Hematopoiesis and Stem Cell Renewal in Long-Term Bone Marrow Cultures Containing Catalase.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 371-371 ◽  
Author(s):  
Rashmi Gupta ◽  
Simon Karpatkin ◽  
Ross Basch
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Bone Marrow Cells ◽  
Fold Increase ◽  
Cell Renewal ◽  
Hematopoietic Stem ◽  
Stem Cell Renewal ◽  
Bone Marrow Cultures

Abstract Many of the events that occur within the bone marrow can be modeled in long-term bone marrow cultures (LTBMC), which are capable of producing stem cells. Although the cultures faithfully replicate the differentiation of many hematopoietic lineages, they are relatively short-lived. The stem cell compartment is rapidly depleted and attempts to achieve expansion of hematopoietic cells in culture have met with limited success. These cultures accumulate large numbers of granulocytes and monocytes capable of producing significant levels of reactive oxygen species (ROS). It has recently become clear that some ROS, including H2O2 can play a critical role in intracellular signalling induced by various growth factors and cytokines. We therefore elected to test the effect of 2 different H2O2 scavenger catalases, (bovine or aspergillosis added on alternate days) on LTBMC hematopoiesis of mouse low density bone marrow cells on irradiated adherent preformed stromal monolayers. Dramatic alterations were noted with either catalase, whereas heat-inactivated catalase had no effect. Initially there is a 5–10 fold increase in the non-adherent granulocytes and their precursors. The increase is relatively short-lived at 3–4 weeks when catalase cultures contain 1/5 as many hematopoietic cells as controls. However these cells contain 5 times the number of myeloid clonal progenitors (CFU-c) than controls. After 4–5 weeks the catalase treated cells become quiescent. When catalase is removed hematopoiesis returns promptly, ruling out a catalase-induced toxic effect. By the 3rd week of catalase treatment >90% of non-adherent cells are Sca-1+ and 36% of them are Lin−. In absolute numbers the Sca-1+ and Lin− population increase 80 fold at 3 weeks. If losses induced by removal of half of the non-adherent cells with each weekly feeding are considered, the absolute increase is >500 fold. Virtually all of the Sca-1+, Lin− cells express C-Kit+. At 2–3 weeks, approximately 15% of cells recovered from the catalase cultures have this stem cell phenotype described for murine cells, which represents a 200 fold increase in stem cells compared to controls. These cells (20,000 Ly 5.1 cells) were then tested for their ability to sustain both short- and long-term hematopoiesis in lethally irradiated Ly 5.2 mice along with 30,000 freshly isolated Ly 5.2 bone marrow cells. The catalase-treated cells showed both short- and long-term repopulating activity. At 3,6 and 10 weeks sorted Sca-1+, Lin− catalase-treated Ly 5.1 cells were 14,20 and 39% respectively of host cells, compared to 1,3 and 5% of cells cultured without catalase. These catalase-treated cells underwent multilinege repopulation granulocytes (Gr-1+), monocytes (mac-1+), T-cells (CD3+) and B− cells (B-220+) in the Ly 5.2 host. Thus, peroxide-sensitive regulatory mechanisms play an important role in regulating hematopoietic stem cell renewal and differentiation. Protected from H2O2, hematopoietic progenitors multiply and become quiescent. These cells are 200–500 fold enriched with functional stem cells. Manipulation of peroxide levels in vitro can dramatically enhance the growth of self-renewing hematopoietic stem cells and may provide a unique source of undifferentiated hematopoietic progenitors.

