Marking of Embryonic Precursors of Adult Hematopoietic Stem Cells At Gestational Day E7.5-E8.5 Using An Abcg2-CreERT2 Lineage Tracing Mouse Model

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1287-1287
Author(s):  
Sheng Zhou ◽  
Soghra Fatima ◽  
Brian P. Sorrentino
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Peripheral Blood ◽  
Lineage Tracing ◽  
Tamoxifen Treatment ◽  
Specific Expression ◽  
Hematopoietic Stem ◽  
Tissue Specific ◽  
Tissue Specific Expression

Abstract Abstract 1287 Abcg2 is a member of the ATP-binding cassette transporter family, is expressed in adult hematopoietic stem cells (HSCs), and is required for the side population phenotype of adult bone marrow HSCs as well as other adult tissue-specific stem cells. We have used these properties of Abcg2 expression for lineage tracing of stem cell development in mice, particularly for HSCs. An ires-CreERT2 cassette was inserted into the Abcg2 locus, down-stream of its stop codon but upstream of its endogenous polyadenylation site, so that both Abcg2 and CreER are co-expressed from a single bicistronic transcript. This design allows for minimum disruption of Abcg2 expression and tissue specific expression of CreERT2 under the control of endogenous Abcg2 transcription elements. The Abcg2CreER/CreER mouse was crossed with flox-STOP-flox-YFP (Rosa26RYFP/YFP) mouse to generate compound heterozygous Abcg2CreER/+ Rosa26RYFP/+ mice. Treatment of adult Abcg2CreER/+ Rosa26RYFP/+ mice with tamoxifen resulted in robust YFP expression in kidney proximal tubule cells and hepatocytes demonstrating the expected tissue-specific expression of the Abcg2CreER allele. We also observed tamoxifen-dependent appearance of YFP+ cells in all hematopoietic lineages in the peripheral blood and bone marrow, confirming our prior observations that Abcg2 is expressed in adult stem cells. Unexpectedly, we observed long term marking in intestinal epithelial cells and in seminiferous tubules 9 to 20 months after tamoxifen treatment, recapitulating classic progeny tracking patterns, proving that intestinal stem cells and spermatogonial stem cells express the Abcg2 marker. Pregnant females were treated with a single dose of 4-hydroxytamoxifen (4-HT) at gestational days E7.5 and E8.5 using overnight timed breeding pairs. We chose 4-HT rather than tamoxifen because 4-HT has been shown to decay relatively quickly in the fetus so that no recombination can occur 24 hours after the pulse. After maternal treatment, mice were born, grew to adulthood, and were analyzed 14–17 weeks after birth for expression of YFP in distinct peripheral blood lineages. In the majority of the 18 mice born from mothers treated with 4-HT at day E7.5 and from 17 mice born of mothers treated at day E8.5, a small but distinct YFP+ subpopulation could be clearly detected in all hematopoietic lineages (Figure A and B). The numbers of marked cells have been stable for approximately 4 months and are strictly dependent on 4-HT treatment of the mother. These results demonstrate that a precursor to adult hematopoietic stem cells exists at gestational day E7.5 to E8.5 and contributes to a stable subpopulation of HSCs well into adulthood. The low level of marking could reflect inefficient recombination due to either relatively low levels of expression of the recombinant allele in these embryonic HSC precursors or due to inefficient nuclear localization with the single 4-HT pulse. Alternatively, these marked embryonic HSC precursors may be generating only a minor population of adult HSCs that are competing against a larger fraction of HSCs that arise from precursors that originate later in gestation after the 24 hour 4-HT washout. We are in the process of determining the embryonic source of the E7.5 – E8.5 adult HSC precursor and have not yet determined whether it originates in the yolk sac, in another extra-embryonic source, or within the embryo proper. We are following these mice for longer periods of time to determine the stability of marking in primary and serial transplant experiments. Altogether, we expect that studies with this novel lineage tracing model will provide a better understanding of steady-state, uninterrupted embryonic hematopoietic development that does not require transplant assays to detect HSC activity. Disclosures: No relevant conflicts of interest to declare.

