Long-term Engrafting Hematopoietic Stem Cells Research Articles

Impact of CRISPR/HDR editing versus lentiviral transduction on long-term engraftment and clonal dynamics of HSPCs in rhesus macaques

For precise genome editing via CRISPR/homology-directed repair (HDR), effective and safe editing of long-term engrafting hematopoietic stem cells (LT-HSCs) is required. The impact of HDR on true LT-HSC clonal dynamics in a relevant large animal model has not been studied. To track the output and clonality of HDR-edited cells and to provide a comparison to lentivirally transduced HSCs invivo, we developed a competitive rhesus macaque (RM) autologous transplantation model, co-infusing HSCs transduced with a barcoded GFP-expressing lentiviral vector (LV) and HDR edited at the CD33 locus. CRISPR/HDR-edited cells showed a two-log decrease by 2months following transplantation, with little improvement via p53 inhibition, in comparison to minimal loss of LV-transduced cells long term. HDR long-term clonality was oligoclonal in contrast to highly polyclonal LV-transduced HSCs. These results suggest marked clinically relevant differences in the impact of current genetic modification approaches on HSCs.

Clonal Dynamics of HDR-Edited HSPCs Targeting the CD33 Locus in Rhesus Macaques

For precise genome editing via CRISPR/homology-directed repair (HDR), effective and safe editing of long-term engrafting hematopoietic stem cells (LT-HSCs) requires both sufficient HDR efficiency and protection of LT-HSC function and number. Loss or damage of LT-HSCs with resultant oligoclonal hematopoiesis can increase the risk of malignant transformation or long-term hematopoietic and immune dysfunction. HDR has adverse impacts on LT-HSC function as revealed by ex vivo studies and murine xenograft models, likely due to p53 activation, intracellular innate immune activation, and/or other pathways. However, the impact of HDR on true LT-HSC clonal dynamics in a relevant large animal model has not previously been studied. We tracked the clonal dynamics of CRISPR/HDR-edited barcoded HSPCs following autologous transplantation in rhesus macaques (RMs). HDR-favored gRNAs targeting the CD33 gene locus and an ssODN containing a 6 bp barcode ID and 14 bp random barcode sequences and flanked by CD33 homology arms were designed. Autologous CD34+ cells were electroporated with the Cas9 RNP and HDR cassette in the presence of i53 mRNA to inhibit p53-mediated cell death and reinfused into animal 1 following myeloablation. In animal 1, CD33 KO neutrophils were prevalent early following engraftment and then rapidly decreased over the next two months. Similar to CD33 expression level, we revealed that HDR knock-ins as well as total indel frequency drastically decreased following transplantation. This was in marked contrast to macaques transplanted with CD34+ HSPCs edited at the CD33 locus only for NHEJ, without inclusion of an HDR cassette (Kim et al, Cell, 2018, and Figure 1 below). We next performed a competitive transplantation comparing clonal dynamics of edited cells to barcoded lentiviral (LV)-transduced cells in two macaques. For each animal, CD34+ cells were fractionated into 2 aliquots, and half were transduced with a barcoded GFP-expressing LV vector and half electroporated with the Cas9 RNP and HDR cassette with higher concentrations of i53 mRNA to enhance protection of CRISPR edited LT-HSCs. The two cell populations were combined and reinfused into myeloablated animals. CD33 KO cells in both animals decreased rapidly, however the higher concentration of i53 mRNA facilitated a less steep reduction in CD33 total indel frequency in vivo over time. However, HDR in these two animals also markedly decreased, similar to the pattern in animal 1. In contrast, LV-transduced GFP+ cell engraftment stabilized at 20% after 2 months. We retrieved embedded barcodes from CRISPR/HDR cells and revealed that a diversity of HDR clones contributed to early hematopoietic reconstitution, with restriction to a much smaller set of long-term clones at steady state, proportional in the 3 animals to the level of HDR overall. The diversity of HDR-edited contributing clones was far less than LV-transduced contributing clones long-term post-transplantation. In conclusion, CRISPR/HDR edited cells were found to decrease rapidly after autologous transplantation in rhesus macaques despite substantial gene editing assessed in the infusion product prior to infusion, in contrast to much more stable LV-transduced cells. Clonality of HDR-edited cells drastically shrank at early stage and then relied on several dominant clones, which was somewhat improved by use of high dose i53 mRNA during editing. It will be important to find improved approaches to retain LT-HSC functional activity during the HDR editing process, or explore targeted gene correction approaches such as base or PRIME editing that may better preserve HSC activity. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A comparison of DNA repair pathways to achieve a site-specific gene modification of the Bruton's tyrosine kinase gene

Gene editing utilizing homology-directed repair has advanced significantly for many monogenic diseases of the hematopoietic system in recent years but has also been hindered by decreases between in vitro and in vivo gene integration rates. Homology-directed repair occurs primarily in the S/G2 phases of the cell cycle, whereas long-term engrafting hematopoietic stem cells are typically quiescent. Alternative methods for a targeted integration have been proposed including homology-independent targeted integration and precise integration into target chromosome, which utilize non-homologous end joining and microhomology-mediated end joining, respectively. Non-homologous end joining occurs throughout the cell cycle, while microhomology-mediated end joining occurs predominantly in the S phase. We compared these pathways for the integration of a corrective DNA cassette at the Bruton’s tyrosine kinase gene for the treatment of X-linked agammaglobulinemia. Homology-directed repair generated the most integration in K562 cells; however, synchronizing cells into G1 resulted in the highest integration rates with homology-independent targeted integration. Only homology-directed repair produced seamless junctions, making it optimal for targets where insertions and deletions are impermissible. Bulk CD34+ cells were best edited by homology-directed repair and precise integration into the target chromosome, while sorted hematopoietic stem cells contained similar integration rates using all corrective donors.

