine different culture conditions on ex vivo expansion and homeobox protein Hox-B4 gene (HOXB4) expression in cord blood CD34+ stem cells. Human cord blood CD34+ HSC were cultured in serum-free medium supplemented with cytokines with and without a feeder layer in normoxia (21% O2) and mild hypoxia (5% O2). At the end of 7 days of culture, the highest number of total nucleated cells (TNC), CD34+ cells, colony forming units (CFUs) and HOXB4 mRNA were observed in a co-culture of HSC with a bone marrow mesenchymal stem cell(MSC) feeder layer at 5% O2.We concluded that the combination of bone marrow (BM)-MSC and mild hypoxia (5% O2) not only improved HSC expansion but also enhanced HOXB4 gene expression as a self-renewal marker of HSC, and better mimicked the niche microenvironment conditions.

TGFβ1 Promotes Breast Cancer Local Invasion and Liver Metastasis by Increasing the CD44high/CD24- Subpopulation.

Objective:Previous studies have shown that the transforming growth factor β1 pathway plays an important role in breast cancer metastasis to the liver. However, the mechanism of this metastasis has not been fully clarified. Cancer stem cells are essential for the initiation and propagation of tumor metastasis. The objective of our current study was to define the role of cancer stem cells in transforming growth factor β1-mediated breast cancer hepatic metastases.Method:Hematoxylin and eosin staining was used to assess the formation of breast cancer liver metastases and local invasion. Cancer stem cells surface markers (CD44, CD24, and Epithelial cell adhesion molecule [EpCAM]), luminal/mesenchymal markers (keratin8 and alpha smooth muscle actin), and proliferation markers (Ki-67 and cyclinD1) were detected by immunohistochemistry assays. Flow cytometry was used to evaluate the effect of transforming growth factor β1 on the CD44+/CD24− cancer stem cell population. Quantitative real-time polymerase chain reaction was employed to assess the gene expression of the stem cell self-renewal markers nanog, pou5f1 (coding for Oct4), and sox2.Results:Transforming growth factor β1 increased the formation of liver metastases by the MDA-MB231 (MDA) breast cancer cell line but did not affect the liver metastasis of CD44+/CD24+ noncancer stem cells. Transforming growth factor β1 treatment did not significantly affect tumor proliferation in vitro or in vivo. Transforming growth factor β1 promoted mammary tumor local invasion. Furthermore, the CD44high/CD24− cancer stem cell population was also significantly increased by transforming growth factor β1 treatment. Besides, the gene expression of the stem cell self-renewal markers (nanog, pou5f1, and sox2) and another stem cell surface marker (EpCAM) was increased by transforming growth factor β1 treatment. Finally, clusters of CD44-positive breast cancer cells were observed in the livers of mice from the control and transforming growth factor β1 pretreatment groups.Conclusion:Our results indicate that transforming growth factor β1 increases the local invasive capacity and liver metastasis of breast cancer cells by inducing the CD44high/CD24− cancer stem cell population.

Autophagy Is Indispensable for the Self-Renewal and Quiescence of Ovarian Cancer Spheroid Cells with Stem Cell-Like Properties.

Epithelial ovarian cancer has the highest mortality rate of all gynecologic cancers. Cancer stem cells are considered to be the initiating cells of tumors. It is known that spheroid culture promotes ovarian cancer cells to acquire stem cell characteristics and to become stem cell-like. But the mechanisms remain largely unclear. Our data show that autophagy is sustainably activated in ovarian cancer spheroid cells. Inhibition of autophagy by knockdown of ATG5 abolishes the self-renewal ability of ovarian cancer spheroid cells. Knockdown of ATG5 prevents ovarian cancer spheroid cells to enter quiescent state. Autophagy is critical for quiescent ovarian cancer spheroid cells to reenter the cell cycle because rapamycin can promote quiescent ovarian cancer spheroid cells to form colonies on soft agar and knockdown of ATG5 can arrest ovarian cancer cells in G0/G1. Autophagy and NRF2 form a positive feedback regulation loop to regulate reactive oxygen species (ROS) levels in ovarian cancer spheroid cells. The optimal ROS level, neither too high nor too low, facilitates the self-renewal marker, NOTCH1, to reach to the highest level. Bafilomycin A1 can impair the self-renewal of ovarian cancer spheroid cells by disturbing ROS levels.

