Convergence Of Hypoxia and TGFβ Pathways On Cell Cycle Regulation In Human Hematopoietic Stem/Progenitor Cells

Abstract Hematopoietic stem cells reside within specialized hypoxic niches in the bone marrow where they are kept in a relative quiescent state. One of the key pathways activated under low oxygen conditions is the Hypoxia Induced Factor (HIF) pathway. HIF1 and HIF2 act as oxygen sensors that are degraded by the Von Hippel Lindau (VHL) tumor suppressor protein under normoxic conditions but not when oxygen levels are low, resulting in stabilization of the HIF proteins, translocation to the nucleus and initiation of target gene transcription. Although it has been shown that HIF1 and 2 fulfill essential roles in the regulation of HSC fate, little is known about the mechanisms that are involved. Here, we set out to investigate the effects of hypoxia, HIF1 and HIF2 on gene transcription in human hematopoietic stem/progenitor cells. Cord blood (CB) CD34+ cells were cultured under low oxygen conditions (2%), or were lentivirally transduced with constitutively active HIF1(P402/564) and HIF2(P405/531) constructs under normoxic conditions and after 24 hrs transcriptome changes were analyzed by Illumina BeadArray analysis. This provided the possibility to identify common hypoxia-HIF1-HIF2 gene signatures, but also the identification of specific target genes that were exclusively regulated by HIF1, HIF2 or hypoxia. Geneset enrichment analysis (GSEA) using Gene Ontology genesets revealed that overexpression of HIF1 and -2 resulted in significant enrichment for known pathways like “hypoxia induced signaling”, but unexpectedly also for the Transforming Growth Factor beta (TGFβ) pathway. GSEA using a published dataset of TGFβ stimulated CB CD34+ cells indeed confirmed a high correlation between hypoxia target genes and TGFβ induced genes. Two of the most significantly upregulated genes in both gene sets were the cyclin dependent kinase inhibitor CDKN1C (p57kip2) and Regulator of G-protein signaling (RGS)1. q-RT-PCR analysis demonstrated enhanced expression of CDKN1C by hypoxia treatment or HIF overexpression under normoxia in combination with TGFβ stimulation. Although it was demonstrated that CD34+cells cultured under hypoxic conditions secreted high levels of latent TGFβ, no rescue of the hypoxia induced cell cycle arrest was demonstrated by knockdown of SMAD4, arguing against direct effects of hypoxia-induced secreted TGFβ on cell cycle quiescence. RGS1 is a member of the RGS family, involved in the negative regulation of G-protein coupled receptor signaling. Overexpression studies under normoxic conditions in CB CD34+cells demonstrated a decrease of SDF1-mediated migration. Furthermore, overexpression of RGS1 attenuated SDF1 and GM-CSF-induced ERK phosphorylation whereas the GM-CSF-induced STAT5 tyrosine phosphorylation was unaffected. These findings indicate that RGS1 can interfere with specific signaling pathways involved in the regulation of cell proliferation and migration. Analysis of the CDKN1C as well as the RGS1 promoters revealed binding sites for both HIF and SMAD2/3/4 in the proximal part, suggesting that both pathways can indeed converge on the regulation of these important proteins that control cell cycle progression and the response to stimulatory cytokines in human stem/progenitor cells. Disclosures: No relevant conflicts of interest to declare.

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ATG5 and ATG7 Fulfill Essential and Non-Redundant Roles in Human Hematopoietic Stem/Progenitor Cells

Blood ◽

10.1182/blood.v124.21.4316.4316 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4316-4316

Author(s):

Hendrik Folkerts ◽

Maria Catalina Gomez Puerto ◽

Albertus T.J. Wierenga ◽

Koen Schepers ◽

Jan Jacob Schuringa ◽

...

Keyword(s):

