scholarly journals Microtubule Polymerization Inhibition Enhances Human Hematopoietic Stem Cell Homing and Engraftment

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3820-3820
Author(s):  
Kuiying Ma ◽  
Riguo Fang ◽  
Lingling Yu ◽  
Chao Li ◽  
Zhongyu Shi ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Small Molecules ◽  
Chemical Compounds ◽  
Post Treatment ◽  
Control Group ◽  
Short Term ◽  
Hematopoietic Stem ◽  
Microtubule Polymerization ◽  
Vinblastine Sulfate

Abstract Hematopoietic stem cells (HSCs) serve as the origin of the hematopoietic system, with the ability to differentiate into all blood cell lineages and self-renewal to sustain the hematopoiesis throughout life. Hematopoietic stem cell transplantation (HSCT) currently represents the most effective therapeutic strategies to treat hematological and non-hematological diseases. However, limited numbers of HSCs or poor homing capabilities into the bone marrow are still major hurdles for successful HSCT. Moreover, graft failure and delayed reconstitution due to inefficient engraftment, remains an important complication because of the high morbidity and mortality. Although ex vivo expansion of HSCs has been well studied for decades, which displays huge potentials for clinical application, exploration of novel targets to improve HSC homing and engraftment will provide new insights to enhance HSCT efficacy. To explore the chemical compounds enhancing the capabilities of homing and engraftment, we used CXCR4 (CD184) as the readout bio-marker, which is considered as the most essential chemokine receptor of HSPCs interacting with CXCL12 (SDF1) secreted in BM niche to support HSPCs homing, migration, proliferation and survival. We first performed chemical screening of 139 small molecules that can increase CD184 expression on cord blood (CB) CD34 + hematopoietic stem and progenitor cells (HSPCs). We concluded that treatment of CB CD34 + HSPCs for 16 hours with Lexibulin (Lex) or Vinblastine Sulfate (VS), both of which were microtubule polymerization (MP) inhibitors, could significantly promote the CD184 expression. Next, we optimized the MP inhibitors treatment conditions including dosage, treatment duration and culture time prior to treatment. The results proved that treatment with Lex or VS for 16 hours at 1μM was the optimal conditions to significantly enhance the CD184 expression of CD34 + HPSCs and LT-HSCs (CD34 +CD90 +CD45RA -), while maintaining robust cell survival, when compared with the DMSO control group. Moreover, we found that only when HSPCs were under culture within two days prior to small molecules treatment, CD184 expression was significantly increased by MP inhibitors while maintaining high viability, compared with DMSO control group. In order to assess the in vivo repopulating potential of the CB CD34 + HSPCs post treatment with MP inhibitors, we transplanted CB-HSPCs 16 hours post-treatment with Lex and VS respectively into irradiated nonobese diabetic (NOD)/Prkdc scid/IL-2Rγ null (NPG) mice. All transplanted mice of MP inhibitors-treated groups presented efficient engraftment, in multiple immune organs at 4-16 weeks post-transplantation, suggesting greater engraftment potential than the mock group, as measured by human CD45 of total CD45. Furthermore, hematopoietic reconstitution analysis indicated that the MP inhibitors -treated cells maintained different lineage distribution in peripheral blood (PB), bone marrow (BM) and spleen. Moreover, the equivalent phenotypes of pre- and post-treatment reveal the better reconstitution by MP inhibitors was independent of HSC-enrichment, Thus, short-term MP inhibitors treatment of CB CD34 + HSPCs enhances their homing and long-term engraftment. In conclusion, we demonstrated that short-term microtubule polymerization inhibition on human CB CD34 + HSPCs could not only enhance CD184 cell surface expression but also the capabilities of in vivo human HSCs homing and reconstitution via screening chemical compounds to increase CD184 expression and the following function evaluation study. Vinblastine Sulfate and Lexbulin were applied or registered as anti-cancer drugs for clinical use. Our study also indicates that MP inhibitors pretreatment of cells possesses significant translational implications, designating MP inhibitors as promising drug candidates to facilitate clinical HSCT. Figure 1 Figure 1. Disclosures Fang: EdiGene, Inc.: Current Employment.

