The TRACE-Seq method tracks recombination alleles and identifies clonal reconstitution dynamics of gene targeted human hematopoietic stem cells

AbstractTargeted DNA correction of disease-causing mutations in hematopoietic stem and progenitor cells (HSPCs) may enable the treatment of genetic diseases of the blood and immune system. It is now possible to correct mutations at high frequencies in HSPCs by combining CRISPR/Cas9 with homologous DNA donors. Because of the precision of gene correction, these approaches preclude clonal tracking of gene-targeted HSPCs. Here, we describe Tracking Recombination Alleles in Clonal Engraftment using sequencing (TRACE-Seq), a methodology that utilizes barcoded AAV6 donor template libraries, carrying in-frame silent mutations or semi-randomized nucleotides outside the coding region, to track the in vivo lineage contribution of gene-targeted HSPC clones. By targeting the HBB gene with an AAV6 donor template library consisting of ~20,000 possible unique exon 1 in-frame silent mutations, we track the hematopoietic reconstitution of HBB targeted myeloid-skewed, lymphoid-skewed, and balanced multi-lineage repopulating human HSPC clones in mice. We anticipate this methodology could potentially be used for HSPC clonal tracking of Cas9 RNP and AAV6-mediated gene targeting outcomes in translational and basic research settings.

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TRACE-Seq Reveals Clonal Reconstitution Dynamics of Gene Targeted Human Hematopoietic Stem Cells

10.1101/2020.05.25.115329 ◽

2020 ◽

Author(s):

Rajiv Sharma ◽

Daniel P Dever ◽

Ciaran M Lee ◽

Armon Azizi ◽

Yidan Pan ◽

...

Keyword(s):

Viral Vector ◽

Genetic Diseases ◽

Basic Research ◽

Gene Correction ◽

Coding Region ◽

Hematopoietic Stem ◽

Immunodeficient Mice ◽

Nucleotide Resolution ◽

Donor Template

AbstractTargeted DNA correction of disease-causing mutations in hematopoietic stem and progenitor cells (HSPCs) may usher in a new class of medicines to treat genetic diseases of the blood and immune system. With state-of-the-art methodologies, it is now possible to correct disease-causing mutations at high frequencies in HSPCs by combining ribonucleoprotein (RNP) delivery of Cas9 and chemically modified sgRNAs with homologous DNA donors via recombinant adeno-associated viral vector serotype six (AAV6). However, because of the precise nucleotide-resolution nature of gene correction, these current approaches do not allow for clonal tracking of gene targeted HSPCs. Here, we describe Tracking Recombination Alleles in Clonal Engraftment using sequencing (TRACE-Seq), a novel methodology that utilizes barcoded AAV6 donor template libraries, carrying either in-frame silent mutations or semi-randomized nucleotide sequences outside the coding region, to track the in vivo lineage contribution of gene targeted HSPC clones. By targeting the HBB gene with an AAV6 donor template library consisting of ∼20,000 possible unique exon 1 in-frame silent mutations, we track the hematopoietic reconstitution of HBB targeted myeloid-skewed, lymphoid-skewed, and balanced multi-lineage repopulating human HSPC clones in immunodeficient mice. We anticipate that this methodology has the potential to be used for HSPC clonal tracking of Cas9 RNP and AAV6-mediated gene targeting outcomes in translational and basic research settings.

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Organ-Selective Homing Defines Engraftment Kinetics of Murine Hematopoietic Stem Cells and Is Compromised by Ex Vivo Expansion

Blood ◽

10.1182/blood.v93.5.1557 ◽

1999 ◽

Vol 93 (5) ◽

pp. 1557-1566 ◽

Cited By ~ 113

Author(s):

Stephen J. Szilvassy ◽

Michael J. Bass ◽

Gary Van Zant ◽

Barry Grimes

Keyword(s):