Cell Cycle ◽

Progenitor Cells ◽

Target Genes ◽

Kinase Inhibitor ◽

Conflicts Of Interest ◽

Erythroid Progenitor ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

Human Hscs

Abstract Macroautophagy is a catabolic process by which intracellular contents are delivered to lysosomes for degradation. ATG5 and ATG7 play an essential role in this process. Recent studies have shown that mouse hematopoietic stem cells (HSCs) lacking ATG7 were unable to survive in vivo, however, the role of macroautophagy in proliferation and survival of human HSCs has not yet been defined. Here, we demonstrate that autophagy is functional in human hematopoietic stem/progenitor cells. Robust accumulation of the autophagy markers LC3 and p62 were observed in cord blood (CB)-derived CD34+ cells treated with bafilomycin-A1 (BAF) or hydroxychloroquine (HCQ), as defined by Western blotting. When these cells were subsequently differentiated towards the myeloid or erythroid lineage, a decreased accumulation of LC3 was observed. In addition, CB CD34+CD38- cells showed enhanced accumulation of cyto-ID (a marker for autophagic vesicles) compared to CD34+CD38+ progenitor cells upon BAF or HCQ treatment. In line with these results, also more mature CB CD33+ and CD14+ myeloid cells or CD71+CD235+ erythroid cells showed reduced levels of cyto-ID accumulation upon BAF or HCQ treatment. These findings indicate that human hematopoietic stem and progenitor cells (HSPCs) have a higher basal autophagy flux compared to more differentiated cells. To study the functional consequences of autophagy in human HSCs and their progeny, ATG5 and ATG7 were downregulated in CB-derived CD34+ cells, using a lentiviral shRNA approach which resulted in 80% and 70% reduced expression, respectively. Downmodulation of ATG5 or ATG7 in CB CD34+ cells resulted in a significant reduction of erythroid progenitor frequencies, as assessed by colony forming cell (CFC) assays (shATG5 2.2 fold, p<0.05 or shATG7 1.4 fold p<0.05). Additionally, a strong reduction in expansion was observed when transduced cells were cultured under myeloid (shATG5 17.9 fold, p<0.05 or shATG7 12.3 fold, p<0.05) or erythroid permissive conditions (shATG5 6.7 fold, p<0.05 or shATG7 1.7 fold, p<0.05), whereby differentiation was not affected. The phenotype upon knockdown of ATG5 or ATG7 could not be reversed by culturing the cells on a MS5 stromal layer. In addition to progenitor cells, HSCs were also affected since long term culture-initiating cell (LTC-IC) assays in limiting dilution revealed a 3-fold reduction in stem cell frequency after ATG5 and ATG7 knockdown. The inhibitory effects of shATG5 and shATG7 in cultured CD34+ cells were at least in part due to a decline in the percentage of cells in S phase and (shATG5 1.4 fold, p<0.01 and shATG7 1.3 fold, p<0.01) and an increase of Annexin V positive cells. The changes in cell cycle and apoptosis coincided with a marked increase in expression of the cell cycle-dependent kinase inhibitor p21, an increase in p53 levels, and an increase in proapoptotic downstream target genes BAX, PUMA and PHLDA3. Additionally, ROS levels were increased after ATG5 and ATG7 knockdown. The increased apoptosis in shATG5 and shATG7 transduced cells might be triggered by elevated ROS levels. Taken together, our data demonstrate that autophagy is an important survival mechanism for human HSCs and their progeny. Disclosures No relevant conflicts of interest to declare.

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Enforced Expression of GATA-2 Confers Quiescence on Human Hematopoietic Stem and Progenitor Cells In Vitro and In Vivo.

Blood ◽

10.1182/blood.v108.11.83.83 ◽

2006 ◽

Vol 108 (11) ◽

pp. 83-83

Author(s):

Alex J. Tipping ◽

Cristina Pina ◽

Anders Castor ◽

Ann Atzberger ◽

Dengli Hong ◽

...

Keyword(s):

Cell Cycle ◽

Progenitor Cells ◽

Zinc Finger ◽

Human Cord Blood ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

Stem And Progenitor Cells ◽

Colony Number

Abstract Hematopoietic stem cells (HSCs) in adults are largely quiescent, periodically entering and exiting cell cycle to replenish the progenitor pool or to self-renew, without exhausting their number. Expression profiling of quiescent HSCs in our and other laboratories suggests that high expression of the zinc finger transcription factor GATA-2 correlates with quiescence. We show here that TGFβ1-induced quiescence of wild-type human cord blood CD34+ cells in vitro correlated with induction of endogenous GATA-2 expression. To directly test if GATA-2 has a causative role in HSC quiescence we constitutively expressed GATA-2 in human cord blood stem and progenitor cells using lentiviral vectors, and assessed the functional output from these cells. In both CD34+ and CD34+ CD38− populations, enforced GATA-2 expression conferred increased quiescence as assessed by Hoechst/Pyronin Y staining. CD34+ cells with enforced GATA-2 expression showed reductions in both colony number and size when assessed in multipotential CFC assays. In CFC assays conducted with more primitive CD34+ CD38− cells, colony number and size were also reduced, with myeloid and mixed colony number more reduced than erythroid colonies. Reduced CFC activity was not due to increased apoptosis, as judged by Annexin V staining of GATA-2-transduced CD34+ or CD34+ CD38− cells. To the contrary, in vitro cultures from GATA-2-transduced CD34+ CD38− cells showed increased protection from apoptosis. In vitro, proliferation of CD34+ CD38− cells was severely impaired by constitutive expression of GATA-2. Real-time PCR analysis showed no upregulation of classic cell cycle inhibitors such as p21, p57 or p16INK4A. However GATA-2 expression did cause repression of cyclin D3, EGR2, E2F4, ANGPT1 and C/EBPα. In stem cell assays, CD34+ CD38− cells constitutively expressing GATA-2 showed little or no LTC-IC activity. In xenografted NOD/SCID mice, transduced CD34+ CD38−cells expressing high levels of GATA-2 did not contribute to hematopoiesis, although cells expressing lower levels of GATA-2 did. This threshold effect is presumably due to DNA binding by GATA-2, as a zinc-finger deletion variant of GATA-2 shows contribution to hematopoiesis from cells irrespective of expression level. These NOD/SCID data suggest that levels of GATA-2 may play a part in the in vivo control of stem and progenitor cell proliferation. Taken together, our data demonstrate that GATA-2 enforces a transcriptional program on stem and progenitor cells which suppresses their responses to proliferative stimuli with the result that they remain quiescent in vitro and in vivo.

