HoxA11 Is Expressed in the Developing Hemangioblast as Well as Early Hematopoietic Precursor Stem Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4206-4206
Author(s):  
Regina D. Horvat-Switzer ◽  
Alexis A. Thompson
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mrna Expression ◽  
Progenitor Cells ◽  
Hox Genes ◽  
Es Cells ◽  
Critical Role ◽  
Embryonic Stem ◽  
Rt Pcr ◽  
Hematopoietic Precursor

Abstract Congenital amegakaryocytic thrombocytopenia with radio-ulnar synostosis is associated with mutations in the HOXA11 gene, suggesting that HoxA11 may play a role in megakaryocytic lineage commitment or differentiation. The HOX genes encode transcription factors that are involved in cellular differentiation in embryonic as well as adult tissues. Numerous studies have identified HOX genes as important regulators of various aspects of hematopoiesis including self-renewal, proliferation, differentiation and leukemogenesis. Our initial studies failed to identify the expression of HoxA11 in platelets, TPO-induced CD34+ umbilical cord stem cells or normal bone marrow. More recently our lab has detected a small amount of HoxA11 mRNA in cells isolated from unfractionated human cord blood, suggesting the expression of HoxA11 may occur in a small subset of early hematopoietic or stromal cells. To test this hypothesis we have employed a murine embryonic stem (ES) cell culture system. Co-culture of ES cells and the bone marrow stromal cell line, OP9, can give rise to primitive as well as definitive hematopoietic progenitors in the absence of leukemia inhibitory factor (LIF). By day 6, ES cells on OP9 can differentiate into mesodermal colonies, which contain a bi-potential progenitor known as the hemangioblast. The hemangioblast can further differentiate into either a hematopoietic or endothelial lineage. To determine when HoxA11 is expressed we have employed this model using green fluorescent protein (GFP) expressing ES cells grown on OP9 and differentiated into hematopoietic precursors in the absence of LIF. Nested RT-PCR revealed that HoxA11 mRNA is highly expressed in ES cells following 6 days (D6) on OP9. HoxA11 expression was restricted to D6 ES cells, as HoxA11 mRNA was not found in OP9 cells alone or ES cells differentiated on OP9 for 0, 3, or 9 days. RT-PCR revealed HoxA11 mRNA expression coincided with the expression of flk-1, a marker for the hemangioblast. Since HoxA11 expression is concurrent with hemangioblast differentiation, we sought to determine if the hemangioblast is the cell that expressed HoxA11. Using flow cytometery and fluorescence activated cell sorting (FACS) analysis we separated D6 ES cells into flk-1 positive (flk-1+) and negative (flk-1−) populations and investigated which population expressed HoxA11. Nested RT-PCR revealed that HoxA11 mRNA expression is found in both the flk-1+ and flk-1- fractions. We further analyzed these fractions by RT-PCR for SCL/Tal-1. SCL/Tal-1 is a transcription factor that plays a critical role in the commitment of mesoderm into hematopoietic progenitor cells. We find SCL/Tal-1 mRNA also expressed in both flk-1+ and flk-1- fractions, which parallels HoxA11 mRNA expression. These data suggest HoxA11 expression occurs in the flk-1+ hemangioblast but also possibly in a flk-1-/SCL+ hematopoietic precursor cell population. Current studies are underway to determine the cell fate and role of the HoxA11 expressing progenitor cell. Taken together, these data are the first findings of HoxA11 expression in early progenitor cells as well as the first evidence of controlled HoxA11 regulation during early hematopoietic development.