Ex Vivo ◽

Ex Vivo Expansion ◽

Hematopoietic Stem ◽

Murine Bone Marrow ◽

Hematopoietic Reconstitution ◽

Dramatic Decline ◽

Stem And Progenitor Cells ◽

Clonogenic Cells

Abstract Hematopoietic reconstitution of ablated recipients requires that intravenously (IV) transplanted stem and progenitor cells “home” to organs that support their proliferation and differentiation. To examine the possible relationship between homing properties and subsequent engraftment potential, murine bone marrow (BM) cells were labeled with fluorescent PKH26 dye and injected into lethally irradiated hosts. PKH26+ cells homing to marrow or spleen were then isolated by fluorescence-activated cell sorting and assayed for in vitro colony-forming cells (CFCs). Progenitors accumulated rapidly in the spleen, but declined to only 6% of input numbers after 24 hours. Although egress from this organ was accompanied by a simultaneous accumulation of CFCs in the BM (plateauing at 6% to 8% of input after 3 hours), spleen cells remained enriched in donor CFCs compared with marrow during this time. To determine whether this differential homing of clonogenic cells to the marrow and spleen influenced their contribution to short-term or long-term hematopoiesis in vivo, PKH26+ cells were sorted from each organ 3 hours after transplantation and injected into lethally irradiated Ly-5 congenic mice. Cells that had homed initially to the spleen regenerated circulating leukocytes (20% of normal counts) approximately 2 weeks faster than cells that had homed to the marrow, or PKH26-labeled cells that had not been selected by a prior homing step. Both primary (17 weeks) and secondary (10 weeks) recipients of “spleen-homed” cells also contained approximately 50% higher numbers of CFCs per femur than recipients of “BM-homed” cells. To examine whether progenitor homing was altered upon ex vivo expansion, highly enriched Sca-1+c-kit+Lin−cells were cultured for 9 days in serum-free medium containing interleukin (IL)-6, IL-11, granulocyte colony-stimulating factor, stem cell factor, flk-2/flt3 ligand, and thrombopoietin. Expanded cells were then stained with PKH26 and assayed as above. Strikingly, CFCs generated in vitro exhibited a 10-fold reduction in homing capacity compared with fresh progenitors. These studies demonstrate that clonogenic cells with differential homing properties contribute variably to early and late hematopoiesis in vivo. The dramatic decline in the homing capacity of progenitors generated in vitro underscores critical qualitative changes that may compromise their biologic function and potential clinical utility, despite their efficient numerical expansion.

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Xenotransplantation of Primary CD34+CD38- Hematopoietic Stem Cells Results in an In Vivo Model of Idiopathic Myelofibrosis.

Blood ◽

10.1182/blood.v108.11.666.666 ◽

2006 ◽

Vol 108 (11) ◽

pp. 666-666

Author(s):

Noriyuki Saito ◽

Fumihiko Ishikawa ◽

Kazuya Shimoda ◽

Shuro Yoshida ◽

Yoriko Saito ◽

...

Keyword(s):

Stem Cells ◽

In Vivo Model ◽

Idiopathic Myelofibrosis ◽

Hematopoietic Stem ◽

Hematopoietic Reconstitution ◽

Cd34 Cells ◽

Normal Human ◽

Myelomonocytic Cells ◽

Xenotransplantation Model

Abstract Idiopathic myelofibrosis (IMF) is characterized by clonal proliferation of abnormal myelomonocytic cells and megakaryocytes. These abnormal cells secrete various cytokines resulting in reactive fibrosis and increased collagen content in the bone marrow (BM), and lead to extramedullary hematopoiesis and the appearance of CD34+ cells in the peripheral blood (PB). Although IMF is thought to originate at the level of hematopoietic stem cell (HSC), this has not been demonstrated directly in primary human IMF. To demonstrate the involvement of HSCs in the pathogenesis of IMF and to establish an in vivo model of IMF, we used the newborn NOD/SCID/IL2rg-null xenotransplantation model. We purified PB CD34+ cells from six IMF patients, transplanted 1–10 x10e4 cells intravenously into newborn NOD/SCID/IL2rg-null recipients and analyzed PB and BM human CD45+ hematopoietic cell chimerism, degree of suppression of murine hematopoiesis, presence of hallmark BM fibrosis and plasma TGF-b1 levels in the recipients at 6 months post-transplantation. Primary IMF PB CD34+ cells from five out of six patients engrafted in twelve out of twelve recipients. BM of all engrafted recipients demonstrated fibrotic changes associated with increased proliferation of murine fibroblasts, the presence of human megakaryocytes and elevated plasma TGF-b1 levels, recapitulating the clinical features of IMF. Three distinct patterns of human hematopoietic reconstitution were observed among the engrafted recipients: Predominantly malignant myelomonocytic engraftment in the PB and BM (n=4), Reconstitution of both normal human hematopoiesis (with mature B and T cells, myeloid cells and platelets) and malignant myelomonocytic cells (n=6) and Development of acute leukemia (n=2). Fibrotic change was seen even in the BM of recipients that showed normal human hematopoietic reconstitution, showing that in IMF, there is co-existence of both normal and malignant hematopoietic stem/progenitor cells in the PB CD34+ fraction. Furthermore, when 5–10 x 10e3 sorted PB CD34+CD38– cells from three patients were transplanted into six newborn NOD/SCID/IL2rg-null recipients, reconstitution with human myelomonocytic cells associated with BM fibrosis was demonstrated in all recipients, with compatible level of PB and BM chimerism with those transplanted with PB CD34+ cells. These findings demonstrate that the IMF-initiating cells are contained within the HSC fraction. The newborn NOD/SCID/IL2rg-null xenotransplantation model provides an in vivo model of primary human IMF that may lead to better understanding of the mechanisms of IMF pathogenesis including the identification of IMF stem cells and may be useful for development of novel therapeutic agents for IMF.