Combinatorial effects of an epigenetic inhibitor and ionizing radiation contribute to targeted elimination of pancreatic cancer stem cell.

Pancreatic cancer is associated with a high mortality rate, owing to de novo and acquired drug resistance, thereby leading to highly invasive and metastatic pancreatic cancer cells. Therefore, targeting pancreatic cancer stem cells (CSCs) may be a novel therapeutic strategy for the treatment of pancreatic cancer. Here, we combined a DNA methylation inhibitor (5-aza-2’-deoxycytidine; 5-aza-dC) and ionizing radiation (IR) to improve anti-cancer effects by inhibiting growth and proliferation and promoting apoptosis of pancreatic cancer cells in vitro and in vivo. Importantly, the combinatorial effect of 5-aza-dC with IR on sphere-forming pancreatic cancer cells was preferentially targeted toward CSCs through the downregulation of regulatory factors of self-renewal and CSC surface markers. We next performed the RNA sequencing to understand the underlying cellular mechanisms of the combined treatment with IR and 5-aza-dC in pancreatic cancer cells. Global transcriptome profiling indicated that the expression of the Oct4-centered transcriptional network of genes was significantly downregulated in cells with combination treatment. Our data suggested that combination treatment with DNA methylation inhibitor and IR may be a novel therapeutic strategy for pancreatic cancer. Overall, these findings support the use of epigenetic therapy in combination with radiotherapy to improve therapeutic efficacy by targeting and eradicating pancreatic CSCs.

Bisphenol A Affects on the Functional Properties and Proteome of Testicular Germ Cells and Spermatogonial Stem Cells in vitro Culture Model

The endocrine disruptor bisphenol A (BPA) is well known for its adverse effect on male fertility. Growing evidence suggests that BPA may interact with testicular germ cells and cause infertility as a result of its estrogenic activity. Objective of current in vitro study was to investigate the proliferation, survivability and stemness properties of mouse testicular germ cells exposed to BPA, and to evaluate possible expression of cellular proteome. Our results showed that germ cell viability and proliferation were not affected by low concentrations (0.01, 0.1, 1, and 10 µM) although significant reduction observed at 100 µM BPA. Germ cell self-renewal and differentiation related marker proteins expression found unchanged at those concentrations. When BPA-exposed germ cells were transplanted into recipient testes, we observed fewer colonies at higher concentrations (10 and 100 µM). Additionally, a significant frequency of recombination failure during meiosis was observed in 10 µM BPA-exposed germ cell transplanted recipient. Moreover, experiment on continuous BPA-exposed and 100 µM BPA-recovered germ cells suggested that spermatogonial stem cells are more potential to survive in adverse environment. Finally, scrutinizing differentially expressed cellular proteins resulted from our proteomic analysis, we conclude that BPA exposure might be associated with several health risks and infertility.

Cancer stem cell-related gene expression as a potential biomarker of response for first-in-class imipridone ONC201 in solid tumors.

Cancer stem cells (CSCs) correlate with recurrence, metastasis and poor survival in clinical studies. Encouraging results from clinical trials of CSC inhibitors have further validated CSCs as therapeutic targets. ONC201 is a first-in-class small molecule imipridone in Phase I/II clinical trials for advanced cancer. We have previously shown that ONC201 targets self-renewing, chemotherapy-resistant colorectal CSCs via Akt/ERK inhibition and DR5/TRAIL induction. In this study, we demonstrate that the anti-CSC effects of ONC201 involve early changes in stem cell-related gene expression prior to tumor cell death induction. A targeted network analysis of gene expression profiles in colorectal cancer cells revealed that ONC201 downregulates stem cell pathways such as Wnt signaling and modulates genes (ID1, ID2, ID3 and ALDH7A1) known to regulate self-renewal in colorectal, prostate cancer and glioblastoma. ONC201-mediated changes in CSC-related gene expression were validated at the RNA and protein level for each tumor type. Accordingly, we observed inhibition of self-renewal and CSC markers in prostate cancer cell lines and patient-derived glioblastoma cells upon ONC201 treatment. Interestingly, ONC201-mediated CSC depletion does not occur in colorectal cancer cells with acquired resistance to ONC201. Finally, we observed that basal expression of CSC-related genes (ID1, CD44, HES7 and TCF3) significantly correlate with ONC201 efficacy in >1000 cancer cell lines and combining the expression of multiple genes leads to a stronger overall prediction. These proof-of-concept studies provide a rationale for testing CSC expression at the RNA and protein level as a predictive and pharmacodynamic biomarker of ONC201 response in ongoing clinical studies.

