Maintenance Of Stem Cell Phenotype Research Articles

Characterizing the Effects of Synergistic Thermal and Photo-Cross-Linking during Biofabrication on the Structural and Functional Properties of Gelatin Methacryloyl (GelMA) Hydrogels.

Gelatin methacryloyl (GelMA) hydrogels have emerged as promising and versatile biomaterial matrices with applications spanning drug delivery, disease modeling, and tissue engineering and regenerative medicine. GelMA exhibits reversible thermal cross-linking at temperatures below 37 °C due to the entanglement of constitutive polymeric chains, and subsequent ultraviolet (UV) photo-cross-linking can covalently bind neighboring chains to create irreversibly cross-linked hydrogels. However, how these cross-linking modalities interact and can be modulated during biofabrication to control the structural and functional characteristics of this versatile biomaterial is not well explored yet. Accordingly, this work characterizes the effects of synergistic thermal and photo-cross-linking as a function of GelMA solution temperature and UV photo-cross-linking duration during biofabrication on the hydrogels' stiffness, microstructure, proteolytic degradation, and responses of NIH 3T3 and human adipose-derived stem cells (hASC). Smaller pore size, lower degradation rate, and increased stiffness are reported in hydrogels processed at lower temperature or prolonged UV exposure. In hydrogels with low stiffness, the cells were found to shear the matrix and cluster into microspheroids, while poor cell attachment was noted in high stiffness hydrogels. In hydrogels with moderate stiffness, ones processed at lower temperature demonstrated better shape fidelity and cell proliferation over time. Analysis of gene expression of hASC encapsulated within the hydrogels showed that, while the GelMA matrix assisted in maintenance of stem cell phenotype (CD44), a higher matrix stiffness resulted in higher pro-inflammatory marker (ICAM1) and markers for cell-matrix interaction (ITGA1 and ITGA10). Analysis of constructs with ultrasonically patterned hASC showed that hydrogels processed at higher temperature possessed lower structural fidelity but resulted in more cell elongation and greater anisotropy over time. These findings demonstrate the significant impact of GelMA material formulation and processing conditions on the structural and functional properties of the hydrogels. The understanding of these material-process-structure-function interactions is critical toward optimizing the functional properties of GelMA hydrogels for different targeted applications.

A Regulatory Loop Involving Notch and Wnt Signaling Maintains Leukemia Stem Cells in T-Cell Acute Lymphoblastic Leukemia.

Leukemia-initiating cells play critical role in relapse, resistance to therapies and metastases but the mechanism remains largely elusive. We report that β-catenin is over-expressed in almost all T-ALL patients and flow sorted β-cateninhigh fractions are highly resistant to therapy, leading to liver metastases in nude mice as well as dysregulated lncRNAs. Pharmacological inhibition through XAV-939 as well as si-RNA mediated inhibition of β-catenin is initially effective in re-sensitization to therapy, however, prolonged inhibition shifts dependency from β-catenin to Notch signaling, with particularly high levels of receptors Notch 1 and Notch 2. The results are verifiable in a cohort of T-ALL patients comprising of responders vs. those who have progressed, with β-catenin, Notch 1 and Notch 2 elevated in progressed patients. Further, in patients-derived cells, silencing of Notch 1 or Notch 2 does not counter resistance to β-catenin inhibition, rather pharmacological pan-Notch inhibition is needed to overcome resistance and its effect on in vitro tumor sphere formations as well as in vivo liver metastases. Thus, wnt and Notch signaling are part of a regulatory loop mutually compensating for each other in T-ALL, while ensuring the maintenance of stem cell phenotype.

Chlorella vulgaris Extract as a Serum Replacement That Enhances Mammalian Cell Growth and Protein Expression.

