scholarly journals Reduction in Bone Marrow Insulin-like Growth Factor-1 Levels Enhances Long-Term Hematopoietic Stem Cell Engraftment Following Bone Marrow Transplantation

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. 68-68
Author(s):  
Timothy S. Olson ◽  
Ji Zha ◽  
Lori Kunselman ◽  
Jian-Meng Fan ◽  
Edwin M. Horwitz
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Bone Marrow Transplantation ◽  
Cell Expansion ◽  
Critical Role ◽  
Fold Increase ◽  
Hematopoietic Stem ◽  
Protein Levels ◽  
Osteolineage Cells

Abstract Efficient donor hematopoietic stem cell (HSC) engraftment is critical for successful bone marrow transplantation (BMT) and requires interactions between donor HSC and host-derived HSC niche elements. Due to disruption of perivascular stem cell niches induced by myeloablative BMT conditioning, initial engraftment of HSC following BMT occurs in niches located near the endosteum. We have previously shown that myeloablative BMT conditioning with total body irradiation (TBI) induces expansion of endosteal niche osteolineage cells in a time course correlating with increases in bone marrow (BM) levels of insulin-like growth factor (IGF)-1. We also have shown that inhibition of IGF-1 Receptor (IGF1R) signaling blocks osteolineage cell expansion after TBI, resulting in poor engraftment of long-term (LT)-HSC after BMT and suggesting a critical role for IGF-1/IGF1R interactions in regulating engraftment after BMT. We now present data in which we have defined the cellular sources of BM IGF-1 following TBI and have surprisingly discovered that reduction in BM IGF-1 levels promotes LT-HSC retention at baseline and donor LT-HSC engraftment following BMT. Using mice with floxed IGF-1 alleles (IGF1L/L) and with tissue-specific expression of Cre-recombinase, we generated models with deletion of IGF-1 derived from mature osteolineage cells (Col1A1CreIGF1Exc), hematopoietic cells (CD45CreIGF1Exc), or liver (AlbCreIGF1Exc). We additionally generated a conditional IGF-1 deletion model (MX1CreIGF1Exc) in which polyI:polyC (pI:pC) treatment disrupts IGF-1 production from all 3 of the above sources. By qPCR analysis, CD45CreIGF1Exc BM showed marked reduction (>95%) in BM IGF-1 mRNA at baseline and following TBI, suggesting that hematopoietic cells produce most of the locally derived IGF-1 in BM. Unexpectedly however, neither deletion of osteoblast-derived (Col1A1CreIGF1Exc) nor hematopoietic cell-derived (CD45CreIGF1Exc) IGF-1 significantly reduced BM extracellular or intracellular IGF-1 protein levels at baseline, or extracellular BM IGF-1 levels following TBI. In contrast, elimination of liver-derived IGF-1 in AlbCreIGF1Exc mice not only reduced plasma IGF-1 levels by 80%, but surprisingly reduced BM extracellular IGF-1 protein levels by ≥80% both at baseline and 48 hours post-TBI, and reduced BM intracellular IGF-1 protein levels by >80% and >60% at baseline and post-TBI, respectively. These data suggest that most IGF-1 protein in BM during BMT is derived from liver. Conditional IGF-1 deletion in MX1CreIGF1Exc mice resulted in severe reduction of both BM IGF-1 mRNA and protein levels, combining effects seen in CD45CreIGF1Exc and AlbCreIGF1Exc mice. None of these models, however, showed deficits in TBI-induced endosteal cell expansion, suggesting that other ligands or perhaps only low levels of IGF-1 are required to induce IGFR1 signaling-dependent endosteal cell expansion. Unexpectedly, reduction of BM IGF-1 in ALBCreIGF1Exc and MX1CreIGF1Exc mice caused a 3-4 fold increase (% of total BM) in BM Lin-cKit+Sca1+CD48-CD150+ or Lin-cKit+Sca1+CD34-CD135- HSC. ALBCreIGF1Exc BM displayed enhanced multi-lineage long-term repopulating capacity in competitive transplant assays, proving that these cells are functional LT-HSC. Given this baseline LT-HSC enhancement, we asked if reduction in BM IGF-1 levels impacted capacity of ALBCreIGF1Exc BM niches to engraft donor HSC after BMT. Using competitive secondary transplantation assays to assess numbers of engrafted WT GFP+ LT-HSC 3 weeks after primary BMT into ALBCreIGF1Exc or control mice, we found that reduced IGF-1 in the ALBCreIGF1Exc BM microenvironment resulted in >2.5 fold enhancement of donor LT-HSC engraftment. In an initial search of downstream mediators of this effect, we discovered that reduction of IGF-1 levels in MX1CreIGF1Exc BM results in a 2.5-fold increase in post-TBI CXCL12 mRNA expression, suggesting that reducing BM IGF-1 may enhance donor LT-HSC engraftment by promoting LT-HSC homing and maintenance within HSC niches. Taken together our data define a critical role for IGF-1/IGF1R interactions in regulating the efficiency of donor HSC engraftment following BMT. Therapeutic strategies to reduce BM IGF-1 may prove to be a valuable approach to improving engraftment following clinical BMT. Disclosures No relevant conflicts of interest to declare.