Lentiviral gene transfer regenerates hematopoietic stem cells in a mouse model for Mpl-deficient aplastic anemia

Blood ◽  
2011 ◽  
Vol 117 (14) ◽  
pp. 3737-3747 ◽  
Author(s):  
Dirk Heckl ◽  
Daniel C. Wicke ◽  
Martijn H. Brugman ◽  
Johann Meyer ◽  
Axel Schambach ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mouse Model ◽  
Hematopoietic Stem Cells ◽  
Aplastic Anemia ◽  
Bone Marrow Cells ◽  
Specific Expression ◽  
Hematopoietic Stem ◽  
Egfp Reporter

AbstractThpo/Mpl signaling plays an important role in the maintenance of hematopoietic stem cells (HSCs) in addition to its role in megakaryopoiesis. Patients with inactivating mutations in Mpl develop thrombocytopenia and aplastic anemia because of progressive loss of HSCs. Yet, it is unknown whether this loss of HSCs is an irreversible process. In this study, we used the Mpl knockout (Mpl−/−) mouse model and expressed Mpl from newly developed lentiviral vectors specifically in the physiologic Mpl target populations, namely, HSCs and megakaryocytes. After validating lineage-specific expression in vivo using lentiviral eGFP reporter vectors, we performed bone marrow transplantation of transduced Mpl−/− bone marrow cells into Mpl−/− mice. We show that restoration of Mpl expression from transcriptionally targeted vectors prevents lethal adverse reactions of ectopic Mpl expression, replenishes the HSC pool, restores stem cell properties, and corrects platelet production. In some mice, megakaryocyte counts were atypically high, accompanied by bone neo-formation and marrow fibrosis. Gene-corrected Mpl−/− cells had increased long-term repopulating potential, with a marked increase in lineage−Sca1+cKit+ cells and early progenitor populations in reconstituted mice. Transcriptome analysis of lineage−Sca1+cKit+ cells in Mpl-corrected mice showed functional adjustment of genes involved in HSC self-renewal.

Multifunctional Role of Caspase-3 in Regulating Hematopoietic Stem Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 861-861 ◽  
Author(s):  
Viktor Janzen ◽  
Heather E. Fleming ◽  
Michael T. Waring ◽  
Craig D. Milne ◽  
David T. Scadden
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Peripheral Blood ◽  
Caspase 3 ◽  
Brdu Incorporation ◽  
Hematopoietic Stem ◽  
Regulatory Pathways

Abstract The processes of cell cycle control, differentiation and apoptosis are closely intertwined in controlling cell fate during development and in adult homeostasis. Molecular pathways connecting these events in stem cells are poorly defined and we were particularly interested in the cysteine-aspartic acid protease, Caspase-3, an ‘executioner’ caspase also implicated in the regulation of the cyclin dependent kinase inhibitors, p21Cip1 and p27Kip1. These latter proteins are known to participate in primitive hematopoietic cell cycling and self-renewal. We demonstrated high levels of Caspase-3 mRNA and protein in immunophenotypically defined mouse hematopoietic stem cells (HSC). Using mice engineered to be deficient in Caspase-3, we observed a consistent reduction of lymphocytes in peripheral blood counts and a slight reduction in bone marrow cellularity. Notably, knockout animals had an increase in the stem cell enriched Lin−cKit+Sca1+Flk2low (LKSFlk2lo) cell fraction. The apoptotic rates of LKS cells under homeostatic conditions as assayed by the Annexin V assay were not significantly different from controls. However, in-vitro analysis of sorted LKS cells revealed a reduced sensitivity to apoptotic cell death in absence of Caspase-3 under conditions of stress (cytokine withdrawal or gamma irradiation). Primitive hematopoietic cells displayed a higher proliferation rate as demonstrated by BrdU incorporation and a significant reduction in the percentage of cells in the quiescent stage of the cell cycle assessed by the Pyronin-Y/Hoechst staining. Upon transplantation, Caspase-3−/− stem cells demonstrated marked differentiation abnormalities with significantly reduced ability to differentiate into multiple hematopoietic lineages while maintaining an increased number of primitive cells. In a competitive bone marrow transplant using congenic mouse stains Capase-3 deficient HSC out-competed WT cells at the stem cell level, while giving rise to comparable number of peripheral blood cells as the WT controls. Transplant of WT BM cells into Caspase-3 deficient mice revealed no difference in reconstitution ability, suggesting negligible effect of the Caspase-3−/− niche microenvironment to stem cell function. These data indicate that Caspase-3 is involved in the regulation of differentiation and proliferation of HSC as a cell autonomous process. The molecular bases for these effects remain to be determined, but the multi-faceted nature of the changes seen suggest that Caspase-3 is central to multiple regulatory pathways in the stem cell compartment.

