Differential Role of the Transcription Factor ZBP-89 in Hemangioblast Fate Determination: ZBP-89 Is a Direct Regulator of SCL.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1253-1253
Author(s):  
Xiangen Li ◽  
Carl Simon Shelley ◽  
M. Amin Arnaout
Keyword(s):  
Stem Cells ◽  
Transcription Factor ◽  
Endothelial Cell ◽  
Embryonic Stem Cells ◽  
Flow Cytometric Analysis ◽  
Ectopic Expression ◽  
Embryonic Stem ◽  
Hematopoietic Stem ◽  
Fate Determination

Abstract Several molecular pathways have been identified that regulate distinct stages in the developmental progression from mesoderm to the formation of the hematopoietic and vascular lineages. Our previous work indicated that ectopic expression of the zinc finger transcription factor ZBP-89 promotes hematopoietic lineage development and represses endothelial cell lineage differentiation from hemangioblasts in murine embryonic stem cells. Here we evaluated the functional consequences of stable knockdown of ZBP-89 in embryonic stem cells (ESC) on hematopoietic and vascular development. Stable knock down of ZBP-89 in ESC significantly decreased the number of Blast Colony Forming Cells (BL-CFC) hemangioblasts, as well as primitive and definitive hematopoietic progenitor colonies BFU-E, GM-CFU, G-CFU, M-CFU and GEMM-CFU in vitro. In contrast, sprouting angiogenesis was markedly increased in EB cultures. Flow cytometric analysis of the lineages derived from ZBP-89 deficient EB cultures showed that the early (C-kit+Sca-1+) and definitive (CD45+) hematopoietic stem cells populations were reduced, but the endothelial cell population (CD31+ VE-Cadherin+) was increased. RT-PCR analysis of EB cultures revealed a direct correlation between the expression levels of ZBP-89 and hematopoietic markers (including SCL and Runx1) but an inverse correlation with the vascular marker CD31, with no change in Oct4 expression level. To investigate the mechanism underlying the role of ZBP-89 in hematopoiesis, the effect of ZBP-89 on expression of SCL, a master regulator of hematopoiesis, was examined. The murine SCL promoter transduced into the ZBP-89-expressing MEL cell line drove luciferase gene expression. ZBP-89 knockdown in MEL cells markedly reduced SCL expression. ChIP analysis showed that endogenous ZBP-89 protein bound directly to the murine SCL promoter in MEL cells. Thus ZBP-89 plays a central role in fate determination of hemangioblasts; its induction suppresses angiogenesis but enhances differentiation of hemangioblasts along the hematopoietic pathway, an effect mediated through the regulated expression of SCL.

scholarly journals CtBP-interacting BTB Zinc Finger Protein (CIBZ) Promotes Proliferation and G1/S Transition in Embryonic Stem Cells via Nanog

2012 ◽  
Vol 287 (15) ◽  
pp. 12417-12424 ◽  
Author(s):  
Tomonori Nishii ◽  
Yu Oikawa ◽  
Yasumasa Ishida ◽  
Masashi Kawaichi ◽  
Eishou Matsuda
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Zinc Finger ◽  
Zinc Finger Protein ◽  
Ectopic Expression ◽  
Embryonic Stem ◽  
Btb Domain ◽  
Constitutive Overexpression ◽  
Sirna Knockdown

Mouse embryonic stem cells (ESCs) require transcriptional regulation to ensure rapid proliferation that allows for self-renewal. However, the molecular mechanism by which transcriptional factors regulate this rapid proliferation remains largely unknown. Here we present data showing that CIBZ, a BTB domain zinc finger transcriptional factor, is a key transcriptional regulator for regulation of ESC proliferation. Here we show that deletion or siRNA knockdown of CIBZ inhibits ESC proliferation. Cell cycle analysis shows that loss of CIBZ delays the progression of ESCs through the G1 to S phase transition. Conversely, constitutive ectopic expression of exogenous CIBZ in ESCs promotes proliferation and accelerates G1/S transition. These findings suggest that regulation of the G1/S transition explains, in part, CIBZ-associated ESC proliferation. Our data suggest that CIBZ acts through the post-transcriptionally regulates the expression of Nanog, a positive regulator of ESC proliferation and G1/S transition, but does not affect Oct3/4 and Sox2 protein expression. Notably, constitutive overexpression of Nanog partially rescued the proliferation defect caused by CIBZ knockdown, indicating the role of CIBZ in ESC proliferation and G1/S transition at least in part depends on the Nanog protein level.

scholarly journals A New Entity of Acute Myeloid Leukemia Driven By Epigenetic and Somatic Dis-Regulation of Uncx, a Novel Homeobox Transcription Factor Gene