Altered colony-forming activities of bone marrow hematopoietic stem cells in mice following short-term in vivo exposure to parathion

1987 ◽  
Vol 5 (3) ◽  
pp. 231-241 ◽  
Author(s):  
Vincent S. Gallicchio ◽  
Thomas D. Watts ◽  
George P. Casale ◽  
Philip M. Bartholomew
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Short Term ◽  
Hematopoietic Stem

Flt3 Ligand Negatively Regulates In Vitro Migration, Intracellular Signaling Activated by SDF-1α/CXCR4 and In Vivo Homing of Hematopoietic Progenitor Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2674-2674
Author(s):  
Seiji Fukuda ◽  
Hal E. Broxmeyer ◽  
Louis M. Pelus
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Hematopoietic Progenitor Cells ◽  
Leukemia Cells ◽  
Flt3 Ligand ◽  
Hematopoietic Progenitor ◽  
Short Term ◽  
Hematopoietic Stem

Abstract The Flt3 receptor tyrosine kinase (Flt3) is expressed on primitive normal and transformed hematopoietic cells and Flt3 ligand (FL) facilitates hematopoietic stem cell mobilization in vivo. The CXC chemokine SDF-1α(CXCL12) attracts primitive hematopoietic cells to the bone marrow microenvironment while disruption of interaction between SDF-1α and its receptor CXCR4 within bone marrow may facilitate their mobilization to the peripheral circulation. We have previously shown that Flt3 ligand has chemokinetic activity and synergistically increases migration of CD34+ cells and Ba/F3-Flt3 cells to SDF-1α in short-term migration assays; this was associated with synergistic phosphorylation of MAPKp42/p44, CREB and Akt. Consistent with these findings, over-expression of constitutively active ITD (internal tandem duplication) Flt3 found in patients with AML dramatically increased migration to SDF-1α in Ba/F3 cells. Since FL can induce mobilization of hematopoietic stem cells, we examined if FL could antagonize SDF-1α/CXCR4 function and evaluated the effect of FL on in vivo homing of normal hematopoietic progenitor cells. FL synergistically increased migration of human RS4;11 acute leukemia cells, which co-express wild-type Flt3 and CXCR4, to SDF-1α in short term migration assay. Exogenous FL had no effect on SDF-1α induced migration of MV4-11 cells that express ITD-Flt3 and CXCR4 however migration to SDF-1α was partially blocked by treatment with the tyrosine kinase inhibitor AG1296, which inhibits Flt3 kinase activity. These results suggest that FL/Flt3 signaling positively regulates SDF-1α mediated chemotaxis of human acute leukemia cells in short-term assays in vitro, similar to that seen with normal CD34+ cells. In contrast to the enhancing effect of FL on SDF-1α, prolonged incubation of RS4;11 and THP-1 acute myeloid leukemia cells, which also express Flt3 and CXCR4, with FL for 48hr, significantly inhibited migration to SDF-1α, coincident with reduction of cell surface CXCR4. Similarly, prolonged exposure of CD34+ or Ba/F3-Flt3 cells to FL down-regulates CXCR4 expression, inhibits SDF-1α-mediated phosphorylation of MAPKp42/p44, CREB and Akt and impairs migration to SDF-1α. Despite reduction of surface CXCR4, CXCR4 mRNA and intracellular CXCR4 in Ba/F3-Flt3 cells were equivalent in cells incubated with or without FL, determined by RT-PCR and flow cytometry after cell permeabilization, suggesting that the reduction of cell surface CXCR4 expression is due to accelerated internalization of CXCR4. Furthermore, incubation of Ba/F3-Flt3 cells with FL for 48hr or over-expression of ITD-Flt3 in Ba/F3 cells significantly reduced adhesion to VCAM1. Consistent with the negative effect of FL on in vitro migration and adhesion to VCAM1, pretreatment of mouse bone marrow cells with 100ng/ml of FL decreased in vivo homing of CFU-GM to recipient marrow by 36±7% (P<0.01), indicating that FL can negatively regulate in vivo homing of hematopoietic progenitor cells. These findings indicate that short term effect of FL can provide stimulatory signals whereas prolonged exposure has negative effects on SDF-1α/CXCR4-mediated signaling and migration and suggest that the FL/Flt3 axis regulates hematopoietic cell trafficking in vivo. Manipulation of SDF-1α/CXCR4 and FL/Flt3 interaction could be clinically useful for hematopoietic cell transplantation and for treatment of hematopoietic malignancies in which both Flt3 and CXCR4 are expressed.