Correlative Lightsheet and Electron Microscopy Applied to a Zebrafish Model of Fetal Bone Marrow: Resolving the Ultrastructure of a Single Hematopoietic Stem Cell in the Niche

Hematopoietic stem and progenitor cells (HSPC) emerge from the hemogenic endothelium of the dorsal aorta. Next, HSPC migrate to the fetal liver where they expand exponentially before migrating again to the fetal bone marrow. This early stage of bone marrow colonization is poorly understood. Current imaging technology does not allow direct visualization of the fetal bone marrow niche. Zebrafish provide a model to observe these events because they develop externally, are transparent, and ontogeny of the hematopoietic system is highly conserved. The adult marrow niche in zebrafish is in the kidney and is colonized 4 to 5 days post fertilization. To track HSPC during this dynamic process we used our previously characterized HSPC-specific transgenic reporter lines (Runx:GFP and Runx:mCherry). We demonstrated by transplantation these cells mark a highly pure population of long-term engrafting hematopoietic stem cells. To image the kidney marrow live we applied lightsheet microscopy. This technology uses three objective lenses: two that illuminate the sample in the XY plane, and one that takes images in the Z plane. This allows hundreds of optical sections to be captured in seconds. The temporal and spatial resolution of this technique produces a highly dynamic view of the entire developing marrow niche. By imaging Runx+ HSPC together with flk+ endothelial cells in a double transgenic embryo, we could track interactions between HSPC and the perivascular niche. Previously, we showed that HSPC arrival in the caudal hematopoietic tissue, the zebrafish equivalent of the fetal liver, triggers remodeling of endothelial cells into a surrounding pocket. In this study, we found a similar cellular structure forms in the kidney marrow niche. We wanted to resolve the ultrastructure of the HSPC and surrounding cells in the context of the larger developing kidney organ. To do this we applied Correlative Light and Electron Microscopy (CLEM) to match fluorescent cells tracked by lightsheet in the same embryo fixed and stained for EM. Micro-CT was used to orient and trim the embedded sample to isolate the kidney niche. Serial block-face scanning EM was used to capture high resolution serial sections (volume dimensions: x, y, z (um) = 236, 114, 105; pixel size: x, y, z (nm) = 9.5, 9.5, 70). Blood vessels observed in light and EM 3D data sets provided the anatomical markers needed to match the position of cells found using both techniques. As we previously found in the caudal hematopoietic tissue, a single round HSPC with a large nucleus, little cytoplasm, and a ruffled membrane, was lodged just under the vessel wall. This single HSPC was in a distinct anatomical location compared to larger clusters of HSPC in an abluminal space. This observation is suggestive of a marrow with distinct anatomical niche sites that may support different functions, such as proliferation versus quiescence. This unique system for analysis of the ultrastructure of an endogenous HSPC in an unperturbed niche will allow further functional and mechanistic studies at the subcellular level. Our goal is to gain novel insights into intercellular interactions between HSPC and the surrounding supportive cells in the perivascular niche. DisclosuresNo relevant conflicts of interest to declare.

Integrated Single Cell Transcriptomics Defines an Engineered Niche Supporting Hematopoietic Stem Cell Development Ex Vivo

During embryonic development, hematopoietic stem cells (HSC) arise from hemogenic endothelial cells (HEC) within arterial vessels such as the aorta of the AGM (aorta-gonad-mesonephros) region, in a process referred to as the endothelial to hematopoietic transition (EHT). Although numerous signal pathways have been implicated in EHT, the precise combination of niche-derived signals required to support the generation and self-renewal of functional, long-term engrafting HSC remains poorly defined. To elucidate the niche signals regulating HSC emergence, we used single cell RNA-sequencing to simultaneously analyze the global transcriptional profiles of HEC during their transition to HSC and the AGM-derived endothelial cell stroma (AGM-EC) that supports the generation and expansion of functional HSC. Trajectory analysis of single cell transcriptomes enabled reconstruction of EHT in pseudotime, revealing dynamics of gene expression, including genes encoding cell surface receptors and downstream pathways, during the process of HSC genesis and self-renewal in vivo and in vitro. Transcriptional profiles of niche AGM-EC enabled identification of corresponding ligands which serve to activate these receptors during HSC generation. We integrated this knowledge to engineer a stromal cell-free niche for generation of engrafting HSC from hemogenic precursors in vitro. Specifically, we defined serum-free conditions combining immobilized Notch1 and Notch2-specific antibodies to activate Notch receptors, recombinant VCAM1-Fc chimera or fibronectin fragment to bind VLA-4 integrin, recombinant interleukin-3, stem cell factor, thrombopoietin, and CXCL12 to activate their respective cytokine/chemokine receptors, and small molecule inhibition of TGF-β Receptor 1. We demonstrated that this engineered niche is sufficient to support the generation of functional HSC, as measured by long-term (24 week) multilineage engraftment after transplantation to immune-competent, lethally irradiated adult recipient mice, following culture of hemogenic precursors isolated from E9.5 to E10.5 murine embryos. The observed efficiency of generating long-term engrafting HSC, particularly from precursors derived from early embryonic stages before E10, was lower in engineered conditions compared with AGM-EC stroma, suggesting additional niche signal factors remain to be defined to optimally support HSC maturation and self-renewal in the engineered niche. Single cell RNA-sequencing of hematopoietic progeny generated following culture in the engineered niche demonstrated the formation of populations with transcriptional signatures of HSC, as well as multipotent and lineage-specific progenitors, comparable to those generated following co-culture with niche AGM-EC stroma. However, we observed relative overexpression of Notch target genes promoting early T-lymphoid fate in cells generated from the engineered niche compared to those from AGM-EC stroma. Incorporating stage-specific attenuation of Notch1 receptor activation with soluble Notch1 blocking antibody during culture was sufficient to limit markers of early T-cell precursors, suggesting that temporal titration of Notch signal activation could be used to further modulate HSC and T-lymphoid output in the engineered niche. Altogether, these studies enhance our understanding of the core signal pathways necessary for the embryonic development of functional HSC, with the potential to advance in vitro engineering of therapeutically relevant pluripotent stem cell-derived HSC in stromal cell-free culture. Disclosures Bernstein: Lyell Immunopharma: Consultancy, Equity Ownership, Patents & Royalties, Research Funding; Nohla Therapeutics: Consultancy, Equity Ownership, Patents & Royalties, Research Funding.

