scholarly journals Use of 5-fluorouracil to analyze the effect of macrophage inflammatory protein-1 alpha on long-term reconstituting stem cells in vivo

Blood ◽  
1993 ◽  
Vol 81 (6) ◽  
pp. 1497-1504 ◽  
Author(s):  
VF Quesniaux ◽  
GJ Graham ◽  
I Pragnell ◽  
D Donaldson ◽  
SD Wolpe ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Inhibitory Effect ◽  
In Vivo Model ◽  
Hematopoietic Progenitors ◽  
Inflammatory Protein ◽  
Macrophage Inflammatory Protein

A macrophage-derived inhibitor of early hematopoietic progenitors (colony-forming unit-spleen, CFU-A) called stem cell inhibitor was found to be identical to macrophage inflammatory protein-1 alpha (MIP-1 alpha). We investigated the effect of MIP-1 alpha on the earliest stem cells that sustain long-term hematopoiesis in vivo in a competitive bone marrow repopulation assay. Because long-term reconstituting (LTR) stem cells are normally quiescent, an in vivo model was first developed in which they are triggered to cycle. A first 5-fluorouracil (5-FU) injection was used to eliminate later progenitors, causing the LTR stem cells, which are normally resistant to 5-FU, to enter the cell cycle and become sensitive to a second 5-FU injection administered 5 days later. Human MIP-1 alpha administered from day 0 to 7 was unable to prevent the depletion of the LTR stem cells by the second 5-FU treatment, as observed on day 7 in this model, suggesting that the LTR stem cells were not prevented from being triggered into cycle despite the MIP-1 alpha treatment. However, the MIP-1 alpha protocol used here did substantially decrease the number of more mature hematopoietic progenitors (granulocyte-macrophage colony-forming cells [CFC], burst- forming unit-erythroid, CFCmulti, and preCFCmulti) recovered in the bone marrow shortly after a single 5-FU injection. In vitro, MIP-1 alpha had no inhibitory effect on the ability of these progenitors to form colonies. This study confirms the in vivo inhibitory effect of MIP- 1 alpha on subpopulations of hematopoietic progenitors that are activated in myelodepressed animals. However, MIP-1 alpha had no effect on the long-term reconstituting stem cells in vivo under conditions in which it effectively reduced all later progenitors.

scholarly journals Use of 5-fluorouracil to analyze the effect of macrophage inflammatory protein-1 alpha on long-term reconstituting stem cells in vivo

Blood ◽  
1993 ◽  
Vol 81 (6) ◽  
pp. 1497-1504 ◽  
Author(s):  
VF Quesniaux ◽  
GJ Graham ◽  
I Pragnell ◽  
D Donaldson ◽  
SD Wolpe ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Inhibitory Effect ◽  
In Vivo Model ◽  
Hematopoietic Progenitors ◽  
Inflammatory Protein ◽  
Macrophage Inflammatory Protein

Abstract A macrophage-derived inhibitor of early hematopoietic progenitors (colony-forming unit-spleen, CFU-A) called stem cell inhibitor was found to be identical to macrophage inflammatory protein-1 alpha (MIP-1 alpha). We investigated the effect of MIP-1 alpha on the earliest stem cells that sustain long-term hematopoiesis in vivo in a competitive bone marrow repopulation assay. Because long-term reconstituting (LTR) stem cells are normally quiescent, an in vivo model was first developed in which they are triggered to cycle. A first 5-fluorouracil (5-FU) injection was used to eliminate later progenitors, causing the LTR stem cells, which are normally resistant to 5-FU, to enter the cell cycle and become sensitive to a second 5-FU injection administered 5 days later. Human MIP-1 alpha administered from day 0 to 7 was unable to prevent the depletion of the LTR stem cells by the second 5-FU treatment, as observed on day 7 in this model, suggesting that the LTR stem cells were not prevented from being triggered into cycle despite the MIP-1 alpha treatment. However, the MIP-1 alpha protocol used here did substantially decrease the number of more mature hematopoietic progenitors (granulocyte-macrophage colony-forming cells [CFC], burst- forming unit-erythroid, CFCmulti, and preCFCmulti) recovered in the bone marrow shortly after a single 5-FU injection. In vitro, MIP-1 alpha had no inhibitory effect on the ability of these progenitors to form colonies. This study confirms the in vivo inhibitory effect of MIP- 1 alpha on subpopulations of hematopoietic progenitors that are activated in myelodepressed animals. However, MIP-1 alpha had no effect on the long-term reconstituting stem cells in vivo under conditions in which it effectively reduced all later progenitors.