Long-Term Follow-Up of Serially Transplanted Mice Receiving Extended Reduced Intensity Selection of MGMT-P140K Expressing Murine Repopulating Stem Cells Demonstrates Stable Oligo- to Polyclonal Hematopoiesis without Clonal Exhaustion.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1286-1286
Author(s):  
Claudia Ball ◽  
Manfred Schmidt ◽  
Ingo Pilz ◽  
Monika Schrempp ◽  
Christof von Kalle ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Gene Transfer ◽  
Blood Cells ◽  
Bone Marrow Cells ◽  
Hematopoietic Stem ◽  
Reduced Intensity ◽  
Selection Of

Abstract In vivo selection of gene modified hematopoietic stem cells permanently increases the relative proportion of blood cells that carry a therapeutic transgene despite initially low gene transfer efficiency, thereby decreasing the likelihood of insertional mutagenesis and avoiding the need of myeloablative conditioning regimens. P140K Mutant O6-methylguanine-DNA methyltransferase (MGMT) enzyme confers resistance to the combination of the MGMT inhibitor O(6)-benzylguanine (O(6)BG) and nitrosourea drugs such as 1,3-bis-(2 chloroethyl)-1-nitrosourea (BCNU). We have previously shown that reduced intensity and toxicity BCNU/O6-BG selection allows efficient selection of MGMT-P140K expressing oligoclonal murine hematopoiesis. Nevertheless, whether long-term selection and the associated proliferative stress impairs long-term differentiation and proliferation of MGMT-P140K expressing stem cell clones is currently unknown and remains a major concern in the clinical application of MGMT selection. To address this question, serial transplantations of murine MGMT-P140K expressing hematopoiesis combined with repeated administrations of O6-BG and BCNU were done. After ex vivo gene transfer of an MGMT/IRES/eGFP encoding retroviral vector, bone marrow cells were transplanted into syngeneic C57 BL/6J mice and primary, secondary and tertiary recipient mice were subsequently treated every four weeks in order to exaggerate potential effects on long-term clonal behaviour. Lineage contribution of the transduced hematopoiesis was monitored by FACS over a total of 14 rounds of selection and clonality by LAM-PCR over a total of 12 rounds of selection. In primary mice the percentage of transduced blood cells increased from 4.7 ± 0.8 % to 36.4 ± 9.8 % (n=12) and in secondary mice from 29.9 ± 7.2 % to 65.1 ± 8.7 % (n=18) after selection without persisting peripheral blood cytopenia. Lineage analysis showed an unchanged multilineage differentiation potential of transduced cells in 1st, 2nd and 3rd generation animals. LAM PCR analysis of peripheral blood samples revealed stable oligo- to polyclonal hematopoiesis in primary and secondary mice. Evidence for predominant clones or clonal exhaustion was not observed despite up to 12 rounds of BCNU/O6-BG treatment. Interestingly, pairs of secondary transplanted mice that received bone marrow cells from identical donors showed very similar clonal composition, engraftment kinetics under selection and lineage contribution of the transduced hematopoiesis, indicating extensive self-renewal of transplantable stem cells in the primary mice resulting in a net symmetric refilling of the stem cell compartment. In summary, we demonstrate that even extended selection of MGMT-P140K expressing hematopoietic stem cells by repetitive chemotherapy does not affect their differentiation or proliferation potential and does not result in clonal exhaustion. Our results have important implications for the clinical use of MGMT selection strategies for the amplification of a limited number of gene corrected clones in clinical gene therapy.

scholarly journals Searching for hematopoietic stem cells: evidence that Thy-1.1lo Lin- Sca-1+ cells are the only stem cells in C57BL/Ka-Thy-1.1 bone marrow.

1992 ◽  
Vol 175 (1) ◽  
pp. 175-184 ◽  
Author(s):  
N Uchida ◽  
I L Weissman
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Bone Marrow Cells ◽  
Cell Activity ◽  
Hematopoietic Stem ◽  
Stem Cell Activity ◽  
Marrow Cells