Prostate Tumor Overexpressed-1 (PTOV1) promotes docetaxel-resistance and survival of castration resistant prostate cancer cells.

Metastatic prostate cancer is presently incurable. The oncogenic protein PTOV1, first described in prostate cancer, was reported as overexpressed and significantly correlated with poor survival in numerous tumors. Here, we investigated the role of PTOV1 in prostate cancer survival to docetaxel and self-renewal ability. Transduction of PTOV1 in docetaxel-sensitive Du145 and PC3 cells significantly increased cell survival after docetaxel exposure and induced docetaxel-resistance genes expression (ABCB1, CCNG2 and TUBB2B). In addition, PTOV1 induced prostatospheres formation and self-renewal genes expression (ALDH1A1, LIN28A, MYC and NANOG). In contrast, Du145 and PC3 cells knockdown for PTOV1 significantly accumulated in the G2/M phase, presented a concomitant increased subG1 peak, and cell death by apoptosis. These effects were enhanced in docetaxel-resistant cells. Analyses of tumor datasets show that PTOV1 expression significantly correlated with prostate tumor grade, drug resistance (CCNG2) and self-renewal (ALDH1A1, MYC) markers. These genes are concurrently overexpressed in most metastatic lesions. Metastases also show PTOV1 genomic amplification in significant co-occurrence with docetaxel-resistance and self-renewal genes. Our findings identify PTOV1 as a promoter of docetaxel-resistance and self-renewal characteristics for castration resistant prostate cancer. The concomitant increased expression of PTOV1, ALDH1A1 and CCNG2 in primary tumors, may predict metastasis and bad prognosis.

Abstract 4810: Loss of Cav-1 is associated with manifestation of stem cell-like properties via Src-mediated phosphorylation in human breast MDA-MB-231 cancer cells

Abstract A subpopulation of cancer cells, termed cancer stem cells (CSCs), has self-renewal and differentiation potential, which contributes to cancer recurrence and tumor resistance. Caveolin-1 (Cav-1), the major structural protein of flask-shaped plasma membrane invaginations called caveolae, functions as a tumor suppressor or an oncogene depending on the tumor type and the grade. Caveolae participate in various cellular functions, such as vesicle trafficking, cholesterol homeostasis, signal transduction, and tumor progression. In the present study, we investigated the role of Cav-1 on the stemness of human breast cancer (MDA-MB-231) cells. A sphere formation assay has been widely used to enrich for CSCs through serial passaging. A level of Cav-1 protein expression decreased in third-generation mammospheres as compared to parent MDA-MB-231 cells. Under the same experimental conditions, however, there was no concurrent decrease in the Cav-1 mRNA level. Therefore, it is likely that stability of Cav-1 protein is decreased during mammosphere formation. Recently, it has been reported that sustained phosphorylation of Cav-1 promotes its ubiquitination and degradation by the proteasomes. We observed that PP2, an inhibitor of Src, suppressed phosphorylation of Cav-1 in MDA-MB-231-derived mammosphere cells, which in turn increases its protein stability. Breast CSCs are enriched in cells with enhanced expression of phenotypic markers, such as CD44+/CD24-. We found that Cav-1 gene silencing using siRNA enhanced the proportion of CD44+/CD24- MDA-MB-231 cells. In addition, Cav-1 knockdown increased the number and the size of secondary mammospheres, and this was accompanied by enhanced expression of Bmi-1 and Sox-2, which are representative self-renewal markers. Moreover, epithelial-mesenchymal transition (EMT) was decreased by knockdown of Cav-1 in MDA-MB-231 cells. Taken together, these findings suggest that loss of Cav-1 protein stability facilitates the formation of mammospheres through upregulation of self-renewal and EMT markers in breast CSCs. Citation Format: Hyo-Jin YOON, Do-Hee KIM, Young-Nam CHA, Young-Joon SURH. Loss of Cav-1 is associated with manifestation of stem cell-like properties via Src-mediated phosphorylation in human breast MDA-MB-231 cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4810. doi:10.1158/1538-7445.AM2017-4810