Pleiotrophin Signaling Is Necessary and Sufficient for Hematopoietic Stem Cell Self-Renewal In Vivo

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 404-404 ◽  
Author(s):  
Heather A Himburg ◽  
Pamela Daher ◽  
J. Lauren Russell ◽  
Phuong Doan ◽  
Mamle Quarmyne ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Signaling Pathways ◽  
Fold Increase ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Human Hscs ◽  
Necessary And Sufficient

Abstract Abstract 404 Several signaling pathways have been elucidated which regulate hematopoietic stem cell self-renewal, including the Notch, Wnt, HOX and BMP signaling pathways. However, several of these pathways (e.g. Notch, Wnt) may not be necessary for maintenance of HSCs in vivo. We recently demonstrated that treatment of murine and human HSCs with the heparin binding growth factor, pleiotrophin (PTN), was sufficient to induce self-renewal of murine and human HSCs in culture (Himburg, Nat Med, 2010). In order to determine if PTN signaling is necessary for HSC self renewal and normal hematopoiesis in vivo, we examined the bone marrow HSC content and hematopoietic profile of mice bearing a constitutive deletion of PTN (PTN−/− mice) as well as mice bearing constitutive deletion of the PTN receptor, receptor protein tyrosine phosphatase β/ζ (RPTPβ/ζ) (courtesy of Dr. Gonzalo Herradon, Spain and Dr. Sheila Harroch, L'Institut Pasteur, Paris, FR). PTN−/− mice demonstrated no significant differences in total bone marrow (BM) cells or BM colony forming cells (CFCs) but had significantly decreased bone marrow CD34(-)c-kit(+)sca-1(+)lin(-) (34-KSL) cells compared to littermate controls which retained PTN (PTN+/+) mice (0.007% vs. 0.02%, p=0.03). Consistent with this phenotype, PTN−/− mice also contained 2–fold decreased CFU-S12 compared to control PTN+/+ mice (p= 0.003). PTN−/− mice also demonstrated an 11-fold reduction in long-term repopulating HSC content compared to PTN+/+ mice as measured via competitive repopulating assay (12 week CRU frequency: 1 in 6 cells vs. 1 in 66 cells). Taken together, these data demonstrate that PTN signaling is necessary for maintenance of the BM HSC pool in vivo. Since PTN is known to antagonize the phosphatase activity of RPTPβ/ζ, we hypothesized that deletion of RPTPβ/ζ would increase BM HSC self-renewal and result in expansion of the BM HSC pool in vivo. Consistent with this hypothesis, RPTPβ/ζ−/− mice displayed a 1.3-fold increase in total BM cells (p= 0.04), 1.8-fold increase in BM 34-KSL cells (p=0.03), 1.6-fold increase in BM CFCs (p= 0.002) and 1.6–fold increase in BM CFU-S (p< 0.0001). RPTPβ/ζ−/− mice also demonstrated 1.4–fold higher long-term repopulating capacity (12 weeks) following competitive repopulating assay compared to RPTPβ/ζ+/+ mice (Donor CD45.1+ cell engraftment: 4.2% vs. 1.5%). Interestingly, RPTPβ/ζ −/− mice had significantly increased PB white blood cell counts, hemoglobin and platelet counts compared to RPTPβ/ζ+/+ mice coupled with splenomegaly. The RPTPβ/ζ−/− mice also had significantly increased BM vascular density (via quantitative mouse endothelial cell antigen staining) compared to RPTPβ/ζ+/+ mice, suggesting that PTN/RPTPβ/ζ signaling may augment the HSC pool size directly and also indirectly via activation of the BM vascular niche. These results demonstrate that PTN signaling is necessary and sufficient for induction of HSC self-renewal in vivo. Disclosures: No relevant conflicts of interest to declare.