Therapeutic Base Editing of Human Hematopoietic Stem Cells

Base editing with fusions of RNA-guided DNA-binding proteins and nucleotide deaminases represents a promising approach to permanently remedy genetic blood disorders without obligatory double strand breaks, however its application in engrafting hematopoietic stem cells (HSCs) remains unexplored. Here we purified A3A(N57Q)-BE3 protein for RNP electroporation of human peripheral blood (PB) mobilized CD34+ hematopoietic stem and progenitor cells (HSPCs). We found that sgRNAs targeting for cytidine base editing the core GATA1 binding motif of the BCL11A +58 erythroid enhancer resulted in efficient on-target base edits (81% allele frequency) with low indels. There was similar HbF induction in erythroid progeny as compared to Cas9:sgRNA RNP nuclease mediated modification of the same target sequence (36% median HbF in base edited cells with 81% allele modifications, 39% HbF in 3xNLS-SpCas9:sgRNA#1617 nuclease edited cells with 99% indels, and 5%HbF in unedited cells). A single therapeutic base edit of the BCL11A enhancer was sufficient to ameliorate the pathobiology of both sickle cell disease (SCD) and beta-thalassemia. Base editing of CD34+ HSPCs from plerixafor mobilized PB SCD patient donors resulted in potent HbF induction (24-31% HbF level in bulk cells with 87-90% on-target allele modifications) and reduced sodium metabisulfite induced sickling (from 84% to 29% of erythrocytes). Base editing of non-mobilized PB CD34+ HSPCs from 3 beta-thalassemia patient donors potently induced gamma-globin and improved ineffective erythropoiesis in vitro, with 59-75% on-target allele modifications. Moreover highly efficient multiplex editing could be achieved in HSPCs. Simultaneous disruption of the BCL11A +58 erythroid enhancer core GATA1 motif and correction of the HBB -28A>G promoter mutation in HSPCs resulted in production of at least one therapeutic allele in 35 of 35 erythroid colonies analyzed, with each colony achieving improved alpha-to-non-alpha globin chain balance. Finally we found that base editing could be efficiently produced in engrafting HSCs. Using 3 donors, we observed 62-64% base edits at the BCL11A +58 enhancer GATA1 motif in input HSPCs with one round of electroporation and 73-85% base edits with two rounds of electroporation (separated by 24 hours), albeit with modest reduction of cell viability with tandem electroporation. We observed 70-94% human chimerism and 35-82% base edits in engrafting human cells in primary recipients and 11-61% base edits in secondary recipients as measured 16 weeks following cell infusion. Similar edit frequencies were observed across all assayed hematopoietic lineages including engrafting B-lymphocytes, erythroid precursors, and HSPCs. We observed that compared to non-engrafting progenitors, long-term engrafting HSCs favored C to T (as compared to C to G/A) editing. There was a strong correlation (Spearman r 0.86, P<0.0001) between base edit frequency and HbF level of engrafting erythroid cells, with in vivo HbF level induced from median 1.8% in unedited recipients to 22% in recipients of tandem base edited cells. Together these results demonstrate, to our knowledge for the first time, the potential of RNP base editing of human HSPCs as an alternative to nuclease editing for HSC-targeted therapeutic genome modification. Disclosures No relevant conflicts of interest to declare.

Genome Editing Via NHEJ-Mediated Targeted Integration Retains Engraftment Potential of Edited Hematopoietic Stem Cells

The ability to edit the genome of long-term-engrafting hematopoietic stem cells (LT-HSCs) would provide a curative therapy for a wide range of hematological disorders. While mixed LT/ST-HSC (ST, short-term) pools edited via non-homologous end joining (NHEJ) yield engraftable transgenic cells, pools made transgenic via homology-dependent repair (HDR) do not. This observation is likely explained by the absence of DNA replication required for HDR in LT-HSCs. Quiescence renders LT-HSCs refractory to transgene insertion via HDR, with the transgene-positive population consisting overwhelmingly of actively dividing, short-term-engrafting HSCs. In contrast to HDR, NHEJ is a DNA repair mechanism active in non-dividing cells. We hypothesized that CRISPR/Cas9-mediated transgenesis of HSC pools via NHEJ-mediated targeted integration (NHEJ-TI) would allow modification of LT-HSCs and produce edited, long-term engrafting progeny.First, we investigated whether NHEJ-TI is a feasible method of genome editing in HSCs. Utilizing CRISPR/Cas9 to induce a DSB in the AAVS1 (PPP1R12C) locus and a self-complementary rAAV6 (scAAV6) to deliver a GFP expression cassette dependent on the endogenous promoter, we optimized RNP nucleofection, scAAV6 infection, and HSC culture conditions for efficient NHEJ-TI in HSCs. Flow cytometry for eGFP and in-out PCR was used to confirm transgene integration at frequencies ranging from 4-6%.Next, we investigated whether the NHEJ-TI edited pool of mixed LT/ST-HSCs contained engraftable, edited cells by injection into irradiated NOD/SCID/IL2Rg-/- (NSG) mice. We compared the engraftment potential of transgenic HSCs cultured for either 2 days (2D) or 2 hours (2H) prior to editing. While the overall editing efficiency was ~20% higher in the 2D condition, the near absence of cell prestimulation in the 2H condition resulted in increased levels of overall engraftment, doubling the total engraftment level of edited hematopoietic cells at 10 weeks post-injection. Importantly, the percentage of edited cells among the engrafted human hematopoietic cell population remained essentially unchanged relative to the starting efficiency (2D: 4.0%, 2H: 3.6%; 10 weeks post-injection). Analysis of bone marrow-resident CD34-positive cells at 16 weeks post-engraftment will reveal the fraction of edited LT-HSCs. Our results show that NHEJ-TI is a feasible method of inserting transgenes into HSCs whilst retaining their engraftment potential. To our knowledge, this is the first study to achieve successful NHEJ-mediated targeted integration in HSCs while retaining engraftment potential and (perhaps) transgenesis as well. DisclosuresUenishi:Casebia Therapeutics: Employment. Abe:Casebia Therapeutics: Employment. Mallari:Casebia Therapeutics: Employment. Scharenberg:Generation Bio: Equity Ownership; Casebia Therapeutics: Employment; Alpine Immune Sciences: Equity Ownership. Cost:Casebia Therapeutics: Employment.