The global cell culture market is experiencing significant growth due to the rapid advancement in antibody-based and cell-based therapies. Both rely on the capacity of different living factories, namely prokaryotic and eukaryotic cells, plants or animals for reliable and mass production. The ability to improve production yield is of important concern. Among many strategies pursued, optimizing the complex nutritional requirements for cell growth and protein production has been frequently performed via culture media component titration and serum replacement. The addition of specific ingredients into culture media to modulate host cells’ metabolism has also recently been explored. In this study, we examined the use of extracted bioactive components of the microalgae Chlorella vulgaris, termed chlorella growth factor (CGF), as a cell culture additive for serum replacement and protein expression induction. We first established a chemical fingerprint of CGF using ultraviolet-visible spectroscopy and liquid chromatography-mass spectrometry and evaluated its ability to enhance cell proliferation in mammalian host cells. CGF successfully promoted the growth of Chinese hamster ovary (CHO) and mesenchymal stem cells (MSC), in both 2D and 3D cell cultures under reduced serum conditions for up to 21 days. In addition, CGF preserved cell functions as evident by an increase in protein expression in CHO cells and the maintenance of stem cell phenotype in MSC. Taken together, our results suggest that CGF is a viable culture media additive and growth matrix component, with wide ranging applications in biotechnology and tissue engineering.

Stem Cell Transcription Factor Sox2 Is Expressed in a Subset of Folliculo-stellate Cells of Growth Hormone-Producing Pituitary Neuroendocrine Tumours and Its Expression Shows No Association with Tumour Size or IGF1 Levels: a Clinicopathological Study of 109 Cases.

Sox2 is one of the transcription factors responsible for the maintenance of stem cell phenotype. It has been implicated as a marker of stem cells in normal pituitaries and pituitary neuroendocrine tumours. To explore the clinical significance of Sox2 expression in histological sections, we performed immunohistochemical detection of Sox2 in 113 pituitary neuroendocrine tumours from 109 patients with acromegaly. In 11 tumours, we performed double immunostaining for Sox2, annexin A1 and S100 protein. Tumours were characterised using the WHO classification system. Proliferative activity and invasion were assessed. The amount of immunoreactive cells was evaluated and correlated with tumour size and biochemical features (levels of IGF1, GH, prolactin, βTSH). Sox2+ cells were identified in 35/38 normal pituitaries adjacent to the tumours. In 36 tumours (33%), ≥ 1% of the cells expressed Sox2, in 24 cases (22%), Sox2+ cells comprised < 1% and 49 cases (45%) were negative. We found no significant differences between Sox2+ and Sox2- groups with respect to the age, initial levels of GH, IGF1, prolactin, βTSH, tumour size, invasion, proliferative activity or histological features. We observed a positive correlation between Sox2+ cell count and βTSH immunoreactive cells (r = 0.459, p< 0.001) that was further verified by multivariate analysis. Using double stain, the majority of Sox2+ cells coexpressed annexin A1 (average 89%) and S100 protein (average 76.2%) and showed morphological features of folliculo-stellate cells. Sox2+ cells are thus commonly present in growth hormone-producing tumours and normal pituitaries, and their amount does not have any prognostic significance. Most of these cells represent a subpopulation of folliculo-stellate cells, pointing out to their role as a possible stem cell population.

Development and characterization of cancer stem cell-based tumoroids as an osteosarcoma model.

