Cells In Undifferentiated State Research Articles

Mouse embryonic fibroblast (MEF)/BMP4-conditioned medium enhanced multipotency of human dental pulp cells.

Dental pulp cells (DPCs) are valuable cell source for dental regeneration, albeit their application is restricted by limited pluripotency due to current culture condition. Mouse embryonic fibroblasts (MEFs) are served as feeder layer to maintain undifferentiated state of iPSCs and ESCs with long-term in vitro culture. Bone morphogenetic protein 4 (BMP4) plays an important role in the regulation of undifferentiated state and lineage commitment of cells through modulation of microenvironment. However, so far little was known how micro environment affect the multipotency of dental derived cells. To demonstrate the effect of optimized culture condition on multipotency of DPCs, cell proliferation and senescence of DPCs with MEF and/or rhBMP4-CM were examined by CCK8, telomerase activity and flow cytometry. Multilineage differentiation was detected by immunofluorescent staining, Real-time PCR and western blot. Expression of BMP4/NFATc1/LIF in the co-culture medium was evaluated by ELISA and expression of Oct-4/Sox2/c-Myc/NFATc1 in co-cultured DPCs was detected by Real-time PCR. NFATc1 inhibitor INCA-6 was applied to DPCs with MEF and/or rhBMP4-CM, expression of NFATc1/Oct-4/Sox2/c-Myc was examined by Realtime PCR and western blot. Our results demonstrated that DPCs cultured with MEF and/or rhBMP4-CM showed increased cell proliferation, telomerase rate and multilineage differentiation capability. MEF-CM enhanced expression of Oct-4/Sox2/c-Myc/NFATc1 in co-cultured DPCs through secretion of BMP4/NFATc1 in the culture medium. INCA-6 effectively restrained the MEF/BMP4-CM induced upregulation of Oct-4/Sox2/c-Myc/NFATc1 in DPCs. These resuts indicate that both MEF-CM and BMP4-CM provided similar efficient culture system to improve the multipotency of DPCs, which might contribute to the application of DPCs in dental regeneration.

The Arabidopsis JAGGED LATERAL ORGANS (JLO) gene sensitizes plants to auxin.

Plant growth and development of new organs depend on the continuous activity of the meristems. In the shoot, patterns of organ initiation are determined by PINFORMED (PIN)-dependent auxin distribution, while the undifferentiated state of meristem cells requires activity of KNOTTED LIKE HOMEOBOX (KNOX) transcription factors. Cell proliferation and differentiation of the root meristem are regulated by the largely antagonistic functions of auxin and cytokinins. It has previously been shown that the transcription factor JAGGED LATERAL ORGANS (JLO), a member of the LATERAL ORGAN BOUNDARY DOMAIN (LBD) family, coordinates KNOX and PIN expression in the shoot and promotes root meristem growth. Here we show that JLO is required for the establishment of the root stem cell niche, where it interacts with the auxin/PLETHORA pathway. Auxin signaling involves the AUX/IAA co-repressor proteins, ARF transcription factors and F-box receptors of the TIR1/AFB1-5 family. Because jlo mutants fail to degrade the AUX/IAA protein BODENLOS, root meristem development is inhibited. We also demonstrate that the expression levels of two auxin receptors, TIR1 and AFB1, are controlled by JLO dosage, and that the shoot and root defects of jlo mutants are alleviated in jlo plants expressing TIR1 and AFB1 from a transgene. The finding that the auxin sensitivity of a plant can be differentially regulated through control of auxin receptor expression can explain how different developmental processes can be integrated by the activity of a key transcription factor.

Level of FACT defines the transcriptional landscape and aggressive phenotype of breast cancer cells.

Although breast cancer (BrCa) may be detected at an early stage, there is a shortage of markers that predict tumor aggressiveness and a lack of targeted therapies. Histone chaperone FACT, expressed in a limited number of normal cells, is overexpressed in different types of cancer, including BrCa. Recently, we found that FACT expression in BrCa correlates with markers of aggressive BrCa, which prompted us to explore the consequences of FACT inhibition in BrCa cells with varying levels of FACT.FACT inhibition using a small molecule or shRNA caused reduced growth and viability of all BrCa cells tested. Phenotypic changes were more severe in high- FACT cells (death or growth arrest) than in low-FACT cells (decreased proliferation). Though inhibition had no effect on the rate of general transcription, expression of individual genes was changed in a cell-specific manner. Initially distinct transcriptional profiles of BrCa cells became similar upon equalizing FACT expression. In high-FACT cells, FACT supports expression of genes involved in the regulation of cell cycle, DNA replication, maintenance of an undifferentiated cell state and regulated by the activity of several proto-oncogenes. In low-FACT cells, the presence of FACT reduces expression of genes encoding enzymes of steroid metabolism that are characteristic of differentiated mammary epithelia.Thus, we propose that FACT is both a marker and a target of aggressive BrCa cells, whose inhibition results in the death of BrCa or convertion of them to a less aggressive subtype.