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Systemic Administration of Pleiotrophin Induces Hematopoietic Stem Cell Regeneration In Vivo.

Blood ◽

10.1182/blood.v114.22.1498.1498 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1498-1498

Author(s):

Heather A Himburg ◽

Pamela Daher ◽

Sarah Kristen Meadows ◽

J. Lauren Russell ◽

Phuong Doan ◽

...

Keyword(s):

Stem Cell ◽

Growth Factor ◽

Progenitor Cells ◽

Progenitor Cell ◽

Fold Increase ◽

Cell Regeneration ◽

Hematopoietic Stem ◽

Hematopoietic Reconstitution

Abstract Abstract 1498 Poster Board I-521 Significant progress has been made toward delineating the intrinsic and extrinsic signaling pathways that regulate hematopoietic stem cell (HSC) self-renewal. However, much less is known regarding the process of HSC regeneration or the extrinsic signals that regulate hematopoietic reconstitution following stress or injury. Elucidation of the microenvironmental signals which promote HSC regeneration in vivo would have important implications for the treatment of patients undergoing radiation therapy, chemotherapy and stem cell transplantation. We recently reported that pleiotrophin, a soluble heparin-binding growth factor, induced a 10-fold expansion of murine long-term repopulating HSCs in short term culture (Himburg et al. Blood (ASH Annual Meeting Abstracts), Nov 2008; 112: 78). Based on this observation, we hypothesized that PTN might also be a regenerative growth factor for HSCs. Here we tested the effect of systemic administration of PTN to non-irradiated and irradiated C57Bl6 mice to determine if PTN could promote HSC regeneration in vivo. C57Bl6 mice were irradiated with 700 cGy total body irradiation (TBI) followed by intraperitoneal administration of 2 μg PTN or saline x 7 days, followed by analysis of BM stem and progenitor cell content. Saline-treated mice demonstrated significant reductions in total BM cells, BM c-kit+sca-1+lin- (KSL) cells, colony forming cells (CFCs) and long term culture-initiating cells (LTC-ICs) compared to non-irradiated control mice. In contrast, PTN-treated mice demonstrated a 2.3-fold increase in total BM cells (p=0.03), a 5.6-fold increase in BM KSL stem/progenitor cells (p=0.04), a 2.9-fold increase in BM CFCs (p=0.004) and an 11-fold increase in LTC-ICs (p=0.03) compared to saline-treated mice. Moreover, competitive repopulating transplantation assays demonstrated that BM from PTN-treated, irradiated mice contained 5-fold increased competitive repopulating units (CRUs) compared to saline-treated, irradiated mice (p=0.04). Taken together, these data demonstrate that the administration of PTN induces BM HSC and progenitor cell regeneration in vivo following injury. Comparable increases in total BM cells, BM KSL cells and BM CFCs were also observed in PTN-treated mice compared to saline-treated controls following 300 cGy TBI, demonstrating that PTN is a potent growth factor for hematopoietic stem/progenitor cells in vivo at less than ablative doses of TBI. In order to determine whether PTN acted directly on BM HSCs to induce their proliferation and expansion in vivo, we exposed mice to BrDU in their drinking water x 7 days and compared the response to saline treatment versus PTN treatment. PTN-treated mice demonstrated a significant increase in BrDU+ BM KSL cells compared to saline-treated controls (p=0.04) and cell cycle analysis confirmed a significant increase in BM KSL cells in S phase in the PTN-treatment group compared to saline-treated controls (p=0.04). These data indicate that PTN serves as a soluble growth factor for BM HSCs and induces their proliferation and expansion in vivo while preserving their repopulating capacity. These results suggest that PTN has therapeutic potential as a novel growth factor to accelerate hematopoietic reconstitution in patients undergoing myelosuppressive radiotherapy or chemotherapy. Disclosures: No relevant conflicts of interest to declare.