Stem/progenitor cells derived from adult tissues: potential for the treatment of diabetes mellitus

2003 ◽  
Vol 284 (2) ◽  
pp. E259-E266 ◽  
Author(s):  
Andreas Lechner ◽  
Joel F. Habener
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Progenitor Cells ◽  
Es Cells ◽  
Embryonic Stem ◽  
Pancreatic Islet Transplantation ◽  
Β Cells ◽  
Β Cell ◽  
Adult Islet

In view of the recent success in pancreatic islet transplantation, interest in treating diabetes by the delivery of insulin-producing β-cells has been renewed. Because differentiated pancreatic β-cells cannot be expanded significantly in vitro, β-cell stem or progenitor cells are seen as a potential source for the preparation of transplantable insulin-producing tissue. In addition to embryonic stem (ES) cells, several potential adult islet/β-cell progenitors, derived from pancreas, liver, and bone marrow, are being studied. To date, none of the candidate cells has been fully characterized or is clinically applicable, but pancreatic physiology makes the existence of one or more types of adult islet stem cells very likely. It also seems possible that pluripotential stem cells, derived from the bone marrow, contribute to adult islet neogenesis. In future studies, more stringent criteria should be met to clonally define adult islet/β-cell progenitor cells. If this can be achieved, the utilization of these cells for the generation of insulin-producing β-cells in vitro seems to be feasible in the near future.

scholarly journals Induction of Recurrent Break Cluster Genes in Neural Progenitor Cells Differentiated from Embryonic Stem Cells In Culture

2019 ◽  
Author(s):  
Aseda Tena ◽  
Yuxiang Zhang ◽  
Nia Kyritsis ◽  
Anne Devorak ◽  
Jeffrey Zurita ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Progenitor Cells ◽  
Es Cells ◽  
Embryonic Stem ◽  
Neural Progenitors ◽  
Neural Genes ◽  
Genome Wide ◽  
Primary Mouse

ABSTRACTMild replication stress enhances appearance of dozens of robust recurrent genomic break clusters, termed RDCs, in cultured primary mouse neural stem and progenitor cells (NSPCs). Robust RDCs occur within genes (“RDC-genes”) that are long and have roles in neural cell communications and/or have been implicated in neuropsychiatric diseases or cancer. We sought to develop an in vitro approach to determine whether specific RDC formation is associated with neural development. For this purpose, we adapted a system to induce neural progenitor cell (NPC) development from mouse embryonic stem cell (ESC) lines deficient for XRCC4 plus p53, a genotype that enhances DNA double-strand break (DSB) persistence to enhance detection. We tested for RDCs by our genome wide DSB identification approach that captures DSBs genome-wide via their ability to join to specific genomic Cas9/sgRNA-generated bait DSBs. In XRCC4/p53-deficient ES cells, we detected 7 RDCs, which were in genes, with two RDCs being robust. In contrast, in NPCs derived from these ES cell lines, we detected 29 RDCs, a large fraction of which were robust and associated with long, transcribed neural genes that were also robust RDC-genes in primary NSPCs. These studies suggest that many RDCs present in NSPCs are developmentally influenced to occur in this cell type and indicate that induced development of NPCs from ES cells provides an approach to rapidly elucidate mechanistic aspects of NPC RDC formation.SIGNIFICANCE STATEMENTWe previously discovered a set of long neural genes susceptible to frequent DNA breaks in primary mouse brain progenitor cells. We termed these genes RDC-genes. RDC-gene breakage during brain development might alter neural gene function and contribute to neurological diseases and brain cancer. To provide an approach to characterize the unknown mechanism of neural RDC-gene breakage, we asked whether RDC-genes appear in neural progenitors differentiated from embryonic stem cells in culture. Indeed, robust RDC-genes appeared in neural progenitors differentiated in culture and many overlapped with robust RDC-genes in primary brain progenitors. These studies indicate that in vitro development of neural progenitors provides a model system for elucidating how RDC-genes are formed.

BMP4 and TGFbeta Differentially Regulate CD34+ Progenitor Development in Human Embryonic Stem Cells through SMAD-Dependent Pathway

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 889-889
Author(s):  
ZacK Z. Wang ◽  
Hao Bai ◽  
Melanie Arzigian ◽  
Yong-Xing Gao ◽  
Wen-Shu Wu
Keyword(s):  
Stem Cells ◽  
Smooth Muscle ◽  
Growth Factors ◽  
Progenitor Cells ◽  
Embryoid Body ◽  
Es Cells ◽  
Embryonic Stem ◽  
Ips Cells ◽  
Bmp Signaling ◽  
Cd34 Cells