Characterizing the adult Hematopoietic Stem cell (HSC) niche in a zebrafish model for fetal bone marrow

Hematopoietic stem and progenitor cells (HSPCs) originate from the dorsal aorta and migrate to the intermediate niche, the fetal liver, where they undergo rapid expansion before migrating to the prospective adult niche, the bone marrow. This early stage of bone marrow colonization is poorly understood. Current imaging technology does not allow direct visualization of the fetal bone marrow niche. Zebrafish embryos can be used to directly visualize HSPC‐niche cell interactions unperturbed because they develop externally and are transparent. Importantly, their HSC ontogeny is highly similar to mammals. The zebrafish kidney is equivalent to the adult bone marrow niche and gets colonized from 4 to 5 days post fertilization (dpf). To track HSPCs during this dynamic process, we used our previously characterized HSPC‐specific transgenic reporter lines (Runx:GFP and Runx:mCherry). We demonstrated by transplantation that these cells mark a highly pure population of long‐term engrafting hematopoietic stem cells. Using a tissue clearing technique that allows visualization of deeper tissues, we detected ~100 HSPCs in the kidney marrow in 5 dpf embryos. These cell numbers could be manipulated through drug treatments and gene knockdowns. For live‐imaging of the kidney marrow, we applied lightsheet microscopy that allows rapid image acquisition with high spatio‐temporal resolution, providing a highly dynamic view of the entire developing niche. By imaging Runx+ HSPC together with flk+ endothelial cells in a double transgenic embryo, we could track interactions between HSPC and the perivascular niche. Previously, we showed that HSPC arrival in the caudal hematopoietic tissue, the zebrafish equivalent of the fetal liver, triggers remodeling of endothelial cells into a surrounding pocket. In this study, we found that a similar cellular structure forms within the kidney marrow niche. To resolve the ultrastructure of the HSPC and its surrounding cells in context of the entire niche, we performed Correlative Light and Electron Microscopy. For this, the embryos imaged on lightsheet were fixed and stained for EM. MicroCT was used to orient and trim the embedded sample to isolate the kidney niche and serial blockface scanning EM to capture high resolution serial sections. By matching anatomical markers such as blood vessels observed in light and EM 3D data sets, a single round HSPC with a large nucleus, little cytoplasm, and a ruffled membrane, was identified lodged just under the vessel wall. This single HSPC was in a distinct anatomical location compared to larger clusters of HSPC in an abluminal space. This is suggestive of distinct anatomical niche sites within the marrow that may support different functions, such as proliferation versus quiescence. Our goal is to gain novel insights into intercellular interactions between HSPC and the surrounding supportive cells in the perivascular niche.Support or Funding InformationASH, NIDDKThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Bone Marrow Characterization in Sickle Cell Disease: Inflammation and Stress Erythropoiesis Lead to Suboptimal CD34 Recovery Compared to Normal Volunteer Bone Marrow