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Zinc Finger Nucleases Targeting The β-Globin Locus Drive Efficient Correction Of The Sickle Mutation In CD34+ Cells

Blood ◽

10.1182/blood.v122.21.2904.2904 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2904-2904 ◽

Cited By ~ 1

Author(s):

Megan D Hoban ◽

Alok V Joglekar ◽

David Gray ◽

Michael L Kaufman ◽

Fabrizia Urbinati ◽

...

Keyword(s):

Gene Therapy ◽

Stem Cell ◽

Zinc Finger Nucleases ◽

Gene Correction ◽

Hematopoietic Stem ◽

Site Specific ◽

Allogeneic Hsct ◽

Cd34 Cells ◽

Cell Source ◽

Donor Template

Abstract Despite major improvements in clinical care and advances in understanding of its complex pathophysiology, sickle cell disease (SCD) continues to be a significant cause of morbidity and early mortality. Allogeneic hematopoietic stem cell transplant (HSCT) can benefit patients with SCD, by providing a source for life-long production of normal red blood cells. However, allogeneic HSCT is limited by the availability of well-matched donors and the immunological complications of graft rejection and graft-versus-host disease that can occur, especially for the more than 80% of patients who lack an HLA-identical sibling donor. Gene therapy could provide a way to cure SCD; however, the current approaches use integrating lentiviral vectors, and therefore carry a risk of insertional oncogenesis. An alternative approach is to use site-specific nucleases to correct the patients’ own cells, obviating the need for allogeneic HSCT and the use of randomly integrating vectors. Zinc finger nucleases (ZFNs) offer a possible way to achieve successful gene therapy by site-specifically and permanently modifying the endogenous gene in hematopoietic stem cells (HSCs). These engineered nucleases create a site-specific, double strand break upon dimerization. If a homologous donor molecule is co-introduced which contains the normal β-globin sequence at the site of the sickle mutation, the cells may undergo homology-directed repair to correct the mutation and restore functional hemoglobin production. With this aim in mind, we have designed and tested ZFN pairs targeting the β-globin locus along with a donor template that restores the normal β-globin gene sequence while simultaneously introducing a silent base pair change that generates a restriction enzyme site for analysis. These components have led to high levels of site-specific base-pair modification in introducing the sickle mutation at the normal β-globin locus in K562 cells (upwards of 45%). Using electroporation, we delivered the ZFNs as mRNA to cord blood-derived (CB) CD34+ cells which resulted in up to 30% allelic disruption as measured by the Surveyor Nuclease assay. To achieve gene correction, the ZFNs were again delivered as mRNA and the donor template was delivered as an integrase defective lentiviral vector (IDLV). Based on pyrosequencing data, this delivery method resulted in up to 10% gene correction (the correct nucleotide replacing the sickle mutation in β-globin). Importantly, in the clinically relevant cell source, namely CD34+ cells isolated from SCD patient bone marrow, these gene modification frequencies were maintained, resulting in up to 7% correction using this multi-modal delivery strategy. These data set the stage for further investigations, including ongoing studies in a humanized mouse model. Efficient correction of the sickle mutation in HSC may provide an excellent stem cell source for autologous transplantation for SCD. Disclosures: Cost: Sangamo BioSciences: Employment, Equity Ownership. Reik:Sangamo BioSciences: Employment. Holmes:Sangamo BioSciences: Employment. Gregory:Sangamo BioSciences: Employment.