scholarly journals Sustained, retransplantable, multilineage engraftment of highly purified adult human bone marrow stem cells in vivo

Blood ◽  
1996 ◽  
Vol 88 (11) ◽  
pp. 4102-4109 ◽  
Author(s):  
CI Civin ◽  
G Almeida-Porada ◽  
MJ Lee ◽  
J Olweus ◽  
LW Terstappen ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Cell Population ◽  
Mononuclear Cells ◽  
Cell Subset ◽  
Fetal Sheep ◽  
Adult Human

Abstract Data from many laboratory and clinical investigations indicate that CD34+ cells comprise approximately 1% of human bone marrow (BM) mononuclear cells, including the progenitor cells of all the lymphohematopoietic lineages and lymphohematopoietic stem cells (stem cells). Because stem cells are an important but rare cell type in the CD34+ cell population, investigators have subdivided the CD34+ cell population to further enrich stem cells. The CD34+/CD38-cell subset comprises less than 10% of human CD34+ adult BM cells (equivalent to < 0.1% of marrow mononuclear cells), lacks lineage (lin) antigens, contains cells with in vitro replating capacity, and is predicted to be highly enriched for stem cells. The present investigation tested whether the CD34+/CD38-subset of adult human marrow generates human hematopoiesis after transfer to preimmune fetal sheep. CD34+/ CD38- cells purified from marrow using immunomagnetic microspheres or fluorescence-activated cell sorting generated easily detectable, long- term, multilineage human hematopoiesis in the human-fetal sheep in vivo model. In contrast, transfer of CD34+/CD38+ cells to preimmune fetal sheep generated only short-term human hematopoiesis, possibly suggesting that the CD34+/CD38+ cell population contains relatively early multipotent hematopoletic progenitor cells, but not stem cells. This work extends the prior in vitro evidence that the earliest cells in fetal and adult human marrow lack CD38 expression. In summary, the CD34+/ CD38-cell population has a high capacity for long-term multilineage hematopoietic engraftment, suggesting the presence of stem cells in this minor adult human marrow cell subset.

CD34+/CD33- cells reselected from macrophage inflammatory protein 1 alpha+interleukin-3--supplemented "stroma-noncontact" cultures are highly enriched for long-term bone marrow culture initiating cells

Blood ◽  
1994 ◽  
Vol 84 (5) ◽  
pp. 1442-1449 ◽  
Author(s):  
CM Verfaillie ◽  
JS Miller
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Antigen Expression ◽  
Hematopoietic Stem ◽  
Interleukin 3 ◽  
Inflammatory Protein ◽  
Hla Dr ◽  
Macrophage Inflammatory Protein