SC1 Promotes MiR124-3p Expression to Maintain the Self-Renewal of Mouse Embryonic Stem Cells by Inhibiting the MEK/ERK Pathway

Background/Aims: Self-renewal is one of the most important features of embryonic stem (ES) cells. SC1 is a small molecule modulator that effectively maintains the self-renewal of mouse ES cells in the absence of leukemia inhibitory factor (LIF), serum and feeder cells. However, the mechanism by which SC1 maintains the undifferentiated state of mouse ES cells remains unclear. Methods: In this study, microarray and small RNA deep-sequencing experiments were performed on mouse ES cells treated with or without SC1 to identify the key genes and microRNAs that contributed to self-renewal. Results: SC1 regulates the expressions of pluripotency and differentiation factors, and antagonizes the retinoic acid (RA)-induced differentiation in the presence or absence of LIF. SC1 inhibits the MEK/ERK pathway through Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis and pathway reporting experiments. Small RNA deep-sequencing revealed that SC1 significantly modulates the expression of multiple microRNAs with crucial functions in ES cells. The expression of miR124-3p is upregulated in SC1-treated ES cells, which significantly inhibits the MEK/ERK pathway by targeting Grb2, Sos2 and Egr1. Conclusion: SC1 enhances the self-renewal capacity of mouse ES cells by modulating the expression of key regulatory genes and pluripotency-associated microRNAs. SC1 significantly upregulates miR124-3p expression to further inhibit the MEK/ ERK pathway by targeting Grb2, Sos2 and Egr1.