Longitudinal Trends in Peripheral Blood Parameters Predict Development of Therapy-Related Myelodysplasia/Acute Myeloid Leukemia (t-MDS/ AML) after Autologous Transplantation for Lymphoma.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2133-2133
Author(s):  
Liton Francisco ◽  
Can-Lan Sun ◽  
Lester Laddaran ◽  
Melanie Sabado ◽  
Alysia Bosworth ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Hodgkin Lymphoma ◽  
Peripheral Blood ◽  
Blood Parameters ◽  
Red Cell ◽  
Hematopoietic Stem ◽  
Time Points ◽  
Over Time

Abstract t-MDS/AML is the most common cause of non-relapse mortality in patients undergoing autologous hematopoietic cell transplantation (aHCT) for Hodgkin lymphoma (HL) or non-Hodgkin lymphoma (NHL). Although t-MDS/AML is known to result from damage to hematopoietic stem cells (HSC) as a result of genotoxic cancer treatment, the sequential cellular and molecular changes leading to its development are not clearly defined. To better understand the pathogenesis of t-MDS/AML, we conducted a prospective study in 179 patients undergoing aHCT for HL (n=41) or NHL (n=138) between 1999 and 2004, who participated in a prospective longitudinal study from pre-aHCT to five years post-aHCT, with a serial collection of bone marrow and peripheral blood samples. The median length of follow-up for this cohort was 3.9 years. This report focuses on alterations in peripheral blood parameters from pre-aHCT to the development of t-MDS/AML, and compares these trends with the patients in this cohort who did not develop t-MDS/AML. A total of 22 patients have developed t-MDS/AML in this longitudinally followed cohort thus far, resulting in a cumulative incidence of 11% at 5 years. Serial evaluation of peripheral blood parameters including hematocrit, mean corpuscular volume (MCV), hemoglobin (HGB), red cell distribution width (RDW), white blood cell (WBC) count, and platelet (PLT) count, were abstracted from medical records for the following time points: pre-aHCT, day 100, 6 month, 1 year, 2 year, 3 year, 4 year and 5 year after aHCT, for a total of 1129 time points. Values of peripheral blood parameters associated with post-aHCT relapse or persistence of the primary lymphoma or from 3 months prior to development of t-MDS/AML, were excluded from analysis. As shown in the Figure, comparison of the peripheral blood parameters in subjects who developed t-MDS/AML (cases; n=22) with those who did not (controls; n=157) revealed that hematocrit values were lower for cases compared to controls at all post-aHCT time points. HGB values were lower among cases compared to controls at all post-aHCT time points. The RDW values were higher for cases compared to controls at day 100, 6 months and 1 year post-aHCT. MCV values did not differ between cases and controls at any of the time points. WBC counts for the cases were lower than controls pre-aHCT and also at all time points from 6 months post-aHCT onwards. PLT counts for cases were lower than controls at all time points pre- and post-aHCT. A fixed effect growth curve model was fitted to the data from day 100 to 5 years post-aHCT after adjusting for age at aHCT, primary diagnosis, race/ethnicity, and sex, to examine the rate of change in the peripheral blood parameters over time. Results revealed a significantly sharper decline in MCV for cases (β per 100 days = −0.43) over time as compared to controls (β =−0.15; p = 0.006). Although hematocrit increased with time for both cases and controls, the slope for the cases was significantly less steep (controls: β per 100 days=0.31 vs. cases: β per 100 days=0.12; p =0.01). In summary, we consistently observed lower values for red cell parameters, WBC, and platelets in patients with t-MDS/ AML as compared to controls across multiple timepoints post-aHCT. These differences appeared soon after HCT, were persistent, and preceded the development of t-MDS/AML. Our previous studies indicate that there is increased turnover and reduced regenerative capacity of premalignant hematopoietic stem cells at early stages of development of t-MDS/AML. The early and persistent reduction in peripheral blood parameters observed here provides further evidence that bone marrow injury and ineffective hematopoiesis long predate the development of t-MDS/AML after aHCT. Poor hematocrit recovery and enhanced decline in MCV after aHCT were independently associated with increased risk of t-MDS/AML and warrant further development as readily applied biomarkers for disease and the need for close monitoring. Figure Figure