IntroductionGene therapy for sickle cell disease (SCD) requires modification of a high number of long term engrafting hematopoietic stem cells (LT-HSCs) sufficient to sustain production of the gene of interest at levels capable of overcoming the pathogenic HbSS phenotype. Unlike β-Thalassemia, the inflammatory bone marrow (BM) environment and stress erythropoiesis associated with SCD may have significant impacts on HSC quality and yield necessary for disease amelioration. Important work to optimize gene therapy through improvement in gene transfer efficiency, editing strategies, or transplant conditioning can only improve gene therapy in SCD if enough autologous HSCs are LT-HSCs, thus characterization of SCD BM and CD34+ HSCs is required. Collection type, storage, and delays in processing may further impact CD34+ recovery and should be investigated as a strategy to maximize LT-HSC recovery.MethodsTwenty milliliters of BM from subjects with SCD (HbSS genotype) and normal volunteers was collected in different anticoagulants (Heparin, ACD-A) and processed immediately(day 0) or stored at 40C and processed the following day(day 1). After isolation via Ficoll density gradient centrifugation, the mononuclear (MN) layer was stained with antibodies against inflammatory markers (CD36, CD35, CD11b, CD62L, CD62P), non-MN cells (GPA, CD66b, CD41/61), or processed for CD34+ selection using a magnetic microbead CD34+ selection kit and stained for CD34, CD45, and GPA expression. Data were analyzed by conventional and imaging flow cytometry, the latter confirming post-CD34+ selection flow data and demonstrating antibody intensity as a characterization of HSC heterogeneity and progenitor lineage. Complete blood count and hemoglobin (Hb) electrophoresis were obtained at the time of BM collection. Statistical analyses were performed using unpaired t-tests.ResultsBM was collected from 18 subjects (16 with SCD; 11M; age 21-41 years). Median Hb (8.6 vs. 13.5 gm/dL, p<0.01) and white blood cell count (8.8 vs. 4.2 K/mcL, p<0.05) differed significantly between SCD and non-SCD subjects. Median percent sickle Hb in SCD subjects was 62%. Inflammatory markers and contamination with red cell and platelet markers in the post-Ficoll MN layer were higher in SCD vs. non-SCD BM regardless of anticoagulant (CD35 24% vs. 13%, p<0.05; CD36 22% vs. 11%, p<0.05; CD62P 16% vs. 3%, p<0.05; GPA 16% vs. 4%, p<0.05; CD41/61 19% vs. 3%, p<0.05), and trended higher on day 1 in SCD BM in both anticoagulants, significantly in Heparin (GPA 23% vs. 33% on day 1, p<0.05). Total CD45 expression was lower in SCD vs. non-SCD BM in both anticoagulants (p<0.05) and on day 0 (p<0.05) and 1 (p<0.01), with Amnis data confirming a higher CD34+CD45- population in SCD BM (4 ± 2% vs. 0.5 ± 0.3%, p<0.05). While there was no significant difference in total CD34+ cell count between SCD and non-SCD BM after selection post-Ficoll, there was a trend for lower CD34+ count in SCD in both anticoagulants (2.6x10^5 vs. 4.7x10^5, p=0.1). SCD CD34+ cells were characterized by higher GPA expression (28 ± 5% vs. 13 ± 3% in non-SCD BM, p<0.01) that worsened in Heparin on day 1 (22 ± 6.3% vs. 35 ± 12.4%, p<0.05). Image cytometry confirmed a majority of GPA expression in SCD BM is from single cell CD34+CD45+GPA+ and CD34+CD45-GPA+ HSCs in addition to red cell aggregates, with an increase in CD34+CD45-GPA+ HSCs on day 1 (10 ± 5% vs. 0.6 ± 0.2 % on day 0, p<0.05). Furthermore, the percentage of CD34hi HSCs was lower in SCD vs. non-SCD BM, with >50% SCD HSCs characterized as CD34dim (56% vs. 4% in non-SCD BM, p<0.001). Lastly, the purity of CD34+ selection worsened from day 0 to day 1 in SCD BM in heparin (94% vs. 68 ± 8%, p<0.05) and ACD-A (88% vs. 68 ± 0.7%, p<0.05).ConclusionsSCD BM is characterized by increased inflammation and cell contamination in the MN layer regardless of anticoagulant that worsens over time in Heparin more significantly than in ACD-A. Compared to non-SCD BM, CD34+ HSC yield post-Ficoll is lower in SCD subjects, and is characterized by a larger proportion of CD34+CD45+GPA+ and CD34+CD45-GPA+ HSCs that rise with delays in processing. This indication of early differentiation along the erythroid lineage, with more than 50% of HSCs losing CD34+ intensity suggesting they are not LT-HSCs, suggests suppression of inflammation and stress erythropoiesis, combined with early cell processing may be critical for maximal HSC recovery necessary for successful gene therapy in SCD. [Display omitted] DisclosuresLuo:bluebird bio Inc.: Employment. Pierciey:bluebird bio: Employment.

Lentivirus-Mediated Platelet Gene Therapy Corrects Bleeding Diathesis and Induces Immune Tolerance in Murine Hemophilia B Mice

Abstract 1101While data from the clinical trials using AAV vector expression FIX in hemophilia B gene therapy in humans are very encouraging, for individuals with severe liver disease or neutralizing antibodies to AAV, an alternative gene therapy approach might be desired. Our previous studies have demonstrated that lentivirus-mediated platelet gene therapy can correct murine hemophilia A phenotype, but this approach has not been explored for hemophilia B. In the current study, we developed a clinical translatable approach for platelet gene therapy of hemophilia B. Platelet-FIX (2bF9) expression in hemophilia B (FIXnull) mice was introduced by transplantation of hematopoietic stem cells (HSCs) transduced with 2bF9 lentivirus (LV). The recipients were analyzed beginning at 3 weeks after bone marrow (BM) transplantation. Expression of the 2bF9 product was detected by PCR in all recipients that received 2bF9 LV-transduced BM cells, indicating viable engraftment of BM genetically modified with the 2bF9 LV transfer vector. The expression of the hFIX transgene protein in the transduced platelets was confirmed by immunofluorescent confocal microscopy. Flow cytometry showed that there were 20.8 ± 12.1% (n = 7) and 14.8 ± 10.7% (n = 6) 2bF9 LV-transduced platelets respectively in the recipients preconditioned with 1100 cGy or 660 cGy. The antigen levels of FIX (FIX:Ag) were 2.89 ± 1.75 mU/108 platelets (n = 9) in the recipients preconditioned with 1100 cGy and 1.87 ± 1.30 mU/108 platelets (n = 7) in the 660 cGy group, while the activity (FIX:C) levels were 1.67 ± 1.15 and 1.13 ± 0.85 mU/108 platelets respectively. There was a small amount of FIX detected in the 2bF9 LV-transduced recipient plasma with the average levels of 2.22 mU/ml in 1100 cGy group and 1.44 mU/ml in 660 cGy group. To analyze the distribution of the FIX between platelets and plasma, we normalized FIX levels to total whole blood FIX content. The results demonstrated that 90% to 95% of whole blood FIX was stored in platelets. The tail clip survival test demonstrated that 15 out of 16 mice that received 2bF9 LV-transduced HSCs survived the tail clip challenge, while 8 out of 10 FIXnull control mice died after tail clipping. Nine months after transplantation, sequential transplantation was performed on some of the primary recipients. Platelet-hFIX expression in the secondary recipients was sustained, leading to phenotypic correction and confirming that long-term engrafting HSCs were successfully transduced with 2bF9 LV. Notably, none of the transduced recipients developed anti-FIX antibodies after platelet gene therapy. To investigate whether immune tolerance was induced in 2bF9 LV-transduced recipients, we challenged the recipients with recombinant human FIX (rhFIX) in the presence of adjuvant. Only 1 out of 9 2bF9 LV-transduced recipients developed a low titer of inhibitory antibodies (1.6 BU/ml) as measured by a modified Bethesda assay. In contrast, all of the FIXnull controls developed inhibitory antibodies ranging from 17 – 37 BU/ml after the same challenge (n = 5). To ensure that the immune system was not defective in the 2bF9 LV-transduced recipients and that the tolerance induction is FIX antigen-specific, we further challenged the animals with ovalbumin (OVA) absorbed on Alum. Both the 2bF9 LV-transduced and FIXnull control mice developed high-titer of anti-OVA antibodies. The levels of anti-OVA IgG in the 2bF9 transduced recipients were not significantly different from FIXnull mice after the OVA immunization, confirming that tolerance induction in 2bF9 LV-transduced mice is FIX-specific. Taken together, our data suggest that lentivirus-mediated bone marrow transduction and transplantation can not only provide sustained phenotypic correction, but also induce immune tolerance in hemophilia B mice, indicating that this approach may be a promising strategy for gene therapy of hemophilia B in humans. Disclosures:No relevant conflicts of interest to declare.