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Evaluation of persistence and fate of ex vivo edited-HSC modified with donor template and Its role in correcting Sickle Cell Disease

10.1101/2021.06.30.450644 ◽

2021 ◽

Author(s):

Sowmya Pattabhi ◽

Samantha N Lotti ◽

Mason P Berger ◽

David J Rawlings

Keyword(s):

Sickle Cell Disease ◽

Sickle Cell ◽

Ex Vivo ◽

Cell Disease ◽

Gene Correction ◽

Healthy Human ◽

Peripheral Blood Stem Cells ◽

Donor Template

Sickle cell disease (SCD) is caused by a single nucleotide transversion in exon 1 of the HBB gene that changes the hydrophobicity of adult globin (βA), leading to substantial morbidity and reduced lifespan. Ex vivo autologous gene editing utilizing co-delivery of a designer nuclease along with a DNA donor template allows for precise homology-directed repair (HDR). These gene corrected cells when engrafted into the bone marrow (BM) can prove to be therapeutic and serves as an alternative to HLA-matched BM transplantation. In the current study, we extensively explored the role of single stranded oligonucleotide (ssODN) and recombinant adeno-associated 6 (rAAV6) donor template delivery to introduce a codon-optimized change (E6optE) or a sickle mutation (E6V) change following Crispr/Cas9-mediated cleavage of HBB in healthy human mobilized peripheral blood stem cells (mPBSCs). We achieved efficient HDR in vitro in edited cells and observed robust human CD45+ engraftment in the BM of NBSGW mice at 16-17 weeks. Notably, recipients of ssODN-modified HSC exhibited a significantly higher proportion of HDR-modified cells within individual BM, CD34+ and CD235+ compartments of both E6optE and E6V cohorts. We further assessed key functional outcomes including RNA transcripts analysis and globin sub-type expression. Our combined findings demonstrate the capacity to achieve clinically relevant HDR in vitro and in vivo using both donor template delivery method. The use of ssODN donor template-delivery is consistently associated with higher levels of gene correction in vivo as demonstrated by sustained engraftment of HDR-modified HSC and erythroid progeny. Finally, the HDR-based globin protein expression was significantly higher in the E6V ssODN-modified animals compared to the rAAV6-modified animals confirming that the ssODN donor template delivery outperforms rAAV6-donor template delivery.

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S. Aureus Cas9 Leads to Higher Sickle Mutation Correction Compared to S. Pyogenes Cas9 in Patient Hematopoietic Stem and Progenitor Cells

Blood ◽

10.1182/blood-2021-150830 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 1861-1861

Author(s):

Byoungyong Yoo ◽

So Hyun Julie Park Park ◽

Yankai Zhang ◽

Vivien A. Sheehan ◽

Gang Bao

Keyword(s):