Abstract Pluripotent stem cells derived from patients, including embryonic stem (ES) cells and “induced pluripotent stem” (iPS) cells, are a promising area of regenerative medical research. A major roadblock toward human clinical therapies using ES cells or iPS cells is to define the factors that direct ES cell differentiation into lineage specific cells. We previously established a simple and efficient human embryonic stem cell (hESC) differentiation system to generate CD34+/CD31+ progenitor cells that gave rise to hematopoietic and endothelial cells (Nat Biotech.25:317, 2007). To advance potential clinical application and to define the effects of growth factors on hematopoietic and vascular differentiation, we assessed hESC differentiation on human feeder cells in serum-free condition without intermediate embryoid body (EB) formation. We investigated the roles of BMPs, TGFbeta, VEGF, and FGF2 in directing hESC differentiation. Growth factors were added into culture at different time points to test their stage specific roles. Our study demonstrated that BMP proteins, including BMP2, BMP4, and BMP7, but not BMP9, had synergic effects to VEGF and FGF-2 on hESC differentiation to CD34+/CD31+ progenitor cells. BMP4 was essential to initial CD34+/CD31+ cell development, whereas VEGF and FGF2 promoted the differentiation in later stage, suggesting the sequential roles of BMP4, VEGF and FGF2 in directing hESC differentiation to CD34+/CD31+ progenitor cells. TGFbeta or activin promoted hESC differentiation into CD34+/CD31− cells that were unable to give rise to hematopoietic, endothelial, and smooth muscle cells. Furthermore, TGFbeta or activin activated Smad2/3 signaling, and suppressed BMP4-induced CD34+/CD31+ cells. Microarray analysis revealed that BMP4-induced CD34+ cells expressed hematopoietic, endothelial and smooth muscle genes, including GATA2, gamma globins, VE-Cad, KDR, CD31, Tie2, and aortic smooth muscle actin, whereas TGFbeta-induced CD34+ cells expressed pluripotent markers and endoderm markers, including Oct3/4, Sox2, and Nanog, HHEX, GATA6, and FoxA2. Both canonical BMP signaling (Smad1/5/8-dependent) and non-canonical BMP signaling (p38 MAPK and p42 ERK pathway) were activated by BMP4 in hESCs. Dorsomorphin specifically inhibited BMP4-mediated phosphorylation of Smad1/5/8, and blocked hESC differentiation into CD34+/CD31+ cells. In summary, BMPs and TGFbeta regulate distinct populations of CD34+ cells in hESCs. BMP-Smad1/5/8 pathway is critical for hematopoietic and vascular progenitor development.

Bloodstream Progenitor-Cell Traffic In Primary Myelofibrosis Reveals Cell Subsets Showing Some Features Of Very-Small Embryonnic-Like Stem Cells (VSELs), Along With Endothelial (PEC), Mesenchymal (MPC) and Hematopoietic (HPC) Progenitor Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 5265-5265 ◽  
Author(s):  
Mario Ojeda-Uribe ◽  
Hanna Sovalat ◽  
Laura Jung ◽  
Christophe Desterke ◽  
Sylvie Thiebault ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Hot Spot ◽  
Embryonic Stem ◽  
Primary Myelofibrosis ◽  
Rt Pcr ◽  
In Vitro Development ◽  
Oct4 Expression ◽  
Vitro Development