scholarly journals Hydroxyurea Can Be Used to Increase Mouse c-kit+Thy-1.1loLin−/loSca-1+ Hematopoietic Cell Number and Frequency in Cell Cycle In Vivo

Blood ◽  
1997 ◽  
Vol 90 (11) ◽  
pp. 4354-4362 ◽  
Author(s):  
Nobuko Uchida ◽  
Annabelle M. Friera ◽  
Dongping He ◽  
Michael J. Reitsma ◽  
Ann S. Tsukamoto ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Bone Marrow ◽  
Cell Number ◽  
Short Term ◽  
Synthesis Inhibitor ◽  
Hematopoietic Stem ◽  
Cell Cycle Status

Abstract The DNA synthesis inhibitor hydroxyurea (HU) was administered to determine whether it induces changes in the cell-cycle status of primitive hematopoietic stem cells (HSCs)/progenitors. Administration of HU to mice leads to bone marrow accumulation of c-kit+Thy-1.1loLin−/loSca-1+ (KTLS) cells in S/G2/M phases of the cell cycle. HU is a relatively nontoxic, reversible cell-cycle agent that can lead to approximately a threefold expansion of KTLS cells in vivo and approximately an eightfold increase in the number of KTLS cells in S/G2/M. HSCs in HU-treated mice have undiminished multilineage long-term and short-term clonal reconstitution activity.

scholarly journals In vivo prostaglandin E2 treatment alters the bone marrow microenvironment and preferentially expands short-term hematopoietic stem cells

Blood ◽  
2009 ◽  
Vol 114 (19) ◽  
pp. 4054-4063 ◽  
Author(s):  
Benjamin J. Frisch ◽  
Rebecca L. Porter ◽  
Benjamin J. Gigliotti ◽  
Adam J. Olm-Shipman ◽  
Jonathan M. Weber ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Prostaglandin E2 ◽  
Bone Marrow Cells ◽  
Negative Impact ◽  
Hematopoietic Progenitors ◽  
Short Term ◽  
Hematopoietic Stem ◽  
Marrow Cells

Abstract Microenvironmental signals can determine hematopoietic stem cell (HSC) fate choices both directly and through stimulation of niche cells. In the bone marrow, prostaglandin E2 (PGE2) is known to affect both osteoblasts and osteoclasts, whereas in vitro it expands HSCs and affects differentiation of hematopoietic progenitors. We hypothesized that in vivo PGE2 treatment could expand HSCs through effects on both HSCs and their microenvironment. PGE2-treated mice had significantly decreased number of bone trabeculae, suggesting disruption of their microarchitecture. In addition, in vivo PGE2 increased lineage− Sca-1+ c-kit+ bone marrow cells without inhibiting their differentiation. However, detailed immunophenotyping demonstrated a PGE2-dependent increase in short-term HSCs/multipotent progenitors (ST-HSCs/MPPs) only. Bone marrow cells transplanted from PGE2 versus vehicle-treated donors had superior lymphomyeloid reconstitution, which ceased by 16 weeks, also suggesting that ST-HSCs were preferentially expanded. This was confirmed by serial transplantation studies. Thus in vivo PGE2 treatment, probably through a combination of direct and microenvironmental actions, preferentially expands ST-HSCs in the absence of marrow injury, with no negative impact on hematopoietic progenitors or long-term HSCs. These novel effects of PGE2 could be exploited clinically to increase donor ST-HSCs, which are highly proliferative and could accelerate hematopoietic recovery after stem cell transplantation.

