Expression of Runx1 Isoforms in Sub-Populations of Human Blood and Bone Marrow Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4244-4244
Author(s):  
Maxim Yankelevich ◽  
Judith F. Margolin ◽  
Margaret A. Goodell
Keyword(s):  
Bone Marrow ◽  
Peripheral Blood ◽  
Progenitor Cell ◽  
Bone Marrow Cells ◽  
Hematopoietic Cells ◽  
Pcr Analysis ◽  
Cell Populations ◽  
Total Rna ◽  
Isoform Expression ◽  
A Minor

Abstract Runx1 is a pivotal regulator of hematopoiesis. Deficiency of Runx1 leads to complete block of definitive hematopoiesis in mouse embryos. Runx1 protein forms a heterodimer with CBFβ and binds to the specific DNA sequence TGT/cGGT to regulate the expression of a number of hematopoietic genes. Runx 1 has been recognized to have three different isoforms (a, b, and c) due to differential splicing and promoter utilization. The distal (Runx1c) promoter region was shown to have binding sites mediating repression of Runx1 transcription. Data on the pattern of Runx1 isoforms expression in different hematopoietic lineages are scant. The functional impact of different Runx1 isoforms on commitment and differentiation of blood cells is also essentially unknown. To further explore this, we studied Runx1 isoform expression in different subpopulations of human hematopoietic cells. Total RNA was extracted from peripheral blood and unstimulated bone marrow. Also, Lin-CD34+CD38+, and Lin-CD34+CD38− cell populations were sorted from G-CSF-stimulated bone marrow and total RNA extracted. Expression of Runx1 isoforms was assessed by semiquantitative RT-PCR analysis, using GAPDH as a control. All 3 isoforms were present in all studied cell populations. Runx1a was uniformly a minor form with lowest expression. Runx1c isoform was a dominant one in peripheral blood as opposed to Runx1b in bone marrow. Most interestingly, the ratio of Runx1b to other isoforms was significantly increased in Lin-CD34+CD38- subpopulation compared to Lin-CD34+CD38+ cells. The prevalence of Runx1c isoform in peripheral blood as compaired to bone marrow is most likely related to a different cellular composition of these tissues and suggests that Runx 1c isoform may be predominantly expressed in some of the mature cell lineages. Since Lin-CD34+CD38− cells represent most primitive progenitor cell population with multi-lineage repopulating ability, the observed overexpression of Runx1b may be related to transcriptional accessibility of these cells. Upregulation of Runx 1b with G-CSF in this subpopulation can not be ruled out as well. These preliminary data suggest that the expression of Runx1 alternative splicing isoforms is both lineage and differentiation stage-specific in human hematopoietic cells. We will also test whether Runx1 isoform expression has a consistent pattern in different types of leukemia and if it is resembles such of mature blood counterparts or primitive progenitors. These studies, as well as the studies of differential expression of Runx1 isoforms in different mature blood lineages and progenitor cell sub-populations are underway and results will be presented. These data may further shed a light on functional differences in Runx1 isoforms and mechanisms of hematopoiesis.

scholarly journals Detection of chromosome 20q deletions in bone marrow metaphases but not peripheral blood granulocytes in patients with myeloproliferative disorders or myelodysplastic syndromes

Blood ◽  
1996 ◽  
Vol 87 (4) ◽  
pp. 1561-1570 ◽  
Author(s):  
FA Asimakopoulos ◽  
TL Holloway ◽  
EP Nacheva ◽  
MA Scott ◽  
P Fenaux ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Myelodysplastic Syndromes ◽  
Peripheral Blood ◽  
Myeloproliferative Disorders ◽  
Pcr Analysis ◽  
Multipotent Progenitors ◽  
The Common ◽  
Polymerase Chain ◽  
A Minor

Myeloproliferative disorders and myelodysplastic syndromes arise in multipotent progenitors and may be associated with chromosomal deletions that can be detected in peripheral blood granulocytes. We present here seven patients with myeloproliferative disorders or myelodysplastic syndromes in whom a deletion of the long arm of chromosome 20 was detectable by G-banding and/or fluorescence in situ hybridization in most or all bone marrow metaphases. However, in each case, microsatellite polymerase chain reaction (PCR) using 15 primer pairs spanning the common deleted region on 20q showed that the deletion was absent from most peripheral blood granulocytes. The human androgen receptor clonality assay was used to show that the vast majority of peripheral blood granulocytes were clonal in all four female patients. This represents the first demonstration that the 20q deletion can arise as a second event in patients with pre-existing clonal granulopoiesis. Microsatellite PCR analysis of whole bone marrow from two patients was consistent with cytogenetic studies, a result that suggests that cytogenetic analysis was not merely selecting for a minor subclone of cells carrying the deletion. Furthermore, in one patient, the deletion was present in both erythroid and granulocyte/monocyte colonies. This implies that the absence of the deletion in most peripheral blood granulocytes did not reflect lineage restriction of the progenitors carrying the deletion but may instead result from other selective influences such as preferential retention/destruction within the bone marrow of granulocytes carrying the deletion.

