Early Passage Cells Research Articles

Cyclin A1 Is Essential for Setting the Pluripotent State and Reducing Tumorigenicity of Induced Pluripotent Stem Cells

The proper differentiation and threat of cancer rising from the application of induced pluripotent stem (iPS) cells are major bottlenecks in the field and are thought to be inherently linked to the pluripotent nature of iPS cells. To address this question, we have compared iPS cells to embryonic stem cells (ESCs), the gold standard of ground state pluripotency, in search for proteins that may improve pluripotency of iPS cells. We have found that when reprogramming somatic cells toward pluripotency, 1%-5% of proteins of 5 important cell functions are not set to the correct expression levels compared to ESCs, including mainly cell cycle proteins. We have shown that resetting cyclin A(1) protein expression of early-passage iPS cells closer to the ground state pluripotent state of mouse ESCs improves the pluripotency and reduces the threat of cancer of iPS cells. This work is a proof of principle that reveals that setting expression of certain proteins correctly during reprogramming is essential for achieving ESC-state pluripotency. This finding would be of immediate help to those researchers in different fields of iPS cell work that specializes in cell cycle, apoptosis, cell adhesion, cell signaling, and cytoskeleton.

Dnmt3a regulates both proliferation and differentiation of mouse neural stem cells

DNA methylation is known to regulate cell differentiation and neuronal function in vivo. Here we examined whether deficiency of a de novo DNA methyltransferase, Dnmt3a, affects in vitro differentiation of mouse embryonic stem cells (mESCs) to neuronal and glial cell lineages. Early-passage neural stem cells (NSCs) derived from Dnmt3a-deficient ESCs exhibited a moderate phenotype in precocious glial differentiation compared with wild-type counterparts. However, successive passaging to passage 6 (P6), when wild-type NSCs become gliogenic, revealed a robust phenotype of precocious astrocyte and oligodendrocyte differentiation in Dnmt3a(-/-) NSCs, consistent with our previous findings in the more severely hypomethylated Dnmt1(-/-) NSCs. Mass spectrometric analysis revealed that total levels of methylcytosine in Dnmt3a(-/-) NSCs at P6 were globally hypomethylated. Moreover, the Dnmt3a(-/-) NSC proliferation rate was significantly increased compared with control from P6 onward. Thus, our work revealed a novel role for Dnmt3a in regulating both the timing of neural cell differentiation and the cell proliferation in the paradigm of mESC-derived-NSCs.

The neural plasticity of early-passage human bone marrow-derived mesenchymal stem cells and their modulation with chromatin-modifying agents

Mesenchymal stem cells (MSCs) in their immature state express a variety of genes of the three germ layers at relatively low or moderate levels that might explain their phenomenal plasticity. Numerous recent studies have demonstrated that under the appropriate conditions in vitro and in vivo the expression of different sets of these genes can be upregulated, turning MSCs into variety of cell lineages of mesodermal, ectodermal and endodermal origin. While transdifferentiation of MSCs is still controversial, these unique properties make MSCs an ideal autologous source of easily reprogrammable cells. Recently, using the approach of cell reprogramming by biological active compounds that interfere with chromatin structure and function, as well as with specific signalling pathways that promote neural fate commitment, we have been able to generate neural-like cells from human bone marrow (BM)-derived MSCs (hMSCs). However, the efficiency of neural transformation of hMSCs induced by this approach gradually declined with passaging. To elucidate the mechanisms that underlie the higher plasticity of early-passage hMSCs, comparative analysis of the expression levels of several pluripotent and neural genes was conducted for early- and late-passage hMSCs. The results demonstrated that early-passage hMSCs expressed the majority of these genes at low and moderate levels that gradually declined at late passages. Neural induction further increased the expression of some of these genes in hMSCs, accompanied by morphological changes into neural-like cells. We concluded that low and moderate expression of several pluripotent and neural genes in early-passage hMSCs could explain their higher plasticity and pliability for neural induction.

Role of cytokine-induced plasticity in melanoma on invasive, therapy-resistant cells that express EMT-related genes and pathways.