Abstract LB-227: Combined treatment with radiation and epigenetic regulation improve to targeting cancer stem cells in pancreatic cancer

Abstract Increasing evidences suggests that various cancer types contain a population of cells that display stem cell properties. These cells are called cancer stem cells (CSCs). They are virtually resistant to radiation, and may contribute to treatment resistance and recurrence. Therefore, therapies specifically targeting CSCs will likely be needed for complete tumor eradication. Exposure of cells to ionizing radiation (IR) induces, not only, activation of multiple signaling pathways that play critical roles in cell fate determination, but also alteration of molecular pathways involved in cell death or survival. Recently, DNA methylation has been established as a critical epigenetic process involved in the regulation of gene expression in cancer cells, suggesting that DNA methylation inhibition may be an effective cancer treatment strategy. We therefore investigated the combination effect of a DNA methyltransferase inhibitor, 5-aza-2’-deoxycytidine (5-aza-dC), and radiation treatment in pancreatic cancer. Pancreatic cancer cell lines were initially tested for radiation sensitivity by ionizing radiation (IR) in vitro and were treated with 5-aza-dC. The effects of 5-aza-dC along with irradiation on cell growth, cell cycle distribution, apoptosis, and apoptosis-related gene expression were examined. Combination irradiation treatment with 5-aza-dC significantly decreased growth activity compared with irradiation or 5-aza-dC treatment alone. To understand whether combination treatment can target to pancreatic cancer stem cells, we observed that combination treatment can significantly reduce Oct4, Nanog, Sox2, CD133, and CD44 levels which are key factors for self-renewal pathway or cancer stem cell surface markers in pancreatic cancer cells, respectively. Using transcriptome analysis, OCT4 target genes showed differential expression pattern between irradiation or 5-aza-dC treatment alone and combination treatment. In addition, sphere forming assay supports these data in vitro and in vivo, describing that combination effect of IR and 5-aza-dC can reduce significantly sphere forming ability and proportion of side population (putative CSCs) significantly decreased in combination treated cells compared to control or single treatment. Therefore combined treatment of IR and 5-aza-dC is capable of eliminating pancreatic CSCs. Further investigation of this useful approach may lead to the development of a novel intervention for the cancer therapeutic strategy. Citation Format: Joo Mi Yi, Hyun-Mi Kwon, Eun-Jin Kang, Kwangmo Yang. Combined treatment with radiation and epigenetic regulation improve to targeting cancer stem cells in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-227. doi:10.1158/1538-7445.AM2017-LB-227

The molecular characterization of porcine egg precursor cells.

Female-factor infertility can be caused by poor oocyte quality and depleted ovarian reserves. Egg precursor cells (EPCs), isolated from the ovarian cortex, have the potential to be used to overcome female infertility. We aimed to define the origins of EPCs by analyzing their gene expression profiles and mtDNA content using a mini-pig model. We characterized FAC-sorted DDX4+-derived porcine EPCs by performing RNA-sequencing and determined that they utilize pathways important for cell cycle and proliferation, which supports the existence of adult mitotically active oogonial cells. Expression of the pluripotent markers Sox2 and Oct4, and the primitive germ cell markers Blimp1 and Stella were not detected. However, Nanog and Ddx4 were expressed, as were the primitive germ cell markers Fragilis, c-Kit and Tert. Moreover, porcine EPCs expressed self-renewal and proliferation markers including Myc, Esrrb, Id2, Klf4, Klf5, Stat3, Fgfr1, Fgfr2 and Il6st. The presence of Zp1, Zp2, Zp3 and Nobox were not detected, indicating that porcine EPCs are not indicative of mature primordial oocytes. We performed mitochondrial DNA Next Generation Sequencing and determined that one mtDNA variant harbored by EPCs was present in oocytes, preimplantation embryos and somatic tissues over three generations in our mini-pig model indicating the potential germline origin of EPCs.