Three-dimensional (3D) cancer tumor models are becoming vital approaches for high-throughput drug screening, drug targeting, development of novel theranostic systems, and personalized medicine. Yet, it is becoming more evident that the tumor progression and metastasis is fueled by a subpopulation of stem-like cells within the tumor that are also called cancer stem cells (CSCs). This study aimed to develop a tumoroid model using CSCs. For this purpose CD133+ cells were isolated from SaOS-2 osteosarcoma cell line with magnetic-activated cell sorting. To evaluate tumoroid formation ability, the cells were incubated in different cell numbers in agar gels produced by 3D Petri Dish® method. Subsequently, CD133+ cells and CD133- cells were co-cultured to investigate CD133+ cell localization in tumoroids. The characterization of tumoroids was performed using Live&Dead staining, immunohistochemistry, and quantitative polymerase chain reaction analysis. The results showed that, CD133+ , CD133- and SaOS-2 cells were all able to form 3D tumoroids regardless of the initial cell number, but, while 72 hr were needed for CD133+ cells to self-assemble, 24 hr were enough for CD133- and SaOS-2 cells. CD133+ cells were located within tumoroids randomly with high cell viability. Finally, when compared to two-dimensional (2D) cultures, there were 5.88, 4.14, 6.95, and 1.68-fold higher messenger RNA expressions for Sox2, OCT3/4, Nanog, and Nestin, respectively, in CD133+ cells that were cultured within 3D tumoroids, showing longer maintenance of stem cell phenotype in 3D, that can allow more relevant screening and targeting efficiency in pharmaceutical testing. It was concluded that CSC-based tumoroids are propitious as 3D tumor models to fill the gap between conventional 2D in vitro culture and in vivo animal experiments for cancer research.

Abstract 3117: Eribulin is a novel TERT-targeting inhibitor that penetrates into intracerebrally grown glioblastomas and suppresses their growth in mice

Abstract [Introduction] No effective molecular targeting therapy has been clinically developed for patients with glioblastoma (GBM) despite of intense genome analysis. Approximately 60-80% of GBM harbor mutations in the promoter region of TERT that leads to its upregulation, making it the most common single genetic abnormalities in GBM. TERT, a reverse-transcriptase subunit of telomerase, has been an attractive candidate for therapy target because of their observed upregulation in a wide variety of cancers, including GBMs, a phenomenon that presumably helps maintain telomeres for their indefinite proliferation. It has been reported recently that TERT has an RNA-dependent RNA polymerase (RdRP) activity, with which TERT is involved in maintenance of stem cell phenotype and mitotic progression. We have previously identified eribulin as a specific inhibitor of RdRP activity of TERT through drug screening and shown that eribulin suppressed growth of ovarian cancers with high TERT expression. In order to investigate an efficacy of eribulin as a novel TERT-targeted therapy against GBM, we investigated the antitumor effect of eribulin in cultured cell and brain tumor model using GBM cell lines. [Methods] The TERT promoter status was examined in 20 glioma cell lines. Seven cell lines were subjected to in vitro cytotoxicity assay. U87MG was either subcutaneously or intracranially transplanted in athymic mice. Tissue or plasma concentration of eribulin was measured by LC-MS/MS. [Results] All GBM cell lines tested that harbored TERT promoter mutations including U87MG, U251MG, U118MG as well as several patient-derived glioblastoma sphere culture cells were highly sensitive to eribulin, IC50 below 1nM. Intraperitoneal administration of eribulin completely suppressed the growth of U87MG subcutaneous tumor in nude mice, and the RdRP activities in treated tumors were significantly decreased in a dose-dependent manner. In the brain tumor model, a high concentration of eribulin was detected in the brain tumor tissues as early as 15 minutes after intravenous injection of eribulin, which remained stable even 24 hours later when the plasma concentration of eribulin became undetectable. Finally, intraperitoneal administration of eribulin significantly prolonged the survival of mice with intracerebrally transplanted U87MG xenograft (p&amp;lt;0.001). [Conclusion] Our results showed that eribulin efficiently transfers into brain tumor tissues and has a strong antitumor effect against GBM cells through inhibition of RdRP activity. Eribulin thus appears to be a promising novel TERT-targeting therapeutic agent against GBM. A clinical trial is being scheduled. Citation Format: Masamichi Takahashi, Shunichiro Miki, Yuko Matsushita, Kohei Fukuoka, Yoshiko Maida, Mami Yasukawa, Mitsuhiro Hayashi, Akinobu Hamada, Akitake Mukasa, Ryo Nishikawa, Kenji Tamura, Yoshitaka Narita, Kenkichi Masutomi, Koichi Ichimura. Eribulin is a novel TERT-targeting inhibitor that penetrates into intracerebrally grown glioblastomas and suppresses their growth in mice [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 3117. doi:10.1158/1538-7445.AM2017-3117