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1356-1356
Author(s):  
Giulia Daniele ◽  
Clelia Tiziana Storlazzi ◽  
Cristina Papayannidis ◽  
Ilaria Iacobucci ◽  
Angelo Lonoce ◽  
...  
Keyword(s):  
Stem Cells ◽  
Acute Myeloid Leukemia ◽  
Transcription Factor ◽  
Myeloid Leukemia ◽  
De Novo ◽  
Ectopic Expression ◽  
Response To Therapy ◽  
Hematopoietic Stem ◽  
Acute Myeloid

Abstract We describe a new AML entity, occurring in 30% of de novo acute myeloid leukemia, due to structural and epigenetic deregulation of the UNCX homeobox (HB) gene. By molecular approaches, we identified a M5 AML patient with a t(7;10)(p22;p14) translocation as the sole cytogenetic anomaly and showing ectopic expression of UNCX (7p22.3), which encode for a transcription factor involved in somitogenesis and neurogenesis. Since UNCX was never reported in association with cancer but only with common myeloid cell proliferation and regulation of cell differentiation, we decided to investigate its contribution to leukemogenesis. We observed UNCX ectopic expression in 32.3% (20/62) and in 8% (6/75) of acute myeloid leukemia (AML) patients and cell lines, respectively. Notably, retroviral-mediated UNCX transfer in CD34+ HSCs induced a slow-down in their proliferation and differentiation and transduced cells showed a lower growth rate but a higher percentage of CD34+ stem cells in liquid culture than controls. Additionally, UNCX infected cells displayed a decrease of MAP2K1 proliferation marker but increase of KLF4, HOXA10, and CCNA1, associated with impaired differentiation and pluripotency. Similarly, UNCX-positive patients revealed alteration of gene pathways involved in proliferation, cell cycle control and hematopoiesis. Since HB genes encode for transcription factors showing a crucial role in normal hematopoiesis and in leukemogenesis, we focused our attention on the role of altered UNCX expression level. Of note, its murine ortholog, (Uncx) was previously described as embedded within a low-methylated regions (≤ 10%) called "canyon" and dysregulated in murine hematopoietic stem cells (HSCs) as a consequence of altered methylation at canyons edges (borders) due to Dnmt3a inactivation. In our hands, UNCX activation was accompanied by methylation changes at both its canyon borders, clearly indicating an epigenetic regulation of this gene, although not induced by DNMT3A mutations. Clinical parameters and correlation with response to therapy will be presented. Taken together, our results indicate that more than 30% of de novo AML have a novel entity with a putative leukemogenic role of UNCX, whose activation may be ascribed to epigenetic regulators. Acknowledgments: MG, CP, GS, and AP(2) and this work was supported by ELN, AIL, AIRC, progetto Regione-Università 2010-12 (L. Bolondi), Fondazione del Monte di Bologna e Ravenna, FP7 NGS-PTL project. CTS, GD and AL are supported by Associazione Italiana Ricerca sul Cancro (AIRC) funding. Disclosures Nadarajah: MLL Munich Leukemia Laboratory: Employment. Martinelli:MSD: Consultancy; Novartis: Consultancy, Speakers Bureau; Ariad: Consultancy; BMS: Consultancy, Speakers Bureau; Pfizer: Consultancy; AMGEN: Consultancy; ROCHE: Consultancy.

Role of voltage-gated potassium channels in the fate determination of embryonic stem cells

2010 ◽  
pp. n/a-n/a ◽  
Author(s):  
Sze-Ying Ng ◽  
Chi-Hou Chin ◽  
Yuen-Ting Lau ◽  
Jialie Luo ◽  
Chun-Kit Wong ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Potassium Channels ◽  
Embryonic Stem ◽  
Fate Determination ◽  
Voltage Gated

In vitro generation of HSC-like cells from murine ESCs/iPSCs by enforced expression of LIM-homeobox transcription factor Lhx2

Blood ◽  
2011 ◽  
Vol 117 (14) ◽  
pp. 3748-3758 ◽  
Author(s):  
Kenji Kitajima ◽  
Ken-ichi Minehata ◽  
Kenji Sakimura ◽  
Toru Nakano ◽  
Takahiko Hara
Keyword(s):  
Stem Cells ◽  
Transcription Factor ◽  
Ectopic Expression ◽  
Hematopoietic Cells ◽  
Embryonic Stem ◽  
Hematopoietic Stem ◽  
Homeobox Transcription Factor ◽  
Induced Pluripotent