Hematopoietic stem cells (HSCs) are defined in mice by three activities: they must rescue lethally irradiated mice (radioprotection), they must self-renew, and they must restore all blood cell lineages permanently. We initially demonstrated that HSCs were contained in a rare (approximately 0.05%) subset of bone marrow cells with the following surface marker profile: Thy-1.1lo Lin- Sca-1+. These cells were capable of long-term, multi-lineage reconstitution and radioprotection of lethally irradiated mice with an enrichment that mirrors their representation in bone marrow, namely, 1,000-2,000-fold. However, the experiments reported did not exclude the possibility that stem cell activity may also reside in populations that are Thy-1.1-, Sca-1-, or Lin+. In this article stem cell activity was determined by measuring: (a) radioprotection provided by sorted cells; (b) long-term, multi-lineage reconstitution of these surviving mice; and (c) long-term, multi-lineage reconstitution by donor cells when radioprotection is provided by coinjection of congenic host bone marrow cells. Here we demonstrate that HSC activity was detected in Thy-1.1+, Sca-1+, and Lin- fractions, but not Thy-1.1-, Sca-1-, or Lin+ bone marrow cells. We conclude that Thy-1.1lo Lin- Sca-1+ cells comprise the only adult C57BL/Ka-Thy-1.1 mouse bone marrow subset that contains pluripotent HSCs.

scholarly journals Hematopoiesis and stem cell renewal in long-term bone marrow cultures containing catalase

Blood ◽  
2006 ◽  
Vol 107 (5) ◽  
pp. 1837-1846 ◽  
Author(s):  
Rashmi Gupta ◽  
Simon Karpatkin ◽  
Ross S. Basch
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Ex Vivo ◽  
Mouse Bone Marrow ◽  
Cell Renewal ◽  
Hematopoietic Stem ◽  
Stem Cell Renewal

Culturing mouse bone marrow in the presence of catalase dramatically alters hematopoiesis. Granulocyte output is initially increased 4- to 5-fold. This increase is transient and granulocyte production declines as immature (Sca-1+/LIN-) cells accumulate. One third of these immature cells have a phenotype (Sca-1+/c-Kit+) characteristic of hematopoietic stem cells. At 2 to 3 weeks there are greater than 200-fold more Sca-1+/c-Kit+/LIN- cells in treated cultures than in controls. This population contains functional stem cells with both short-term and long-term bone marrow repopulating activity. In addition to myeloid progenitors, this Sca-1+/LIN- population contains a large number of cells that express CD31 and CD34 and have an active Tie-2 promoter, indicating that they are in the endothelial lineage. After 3 to 4 weeks hematopoiesis in treated cultures wanes but if catalase is removed, hematopoiesis resumes. After 7 to 10 days the cultures are indistinguishable from untreated controls. Thus, protected from H2O2, hematopoietic progenitors multiply and become quiescent. This sequence resembles in vivo development in normal marrow. These results make it clear that peroxide-sensitive regulatory mechanisms play an important role in controlling hematopoiesis ex vivo and presumably in vivo as well. They also indicate that manipulation of the peroxide levels can be used to enhance the growth of hematopoietic stem cells in culture.

Genistein Protects Hematopoietic Stem Cells Against G-CSF Induced DNA Damage

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3486-3486
Author(s):  
Liliana Souza ◽  
Erica Silva ◽  
Elissa Calloway ◽  
Michael Rossi ◽  
Omer Kucuk ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Bone Marrow ◽  
Dna Damage ◽  
Stem Cell ◽  
Bone Marrow Cells ◽  
Fold Increase ◽  
Hematopoietic Stem ◽  
Oral Supplementation ◽  
Marrow Cells