Cripto Selectively Expands a Distinct Population of Hematopoietic Stem Cells Expressing the Cell Surface Receptor GRP78 and Strongly Induces An Immature Phenotype In Vivo After Ex Vivo Culture

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 405-405
Author(s):  
Kenichi Miharada ◽  
Göran Karlsson ◽  
Jonas Larsson ◽  
Emma Larsson ◽  
Kavitha Siva ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Peripheral Blood ◽  
Distinct Population ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Total Bone Marrow

Abstract Abstract 405 Cripto is a member of the EGF-CFC soluble protein family and has been identified as an important factor for the proliferation/self-renewal of ES and several types of tumor cells. The role for Cripto in the regulation of hematopoietic cells has been unknown. Here we show that Cripto is a potential new candidate factor to increase self-renewal and expand hematopoietic stem cells (HSCs) in vitro. The expression level of Cripto was analyzed by qRT-PCR in several purified murine hematopoietic cell populations. The findings demonstrated that purified CD34-KSL cells, known as highly concentrated HSC population, had higher expression levels than other hematopoietic progenitor populations including CD34+KSL cells. We asked how Cripto regulates HSCs by using recombinant mouse Cripto (rmCripto) for in vitro and in vivo experiments. First we tested the effects of rmCripto on purified hematopoietic stem cells (CD34-LSK) in vitro. After two weeks culture in serum free media supplemented with 100ng/ml of SCF, TPO and 500ng/ml of rmCripto, 30 of CD34-KSL cells formed over 1,300 of colonies, including over 60 of GEMM colonies, while control cultures without rmCripto generated few colonies and no GEMM colonies (p<0.001). Next, 20 of CD34-KSL cells were cultured with or without rmCripto for 2 weeks and transplanted to lethally irradiated mice in a competitive setting. Cripto treated donor cells showed a low level of reconstitution (4–12%) in the peripheral blood, while cells cultured without rmCripto failed to reconstitute. To define the target population and the mechanism of Cripto action, we analyzed two cell surface proteins, GRP78 and Glypican-1, as potential receptor candidates for Cripto regulation of HSC. Surprisingly, CD34-KSL cells were divided into two distinct populations where HSC expressing GRP78 exhibited robust expansion of CFU-GEMM progenitor mediated by rmCripto in CFU-assay whereas GRP78- HSC did not respond (1/3 of CD34-KSL cells were GRP78+). Furthermore, a neutralization antibody for GRP78 completely inhibited the effect of Cripto in both CFU-assay and transplantation assay. In contrast, all lineage negative cells were Glypican-1 positive. These results suggest that GRP78 must be the functional receptor for Cripto on HSC. We therefore sorted these two GRP78+CD34-KSL (GRP78+HSC) and GRP78-CD34-KSL (GRP78-HSC) populations and transplanted to lethally irradiated mice using freshly isolated cells and cells cultured with or without rmCripto for 2 weeks. Interestingly, fresh GRP78-HSCs showed higher reconstitution than GRP78+HSCs (58–82% and 8–40%, p=0.0038) and the reconstitution level in peripheral blood increased rapidly. In contrast, GRP78+HSC reconstituted the peripheral blood slowly, still at a lower level than GRP78-HSC 4 months after transplantation. However, rmCripto selectively expanded (or maintained) GRP78+HSCs but not GRP78-HSCs after culture and generated a similar level of reconstitution as freshly transplanted cells (12–35%). Finally, bone marrow cells of engrafted recipient mice were analyzed at 5 months after transplantation. Surprisingly, GRP78+HSC cultured with rmCripto showed higher reconstitution of the CD34-KSL population in the recipients' bone marrow (45–54%, p=0.0026), while the reconstitution in peripheral blood and in total bone marrow was almost the same. Additionally, most reconstituted CD34-KSL population was GRP78+. Interestingly freshly transplanted sorted GRP78+HSC and GRP78-HSC can produce the GRP78− and GRP78+ populations in the bone marrow and the ratio of GRP78+/− cells that were regenerated have the same proportion as the original donor mice. Compared to cultured cells, the level of reconstitution (peripheral blood, total bone marrow, HSC) in the recipient mice was almost similar. These results indicate that the GRP78 expression on HSC is reversible, but it seems to be “fixed” into an immature stage and differentiate with lower efficiency toward mature cells after long/strong exposure to Cripto signaling. Based on these findings, we propose that Cripto is a novel factor that maintains HSC in an immature state and may be a potent candidate for expansion of a distinct population of GRP78 expressing HSC. Disclosures: No relevant conflicts of interest to declare.

