199 DIFFERENTIATION OF HIGHLY ENRICHED OLIGODENDROCYTE PRECURSORS AND MATURE OLIGODENDROCYTES FROM RHESUS MONKEY EMBRYONIC STEM CELLS

2006 ◽  
Vol 18 (2) ◽  
pp. 207
Author(s):  
T. Li ◽  
Y. Xie ◽  
W. Ji
Keyword(s):  
Stem Cells ◽  
Cell Differentiation ◽  
Growth Factor ◽  
Rhesus Monkey ◽  
Molecular Mechanisms ◽  
Es Cells ◽  
Embryonic Stem ◽  
Oligodendrocyte Differentiation ◽  
Culture Systems ◽  
Oligodendrocyte Precursors

Generating homologous oligodendrocytes are required for studying the molecular mechanisms of oligodendrogliogenesis and for providing donor cells for transplantation therapies. Previous studies have shown that embryonic stem (ES) cells can be induced to generate neural stem cells with many kinds of culture systems; however, few or no oligodendrocytes were obtained from these culture systems. Here we present a simple method containing five steps for obtaining highly enriched oligodendrocyte precursors (75 � 6.8%) and mature oligodendrocytes (81 � 8.6%) from rhesus monkey embryonic stem (rES) cells. We expanded rES cells on a feeder layer of irradiated MESF (ear skin fibroblasts from a one-week-old rhesus monkey), formed embryoid bodies (EBs), promoted Day 9 (3 days in hanging drop and 6 days in suspension) differentiation into highly enriched (90.2 � 6.1%) neural progenitors (NPs) with hepatocyte growth factor (HGF) and G5 supplement [containing 5 ng/mL (bFGF) and 10 ng/mL epidermal growth factor (EGF)], purified NPs with 0.0625% trypsin in 0.04% EDTA (98% of cells were nestin-positive), amplified those progenitors in HGF and G5 media for two months, and then induced oligodendrocyte precursors differentiation in the absence of G5, but in the presence of 20 ng/mL HGF for 2 days. To obtain terminal oligodendrocytes, neurospheres cultured for 2 months were plated on laminin-coated plates for 3 weeks in the presence of HGF. The results showed that differentiated cells expressed myelin basic protein (MBP) and had typical mature oligodendrocyte morphology. Our studies also revealed that HGF significantly increased the NP proliferation speed (P < 0.05) by both decreasing cell apoptosis rate (P < 0.05) and shortening cell cycle time (P < 0.05) in the presence of G5. Additionally, HGF promoted oligodendrocyte maturation by increasing the length and number of branches and the expression of MBP. To test whether the original HGF had similar functions for oligodendrocyte specification, a series of experiments were evaluated by adding HGF or G5 to differentiation or expansion media at different differentiation stages. The results demonstrated that the ability of HGF responsiveness to initiate oligodendrocyte differentiation was regulated by G5 and by HGF alone without G5-induced rES cell differentiation into neurons. Further studies showed that the crucial time point of G5 action was from EBs to NPs; the early addition of HGF to EBs in the presence of G5 increased oligodendrocyte differentiation rate, but was not necessary, and the treatment during the first 2 days was enough to produce a similar effect; and HGF was required for terminal oligodendrocyte differentiation from NPs. Taken together, these results showed that HGF and G5 cooperatively promote rES cell differentiation into highly enriched oligodendrocyte precursors and mature oligodendrocytes.These observations set the method for obtaining highly enriched oligodendrocytes from ES cells in the nonhuman primate for clinical application and provide a platform to probe the molecular mechanisms that control oligodendrocyte differentiation.

scholarly journals Isolation and Initial Application of a Novel Peptide That Specifically Recognizes the Neural Stem Cells Derived from Rhesus Monkey Embryonic Stem Cells