Establishment of a tumor sphere cell line from a metastatic brain neuroendocrine tumor.

Neuroendocrine tumors are rare, and little is known about the existence of cancer stem cells in this disease. Identification of the tumorigenic population will contribute to the development of effective therapies targeting neuroendocrine tumors. Surgically resected brain metastases from a primary neuroendocrine tumor of unknown origin were dissociated and cultured in serum-free neurosphere medium. Stem cell properties, including self-renewal, differentiation potential, and stem cell marker expression, were examined. Tumor formation was evaluated using intracranial xenograft models. The effect of temozolomide was measured in vitro by cell viability assays. We established the neuroendocrine tumor sphere cell line ANI-27S, which displayed stable exponential growth, virtually unlimited expansion in vitro, and expression of stem-cell markers such as CD133, nestin, Sox2, and aldehyde dehydrogenase. FBS-induced differentiation decreased Sox2 and nestin expression. On the basis of real-time PCR, ANI-27S cells expressed the neuroendocrine markers synaptophysin and chromogranin A. Intracranial xenotransplanted brain tumors recapitulated the original patient tumor and temozolomide exhibited cytotoxic effects on tumor sphere cells. For the first time, we demonstrated the presence of a sphere-forming, stem cell-like population in brain metastases from a primary neuroendocrine tumor. We also demonstrated the potential therapeutic effects of temozolomide for this disease.

Spatial development of gingival fibroblasts and dental pulp cells: Effect of extracellular matrix.

Cells sensing changes in their microenvironmental stiffness and composition alter their responses, accordingly. This study determines whether gingival fibroblasts (GFs) and dental pulp mesenchymal stem cells (DPMSCs) support the formation of continuous layers in vitro by mimicking the stiffness and protein composition of their native extracellular matrix (ECM). Immortalized cells were incubated with (i) 0-100% Matrigel-ECM (M-ECM) for 7-28d, and with (ii) collagen and fibrin matrices for 14d. Cultures were analyzed by phase-contrast, fluorescence and confocal microscopies. The diameters and surface areas were measured via ImageJ. Self-renewal markers were detected by RT-PCR and immunocytochemistry assays. GFs and DPMSCs developed spheroids interconnected by elongated cell bundles or layers, respectively, expressing the self-renewal markers. Increased matrix stiffness resulted in spheroids replacement by the interconnecting cells/layers. Both cells required 100% M-ECM to reduce their spheroid diameter. However, it reduced the surface area of the interconnecting layers. Those differences led to extended, spindle-shaped GFs vs. compact, ring-shaped DPMSCs constructs. Collagen and fibrin matrices developed continuous layers of tightly connected cells vs. distinctive scattered cell aggregates, respectively. The ability of GFs and DPMSCs to create tissue-like multicellular layers at various matrix conditions may be imprinted by cells' adaptation to mechanical forces and composition in vivo.

HPaf1/PD2 interacts with OCT3/4 to promote self-renewal of ovarian cancer stem cells.