In Utero Depletion Of Fetal Host Hematopoietic Stem Cells Improves Engraftment Following Neonatal Transplantation In Mice

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3240-3240
Author(s):  
Chris Derderian ◽  
Charmin King ◽  
Priya Togarrati ◽  
Agnieszka Czechowicz ◽  
Ninnia Lescano ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Peripheral Blood ◽  
Hematopoietic Cell Transplantation ◽  
Mononuclear Cells ◽  
Fetal Liver ◽  
In Utero ◽  
Hematopoietic Stem ◽  
In Utero Transplantation

Abstract Introduction In utero hematopoietic cell transplantation (IUHCTx) is a promising strategy to treat congenital disorders as the fetal host can potentially be tolerized to transplanted cells early in gestation. However, levels of engraftment have been low and fetal host conditioning strategies to increase space in hematopoietic niches have not been widely explored. We hypothesized that depletion of fetal host hematopoietic stem cells (HSC) using an antibody against the c-kit receptor (ACK2), a strategy which selectively depletes HSC by disrupting stem cell factor (SCF) signaling, would improve engraftment after HSC transplantation. Methods Fetal C57B6.CD45.2 (B6) mice were injected with increasing doses of ACK2 (2.5-50 µg/fetus) or isotype control antibody on E14.5 and surviving pups were transplanted with congenic B6.CD45.1 fetal liver mononuclear cells (2.5×106 cells/pup) on day of life 1 (P1, 7 days after in utero injection), allowing post-transplantation host monitoring. Host HSC depletion and residual serum ACK2 concentration were examined on P1. Peripheral blood chimerism, defined as donor/(donor+host) CD45 cells, as well as the lineage distribution of chimeric cells, were determined beginning 4 weeks after transplantation. Results Survival to birth among fetuses injected with 2.5, 5, or 10 µg of ACK2 was similar to controls (control: 74%; 2.5 µg: 80%; 5 µg: 71%; 10 µg: 60%, p=0.2 by chi-square test, n≥45/group) but was significantly lower at higher concentrations (20 µg: 37%; 50 µg: 31%, p<0.001 vs. control, n≥70/group). Transient anemia and leukopenia were observed on P1 with doses ≥ 5 µg which resolved by P7 (n=17). Four of 19 pups previously treated with ACK2 (2.5-10 µg) and observed long-term had patchy coat discoloration, possibly a manifestation of disruption of C-kit+ melanocyte migration. In utero ACK2 treatment resulted in significant and dose-dependent depletion of host HSCs (defined as Lin-Sca-1+C-kit+, KLS) in the bone marrow of treated animals by P1 (Figure 