Abstract Abstract 3486 Granulocyte colony-stimulating factor (G-CSF) is widely utilized in multiple clinical settings to lessen the effects of neutropenia. Although clearly beneficial, there are concerns about the long term effects of G-CSF. A particular concern is that G-CSF therapy may increase the risk of MDS and or AML. The most striking example is that of Severe Congenital Neutropenia (SCN). While G-CSF clearly improves survival, there are several lines of evidence to suggest that G-CSF treatment contributes to development of leukemia in these patients. First, the risk of leukemia appears to correlate with the cumulative dose of G-CSF. Second, of all the congenital marrow failure syndromes predisposed to AML, SCN alone does not appear to be a hematopoietic stem cell disorder. Since AML appears to rise from sequential mutations in hematopoietic stem cells, this would suggest that therapy, not the intrinsic cell defect is causal. It has been demonstrated that G-CSF does initiate signaling pathways in hematopoietic stem cell (HSC). We hypothesize that G-CSF induced excessive HSC proliferation can lead to DNA damage and genome instability. To test our premise, mice were treated with G-CSF for 4 months and bone marrow cells were analyzed. Our results demonstrated a 3 fold increase in linage negative, Sca positive and cKit positive (LSK) population and a 2 fold increase in the amount of DNA double strand breaks via the presence of nuclear pH2AX in the LSK population. To determine if the G-CSF induced proliferation lead to chromosome alterations, we performed array-comparative genomic hybridization analyses (CGH). DNA from lineage negative bone marrow cells from animals treated with G-CSF for 4 months were compared to untreated mice. Our results demonstrate variations in gains and losses of several chromosome regions. Fluorescence in situ hybridization (FISH) of Lin-Sca+ bone marrow cells confirmed loss on regions of chromosome 2 (6%) and 17 (30%). Since prolonged G-CSF exposure promotes genomic instability in HSCs we hypothesize that an alternative strategy would be to co-administer a drug that selectively blocks the effect of G-CSF on HSCs. Previous studies suggested genistein as an attractive compound. Genistein is a natural soy isoflavone with excellent bioavalibity that has anti-oxidant and anti-proliferative properties. In this study, we utilized a dose of genistein that can easily be obtained through oral supplementation. Mice were concomitant treated with G-CSF and genistein 3 times a week. Genistein partially blocked the G-CSF induced expansion of LSK cells and reduced pH2AX levels in this population by 40%. This was also accompanied by a reduction in LSK cells with an abnormal FISH signal (50% reduction). Importantly, genistein did not block the G-CSF driven expansion of mature neutrophils as total number of neutrophils in mice treated with G-CSF and genistein are the same as those treated with G-CSF alone. Our results suggest that genistein's effects are mediated primarily through inhibition of HSC proliferation. We demonstrate that G-CSF treatment induces GSK3β phosphorylation and Cyclin D1 and D3 expression. Genistein blocked GSK3β phosphorylation and Cyclin D1 and D3 induction. Inhibition of GSKβ3 has been demonstrated to delay HSC entry into cell cycle by promoting degradation of β-catenin, while HSCs from the triple cyclin knock out mouse (Cyclins D1, D2, and D3) display delayed cell cycle entry. Collectively, our results imply that prolonged G-CSF treatment induces DNA damage in HSCs by initiating cell cycle progression. HSCs are long lived, quiescent cells that preferentially utilize non-homologus end joining for DNA repair when progressing from G0 to G1. NHEJ is a relatively error prone DNA repair mechanism. Its preferential use by HSCs has been postulated as reason chromosomal deletions and translocations are often seen and many times are causal in the development of acute leukemia. Importantly, we demonstrate, that genistein, at levels obtainable through oral supplementation, is able to reduce DNA damage by attenuating G-CSF induced HSC proliferation without compromising G-CSFs ability to accelerate terminal neutrophilic differentiation. These results suggest that genistein may be an effective therapeutic agent in patients with SCN who require prolonged G-CSF support. Disclosures: No relevant conflicts of interest to declare.

SIRT1 Deacetylase Is Essential for Hematopoietic Stem Cell Activity Via Regulation of Foxo3.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2315-2315 ◽  
Author(s):  
Pauline Rimmele ◽  
Carolina L. Bigarella ◽  
Valentina d'Escamard ◽  
Brigitte Izac ◽  
David Sinclair ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Bone Marrow Cells ◽  
Leukemic Stem Cells ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Marrow Cells