Progenitor Cells ◽

Research Funding ◽

Gene Editing ◽

Cell Phenotype ◽

High Morbidity ◽

Gene Correction ◽

Hematopoietic Stem ◽

Nucleotide Mutation ◽

Stem And Progenitor Cells ◽

Donor Template

Abstract Introduction: Sickle cell disease (SCD) is a red blood cell disorder caused by a single nucleotide mutation in the β-globin gene (HBB). Allogeneic hematopoietic stem cell transplantation (HSCT) is the only available cure, but is available to only a minority of patients and can be associated with high morbidity and mortality. CRISPR/Cas9 mediated genome editing may provide a permanent cure for SCD patients by correcting the sickle mutation in HBB in hematopoietic stem and progenitor cells (HSPCs). Previously, we achieved ~39% sickle mutation correction in SCD HSPCs by delivering S. pyogenes (Spy) Cas9/R-66S gRNA as ribonucleoprotein (RNP) and single-stranded oligodeoxynucleotides (ssODN) corrective donor template. S. aureus (Sau) Cas9 has potentially advantageous properties to improve therapeutic gene editing efficiency and safety, including smaller size allowing for efficient in vivo delivery and longer Protospacer Adjacent Motif (PAM) sequence for higher specificity. However, although in general, the cutting efficiency of SauCas9 is lower than SpyCas9, the differences in gene correction and other gene-editing outcomes between SpyCas9 and SauCas9 have not been well studied. Methods: With our R-66S gRNA sequence targeting the sickle mutation, the PAM sequence of SauCas9 (NGGRRT) is mutually permissive with that of SpyCas9 (NGG), allowing the same sequence to be targeted by both Cas9 nucleases. We delivered R-66S gRNA with SpyCas9 and SauCas9 respectively as RNP, along with corrective ssODN donor template into SCD HSPCs. We analyzed sickle mutation correction rate and small insertions and deletions (INDELs) profile by Next Generation Sequencing (NGS). Results/discussions: We found that although the INDEL rate of SpyCas9 is higher than SauCas9 at the same molar concentration of RNP, SauCas9 gave 43% sickle mutation correction, slightly higher than SpyCas9 (39%), demonstrating efficient homology-directed repair (HDR) mediated gene correction by SauCas9. To further investigate the potential for clinical translation, we will perform in-depth efficiency and safety characterization comparing SauCas9 and SpyCas9 mediated sickle mutation correction therapy in SCD HSPCs. Conclusion: In this work, we showed that, compared with the highly-optimized and widely-used SpyCas9, SauCas9 leads to a higher sickle mutation correction in SCD HSPCs, demonstrating the therapeutic potential of SauCas9 for treating SCD. We will further investigate the efficiency and safety of gene-edited therapy mediated by these two Cas9 orthologs, including in-depth characterization of off-target effects, chromosomal rearrangement and aberrations, and large genomic modifications. We will differentiate gene-corrected SCD HSPCs to study erythropoiesis and red cell phenotype, including normal hemoglobin production and reduced sickling under hypoxic conditions. Lastly, we will evaluate the engraftment efficiency of gene-edited cells in Nonirradiated NOD,B6.SCID Il2rγ -/- Kit (W41/W41) (NBSGW) mice that support the engraftment of human hematopoietic stem cells. Disclosures Sheehan: Forma Therapeutics: Research Funding; Beam Therapeutics: Research Funding; Novartis: Research Funding.

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Phase I/II Gene Therapy Study for Chronic Granulomatous Disease: Results, Lessons and Perspectives.

Blood ◽

10.1182/blood.v110.11.503.503 ◽

2007 ◽

Vol 110 (11) ◽

pp. 503-503

Author(s):

Marion G. Ott ◽

Stefan Stein ◽

Stephan Schultze-Strasser ◽

Anna Jauch ◽

Barbara Burwinkel ◽

...

Keyword(s):

Gene Expression ◽

Gene Therapy ◽

Side Effects ◽

Chronic Granulomatous Disease ◽

Genetic Diseases ◽

Cpg Methylation ◽

Granulomatous Disease ◽

Gene Correction ◽

Hematopoietic Stem ◽

Cpg Dinucleotides

Abstract Gene transfer into hematopoietic stem cells has been envisaged as an alternative to stem cell transplantation for the treatment of many genetic diseases of the blood system. In 2004 we initiated a gene therapy trial aimed at the correction of Chronic Granulomatous Disease (CGD), a rare inherited immunodeficiency caused by a functional defect in the microbial killing activity of phagocytes. Gene marking and functional correction of phagocytes were high shortly after transplantation of gene modified cells, leading to the eradication of therapy resistant infections from which patients had suffered for many years. However, one of our patients died 27 months after treatment due to a severe sepsis with multiorgan dysfunction. Although gene marking was still high at this time point, expression of the therapeutic gene, gp91phox, was minimal. This down regulation of transgene expression was due to CpG methylation within the viral LTR. Similar effects were observed in a second patient treated more than 3 years ago. In both patients CpG methylation was restricted to the promoter region of the viral LTR, while CpG dinucleotides within the enhancer region of the viral LTRs were not methylated. As a consequence, gp91phox gene expression was suppressed but the capacity of the viral LTRs to transactivate nearby sequences is still intact. Indeed we were able to detect cellular transcripts at predominant retroviral integration sites leading to an unbalanced clonal distribution of gene marked cells in peripheral blood and bone marrow. A third patient, a 5 years old child was treated in a similar way in Zurich. In this case only low levels of engraftment and gene correction were achieved. Although the treatment has been beneficial for all treated patients, the side effects observed in the two adults demand modifications in vector design for sustained gene expression and long term correction of the disease without side effects.