Abstract Introduction Primary myelofibrosis (PMF) is accompanied by an increase in the bloodstream circulation of some adult progenitor cells. Extramedullary hematopoiesis observed in this setting might remind some features related to foetal hematopoiesis. Material and methods We looked for evidence in favour of this hypothesis in blood samples of a small cohort of untreated patients with PMF (4 pre-fibrotic (PF) and 4 fibrotic (F), defined according to the WHO and Thiele's histopathology score (Blood, 2011)). Patient baseline characteristics are shown below. We performed a) flow-cytometric analysis for cell subsets related to VSEL, PEC, MPC, HPC; b) RT-PCR for embryonic transcriptional factors NANOG, OCT4, SOX2, LIN28 from MNC fraction (positive control hES, negative control CPRE2 c) in-vitro development of embryonic stem like cells (ESlC) under specific culture conditions. In addition we looked for SRSF2 mutations in order to better characterize PMF stages. Results As expected we detected high numbers of circulating CD34+ cells (HPC) (mean 233083±307148/ml (range 4600-783000), with similar numbers in PF- (231125±289553/ml) and F-PMF (235040 ±369156/ml). We were able to detect small numbers of the following cell subsets related to VSEL (size 2-4m) (Fig 1) Lin-/CD45-/CD34+ (mean 124±239/ml), Lin-/CD45-/CD133+ (mean 1178±971/ml), Lin-/CD45-/CXCR4+ (mean 1572±1622/ml). Lin-/CD45-CD34+AC133+CXCR4+ cells were detected in 6 of 8 patients (mean 186±375/ml) with F-PMF patients showing higher numbers (279±416/ml) than PF-PMF (63±71/ml). NANOG and OCT4 expression was detected by RT-PCR in all the patients tested. Mean OCT4 expression was about 50% the level of hES, but F-PMF showed higher levels. NANOG expression was similar to that of hES, whereas Sox2 and Lin28 were not expressed in most patients. We failed to observe the in-vitro development of ESlC in the 2 tested patients. PEC (Lin-/CD45-CD34+AC133+KDR+) were detected in all the PF-PMF (185±332/ml) and in 1 of 4 F-PMF (mean 9±18/ml). MPC (Lin-/CD45-CD90+CD105+) were detected in higher numbers in PF-PMF (mean 413±528/ml) than in F-PMF (mean 157±216/ml). We were not able to detect mutations in the hot spot of SRSF2 (codons 93,94,95). Conclusions Small numbers of cell subsets displaying morphologic and immunophenotypic features of VSELs were detected in PMF patients. However, we are not able to define these as fully specific VSELs according to previous works that defined them (Kucia, Leukemia 2006). Interestingly Lin-/CD45-CD34+AC133+CXCR4+ cells were observed in higher numbers in F-PMF, supporting in part our hypothesis that PMF evolution can be associated to the recruitment and circulation of some primitive progenitors (dormant in the adult life) as it can be observed during the foetal period. This recruitment also involves HPC. Moreover although all patients expressed OCT4 and NANOG, OCT4 expression was higher in F-PMF. As expected PEC circulate in higher numbers in PF-PMF compared to F-PMF. Interestingly both F-PMF and PF-PMF were associated to the circulation of significant numbers of MPC but higher numbers observed in PF-MFP might be interpreted either as a necessary recruitment to establish extra-medullary stroma or due to the exit from bone marrow of highly proliferative MPC. Whether all these different circulating progenitor cells, are clonally or not clonally related to the PMF pathogenesis or to unspecific mobilisation secondary to bone marrow microenvironment injury cannot be determined from these preliminary results. Disclosures: No relevant conflicts of interest to declare.

Exosomes Derived from AML/MDS Cells Is Involved in Stromal Dysfunction and Bone Marrow Failure

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4627-4627
Author(s):  
Hiroto Horiguchi ◽  
Masayoshi Kobune ◽  
Shohei Kikuchi ◽  
Wataru Jomen ◽  
Kazuyuki Murase ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mrna Expression ◽  
Progenitor Cells ◽  
Stromal Cells ◽  
Leukemic Cells ◽  
Boyden Chamber ◽  
Hematopoietic Stem ◽  
Coculture System ◽  
Qpcr Array