8542 Background: The acquisition of new mutations can explain resistance to targeted therapies; however resistance also develops without demonstrable new mutations. To better understand potential mechanisms for treatment failure we studied subpopulations of chemo-resistant cells that can be identified in early passage cell lines and the cellular plasticity that yields these. Methods: Melanoma subpopulations were identified and sorted on the basis of functional assays. Gene expression was evaluated with Illumina HT12 arrays and qPCR to identify potential mechanisms and targets. Sorted cells were evaluated in vitro for the induction of phenotype switching and effects of target inhibition. Results: A subset of slow proliferating label-retaining cells (LRC) was identified using a membrane dye. Direct isolation of LRC from a pt showed these were not an in vitro artefact. LRC isolated from multiple cell lines comprised the majority of cells that resisted cytotoxic drugs and could invade through an artificial extracellular membrane. Gene expression profiling identified a network of over-expressed genes related to epithelial-to-mesenchymal transition (EMT) notable for the expression of Thrombospondin-1 (TSP-1), Transforming-Growth Factor, Beta Induced, (TGFBI) and other extracellular matrix molecules. Initial studies show that LRC shared gene expression characteristics with PLX4032-resistant cells. Although interrogating LRC by qPCR showed up-regulation of the putative melanoma stem cell marker ABCB5 and the lysine specific demethylase Jarid1B, cell sorting and serial re-labelling experiments revealed a dynamic and interchangeable phenotype for these cells, challenging the hierarchical stem cell model for melanoma. The observed slow-cycling and chemo-resistant phenotype could be induced in vitro through TGF beta-mediated phenotype switching and cellular invasion was blocked using an antibody against TSP-1. Conclusions: Cytokine-induced plasticity in melanoma yields invasive, therapy-resistant cells which express EMT-related genes and pathways. We hypothesize that these cells are a source of treatment resistance in vivo and blocking their emergence may prevent treatment failure.

Abstract 1496: Changes in white adipose tissue progenitor populations with ovarian cancer

Abstract Ovarian cancer is the fourth leading cause of cancer death in women and has the highest mortality rate of all gynecological malignancies. Among the greatest risk factors are age, reproductive history, hormone therapy, and more recently suggested, obesity. Currently, the mechanism for how obesity contributes to ovarian cancer is unknown, but there is potential for adipose tissue-associated cells to be key players in this pathogenesis. The stromal vascular fraction (SVF) of white adipose tissue is a rich source of various stem and progenitor cell populations, which can be recruited by cancer cells to enhance cancer progression and metastatic potential. Studies have shown that there is an increase in progenitor populations within the adipose tissue with obesity and age, two risk factors for ovarian cancer. While it has been shown that SVF cells can contribute to cancer, it has not been determined what impact cancer progression has on adipose tissue. It is plausible that ovarian cancer has systemic effects, which may influence cell populations within adipose tissue to promote metastases. Our lab has developed a spontaneously transformed murine ovarian surface epithelial (MOSE) cell model that becomes increasingly more aggressive with repeated passaging. Late passage cells rapidly form tumors when injected into mice, while earlier passage cells do not. Using this model, we investigated the effects of ovarian cancer on systemic changes in progenitor populations. Firefly luciferase-expressing MOSE-L TICv cells, a highly aggressive, metastatic tumor-initiating cell variant were injected into the peritoneal cavity of C57BL/6 mice and stem/progenitor populations and various immune cell populations within the perigonadal white adipose tissue, blood, ascites, and omentum were immunophenotyped by flow cytometry. Changes in gene expression profiles were also analyzed using quantitative real-time PCR. Interestingly, peritoneal dissemination changed the profile and composition of progenitor populations in the various tissues, suggesting that ovarian cancer does have a more widespread impact, affecting the blood and perigonadal adipose tissue. Additionally, co-culture studies demonstrate that MOSE cells and SVF associated cells positively interact enhancing tumor spheroid growth and provide survival signals. This suggests a synergistic relationship between the MOSE and SVF cells. These discoveries may provide a mechanistic link for the detrimental impact of obesity on ovarian cancer progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1496. doi:1538-7445.AM2012-1496

