550. Primary Human CD34+ Hematopoietic Stem/Progenitor Cell (HSPC) Transduction By AAV6 Serotype Vectors: Strategies for Overcoming the Donor-Variation

Abstract SHP-2 (ptpn11), a Src homology 2 (SH2) domain-containing protein-tyrosine phosphatase, is expressed at high levels in hematopoietic cells and regulates downstream signaling from growth factor (GF) receptors. SHP-2 has been shown to play an important role in murine hematopoiesis. Moreover, several SHP-2 activating mutations have been identified in myeloid malignancies and there is interest in the development of SHP-2 inhibitors for cancer treatment. On the other hand previous report suggested that SHP-2 inhibition was associated with enhanced GF responsiveness in human hematopoietic cell lines. However the role of SHP-2 signaling in normal human hematopoietic stem and progenitor cell growth has not been studied. Here we investigated the function of SHP-2 in normal human hematopoiesis by inhibiting SHP-2 expression in cord blood (CB) CD34+ cells with stable SHP-2 shRNA expression. We transduced CB CD34+ cells with lentivirus vectors coexpressing SHP-2 specific shRNAs (Si-1 or Si-2) or a control shRNA (Ctrl) and RFP and selected RFP expressing CD34+ cells by flow cytometry sorting. We observed >80% inhibition of SHP-2 expression by Western blotting in Si-1 or Si-2 shRNA transduced cells compared with Ctrl shRNA transduced cells. We observed that culture with increasing concentrations of GF was associated with markedly reduced GF-induced stimulation of proliferation of SHP-2-knockdown CD34+ cells compared to controls. In addition we observed significantly increased apoptosis of SHP-2-knockdown CD34+ cells cultured under low and high GF conditions compared to controls, but little increase in apoptosis in GF-deprived cells, indicating markedly reduced response of SHP-2-knockdown cells to GF-mediated promotion of cell survival. SHP-2-knockdown CD34+ cells also demonstrated significantly reduced expansion in cell numbers following culture in high GF conditions compared with controls (115.3, 25.5 and 10.4 fold expansion for Ctrl, Si-1 and Si-2 at day 7). Analysis of the nature of cells generated in GF culture showed significantly reduced generation of both myeloid (CD33+, CD11b+ and CD14+) and erythroid cells from SHP-2-knockdown CD34+ cells compared with controls, with relatively greater inhibition of myeloid compared with erythroid differentiation. On the other hand CD34+ cell numbers were retained at levels similar to controls after culture. We also observed significantly reduced cell expansion and differentiation and higher apoptotic rates of SHP-2-knockdown cells cultured under either myeloid promoting (IL-3+SCF+G-CSF+GM-CSF) or erythroid promoting (SCF+EPO) GF conditions. SHP-2-knockdown cells demonstrated reduced activation of MAPK and STAT5 but not Akt on Western blotting that was associated with reduced MCL-1 expression, consistent with their reduced GF mediated proliferation and survival. Expression of the transcription factors SCL1, GATA-1, NF-E2 and FOG-1 was increased in SHP-2 knockdown CD34+ cells compared to controls, consistent with the relatively higher retention of CD34+ and erythroid cells compared with myeloid cells after culture. In conclusion, we show that SHP-2 knockdown in human CD34+ cells results in markedly decreased responsiveness to GF stimulation with significantly increased apoptosis, markedly diminished proliferation and reduced generation of differentiated cells during GF culture. A relative retention of the CD34+ cell population was seen despite increased apoptosis, which may be the result of reduced cell turnover and altered transcription factor expression in SHP-2-knockdown cells, and is in contrast to reduced stem cell self-renewal observed following SHP-2 knockdown in murine models. These results indicate a critical role for SHP-2 in GF mediated signaling responses in human hematopoietic stem/progenitor cells. These studies also caution that therapeutic SHP-2 inhibition could be associated with significant hematopoietic toxicity.

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The human CD34 hematopoietic stem cell antigen promoter and a 3' enhancer direct hematopoietic expression in tissue culture

Blood ◽

10.1182/blood.v80.12.3051.3051 ◽

1992 ◽

Vol 80 (12) ◽

pp. 3051-3059 ◽

Cited By ~ 26

Author(s):