Abstract Human hematopoietic stem cells are thought to express the CD34 stem cell antigen, low numbers of HLA-DR and Thy1 antigens, but no lineage commitment antigens, CD38, or CD45RA antigens. However, fluorescence- activated cell sorted CD34+ subpopulations contain not more than 1% to 5% primitive progenitors capable of initiating and sustaining growth in long-term bone marrow culture initiating cells (LTBMC-ICs). We have recently shown that culture of fresh human marrow CD34+/HLA-DR- cells separated from a stromal layer by a microporous membrane (“stroma- noncontact” culture) results in the maintenance of 40% of LTBMC-ICs. We hypothesized that reselection of CD34+ subpopulations still present after several weeks in stroma-noncontact cultures may result in the selection of cells more highly enriched for human LTBMC-ICs. Fresh marrow CD34+/HLA-DR- cells were cultured for 2 to 3 weeks in stroma- noncontact cultures. Cultured progeny was then sorted on the basis of CD34, HLA-DR, or CD33 antigen expression, and sorted cells evaluated for the presence of LTBMC-ICs by limiting dilution analysis. We show that (1) LTBMC-ICs are four times more frequent in cultured CD34+/HLA- DR- cells (4.6% +/- 1.7%) than in cultured CD34+/HLA-DR- cells (1.3% +/- 0.4%). This suggests that HLA-DR antigen expression may depend on the activation status of primitive cells rather than their lineage commitment. We then sorted cultured cells on the basis of the myeloid commitment antigen, CD33. (2) These studies show that cultured CD34+/CD33- cells contain 4% to 8% LTBMC-ICs, whereas cultured CD34+/CD33+bright cells contain only 0.1% +/- 0.03% LTBMC-ICs. Because LTBMC-ICs are maintained significantly better in stroma-noncontact cultures supplemented with macrophage inflammatory protein 1 alpha (MIP- 1 alpha) and interleukin-3 (IL-3) (Verfaillie et al, J Exp Med 179:643, 1994), we evaluated the frequency of LTBMC-ICs in CD34+/CD33- cells present in such cultures. (3) CD34+/CD33- cells present in MIP-1 alpha + IL-3-supplemented cultures contain up to 30% LTBMC-ICs. The increased frequency of LTBMC-ICs in cultured CD34+ subpopulations may be the result of terminal differentiation of less primitive progenitors, loss of cells that fail to respond to the culture conditions or recruitment of quiescent LTBMC-ICs. The capability to select progenitor populations containing up to 30% LTBMC-ICs should prove useful in studies examining the growth requirements, self-renewal, and multilineage differentiation capacity of human hematopoietic stem cells at the single-cell level.

scholarly journals Local clonal analysis of the hematopoietic system shows that multiple small short-living clones maintain life-long hematopoiesis in reconstituted mice

Blood ◽  
1996 ◽  
Vol 88 (8) ◽  
pp. 2927-2938 ◽  
Author(s):  
NJ Drize ◽  
JR Keller ◽  
JL Chertkov
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Clonal Analysis ◽  
Hematopoietic Progenitors ◽  
Hematopoietic System ◽  
Colony Forming Unit ◽  
Cell Clones

We describe here a technique to study the clonal contribution of primitive stem cells that account for long-term hematopoiesis in the same mouse over a 14-month period. Specifically, irradiated recipient female mice were transplanted with retrovirally marked male hematopoietic progenitors. Bone marrow was then collected repeatedly from local sites from the same mice throughout a 14-month period and injected into secondary irradiated recipients for analysis of donor retrovirally marked day-11 colony-forming unit-spleen (CFU-S-11). We have tracked the temporal in vivo fate of 194 individual CFU-S-derived cell clones in 38 mice reconstituted with such retrovirally marked bone marrow cells. Our data show that long-term hematopoiesis is maintained by a large number of simultaneously functioning small, shortlived (1 to 3 months) clones that usually grow locally with little or no dispersion between different regions of the hematopoietic system. Furthermore, the clones that disappeared were never detected again. The data suggest that normal hematopoiesis is supported by the sequential recruitment of marrow repopulating cells into a differentiation mode.