(Invited) Application of Ferrite Nanoparticles to Magnetic Hyperthermia

Hyperthermia (thermotherapy), in which cancer cells are treated with heat of approximately 43°C, has recently received attention as a promising approach to cancer therapy because hyperthermia is non-invasive and has very few side effects, compared to surgical resection, chemotherapy, and radiation therapy. In hyperthermia using magnetic nanoparticles and an alternating magnetic field of a few hundred kHz, heat generation is derived just from their magnetic property. Magnetic nanoparticles, in particular iron oxide such as magnetite (Fe­ 3O4) or maghemite (γ-Fe2O3), are generally used as heat sources as they have the advantages of high biocompatibility and relatively large magnetization. We have been investigating the synthesis of ferrite nanoparticles and their in vitro and in vivo evaluation for magnetic hyperthermia. We demonstrated the synthesis of Fe3O4 nanoparticles with the size tuned in the range of 10 to 40 nm in mean diameter by chemical reaction in an aqueous solution containing iron(II) and iron(III) salts at various ratios with 1,6-hexanediamine as a base [1]. Here organic amines are considered to serve not only as a base but also as a protective reagent. Magnetic properties of Fe3O4 nanoparticles can be controlled by the particle diameter [1]. Considering to their biomedical application, Fe3O4 nanoparticles were evaluated for their safety by using mouse embryonic stem (mES) cells from the viewpoint of viability and keeping undifferentiated state of mES cells [2]. In addition, we demonstrated that the use of spermine instead of 1,6-hexanediamine is also useful for the synthesis and found that Fe3O4 nanoparticles with diameter of approximately 40 nm prepared with 1,6-hexanediamine and spermine possess negative and positive charge, respectively [3]. With human breast cancer MCF-7 cells, we demonstrated that the nanoparticles with positive charge showed higher internalization into MCF-7 cells than the nanoparticles with negative charge [3], and that ~40-nm Fe3O4 nanoparticles showed a higher heating efficacy than did ~10-nm Fe3O4 nanoparticles when MCF-7 cells containing nanoparticles were subjected to alternating magnetic field [4]. With mesothelioma cells that are of an asbestos-related fatal cancer with no effective treatment, we demonstrated the potential of Fe3O4 nanoparticles for future application to mesothelioma treatment via the following two approaches: (i) the use of specific apoptotic effect of Fe3O4 nanoparticles on biphasic MSTO-211H cells and (ii) the use of heat generation by Fe3O4 nanoparticles subjected to alternating magnetic field, which induced a high degree of cell mortality in all three major histologic subtypes of mesothelioma cells (epithelioid NCI-H28, sarcomatoid NCI-H2052, and biphasic MSTO-211H cells) [5]. For in vivo investigation, we designed optimized multifunctional Fe3O4 nanoparticles, composed of Fe3O4 nanoparticles and photosensitizer conjugated hyaluronic acid, to achieve enhanced tumor diagnosis and therapy. We successfully detected tumors implanted in mice via magnetic resonance imaging and optical imaging, and demonstrated the photodynamic/hyperthermia-combined therapeutic efficacy of the functionalized nanoparticles with synergistically enhanced efficacy against cancer [6]. Aiming to effective heat generation, we focused our attention on other ferrite nanoparticles. In CoFe2O4 nanoparticles, which were synthesized by chemical reaction in an aqueous solution containing cobalt(II) and iron(III) salts with spermine as a base, we demonstrated that an increase in coercivity derived from the substitution of Fe2+ in Fe3O4 with Co2+ facilitated the ability of the nanoparticles to induce cell death in human breast cancer cells under alternating magnetic field [7]. [1] H. Iida, K. Takayanagi, T. Nakanishi, T. Osaka, J. Colloid Interface Sci., 314 (2007) 274. [2] C. Shundo, H. Zhang, T. Nakanishi, T. Osaka, Colloids Surf. B, 97 (2012) 221. [3] T. Osaka, T. Nakanishi, S. Shanmugam, S. Takahama, H. Zhang, Colloids Surf. B, 71 (2009) 325. [4] D. Baba, Y. Seiko, T. Nakanishi, H. Zhang, A. Arakaki, T. Matsunaga, T. Osaka, Colloids Surf. B, 95 (2012) 254. [5] S. Matsuda, A. Hitsuji, T. Nakanishi, H. Zhang, A. Tanaka, H. Matsuda, T. Osaka, ACS Biomater. Sci. Eng., 1 (2015) 632. [6] K.S. Kim, J.Y. Kim, J.Y. Lee, S. Matsuda, S. Hideshima, Y. Mori, T. Osaka, K. Na, submitted. [7] S. Matsuda, T. Nakanishi, K. Kaneko, T. Osaka, Electrochim. Acta, 183 (2015) 153.

Abstract 2877: Effects of metabolic stressors on cellular differentiation

Abstract Poor differentiation (the divergence of tumor cell morphology from the cellular morphology of the normal tissue of origin) is associated with unfavorable responses to chemotherapy and poor clinical outcomes in solid tumors. Recurrent somatic mutations in the genes encoding for the metabolic enzymes isocitrate dehydrogenase (IDH) 1 and 2 have recently been identified in patients with acute myeloid leukemias, chrondrosarcomas, cholangiocarcinomas and glioblastomas. Point mutations in IDH lead to production of 2-hydroxyglutarate (2HG). 2HG acts as a potent inhibitor of enzymes involved in the removal of chromatin marks associated with transcriptional repression (DNA and Histone H3K9 methylation). Excess 2HG leads to impaired differentiation but very little is known about the underlying mechanisms. Here we use a well established model of cellular differentiation, the differentiation of 10T1/2 fibroblasts into myofibers by the lineage-specifying transcription factor MyoD to study the mechanisms by which IDH mutations impair differentiation. We show that 5-azacytidine induced differentiation of 10T1/2 cells is impaired when mutant IDH2 is expressed and that IDH2 mutations impair the ability of MyoD expression to reprogram 10T1/2 cells into myofibers. We propose that the metabolic landscape of undifferentiated cells determines the ability of the lineage determining factor MyoD to trigger differentiation and that the undifferentiated state of cancer cells is dependent on their unique metabolic profile. Further work aims to a) define the effects of mutant IDH1/2 on the ability of MyoD to program muscle differentiation, b) determine the nature of the tumorigenic effects of IDH1/2 mutations in 10T1/2 cells and c) identify molecular pathways that can overcome differentiation blocks upon activation. Insight from these experiments will outline the mechanism(s) by which metabolic alterations, in the form of 2HG, impair differentiation and drive tumorigenesis. They will also serve as a platform for the identification of metabolic alterations that trigger differentiation. These mechanisms could prove to be novel and readily targetable processes in an array of poorly differentiated and aggressive cancer subtypes where other pharmacological approaches have failed. Citation Format: Juan Manuel Schvartzman, Craig B. Thompson. Effects of metabolic stressors on cellular differentiation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2877.