Laminin Enriched Scaffolds for Tissue Engineering Applications

Laminin LM is heterotrimeric large molecular weight glycoprotein that forms a crucial component of the basal lamina or basement membrane of most tissues LMs are integral for cell adhesion proliferation survival migration and differentiation Several studies have used a LM coated D substrate for the culture and maintenance of stem cell phenotype in vitro Recent studies have reported that LM can also be incorporated in D scaffolds for tissue engineering applications This article illustrates the bioactivity and regenerative potential of LM protein in D scaffolds for skeletal muscle nerve vascular and intervertebral disc regeneration

Human embryonic and induced pluripotent stem cells maintain phenotype but alter their metabolism after exposure to ROCK inhibitor

Human pluripotent stem cells (hPSCs) are adhesion-dependent cells that require cultivation in colonies to maintain growth and pluripotency. Robust differentiation protocols necessitate single cell cultures that are achieved by use of ROCK (Rho kinase) inhibitors. ROCK inhibition enables maintenance of stem cell phenotype; its effects on metabolism are unknown. hPSCs were exposed to 10 μM ROCK inhibitor for varying exposure times. Pluripotency (TRA-1-81, SSEA3, OCT4, NANOG, SOX2) remained unaffected, until after prolonged exposure (96 hrs). Gas chromatography–mass spectrometry metabolomics analysis identified differences between ROCK-treated and untreated cells as early as 12 hrs. Exposure for 48 hours resulted in reduction in glycolysis, glutaminolysis, the citric acid (TCA) cycle as well as the amino acids pools, suggesting the adaptation of the cells to the new culture conditions, which was also reflected by the expression of the metabolic regulators, mTORC1 and tp53 and correlated with cellular proliferation status. While gene expression and protein levels did not reveal any changes in the physiology of the cells, metabolomics revealed the fluctuating state of the metabolism. The above highlight the usefulness of metabolomics in providing accurate and sensitive information on cellular physiological status, which could lead to the development of robust and optimal stem cell bioprocesses.

P08.41 Development of a novel TERT-targeting therapy for glioblastomas

Introduction: Approximately 60–80% of GBM harbour mutations in the promoter region of TERT that leads to its upregulation, making it the most common single genetic abnormalities in GBM. TERT, a reverse-transcriptase subunit of telomerase, has been an attractive candidate for therapy target because of their observed upregulation in a wide variety of cancers, including GBMs, a phenomenon that presumably helps maintain telomeres for their indefinite proliferation. However, no targeted therapy against telomerase has been successful so far, possibly because telomerase inhibition may not be necessarily expected to immediately suppress proliferation when telomere has already been elongated. It has been reported recently that TERT has an RNA-dependent RNA polymerase (RdRP) activity independent of its function at telomeres. The TERT-RdRP activity appears to be involved in controlling cell cycle and maintenance of stem cell phenotype, suggesting that TERT may be directly targeted to suppress tumour cell proliferation. Through drug screening, we have previously identified eribulin as a specific inhibitor for RdRP activity of TERT. Eribulin does not affect the telomerase activity at the concentration to inhibit the RdRP activity. In order to develop a novel TERT-targeted therapy against GBM, we investigated the anti-tumour efficacy of eribulin in cultured and xenografted GBM cells.

The Wnt signaling pathway: prospects for pharmacological regulation

Wnt cascade is one of the most important signal pathways in the cell, which is required for normal embryonic development, differentiation, maintenance of stem cell phenotype and migration. Mutations in this pathway are associated with tumor growth (especially with colon cancers, hepatocarcinomas and leukemias), where they participate in maintenance of tumor-initiating cells and metastasing. Because of that there is a considerable interest to develop inhibitors of Wnt pathway as anti-tumor agent. But this inhibitors are still only in early stages of clinical investigations. In this article we review the main targets for this anti-cancer agents and their preclinical and clinical status.