In Vivo Selection and Long-Term Engraftment Of Hematopoietic Stem Cells Generated Via Vascular Niche Induction Of Nonhuman Primate Induced Pluripotent Stem Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 466-466
Author(s):  
Jennifer L Gori ◽  
Jason M Butler ◽  
Devikha Chandrasekaran ◽  
Brian C Beard ◽  
Daniel J Nolan ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Nonhuman Primate ◽  
Fold Increase ◽  
Hematopoietic Progenitors ◽  
Hematopoietic Stem ◽  
In Vivo Selection ◽  
Selection Of

Clinical use of human pluripotent stem cell (PSC)-hematopoietic stem cells (HSCs) is impeded by low engraftment potential. This block suggests that additional vascular derived angiocrine signals and hematopoietic cues must be provided to produce authentic HSCs. In addition, gene modification of induced (i)PSCs with a chemotherapy resistance transgene would provide a selective mechanism to stabilize or increase engraftment of HSCs. We therefore hypothesized that modifying iPSCs to express the O6-benzylguanine (O6BG)-resistant P140K variant of methylguanine methyltransferase (MGMT), would support in vivo selection of early-engrafted iPSC-HSCs. We further postulated that Akt-activated human endothelial cells afforded by transduction of the E4ORF1 gene (E4ORF1+ECs) through angiocrine upregulation of Notch and IGF ligands would provide the necessary signals under xenobiotic-free conditions to promote definitive hematopoiesis. This vascular induction platform could drive the emergence of true HSCs. We focused on pigtail macaque (Mn)iPSCs, as a scalable, clinically relevant nonhuman primate model. MniPSCs modified to express P140K had 15-fold higher MGMT levels compared to levels in human peripheral blood mononuclear cells. P140K-MniPSCs differentiated into chemoresistant CD34+ hematopoietic progenitors (50% CD34+) with a predominant long-term (LT)-HSC-like phenotype (CD34+CD38-Thy1+CD45RA-CD49f+). Hematopoietic progenitors maintained colony forming potential after O6BG and bis-chloroethylnitrosourea (BCNU) treatment. HSCs expanded on E4ORF1+ECs maintained colony forming potential, in contrast to cells cultured with cytokines alone, with a 22-fold increase in CD34+ cell content and 10-fold increase in LT-HSC-like cells. Importantly, MniPSC-HSCs expanded with the E4ORF1+ECs had long-term engraftment in NSG mice at levels comparable to Mn bone marrow HSC engrafted mice. O6BG/BCNU treatment increased engraftment to 35% CD45+ cells the blood of mice transplanted with E4ORF1+EC expanded P140K-MniPSC-HSCs, which was maintained 16 weeks post transplantation. Primate CD45+ cell levels in the blood after selection were significantly higher for this cohort compared to mice transplanted with P140K-MniPSC-HSCs expanded in the “cytokines alone” condition (18% vs. 3% CD45+, P<0.05). On average, 15% CD34+ and 37% CD45+ cells were detected in the bone marrow of mice transplanted with E4ORF1+EC-expanded P140K-MniPSC HSCs, which is significantly higher than levels detected in the other cohorts (Table 1). CD45+ cells in the marrow were predominantly myeloid but lymphoid subsets were also present (10-25% CD3+ cells). Remarkably, the level of gene marking in CFCs and number of gene marked CFCs from mouse bone marrow was substantially higher for mice transplanted with E4ORF1+EC expanded compared to cytokine expanded P140K-MniPSC-HSCs (Table 1). Finally, to confirm engraftment of authentic HSCs, secondary transplants were established. Although engraftment was achieved in all secondary transplanted cohorts, the level of nonhuman primate cells detected was significantly higher in animals transplanted with E4ORF1+EC expanded P140K-MniPSC-HSCs. Significantly more lymphocytes (CD45+CD3+ and CD45+CD56+) and monocytes (CD45+CD14+) were detected in the blood of these secondary transplant recipients. These findings confirm generation of bona fide HSCs derived from nonhuman primate iPSCs and demonstrate that O6BG/BCNU chemotherapy supports in vivo selection of P140K-MniPSC-HSCs generated by co-culture with the E4ORF1+EC vascular platform. Our studies mark a significant advance toward clinical translation of PSC-based blood therapeutics and the development of a nonhuman primate preclinical model. Table 1 CD34+ and CD45+ engraftment and gene marking in the bone marrow of mice transplanted with nonhuman primate HPSCs from MniPSCs and bone marrow. HSCs E4ORF1+ECs O6BG/BCNU Mean %CD34+ Mean %CD45+ % gene marking in CFCs (lentivirus+) total lentivirus+ CFCs per 105 cells GFP-MniPSC + - 3 16 9 ± 2 13 ± 2 P140K-MniPSC + - 4 19 12 ± 5 17 ± 7 P140K-MniPSC - + 0.4 24 3 ± 2 2 ± 1 P140K-MniPSC + + 15 37 27 ± 24 111 ± 96 Mn BM CD34+ - - 2 21 0 0 Disclosures: Nolan: Angiocrine Bioscience: Employment. Ginsberg:Angiocrine Bioscience: Employment. Rafii:Angiocrine Bioscience: Founder Other.