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Tie2+ Bone Marrow Endothelial Cells Regulate Hematopoietic Reconstitution In Vivo.

Blood ◽

10.1182/blood.v114.22.248.248 ◽

2009 ◽

Vol 114 (22) ◽

pp. 248-248

Author(s):

Phuong L. Doan ◽

J. Lauren Russell ◽

Sarah K. Meadows ◽

Heather A Himburg ◽

Pamela Daher ◽

...

Keyword(s):

Progenitor Cells ◽

Knockout Mice ◽

Fold Increase ◽

Chimeric Mice ◽

Wild Type ◽

Hematopoietic Stem ◽

Hematopoietic Reconstitution ◽

Total Body ◽

Hematopoietic Response

Abstract Abstract 248 Radiation and chemotherapy cause myelosuppression in patients via damage to bone marrow (BM) hematopoietic stem cells (HSCs) and progenitor cells. It has been shown that BM HSCs reside in close association with osteoblasts and that BM osteoblasts regulate the maintenance of quiescent HSCs in vivo. HSCs also reside in proximity to BM sinusoidal vessels but the function of BM endothelial cells (BM ECs) in regulating HSC fate in vivo remains less well understood. We hypothesized that BM ECs play a critical role in regulating BM hematopoietic reconstitution following stress. In order to test this hypothesis, we utilized Cre-LoxP recombination to generate mice bearing targeted deletion of the pro-apoptotic genes, Bak and Bax, in Tie2+ BM ECs (Tie2Cre;Bak−/− ;BaxFl/− mice) and measured their hematopoietic response to total body irradiation (TBI). Tie2Cre;Bak−/−;BaxFl/− mice (EC-Bak;Bax knockouts) were compared with Tie2Cre;Bak−/−;BaxFl/+ mice (EC-Bak;Bax (+) mice) which have constitutive Bak deletion but retain Bax in Tie2+ BM ECs. EC-Bak;Bax knockout mice had no detectable Bak or Bax by Western Blot in vascular tissues. After exposure to 100 cGy total body irradiation (TBI), EC-Bak;Bax knockout mice displayed a 2-fold increase in total viable BM cells (p=0.04), a 3-fold increase in BM ckit+sca+lineage- (KSL) progenitor cells, a 3-fold increase in colony-forming unit-spleen day 12 (CFU-S12) content (p=0.0003) and a 2-fold increase in 4 week competitive repopulating units (CRUs) compared to EC-Bak;Bax (+) mice. After 300 cGy TBI, comparable radioprotection was observed in the EC-Bak;Bax knockout mice, with a 1.9-fold increase in total BM cells, a 2.4-fold increase in CFU-S (p=0.01) and a 3.6-fold increase in 4 week CRU compared to EC-Bak;Bax (+) mice. Taken together, these results suggested that targeted protection of Tie2+ BM ECs from the intrinsic pathway of apoptosis mediated protection of BM hematopoietic stem/progenitor cells following TBI. Since Tie2 is expressed by BM ECs and a small subset of quiescent BM HSCs, we carried out experiments to determine whether the radioprotection we observed in EC-Bak;Bax knockout mice was caused autonomously by protection of Tie2+ BM ECs or Tie2+ HSCs. We transplanted 4 × 106 BM cells from EC-Bak;Bax knockout mice into lethally irradiated (950 cGy) wild type (WT) B6.SJL mice such that, after 12 weeks post-transplant, the recipient mice were chimeric for Bak and Bax deletions only in hematopoietic cells while retaining a wild type BM microenvironment (HSC-Bak;Bax knockout;EC-wild type), verified by qRTPCR. At 16 weeks post-transplant, the chimeric recipient mice were then exposed to 100 and 300 cGy TBI and we compared the hematopoietic response of these mice to that of EC-Bak;Bax knockout mice. Interestingly, after 100 cGy TBI, the hematopoietic response of the chimeric mice was comparable to that of wild type C57Bl6 mice and revealed a significant reduction in total viable BM cells (1.3-fold), BM KSL cells (7.4 fold) and BM CRU-4 weeks (9-fold) compared to 100 cGy-irradiated EC-Bak;Bax knockout mice. Significant reductions in total BM cells and BM CRU content were also observed in chimeric mice compared to EC-Bak;Bax knockout mice following 300 cGy TBI. These results suggest that deletion of Bak and Bax-mediated apoptosis in Tie2+ BM ECs protects BM hematopoietic stem and progenitor cells from ionizing radiation damage and this protection is largely autonomous to Tie2+ BM ECs. More generally, these data demonstrate that protection of BM ECs from the deleterious effects of ionizing radiation results in augmented hematopoietic reconstitution following TBI. This study demonstrates that Tie2+ BM ECs have an essential role in regulating hematopoietic regeneration following injury and reveals that BM ECs are a novel and attractive therapeutic target to augment hematopoietic reconstitution in vivo. Disclosures: No relevant conflicts of interest to declare.