Abstract Abstract 2315 SIRT1 is a member of the NAD-dependent family of sirtuin deacetylases with critical functions in cellular metabolism, response to stress and aging. Although SIRT1 is clearly a regulator of embryonic stem cells, reports on the function of SIRT1 in adult hematopoietic stem cell (HSC) have been conflicting. While SIRT1 was positively associated with HSC activity on a genetic screen, using a germline deletion of SIRT1 three groups found SIRT1 to be dispensable for adult HSC. Here, we first showed that nuclear SIRT1 expression is enriched in bone marrow-derived Lin−Sca1+cKit+ (LSK) cells, as compared to total bone marrow cells. Germline deletion of SIRT1 is associated with developmental defects and high perinatal mortality resulting in only 10% of mice reaching adulthood. To circumvent the potential developmental adaptation of these mice, we used an adult-tamoxifen inducible SIRT1 knockout mouse model. Full-length SIRT1 protein was nearly undetectable in the bone marrow and spleen of SIRT1−/− mice. Analysis of wild type and SIRT1−/− bone marrow cells, 4 weeks after tamoxifen treatment, showed that loss of SIRT1 increased the size and frequency of the LSK compartment. Interestingly, this was associated with a significant decrease in the frequency of long-term repopulating HSC as determined by SLAM markers (CD48−CD150+LSK) within LSK cells. This decrease was even more pronounced with time. In agreement with these results, the long-term repopulation ability of CD48−CD150+LSK cells is severely compromised in SIRT1−/− mice as measured 16 weeks after transplantation, strongly suggesting that SIRT1 is essential for long-term HSC function. Thus, loss of SIRT1 results in loss of long-term repopulating stem cells in favor of total LSK cells that is a more heterogeneous population of stem cells. SIRT1 has several substrates with a potential function in HSC. Among these, we focused on Foxo3 Forkhead transcription factor which is essential for the maintenance of hematopoietic and leukemic stem cell pool. Despite the importance of Foxo3 to the control of HSC function, mechanisms that regulate Foxo3 activity in HSC remain unknown. Negative regulation of FoxOs by AKT phosphorylation promotes their cytosolic localization in response to growth factors stimulation. Interestingly, Foxo3 is constitutively nuclear in bone marrow LSK and in leukemic stem cells, strongly suggesting that negative phosphorylation may not be the sole Foxo3 regulatory mechanism in these stem cells. FoxO proteins are regulated by several post-translational modifications including acetylation in addition to phosphorylation, although the impact of acetylation on Foxo3 function remains unresolved. Therefore, we asked whether regulation of adult HSC activity by SIRT1 deacetylase is mediated by Foxo3. The in vivo injection of sirtinol, a SIRT1 inhibitor, for 3 weeks compromised significantly the long-term repopulation capacity of wild type but not Foxo3−/− HSC as measured by the repopulation ability of CD48−CD150+LSK cells in lethally irradiated mice after 16 weeks. These results suggest that Foxo3 is likely to be required for SIRT1 regulation of HSC activity. In agreement with this, we showed that in contrast to wild type LSK cells, Foxo3 is mostly cytoplasmic in SIRT1−/− LSK cells, indicating that loss of SIRT1 is sufficient to translocate Foxo3 to the cytosol and presumably inhibit its activity. We further showed that ectopically expressed acetylation-mimetic mutant of Foxo3 where all putative acetyl-lysine residues are mutated to glutamine, in bone marrow mononuclear cells, is mostly localized in the cytosol in contrast to wild type Foxo3 protein and results in significant decrease of colony-forming unit-spleen (CFU-S) activity. Using pharmacological antagonism as well as conditional deletion of SIRT1 in adult HSC, we identified a critical function for SIRT1 in the regulation of long-term HSC activity. Our results contrast with previously published data obtained from germline deleted SIRT1 mice, and suggest that the use of a conditional approach is essential for unraveling SIRT1 function in adult tissues. Our data also suggest that SIRT1 regulation of HSC activity is through activation of Foxo3. These findings are likely to have an important impact on our understanding of the regulation of hematopoietic and leukemic stem cells and may be of major therapeutic value for hematological malignancies and disorders of stem cells and aging. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Myelosuppressive conditioning is required to achieve engraftment of pluripotent stem cells contained in moderate doses of syngeneic bone marrow