Abstract 3322: Phenotypic plasticity in the normal breast

Abstract Metaplasia is observed in almost all epithelial cancers. The origins of metaplasia are obscure although chronic inflammation is thought to be one etiology. We present data that suggests that an origin of metaplasia is the normal resident stem cell population. Methods: Starting from 10 gauge tissue cores of normal breast donated to the Komen Tissue Bank, 28 normal mammary epithelial (HME) and 33 normal stromal (HMS) cell lines were established using an organoid isolation method after digestion with enzymes for 24 hours. The HME cell lines were characterized by immunohistochemistry (IHC). Ploidy was assayed. Cellular morphology was observed both on two-dimensional and in three-dimensional culture systems. The HME cells were subjected to FACS analysis using multiple antibodies. Results: 96.9% of early passage cells are diploid. The HME cells express vimentin, CK 5/6, p63, CD 10, CK 18, and HER-1 when grown on two dimensional plastic surfaces. Cells placed in the center of a sandwich of Matrigel uniformly make spheres 37mm-325mm in diameter. Hematoxylin and eosin staining of the formalin-fixed and paraffin-embedded sections of these spheres reveal keratinized squamous differentiation. When the cells are grown on Laminin, Collagen Type IV, or Fibronectin surfaces multiple cell types are observed including osteoclasts, distinguished by the presence of Tartrate Resistant Acid Phospatase; and chondrocytes, confirmed by staining with Alcian Blue. Other cells with a spindle-shape and cytoplasmic vacuoles turn a dark reddish-brown color when stained with Oil Red O, characteristics of adipocytes. In other areas of the culture, the cells form a syncitium and they express the protein MyoD, a marker of immature muscle. Finally, there are numerous cells with long, dendritic processes. These cells express Nestin, glial fibrillary acidic protein (GFAP), and beta-III tubulin. Using FACS, the HME cells were found to be CD49f positive and EpCAM negative. Multiple nucleoli were confirmed using anti-Nucleostemin IHC. Conclusions: Phenotypic plasticity is common to all the HME cell lines characterized to date. Differentiation into cells of mesodermal and ectodermal origin, CD49+/EpCAM- by FACS, and the presence of multiple nucleoli suggest that the isolated cells are a multipotent/stem cell residing in the normal adult breast. These cells, through a series of yet to be elucidated events, may be the cells of origin of both benign and malignant metaplasia observed in breast lesions. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3322. doi:1538-7445.AM2012-3322

Decrease in nicotinamide adenine dinucleotide dehydrogenase is related to skin pigmentation

Skin pigmentation is caused by various physical and chemical factors. It might also be influenced by changes in the physiological function of skin with aging. Nicotinamide adenine dinucleotide (NADH) dehydrogenase is an enzyme related to the mitochondrial electron transport system and plays a key role in cellular energy production. It has been reported that the functional decrease in this system causes Parkinson's disease. Another study reports that the amount of NADH dehydrogenase in heart and skeletal muscle decreases with aging. A similar decrease in the skin would probably affect its physiological function. However, no reports have examined the age-related change in levels of NADH dehydrogenase in human skin. In this study, we investigated this change and its effect on skin pigmentation using cultured human epidermal keratinocytes. The mRNA expression of NDUFA1, NDUFB7, and NDUFS2, subunits of NADH dehydrogenase, and its activity were significantly decreased in late passage keratinocytes compared to early passage cells. Conversely, the mRNA expression of melanocyte-stimulating cytokines, interleukin-1 alpha and endothelin 1, was increased in late passage cells. On the other hand, the inhibition of NADH dehydrogenase upregulated the mRNA expression of melanocyte-stimulating cytokines. Moreover, the level of NDUFB7 mRNA was lower in pigmented than in nonpigmented regions of skin in vivo. These results suggest the decrease in NADH dehydrogenase with aging to be involved in skin pigmentation.

P5-05-02: Phenotypic Plasticity in the Normal Breast.

Abstract Background: The cell of origin of metaplastic carcinoma of the breast (MCB) is an enigma. MCB comprises less than 4% of all breast cancers, and is a member of the subtype of breast cancer referred to as Triple Negative Breast Cancer (TNBC). It is aggressive with a prognosis worse than that that for invasive ductal carcinoma NOS (not otherwise specified), as well as other TNBCs. Identification of the histogenesis of MCB is likely to provide clues to its oncogenesis. Differentiation along non-epithelial lineages is also observed in benign lesions of the breast. Adenomyoepitheliomas, thought to arise from myoepithelial cells, have been observed to contain areas of cartilaginous, sebaceous, squamous, and osseous differentiation. Methods: Healthy woman volunteers from central Indiana were invited to donate breast tissue to the Susan G. Komen for the Cure® Tissue Bank at the IU Simon Cancer Center. Starting from the 10 gauge tissue cores, 28 normal mammary epithelial (HME) and 33 normal stromal (HMS) cell lines were established using an organoid isolation method after digestion with enzymes for 24 hours. The HME cell lines were characterized by immunohistochemistry (IHC). Ploidy was determined by karyotype and Interphase FISH mapping with centromere probes for Chromosomes X and 17. Cellular morphology was observed both on two-dimensional and in three-dimensional culture systems. The HME cells were subjected to FACS analysis using multiple antibodies including CD24, CD44, Muc1, CD49f, and EpCAM. Results: 96.9% of early passage cells are diploid. The HME cells express vimentin, CK 5/6, p63, CD 10, CK 18, and HER-1 when grown on two dimensional plastic surfaces. Cells placed in the center of a sandwich of Matrigel® uniformly form spheres 37mm-325mm in diameter. Hematoxylin and eosin staining of the formalin-fixed and paraffin-embedded sections of these spheres reveal keratinized squamous differentiation. When the cells are grown on Laminin, Collagen IV, or Fibronectin surfaces multiple cell types are observed including osteoclasts, distinguished by the presence of Tartrate Resistant Acid Phospatase; and chondrocytes, confirmed by staining with Alcian Blue. Other cells with a spindle-shape and cytoplasmic vacuoles turn a dark reddish-brown color when stained with Oil Red O, characteristics of adipocytes. In other areas of the culture, the cells form a syncitium and they express the protein MyoD, a marker of immature muscle. Finally, there are numerous cells with long, dendritic processes. These cells express Nestin, a marker of neural stem cells; glial fibrillary acidic protein (GFAP), expressed by mature astrocytes; and beta-III tubulin produced by differentiated neurons. Using FACS, the HME cells were found to be CD49f positive and EpCAM negative. Multiple nucleoli were confirmed using anti-Nucleostemin IHC. Conclusions: Phenotypic plasticity is common to all the HME cell lines characterized to date. Differentiation into cells of mesodermal and ectodermal origin, CD49+/EpCAM- by FACS, and the presence of multiple nucleoli suggest that the isolated cells are a multipotent/stem cell residing in the normal adult breast. These cells, through a series of yet to be elucidated events, may be the cells of origin of MCB. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-05-02.