The Bone Marrow "Mystery Population" of Stem Cells 20 Years Later - a Puzzle Solved?

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2392-2392
Author(s):  
Malwina Suszynska ◽  
Daniel Pedziwiatr ◽  
Magdalena J Kucia ◽  
Mariusz Z Ratajczak ◽  
Janina Ratajczak
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Receptor Expression ◽  
Stem Cell Population ◽  
Hematopoietic Stem ◽  
Murine Bone Marrow ◽  
Low Levels

Abstract Background . Almost 20 years ago, a "mystery" population of small stem cells with many of the phenotypic characteristics attributed to resting hematopoietic stem cells was identified in murine bone marrow (BM) (Stem Cells 1998, 16, 38-48). These cells expressed high levels of Sca-1, H-2K, and CD38 and low levels of Thy-1.1; they expressed CD45 antigen but were lineage-negative (lin-) for other hematopoietic markers. These cells incorporated only low levels of Rh123 and were resistant to the cytotoxic effects of 5-fluorouracil. The only phenotypic characteristic that distinguishes these cells from Sca-1+, Lin-, CD45+ Thy-1.1low long-term-reconstituting hematopoietic stem cell population is the lack of c-kit expression. In sum, this "mystery" population of small Sca-1+, lin-, c-kit- but CD45+ stem cells do not respond to hematopoietic growth factors in vitro, form in vivo spleen colonies, or reconstitute lethally irradiated mice. With our discovery of Sca-1+ Lin- CD45- very small embryonic-like stem cells (VSELs) in murine bone marrow (BM) (Leukemia 2006, 20, 857-869), we became interested in this "mystery" population of stem cells. VSELs, like the "mystery" population, are c-kit - and, if freshly isolated from BM, do not show any hematopoietic activity in standard in vitro and in vivo assays. In order to become specified to hematopoiesis, they need to be expanded over an OP-9 stromal support (Exp Hematol 2011;39:225-237). Hypothesis. Since (1) very small CD45- VSELs can be specified in OP-9 co-cultures into long-term reconstituting CD45+ HSCs, (2) the size of the "mystery" population is intermediate between VSELs and HSCs, and (3) VSELs and HSCs differ in cell surface receptor expression, we hypothesized that the "mystery" population is a missing developmental intermediate between VSELs and HSCs. Materials and Methods . Multicolor FACS analysis was employed to compare size and expression of surface markers between murine BM HSCs, the unknown population of stem cells, and VSELs. Next, the populations of small Sca-1+ H2-K+ lin- c-kit+ CD38+/- CD45+ cells (HSCs), smaller Sca-1+ H-2K+ lin- c-kit- CD38+ CD45+ cells (the "mystery" population), and very small in size Sca-1+ H-2K+ lin- c-kit- CD38+/- CD45- cells (VSELs) were purified by FACS from BM (Figure 1) and tested for in vitro colony formation. All these cell populations were primed/expanded over OP-9 support and subsequently evaluated for their hematopoietic potential after passaging in consecutive methylocellulose cultures (passages 1-4). RQ-PCR analysis was employed for detection of pluripotency marker expression as well as hematopoietic gene expression. Results . We found that, in contrast to HSCs, neither freshly sorted stem cells from the "mystery" BM population nor, as expected, VSELs grew hematopoietic colonies in standard methylcellulose cultures. This was also an important step in excluding contamination of our sorted populations with clonogenic cells. We also found that, while VSELs highly expressed Oct-4, this transcription factor was expressed at very low levels in the "mystery" population and was not detectable in HSCs. The most important observation was that the "mystery" population of stem cells became specified in OP-9-supported cultures into clonogenic HSPCs, and this specification occurred faster than the delayed specification of VSELs. VSELs first became enriched for HSPCs after acquiring CD45 antigen expression. Conclusions . Based on the results presented, we propose that the "mystery" population in murine BM is a population of stem cells intermediate between the most primitive population of BM-residing stem cells (VSELs) and the population of stem cells already specified to lympho-hematopoietic development (HSCs). Disclosures No relevant conflicts of interest to declare.