Enhancement of Satellite Cell Transplantation Efficiency by Leukemia Inhibitory Factor.

Background and Objectives: Cell transplantation is a promising therapy for several muscle diseases, including Duchenne muscular dystrophy. Satellite cells are stem cells in skeletal muscle that provide an important cell source for transplantation therapy. However, culture of satellite cells in vitro causes them to lose their undifferentiated state, associated with reduced transplantation efficiency. It is therefore necessary to develop optimal culture conditions for maintaining the undifferentiated state of satellite cells.Methods: Primary satellite cells were cultured with or without leukemia inhibitory factor (LIF). The expression of undifferentiation and differentiation markers, and the transplantation efficiency were analyzed.Results: LIF-treated satellite cells showed increased expression of Pax7, and enhanced transplantation efficiency in a mouse model of Duchenne muscular dystrophy.Conclusions: Our study showed that the treatment with LIF effectively maintained the undifferentiated state of satellite cells, and enhanced their transplantation efficiency. These results will contribute to the optimization of culture conditions for cell transplantation therapy.

Comprehensive Identification of Krüppel-Like Factor Family Members Contributing to the Self-Renewal of Mouse Embryonic Stem Cells and Cellular Reprogramming.

Pluripotency is maintained in mouse embryonic stem (ES) cells and is induced from somatic cells by the activation of appropriate transcriptional regulatory networks. Krüppel-like factor gene family members, such as Klf2, Klf4 and Klf5, have important roles in maintaining the undifferentiated state of mouse ES cells as well as in cellular reprogramming, yet it is not known whether other Klf family members exert self-renewal and reprogramming functions when overexpressed. In this study, we examined whether overexpression of any representative Klf family member, such as Klf1–Klf10, would be sufficient for the self-renewal of mouse ES cells. We found that only Klf2, Klf4, and Klf5 produced leukemia inhibitory factor (LIF)-independent self-renewal, although most KLF proteins, if not all, have the ability to occupy the regulatory regions of Nanog, a critical Klf target gene. We also examined whether overexpression of any of Klf1-Klf10 would be sufficient to convert epiblast stem cells into a naïve pluripotent state and found that Klf5 had such reprogramming ability, in addition to Klf2 and Klf4. We also delineated the functional domains of the Klf2 protein for LIF-independent self-renewal and reprogramming. Interestingly, we found that both the N-terminal transcriptional activation and C-terminal zinc finger domains were indispensable for this activity. Taken together, our comprehensive analysis provides new insight into the contribution of Klf family members to mouse ES self-renewal and cellular reprogramming.

Antidifferentiation Noncoding RNA Regulates the Proliferation of Osteosarcoma Cells.

Antidifferentiation noncoding RNA (ANCR), a newly identified long noncoding RNA (lncRNA), plays a critical role for stem cells to maintain undifferentiated cell state. However, the functions of ANCR in human cancers have not been reported. This study is designed to explore the role of ANCR in osteosarcoma. Lentivirus-mediated (shRNA) was applied to silence ANCR in the human osteosarcoma cell lines U2OS and Saos-2. Cell viability was measured by MTT assay. Colony-forming ability was measured by colony formation assay. Cell cycle progression was determined by flow cytometry with propidium iodide staining. In addition, cell cycle makers, including p21, CDK2, and CDK4, were investigated in ANCR silencing U2OS cells by real time PCR (RT-PCR) analysis. In this study, we first proved that lentivirus-mediated shRNA specifically suppressed the expression level of ANCR in U2OS and Saos-2 cells. Further investigations revealed that knockdown of ANCR significantly inhibited the proliferation of U2OS and Saos cells and colony formation of U2OS cells. Moreover, the cell cycle of U2OS cells was arrested at G0/G1 phase after ANCR knockdown. Furthermore, the expression level of p21 was increased and CDK2 was decreased in ANCR knock-down cells. Our data indicated that ANCR might be an oncogenic lncRNA that promoted proliferation of osteosarcoma. The potential application of ANCR-targeted therapy using the lentivirus-mediated shRNA approach is worth further investigations in preclinical and clinical studies.

Comparison between the cultures of human induced pluripotent stem cells (hiPSCs) on feeder-and serum-free system (Matrigel matrix), MEF and HDF feeder cell lines.