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The Histone Deacetylase Inhibitor LAQ824 Maintains Normal Hematopoietic Progenitor cells (HPC) Associated with Induction of Notch Target Genes and Does Not Eliminate Leukemic HPC in Vitro.

Blood ◽

10.1182/blood.v112.11.1352.1352 ◽

2008 ◽

Vol 112 (11) ◽

pp. 1352-1352

Author(s):

Kerstin Schwarz ◽

Oliver Ottmann ◽

Annette Romanski ◽

Anja Vogel ◽

Jeffrey W. Scott ◽

...

Keyword(s):

Gene Expression ◽

Cell Cycle ◽

Progenitor Cells ◽

Gene Expression Analysis ◽

Target Genes ◽

Notch Pathway ◽

Cycle Arrest ◽

Cd34 Cells ◽

20 Nm

Abstract Introduction: Histone deacetylase inhibitors (DACi) have shown promising antileukemic activity by overcoming the differentiation block and inducing apoptosis in AML blasts. Recent data demonstrating enhanced maintenance and functional capacity of normal, but also leukemic hematopoietic progenitor cells (HPC) by the selective class I DACi valproic acid (VPA) have raised concerns about VPA in AML therapy. As more potent pan-DACi have entered clinical trials, we analysed the impact of the hydroxamic acid LAQ824 on phenotype and function of normal and leukemic CD34+ HPC and studied LAQ824- induced gene expression in the most primitive CD34+CD38- population of normal HPC. Methods: Differentiation and proliferation of CD34+ cells of bone marrow of healthy donors and peripheral blood samples of newly diagnosed AML patients were evaluated after one week of culture in presence of SCF, FLT3 ligand, TPO, IL-3 +/− LAQ824. The effect of LAQ824 on gene expression profiles in normal CD34+CD38− cells was assessed in three independent cell samples following incubation with cytokines +/− LAQ824 for 48 hours using Affymetrix GeneChip Human Genome U133 Plus 2.0 and Gene Spring Software. Serial replating of murine Sca1+Lin- HPC was performed in the presence of SCF, G-CSF, GM-CSF, IL-3, IL-6 +/− LAQ824. Results: Treatment of murine Sca1+Lin- HPC with LAQ824 (10 nM) significantly augmented colony numbers (p<0.01; n=3), and supported colony growth after four cycles of replating whereas no colonies developed in its absence beyond the second plating indicating preservation of functionally active multipotent progenitor cells. LAQ824 (10–20 nM) mediated acetylation of histone H3 in human normal and leukemic HPC. In normal HPC, LAQ824 (0–20 nM) lead to a dose-dependent increase in the proportion of CD34+ cells (20% w/o LAQ824 vs. 36% with LAQ824 20nM, p=0.07) and a significant reduction of CD14+ monocytes (18% vs. 3%, p= 0.02; n=3). The total number of CD34+ cells remained stable up to 10 nM and decreased at 20 nM. Gene expression analysis showed, that LAQ824 (20 nM) lead to an at least 3-fold up-regulation of 221 genes in all three HPC samples tested including HDAC11 and the cell cycle inhibitor p21waf1/cip1 known to be induced by most DACi in HPC. We identified several members of the notch pathway such as mastermind-like protein 2 (MAML2, a component of the active notch transcriptional complex) and notch target genes including the transcription factors HES1, HEY1 and HOXA10 and confirmed increase of protein levels by Western blotting. Reduced gene expression of mini-chromosome-maintenance (MCM) protein family members was observed which - in addition to up-regulation of p21 - has previously been associated with notch-mediated cell cycle arrest. To compare the effect of LAQ824 (20 nM) with VPA (150 ng/ml) on leukemic HPC, cells were cultured for one week with or w/o DACi. Of note, LAQ824 resulted in a 0.8-fold reduction of CD34+ leukemic HPC, while VPA expanded this population 2.2-fold compared with cytokine-treated controls (p=0.03; n=12). CFU numbers growing from CD34+ leukemic HPC in presence of LAQ824 did not differ significantly from controls (n=9). Conclusion: LAQ824 seems to diminish, but not eliminate normal as well as leukemic HPC as determined by phenotypic and functional in vitro analyses. Our gene expression analysis suggested an association with coactivator and target genes of the notch pathway and cell cycle arrest-inducing genes. In contrast to VPA, LAQ824 does not seem to support growth of leukemic HPC which may contribute to its more potent antileukemic effect.