Targeting the SIN3A-PF1 interaction inhibits epithelial to mesenchymal transition and maintenance of a stem cell phenotype in triple negative breast cancer.

Triple negative breast cancer (TNBC) is characterized by a poorly differentiated phenotype and limited treatment options. Aberrant epigenetics in this subtype represent a potential therapeutic opportunity, but a better understanding of the mechanisms contributing to the TNBC pathogenesis is required. The SIN3 molecular scaffold performs a critical role in multiple cellular processes, including epigenetic regulation, and has been identified as a potential therapeutic target. Using a competitive peptide corresponding to the SIN3 interaction domain of MAD (Tat-SID), we investigated the functional consequences of selectively blocking the paired amphipathic α-helix (PAH2) domain of SIN3. Here, we report the identification of the SID-containing adaptor PF1 as a factor required for maintenance of the TNBC stem cell phenotype and epithelial-to-mesenchymal transition (EMT). Tat-SID peptide blocked the interaction between SIN3A and PF1, leading to epigenetic modulation and transcriptional downregulation of TNBC stem cell and EMT markers. Importantly, Tat-SID treatment also led to a reduction in primary tumor growth and disseminated metastatic disease in vivo. In support of these findings, knockdown of PF1 expression phenocopied treatment with Tat-SID both in vitro and in vivo. These results demonstrate a critical role for a complex containing SIN3A and PF1 in TNBC and provide a rational for its therapeutic targeting.

SOX17 is critical for the maintenance of stem cell phenotype of intra-aortic hematopoietic clusters of cells in the aorta-gonad-mesonephros region

SOX17 is critical for the maintenance of stem cell phenotype of intra-aortic hematopoietic clusters of cells in the aorta-gonad-mesonephros region

Natural polymers for the microencapsulation of cells.

The encapsulation of living mammalian cells within a semi-permeable hydrogel matrix is an attractive procedure for many biomedical and biotechnological applications, such as xenotransplantation, maintenance of stem cell phenotype and bioprinting of three-dimensional scaffolds for tissue engineering and regenerative medicine. In this review, we focus on naturally derived polymers that can form hydrogels under mild conditions and that are thus capable of entrapping cells within controlled volumes. Our emphasis will be on polysaccharides and proteins, including agarose, alginate, carrageenan, chitosan, gellan gum, hyaluronic acid, collagen, elastin, gelatin, fibrin and silk fibroin. We also discuss the technologies commonly employed to encapsulate cells in these hydrogels, with particular attention on microencapsulation.

DEK oncogene expression during normal hematopoiesis and in Acute Myeloid Leukemia (AML)

DEK is important in regulating cellular processes including proliferation, differentiation and maintenance of stem cell phenotype. The translocation t(6;9) in Acute Myeloid Leukemia (AML), which fuses DEK with NUP214, confers a poor prognosis and a higher risk of relapse. The over-expression of DEK in AML has been reported, but different studies have shown diminished levels in pediatric and promyelocytic leukemias. This study has characterized DEK expression, in silico, using a large multi-center cohort of leukemic and normal control cases. Overall, DEK was under-expressed in AML compared to normal bone marrow (NBM). Studying specific subtypes of AML confirmed either no significant change or a significant reduction in DEK expression compared to NBM. Importantly, the similarity of DEK expression between AML and NBM was confirmed using immunohistochemistry analysis of tissue mircorarrays. In addition, stratification of AML patients based on median DEK expression levels indicated that DEK showed no effect on the overall survival of patients. DEK expression during normal hematopoiesis did reveal a relationship with specific cell types implicating a distinct function during myeloid differentiation. Whilst DEK may play a potential role in hematopoiesis, it remains to be established whether it is important for leukemagenesis, except when involved in the t(6;9) translocation.

Abstract 2997: SOX2 contributes towards invasive phenotype of serous ovarian cancer by up-regulating MMP2.