2010 ◽  
Vol 15 (6) ◽  
pp. 687-694 ◽  
Author(s):  
Wenxiu Zhao ◽  
Hang Yuan ◽  
Xing Xu ◽  
Lan Ma
Keyword(s):  
Stem Cells ◽  
Cell Differentiation ◽  
Rhesus Monkey ◽  
Neural Stem Cells ◽  
Phage Display ◽  
Es Cells ◽  
Embryonic Stem ◽  
Peptide Sequence ◽  
Enzyme Linked Immunosorbent Assay ◽  
Es Cell

The search for new receptor ligands is important in the study of embryonic stem (ES) cell differentiation processes. In this study, a novel peptide (HGE VPRFHAVHL) with a specific ability to bind with neural stem cells derived from rhesus monkey ES cells was successfully screened out using a Ph.D-12 peptide phage display library. High affinity and specificity of the HGE phage were shown in an enzyme-linked immunosorbent assay. The binding ability of the phage could be matched with that of a chemically synthesized peptide with a sequence identical to that displayed by the phage, indicating that this binding capability manifests a peptide sequence. Combined with quantum dots, the HGE peptide can be used as a direct tool to show optical imaging of specific binding on a single cell membrane. Further results of Western blot showed that the HGE peptide interacted with 48/34-kDa proteins on the membrane of neural stem cells. This work is the first time that a phage display technique has been applied in ES cell differentiation studies. The findings extend the utilization of a targeting agent for neural stem cells and can also be used as a research tool in studying other cell lineages derived from ES cells.

scholarly journals Maturation of Embryonic Stem Cells Into Endothelial Cells in an In Vitro Model of Vasculogenesis

Blood ◽  
1999 ◽  
Vol 93 (4) ◽  
pp. 1253-1263 ◽  
Author(s):  
Masanori Hirashima ◽  
Hiroshi Kataoka ◽  
Satomi Nishikawa ◽  
Norihisa Matsuyoshi ◽  
Shin-Ichi Nishikawa
Keyword(s):  
Cell Adhesion ◽  
Endothelial Cell ◽  
Growth Factor ◽  
Culture System ◽  
Molecular Mechanisms ◽  
Es Cells ◽  
Embryonic Stem ◽  
Vascular Endothelial ◽  
Cell Cell

A primitive vascular plexus is formed through coordinated regulation of differentiation, proliferation, migration, and cell-cell adhesion of endothelial cell (EC) progenitors. In this study, a culture system was devised to investigate the behavior of purified EC progenitors in vitro. Because Flk-1+ cells derived from ES cells did not initially express other EC markers, they were sorted and used as EC progenitors. Their in vitro differentiation into ECs, via vascular endothelial-cadherin (VE-cadherin)+ platelet-endothelial cell adhesion molecule-1 (PECAM-1)+ CD34−to VE-cadherin+ PECAM-1+CD34+ stage, occurred without exogenous factors, whereas their proliferation, particularly at low cell density, required OP9 feeder cells. On OP9 feeder layer, EC progenitors gave rise to sheet-like clusters of Flk-1+ cells, with VE-cadherin concentrated at the cell-cell junction. The growth was suppressed by Flt-1-IgG1 chimeric protein and dependent on vascular endothelial growth factor (VEGF) but not placenta growth factor (PIGF). Further addition of VEGF resulted in cell dispersion, indicating the role of VEGF in the migration of ECs as well as their proliferation. Cell-cell adhesion of ECs in this culture system was mediated by VE-cadherin. Thus, the culture system described here is useful in dissecting the cellular events of EC progenitors that occur during vasculogenesis and in investigating the molecular mechanisms underlying these processes.

scholarly journals Loss of Function of E-Cadherin in Embryonic Stem Cells and the Relevance to Models of Tumorigenesis

2011 ◽  
Vol 2011 ◽  
pp. 1-19 ◽  
Author(s):  
Lisa Mohamet ◽  
Kate Hawkins ◽  
Christopher M. Ward
Keyword(s):  
Stem Cells ◽  
Growth Factor ◽  
Epithelial Mesenchymal Transition ◽  
Es Cells ◽  
Embryonic Stem ◽  
Loss Of Function ◽  
Mesenchymal Transition ◽  
Cell Metastasis ◽  
Patient Prognosis ◽  
E Cadherin