CD26 Inhibitor Treated and CD26−/− Recipient Mice Exhibit Improved Transplant Efficiency as Quantitated by Increased Short-Term Homing and Long-Term Engraftment

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 361-361 ◽  
Author(s):  
Laura A. Paganessi ◽  
Stephanie A. Gregory ◽  
Henry C. Fung ◽  
Kent W. Christopherson
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Mononuclear Cells ◽  
Short Term ◽  
Hematopoietic Stem ◽  
Post Transplant ◽  
Inhibitor Treatment

Abstract A firm understanding of the biology of hematopoietic stem and progenitor cell (HSC/ HPC) trafficking is believed to be critical for the development of methodologies to improve transplant efficiency and subsequently immune reconstitution during hematopoietic stem cell transplantation in the clinical setting. Through the use of CD26 inhibitors and CD26 deficient mice (CD26−/−), we have previously generated data in mice suggesting that suppression of CD26/DPPIV (dipeptidylpeptidase IV) enzymatic activity on the transplant donor cell population can be utilized as a method of increasing transplant efficiency (Christopherson, KW 2nd, et al, Science 2004. 305:1000–3). However, the clinical importance of the transplant recipient should not to be overlooked given the potential importance of the bone marrow microenvironment in regulating the transplant process. We therefore investigated here whether inhibition or loss of CD26 activity in recipient mice would have an effect on transplant efficiency utilizing an in vivo congenic mouse model of transplantation. The short-term homing and long-term engraftment of BoyJ donor cells (expressing CD45.1+) into lethally irradiated control C57BL/6, CD26 inhibitor (Diprotin A) treated C57BL/6, or CD26−/− mice (expressing CD45.2+) was monitored by flow cytometric analysis of the bone marrow and peripheral blood at 24 hours and 6 months post-transplant respectively. Twenty-four hours post-transplant of 20×106 BoyJ mononuclear cells, we observed 8.85±0.58%, 10.69±1.01%, and 12.45±1.33% donor derived Sca-1+lin− cells in the bone marrow of recipient mice for control, Diprotin A treated, and CD26−/− recipient mice respectively. As compared to control mice, this represents a 20.8% increase (p=0.01) with CD26 inhibitor treatment and a 40.7% increase (p£0.05) resulting from the use of a CD26−/− recipient in short-term homing (N=5 mice per group). Six months post-transplant of 1×105 BoyJ mononuclear cells, we observed 39.90± 4.38%, 70.22± 3.72%, and 92.51± 1.04% donor contribution to hematopoiesis in the peripheral blood of control, Diprotin A treated, and CD26−/− recipient mice respectively. This represents a 76.0% increase (p£0.01) with CD26 inhibitor treatment and a 131.9% increase (p£0.01) as a result of the CD26−/− recipient in long-term engraftment as compared to control recipient mice (N=14 mice per group). These results provide pre-clinical evidence of the importance of CD26 expression within the transplant recipient with regard to regulating hematopoietic stem cell homing and engraftment. Our results also support the potential use of CD26 inhibitors to treat transplant patients during hematopoietic stem cell transplantation as a method of improving transplant efficiency. Lastly, our use of inhibitor treated C57BL/6 and CD26−/− recipient mice, which are also on a C57BL/6 background, in conjunction with a congenic model of transplantation provides a accurate and convenient model system for the in vivo testing of the efficacy of existing and new CD26 inhibitors in transplant recipients.