Human induced pluripotent stem cells (hiPSCs) are a type of pluripotent stem cells artificially derived from an adult somatic cell (typically human fibroblast) by forced expression of specific genes. In recent years, different feeders like inactivated mouse embryonic fibroblasts (MEFs), human dermal fibroblasts (HDFs), and feeder free system have commonly been used for supporting the culture of stem cells in undifferentiated state. In the present work, the culture of hiPSCs and their characterizations on BD Matrigel (feeder-and serum-free system), MEF and HDF feeders using cell culture methods and molecular techniques were evaluated and compared. The isolated HDFs from foreskin samples were reprogrammed to hiPSCs using gene delivery system. Then, the pluripotency ability of hiPSCs cultured on each layer was determined by teratoma formation and immunohistochemical staining. After EBs generation the expression level of three germ layers genes were evaluated by Q-real-time PCR. Also, the cytogenetic stability of hiPSCs cultured on each condition was analyzed by karyotyping and comet assay. Then, the presence of pluripotency antigens were confirmed by Immunocytochemistry (ICC) test and alkaline phosphatase staining. This study were showed culturing of hiPSCs on BD Matrigel, MEF and HDF feeders had normal morphology and could maintain in undifferentiated state for prolonged expansion. The hiPSCs cultured in each system had normal karyotype without any chromosomal abnormalities and the DNA lesions were not observed by comet assay. Moreover, up-regulation in three germ layers genes in cultured hiPSCs on each layer (same to ESCs) compare to normal HDFs were observed (p < 0.05). The findings of the present work were showed in stem cells culturing especially hiPSCs both MEF and HDF feeders as well as feeder free system like Matrigel are proper despite benefits and disadvantages. Although, MEFs is suitable for supporting of stem cell culturing but it can animal pathogens transferring and inducing immune response. Furthermore, HDFs have homologous source with hiPSCs and can be used as feeder instead of MEF but in therapeutic approaches the cells contamination is a problem. So, this study were suggested feeder free culturing of hiPSCs on Matrigel in supplemented media (without using MEF conditioned medium) resolves these problems and could prepare easy applications of hiPSCs in therapeutic approaches of regenerative medicine such as stem-cell therapy and somatic cell nuclear in further researches.

Vasa, PL10, and Piwi gene expression during caudal regeneration of the polychaete annelid Alitta virens

Polychaetes are famous for their outstanding ability to regenerate lost body parts. Moreover, these worms possess a number of ancestral features in anatomy, development, and genetics, making them particularly suitable for comparative studies. Thus, fundamental as well as new undisclosed so far features of regenerative processes may be revealed, using polychaetes as a model. In the present work, we aimed to analyze the molecular basis of caudal regeneration in the nereid polychaete Alitta virens (formerly Nereis virens). We focused on homologues genes of RNA helicases Vasa and PL10 and ncRNA-binding proteins Piwi. These markers are suggested to play a significant role in maintenance of undifferentiated state of primordial germ cells and multipotent stem cells across invertebrates. In normal conditions, A. virens homologues of Vasa, PL10, and Piwi were differentially expressed in the subterminal growth zone and germline cells. Caudal amputation induced expression of studied genes de novo, which further accompanies all steps of regeneration. An early appearance of the transcripts in wound epithelium and internal blastemal cells suggests involvement of these genes in the well-known cell dedifferentiation events that assure polychaete regeneration. Provided interpretation of the gene expression dynamics implies the primary restoration of the pygidium and growth zone, which promotes following segment formation. Obtained results are valuable as a molecular fingerprint of the alterations occurring in regulatory state of locally regenerating tissues.

SNAI2 controls the undifferentiated state of human epidermal progenitor cells.

The transcription factor, SNAI2, is an inducer of the epithelial to mesenchymal transition (EMT) which mediates cell migration during development and tumor invasion. SNAI2 can also promote the generation of mammary epithelial stem cells from differentiated luminal cells when overexpressed. How SNAI2 regulates these critical and diverse functions is unclear. Here, we show that the levels of SNAI2 expression are important for epidermal cell fate decisions. The expression of SNAI2 was found to be enriched in the basal layer of the interfollicular epidermis where progenitor cells reside and extinguished upon differentiation. Loss of SNAI2 resulted in premature differentiation whereas gain of SNAI2 expression inhibited differentiation. SNAI2 controls the differentiation status of epidermal progenitor cells by binding to and repressing the expression of differentiation genes with increased binding leading to further transcriptional silencing. Thus, the levels of SNAI2 binding to genomic targets determine the differentiation status of epithelial cells with increased levels triggering EMT and dedifferentiation, moderate (physiological) levels promoting epidermal progenitor function, and low levels leading to epidermal differentiation.