scholarly journals IGF-1-mediated osteoblastic niche expansion enhances long-term hematopoietic stem cell engraftment after murine bone marrow transplantation

Stem Cells ◽  
2013 ◽  
Vol 31 (10) ◽  
pp. 2193-2204 ◽  
Author(s):  
Anna Caselli ◽  
Timothy S. Olson ◽  
Satoru Otsuru ◽  
Xiaohua Chen ◽  
Ted J. Hofmann ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Bone Marrow Transplantation ◽  
Hematopoietic Stem Cell ◽  
Marrow Transplantation ◽  
Hematopoietic Stem ◽  
Niche Expansion ◽  
Murine Bone Marrow ◽  
Cell Engraftment

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.

A Functional In Vivo RNAi screen Involving Jumonji C Domain Containing Candidates Unravels Kdm5b As a Negative Modulator of Hematopoietic Stem Cell Self-Renewal

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 385-385
Author(s):  
Sonia Cellot ◽  
Kristin J Hope ◽  
Martin Sauvageau ◽  
Jalila Chagraoui ◽  
Eric Deneault ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Cell Expansion ◽  
Rt Pcr ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Stem Cell Expansion ◽  
Time Point

Abstract Abstract 385 Epigenetic modifications influence chromatin accessibility, impacting on cell fate decisions, such as stem cell self-renewal and differentiation, in both normal and leukemic stem cells (LSC). To investigate the putative role of histone demethylases (HDM) in modulating primary hematopoietic stem cell (HSC) fate, an in vivo functional screen was performed, using an RNAi based strategy, involving 25 members of the Jumonji (JmjC) domain protein family. As a first step, expression profile studies of these gene candidates were undertaken. Transcripts of all these enzymes were detected in isolated HSC populations (frequency 1:2) from fetal liver (n=1) and bone marrow (n=2), except for Hairless. As compared to unsorted bone marrow (BM), stem cells harboured higher expression of Jarid1b (relative-fold enrichment (RQ) of 3.9±1.7), Jmjd2d (RQ3.8±1.9), and Jhdm1b (3.1±1.7). Next, 5shRNA were designed against each of the 25 JmjC containing proteins, and cloned into a retroviral LMP vector encoding GFP to permit tracking of transduced cells in vivo. HSC-enriched CD150+CD48−Lin−cells (∼60 LT-HSC) were infected over 5 days by co-culture with retroviral producer cells in an arrayed 96-well format, with one shRNA per well. Directly after infection, the in vivo reconstituting potential of ¼ of each well was evaluated through duplicate competitive repopulation assays involving the co-transplantation of 1.5 × 105 congenic BM competitor cells into irradiated recipients. The remaining cell fraction served to asses gene transfer by GFP epifluorescence measurements, and RNA isolated from sorted GFP+ cells was used to evaluate gene knockdown levels by Q-RT-PCR analysis. Blood reconstitution was evaluated at an early (4wks) and late time point (16–20wks), tracking the contribution of the donor CD45.1+ transduced (GFP+) cells to recipient hematopoiesis over time. As baseline references, sh-RNA to Luciferase (no effect) and the histone acetyl transferase Myst3 (stem cell loss) were used, as well as Hoxb4 over-expression (stem cell expansion). The primary screen, followed by validation experiments, unravelled one positive (Jhdm1f/Phf8) and two negative (Jarid1b, Hif1an) regulators of HSC activity. The strongest impact was seen with Jarid1b knockdown, and the resulting gain in HSC activity. As a confirmation step, cells were kept in culture for one week, to better contrast an increase in HSC activity, compared to control HSC. After 7 days in vitro, 1/8 equivalents of single well cultures were transplanted into 3 mice, and blood