Efficient Expression of a Kit/GFP Transgene in Hematopoietic Stem Cells of Mouse Fetal Liver and Bone Marrow.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4159-4159
Author(s):  
Francesco Cerisoli ◽  
Letizia Cassinelli ◽  
Giuseppe Lamorte ◽  
Stefania Citterio ◽  
Maria Cristina Magli ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Fetal Liver ◽  
Hematopoietic Progenitors ◽  
Hematopoietic Stem ◽  
Adult Bone Marrow ◽  
Gfp Fluorescence ◽  
Adult Bone

Abstract The hierarchy of transcription factors and signalling molecules involved in hematopoietic development has been dissected through transgenic and knock-out experiments, leading to the identification of several important genes. Less well known are the networks of transcription factors which regulate the activities of the main genes identified. Kit, encoding the membrane receptor of Stem Cell Factor (SCF), is a critical molecule for Hematopoietic Stem Cells (HSC) and some early progenitors, in which it is expressed. In a previous work (Cairns et al., Blood102, 3954;2003), we used mouse lines expressing transgenic Green Fluorescent Protein (GFP) under the control of Kit regulatory elements to investigate Kit regulation in different cell systems such as the hematopoietic and germ cell lineages. We generated a mouse Kit transgene capable of efficiently driving GFP expression both in PGC and in hematopoietic progenitors, such as CFU-Mix and BFU-Es. In the present work, we evaluated the functional efficiency of the same transgene also in HSC residing in the Fetal Liver (FL) and adult Bone Marrow (BM). To test if the construct is expressed in HSC, we transplanted FL or BM cells, fractionated on the basis of Kit expression and the level of GFP fluorescence, into irradiated non-transgenic mice. At the same time, the proportion of hematopoietic progenitors in the various fractions was assessed by in vitro colony assays. Following long term hematological reconstitution, the contribution of transplanted GFP cells was evaluated by the proportion of fluorescent mixed colonies in colture as well as by the proportion of fluorescent bone marrow cells, as assessed by FACS analysis. Long term reconstitution was confirmed by secondary transplants. Results show that the repopulating cells derived from fetal liver and adult bone marrow reside in a fraction of Kit+ cells with intermediate GFP fluorescence level, whereas CFU-Mix and BFU-E are in the highly GFP fluorescent fraction. Furthermore, flow cytometry of fetal liver shows that the intermediate fluorescence fraction is highly enriched in Kit+, Sca1+, CD11b+ cells (the expected HSC immunophenotype), whereas the high fluorescence fraction contains mainly Kit+, Sca1−, CD11b− cells. Similarly, the HSC-enriched tip of the Side Population (SP) of adult bone marrow is highly enriched in Kit+, Sca1+ cells of intermediate GFP fluorescence, whereas the upper part of the SP is enriched in Kit+, Sca1− cells of high GFP fluorescence. Our results indicate that the transgene (and possibly the endogenous Kit gene as well) might be transcribed at relatively low levels in HSC versus other progenitors. Noteworthy, the same transgene is also highly expressed in PGC and in Cardiac Stem Cells (CSC) (Messina et al., Circ. Res. 95,911;2004) and in blastocyst inner mass grown in vitro, indicating that the most 5′ part of the intron (4kb), added to the otherwise inactive promoter might include sites regulating Kit expression in multiple stem cell types.