Single Cell Level Genome-Wide Gene Expression Analysis of Bone Marrow-Derived Oct-4+ very Small Embryonic-Like Stem Cells (VSELs) Revealed That a Polycomb Group Protein Ezh2 Regulates VSELs Pluripotency by Maintaining Bivalent Domains At Promoters of Important Homeodomain-Containing Developmental Transcription Factors

Abstract 2345Recently, we identified a population of very small embryonic-like (VSEL) stem cells (SCs) in adult bone marrow (BM) (Leukemia 2006:20;857). These Oct4+CXCR4+SSEA-1+Sca-1+CD45−Lin− VSELs are capable of differentiation in vitro into cells from all three germ lineages and in in vivo animal models they can be specified into mesenchymal stem cells (MSCs) (Stem Cells Dev 2010:19;1557), cardiomyocytes (Stem Cell 2008:26;1646), and long-term engrafting hematopoietic stem cells (HSCs) (Exp Hematol 2011:39;225). Be employing gene-expression and epigenetic profiling studies we reported that VSELs in BM have germ-line stem cell like epigenetic features including i) open/active chromatin structure in Oct4 promoter, ii) parent-of-origin specific reprogramming of genomic imprinting (Leukemia 2009, 23, 2042–2051), and iii) that they share several markers with epiblast-derived primordial germ cells (PGCs), in particular with migratory PGCs (Leukemia 2010, 24, 1450–1461). However, it was not clear how VSELs maintain pluripotent state. To address this issue we recently employed single cell-based genome-wide gene expression analysis and found that, Oct4+ VSELs i) express a similar, yet nonidentical, transcriptome as embryonic stem-cells (ESCs), ii) up-regulate cell-cycle checkpoint genes, and iii) down-regulate genes involved in protein turnover and mitogenic pathways. Interestingly, our single cell library studies also revelaed that Ezh2, a polycomb group protein, is highly expressed in VSELs. This protein is well known to be involved in maintaining a bivalent domains (BDs) at promoters of important homeodomain-containing developmental transcription factors. Of note a presence of BDs is characteristic for pluripotent stem cells (e.g., ESCs) and as result of Ezh2 overexpression, VSELs, like ESCs, exhibit BDs - bivalently modified nucleosomes (trimethylated H3K27 and H3K4) at promoters of important homeodomain-containing developmental transcription factors (Sox21 Nkx2.2 Dlx1 Zfpm2 Irx2 Lbx1h Hlxb9 Pax5 HoxA3). Of note, spontaneous (as seen during differentiation) or RNA interference-enforced down-regulation of Ezh2 removes BDs what, results in lose of their plurioptentiality and de-repression of several BD-regulated genes that control their tissue commitment. In conclusion, Our results show for first time that in addition to the expression of pluripotency core transcription factor Oct-4, VSELs, like other pluripotent stem-cells, maintain their pluripotent state through an Ezh2-dependent BD-mediated epigenetic mechanism. Based on this our genome-wide gene expression study not only advances our understanding of biological processes that govern VSELs pluripotency, differentiation, and quiescence but will also help to develop better protocols for ex vivo expansion of these promising cells for potential application in regenerative medicine. Disclosures:Ratajczak:Neostem Inc: Consultancy, Research Funding.

An In Vivo Evidence That the CD45negative Adult Marrow-Derived CXCR4+ SSEA-1+ OCT-4+ Very Small Embryonic-Like (VSEL) Stem Cells May Differentiate into CD45positive Long Term Repopulating Hematopoietic Stem Cells.