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Culture of Mobilized Human CD34+ Cells in Hypoxic Conditions Improves Lentiviral Transduction Efficiency in SCID-Repopulating Cells

Blood ◽

10.1182/blood.v112.11.3545.3545 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3545-3545

Author(s):

Andre Larochelle ◽

Hezhi Gan ◽

Joshua R. Clevenger ◽

Cynthia E. Dunbar

Keyword(s):

Cell Cycle ◽

Transduction Efficiency ◽

Low Oxygen ◽

Lentiviral Transduction ◽

Hypoxic Conditions ◽

Human Cd34 ◽

Cd34 Cells ◽

Oxygen Conditions ◽

Cells Cultured

Abstract Under normal physiological conditions, hematopoietic stem cells (HSC) are sequestered in a hypoxic microenvironment in the bone marrow (BM), suggesting that low oxygen levels may play a fundamental role in the maintenance of normal stem cell function and protect these cells from the damaging effects of reactive oxygen species (ROS). In vitro culture of human BM CD34+ cells under hypoxic conditions has been shown to result in expansion of SCID-repopulating cells (SRC) as compared to culture under normoxic conditions (JCI112 (1); 126, 2003). We investigated whether culture of human mobilized CD34+ cells under low oxygen conditions (5% O2) could improve lentiviral transduction efficiency in SRC compared with culture under atmospheric O2 conditions (21%). G-CSF mobilized CD34+ cells from 4 healthy volunteers were prestimulated for 48 hours in the presence of cytokines (SCF, Flt-3 ligand and thrombopoietin) and subsequently transduced in fibronectin coated plates for 24 hours with SIN-lentiviral vectors carrying the GFP gene under the control of an EF1α promoter. In 3 experiments, cells were used for in vitro assays, including ROS, phenotypic, cell cycle, clonogenic and apoptosis assays. In one experiment, cells were injected intravenously in the tail vein of sublethally irradiated NOD/SCID IL2rγ −/− mice after transduction. Intracellular ROS levels increased more significantly in human CD34+ cells cultured for 3 days in 21% O2 compared with cells cultured in 5% O2. When cultures were maintained more than 3 days, ROS levels were similar between the 2 conditions. The levels of expansion of CD34+ cells compared with baseline were similar in hypoxia (3.9-fold) and normoxia (3.5-fold) (p=0.47). In contrast, the expansion of CD34+CD38− cells, a subpopulation enriched in HSCs, was greater in hypoxia (3.8-fold) than in normoxia (2.2-fold) (p=0.02). After 3 days of culture, the total number of colony-forming cells (CFC) increased 1.1-fold and 1.3-fold under hypoxic and normoxic conditions, respectively (p=0.32) compared with freshly isolated CD34+ cells. The level of O2 had no significant effect on lineage commitment of the CFC. At baseline, the majority (59.5%) of the CD34+ cells were in the G0 phase of the cell cycle. After 3 days in culture under hypoxic or normoxic conditions, the percentages of cells in G0 were 5.5% and 3.5%, respectively (p=0.03). The differences in percentages of cells in the G1 and G2/S/M phases of the cell cycle were not statistically different. The percentages of CD34+ apoptotic cells were similar between hypoxic (32.8%) and normoxic (29.5%) conditions (p=0.18). The pO2 also had no impact on CD34+ cell death (12.2% at 5% O2 and 11.7% at 21% O2, p=0.9). When considering the bulk of CD34+ cells after transduction with GFP-lentiviral vectors, there was no statistically significant difference in the percentages of GFP+ cells under hypoxia (22.3%) or normoxia (21%) (p=0.88). In contrast, when CD34+ cells cultured under hypoxia were injected into NOD/SCID IL2rγ −/− mice at the end of the transduction period, improved human cell engraftment and lentiviral transduction efficiency were detected 2 months after transplantation compared with CD34+ cells cultured under normoxia. Human cell engraftment in the mouse BM, as determined by flow cytometry using a human specific CD45 antibody, was 84% in the hypoxic group (n=4) and 54% in the normoxic group (n=4) (p=0.04). The level of O2 had no significant impact on the lineage commitment of the SRC, with a majority of CD45+CD15+ granulocytes in both groups. The percentage of GFP+CD45+ cells was 54% (hypoxia) and 43% (normoxia) (p=0.02), indicating an improved transduction efficiency of SRC under hypoxic conditions. Overall, these data indicate that human CD34+ cells cultured under low oxygen conditions maintain a more primitive phenotype and have an increased susceptibility to lentiviral transduction compared with cells cultured in 21% O2 conditions. Improved engraftment and transduction efficiency do not appear to be related to decreased apoptosis in lower O2 concentrations; instead, increased ROS production in higher O2 concentrations could lead to increased cell signaling and differentiation. Use of low O2 levels for in vitro transduction of human CD34+ cells could have important clinical implications in gene therapy.