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VE Cadherin Positive Endothelial Cells Regulate Hematopoietic Reconstitution In Vivo.

Blood ◽

10.1182/blood.v116.21.3734.3734 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3734-3734

Author(s):

J. Lauren Russell ◽

Phuong Doan ◽

Heather A Himburg ◽

Sarah K. Meadows ◽

Pamela Daher ◽

...

Keyword(s):

Bone Marrow ◽

Endothelial Cells ◽

Expression Profiles ◽

Gene Expression Profiles ◽

Fold Increase ◽

Small Subset ◽

High Dose ◽

Hematopoietic Stem ◽

Hematopoietic Reconstitution

Abstract Abstract 3734 We have recently demonstrated that targeted deletion of the pro-apoptotic genes, Bak and Bax, in Tie2+ bone marrow endothelial cells (BM ECs) causes the protection of the BM sinusoidal vasculature, and the BM hematopoietic stem cell (BM HSC) pool following high dose total body irradiation (TBI). Since Tie2 is expressed on BM ECs and a small subset of quiescent BM HSCs, we developed a complementary model utilizing VE-cadherin Cre mice to more specifically confirm the function of BM ECs in regulating hematopoietic reconstitution in vivo. VE-cadherinCre;Bak−/−;BaxFl/− mice, which bear deletion of Bak and Bax in VE-cadherin+ ECs, and VE-cadherinCre;Bak−/−;BaxFl/+ control mice were generated and we compared the hematopoietic responses of these animals to high dose TBI. At steady state, these mice showed no difference in total BM cells, bone marrow ckit+sca+lineage- (KSL) progenitor cells, BM colony forming cells (CFCs), or BM colony forming unit spleen day 12 (CFU-S12) counts. At 2 hours after exposure to 500cGy TBI, the mice also did not demonstrate any significant differences in total BM cells, BM KSL cells, CFCs, or CFU-S12. However, 7 days after exposure to 500cGy TBI, VE-cadherin;Bak−/−;BaxFl/− mice displayed a 2-fold increase in total BM cells (p=0.006), a 3-fold increase in BM CFCs (p=0.0009), and 4-fold increase in BM CFU-S12 (p=0.079) compared to VE-cadherinCre;Bak−/−;BaxFl/+ control mice. Microscopic examination confirmed severe hypocellularity and corruption of the BM sinusoidal vasculature at day +7 post-TBI in VE-cadherinCre;Bak−/−;BaxFl/+ mice, whereas VE-cadherinCre;Bak−/−;BaxFl/− mice displayed nearly normal cellularity and preserved BM sinusoidal vessels. Taken together, these data demonstrate that VE-cadherin+ BM ECs regulate hematopoietic reconstitution in vivo and that targeted therapies aimed at augmentation of BM EC function can accelerate hematopoietic regeneration in vivo. Currently, we are comparing the cytokine production and gene expression profiles of VE-cadherinCre;Bak−/−;BaxFl/− BM ECs versus VE-cadherinCre;Bak−/−;BaxFl/+ BM ECs to identify candidate BM EC-derived proteins which are responsible for the protection of the BM stem/progenitor cell pool from radiation injury. VE-cadherin+ BM ECs represent an attractive mechanistic target for the identification of signaling pathways that regulate hematopoietic regeneration following injury. Disclosures: Chao: Genzyme: Research Funding.

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