The miR-302 cluster transcriptionally regulated by POUV, SOX and STAT5B controls the undifferentiated state through the post-transcriptional repression of PBX3 and E2F7 in early chick development.

Early chick development is a systematic process governed by the concerted action of multiple mechanisms that regulate transcription and post-transcriptional processes. Post-transcriptional microRNA-mediated regulation, with regard to lineage specification and differentiation in early chick development, requires further investigation. Here, we characterize the transcriptional and post-transcriptional regulation mechanisms in undifferentiated chick blastodermal cells. Expression of the miR-302 cluster, POUV, SOX2, and STAT5B decreased in a time-dependent manner in early chick development. We found that POUV, SOX2, and STAT5B regulate the transcription of the miR-302 cluster, as its 5'-flanking region contains binding elements for each transcription factor. Additionally, POUV, SOX2, and STAT5B maintain pluripotency by regulating genes containing the miR-302 cluster target sequence. For example, microRNAs from the miR-302 cluster can bind to PBX3 and E2F7 transcripts, thus acting as a post-transcriptional regulator that maintains the undifferentiated state of blastodermal cells by balancing the expression of genes related to pluripotency and differentiation. Based on these results, we suggest that both transcriptional and post-transcriptional regulation of the miR302 cluster is critical for intrinsically controlling the undifferentiated state of chick embryonic blastodermal cells. These findings may help our understanding of the cellular and molecular mechanisms that underlie developmental decisions during early chick development.

H19 long noncoding RNA controls the mRNA decay promoting function of KSRP.

Long noncoding RNAs (lncRNAs) interact with protein factors to regulate different layers of gene expression transcriptionally or posttranscriptionally. Here we report on the functional consequences of the unanticipated interaction of the RNA binding protein K homology-type splicing regulatory protein (KSRP) with the H19 lncRNA (H19). KSRP directly binds to H19 in the cytoplasm of undifferentiated multipotent mesenchymal C2C12 cells, and this interaction favors KSRP-mediated destabilization of labile transcripts such as myogenin. AKT activation induces KSRP dismissal from H19 and, as a consequence, myogenin mRNA is stabilized while KSRP is repurposed to promote maturation of myogenic microRNAs, thus favoring myogenic differentiation. Our data indicate that H19 operates as a molecular scaffold that facilitates effective association of KSRP with myogenin and other labile transcripts, and we propose that H19 works with KSRP to optimize an AKT-regulated posttranscriptional switch that controls myogenic differentiation.

Effects of Feeder Layers, Culture Media, Conditional Media, Growth Factors, and Passages Number on Stem Cell Optimization

Stem cells are undifferentiated and self-renewal cells which could be obtained from the body or artificially derived from an adult somatic cell by forced expression of specific genes. In recent years stem cells are widely used in laboratory for tissue engineering and therapeutic applications. There are different factors and conditions that affect the stem cell culture such as feeder layers, atmosphere, kind of medium, growth factors, passages number, and conditional media with animal or human sources. Optimization of stem cell culture for medical approaches and regenerative medicine is important. Therefore, in the present study, the effect of these factors and agents on optimization of stem cell culture has been discussed. This review study showed that optimization of feeder layer, atmosphere, and using supplemented media with essential growth factors could help in maintaining the stem cells in undifferentiated state in vitro. The present study indicated that optimization of stem cell culture depends on the kind of each cell type and using stem cells in low passage number could decrease chromosomal abnormalities and DNA damages. For inhibiting the stem cell contamination by feeder cell lines, culture of these cells on feeder free systems like Matrigel matrix in conditioned media supplemented with essential growth factors is useful. Also, for eradicating immune system responses and reducing the risk of animal pathogen transfer, culture of stem cells on human feeder in optimized media is suitable for therapeutic approaches and regenerative medicine.

Kit transduced signals counteract erythroid maturation by MAPK-dependent modulation of erythropoietin signaling and apoptosis induction in mouse fetal liver.