scholarly journals Impaired bone marrow homing of cytokine-activated CD34+ cells in the NOD/SCID model

Blood ◽  
2004 ◽  
Vol 103 (6) ◽  
pp. 2079-2087 ◽  
Author(s):  
Forhad Ahmed ◽  
Stuart J. Ings ◽  
Arnold R. Pizzey ◽  
Michael P. Blundell ◽  
Adrian J. Thrasher ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cultured Cells ◽  
Culture Conditions ◽  
Short Term ◽  
Homing Behavior ◽  
Hematopoietic Stem ◽  
Cycle Arrest ◽  
Nonobese Diabetic ◽  
Cd34 Cells

Abstract The reduced engraftment potential of hematopoietic stem/progenitor cells (HSPCs) after exposure to cytokines may be related to the impaired homing ability of actively cycling cells. We tested this hypothesis by quantifying the short-term homing of human adult CD34+ cells in nonobese diabetic/severe combined immunodeficient (NOD/SCID) animals. We show that the loss of engraftment ability of cytokine-activated CD34+ cells is associated with a reduction in homing of colony-forming cells (CFCs) to bone marrow (BM) at 24 hours after transplantation (from median 2.8% [range, 1.9%-6.1%] to 0.3% [0.0%-0.7%]; n = 3; P < .01), coincident with an increase in CFC accumulation in the lungs (P < .01). Impaired BM homing of cytokine-activated cells was not restored by using sorted cells in G0G1 or by inducing cell cycle arrest at the G1/S border. Blocking Fas ligation in vivo did not increase the BM homing of cultured cells. Finally, we tested cytokine combinations or culture conditions previously reported to restore the engraftment of cultured cells but did not find that any of these was able to reverse the changes in homing behavior of cytokine-exposed cells. We suggest that these changes in homing and, as a consequence, engraftment result from the increased migratory capacity of infused activated cells, leading to the loss of selectivity of the homing process.

scholarly journals Cohesin Genes Are Negative Regulators of HSC Renewal

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 605-605
Author(s):  
Roman Galeev ◽  
Aurelie Baudet ◽  
Anders Kvist ◽  
Therese Törngren ◽  
Shamit Soneji ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Control Group ◽  
Hematopoietic Stem ◽  
Myeloid Malignancies ◽  
Genome Wide ◽  
Cd34 Cells ◽  
Recurrent Mutations ◽  
Major Player