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R4 RGS proteins suppress engraftment of human hematopoietic stem/progenitor cells by modulating SDF-1/CXCR4 signaling

Blood Advances ◽

10.1182/bloodadvances.2020003307 ◽

2021 ◽

Author(s):

Kathy Chan ◽

Chi Zhang ◽

Yorky T. S. Wong ◽

Xiao-Bing Zhang ◽

Chi Chiu Wang ◽

...

Keyword(s):

Stem Cell ◽

Progenitor Cells ◽

G Protein ◽

Ex Vivo ◽

Cell Trafficking ◽

Rgs Proteins ◽

Hematopoietic Stem ◽

Cell Functions ◽

Cd34 Cells

Homing and engraftment of hematopoietic stem/progenitor cells (HSPCs) into the bone marrow (BM) microenvironment are tightly regulated by the chemokine SDF-1 and its G-protein-coupled receptor CXCR4, which on engagement with G-protein subunits, trigger downstream migratory signals. Regulators of G-protein signaling (RGS) are GTPase-accelerating protein of the Gα subunit and R4 subfamily members have been implicated in SDF-1-directed trafficking of mature hematopoietic cells, yet their expression and influence on HSPCs remain mostly unknown. Here, we demonstrated that human CD34+ cells expressed multiple R4 RGS genes, of which RGS1, RGS2, RGS13,and RGS16 were significantly upregulated by SDF-1 in a CXCR4-dependent fashion. Forced overexpression of RGS1, RGS13, or RGS16 in CD34+ cellsnot only inhibited SDF-1-directed migration, calcium mobilization, and phosphorylation of AKT, ERK, and STAT3 in vitro, but also markedly reduced BM engraftment in transplanted NOD/SCID mice. Genome-wide microarray analysis of RGS-overexpressing CD34+ cells detected downregulation of multiple effectors with established roles in stem cell trafficking/maintenance. Convincingly, gain-of-function of selected effectors or ex vivo priming with their ligands significantly enhanced HSPC engraftment. We also constructed an evidence-based network illustrating the overlapping mechanisms of RGS1, RGS13 and RGS16 downstream of SDF-1/CXCR4 and Gαi. This model shows that these RGS members mediate compromised kinase signaling and negative regulation of stem cell functions, complement activation, proteolysis and cell migration. Collectively, this study uncovers an essential inhibitory role of specific R4 RGS proteins in stem cell engraftment, which could potentially be exploited to develop improved clinical HSPC transplantation protocols.

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Precise and Efficient Crispr/Cas9 Mediated Gene Editing in Long-Term Engrafting Human Hematopoietic Stem/Progenitor Cells

Blood ◽

10.1182/blood.v128.22.2312.2312 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2312-2312

Author(s):

Jack M Heath ◽

Aditi Chalishazar ◽

Christina S Lee ◽

William Selleck ◽

Cecilia Cotta-Ramusino ◽

...

Keyword(s):

Progenitor Cells ◽

Gene Editing ◽

Target Genes ◽

High Efficiency ◽

Hematopoietic Stem ◽

Cell Therapies ◽

Immunodeficient Mice ◽

Cd34 Cells

Abstract Transplantation of gene-modified autologous hematopoietic stem/progenitor cells (HSPCs) is an effective treatment for several hematologic diseases. However, a number of blood disorders may not be amenable to gene augmentation-based therapeutics. Targeted genome editing in human HSPCs could provide a therapeutic approach for these otherwise untreatable diseases. Here we demonstrate that CRISPR/Cas9 ribonucleoprotein (RNP) edits target genes in human HSPCs with high efficiency and precision. Human adult and umbilical cord blood (CB) CD34+ cells from 20 donors were electroporated with S. pyogenes or S. aureus Cas9 RNP targeting HBB, AAVS1, or CXCR4. Sequence analysis demonstrated up to 80% editing in CB CD34+ cells (mean±s.d: 61%±9%) and up to 57% in adult CD34+ cells (39%±13%). Delivery of Cas9 RNP and a single-stranded oligodeoxynucleotide donor (ssODN) led to up to 12% ssODN-mediated homology directed repair (HDR) and also led to a 20% increase in total gene editing (HDR+NHEJ)(RNP: 48%±15%; RNP+ssODN: 69%±8%). Both Cas9 RNP gene-edited CD34+ cells and donor-matched untreated control CD34+ cells reconstituted human hematopoiesis in primary and secondary recipient immunodeficient mice, with ~85% human CD45+ cell peripheral blood reconstitution 4 months after primary transplantation. Human T and B lymphoid, erythroid, and myeloid cells were detected in the spleen, thymus, and bone marrow with 20% CD34+ cell engraftment in the marrow of mice transplanted with RNP gene-edited or control CD34+ cells. The level of targeted gene editing in human erythroid, myeloid, and CD34+ cells that were recovered and enriched from the hematopoietic organs of primary recipients (~50%) was similar to the level of gene editing detected in the pre-infusion product (~60%). In summary, these results indicate that Cas9 gene-edited human HSPCs retain long-term engraftment potential and support multilineage blood reconstitution in vivo, thus supporting further investigation of CRISPR/Cas9 mediated gene-edited hematopoietic stem/progenitor cell therapies. Disclosures Heath: Editas Medicine: Employment. Chalishazar:Editas Medicine: Employment. Lee:Editas Medicine: Employment. Selleck:Editas Medicine: Employment. Cotta-Ramusino:Editas Medicine: Employment. Bumcrot:Editas Medicine: Employment. Gori:Editas Medicine: Employment.