Forced Overexpression of Ahi-1 Confers a Proliferative Advantage on Primitive Murine Hematopoietic Cells and Enhances the Effects of BCR-ABL.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2573-2573
Author(s):  
Leon Zhou ◽  
Ashley Ringrose ◽  
Xiaoyan Jiang
Keyword(s):  
Bone Marrow ◽  
Growth Factor ◽  
Proliferative Activity ◽  
Insertional Mutagenesis ◽  
Bone Marrow Cells ◽  
Integration Site ◽  
Hematopoietic Cells ◽  
Chronic Phase ◽  
Biological Behavior ◽  
Pcr Analysis

Abstract Ahi-1 (Abelson helper integration site-1) is a novel gene that is commonly activated by proviral insertional mutagenesis in v-abl or myc-induced murine leukemias and lymphomas. Ahi-1 encodes a unique protein with known signaling features including SH3 and WD40-repeat domains but its normal function is unknown. Involvement of Ahi-1 in leukemogenesis is suggested by the high frequency of Ahi-1 mutations seen in certain virus-induced murine leukemias and by the gross perturbations seen in the expression of human AHI-1 and its isoforms in several human leukemic cell lines, as well as in the primary lin−CD34+CD38− leukemic stem cell-enriched population in patients with chronic phase CML. To further investigate the role of Ahi-1 as a potential co-operating oncogene relevant to BCR-ABL-mediated leukemogenesis, we compared the biological behavior of primitive murine hematopoietic cells from the bone marrow of 5-FU-treated adult C57BL/6 mice after their transduction with MSCV-Ahi-1-IRES-YFP, MSCV-BCR-ABL-IRES-GFP retroviruses, either alone or in combination. Quantitative real-time RT-PCR analysis of RNA from FACS-purified lin−YFP+ (Ahi-1+), lin−GFP+ (BCR-ABL+) and lin−YFP+GFP+ (Ahi-1+ + BCR-ABL+)-transduced bone marrow cells showed that Ahi-1 transcripts were present at 40-fold higher levels in the Ahi-1-transduced cells by comparison to the control cells transduced with the empty MIY vector. Immediately post-transduction, the Ahi-1-transduced cells produced a similar number of colonies as the MIY-transduced control cells in semi-solid cultures containing Steel factor (SF) + IL-3 + IL-6 + EPO, although a small proportion of the Ahi-1+ CFCs (~10%) were already growth factor-independent. In addition, the proliferative activity of the FACS-purified lin−YFP+ (Ahi-1+) cells (as indicated by the rate of expansion of viable cells in a week in liquid cultures containing SF, IL-3, and IL-6) was ~3-fold higher than in cultures initiated with control (lin−YFP+) cells. Moreover, after 4 weeks in longterm culture-initiating cell (LTC-IC) assays, the Ahi-1+ cells produced 2x more CFCs than the control cells. All of these endpoints (proliferative activity, growth factor-dependence and CFC output in LTC-IC assays) are also perturbed by BCR-ABL transduction. Interestingly, in cells that were co-transduced with Ahi-1 and BCR-ABL, all of these effects were further enhanced as compared to cells transduced with either BCR-ABL alone (2–4-fold) or Ahi-1 alone (3–6-fold). Thus, overexpression of Ahi-1 alone deregulates the proliferation control of primitive murine hematopoietic cells and this is additive with the effects of BCR-ABL, suggesting that Ahi-1 and BCR-ABL can cooperate to promote the progression of BCR-ABL-associated diseases like CML.

In vivo efficacy of anti-MPL agonist antibody in promoting primary human hematopoietic cells

Blood ◽  
2009 ◽  
Vol 113 (10) ◽  
pp. 2213-2216 ◽  
Author(s):  
Masayuki Kai ◽  
Tetsuya Hagiwara ◽  
Chie Emuta ◽  
Yukiko Chisaka ◽  
Kumi Tsuruhata ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Peripheral Blood ◽  
Bone Marrow Cells ◽  
Therapeutic Potential ◽  
Hematopoietic Cells ◽  
Human Cord Blood ◽  
Cd34 Cells ◽  
Total Body