Cancer stem cells (CSCs), which mediate drug resistance and disease recurrence in several cancers, are therapeutically relevant to ovarian cancer (OC), wherein approximately 80% of patients manifest with tumor recurrence. While there are several markers for ovarian CSCs (OCSCs), the mechanism for their self-renewal maintenance by unique driver/markers is poorly understood. Here, we evaluated the role of hPaf1/PD2, a core component of RNA Polymerase II-Associated Factor (PAF) complex, in self-renewal of OCSCs through marker and functional analyses, including CRISPR/Cas9-silencing of hPaf1/PD2 in OCSCs and provided a possible mechanism for maintenance of OCSCs. Expression of hPaf1/PD2 showed moderate to intense staining in 32.4% of human OC tissues, whereas 67.6% demonstrated basal expression by immunohistochemistry analysis, implying that the minor proportion of cells overexpressing hPaf1/PD2 could be putative OCSCs. Isolated OCSCs showed higher expression of hPaf1/PD2 along with established CSC and self-renewal markers. Knockdown of hPaf1/PD2 in OCSCs resulted in a significant downregulation of CSC and self-renewal markers, and impairment of in vitro tumor sphere (P < 0.05) and colony formation (P = 0.013). Co-immunoprecipitation revealed that OCT3/4 specifically interacts with hPaf1/PD2, and not with other PAF components (Ctr9, Leo1, Parafibromin) in OCSCs, suggesting a complex-independent role for hPaf1/PD2 in OCSC maintenance. Moreover, there was a significant overexpression and co-localization of hPaf1/PD2 with OCT3/4 in OC tissues compared to normal ovary tissues. Our results indicate that hPaf1/PD2 is overexpressed in OCSCs and maintains the self-renewal of OCSCs through its interaction with OCT3/4; thus, hPaf1/PD2 may be a potential therapeutic target to overcome tumor relapse in OC.

Evidence of functional duplicity of Nestin expression in the adult mouse midbrain

Whether or not neurogenesis occurs in the adult substantia nigra pars compacta (SNc) is an important question relevant for developing better treatments for the motor symptoms of Parkinson's disease (PD). Although controversial, it is generally believed that dividing cells here remain undifferentiated or differentiate into glia, not neurons. However, there is a suggestion that Nestin-expressing neural precursor cells (NPCs) in the adult SNc have a propensity to differentiate into neurons, which we sought to confirm in the present study. Adult (>8-weeks old) transgenic NesCreERT2/GtROSA or NesCreERT2/R26eYFP mice were used to permanently label Nestin-expressing cells and their progeny with β-galactosidase (β-gal) or enhanced yellow fluorescent protein (eYFP), respectively. Most β-gal+ or eYFP+ cells were found in the ependymal lining of the midbrain aqueduct (Aq) and in the midline ventral to Aq. Smaller but significant numbers were in the periaqueductal gray (PAG), the ventral tegmental area (VTA), and in SNc. Low-level basal proliferation was evidenced by a modest increase in number of β-gal+ or eYFP+ cells over time, fewer β-gal+ or eYFP+ cells when mice were administered the anti-mitotic agent Cytarabine, and incorporation of the proliferation marker bromodeoxyuridine (BrdU) in a very small number of β-gal+ cells. No evidence of migration was found, including no immunoreactivity against the migration markers doublecortin (DCX) or polysialic acid neural cell adhesion molecule (PSA-NCAM), and no dispersal of β-gal+ or eYFP+ cells through the midbrain parenchyma over time. However, β-gal+ or eYFP+ cells did increase in size and express higher levels of mature neuronal genes over time, indicating growth and neuronal differentiation. In mice whose SNc dopamine neurons had been depleted with 6-hydroxy-dopamine, a model of PD, there were ~2-fold more β-gal+ cells in SNc specifically, although the proportion that were also NeuN+ was not affected. Remarkably, as early as 4days following putative Nestin-expression, many β-gal+ or eYFP+ cells had mature neuronal morphology and were NeuN+. Furthermore, mature neuronal β-gal+ cells were immunoreactive against the self-renewal or pluripotency marker sex determining region Y-box 2 (Sox2). Overall, our data support the notion that some Nestin-expressing, presumably NPCs, have a limited capacity for proliferation, no capacity for migration, and a propensity to generate new neurons within the microenvironment of the adult midbrain. However, our data also suggest that significant numbers of extant midbrain neurons express Nestin and other classical neurogenesis markers in contexts that are presumably not neurogenic. These findings foreshadow duplicitous roles for Nestin and other molecules that are traditionally associated with neurogenesis in the adult midbrain, which should be considered in future PD research.