Abstract The molecular principles regulating hematopoietic stem cells (HSCs) remain incompletely defined. To gain deeper insights into the mechanisms underlying renewal and differentiation of hematopoietic stem and progenitor cells (HSPCs), we have developed global RNAi screens targeted to human cord blood derived CD34+ cells. In previous work such screens have allowed us to identify novel druggable targets to facilitate ex vivo expansion of HSPCs. Recently, we employed a near genome-wide screen (targeting 15 000 genes) to identify genes with an impact on renewal/differentiation of HSPCs, in a completely unbiased manner. Among the most prominent hits from this screen were many transcription factors and epigenetic modifiers and we found a strong enrichment of genes known to be recurrently mutated in hematopoietic neoplasms. A striking finding, was the identification of several members of the cohesin complex (STAG2, RAD21, STAG1 and SMC3) among our top hits (top 0.5%). Cohesin is a multimeric protein complex that mediates adhesion of sister chromatids as well as long-range interactions of chromosomal elements to regulate transcription. Recent large-scale sequencing studies have identified recurrent mutations in the cohesin genes in myeloid malignancies. Upon individual validation and targeting of the cohesin genes by lentiviral shRNA in human CD34+ cells, we found that their knockdown by independent shRNAs led to an immediate and profound expansion of primitive hematopoietic CD34+CD90+ cells in vitro. A similar expansion phenotype was observed in vivo following transplantation to primary and secondary immundeficient mice. Transplantation of CD34+CD38lowCD90+CD45RA- cells transduced with shRNA targeting STAG2 (the cohesin component with the strongest in vitro phenotype) into NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice resulted in a significant increase in human reconstitution in the bone marrow 16 weeks post-transplantation compared to controls (31.3±4.4% vs 11.6±2.8% p=0.001). The engrafted mice showed a marked skewing towards the myeloid lineage as analyzed by CD33/CD15 expression in bone marrow (27.0±5.0% vs 13.0±2.6% p=0.013), as well as an increase in the more primitive CD34+CD38- population (2.8±0.6% vs 1.3±0.4% p=0.036). In secondary transplanted mice, 3/6 recipients in the STAG2 group maintained detectable levels of human chimerism while no engraftment was detected in the control group, indicating an increased expansion of HSPCs in vivo upon knockdown of STAG2. Global transcriptome analysis of cohesin deficient CD34+ cells 36 hours post shRNA transduction showed a distinct up-regulation of HSC specific genes coupled with down-regulation of genes specific for more downstream progenitors, demonstrating an immediate shift towards a more stem-like gene expression signature upon cohesin deficiency. This observation was consistent for all cohesin genes tested (STAG2, RAD21, STAG1 and SMC3). Our findings implicate cohesin as a novel major player in regulation of human HSPCs and, together with the recent discovery of recurrent mutations in myeloid malignancies, point toward a direct role of perturbed cohesin function as a true driver event in myeloid leukemogenesis. Our findings illustrate how global RNAi screens targeted to primary human HSPCs can identify novel modifiers of cell fate and may complement genome-wide sequencing approaches to guide the identification of functionally relevant disease-related genes in hematopoietic malignancies. Disclosures No relevant conflicts of interest to declare.

scholarly journals Hydroxyurea Can Be Used to Increase Mouse c-kit+Thy-1.1loLin−/loSca-1+ Hematopoietic Cell Number and Frequency in Cell Cycle In Vivo

Blood ◽  
1997 ◽  
Vol 90 (11) ◽  
pp. 4354-4362 ◽  
Author(s):  
Nobuko Uchida ◽  
Annabelle M. Friera ◽  
Dongping He ◽  
Michael J. Reitsma ◽  
Ann S. Tsukamoto ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Bone Marrow ◽  
Cell Number ◽  
Short Term ◽  
Synthesis Inhibitor ◽  
Hematopoietic Stem ◽  
Cell Cycle Status

The DNA synthesis inhibitor hydroxyurea (HU) was administered to determine whether it induces changes in the cell-cycle status of primitive hematopoietic stem cells (HSCs)/progenitors. Administration of HU to mice leads to bone marrow accumulation of c-kit+Thy-1.1loLin−/loSca-1+ (KTLS) cells in S/G2/M phases of the cell cycle. HU is a relatively nontoxic, reversible cell-cycle agent that can lead to approximately a threefold expansion of KTLS cells in vivo and approximately an eightfold increase in the number of KTLS cells in S/G2/M. HSCs in HU-treated mice have undiminished multilineage long-term and short-term clonal reconstitution activity.

scholarly journals No Evidence for Hematopoietic Stem Cell Self-Renewal in-Vivo Following Inflammatory Challenge

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 456-456
Author(s):  
Ruzhica Bogeska ◽  
Paul Kaschutnig ◽  
Stella V Paffenholz ◽  
Julia Knoch ◽  
Jan-Philipp Mallm ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Mouse Model ◽  
Hematopoietic Stem Cell ◽  
Normal Aging ◽  
Post Treatment ◽  
Quiescent State ◽  
Self Renewal ◽  
Hematopoietic Stem