Abstract The failure of normal hematopoiesis in myelodysplastic syndrome (MDS)/acute myeloid leukemia (AML) could be induced by a variety of mechanism such as the alteration of property of hematopoietic stem cells and stem cell niche. However, it has not yet been clarified precise mechanism how MDS stem/progenitor cells could replace normal hematopoietic stem/progenitor cells especially regarding involvement of mesenchymal stromal cells (MSCs). To gain insight into the mechanism of stromal dysfunction, comparative analyses of transcriptomes were conducted between normal and MDS/AML-derived MSCs. Further, we attempted to identify certain effectors originated from MDS/AML cells could alter the function of bone marrow (BM) MSCs. The MSCs derived from healthy volunteer (HV)-derived (normal) and MDS/AML-derived stromal cells were established and analyzed mRNA expression by quantitive PCR (qPCR) array. Additionally, the supporting activity of MSCs for BM CD34+ progenitor/stem cells was examined using serum free coculture system. The interaction between MDS/AML cells and MSCs were evaluated by using Boyden Chamber and the changes of mRNA expression were analyzed. The results of qPCR array revealed that the expression of hematopoietic factors was drastically altered in MDS/AML-derived MSCs as compared with normal MSCs. Among these factors, the expression of SCF and JAG1 mRNA were significantly and consistently reduced in all MDS/AML patients examined. Functional assay of these MSCs demonstrated that the number of colony-forming units (CFU) mixed cells (MIXs) and cobblestone area-forming cells (CAFCs) derived from CD34+ cells was significantly reduced after coculture with MDS/AML-derived MSCs as compared with normal MSCs. Even non-contact culture using Boyden Chamber between leukemic cells and MSCs induced the reduction of SCF and JAG1 mRNA, indicating that certain inducers could be soluble factors. Interestingly, this effect of transcriptomes alteration was negated by nSMase2 inhibitor (GW4869). Exosome transfer assay using Boyden Chamber revealed that GFP and PKH26 in leukemic cells transmit onto MSCs in non-contact coculture system and this transfer of exosome was significantly inhibited by GW4869 or nSMase siRNA. The multiple type of microRNA in exosome derived from MDS/AML cells was transferred into MSCs, suggesting that exosome could contribute to the alteration of mRNA expression in stromal cells. Collectively, these results indicated that exosome derived from MDS/AML cells could be involved in the reduction of SCF/JAG mRNA and the stromal supporting activity of normal hematopoietic stem/progenitor cells. Disclosures No relevant conflicts of interest to declare.

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.

scholarly journals The Sox-2 Regulatory Regions Display Their Activities in Two Distinct Types of Multipotent Stem Cells

2004 ◽  
Vol 24 (10) ◽  
pp. 4207-4220 ◽  
Author(s):  
Satoru Miyagi ◽  
Tetsuichiro Saito ◽  
Ken-ichi Mizutani ◽  
Norihisa Masuyama ◽  
Yukiko Gotoh ◽  
...  
Keyword(s):  
Stem Cells ◽  
Neural Stem Cells ◽  
Progenitor Cells ◽  
Es Cells ◽  
Regulatory Region ◽  
Embryonic Stem ◽  
Specific Expression ◽  
Regulatory Regions ◽  
Multipotent Stem Cells ◽  
Differentiated Cells

ABSTRACT The Sox-2 gene is expressed in embryonic stem (ES) cells and neural stem cells. Two transcription enhancer regions, Sox-2 regulatory region 1 (SRR1) and SRR2, were described previously based on their activities in ES cells. Here, we demonstrate that these regulatory regions also exert their activities in neural stem cells. Moreover, our data reveal that, as in ES cells, both SRR1 and SRR2 show their activities rather specifically in multipotent neural stem or progenitor cells but cease to function in differentiated cells, such as postmitotic neurons. Systematic deletion and mutation analyses showed that the same or at least overlapping DNA elements of SRR2 are involved in its activity in both ES and neural stem or progenitor cells. Thus, SRR2 is the first example of an enhancer in which a single regulatory core sequence is involved in multipotent-state-specific expression in two different stem cells, i.e., ES and neural stem cells.

scholarly journals Genome-wide analysis of target genes regulated by HoxB4 in hematopoietic stem and progenitor cells developing from embryonic stem cells

Blood ◽  
2011 ◽  
Vol 117 (15) ◽  
pp. e142-e150 ◽  
Author(s):  
Motohiko Oshima ◽  
Mitsuhiro Endoh ◽  
Takaho A. Endo ◽  
Tetsuro Toyoda ◽  
Yaeko Nakajima-Takagi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Progenitor Cells ◽  
Embryonic Stem ◽  
Es Cell ◽  
Hematopoietic Stem ◽  
Genome Wide Analysis ◽  
Genome Wide ◽  
A Genome