Mounting evidence accumulates that bone marrow (BM) contains a population of pluripotent stem cells (PSC) that give rise to long-term repopulating hematopoietic stem cells (HSC). Recently we identified in murine BM a homogenous population of rare (∼0.01% of BMMNC) and very small (2–4 μm) Sca-1+ lin− CD45− cells that express by RQ-PCR and immunhistochemistry markers of PSC such as SSEA-1, Oct-4, Nanog and Rex-1 and highly express Rif-1 telomerase protein (Leukemia 2006; 20,857–869). Direct electronmicroscopical analysis revealed that these cells display several features typical for primary epiblast-derived embryonic stem cells such as i) a large nuclei surrounded by a narrow rim of cytoplasm, and ii) open-type chromatin (euchromatin). In co-cultures with C2C12 murine sarcoma supportive feeder-layer, these cells grow spheres that are composed of immature CXCR4+SSEA-1+Oct-4+ cells that have large nuclei containing euchromatin and, if plated into cultures promoting tissue differentiation, show pluripotency and expand into cells from all three germ-cell layers. Based on this, we called them very small embryonic like (VSEL) stem cells. However, VSEL isolated freshly from the BM do not posses immediate hematopoietic activity - they neither grow hematopoietic colonies nor radioprotect lethally irradiated recipients. Recently, however, we noticed that if plated over supportive OP9 cell line, these CD45− VSEL give rise to colonies of CD45+CD41+Gr-1−Ter119− cells where their phenotype resembles those of the earliest hematopoietic cells that are derived in vitro from established embryonic cell lines. This hematopoietic differentiation of VSEL was accompanied by upregulation of mRNA for several genes regulating hematopoiesis (e.g. PU-1, c-myb, LMO2, Ikaros). More importantly, the CD45+CD41−Gr-1−Ter119− cells expanded from VSEL isolated from GFP+ mice if transplanted into wild-type animals protected them from lethal irradiation and differentiated in vivo into all major hematopoietic lineages (e.g., Gr-1+, B220+ and CD3+ cells). This hematopoietic activity was maintained after transplantation into secondary recipients. Based on this, we postulate that i) VSEL are PSC that give rise to HSC and ii) that CD45+ cells may derive from a CD45− population. Thus we propose that VSEL are a population of BM-residing PSC that may give rise to long-term engrafting hematopoietic stem cells.

The CD45−LIN− Adult Marrow-Derived CXCR4+ SSEA-1+ Oct-4+ Very Small Embryonic-Like (VSEL) Stem Cells Form In Vitro Spheres Which May Differentiate into CD45+ Hematopoietic Cells.

Recently we identified in murine BM a homogenous population of rare (~0.01% of BMMNC) Sca-1+ lin− CD45− cells that express by RQ-PCR and immunhistochemistry markers of pluripotent stem cells (PSC) such as SSEA-1, Oct-4, Nanog and Rex-1 and highly express Rif-1 telomerase protein (Leukemia 2006; 20,857–869). Direct electronmicroscopical analysis revealed that these cells display several features typical for primary embryonic stem cells such as i) a small size (~2–4 μm in diameter), ii) a large nuclei surrounded by a narrow rim of cytoplasm, and iii) open-type chromatin (euchromatin). These cells isolated freshly from the BM neither grow hematopoietic colonies nor radioprotect lethally irradiated recipients. Recently, however, we noticed that purified VSELs in co-cultures with C2C12 murine sarcoma supportive feeder-layer grow spheres and cells from these VSEL-derived spheres (VSEL-DS) are composed of immature cells with large nuclei containing euchromatin, and similarly as VSELs are CXCR4+SSEA-1+Oct-4+. We found that cells from VSEL-DS after re-plating over C2C12 cells may again (up to 5–7 passages) grow new embryoid-like bodies or if plated into cultures promoting tissue differentiation show pluripotency and expand into cells from all three germ-cell layers. Based on this we tested if CD45− VSEL-DS cells could also differentiate into the hematopoietic lineage. To address this issue we employed similar culture conditions that are employed for hematopoietic differentiation of established embryonic stem cell lines. We noticed that VSEL-DS cells if cultured/passaged in methylocellulose cultures supplemented with hematopoietic growth factors (KL, IL-3, EpO and GM-CSF) give rise to colonies composed of myeloid (CD45+ Gr-1+) and erythroid (Terr-119+) hematopoietic cells. The hematopoietic differentiation of VSEL-DS cells was accompanied by upregulation of mRNA for several genes regulating hematopoiesis (e.g. PU-1, c-myb, LMO2, Ikaros). Based on this we postulate that VSELs that reside in bone marrow may contribute to hematopoiesis. We also provide direct evidence that CD45+ cells may derive from a CD45− population. We postulate that VSELs are closely related to a population of long-term engrafting hematopoietic stem cells and currently we are testing this possibility in animal models.

Human embryonic stem cell–derived hematopoietic cells are capable of engrafting primary as well as secondary fetal sheep recipients

The human/sheep xenograft model has proven valuable in assessing the in vivo hematopoietic activity of stem cells from a variety of fetal and postnatal human sources. CD34+/lineage- or CD34+/CD38- cells isolated from human embryonic stem cells (hESCs) differentiated on S17 feeder layer were transplanted by intraperitoneal injections into fetal sheep. Chimerism in primary transplants was established with polymerase chain reaction (PCR) and flow cytometry of bone marrow and peripheral blood samples. Whole bone marrow cells harvested from a primary recipient were transplanted into a secondary recipient. Chimerism was established as described before. This animal was stimulated with human GM-CSF, and an increase in human hematopoietic activity was noted by flow cytometry. Bone marrow aspirations cultured in methylcellulose generated colonies identified by PCR to be of human origin. We therefore conclude that hESCs are capable of generating hematopoietic cells that engraft primary recipients. These cells also fulfill the criteria for long-term engrafting hematopoietic stem cells as demonstrated by engraftment and differentiation in the secondary recipient.

Human Embryonic Stem Cell (hESC)-Derived Hematopoietic Elements Are Capable of Engrafting Primary as well as Secondary Fetal Sheep Recipients.