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Preservation of Gene Edited Hematopoietic Stem Cells By Transient Overexpression of BCL-2 mRNA

Blood ◽

10.1182/blood.v128.22.3636.3636 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3636-3636

Author(s):

Carmen Flores Bjurström ◽

Michelle Mojadidi ◽

Anastasia Lomova ◽

Stephen Lai ◽

Sorel Fitz-Gibbon ◽

...

Keyword(s):

Cell Cycle ◽

Progenitor Cells ◽

Genome Editing ◽

Gene Correction ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

G2 Phase ◽

Transient Overexpression

Abstract Introduction: Site-specific gene correction of the point mutation causing sickle cell disease (SCD) in hematopoietic stem cells (HSCs) constitutes a precise strategy to generate a life-long source of gene-corrected erythrocytes that do not sickle. However, low efficiency of homology-directed repair (HDR) in primitive reconstituting HSCs is currently a limit to the use of therapeutic genome editing for treatment of severe genetic blood disorders. To identify the mechanism(s) that underlie decreased HDR efficacy in primitive HSCs relative to that in more mature progenitor populations, we assessed: efficiency of gene delivery and expression after electroporation of in vitro transcribed mRNA; functional ZFN-mediated endonuclease activity; cell cycle status; gene expression of key HDR genes; and cytotoxic responses; in the following immunophenotypically-defined human cell populations: HSCs (CD34+/CD38-/CD90+CD45RA-); multipotent progenitors (MPPs) (CD34+/CD38-/CD45RA-/CD90-); and progenitor cells (CD34+/CD38+). Methods: CD34+ cells were enriched from human G-CSF-mobilized peripheral blood and cultured for 1-3 days prior to electroporation of in vitro transcribed mRNA encoding GFP or a pair of zinc finger nucleases (ZFN). The ZFNs, designed to target the sickle mutation in exon 1 of the human beta-globin gene, were co-delivered with one of the homologous donor templates containing the corrective base (A/T): an integrase-deficient lentiviral vector (IDLV) or a 101bp single-stranded oligodeoxynucleotide (oligo). Percentages of alleles containing insertions/deletions (indels) and/or HDR-mediated gene correction were analyzed by high throughput sequencing (HTS). Acute cytotoxicity was determined by flow cytometry, identifying viable cells as 7AAD/AnnexinV neg. cells. To assess HDR-mediated gene correction in vivo after three months, gene-edited cells were transplanted (>1E6 viable CD34+ cells/mouse, I.V.) one day after electroporation into irradiated (250cGy) NOD/SCID/IL2R gamma-/- (NSG) mice. Results: In HSCs, MPPs and progenitor populations, no differences were observed in delivery and expression from electroporated GFP mRNA [%GFP(+) and MFI]. To assess the activity of ZFN mRNA in the stem and progenitor populations, ZFNs were delivered to CD34+ cells through electroporation of in vitrotranscribed mRNA. The CD34+ cells were then FACS-sorted into the respective populations and HTS was used to determine the percentage of alleles containing indels; the frequencies of indels were equivalent among the populations indicating equivalent ZFN mRNA activity. To evaluate the efficacy of site-specific HDR in HSCs and progenitor cells, ZFN mRNA was co-delivered with either an IDLV or an oligodeoxynucleotide donor template to modify the single base-pair involved in SCD. We observed lower percentage of HDR-mediated gene modification in the HSC population compared to progenitors with all donor templates. Due to the cell cycle phase restriction of HDR, we pre-stimulated CD34+ cells for 1-3 days prior to electroporation of ZFN mRNA and the oligo donor, and analyzed the cell cycle phases at the time of electroporation, and the frequencies of HDR and NHEJ produced by HTS. Only a small percentage of the immunophenotypic HSCs were in S/G2 phase after 24 hours of pre-stimulation; no HDR modification was observed in these cells. After 2-3 days of pre-stimulation, the HDR levels increased as the percentage of HSCs in S/G2 phase reached 20%. Importantly, assessment of relative cytotoxicity of the genome editing procedure (electroporation of ZFN mRNA and oligo donor) revealed a heightened sensitivity of HSCs/MPPs compared to progenitors, resulting in ~80% cell death in HSC vs. ~30% in progenitors under the conditions we are using. Transient expression of BCL-2 mRNA, co-electroporated with the genome editing reagents, improved HSC survival and significantly increased the numbers of HDR gene-corrected HSCs both in vitro and in vivo. Conclusions : These data indicate an elevated sensitivity to cytotoxicity from the gene editing process for HSCs compared to the mature progenitor cells under our conditions, which may explain the lower levels of gene modification seen using in vivo compared to in vitro assays. Transient overexpression of BCL-2 mRNA preserves HSC survival after HDR-based gene editing, increasing the frequency of gene-corrected HSCs. Disclosures Bjurström: UCLA: Patents & Royalties: 2016-290. Holmes:Sangamo BioSciences Inc: Employment.