reconstitution levels serially assessed. Cells transduced with sh-RNA against Jarid1b contributed more significantly to host hematopoiesis than sh-RNA Luciferase transduced cells (58±16% vs 26±3% GFP), or Hoxb4 over-expressing cells (37±2% GFP), at comparable gene transfer rates, at the 16 week time point and beyond (3 independent experiments). Long-term HSC frequencies were evaluated from these cultures, and found to be 6–10 fold increased in shJari1d1b-cell cultures. In long-term recipients, differentiation potential of these cells was preserved, as evidenced by CD4+CD8+ thymic cells, B220+ splenic cells and CD11b+ bone marrow cells in the GFP positive contingent. Clonality studies on DNA isolated from these sorted populations confirmed oligoclonality of the stem cell expansion, and HSC pluripotency. There were no cases of leukemic transformation in all of the transplant recipients (n>30). As assessed by Q-RT-PCR, levels of HoxA5, HoxA9, HoxA10 and CxCl5 were increased in day7 sh3Jarib1b-cells (vs ctl), while the levels of the tumor suppressors Cav1, Sash1 and Egr1 were decreased. A more detailed assessment of the HoxA cluster revealed predominant expression of 5' cluster genes in expanding shJarib1b-cells, from HoxA5 to HoxA11, with a concomitant increase in the level of H3K4 tri-methylation, as assessed by ChIP-CHIP. In conclusion, HDM of the JmjC family can modulate HSC activity, both positively and negatively. These data suggest that the H3K4 demethylase Jarid1b (KDM5b) restrains stem cell self-renewal, acting as a co-repressor, possibly via epigenetic regulation of the HoxA gene cluster, among other target genes. This observation could be further exploited as an HSC expansion strategy. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Loss of hematopoietic stem cell self-renewal after bone marrow transplantation

Blood ◽  
1989 ◽  
Vol 74 (2) ◽  
pp. 872-875 ◽  
Author(s):  
P Mauch ◽  
S Hellman
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Bone Marrow Transplantation ◽  
Marrow Transplantation ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Cell Dose ◽  
Dose Dependent Decrease ◽  
Dose Dependent

Abstract The quality of long-term hematopoietic engraftment after bone marrow transplantation (BMT) has not been well characterized. Clinical autologous BMT involves removal of less than 5% of the total content of the recipient marrow followed by ablation of the remaining marrow and reinfusion. To study long-term consequences of transplanting limited numbers of BM stem cells further, we evaluated the hematopoietic reserve in recipient animals after transplantation of varying quantities of BM. Recipient animals demonstrated a donor BM cell dose- dependent decrease in stem cell content and self-renewal capacity that was not reflected in peripheral blood (PB) counts or BM cellularity. This decrease was observed after initial BM recovery and did not change with time after transplantation, demonstrating a permanent loss in BM self-renewal capacity. In addition, animals alive at 3 months, a time selected to allow BM recovery, also demonstrated a donor BM cell dose- dependent decrease in survival at 1 year. These results emphasize the importance of optimizing stem cell number in BMT.

scholarly journals Loss of hematopoietic stem cell self-renewal after bone marrow transplantation

Blood ◽  
1989 ◽  
Vol 74 (2) ◽  
pp. 872-875 ◽  
Author(s):  
P Mauch ◽  
S Hellman
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Bone Marrow Transplantation ◽  
Marrow Transplantation ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Cell Dose ◽  
Dose Dependent Decrease ◽  
Dose Dependent