Signaling by the stem cell factor receptor Kit in hematopoietic stem and progenitor cells is functionally associated with the regulation of cellular proliferation, differentiation and survival. Expression of the receptor is downregulated upon terminal differentiation in most lineages, including red blood cell terminal maturation, suggesting that omission of Kit transduced signals is a prerequisite for the differentiation process to occur. However, the molecular mechanisms by which Kit signaling preserves the undifferentiated state of progenitor cells are not yet characterized in detail. In this study, we generated a mouse model for inducible expression of a Kit receptor carrying an activating mutation and studied its effects on fetal liver hematopoiesis. We found that sustained Kit signaling leads to expansion of erythroid precursors and interferes with terminal maturation beyond the erythroblast stage. Primary KIT(D816V) erythroblasts stimulated to differentiate fail to exit cell cycle and show elevated rates of apoptosis because of insufficient induction of survival factors. They further retain expression of progenitor cell associated factors c-Myc, c-Myb and GATA-2 and inefficiently upregulate erythroid transcription factors GATA-1, Klf1 and Tal1. In KIT(D816V) erythroblasts we found constitutive activation of the mitogen-activated protein kinase (MAPK) pathway, elevated expression of the src kinase family member Lyn and impaired Akt activation in response to erythropoietin. We demonstrate that the block in differentiation is partially rescued by MAPK inhibition, and completely rescued by the multikinase inhibitor Dasatinib. These results show that a crosstalk between Kit and erythropoietin receptor signaling cascades exists and that continuous Kit signaling, partly mediated by the MAPK pathway, interferes with this crosstalk.

Establishment of trophoblast stem cells under defined culture conditions in mice.

The inner cell mass (ICM) and trophoblast cell lineages duet early embryonic development in mammals. After implantation, the ICM forms the embryo proper as well as some extraembryonic tissues, whereas the trophoectoderm (TE) exclusively forms the fetal portion of the placenta and the trophoblast giant cells. Although embryonic stem (ES) cells can be derived from ICM in cultures of mouse blastocysts in the presence of LIF and/or combinations of small-molecule chemical compounds, and the undifferentiated pluripotent state can be stably maintained without use of serum and feeder cells, defined culture conditions for derivation and maintenance of undifferentiated trophoblast stem (TS) cells have not been established. Here, we report that addition of FGF2, activin A, XAV939, and Y27632 are necessary and sufficient for derivation of TS cells from both of E3.5 blastocysts and E6.5 early postimplantation extraembryonic ectoderm. Moreover, the undifferentiated TS cell state can be stably maintained in chemically defined culture conditions. Cells derived in this manner expressed TS cell marker genes, including Eomes, Elf5, Cdx2, Klf5, Cdh1, Esrrb, Sox2, and Tcfap2c; differentiated into all trophoblast subtypes (trophoblast giant cells, spongiotrophoblast, and labyrinthine trophoblast) in vitro; and exclusively contributed to trophoblast lineages in chimeric animals. This delineation of minimal requirements for derivation and self-renewal provides a defined platform for precise description and dissection of the molecular state of TS cells.

Wnt signaling inhibits adrenal steroidogenesis by cell-autonomous and non-cell-autonomous mechanisms.

Wnt/β-catenin (βcat) signaling is critical for adrenal homeostasis. To elucidate how Wnt/βcat signaling elicits homeostatic maintenance of the adrenal cortex, we characterized the identity of the adrenocortical Wnt-responsive population. We find that Wnt-responsive cells consist of sonic hedgehog (Shh)-producing adrenocortical progenitors and differentiated, steroidogenic cells of the zona glomerulosa, but not the zona fasciculata and rarely cells that are actively proliferating. To determine potential direct inhibitory effects of βcat signaling on zona fasciculata-associated steroidogenesis, we used the mouse ATCL7 adrenocortical cell line that serves as a model system of glucocorticoid-producing fasciculata cells. Stimulation of βcat signaling caused decreased corticosterone release consistent with the observed reduced transcription of steroidogenic genes Cyp11a1, Cyp11b1, Star, and Mc2r. Decreased steroidogenic gene expression was correlated with diminished steroidogenic factor 1 (Sf1; Nr5a1) expression and occupancy on steroidogenic promoters. Additionally, βcat signaling suppressed the ability of Sf1 to transactivate steroidogenic promoters independent of changes in Sf1 expression level. To investigate Sf1-independent effects of βcat on steroidogenesis, we used Affymetrix gene expression profiling of Wnt-responsive cells in vivo and in vitro. One candidate gene identified, Ccdc80, encodes a secreted protein with unknown signaling mechanisms. We report that Ccdc80 is a novel βcat-regulated gene in adrenocortical cells. Treatment of adrenocortical cells with media containing secreted Ccdc80 partially phenocopies βcat-induced suppression of steroidogenesis, albeit through an Sf1-independent mechanism. This study reveals multiple mechanisms of βcat-mediated suppression of steroidogenesis and suggests that Wnt/βcat signaling may regulate adrenal homeostasis by inhibiting fasciculata differentiation and promoting the undifferentiated state of progenitor cells.