Abstract In a previous study, we generated novel antithrombopoietin receptor agonist antibodies as therapeutic candidates. In this report, we investigated the in vivo effects of one of these antibodies, MA01G4344U, on primary human hematopoietic cells using xenotransplantation. NOD/Shi-scid, IL-2Rγnull (NOG) mice were pretreated by total-body irradiation and received a transplant of human cord blood–derived CD34+ cells. Weekly intraperitoneal injection of MA01G4344U (100 μg/mouse per week) or Peg-rhMGDF (5 μg/mouse per week) or phosphate-buffered saline (PBS) was performed. Human cells in peripheral blood were analyzed by flow cytometry and bone marrow cells were analyzed by flow cytometry and colony assay. MA01G4344U successfully increased the number of human CD41+ platelets and human CD45+ cells in peripheral blood. In the bone marrow, MA01G4344U increased the number of human CD45+/CD34+ cells, which resulted in more multilineage progenitor cells. The efficacy of MA01G4344U in promoting primary human hematopoietic cells in vivo suggests its therapeutic potential for thrombocytopenic and pancytopenic disorders.

Clonal Marking of Mouse Hematopoietic Stem and Lymphoid Progenitor Cell Populations.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 5544-5544
Author(s):  
Mariluz P. Mojica-Henshaw ◽  
L. Jeanne Pierce ◽  
John D. Phillips ◽  
Vicente Planelles ◽  
Gerald J. Spangrude
Keyword(s):  
Bone Marrow ◽  
Transgene Expression ◽  
Progenitor Cell ◽  
Bone Marrow Cells ◽  
Random Sequence ◽  
Transduction Efficiency ◽  
Cell Populations ◽  
Lentivirus Vector ◽  
Cmv Promoter ◽  
Hematopoietic Stem

Abstract We have developed a method to clonally mark hematopoietic stem and lymphoid progenitor cell populations using a novel sequence tag approach. A library containing an 11-base random sequence tag is cloned into a lentivirus vector, packaged using the VSV-G glycoprotein and HIV-1 capsid, and transduced into freshly isolated mouse hematopoietic stem cell (Thy-1.1lowc-kithigh) or progenitor cell (Thy-1.1negc-kithigh) populations. To minimize artifacts introduced by prolonged culture, we have utilized a 3-hour spinoculation protocol performed in the absence of cytokines. Transduction efficiency was evaluated in vitro by methylcellulose colony assay and liquid cultures, and in vivo by transplanting the transduced cells into lethally irradiated mice. A bicistronic lentivirus vector with a CMV promoter driving expression of a transcript encoding Thy-1.2-IRES-GFP was used to optimize the transduction protocol. Liquid culture assays demonstrated 57% transduction efficiency after 5 days of growth, based on expression of the Thy-1.2 and GFP reporter proteins. Mice transplanted with transduced Thy-1.1negc-kithigh progenitor cells were sacrificed after 16 days, a time at which we have previously observed robust progenitor cell engraftment in the thymus while progeny of Thy-1.1lowc-kithigh HSC have not yet appeared. In 4 of 4 transplanted mice, we observed donor-derived cells in the bone marrow, lymph nodes and thymus. The percentage of total cells expressing the lentivirus-derived transgene ranged from 1.6% of bone marrow cells to 20% of thymocytes. Peripheral blood from mice transplanted with transduced HSC were analyzed and monitored every 4 weeks for transgene expression. We observed that although the Thy-1.2 marker was expressed and maintained up to 14 weeks after HSC transplant, GFP transgene expression was minimal. Based on these preliminary results, we have engineered a new lentivirus vector containing random sequence tags and the Thy-1.2 marker. This strategy provides a simple and efficient way of tracking the progeny of individual cells within a transplanted population, using PCR amplification of the random tags found within mature cell populations derived from the transduced cells. Sequence analysis of individual clones derived from different lineages of cells will enable us to better define the lineage potentials of specific progenitor cell subpopulations.

scholarly journals MicroRNA-126 Regulates HOXA9 by Binding to the Homeobox

2008 ◽  
Vol 28 (14) ◽  
pp. 4609-4619 ◽  
Author(s):  
Wei-Fang Shen ◽  
Yu-Long Hu ◽  
Lalita Uttarwar ◽  
Emmanuelle Passegue ◽  
Corey Largman
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Biological Activity ◽  
Progenitor Cell ◽  
Bone Marrow Cells ◽  
Hematopoietic Cells ◽  
Hematopoietic Stem ◽  
F9 Cells ◽  
Functional Conservation ◽  
Marrow Cells

ABSTRACT The PicTar program predicted that microRNA-126 (miR-126), miR-145, and let-7s target highly conserved sites within the Hoxa9 homeobox. There are increased nucleotide constraints in the three microRNA seed sites among Hoxa9 genes beyond that required to maintain protein identity, suggesting additional functional conservation. In preliminary experiments, forced expression of these microRNAs in Hoxa9-immortalized bone marrow cells downregulated the HOXA9 protein and caused loss of biological activity. The microRNAs were shown to target their predicted sites within the homeobox. miR-126 and Hoxa9 mRNA are coexpressed in hematopoietic stem cells and downregulated in parallel during progenitor cell differentiation; however, miR-145 is barely detectable in hematopoietic cells, and let-7s are highly expressed in bone marrow progenitors, suggesting that miR-126 may function in normal hematopoietic cells to modulate HOXA9 protein. In support of this hypothesis, expression of miR-126 alone in MLL-ENL-immortalized bone marrow cells decreased endogenous HOXA9 protein, while inhibition of endogenous miR-126 increased expression of HOXA9 in F9 cells.