Elevated levels of inflammation have been previously linked to both inherited and acquired bone marrow failure (BMF) syndromes, as well as to normal aging, suggesting a role in the etiology of these conditions. One potential explanation for this phenomenon is that repeated inflammation can promote the suppression of hematopoietic stem cell (HSC) function.We have previously demonstrated that interferon-α can accelerate HSC attrition by driving HSCs out of quiescence, leading to the development of BMF in a mouse model of Fanconi anemia (Walter et al. Nature, 2015). To more broadly address the impact of repetitive inflammatory challenge on HSC regeneration, we challenged C57BL6 wild type (WT) mice with polyinosinic:polycytidylic acid (pI:C), a TLR3 agonist that mimics viral infection. Injection with 1-3 rounds of pI:C (8 injections per round) in WT mice had no sustained impact on hematopoiesis, since peripheral blood (PB) and bone marrow (BM) counts were within normal ranges at 5 weeks (5wk) post-treatment. However, in vitro analysis of the clonal proliferation potential of 411 individual sorted long-term (LT)-HSCs revealed a 2-fold reduction (p<0.0001) in the total number of progeny produced per HSC. Additionally, cell fate tracking experiments showed accelerated entry into first division and differentiation following treatment. In line with this data, competitive repopulation assays demonstrated a progressive depletion of functional HSC numbers, with an approximate 2-fold decrease in multi-lineage competitive repopulating activity with each additional round of inflammatory challenge (p<0.01). In order to assess in vivo recovery of HSCs following inflammatory challenge, competitive and limiting dilution transplantation assays were used to quantify HSC frequencies using BM harvested from mice at 5, 10 or 20wk after 3 rounds of pI:C treatment. In both assays we observed a sustained ~18 fold decrease in functional HSCs, with no evidence of recovery within the 20wk window. To exclude microenvironment effects on HSC function, we performed reverse transplantation experiments in which pI.C challenged WT mice were injected with saturating doses of LT-HSCs from non-treated WT donors, in the absence of additional irradiation conditioning. We observed a durable suppression of endogenous HSCs that was sufficient to facilitate robust engraftment of donor LT-HSCs up to 20wk post-treatment. We next used the inducible transgenic Scl-tTA;H2B-GFP mouse model (Wilson et al., Cell, 2008) in order to prospectively segregate quiescent label retaining LT-HSCs (LRCs) from LT-HSCs that proliferate in vivo in response to pI:C (nonLRCs). Following a single round of pI:C challenge, label retention was reduced as a result of LT-HSC proliferation (Table 1). Importantly, the clonal proliferative potential of individual LRCs was preserved upon pI:C challenge while that of nonLRCs was more than halved. This suggests that LT-HSCs fail to undergo self-renewal divisions in vivo under these conditions but rather are functionally compromised in line with increasing proliferative history. We hypothesized that this apparent progressive irreversible depletion of functional HSCs may eventually lead to compromised hematopoiesis. We therefore assessed the hematologic parameters of aged mice that had been exposed to repetitive pI:C treatment in early to mid-life. While these mice had normal PB counts at 5wk post-treatment, upon reaching 2 years of age, treated mice demonstrated mild PB cytopenias, BM hypocellularity and a relative expansion of BM adipocytes (Table 2). Taken together, our data contradict the canonical view that HSCs demonstrate extensive self-regenerative capacity following injury. Rather, in the context of inflammatory challenge, HSCs are progressively and irreversibly depleted as they are driven out of their quiescent state. These findings have broad implications regarding the role of inflammation in the suppression of hematopoiesis that are likely relevant to BMF and also normal aging. Disclosures Lipka: InfectoPharm GmbH: Employment. Frenette:Pfizer: Consultancy; Cygnal Therapeutics: Equity Ownership; Ironwood Pharmaceuticals: Research Funding; Albert Einstein College of Medicine, Inc: Patents & Royalties.