BRAIN TUMOUR INITIATING CELLS AND TARGETING STAT3 ONCOGENIC SIGNALLING IN GBM

BACKGROUND: Glioblastoma (GBM) is a devastating disease and the most lethal of adult brain tumours. Despite standard surgery, radiation and chemotherapy, the median survival is 15 months, thought to be due, in part, to recurrence from a small reservoir of brain tumour initiating cells. Our laboratory discovered adult neural stem cells, now found to be present in the brains of all adult mammals, through the development of the clonal neurosphere assay. This assay has contributed to the identification of adult human brain tumour initiating cells (BTICs), which may represent a reservoir that leads to GBM recurrence and death. Building upon the identification of growth factors and cytokines that converge on the cytoplasmic signal transducer and activator of transcription 3 (STAT3) to maintain the adult neural stem cell undifferentiated state, and the fact that STAT3 is abnormally active in GBM and may be one of the causes of tumour growth and therapeutic resistance, targeting the janus kinase (JAK)/STAT3 signalling pathway has become a major research focus for our laboratory. Here, we report the GBM translational potential of R333, a JAK/SYK inhibitor in development for other indications by Rigel Pharmaceuticals. METHODS: We have developed a cellular resource of more than 100 BTIC lines derived from GBM patients. Collaborative studies with academic and industry partners has resulted in the identification of promising therapeutic candidates that target JAK/STAT3 signalling in GBM. These compounds are tested for their ability to attenuate growth of BTICs in cell culture, on target activity in vitro and in vivo, and for their impact on the the survival of immunocompromised mice that have received BTIC xenografts. We employed this approach to examine the therapeutic potential of R333. RESULTS: In a collection of diverse patient-derived BTIC lines, R333 demonstrated potent, on-target JAK/STAT3 inhibition at low micromolar doses that effectively decreased BTIC viability and clonogenic potential, without affecting human fetal astrocytes. Importantly, R333 could be co-administered with temozolomide without reducing therapeutic response. Using phosphoproteomics, we confirmed the primary target of R333 in BTSCs as activated STAT3. In a BTIC orthotopic xenograft model, systemic treatment with R348, the pro-drug form of R333, resulted in BBB penetration and significant increased overall median survival without major side effects. CONCLUSIONS: These data suggest that R333 (R348) is a potent, BBB-permeable JAK/STAT3 inhibitor, worthy of further clinical evaluation for GBM treatment. These findings support our overall hypothesis that targeting the STAT3 signalling pathway may represent an effective therapeutic approach to managing GBM. SECONDARY CATEGORY: Tumor Biology.

Differential regulation of B2-type CDK accumulation in Arabidopsis roots

The accumulation of the mitotic B2-type CDK is tightly controlled by multiple pathways in Arabidopsis roots. Root growth depends on cell proliferation in the apices, which determines the root meristem size. The expression of B2-type cyclin-dependent kinase (CDKB2) is known to be restricted to dividing cells in the meristematic region, and therefore, the mechanisms controlling CDKB2 accumulation may be associated with those determining the meristem size. We investigated how CDKB2 expression is controlled in distinct zones of Arabidopsis roots. We found that CDKB2;1 expression was induced by a member of the PLETHORA (PLT) family of transcription factors, which are known to mediate auxin signaling and maintain the undifferentiated state of meristematic cells. When the root meristem was treated with an auxin antagonist, the CDKB2;1 level was reduced not only by transcriptional suppression but also by proteasome-mediated protein degradation. This indicates that auxin promotes CDKB2 accumulation at both mRNA and protein levels in the meristem. In the elongation and differentiation zones, on the other hand, neither the ubiquitin-proteasome system nor the PLT-mediated transcriptional regulation is associated with CDKB2;1 accumulation. Both CDKB2;1 and HIGH PLOIDY2 (HPY2), a SUMO E3 ligase, were ectopically accumulated in the stele when treated with exogenous auxin, suggesting the possibility that CDKB2;1 accumulation is dependent on HPY2-mediated sumoylation, which is usually maintained by a higher auxin level in the meristem. Our results demonstrate that the CDKB2 level is tightly controlled by multiple pathways to maintain the mitotic activity in developing roots.