Blood ◽  
1994 ◽  
Vol 83 (4) ◽  
pp. 939-948 ◽  
Author(s):  
Y Tomita ◽  
DH Sachs ◽  
M Sykes
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Whole Body ◽  
Mixed Chimerism ◽  
Hematopoietic Stem ◽  
Donor Cells ◽  
Donor Type ◽  
Low Levels

Abstract We have investigated the requirement for whole body irradiation (WBI) to achieve engraftment of syngeneic pluripotent hematopoietic stem cells (HSCs). Recipient B6 (H-2b; Ly-5.2) mice received various doses of WBI (0 to 3.0 Gy) and were reconstituted with 1.5 x 10(7) T-cell-depleted (TCD) bone marrow cells (BMCs) from congenic Ly-5.1 donors. Using anti-Ly-5.1 and anti-Ly-5.2 monoclonal antibodies and flow cytometry, the origins of lymphoid and myeloid cells reconstituting the animals were observed over time. Chimerism was at least initially detectable in all groups. However, between 1.5 and 3 Gy WBI was the minimum irradiation dose required to permit induction of long-term (at least 30 weeks), multilineage mixed chimerism in 100% of recipient mice. In these mice, stable reconstitution with approximately 70% to 90% donor-type lymphocytes, granulocytes, and monocytes was observed, suggesting that pluripotent HSC engraftment was achieved. About 50% of animals conditioned with 1.5 Gy WBI showed evidence for donor pluripotent HSC engraftment. Although low levels of chimerism were detected in untreated and 0.5-Gy-irradiated recipients in the early post-BM transplantation (BMT) period, donor cells disappeared completely by 12 to 20 weeks post-BMT. BM colony assays and adoptive transfers into secondary lethally irradiated recipients confirmed the absence of donor progenitors and HSCs, respectively, in the marrow of animals originally conditioned with only 0.5 Gy WBI. These results suggest that syngeneic pluripotent HSCs cannot readily engraft unless host HSCs sustain a significant level of injury, as is induced by 1.5 to 3.0 Gy WBI. We also attempted to determine the duration of the permissive period for syngeneic marrow engraftment in animals conditioned with 3 Gy WBI. Stable multilineage chimerism was uniformly established in 3-Gy-irradiated Ly-5.2 mice only when Ly-5.1 BMC were injected within 7 days of irradiation, suggesting that repair of damaged host stem cells or loss of factors stimulating engraftment may prevent syngeneic marrow engraftment after day 7.

scholarly journals Efficient retroviral gene transfer to purified long-term repopulating hematopoietic stem cells

Blood ◽  
1994 ◽  
Vol 84 (1) ◽  
pp. 74-83 ◽  
Author(s):  
SJ Szilvassy ◽  
S Cory
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Cell Biology ◽  
Bone Marrow Cells ◽  
High Titer ◽  
Marker Gene ◽  
Mouse Bone Marrow ◽  
Hematopoietic Stem

Abstract Efficient gene delivery to multipotential hematopoietic stem cells would greatly facilitate the development of effective gene therapy for certain hematopoietic disorders. We have recently described a rapid multiparameter sorting procedure for significantly enriching stem cells with competitive long-term lymphomyeloid repopulating ability (CRU) from 5-fluorouracil (5-FU)-treated mouse bone marrow. The sorted cells have now been tested as targets for retrovirus-mediated delivery of a marker gene, NeoR. They were cocultured for 4 days with fibroblasts producing a high titer of retrovirus in medium containing combinations of the hematopoietic growth factors interleukin-3 (IL-3), IL-6, c-kit ligand (KL), and leukemia inhibitory factor (LIF) and then injected into lethally irradiated recipients, together with sufficient “compromised” bone marrow cells to provide short-term support. Over 80% of the transplanted mice displayed high levels (> or = 20%) of donor- derived leukocytes when analyzed 4 to 6 months later. Proviral DNA was detected in 87% of these animals and, in half of them, the majority of the hematopoietic cells were marked. Thus, infection of the stem cells was most effective. The tissue and cellular distribution of greater than 100 unique clones in 55 mice showed that most sorted stem cells had lymphoid as well as myeloid repopulating potential. Secondary transplantation provided strong evidence for infection of very primitive stem cells because, in several instances, different secondary recipients displayed in their marrow, spleen, thymus and day 14 spleen colony-forming cells the same proviral integration pattern as the primary recipient. Neither primary engraftment nor marking efficiency varied for stem cells cultured in IL-3 + IL-6, IL-3 + IL-6 + KL, IL-3 + IL-6 + LIF, or all four factors, but those cultured in IL-3 + IL-6 + LIF appeared to have lower secondary engraftment potential. Provirus expression was detected in 72% of the strongly marked mice, albeit often at low levels. Highly efficient retroviral marking of purified lymphomyeloid repopulating stem cells should enhance studies of stem cell biology and facilitate analysis of genes controlling hematopoietic differentiation and transformation.