Five dysfunctional telomeres predict onset of senescence in human cells

Replicative senescence is accompanied by a telomere-specific DNA damage response (DDR). We found that DDR+ telomeres occur spontaneously in early-passage normal human cells and increase in number with increasing cumulative cell divisions. DDR+ telomeres at replicative senescence retain TRF2 and RAP1 proteins, are not associated with end-to-end fusions and mostly result from strand-independent, postreplicative dysfunction. On the basis of the calculated number of DDR+ telomeres in G1-phase cells just before senescence and after bypassing senescence by inactivation of wild-type p53 function, we conclude that the accrual of five telomeres in G1 that are DDR+ but nonfusogenic is associated with p53-dependent senescence.

Telomere Shortening Alters the Kinetics of the DNA Damage Response after Ionizing Radiation in Human Cells

Studies of telomerase-deficient mice and human cell lines have showed that telomere shortening enhances sensitivity to ionizing radiation (IR). The molecular basis for this observation remains unclear. To better understand the connection between telomere shortening and radiation sensitivity, we evaluated components of the DNA damage response pathway in normal human fibroblasts with short and long telomeres. Late-passage cells with short telomeres showed enhanced sensitivity to IR compared with early-passage cells with longer telomeres. Compared with early-passage cells, late-passage cells had a higher baseline level of phosphorylated H2AX protein (γH2AX) before IR but diminished peak levels of H2AX phosphorylation after treatment with IR. Both the appearance and disappearance of γH2AX foci were delayed in late-passage cells, indicative of delayed DNA repair. In contrast to the situation with H2AX, ATM and p53 phosphorylation kinetics were similar in early- and late-passage cells, but phosphorylation of the chromatin-bound ATM targets SMC1 and NBS1 was delayed in late-passage cells. Because impaired phosphorylation associated with short telomeres was restricted to chromatin-bound ATM targets, chromatin structure was assessed. DNA from cells with short telomeres was more resistant to digestion with micrococcal nuclease, indicative of compacted chromatin. Moreover, cells with short telomeres showed histone acetylation and methylation profiles consistent with heterochromatin. Together our data suggest a model in which short telomeres induce chromatin structure changes that limit access of activated ATM to its downstream targets on the chromatin, thereby providing a potential explanation for the increased radiation sensitivity seen with telomere shortening.

Telomere Dysfunction Causes Sustained Inflammation in Chronic Obstructive Pulmonary Disease