Murine Hematopoietic Stem Cells Require Akt1 and Akt2 for Long-Term Self-Renewal

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 502-502
Author(s):  
Marisa M. Juntilla ◽  
Vineet Patil ◽  
Rohan Joshi ◽  
Gary A. Koretzky
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Fetal Liver ◽  
Akt Signaling ◽  
Self Renewal ◽  
Hematopoietic Stem

Abstract Murine hematopoietic stem cells (HSCs) rely on components of the Akt signaling pathway, such as FOXO family members and PTEN, for efficient self-renewal and continued survival. However, it is unknown whether Akt is also required for murine HSC function. We hypothesized that Akt would be required for HSC self-renewal, and that the absence of Akt would lead to hematopoietic failure resulting in developmental defects in multiple lineages. To address the effect of Akt loss in HSCs we used competitive and noncompetitive murine fetal liver-bone marrow chimeras. In short-term assays, Akt1−/−Akt2−/− fetal liver cells reconstituted the LSK compartment of an irradiated host as well or better than wildtype cells, although failed to generate wildtype levels of more differentiated cells in multiple lineages. When placed in a competitive environment, Akt1−/−Akt2−/− HSCs were outcompeted by wildtype HSCs in serial bone marrow transplant assays, indicating a requirement for Akt1 and Akt2 in the maintainance of long-term hematopoietic stem cells. Akt1−/−Akt2−/− LSKs tend to remain in the G0 phase of the cell cycle compared to wildtype LSKs, suggesting the failure in serial transplant assays may be due to increased quiesence in the absence of Akt1 and Akt2. Additionally, the intracellular content of reactive oxygen species (ROS) in HSCs is dependent on Akt signaling because Akt1−/−Akt2−/− HSCs have decreased ROS levels. Furthermore, pharmacologic augmentation of ROS in the absence of Akt1 and Akt2 results in an exit from quiescence and rescue of differentiation both in vivo and in vitro. Together, these data implicate Akt1 and Akt2 as critical regulators of long-term HSC function and suggest that defective ROS homeostasis may contribute to failed hematopoiesis.

Reciprocal expression of CD38 and CD34 by adult murine hematopoietic stem cells

Blood ◽  
2001 ◽  
Vol 97 (9) ◽  
pp. 2618-2624 ◽  
Author(s):  
Fumihito Tajima ◽  
Takao Deguchi ◽  
Joseph H. Laver ◽  
Haiqun Zeng ◽  
Makio Ogawa
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Steady State ◽  
Adult Stem Cells ◽  
Human Stem Cells ◽  
Hematopoietic Stem ◽  
Reciprocal Expression

Abstract The effects of activation of adult murine stem cells on their expression of CD38 were studied using a murine transplantation model. First, the published finding that the majority of long-term engrafting cells from normal adult steady-state marrow are CD38+ was confirmed. Next, it was determined that the majority of stem cells activated in vivo by injection of 5-fluorouracil (5-FU) or mobilized by granulocyte colony-stimulating factor are CD38−. Stem cells that were activated in culture with interleukin-11 and steel factor were also CD38−. Previous studies have shown that expression of CD34 by adult stem cells is also modulated by in vivo or in vitro activation. To determine whether there is reciprocal expression of CD38 and CD34, 4 populations of post–5-FU marrow cells were analyzed. The majority of the stem cells were in the CD38−CD34+ fraction. However, secondary transplantation experiments indicated that when the bone marrow reaches steady state, the majority of the stem cells become CD38+CD34−. In addition, the minority populations of CD34+ stem cells that occur in steady-state bone marrow are CD38−. This reversible and reciprocal expression of CD38 and CD34 by murine stem cells may have implications for the phenotypes of human stem cells.

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