Bone Marrow Niches for Skeletal Progenitor Cells and their Inhabitants in Health and Disease

2019 ◽  
Vol 14 (4) ◽  
pp. 305-319 ◽  
Author(s):  
Marietta Herrmann ◽  
Franz Jakob
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Progenitor Cell ◽  
Adult Stem Cells ◽  
Current Knowledge ◽  
Cell Populations ◽  
Surface Markers ◽  
Bone Marrow Niches

The bone marrow hosts skeletal progenitor cells which have most widely been referred to as Mesenchymal Stem or Stromal Cells (MSCs), a heterogeneous population of adult stem cells possessing the potential for self-renewal and multilineage differentiation. A consensus agreement on minimal criteria has been suggested to define MSCs in vitro, including adhesion to plastic, expression of typical surface markers and the ability to differentiate towards the adipogenic, osteogenic and chondrogenic lineages but they are critically discussed since the differentiation capability of cells could not always be confirmed by stringent assays in vivo. However, these in vitro characteristics have led to the notion that progenitor cell populations, similar to MSCs in bone marrow, reside in various tissues. MSCs are in the focus of numerous (pre)clinical studies on tissue regeneration and repair.Recent advances in terms of genetic animal models enabled a couple of studies targeting skeletal progenitor cells in vivo. Accordingly, different skeletal progenitor cell populations could be identified by the expression of surface markers including nestin and leptin receptor. While there are still issues with the identity of, and the overlap between different cell populations, these studies suggested that specific microenvironments, referred to as niches, host and maintain skeletal progenitor cells in the bone marrow. Dynamic mutual interactions through biological and physical cues between niche constituting cells and niche inhabitants control dormancy, symmetric and asymmetric cell division and lineage commitment. Niche constituting cells, inhabitant cells and their extracellular matrix are subject to influences of aging and disease e.g. via cellular modulators. Protective niches can be hijacked and abused by metastasizing tumor cells, and may even be adapted via mutual education. Here, we summarize the current knowledge on bone marrow skeletal progenitor cell niches in physiology and pathophysiology. We discuss the plasticity and dynamics of bone marrow niches as well as future perspectives of targeting niches for therapeutic strategies.

scholarly journals Primitive Human Hematopoietic Cells Are Enriched in Cord Blood Compared With Adult Bone Marrow or Mobilized Peripheral Blood as Measured by the Quantitative In Vivo SCID-Repopulating Cell Assay

Blood ◽  
1997 ◽  
Vol 89 (11) ◽  
pp. 3919-3924 ◽  
Author(s):  
Jean C.Y. Wang ◽  
Monica Doedens ◽  
John E. Dick
Keyword(s):  
Bone Marrow ◽  
Cord Blood ◽  
Peripheral Blood ◽  
Hematopoietic Cells ◽  
Allogeneic Transplantation ◽  
Functional Assay ◽  
Hematopoietic Stem ◽  
Mobilized Peripheral Blood

Abstract We have previously reported the development of in vivo functional assays for primitive human hematopoietic cells based on their ability to repopulate the bone marrow (BM) of severe combined immunodeficient (SCID) and nonobese diabetic/SCID (NOD/SCID) mice following intravenous transplantation. Accumulated data from gene marking and cell purification experiments indicate that the engrafting cells (defined as SCID-repopulating cells or SRC) are biologically distinct from and more primitive than most cells that can be assayed in vitro. Here we demonstrate through limiting dilution analysis that the NOD/SCID xenotransplant model provides a quantitative assay for SRC. Using this assay, the frequency of SRC in cord blood (CB) was found to be 1 in 9.3 × 105 cells. This was significantly higher than the frequency of 1 SRC in 3.0 × 106 adult BM cells or 1 in 6.0 × 106 mobilized peripheral blood (PB) cells from normal donors. Mice transplanted with limiting numbers of SRC were engrafted with both lymphoid and multilineage myeloid human cells. This functional assay is currently the only available method for quantitative analysis of human hematopoietic cells with repopulating capacity. Both CB and mobilized PB are increasingly being used as alternative sources of hematopoietic stem cells in allogeneic transplantation. Thus, the findings reported here will have important clinical as well as biologic implications.

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