Chronic obstructive pulmonary disease (COPD) is associated with chronic inflammation of unknown pathogenesis. To investigate whether telomere dysfunction and senescence of pulmonary vascular endothelial cells (P-ECs) induce inflammation in COPD. Prospective comparison of patients with COPD and age- and sex-matched control smokers. Investigation of mice null for telomerase reverse transcriptase (Tert) or telomerase RNA component (Terc) genes. In situ lung specimen studies showed a higher percentage of senescent P-ECs stained for p16 and p21 in patients with COPD than in control subjects. Cultured P-ECs from patients with COPD exhibited early replicative senescence, with decreased cell-population doublings, a higher percentage of β-galactosidase-positive cells, reduced telomerase activity, shorter telomeres, and higher p16 and p21 mRNA levels at an early cell passage compared with control subjects. Senescent P-ECs released cytokines and mediators: the levels of IL-6, IL-8, monocyte chemotactic protein (MCP)-1, Hu-GRO, and soluble intercellular adhesion molecule (sICAM)-1 were elevated in the media of P-ECs from patients compared with control subjects at an early cell passage, in proportion to the senescent P-EC increase and telomere shortening. Up-regulation of MCP-1 and sICAM-1 led to increased monocyte adherence and migration. The elevated MCP-1, IL-8, Hu-GROα, and ICAM-1 levels measured in lungs from patients compared with control subjects correlated with P-EC senescence criteria and telomere length. In Tert(-/-) and/or Terc(-/-) mouse lungs, levels of the corresponding cytokines (MCP-1, IL-8, Hu-GROα, and ICAM-1) were also altered, despite the absence of external stimuli and in proportion to telomere dysfunction. Telomere dysfunction and premature P-EC senescence are major processes perpetuating lung inflammation in COPD.

Pulmonary Artery Smooth Muscle Cell Senescence Is a Pathogenic Mechanism for Pulmonary Hypertension in Chronic Lung Disease

Senescence of pulmonary artery smooth muscle cells (PA-SMCs) caused by telomere shortening or oxidative stress may contribute to pulmonary hypertension associated with chronic lung diseases. To investigate whether cell senescence contributes to pulmonary vessel remodeling and pulmonary hypertension in chronic obstructive pulmonary disease (COPD). In 124 patients with COPD investigated by right heart catheterization, we found a negative correlation between leukocyte telomere length and pulmonary hypertension severity. In-depth investigations of lung vessels and derived cultured PA-SMCs showed greater severity of remodeling and increases in senescent p16-positive and p21-positive PA-SMCs and proliferating Ki67-stained cells in 14 patients with COPD compared to 13 age-matched and sex-matched control subjects who smoke. Cultured PA-SMCs from COPD patients displayed accelerated senescence, with fewer cell population doublings, an increased percentage of β-galactosidase-positive cells, shorter telomeres, and higher p16 protein levels at an early cell passage compared to PA-SMCs from controls. Both in situ and in vitro PA-SMC senescence criteria correlated closely with the degree of pulmonary vessel wall hypertrophy. Because senescent PA-SMCs stained for p16 and p21 were virtually confined to the media near the Ki67-positive cells, which predominated in the neointima and hypertrophied media, we evaluated whether senescent cells affected normal PA-SMC functions. We found that senescent PA-SMCs stimulated the growth and migration of normal target PA-SMCs through the production and release of paracrine soluble and insoluble factors. PA-SMC senescence is an important contributor to the process of pulmonary vascular remodeling that underlies pulmonary hypertension in chronic lung disease.

Diversity of Murine Norovirus Strains Isolated from Asymptomatic Mice of Different Genetic Backgrounds within a Single U.S. Research Institute

Antibody prevalence studies in laboratory mice indicate that murine norovirus (MNV) infections are common, but the natural history of these viruses has not been fully established. This study examined the extent of genetic diversity of murine noroviruses isolated from healthy laboratory mice housed in multiple animal facilities within a single, large research institute- the National Institute of Allergy and Infectious Diseases of the National Institutes of Health (NIAID-NIH) in Bethesda, Maryland, U.S. Ten distinct murine norovirus strains were isolated from various tissues and feces of asymptomatic wild type sentinel mice as well as asymptomatic immunodeficient (RAG 2−/−) mice. The NIH MNV isolates showed little cytopathic effect in permissive RAW264.7 cells in early passages, but all isolates examined could be adapted to efficient growth in cell culture by serial passage. The viruses, although closely related in genome sequence, were distinguishable from each other according to facility location, likely due to the introduction of new viruses into each facility from separate sources or vendors at different times. Our study indicates that the murine noroviruses are widespread in these animal facilities, despite rigorous guidelines for animal care and maintenance.