E-cadherin is the primary cell adhesion molecule within the epithelium, and loss of this protein is associated with a more aggressive tumour phenotype and poorer patient prognosis in many cancers. Loss of E-cadherin is a defining characteristic of epithelial-mesenchymal transition (EMT), a process associated with tumour cell metastasis. We have previously demonstrated an EMT event during embryonic stem (ES) cell differentiation, and that loss of E-cadherin in these cells results in altered growth factor response and changes in cell surface localisation of promigratory molecules. We discuss the implication of loss of E-cadherin in ES cells within the context of cancer stem cells and current models of tumorigenesis. We propose that aberrant E-cadherin expression is a critical contributing factor to neoplasia and the early stages of tumorigenesis in the absence of EMT by altering growth factor response of the cells, resulting in increased proliferation, decreased apoptosis, and acquisition of a stem cell-like phenotype.

Suppression of TGF-β and ERK Signaling Pathways as a New Strategy to Provide Rodent and Non-Rodent Pluripotent Stem Cells

2019 ◽  
Vol 14 (6) ◽  
pp. 466-473 ◽  
Author(s):  
Maryam Farzaneh ◽  
Zahra Derakhshan ◽  
Jamal Hallajzadeh ◽  
Neda Hosseini Sarani ◽  
Armin Nejabatdoust ◽  
...  
Keyword(s):  
Stem Cells ◽  
Growth Factor ◽  
Small Molecules ◽  
Signaling Pathways ◽  
Pluripotent Stem Cells ◽  
Transforming Growth Factor ◽  
Es Cells ◽  
Embryonic Stem ◽  
Erk Signaling ◽  
New Strategy

Stem cells are unspecialized cells and excellent model in developmental biology and a promising approach to the treatment of disease and injury. In the last 30 years, pluripotent embryonic stem (ES) cells were established from murine and primate sources, and display indefinite replicative potential and the ability to differentiate to all three embryonic germ layers. Despite large efforts in many aspects of rodent and non-rodent pluripotent stem cell culture, a number of diverse challenges remain. Natural and synthetic small molecules (SMs) strategy has the potential to overcome these hurdles. Small molecules are typically fast and reversible that target specific signaling pathways, epigenetic processes and other cellular processes. Inhibition of the transforming growth factor-&#946; (TGF-&#946;/Smad) and fibroblast growth factor 4 (FGF4)/ERK signaling pathways by SB431542 and PD0325901 small molecules, respectively, known as R2i, enhances the efficiency of mouse, rat, and chicken pluripotent stem cells passaging from different genetic backgrounds. Therefore, the application of SM inhibitors of TGF-&#946; and ERK1/2 with leukemia inhibitory factor (LIF) allows the cultivation of pluripotent stem cells in a chemically defined condition. In this review, we discuss recently emerging evidence that dual inhibition of TGF-&#946; and FGF signaling pathways plays an important role in regulating pluripotency in both rodent and non-rodent pluripotent stem cells.

Abstract P001: HDAC1 Plays an Important Role in the Differentiation of Embryonic Stem Cells and Induced Pluripotent Stem Cells into Cardiovascular Lineages

2011 ◽  
Vol 109 (suppl_1) ◽  
Author(s):  
Eneda Hoxha ◽  
Erin Lambers ◽  
Veronica Ramirez ◽  
Prasanna Krishnamurthy ◽  
Suresh Verma ◽  
...  
Keyword(s):  
Stem Cells ◽  
Molecular Mechanisms ◽  
Es Cells ◽  
Critical Role ◽  
Embryonic Stem ◽  
Ips Cells ◽  
Full Potential ◽  
Early Differentiation ◽  
Increased Risk ◽  
Mes Cells