Abstract Identification of genes involved in in vitro differentiation induction of embryonic stem cells (ESCs) into hematopoietic stem cells (HSCs) has been challenged during last decade. To date, a homeobox transcription factor Hoxb4 has been only demonstrated to possess such an effect in mice. Here, we show that HSC-like cells were efficiently induced from mouse ESCs by enforced expression of Lhx2, a LIM-homeobox transcription factor. Transduction of Lhx2 into ESC-derived mesodermal cells resulted in robust differentiation of c-Kit+/Sca-1+/Lineage− (KSL) cells in vitro. The KSL cell induction frequency was superior to the case of Hoxb4. Furthermore, transplantation of Lhx2-transduced hematopoietic cells into lethally irradiated mice resulted in multilineage repopulation of hematopoietic cells over 4 months. Transduction of Lhx2 into induced pluripotent stem cells (iPSCs) was also effective in generating KSL cells in vitro, as well as HSC-like activities in vivo. These results demonstrate that ectopic expression of Lhx2 confers an in vivo engrafting capacity to ESC/iPSC-derived hematopoietic cells and in vivo behavior of iPSC-derived hematopoietic cells is almost identical to that of ESC-derived cells.

scholarly journals Generation of hematopoietic repopulating cells from human embryonic stem cells independent of ectopic HOXB4 expression

2005 ◽  
Vol 201 (10) ◽  
pp. 1603-1614 ◽  
Author(s):  
Lisheng Wang ◽  
Pablo Menendez ◽  
Farbod Shojaei ◽  
Li Li ◽  
Frederick Mazurier ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Human Embryonic Stem Cells ◽  
Ectopic Expression ◽  
Hematopoietic Cells ◽  
Embryonic Stem ◽  
Gene Expression Pattern ◽  
Gene Clusters ◽  
Hematopoietic Stem

Despite the need for alternative sources of human hematopoietic stem cells (HSCs), the functional capacity of hematopoietic cells generated from human embryonic stem cells (hESCs) has yet to be evaluated and compared with adult sources. Here, we report that somatic and hESC-derived hematopoietic cells have similar phenotype and in vitro clonogenic progenitor activity. However, in contrast with somatic cells, hESC-derived hematopoietic cells failed to reconstitute intravenously transplanted recipient mice because of cellular aggregation causing fatal emboli formation. Direct femoral injection allowed recipient survival and resulted in multilineage hematopoietic repopulation, providing direct evidence of HSC function. However, hESC-derived HSCs had limited proliferative and migratory capacity compared with somatic HSCs that correlated with a distinct gene expression pattern of hESC-derived hematopoietic cells that included homeobox (HOX) A and B gene clusters. Ectopic expression of HOXB4 had no effect on repopulating capacity of hESC-derived cells. We suggest that limitations in the ability of hESC-derived HSCs to activate a molecular program similar to somatic HSCs may contribute to their atypical in vivo behavior. Our study demonstrates that HSCs can be derived from hESCs and provides an in vivo system and molecular foundation to evaluate strategies for the generation of clinically transplantable HSC from hESC lines.

Efficient Differentiation of Common Marmoset Embryonic Stem Cells Into Hemangioblast-Like Cells by the Inhibition of PI3K-AKT Pathway

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2206-2206
Author(s):  
Takenobu Nii ◽  
Tomotoshi Marumoto ◽  
Hirotaka Kawano ◽  
Saori Yamaguchi ◽  
Yoko Nagai ◽  
...  
Keyword(s):  
Stem Cells ◽  
Endothelial Cell ◽  
Embryonic Stem Cells ◽  
Regenerative Medicine ◽  
Embryonic Stem ◽  
Akt Pathway ◽  
Hematopoietic Stem ◽  
Cell Markers ◽  
Novel Technology