1A). There was no depletion of KLS cells in the liver. Residual ACK2 antibody was undetectable in the serum by P1, validating our strategy of in utero depletion and neonatal transplantation. In animals receiving neonatal transplantation, ACK2 depletion resulted in a significant increase in levels of engraftment 4 weeks after transplantation compared to controls (control: 3.3±0.3%; 2.5 µg: 13±1.4%; 5 µg: 10±2.4%; 10 µg: 11±2.0%, p<0.05 for each dose vs control by ANOVA). Accordingly, we detected an increased number total bone marrow KLS cells 7 days after transplantation in ACK2 treated animals compared to controls (412±45.9 vs. 933±112 cells, p=0.01, n≥3/group). Moreover, levels of chimerism increased over time in treated animals (Figure 1B; 12 weeks: 2.5 µg: 190%; 5 µg: 170%; 10 µg: 160%) while they remained unchanged in controls. Overall, levels of chimerism achieved with ACK2 treatment were significantly higher than that observed in animals that received in utero transplantation without ACK2 depletion. Lineage analysis of peripheral blood for granulocytes, B cells, and T cells indicated an equal increase in all lineages, suggesting ACK2 depletes true HSCs and not committed progenitors. Interestingly, ACK2 depletion at doses 2.5-10 µg did not result in engraftment of allogeneic BALB/c cells (n=11), indicating that allogeneic neonatal transplantation, unlike in utero transplantation, is limited by a host immune response which is unaffected by ACK2. Conclusion We have demonstrated that fetal HSC depletion using ACK2 can lead to clinically relevant levels of donor cell engraftment with minimal toxicity. In previous studies with this antibody, host HSC depletion required either immunodeficient animals or concurrent irradiation, whereas we achieved depletion in wild-type fetal hosts, suggesting differences in fetal vs. adult HSC sensitivity to SCF signaling. Future studies should explore this strategy to improve engraftment in large animals models of IUHCTx. Disclosures: Weissman: Amgen, Systemix, Stem cells Inc, Cellerant: Consultancy, Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees.

Different kinases acting on stathmin (oncoprotein, Op18) in human healthy and malignant hematopoietic stem cells

2007 ◽  
Vol 25 (18_suppl) ◽  
pp. 17527-17527
Author(s):  
H. Lannert ◽  
T. Able ◽  
S. Leicht ◽  
R. Saffrich ◽  
V. Eckstein ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Expression Profiles ◽  
Proteome Analysis ◽  
Becton Dickinson ◽  
Hematopoietic Stem ◽  
Cd34 Cells