Despite advancements in the treatment of myocardial infarction (MI), the majority of patients are at increased risk for developing heart failure due to the loss of cardiomyocytes and microvasculature. Some of the main obstacles in the realization of the full potential of iPS/ES cells arise from incomplete and poorly understood molecular mechanisms and epigenetic modifications that govern their pluripotency and directed differentiation. Real-time array experiments revealed that HDAC1 is highly expressed in pluripotent cells. Additionally the lack of this molecule is embryonic lethal, suggesting it plays a key role in development. Thus, we hypothesized that HDAC1 plays a critical role in directing cardiovascular differentiation of mES and iPS cells in vitro. HDAC1 was knocked down in mES cells (C57BL/6) and iPS cells using a shRNA vector. Differentiation through embryoid body (EB) was induced in wild type mES cells and iPS cells and in their HDAC1-null counterparts and the ability of these cells to differentiate into three early embryonic lineages and more specifically cardiovascular lineage was monitored. EBs lacking HDAC1 differentiated slower and showed delayed suppression of pluripotent genes such as Oct4 and Sox2. ChiP experiments revealed high histone acetylation levels at the promoter regions of these genes during early differentiation. In addition cells lacking HDAC1 showed reduced expression of early markers for all three germ layers. HDAC1-null EBs also showed delayed and reduced spontaneous beating. Expression of cardiomyocite markers as well as markers of other cardiovascular lineages was repressed in HDAC1 -null cells. However, supplementation with BMP2 during early differentiation recovered the ability in the HDAC1-null cells to differentiate into endodermal and mesodermal lineages, but not ectodermal. We propose that HDAC1 plays a critical role in early development and cardiovascular differentiation of mES and iPS cells by repressing pluripotent genes and allowing for expression of early developmental genes such as SOX17 and BMP2. Further research in the molecular mechanisms involved in this process will greatly aid our understanding of the epigenetic circuitry of pluripotency and differentiation in ES and iPS cells.

Role of TPO in the Differentiation of Embryonic Stem Cells into Hematopoetic Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4202-4202
Author(s):  
Zheng Wang ◽  
Pramono Andri ◽  
Skokowa Julia ◽  
Welte Karl
Keyword(s):  
Stem Cell ◽  
Cell Differentiation ◽  
Rhesus Monkey ◽  
Embryonic Stem Cell ◽  
Mrna Expression ◽  
Es Cells ◽  
Embryonic Stem ◽  
Stem Cell Differentiation ◽  
Embryonic Stem Cell Differentiation

Abstract Thrombopoetin (TPO) is a primary regulator of megakaryocyte and platelet production. However, studies in c-mpl-deficient mice and in congenital amegakaryocytic thrombocytopenia-patients with non-sense c-mpl mutation who develop pancytopenia during the first years of life suggest that TPO also play an important role on early hematopoesis. We demonstrated that TPO enhances FLK-1 (VEGF-receptor) expression on hemangioblasts during murine embryonic stem cell differentiation in embryoid body-liquid cultures (up to 73%). To extend our studies, we investigated the TPO signaling in FLK-1 positive cells. ES cells at different time point of differentiation showed that TPO enhances c-mpl-, BMP4-, Notch-, HOXB4-, HOXB9-, HOXA10-, Runx1-and CD133- mRNA expression. To investigate mesoderm formation, we also analyzed GATA-4 and T-brachyury mRNA level expression. Interestingly, we found that TPO alone did not increase GATA-4- and T-brachyury- mRNA expression, suggesting that TPO requires other cytokines to form the mesoderm. We also found that TPO could maintain VEGF-A mRNA expression level during differentiation of ES-cells. We hypothesize that VEGF expression together with c-mpl expression is required in hematopoetic differentiation of ES cell. This activity of Tpo was also observed during Rhesus monkey embryonic stem cell differentiation into hematopoetic cell. Only combinations of TPO and VEGF were capable of increasing CD34 positive hematopoietic progenitor cells (up to 8%), but TPO alone failed to induce high levels of CD34+ cell. In addition, analysis of gene expression during hemangioblast development demonstrated that TPO was capable of increasing the expression of VEGF receptors (FLK-1) and TPO receptors (c-mpl) in mice and primates. The in-vitro differentiation of mouse and rhesus monkey ES cells provides an opportunity to better understand the role of TPO in the early stage of hematopoietic development from ES cells to mature hematopoietic cells.