Abstract 2359: Regulation of cytoskeleton organization by sphingosine in a mouse cell model of ovarian cancer progression

Abstract Ovarian cancer is the fifth leading cause of cancer deaths among women mainly because of late detection of the disease due the lack of symptoms and reliable biomarkers. Approximately 90% of ovarian cancers are derived from the surface epithelium of the ovary. To further investigate the events that lead to progression of epithelial ovarian cancer, we previously developed a cell model of ovarian cancer. Mouse ovarian surface epithelial (MOSE) cells that have undergone spontaneous transformation in cell culture were categorized into distinct morphological and phenotypical stages based on passage history: early passage cells (MOSE-E) represent a pre-neoplastic, non-tumorigenic phenotype whereas late passage cells (MOSE-L) exhibit a highly aggressive malignant phenotype both in vitro and in vivo. In depth studies of genetic changes during MOSE cell progression in our labs have identified the cytoskeleton and its regulatory genes as critical early progression events. These changes in gene expression led to an increasingly dysregulated cytoskeleton which may affect the intracellular localization of signaling intermediates, and, thereby, important downstream signaling events involved in cancer progression. Sphingosine (SO), a bioactive metabolite of complex sphingolipids, has been shown to have chemopreventive activity against cancer, including cancer of the colon, breast and ovarian. In the present study, we have investigated the hypothesis that SO prevents or reverses the observed aberrant cytoskeleton organization, and thereby affects the cells metastatic potential. In MOSE-L cells, SO treatment did not significantly influence the gene expression or protein levels of several cytoskeleton components or regulatory proteins. However, immunostaining of SO-treated MOSE-L cells revealed a reversal of the aberrant cytoskeleton organization and mislocalization of regulatory proteins, many of which are associated with cell migration. This corresponds to observations that SO reduces cell motility in MOSE cells. Our data indicates that cytoskeleton re-organization induced by exogenous SO treatment may be involved in the regulation of signaling pathways that reduce proliferation, migration and invasion of the MOSE cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2359. doi:10.1158/1538-7445.AM2011-2359

Abstract 4295: High-throughput screening of osteosarcoma progression: A zebrafish model

Abstract Background: Osteosarcoma is the most common primary malignant bone tumour of non-hematologic origin. The survival of the patients has reached a plateau phase partly due to a poor understanding of osteosarcoma tumorigenesis. Previously we identified murine mesenchymal stem cells (mMSCs) that formed aggressive osteosarcoma-like lesions after injection in mice (Mohseny et al. J Pathol. 2009 Nov;219(3):294-305). In the current study we aimed to set up a high-throughput model to confirm the mMSCs transformation and to investigate further progression of these cells in vivo. Methods: The mMSCs were isolated from the bone marrow of C57BL/6 as well as BALB/c mice and cultured for 8 months. Cells of early and late passages were labeled with a fluorescent dye (CM- Dil) and injected into the yolk sacs of 2-days old transgenic zebrafish embryos with GFP labeled vascular system. The embryos were subjected to live imaging for 3 days to screen for cell homing, proliferation, migration and angiogenesis. Subsequently genome wide mRNA expression analysis for both zebrafish and mouse transcripts was performed. Results: The mMSCs were confirmed to transform in vitro after a period of crisis. High passage mMSCs, i.e. after the transformation, could proliferate, migrate into the fish body and induce angiogenesis whereas low passage MSCs, i.e. prior to transformation, did not. The microarray expression study was technically feasible as mouse transcripts from injected cells were detectable among the more abundant zebrafish RNA. Conclusions: The present study corroborates mMSCs transformation during long-term in vitro culturing. Furthermore it provides an efficient zebrafish model for studying mMSCs tumourigenic progression to characterize clinical relevant processes like migration and angiogenesis. After analyzing both mMSCs and zebrafish embryos on gene-expression level, the differential behavior of normal and transformed mMSCs as well as the host response can provide new insights into osteosarcoma progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4295. doi:10.1158/1538-7445.AM2011-4295

Abstract 3930: Integrative genomic analysis reveals SWI/SNF chromatin remodeling complex to be a TGFβ-mediating tumor suppressor pathway in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) remains a leading cause of cancer deaths in the developed world. A more complete characterization of molecular alterations may suggest new avenues for targeted therapy. Here we report the high-resolution genomic and transcriptional profiling of seventy PDAC early-passage tumor xenografts and cell lines – the largest PDAC dataset currently available of this sample size and resolution. Notable among novel loci, genomic deletion/mutation frequently targeted components of the SWI/SNF chromatin remodeling complex, including ARID1A, ARID1B, PBRM1, SMARCA2 and SMARCA4. Overall, focal deletion/mutation of a SWI/SNF component occurred in at least one-third of all PDAC specimens. In cell culture, RNAi-mediated knockdown of SWI/SNF subunits enhanced cell growth, growth-factor independence, and resistance to TGFβ growth-inhibition. Previously, SWI/SNF was reported to mediate TGFβ signaling. Remarkably, we found that five of the top ten SWI/SNF-mediated TGFβ-downregulated genes were focally amplified (MYC, BIRC3, PARD6B, KLF5, and CEBPD), while two of the top ten upregulated genes were focally deleted (PPAP2B, TNC), implying that its role in TGFβ growth-inhibitory signaling drives SWI/SNF loss in PDAC. In summary, we report the SWI/SNF chromatin remodeling complex, likely through mediating TGFβ signaling, to be a major tumour suppressor pathway in PDAC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3930. doi:10.1158/1538-7445.AM2011-3930