17527 Background: Stathmin/Op18 is a cytosolic phosphoprotein which regulates the dynamics of microtubules. This regulation is important in mitosis during cell division and in the migration of cells in modification of the cytoskeleton. The process of tumor proliferation and metastasis is characterized by high rates of mitosis and migration into distant tissues. Stathmin itself is regulated by kinases through phosphorylation of mainly 4 different serin sides. In this study, we investigated stathmin- and its kinases expression in native hematopoietic CD34+ stem cells (HSCs) from bone marrow (BM) in comparison to mobilized peripheral blood stem cells (mPBSCs) from G-CSF stimulated donors and leukemic CD34+ cells from patients with AML. Methods: Mononuclear cells were isolated by a standard Ficoll-Hypaque gradient separation method from the different blood sources. An Auto-MACS (Miltenyi) and FACS Vantage SE cell sorter (Becton Dickinson) was used to highly enrich (>99%) CD34+ cells fractions. In comparative proteome analysis, we detected the protein expression of stathmin in mPBSCs, AML CD34+ cells, and in native HSCs from BM. We performed microarray-based gene expression profiles of these cells and focused on kinases regulating stathmin’s activity. Furthermore, we monitored stathmin and its relevant kinases by FACS analyses of the enriched cell fractions and by fluorescence microscopy of bone marrow smears and cytospins. Results: In this study, we have shown in comparative proteome analysis (Q-TOF-MS/MS) that stathmin is expressed in G-CSF mobilized hematopoietic stem cells for the first time and in AML cells. In microarray analysis we indentified up- and down-regulated kinases: MAPK, PAK1, PKC beta/zeta, MEKK3 and CDKs. Accordingly, we demonstrated in FACS analyses and in immunofluorescence microscopy the high intracellular expression of PKCzeta in AML cells and MEKK3 as well PAK1 in mPBSCs. Conclusions: Our findings show that G-CSF stimulates Stathmin expression in mPBSCs and plays a key role in migration into peripheral blood. Furthermore, we show the different expression of kinases acting on stathmin in mPBSCs and AML cells. Consequently, stathmin and its relevant kinases promise to become a future target in therapies of malignant processes. No significant financial relationships to disclose.

scholarly journals Enrichment and characterization of murine hematopoietic stem cells that express c-kit molecule

Blood ◽  
1991 ◽  
Vol 78 (7) ◽  
pp. 1706-1712 ◽  
Author(s):  
S Okada ◽  
H Nakauchi ◽  
K Nagayoshi ◽  
S Nishikawa ◽  
S Nishikawa ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Peripheral Blood ◽  
Bone Marrow Cells ◽  
Flow Cytometric Analysis ◽  
Tyrosine Kinase Receptor ◽  
Dependent Manner ◽  
Hematopoietic Stem ◽  
Marrow Cells

The proto-oncogene c-kit encodes a transmembrane tyrosine kinase receptor for stem cell factor (SCF). The c-kit/SCF signal is expected to have an important role in hematopoiesis. A monoclonal antibody (ACK- 2) against the murine c-kit molecule was prepared. Flow cytometric analysis showed that the bone marrow cells that expressed the c-kit molecule (approximately 5%) were B220(B)-, TER119(erythroid)-, Thy1negative-low, and WGA+. A small number of Mac-1(macrophage)+ or Gr- 1(granulocyte)+ cells were c-kit-low positive. Colony-forming unit in culture (CFU-C) and day-8 and day-12 CFU-spleen (CFU-S) existed exclusively in the c-kit-positive fraction. About 20% of the Lin(lineage)-c-kit+ cells were rhodamine-123low and this fraction contained more day-12 CFU-S than day-8 CFU-S. On the basis of these findings, murine hematopoietic stem cells were enriched with normal bone marrow cells. One of two and one of four Thy-1lowLin-WGA+c-kit+ cells were CFU-C and CFU-S, respectively. Long-term repopulating ability was investigated using B6/Ly5 congenic mice. Eight and 25 weeks after transplantation of Lin-c-kit+ cells, donor-derived cells were found in the bone marrow, spleen, thymus, and peripheral blood. In peripheral blood, T cells, B cells, and granulocyte-macrophages were derived from donor cells. Injection of ACK-2 into the irradiated mice after bone marrow transplantation decreased the numbers of day-8 and day-12 CFU-S in a dose-dependent manner. Day-8 spleen colony formation was completely suppressed by the injection of 100 micrograms ACK-2, but a small number of day-12 colonies were spared. Our data show that the c- kit molecule is expressed in primitive stem cells and plays an essential role in the early stages of hematopoiesis.

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