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Gene expression profiling identifies significant differences between the molecular phenotypes of bone marrow–derived and circulating human CD34+ hematopoietic stem cells

Blood ◽

10.1182/blood.v99.6.2037 ◽

2002 ◽

Vol 99 (6) ◽

pp. 2037-2044 ◽

Cited By ~ 113

Author(s):

Ulrich Steidl ◽

Ralf Kronenwett ◽

Ulrich-Peter Rohr ◽

Roland Fenk ◽

Slawomir Kliszewski ◽

...

Keyword(s):

Gene Expression ◽

Cell Cycle ◽

Stem Cells ◽

Bone Marrow ◽

Dna Synthesis ◽

Progenitor Cells ◽

Hematopoietic Stem ◽

Human Cd34 ◽

Cd34 Cells ◽

Stem And Progenitor Cells

Abstract CD34+ hematopoietic stem cells are used clinically to support cytotoxic therapy, and recent studies raised hope that they could even serve as a cellular source for nonhematopoietic tissue engineering. Here, we examined in 18 volunteers the gene expressions of 1185 genes in highly enriched bone marrow CD34+(BM-CD34+) or granulocyte–colony-stimulating factor–mobilized peripheral blood CD34+(PB-CD34+) cells by means of cDNA array technology to identify molecular causes underlying the functional differences between circulating and sedentary hematopoietic stem and progenitor cells. In total, 65 genes were significantly differentially expressed. Greater cell cycle and DNA synthesis activity of BM-CD34+ than PB-CD34+ cells were reflected by the 2- to 5-fold higher expression of 9 genes involved in cell cycle progression, 11 genes regulating DNA synthesis, and cell cycle–initiating transcription factor E2F-1. Conversely, 9 other transcription factors, including the differentiation blocking GATA2 and N-myc, were expressed 2 to 3 times higher in PB-CD34+ cells than in BM-CD34+cells. Expression of 5 apoptosis driving genes was also 2 to 3 times greater in PB-CD34+ cells, reflecting a higher apoptotic activity. In summary, our study provides a gene expression profile of primary human CD34+ hematopoietic cells of the blood and marrow. Our data molecularly confirm and explain the finding that CD34+ cells residing in the bone marrow cycle more rapidly, whereas circulating CD34+ cells consist of a higher number of quiescent stem and progenitor cells. Moreover, our data provide novel molecular insight into stem cell physiology.

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Single-Cell STAT5 Signal Transduction Profiling in Normal and Leukemic Stem and Progenitor Cell Populations Reveals Highly Distinct Cytokine Responses.

Blood ◽

10.1182/blood.v114.22.2510.2510 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2510-2510

Author(s):

Lina Han ◽

Albertus T.J. Wierenga ◽

Marjan Rozenveld-Geugien ◽

Kim van de Lande ◽

Edo Vellenga ◽

...

Keyword(s):