We used transplantation into 10 and 20 pre-immune fetal sheep recipients (55–65 days-old, term: 145 days) to evaluate the in vivo potential of hematopoietic elements derived from hESC. The in utero human/sheep xenograft model has proven valuable in assessing the in vivo hematopoietic activity of stem cells from a variety of fetal and post-natal human sources. Five transplant groups were established. Non-differentiated hESC were injected in one group. In the second and third group, embroid bodies differentiated for 8 days were injected whole or CD34+ cells were selected for injection. In the fourth and fifth group, hESC were differentiated on S17 mouse stroma layer and injected whole or CD34+ cells were selected for injection. The animals were allowed to complete gestation and be born. Bone marrow and peripheral blood samples were taken periodically up to over 12 months after injection, and PCR and flowcytometry was used to determine the presence of human DNA/blood cells in these samples. A total of 30 animals were analyzed. One primary recipient that was positive for human hematopoietic activity was sacrificed and whole bone marrow cells were transplanted into a secondary recipient. We analyzed the secondary recipient at 9 months post-injection by PCR and found it to be positive for human DNA in its peripheral blood and bone marrow. This animal was further challenged with human GM-CSF and human hematopoietic activity was noted by flowcytometry analyses of bone marrow and peripheral blood samples. Further, CD34+ cells enriched from its bone marrow were cultured in methylcellulose and human colonies were identified by PCR. We therefore conclude that hESC are capable of generating hematopoietic cells that engraft in 1° sheep recipients. These cells also fulfill the criteria for long-term engrafting hematopoietic stem cells as demonstrated by engraftment and differentiation in the 20 recipient.

Reversible expression of CD34 by adult human bone marrow long-term engrafting hematopoietic stem cells.

We previously reported that CD34(-) population of bone marrow (BM) cells from adult humans contains cells capable of engraftment and multilineage differentiation. We also reported on the reversibility of CD34 expression by murine hematopoietic stem cells. Based on long-term observations in primary, secondary, and tertiary sheep recipients, we now present definitive evidence for the long-term engrafting capability of human BM CD34(-) cells, and the reversibility of CD34 expression by human BM hematopoietic stem cells (HSC) in vivo. We used serial transplantations into primary, secondary, and tertiary preimmune fetal sheep recipients to evaluate and compare the long-term engraftment and differentiation of adult human bone marrow-derived CD34(-) and CD34(+) cells in vivo. In primary hosts CD34(-) or CD34(+) cells produced multilineage human cell activity that persisted for 31 months. To confirm the long-term engrafting characteristics of CD34(-) cells and determine whether CD34 expression on human HSC is reversible, we transplanted human CD34(-) and CD34(+) cells obtained from primary hosts into secondary sheep recipients. Multilineage engraftment occurred in all secondary hosts, and in tertiary hosts transplanted with CD34(-) or CD34(+) cells obtained from BM of secondary recipients. These results demonstrate that human BM CD34(-) cells are capable of long-term multilineage engraftment in vivo. The finding that both CD34(-) and CD34(+) cells from primary/secondary groups engraft secondary/tertiary hosts indicates that CD34 expression on human HSC is reversible, a process that does not impair HSC function in vivo.

Differentiative potential of human metanephric mesenchymal cells.

To evaluate the ability of mesenchymal cells derived from nonhematopoietic organs to form blood and other tissues in vitro and in vivo. Because of its mesodermic derivation, human fetal kidney was used as a source of mesenchymal cells. Two populations of kidney cells were studied at a nonclonal level: a crude preparation, and an adherent fraction that was derived from the first by propagation in vitro (MNMC). Both populations were transplanted into sheep fetuses and analyzed at intervals for the presence of human cells in different organs by flow cytometry, PCR, immunohistochemistry, and in situ hybridization. Secondary transplantation studies were performed using human hematopoietic cells obtained from the bone marrow (BM) of primary recipients. MNMC were Thy-1(+), CD51(+), CD44(+), CD45(-), and vimentin(+), a phenotype consistent with that of metanephric mesenchyme. The crude population displayed the same phenotype but was contaminated with 0.4% CD34(+)CD45(+) cells. Cells with hepatocyte-like morphology and phenotype were obtained from the MNMC after culture in specific inducing media. After transplantation, both populations of cells produced multilineage hematopoietic engraftment and gave rise to CD34(+) cells. Successful hematopoietic engraftment in secondary recipients demonstrated the generation of long-term engrafting hematopoietic stem cells from MNMC. PCR analysis confirmed human hematopoietic engraftment and revealed that human cells were also present within other organs. Liver sections of transplanted animals contained human albumin-producing hepatocyte-like cells. A human metanephric mesenchymal cell population simultaneously gave rise to human blood and liver-like cells, suggesting that mesenchymal cells may represent a broad population of putative stem cells in multiple adult organs.

Transduction of long-term-engrafting human hematopoietic stem cells by retroviral vectors.

Gene therapy using retroviral vectors to transfer functional exogenous genes into hematopoietic stem cells (HSCs) promises to provide a permanent cure for a wide array of both hematopoietic and nonhematopoietic disorders by virtue of the fact that retroviral vectors permanently integrate into the host cell genome and HSCs are able to self-renew and give rise to differentiated progeny throughout the life span of the patient. However, for transduction and genomic integration to occur, the target cells must undergo cell division and express the appropriate retroviral receptor, requirements that have thus far hindered attempts at inserting exogenous genes into human HSCs in vitro. In the present studies, we used the fetal sheep xenograft model of human hematopoiesis to evaluate whether human long-term engrafting HSCs could be transduced in vivo, within a fetal microenvironment. We transplanted adult human bone marrow-derived CD34(+)Lin(-) cells into preimmune fetal sheep recipients and subsequently (19 days later) administered clinical-grade murine retroviral vector supernatants to these fetal hematopoietic chimeras. Our results demonstrate that this approach successfully transduced adult human HSCs within all seven sheep that survived the procedure, and that these transduced HSCs had the ability to serially engraft primary, secondary, and tertiary fetal sheep recipients. Transgene expression persisted throughout the serial transplantation. The successful in vivo transduction of long-term engrafting human HSCs with the existing generation of murine retroviral vectors has significant implications for developing new approaches to pre- and postnatal gene therapy.