In Vivo Treatment with Prostaglandin E2 (PGE2) Selectively Expands Short-Term Hematopoietic Stem Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1254-1254
Author(s):  
Benjamin J. Frisch ◽  
Jonathan M. Weber ◽  
Rebecca L. Porter ◽  
Benjamin J. Gigliotti ◽  
Julianne N. Smith ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Prostaglandin E2 ◽  
Mononuclear Cells ◽  
Surprising Result ◽  
Short Term ◽  
Hematopoietic Stem ◽  
Donor Cells

Abstract Parathyroid Hormone (PTH) expands hematopoietic stem cells (HSC) through activated osteoblasts in the bone marrow (BM). Since PTH stimulates osteoblastic production of Prostaglandin E2 (PGE2), we hypothesized that PGE2 could also regulate HSC. In vivo PGE2 treatment demonstrated a time and dose dependent increase in BM lineage− Sca-1+ c-kit+ (LSK) BM mononuclear cells (BMMC) from PGE2 vs. vehicle treated mice (0.11 vs. 0.04% BMMC, P=0.0061, n=8 mice per treatment group), an effect superior to PTH (350 vs. 100% increase in LSK). There were no significant PGE2 effects on CFU-Cs or peripheral Hct, Plts or WBC counts compared to vehicle. Therefore PGE2-dependent cell expansion was not global across differentiated subsets, but was restricted to primitive hematopoietic cells, similar to the effects of PTH treatment. Consistent with a PGE2-dependent HSC increase, cells from PGE2 vs vehicle-treated mice had superior lymphomyeloid reconstitution by competitive repopulation analysis. However, this increase was short-lived: specifically, PGE2-dependent myeloid (CD11b+) reconstitution was no longer superior at 6 weeks, while the PGE2-dependent increase in lymphoid (CD3e+ and B220+) reconstitution ceased by 16 weeks. This surprising result suggests that in vivo PGE2 treatment selectively expands short-term HSC (or ST-HSC), which have highly proliferative properties, but limited self-renewal. To further confirm this targeted PGE2 effect, LSK subset analysis based on Flt3 and Thy1.1 expression was performed. Consistent with the competitive repopulation data, PGE2 treatment significantly increased Flt3+Thy1.1int LSK ST-HSC (0.0273 vs 0.0140% n=4 in each group, p=0.0307) as well as Flt3+Thy1.1− LSK Multipotent Progenitors (0.0305 vs 0.0195% n=4 in each group, p=0.0070), while Flt3−Thy1.1int LSK Long-Term HSC or LT-HSC (0.0126 vs 0.0078% n=4 in each group, p=0.1069) were unchanged compared to vehicle treatment. ST vs LT-HSC activity can also be quantified by the in vivo clonogenic Colony Forming Unit-Spleen (CFU-S) assay, where day 8 CFU-S represent ST-HSC, while day 10–12 CFU-S represent LT-HSC. Consistent with a PGE2-dependent specific ST-HSC increase, BMMC from PGE2 treated mice gave rise to a significantly higher number of CFU-Sd8 compared to cells from vehicle treated mice (10.5 vs 4.75 CFU-S per 60,000 BMMC, n=4 in each group, p=0.0053), while CFU-Sd10 were unchanged (12.5 vs 11.5 CFU-S per 60,000 BMMC, n=6, p=0.4950). Finally, since ST-HSC confer radioprotection, PGE2-dependent ST-HSC expansion would be expected to improve survival of lethally irradiated recipients receiving limiting numbers of BMMC from PGE2 vs vehicle-treated mice. As predicted, recipients of BMMC from PGE2 treated mice had increased survival 30 days after transplantation compared to animals receiving BMMC from vehicle treated donors (150,000 donor cells: 80% vs 0% survival, p=0.0018; 75,000 donor cells: 53% vs 0% survival, p=0.0173). Taken together, these data demonstrate specific PGE2-dependent regulation of ST-HSC, and provide a unique and novel model to define control of HSC subsets. This finding implicates for the first time specialized regulation of HSC subsets. Moreover, these data indicate that selective therapeutic manipulation of ST-HSC could be exploited in clinical situations requiring rapid bone marrow reconstitution, such as in recovery from iatrogenic or pathologic myeloablative injury.

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