Abstract SOX2 encodes a member of the SRY-related HMG-box (SOX) family of transcription factors involved in the regulation of embryonic development and in the determination of cell fate. SOX2 maps to 3q26.33, a region frequently amplified in HGSOvCa as reported in the TCGA and other large array-based CGH studies. However, the role of SOX2 in HGSOvCa oncogenesis and progression has not been studied yet. Given the importance of SOX2 in maintenance of stem cell phenotype and its overexpression in solid tumors, we sought to define the role of SOX2 in HGSOvCa. In order to establish a direct role for SOX2 in vivo, we conducted DNA copy number analysis and qRT-PCR analysis in two independent panels of primary HGSOvCa and 25 serous OVCA lines. We overexpressed SOX2 in T29, OVCA433, A2780, SKOV3 and OVCAR5 cells and/or knockdown SOX2 in UCI101 and OV90 cells. Ectopic expression of SOX2 resulted in decreased cell proliferation, soft agar colony formation, in vivo tumorigenicity but higher degree of invasion relative to cells with empty vectors. SOX2 overexpression increased MMP2 activity as determined by gelatin zymography. We also performed mRNA profiling to identify SOX2 target genes. SOX2 knockdown cells decreased migration,andinvasion but increased tumorigenicity. Differentially expressed genes in cells overexpressing SOX2 will be presented. Increased SOX2 expression in HGSOvCa appears to produce a more invasive yet quiestant cell. Overexpression of SOX2 may induce a stem-like transcriptional program in HGSOvCa. Thus, identifying SOX2 target genes may offer new insight into the understanding of ovarian cancer biology. Citation Format: Gayatry Mohapatra, Junko Kikuchi, Michael J. Birrer. SOX2 contributes towards invasive phenotype of serous ovarian cancer by up-regulating MMP2. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2997. doi:10.1158/1538-7445.AM2013-2997

The expression of functional toll‐like receptor 4 is associated with proliferation and maintenance of stem cell phenotype in endothelial progenitor cells (EPCs)

Endothelial dysfunction is involved in various cardiovascular diseases such as atherosclerosis. Endothelial progenitor cells (EPCs) contribute to re-endothelialization and neo-vascularization, and the increase of EPCs in peripherial circulation benefits the prognosis of cardiovascular disease. However, little is known about the biological stimuli that initiate the proliferation and the maintenance of stem cell phenotype of EPCs. Here we reported that human umbilical vein blood derived EPCs expressed gene transcripts coding for Toll-like receptor (TLR) 1-6, TLR8-10, TLR4 co-receptor CD14, and myeloid differentiation factor 88 (MyD88), a TLR adaptor molecule. Protein expression of TLR2, 4, CD14, and MyD88 was also detected by FACS or Western blot. The activation of TLR4 by LPS modulated the expression of TLRs, induced the phosphorylation of NF-kappaB, P38, and ERK42/44, and up-regulated the gene expression of cytokines IL-8, IFN-alpha, IFN-beta, and TNF-alpha, suggesting EPCs expressed functional TLR4. Unexpectedly LPS treatment failed to induce apoptosis in EPCs, but instead promoted cell proliferation of EPCs. Furthermore, the treatment of EPCs with LPS up-regulated stem cell markers AC133 and CD34 in both mRNA and protein levels, and down-regulated the protein expression of differential marker eNOS. These results suggested that TLR4 functions to maintain the stem cell phenotype of EPCs and enlarge its population, which reveals a novel aspect of the multiple-faced TLR biology, and may open new prospects for using TLR4 agonists to promote the production of EPCs for clinical use.