Copy number variation and selection during reprogramming to pluripotency

The mechanisms underlying the low efficiency of reprogramming somatic cells into induced pluripotent stem (iPS) cells are poorly understood. There is a clear need to study whether the reprogramming process itself compromises genomic integrity and, through this, the efficiency of iPS cell establishment. Using a high-resolution single nucleotide polymorphism array, we compared copy number variations (CNVs) of different passages of human iPS cells with their fibroblast cell origins and with human embryonic stem (ES) cells. Here we show that significantly more CNVs are present in early-passage human iPS cells than intermediate passage human iPS cells, fibroblasts or human ES cells. Most CNVs are formed de novo and generate genetic mosaicism in early-passage human iPS cells. Most of these novel CNVs rendered the affected cells at a selective disadvantage. Remarkably, expansion of human iPS cells in culture selects rapidly against mutated cells, driving the lines towards a genetic state resembling human ES cells.

New Insights on Therapeutic Touch: A Discussion of Experimental Methodology and Design That Resulted in Significant Effects on Normal Human Cells and Osteosarcoma

Our purpose is to discuss the study design and innovative approaches that led to finding significant effects of one energy medicine therapy, Therapeutic Touch (TT), on cells. In the original published studies, TT was shown to significantly increase human osteoblast DNA synthesis, differentiation, and mineralization; increase in a dose-dependent manner the growth of other human cell types; and decrease the differentiation and mineralization of a human osteosarcoma-derived cell line. A unique feature of the study's methodology and design that contributed to the success of the findings was that a basic level of skill and maturity of the TT practitioner was quantified for producing observable and replicable outcomes in a test administered to all TT practitioners. Only those practitioners that passed the test were selected for the study. (2) The practitioners were required to keep a journal, which appeared to promote their ability to stay centered and replicate their treatments over months of cell experimentation. (3) The origin of the cells that the practitioners were treating was explained to them, although they were blinded to cell type during the experiments. (4) Only early passage cells were used to maintain a stable cell phenotype. (5) Standard protocols for performing TT in the room were followed to ensure reproducible conditions. (6) Placebo controls and untreated controls were used for each experiment. (7) The principal investigator and technicians performing the assays were blinded as to the experimental groups, and all assays and procedures were well established in the laboratory prior to the start of the TT experiments. The absence of studies on the human biofield from mainstream scientific literature is also discussed by describing the difficulties encountered in publishing. These roadblocks contribute to our lack of understanding of the human biofield and energy medicine modalities in science. In conclusion, this report seeks to encourage well-designed, evidence-based studies on the human biofield and the therapeutic potential of the human biofield.

55 CONSTRUCTION AND IN VITRO DEVELOPMENT OF FOLLISTATIN TRANSGENIC PORCINE EMBRYO BY SOMATIC CELL NUCLEAR TRANSFER

Data from mice showed that follistatin can block myostatin activity in vivo and follistatin overexpressed in transgenic mice can induce dramatic increases in muscle mass. In order to determine whether this effect of follistatin have the same results in swine, we design experiment to make a follistatin transgenic porcine by somatic cell nuclear transfer. We hope to get a follistatin transgenic porcine that has double muscle mass. The present study aimed to construct follistatin transgenic embryos by somatic cell nuclear transfer. A follistatin (FST) expression vector, pFST-1, which contains a porcine FST cDNA (with modification of codon usage) driven by the human cytomegalovirus (CMV) promoter, was constructed. A SV40-neo expression cassette as a selection marker was inserted into pFST-1 to generate pST101, which was transfected into early-passage male primary porcine fetal fibroblast cells by the lipofection method; the transfected cells were selected with 400 mg mL–1 G418. The G418-resistant colonies were pooled. Cell genome DNA was extracted and PCR analysis showed that CMV promoter, follistatin CDS, and SV40 polyA can be detected, indicating that the expression construct had integrated in cell genome. RT-PCR analysis showed that pST101 cells contained higher amounts of FST cDNA compared with the non-transfected cells, indicating that the follistatin was overexpressed in the primary porcine cells. Then, pST101 cell as donor cell and non-transfected cells as control donor cell were used to construct somatic cell nuclear transfer (SCNT) embryos. All of these SCNT embryos were cultured in Porcine Zygote Medium-3. The cleavage rate and blastocyst rate was assessed on 48 h and Day 6. All data were subjected to a Generalized Linear Model Procedure (PROC-GLM) of Statistical Analysis System (SAS, SAS Institute Inc., Cary, NC, USA). The cleavage rate of pST101 cell and control cell derived SCNT embryos were 83.6 ± 2.0% (267/318) and 84.0 ± 2.2% (139/167) respectively, there were no significant (P > 0.05) differences between them. The blastocyst rate from pST101 cell and control cell derived SCNT embryos were 21.4 ± 1.8% (65/318) and 15.0 ± 3.0% (26/167) respectively, the SCNT embryos derived from pST101 cell showed a higher (P < 0.05) blastocyst rate. These results indicated that follistatin transgenic porcine embryo can be successfully constructed and can develop in vitro to blastocyst with a higher rate than control SCNT embryo. Further study will be focused on the full development in vivo of the transgenic embryos and phenotype of transgenic piglets. This work was supported in part by the National High-tech Research and Development Program of China (2008AA101006) and Major Projects for Transgenic Animals of China (2008ZX08006-002).