Stem Cell ◽

Single Cell ◽

Progenitor Cells ◽

Progenitor Cell ◽

Self Renewal ◽

Hematopoietic Stem ◽

Gm Csf ◽

Cytokine Responses ◽

Cd34 Cells

Abstract Abstract 2510 Poster Board II-487 Signal Transducer and Activator of Transcription 5 (STAT5) plays critical roles in normal and leukemic hematopoiesis. We have observed that introduction of activated STAT5A in human stem/progenitor cells enhances long-term stem cell self-renewal, while lentiviral downmodulation of STAT5 expression in both normal as well as primary leukemic CD34+ cells impairs long-term growth and self-renewal. Many cytokines that act on the immature human stem cell compartment are known to be able to activate STAT5, such as TPO and SCF. Yet, little is known about the specificity and kinetics of STAT5 signaling in response to early-acting and lineage-restricted cytokines in specifically defined stem cell and progenitor compartments, particularly in the case of acute myeloid leukemia. We optimized a multiparametric flow cytometry protocol to analyze STAT5 phosphorylation upon cytokine stimulation in stem and progenitor cell compartments at the single-cell level. In normal cord blood (CB) cells, STAT5 phosphorylation was efficiently induced by TPO, IL-3 and GM-CSF within CD34+CD38− hematopoietic stem cells (HSCs). EPO and SCF-induced STAT5 phosphorylation was largely restricted to the megakaryocyte-erythroid progenitor (MEP) compartment, while G-CSF, as well IL-3 and GM-CSF were most efficient in inducing STAT5 phosphorylation in the myeloid progenitor compartments. Strikingly, mobilized adult peripheral blood (PB) CD34+ cells responded much less efficiently to cytokine-induced STAT5 activation, with the exception of TPO. In leukemic stem and progenitor cells, highly distinct cytokine responses were observed, differing significantly from their normal counterparts. A number of different types of responses were observed, being a) a strong STAT5 activation induced by TPO only; b) a strong STAT5 activation induced by IL3 and GM-CSF, but not TPO; c) STAT5 activation induced by various cytokines; and d) constitutive STAT5 phosphorylation that could not significantly be further induced by cytokines. These responses could not be predicted by the expression level of cytokine receptors. Also, heterogeneity existed in cytokine requirements for long-term expansion of AML CD34+ cells on stroma. In most examined cases multiple cytokines acted in an additive fashion in inducing long-term growth of primary AML CD34+ cells in bone marrow stromal cocultures, and in only 1 out of 10 cases cytokine-independent growth was observed. In none of the cases only IL3 or TPO were sufficient to induced long-term expansion of primary AML CD34+ cells. In conclusion, our study demonstrates variable cytokine responses in STAT5 phosphorylation in both normal and leukemic stem/progenitor cells. Disclosures: No relevant conflicts of interest to declare.

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Isolation of small, primitive human hematopoietic stem cells: distribution of cell surface cytokine receptors and growth in SCID-Hu mice

Blood ◽

10.1182/blood.v86.2.512.bloodjournal862512 ◽

1995 ◽

Vol 86 (2) ◽

pp. 512-523 ◽

Cited By ~ 45

Author(s):

JE Wagner ◽

D Collins ◽

S Fuller ◽

LR Schain ◽

AE Berson ◽

...

Keyword(s):

Cell Cycle ◽

Cell Surface ◽

Cell Population ◽

Size Fraction ◽

Cell Surface Antigens ◽

Inhibitory Protein ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

Cell Fractions

Human CD34+ cells were subfractionated into three size classes using counterflow centrifugal elutriation followed by immunoadsorption to polystyrene cell separation devices. The three CD34+ cell fractions (Fr), Fr 25/29, Fr 33/37, and Fr RO, had mean sizes of 8.5, 9.3 and 13.5 microns, respectively. The majority of cells in the large Fr RO CD34+ cell population expressed the committed stage antigens CD33, CD19, CD38, or HLA-DR and contained the majority of granulocyte- macrophage colony-forming units (CFU-GM), burst-forming units-erythroid (BFU-E), and CFU-mixed lineage (GEMM). In contrast, the small Fr 25/29 CD34+ cells were devoid of committed cell surface antigens and lacked colony-forming activity. When seeded to allogeneic stroma, Fr RO CD34+ cells produced few CFU-GM at week 5, whereas cells from the Fr 25/29 CD34+ cell population showed a 30- to 55-fold expansion of myeloid progenitors at this same time point. Furthermore, CD34+ cells from each size fraction supported ontogeny of T cells in human thymus/liver grafts in severe combined immunodeficient (SCID) mice. Upon cell cycle analyses, greater than 97% of the Fr 25/29 CD34+ cells were in G0/G1 phase, whereas greater proportions of the two larger CD34+ cell fractions were in active cell cycle. Binding of the cytokines interleukin (IL)-1 alpha, IL-3, IL-6, stem cell factor (SCF), macrophage inhibitory protein (MIP)-1 alpha, granulocyte colony- stimulating factor (G-CSF), and granulocyte-macrophage (GM)-CSF to these CD34+ cell populations was also analyzed by flow cytometry. As compared with the larger CD34+ cell fractions, cells in the small Fr 25/29 CD34+ cell population possessed the highest numbers of receptors for SCF, MIP1 alpha, and IL-1 alpha. Collectively, these results indicate that the Fr 25/29 CD34+ cell is a very primitive, quiescent progenitor cell population possessing a high number of receptors for SCF and MIP1 alpha and capable of yielding both myeloid and lymphoid lineages when placed in appropriate in vitro or in vivo culture conditions.

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