Abstract 1410: Bmi-1 enhances the radioresistance of cells by suppressing the expression of NADPH oxidase genes

Abstract Bmi-1 is a polycomb group protein necessary for cellular proliferation and maintenance of stem cell phenotype. The current study was undertaken to determine the radioprotective effects of Bmi-1 in human epithelial cells. Primary normal human keratinocytes (NHK) from oral mucosa or epidermis were infected with retroviral vectors expressing Bmi-1. NHK infected with empty virus (NHK/B0) rapidly underwent premature senescence upon exposure to 5 or 10 Gy of ionizing radiation (IR). However, the cells transduced with Bmi-1 (NHK/Bmi-1) maintained cell proliferation and clonogenicity after IR. To determine the mechanism underlying the radioresistance conferred by Bmi-1, we compared the DNA damage response (DDR), production of reactive oxygen species (ROS), and DNA repair activities in both cell types. NHK/B0 and NHK/Bmi-1 cells demonstrated rapid induction of DDR after 5 Gy irradiation. ROS production was increased by irradiation, but Bmi-1 transduction suppressed the ROS production. Irradiation strongly induced the genes encoding the enzymes involved in ROS production, mainly of NADPH oxidase subunits, but Bmi-1 completely abolished the expression of these genes. Chromatin immunoprecipitation (ChIP) revealed binding of Bmi-1 to the promoter regions of these genes in vivo, suggesting transcriptional repression by Bmi-1. In vitro DNA end joining and in vivo double strand break (DSB) repair activities were enhanced in NHK/Bmi-1 cells compared with NHK/B0. These data indicate that Bmi-1 enhances the radioresistance of NHK in part by mitigating the genotoxicity of radiation. This study was supported in part by the grants, UCLA/CBPR (U19) from NIAID and R01DE18295/K02DE18959 from NIDCR/NIH. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1410.

MicroRNAs, RNA binding proteins and cancer

microRNAs (miRNAs) are genes involved in normal development and cancer. They inhibit gene expression through interaction with 3'-untranslated regions of messenger RNAs, and are thought to control the expression of a large proportion of the protein-coding genome. However, it is becoming apparent that RNA-binding proteins (RBPs) control the biogenesis of miRNAs, their activity and stability. This indicates the existence of interplay between RBPs and miRNAs that affects gene expression and processes ranging from development, maintenance of stem cell phenotype and stress responses. Although miRNAs are prominent factors in cancer, little is known about how RBPs affect their cancerous function.