scholarly journals An Inducible Expression System of the Calcium-Activated Potassium Channel 4 to Study the Differential Impact on Embryonic Stem Cells

2011 ◽  
Vol 2011 ◽  
pp. 1-12 ◽  
Author(s):  
Stefan Liebau ◽  
Michael Tischendorf ◽  
Daniel Ansorge ◽  
Leonhard Linta ◽  
Marianne Stockmann ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Differentiation ◽  
Cell Line ◽  
Es Cells ◽  
Embryonic Stem ◽  
Stem Cell Differentiation ◽  
Pacemaker Cell ◽  
Es Cell ◽  
Sk Channel ◽  
Mouse Es Cell

Rationale. The family of calcium-activated potassium channels consists of four members with varying biological functions and conductances. Besides membrane potential modulation, SK channels have been found to be involved in cardiac pacemaker cell development from ES cells and morphological shaping of neural stem cells.Objective. Distinct SK channel subtype expression in ES cells might elucidate their precise impact during cardiac development. We chose SK channel subtype 4 as a potential candidate influencing embryonic stem cell differentiation.Methods. We generated a doxycycline inducible mouse ES cell line via targeted homologous recombination of a cassette expressing a bicistronic construct encoding SK4 and a fluorophore from the murine HPRT locus.Conclusion. We characterized the mouse ES cell line iSK4-AcGFP. The cassette is readily expressed under the control of doxycycline, and the overexpression of SK4 led to an increase in cardiac and pacemaker cell differentiation thereby serving as a unique tool to characterize the cell biological variances due to specific SK channel overexpression.

scholarly journals Zic3 Is Required for Maintenance of Pluripotency in Embryonic Stem Cells

2007 ◽  
Vol 18 (4) ◽  
pp. 1348-1358 ◽  
Author(s):  
Linda Shushan Lim ◽  
Yuin-Han Loh ◽  
Weiwei Zhang ◽  
Yixun Li ◽  
Xi Chen ◽  
...  
Keyword(s):  
Stem Cells ◽  
Transcription Factors ◽  
Embryonic Stem Cells ◽  
Regulatory Networks ◽  
Molecular Mechanisms ◽  
Es Cells ◽  
Embryonic Stem ◽  
Es Cell ◽  
Lineage Specification ◽  
Mouse Es Cells

Embryonic stem (ES) cell pluripotency is dependent upon sustained expression of the key transcriptional regulators Oct4, Nanog, and Sox2. Dissection of the regulatory networks downstream of these transcription factors has provided critical insight into the molecular mechanisms that regulate ES cell pluripotency and early differentiation. Here we describe a role for Zic3, a member of the Gli family of zinc finger transcription factors, in the maintenance of pluripotency in ES cells. We show that Zic3 is expressed in ES cells and that this expression is repressed upon differentiation. The expression of Zic3 in pluripotent ES cells is also directly regulated by Oct4, Sox2, and Nanog. Targeted repression of Zic3 in human and mouse ES cells by RNA interference–induced expression of several markers of the endodermal lineage. Notably, the expression of Nanog, a key pluripotency regulator and repressor of extraembryonic endoderm specification in ES cells, was significantly reduced in Zic3 knockdown cells. This suggests that Zic3 may prevent endodermal marker expression through Nanog-regulated pathways. Thus our results extend the ES cell transcriptional network beyond Oct4, Nanog, and Sox2, and further establish that Zic3 plays an important role in the maintenance of pluripotency by preventing endodermal lineage specification in embryonic stem cells.

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