Abstract Forced expression of the transcription factor HoxB4 has been shown to enhance the self-renewal capacity of mouse bone marrow hematopoietic stem cells (HSCs) and confer a long-term repopulating capacity to yolk sac and embryonic stem (ES) cell–derived hematopoietic precursors. The fact that ES cell–derived precursors do not repopulate bone marrow without HoxB4 underscores an important role for HoxB4 in the maturation of ES-derived hematopoietic precursors into long-term repopulating HSCs. However, the precise molecular mechanism underlying this process is barely understood. In this study, we performed a genome-wide analysis of HoxB4 using ES cell–derived hematopoietic stem/progenitor cells. The results revealed many of the genes essential for HSC development to be direct targets of HoxB4, such as Runx1, Scl/Tal1, Gata2, and Gfi1. The expression profiling also showed that HoxB4 indirectly affects the expression of several important genes, such as Lmo2, Erg, Meis1, Pbx1, Nov, AhR, and Hemgn. HoxB4 tended to activate the transcription, but the down-regulation of a significant portion of direct targets suggested its function to be context-dependent. These findings indicate that HoxB4 reprograms a set of key regulator genes to facilitate the maturation of developing HSCs into repopulating cells. Our list of HoxB4 targets also provides novel candidate regulators for HSCs.

Cyclin A2 Plays a Critical Role in Proliferation of Lymphoid Progenitors

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 914-914
Author(s):  
Kentaro Kohno ◽  
Hiromi Iwasaki ◽  
Tadafumi Iino ◽  
Shin-ichi Mizuno ◽  
Peter Sicinski ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
B Cell ◽  
Progenitor Cells ◽  
Critical Role ◽  
Embryonic Stem ◽  
Hematopoietic Progenitors ◽  
Hematopoietic Stem ◽  
M Phase ◽  
Cyclin A2

Abstract Abstract 914 Cell cycle regulators could be differentially used among self–renewing stem cells, rapidly expanding progenitor cells, and terminally differentiated cells those clonally replicate. Cyclin A is a regulatory subunit for cyclin dependent kinase (Cdk) 1 and Cdk2, and it drives S phase progression as well as transition to G2/M phase in cell cycle. We have previously reported that cyclin A2 is not required for fibroblast replication but it is indispensable in maintenance of self-renewing stem cells, including embryonic stem cells and hematopoietic stem cells (HSCs) (Cell 138 2009). The question is whether cyclin A2 plays a role in proliferation of hematopoietic progenitors downstream of the HSC. Here, we further assessed the requirement of cyclin A2 in non-self-renewing hematopoietic progenitors. Quantitative RT-PCR analysis showed that cyclin A2 was expressed in hematopoietic progenitor cells as well as stem cells, and its expression level is highest in lymphoid-committed progenitor stages of both T and B cell lineages. Thus, in order to test the role of cylin A2 in early lymphopoiesis, we crossed cyclin A2 floxed mice with Rag1-Cre knock-in mice. Because recombination activating gene (RAG)-1 is essential for generation of pre-BCRs and pre-TCRs that are critical for expansion of B and T lymphoid progenitor cells, respectively, we hypothesized that the requirement of Cyclin A2 in early lymphopoiesis can be assessed in this system. As we expected, the Rag1-Cre cyclin A2 floxed/floxed mice were viable, and have normal numbers of HSCs and myeloid progenitors. They, however, displayed severe reduction of mature T and B cell numbers that were only 1/100 - 1/10 of wild-type controls. The number of common lymphoid progenitor was unchanged, but there were severely reduced preB cells in bone marrow and T cell progenitors from CD4-CD8- double negative stage in thymus. Furthermore, cell cycle analysis shows that the Cyclin A2 disrupted progenitors are unable to progress from S to G2/M phase, and in vitro culture clearly showed that those progenitors are unable to proliferate and resulted in apoptosis. These findings clearly demonstrate that cyclin A2 is indispensable not only for self-renewing HSCs, but also for proliferation of T and B cell progenitors. Disclosures: No relevant conflicts of interest to declare.