The quality of long-term hematopoietic engraftment after bone marrow transplantation (BMT) has not been well characterized. Clinical autologous BMT involves removal of less than 5% of the total content of the recipient marrow followed by ablation of the remaining marrow and reinfusion. To study long-term consequences of transplanting limited numbers of BM stem cells further, we evaluated the hematopoietic reserve in recipient animals after transplantation of varying quantities of BM. Recipient animals demonstrated a donor BM cell dose- dependent decrease in stem cell content and self-renewal capacity that was not reflected in peripheral blood (PB) counts or BM cellularity. This decrease was observed after initial BM recovery and did not change with time after transplantation, demonstrating a permanent loss in BM self-renewal capacity. In addition, animals alive at 3 months, a time selected to allow BM recovery, also demonstrated a donor BM cell dose- dependent decrease in survival at 1 year. These results emphasize the importance of optimizing stem cell number in BMT.

SCL regulates the Quiescence and the Long-Term Competence of Hematopoietic Stem Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2520-2520
Author(s):  
Julie Lacombe ◽  
Sabine Herblot ◽  
Shanti Rojas-Sutterlin ◽  
André Haman ◽  
Stephane Barakat ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Gene Dosage ◽  
Critical Role ◽  
Bhlh Transcription Factor ◽  
Hematopoietic Stem

Abstract Abstract 2520 Poster Board II-497 The life-long production of blood cells depends on the regenerative capacity of a rare bone marrow population, the hematopoietic stem cells (HSCs). In the adult, the majority of HSCs are quiescent while a large proportion of progenitors are more cycling. The state of quiescence in HSCs is reversible and these cells can be triggered into cycle by chemotoxic injuries, exposure to cytokines in vitro, as well as transplantation in vivo. SCL/TAL1 is a bHLH transcription factor that has a critical role in generating HSCs during development. However, the role of SCL in adult HSCs is still a matter of debate. In the present study, we took several approaches to address this question. Scl expression was monitored by quantitative PCR analysis in a population that contains adult long-term reconstituting HSCs (LT-HSCs) at a frequency of 20–50%: Kit+Sca+Lin-CD150+CD48-. RT-PCR results were confirmed by β-galactosidase staining of these cells in Scl-LacZ mice. We show that Scl is highly expressed in LT-HSC and that its expression correlates with quiescence, i.e. Scl levels decrease when LT-HSCs exit the G0 state. In order to assess stem cell function, we performed several transplantation assays with adult bone marrow cells in which SCL protein levels were decreased at least two-fold by gene targeting or by RNA interference. 1) The mean stem cell activity of HSCs transplanted at ∼1 CRU was two-fold decreased in Scl heterozygous (Scl+/−) mice. 2) In competitive transplantation, the contribution of Scl+/− cells to primitive populations as well mature cells in the bone marrow was significantly decreased 8 months after transplantation. 3) In secondary transplantation assays, Scl+/− HSCs were severely impaired in their ability to reconstitute secondary recipient in stem cells and progenitor populations and in almost all mature lineages. 4) Reconstitution of the stem cell pool by adult HSCs expressing Scl-directed shRNAs was significantly decreased compared to controls. We therefore conclude that SCL levels regulate HSC long term competence. Since Scl levels decrease when LT-HSCs exit the G0 state, we addressed the question whether the cell cycle state of LT-HSCs is sensitive to Scl gene dosage. We stained bone marrow cell populations with Hoechst and Pyronin Y. At steady state, percentage LT-HSCs in G1 fraction appears to be significantly increased in mice lacking one allele of Scl. Furthermore, a three-fold increase in G1 fraction was also observed when cells were infected with Scl-directed shRNA, suggesting that a decrease in Scl levels facilitates G0-G1 transition. At the molecular level, we show by chromatin immunoprecipitation that SCL occupies the Cdkn1a and Id1 loci. Furthermore, in purified Kit+Sca+Lin-CD150+CD48- cells, the expression levels of these two regulators of HSC cell cycle and long-term functions are sensitive to Scl gene dosage. Together, our observations suggest that SCL impedes G0-G1 transition in HSCs and regulates their long-term competence. Disclosures: No relevant conflicts of interest to declare.

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