Abstract Abstract 2206 Recently various kinds of functional cells differentiated from embryonic stem cells and induced pluripotent stem cells (ESCs/iPSCs) are expected to be utilized for cell therapy in clinical medicine. Among the transplantable functional cells differentiated from ESCs/iPSCs, endothelial progenitor cells (EPCs) and hematopoietic stem cells (HSCs) are considered to be strong candidate cells for regenerative medicine to cure various diseases such as ischemic disease and hematopoietic malignancy. Although the transplantation of EPCs and HSCs derived from human bone marrow, mobilized peripheral blood, and umbilical cord blood is commonly conducted in clinical settings, their availability for clinical use has often been hampered by both the lack of HLA compatible donor and the insufficient number of the cells. As the in vitro expansion of EPCs and HSCs derived from above sources is very difficult using current technology, it may be easier to expand EPCs and HSCs derived from ESCs/iPSCs in vitro. Hemangioblasts have the ability to differentiate into both EPCs and HSCs. Thus the technology to differentiate hemangioblast from ESCs/iPSCs that possess indefinite proliferative capacity is strongly expected. Differentiation of ESCs/iPSCs to hemangioblasts is best exemplified in recent studies that have used two step procedures to enhance hemangioblast differentiation with embryoid body (EB) formation and blast colony forming cell (BL-CFC) assay (Lu SJ et al., Nat Methods 4: 501–509, 2007). However the efficiency of hemangioblast differentiation by this method was quite low (approximately 0.35 ± 0.01%). PI3K-AKT pathway is well known to regulate various cell functions. In ESCs, PI3K-AKT pathway plays an important role in maintaining the undifferentiated state (Armstrong L et al., Hum Mol Genet 15: 1894–1913, 2006), suggesting that inhibition of PI3K may promote the differentiation of ESCs/iPSCs. Previously, we demonstrated that common marmosets (CM) are suitable laboratory animal models for preclinical studies of hematopoietic stem cell therapies (Hibino H et al., blood 1: 2839–2848, 1999). To develop the method for the more efficient generation of hemangioblasts from ESCs/iPSCs, we promoted the hemangioblast differentiation by the inhibition of PI3K-AKT pathway with the inhibitor, LY294002. CM-ESCs (Cj11 and CM40) were differentiated by EB formation in the presence of LY294002 for 4 days, and the EBs were trypsinized, and the dissociated individual cells were processed for BL-CFC assay in the methylcellulose medium containing various cytokines without LY294002 for 7 days. The number of blast colonies found in the BL-CFC assay significantly increased (approximately 10-fold; 3.5 ± 0.3%, p < 0.001) with the treatment of LY294002 during EB formation compared with control. The colonies formed in the BL-CFC assay were homogeneous and looked like a tuft of grapes which is one of hemangioblast characters, and expressed hemagioblast markers (FLK1+, VE-cadherin+, CD31+ and CD45−), suggesting that the inhibition of PI3K during EB formation promoted the generation of hemangioblast-like cells from CM-ESCs. To determine endothelial potential of these hemangioblast-like cells derived from CM-ESCs, we grew them as adherent layers on gelatin-coated plates in EGM-2 medium. The adherent cells derived from hemangioblast-like cells expressed endothelial cell markers (CD31 and vWF). Next, we also examined hematopoietic potential of hemangioblast-like cells by colony forming unit (CFU) assay. Unexpectedly no colonies were formed regardless of whether LY294002 was added or not during EB formation, indicating that hemangioblast-like cells derived from CM-ESC might be endothelial progenitors rather than hemangioblasts. Our novel technology is 10-fold more efficient in inducing endothelial differentiation from ESCs than previously reported methods. It should be emphasized that these endothelial progenitors are morphologically homogenous and expressed endothelial cell markers in a defined adherent cell culture condition, suggesting that our novel technology will be useful for an efficient generation of homogeneous EPCs for future regenerative medicine against ischemic diseases. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The role of nucleotide excision repair in protecting embryonic stem cells from genotoxic effects of UV-induced DNA damage

1999 ◽  
Vol 27 (16) ◽  
pp. 3276-3282 ◽  
Author(s):  
P. P. H. Van Sloun ◽  
J. G. Jansen ◽  
G. Weeda ◽  
L. H. F. Mullenders ◽  
A. A. van Zeeland ◽  
...  
Keyword(s):  
Stem Cells ◽  
Dna Damage ◽  
Embryonic Stem Cells ◽  
Nucleotide Excision Repair ◽  
Excision Repair ◽  
Embryonic Stem ◽  
Genotoxic Effects ◽  
Nucleotide Excision

scholarly journals Modulation of Differentiation of Embryonic Stem Cells by Polypyrrole: The Impact on Neurogenesis

2021 ◽  
Vol 22 (2) ◽  
pp. 501
Author(s):  
Kateřina Skopalová ◽  
Katarzyna Anna Radaszkiewicz ◽  
Věra Kašpárková ◽  
Jaroslav Stejskal ◽  
Patrycja Bober ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Embryonic Stem ◽  
Stem Cell Differentiation ◽  
Active Role ◽  
Low Molecular Weight ◽  
Conducting Materials ◽  
The Impact ◽  
Erk Kinase

The active role of biomaterials in the regeneration of tissues and their ability to modulate the behavior of stem cells in terms of their differentiation is highly advantageous. Here, polypyrrole, as a representantive of electro-conducting materials, is found to modulate the behavior of embryonic stem cells. Concretely, the aqueous extracts of polypyrrole induce neurogenesis within embryonic bodies formed from embryonic stem cells. This finding ledto an effort to determine the physiological cascade which is responsible for this effect. The polypyrrole modulates signaling pathways of Akt and ERK kinase through their phosphorylation. These effects are related to the presence of low-molecular-weight compounds present in aqueous polypyrrole extracts, determined by mass spectroscopy. The results show that consequences related to the modulation of stem cell differentiation must also be taken into account when polypyrrole is considered as a biomaterial.

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