SDF-1/CXCL12 Enhances Survival of Murine Embryonic Stem Cells and Their Production of Myeloid Progenitors, but Not Hemangioblasts.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1693-1693
Author(s):  
Ying Guo ◽  
Giao Hangoc ◽  
Huimin Bian ◽  
Louis M. Pelus ◽  
Hal E. Broxmeyer
Keyword(s):  
Stem Cells ◽  
Progenitor Cells ◽  
Colony Formation ◽  
Es Cells ◽  
Embryonic Stem ◽  
Receptor Expression ◽  
Hematopoietic Progenitor Cells ◽  
Es Cell ◽  
Hematopoietic Progenitor ◽  
Myeloid Progenitors

Abstract Stromal cell derived factor (SDF)-1/CXCL12 has been shown to promote the survival of embryonic retinal ganglion cells, peritoneal B1a (PerBla) lymphocytes and chronic lymphocytic leukemia B cells. Our previous studies implicated SDF-1 as an important factor in enhancing survival of murine bone marrow (BM) hematopoietic stem cells and, human cord blood and adult human BM myeloid progenitors. Since pluripotent embryonic stem (ES) cells can give rise to differentiated cell types derived from all three primary germ layers (endoderm, mesoderm, and ectoderm) and adult stem cells are generated during embryoid body (EB) formation, we investigated whether SDF-1 has effects on survival of ES cells and EB generation of hematopoietic progenitor cells. In order to establish SDF-1 expression patterns during EB formation, we screened supernatants during day 1–5 EB formation for SDF-1 production by three murine ES cell lines (E14, R1 and CCE). We observed low but detectable SDF-1 secreted in cultures of ES cells and day 1 stage EBs. SDF-1 was increased in the media from day 2 stage EBs and continued to increase through days 4–5. CXCR4, SDF-1 receptor, expression was also analyzed. CXCR4 mRNA expression was low in ES cells and day 1–3 EBs, and increased significantly from Day 4 EBs, reaching maximum levels at day 5, and decreasing after day 6. Surface CXCR4 expression was consistent with mRNA data. To determine if SDF-1 had an effect on ES cell survival, we cultured ES cells without serum, and added serum at either 0, 24, 48 or 96 hrs to each of the following groups: A) Control, B) SDF-1 (100ng/ml) or C) AMD3100 (1 μM), an SDF-1 receptor (CXCR4) antagonist. Colonies were scored 7 days after the addition of serum. SDF-1 enhanced survival of ES cells, while AMD3100 decreased survival. We also checked the apoptosis of ES cells after withdrawing serum for 24, 48, 72 and 96 hours in four groups: A) control, B) SDF-1 (100ng/ml), C) AMD3100 (1 μM) or D) AMD3100 (1 μM) and SDF-1 (100ng/ml). SDF-1 decreased apoptosis and AMD3100 blocked the SDF-1 effect. AMD3100 alone increased apoptosis compared to control. This suggests that AMD3100 blocked endogenous SDF-1 effects. To determine if SDF-1 had an effect on differentiation of hematopoietic progenitor cells, we added SDF-1 (100 ng/ml), AMD3100, or AMD3100 plus SDF-1 (100 ng/ml) at the beginning of EB formation, immediately after removal of LIF, and quantitated primitive erythroid (p-BFU-E), definitive erythroid (d-BFU-E), granulocyte-macrophage (CFU-GM) and multipotential Granulocyte/Erythroid/Macrophage/Megakaryocyte (CFU-GEMM) colony formation. In comparison to control cells (cultured without SDF-1 and AMD3100), SDF-1 increased numbers of p-BFU-E, d-BFU-E, CFU-GM, and CFU-GEMM colonies. Addition of AMD3100 with SDF-1 blocked the enhancing effect of SDF-1. In addition, significantly decreased numbers of colonies were also observed in the presence of AMD3100 alone. This suggests that AMD3100 blocks endogenous SDF-1 actions, consistent with our data on SDF-1 production during EB formation. In order to determine when SDF-1 starts affecting hematopoiesis, hemangioblast colony assays were used. Neither SDF-1 nor AMD3100 influenced hemangioblast colony formation or expression of Flk-1 mRNA, a marker of hemangioblasts. The results suggest a role for SDF-1 in ES cell growth and differentiation.

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