Interactions of the Cellular Components of the Hematopoietic Niche

Hematopoietic Stem Cell (HSC) self-renewal and multilineage differentiation potential is governed by multiple intrinsic and extrinsic parameters. Collectively, these parameters dictate the fate of HSC and underscore the heterogeneity observed within phenotypically defined groups of stem cells. While cell cycle status and the genetic profile of HSCs are critical intrinsic modulators of cell fate, interactions with cytokines, growth factors, and cellular elements of the hematopoietic niche (HN) are key extrinsic regulators of stem cell function. We examined the impact of cellular elements of the HN on stem cell fate and maintenance by analyzing the combined effect of calvaria-derived osteoblasts (OB) and mesenchymal stromal cells (MSC) on cultured murine HSC. Murine bone marrow-derived KSL cells were co-cultured with OB alone, MSC alone, or with mixtures of OB and MSC at different ratios for one week. Cultures were supplemented with SCF, Fl-3, Tpo, IL-3, IL-6, IGF1 & OPN. OB alone, maintained the functional properties of cultured HSCs significantly better than MSC thus corroborating the importance of OB in the overall competence of the HN. On day 7, the fold-increase in the number of LSK cells was 1473 ± 291 in OB cultures, 561 ± 159 in MSC cultures, and 603 ± 263 in OB+MSC cultures (n= 4 for all 3 groups). During the same 7 day-period, the number of CFU in progeny cells expanded 74 ± 15 fold in OB cultures, 23 ± 2 fold in MSC cultures, and 27 ± 15 in OB+MSC cultures (n=3 for all groups). The substantial increase in KSL progeny in OB cultures on day 7 was accompanied by a high percentage of cells in active phases of cell cycle (% G0/G1 = 72.5 ± 7.0, n=3) compared to their counterparts in MSC or OB+MSC cultures. In addition, co-culture of KSL cells with OB resulted in an unexpected higher maintenance of the Sca-1+Lin- phenotype (26.5% ± 2.8%) relative to MSC cultures (4.6% ± 1.0%) and OB+MSC cultures (11.7% ± 1.8%; n=3 for all). Only some of these results were reproduced when KSL cells were cultured in OB-conditioned medium suggesting that cell-to-cell contact may be essential for the observed activities. To assess the in vivo potential of LSK cells maintained in these cultures, the 10-day expansion equivalent of 1,000 LSK cells were competitively transplanted in lethally irradiated congenic mice and chimerism was monitored for the next 4 months. At 1 and 2 months post-transplantation, the level of chimerism sustained by LSK cells maintained in OB cultures for 10 days surpassed or was slightly lower than that observed with freshly isolated LSK cells (72.7% vs 59.7% and 57.4% vs 74.7%, respectively) suggesting that OB culture conditions effectively expanded short-term repopulating cells. At 4 months post-transplantation, mice receiving freshly isolated LSK cells were 83.6% ± 1.8% chimeric compared to 53.7% ± 16.1% for mice transplanted with cells from OB cultures and 31.9% ± 21.4% for mice receiving cells from OB+MSC cultures. Overall, these data suggest that OB-LSK interactions promote the maintenance of both short-term and long-term repopulating cells while MSC suppress the OB-mediated activity. To investigate the mechanism of OB-mediated maintenance of stem cell phenotype and function, we examined Notch signaling using Real-Time Q-PCR on cells maintained in culture for 7 days. Relative to the expression in KSL cells, expression of Notch 2 was elevated in OB cultures and suppressed over 2-fold in cultures of MSC and OB+MSC. Similarly, the expression of Jagged 1 and 2, Delta 1 and 4, Hes 1 and 5, Deltex, and SKP2 was increased in OB cultures and suppressed in MSC and OB+MSC cultures. Collectively, these data illustrate that cell-to-cell contact between OB and KSL cells promotes the in vitro maintenance of long-term and short-term repopulating cells and suggest that this stem cell function-promoting activity is induced in part by the upregulation of Notch-mediated signaling between HSCs and osteoblasts. The suppressive effect imparted by MSC on stem cell maintenance compared to cultures of OB alone suggest that these two cellular elements of the HN have opposite effects on the fate and function of stem cells.

Effects of oxygen on mouse embryonic stem cell growth, phenotype retention, and cellular energetics

Most embryonic stem (ES) cell research is performed with a gas phase oxygen partial pressure (pO(2)) of 142 mmHg, whereas embryonic cells in early development are exposed to pO(2) values of 0-30 mmHg. To understand effects of these differences, we studied murine ES (mES) growth, maintenance of stem cell phenotype, and cell energetics over a pO(2) range of 0-285 mmHg, in the presence or absence of differentiation-suppressing leukemia inhibitory factor (LIF). With LIF, growth rate was sensitive to pO(2) but constant with time, and expression of self-renewal transcription factors decreased at extremes of pO(2). Subtle morphological changes suggested some early differentiation, but cells retained the ability to differentiate into derivatives of all three germ layers at low pO(2). Without LIF, growth rate decreased with time, and self-renewal transcription factor mRNA decreased further. Gross morphological changes occurred, and overt differentiation occurred at all pO(2). These findings suggested that hypoxia in the presence of LIF promoted limited early differentiation. ES cells survived oxygen starvation with negligible cell death by increasing anaerobic metabolism within 48 h of anoxic exposure. Decreasing pO(2) to 36 mmHg or lower decreased oxygen consumption rate and increased lactate production rate. The fraction of ATP generated aerobically was 60% at or above 142 mmHg and decreased to 0% under anoxia, but the total ATP production rate remained nearly constant at all pO(2). In conclusion, undifferentiated ES cells adapt their energy metabolism to proliferate at all pO(2) between 0 and 285 mmHg. Oxygen has minimal effects on undifferentiated cell growth and phenotype, but may exert more substantial effects under differentiating conditions.