Tracking Senescence In Human Bone Marrow Stromal Cell (BMSC) Cultures.

AbstractAbstract 1179Bone marrow stromal cell (BMSC) therapy is effective for treating acute-Graft versus Host Disease (aGVHD) and is being investigated as a therapy for graft failure and organ failure following allogeneic hematopoietic stem cell transplantation. Clinical BMSC products are typically produced by serial passage of adherent cells obtained from marrow aspirates. Multiple passages allow for the production of large quantities of human BMSCs, but BMSC expansion is limited by cell senescence. We assessed the nature of BMSC changes associated with multiple passages in order to better understand the nature and impact of senescence on human BMSCs and to identify potential senescence associated biomarkers. BMSCs from marrow aspirates of 5 healthy subjects were cultured in flasks and passed serially until cell growth stopped between the 8th to 12th passages. Early and late passage cells were compared. The early passage BMSCs were passages 1 and 2; because the onset of senescence as assessed by growth characteristics and morphology varied among the 5 donors, the passages included in the late passage group were variable and ranged from passage 5 to 11. The samples (25 total; 10 early and 15 late) were selected based on the replicative lifespan. Senescence was associated with a change to a flattened morphology and increased senescence associated beta-galactosidase (SA β-gal) staining. Early and late passage BMSCs from each subject were also analyzed by flow cytometry and global transcriptome analysis with an oligonucleotide microarray with >44,000 probes. Unsupervised hierarchical clustering analysis separated the 25 BMSC samples into two clusters, one with all early passage and a second one with all late passage samples. A total of 1,739 genes were differentially expressed between the early and late passage BMSCs. Ingenuity pathway analysis of the differentially expressed genes found that those highly expressed in the early passage cells belonged to several immune pathways: cell-mediated immune response, humoral immune responses, hypersensitive responses, and lymphoid tissue structure and development. Genes highly expressed in the late passage BMSCs belonged to the DNA replicative, recombination and repair and nucleic acid metabolism pathways. Specific genes highly expressed in early passage BMSCs compared to late passage BMSCs included the chemokine receptor CXCR7, Secreted frizzle-related protein 4 (SFRP4), WNT1 induced signaling pathway protein 1 (WISP1), lymphocyte-specific protein 1 (LSP1), and insulin-like growth factor 1 (IGF1). Genes up-regulated in late passage cells included the thrombospondin receptor (CD36), coagulation factor III (F3), and tissue plasminogen activator (PLAT). Among the differentially expressed genes were 347 involved with the cell cycle and apoptosis. Hierarchical clustering analysis of these 347 genes again separated the early and late passage cells into two groups and a node of genes more highly expressed in late passages cells included two genes involved with replication senescence, cyclin-dependent kinase inhibitor 1A (p21, Cip1) (CDKN1A) and cyclin-dependent kinase inhibitor 2A (melanoma, p16, inhibits CDK4) (CDKN2A) and one that inhibits TRALI mediated apoptosis, tumor necrosis factor receptor superfamily, member 10d (TNFRSF10D, DCR2, CD264). Flow cytometry analysis confirmed greater expression of TNFRS10D on late passage cells. BMSC senescence is associated with distinct molecular changes. Early passage BMSCs may have more immune modulatory properties. Further studies are needed to determine more precisely the onset of senescence-associated changes. Disclosures:No relevant conflicts of interest to declare.