scholarly journals Self-repopulating recipient bone marrow resident macrophages promote long-term hematopoietic stem cell engraftment

Blood ◽  
2018 ◽  
Vol 132 (7) ◽  
pp. 735-749 ◽  
Author(s):  
Simranpreet Kaur ◽  
Liza J. Raggatt ◽  
Susan M. Millard ◽  
Andy C. Wu ◽  
Lena Batoon ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Hematopoietic Stem ◽  
Cell Engraftment ◽  
Key Points ◽  
Bone Marrow Engraftment

Key Points Recipient macrophages persist in hematopoietic tissues and self-repopulate via in situ proliferation after syngeneic transplantation. Targeted depletion of recipient CD169+ macrophages after transplant impaired long-term bone marrow engraftment of hematopoietic stem cells.

scholarly journals Long-term monoclonal reconstitution of erythropoiesis in genetically anemic W/Wv mice by injection of 5-fluorouracil-treated bone marrow cells of Pgk-1b/Pgk-1a mice

Blood ◽  
1987 ◽  
Vol 70 (6) ◽  
pp. 1758-1763 ◽  
Author(s):  
T Nakano ◽  
N Waki ◽  
H Asai ◽  
Y Kitamura
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Bone Marrow Cells ◽  
Electrophoretic Pattern ◽  
Phosphoglycerate Kinase ◽  
X Chromosomes ◽  
Hematopoietic Stem ◽  
Colony Forming Units ◽  
Marrow Cells

Abstract The spleen colony-forming assay does not represent the number of hematopoietic stem cells with extensive self-maintaining capacity because five to 50 spleen colony-forming units (CFU-S) are necessary to rescue a genetically anemic (WB X C57BL/6)F1-W/Wv(WBB6F1-W/Wv) mouse. We investigated which is more important for the reconstitution of erythropoiesis, the transplantation of multiple CFU-S or that of a single stem cell with extensive self-maintaining potential. The electrophoretic pattern of hemoglobin was used as a marker of reconstitution and that of phosphoglycerate kinase (PGK), an X chromosome-linked enzyme, as a tool for estimating the number of stem cells. For this purpose, we developed the C57BL/6 congeneic strain with the Pgk-1a gene. Bone marrow cells were harvested after injection of 5- fluorouracil from C57BL/6-Pgk-1b/Pgk-1a female mice in which each stem cell had either A-type PGK or B-type PGK due to the random inactivation of one or two X chromosomes. When a relatively small number of bone marrow cells (ie, 10(3) or 3 X 10(3] were injected into 200-rad- irradiated WBB6F1-W/Wv mice, the hemoglobin pattern changed from the recipient type (Hbbd/Hbbs) to the donor type (Hbbs/Hbbs) in seven of 150 mice for at least 8 weeks. Erythrocytes of all these WBB6F1-W/Wv mice showed either A-type PGK alone or B-type PGK alone during the time of reconstitution, which suggests that a single stem cell with extensive self-maintaining potential may sustain the whole erythropoiesis of a mouse for at least 8 weeks.

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