TC Burn ◽

AB Satterthwaite ◽

DG Tenen

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Tissue Culture ◽

Stem Cell ◽

Transcription Factors ◽

Progenitor Cell ◽

Hematopoietic Stem ◽

Culture Cells ◽

Human Cd34 ◽

Tissue Culture Cells

Abstract The human CD34 hematopoietic stem cell antigen is a highly glycosylated type 1 membrane protein of unknown function. CD34 is expressed on 1% to 4% of bone marrow cells, including pluripotent stem cells and committed progenitors of each hematopoietic lineage. CD34 has also been shown to be expressed on the small vessel endothelium of a variety of tissues and on a subset of bone marrow stromal cells. We have chosen to use the human CD34 gene as model to examine the transcription factors and cis-elements required for stem cell/progenitor cell-specific gene regulation. We show here that the CD34 gene is transcriptionally regulated in tissue culture cells. Using a luciferase reporter gene, we have isolated and characterized an active CD34 promoter. A CD34- luciferase construct, containing 4.5 kb of 5′ flanking DNA from a CD34 genomic clone, was 30-fold more active in CD34+ tissue culture cells than in HeLa cells. Sequences from the 3′ end of the CD34 gene were shown to have enhancing activity in CD34+ T-lymphoblastic RPMI-8402 cells and not in CD34- U937 cells or in nonhematopoietic HeLa cells. We also show that a cytidine-guanosine island in the 5′ end of the CD34 gene is heavily methylated in two CD34- hematopoietic cell lines and demethylated in two CD34+ cell lines. Analysis of the CD34 promoter should result in the identification of stem cell/progenitor cell- specific transcription factors and should provide a means to direct the expression of heterologous genes in hematopoietic stem cells and progenitors.

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10. High-Efficiency Transduction of Primary Human CD34+ Hematopoietic Stem/Progenitor Cells by AAV6 Serotype Vectors: Strategies for Overcoming Donor Variation and Implications in Genome Editing

Molecular Therapy ◽

10.1016/s1525-0016(16)32819-2 ◽

2016 ◽

Vol 24 ◽

pp. S5-S6

Author(s):

Zifei Yin ◽

Kanit Bukhai ◽

George Aslanidi ◽

Chen Ling ◽

Mengqun Tan ◽

...

Keyword(s):

Progenitor Cells ◽

Genome Editing ◽

High Efficiency ◽

Hematopoietic Stem ◽

Human Cd34 ◽

Donor Variation

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Expression of TCF3-ZNF384 in Human Hematopoietic Cells Induces Lineage Disruption in Vitro and Acute Leukemia in Vivo

Blood ◽

10.1182/blood-2018-99-118855 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 550-550 ◽

Cited By ~ 1

Author(s):

Kirsten Dickerson ◽

Hiroki Yoshihara ◽

Laura Janke ◽

Charles G. Mullighan

Keyword(s):

Acute Leukemia ◽

Blood Cells ◽

Research Funding ◽

Progenitor Cell ◽

Wild Type ◽

Hematopoietic Stem ◽

Human Cd34 ◽

Cord Blood Cells

Abstract Introduction: Rearrangement of ZNF384, a transcription factor of poorly characterized function, defines a subtype of acute leukemia that may manifest as either B-ALL with aberrant myeloid marker expression or B/myeloid mixed phenotype acute leukemia (MPAL). Such leukemias are characterized by chromosomal rearrangements that result in the fusion of a diverse group of partners, often transcription factors or epigenetic modifiers, to ZNF384. Our prior studies have shown that ZNF384 B-ALL and B/myeloid MPAL are genomically indistinguishable, and that the fusion may be identified in a subset of hematopoietic stem cells, suggesting that the acquisition of a ZNF384 fusion in a primitive progenitor directly perturbs hematopoietic differentiation. The goals of this study were to determine the effect of expression of ZNF384 rearrangements on human hematopoietic stem and progenitor cell differentiation in vitro and in vivo, using TCF3-ZNF384 as a commonly observed exemplar of this form of leukemia. Methods: For in vitro experiments, human CD34+ cord blood cells were sorted into stem and progenitor populations (hematopoietic stem cell (HSC), multipotent progenitor (MPP), common myeloid progenitor (CMP), granulocyte-macrophage progenitor (GMP), and megakaryocyte-erythroid progenitor (MEP)) and lentivirally infected with wild type ZNF384, TCF3-ZNF384, or vector control. Single cells were sorted onto an MS-5 stromal layer and the immunophenotype of colonies was determined 15 days later by flow cytometry. In vivo studies were performed by sorting human CD34+ cord blood cells into stem-enriched (CD34+CD38-) or progenitor-enriched (CD34+CD38+) populations and lentivirally infecting with wild type ZNF384, TCF3-ZNF384, or vector control. Cells were transplanted into sub-lethally irradiated NOD.Cg-Prkdcscid Il2rgtm1Wjl Tg(CMV-IL3,CSF2,KITLG)1Eav/MloySzJ (NSG-SGM3) mice. Results: Single-cell MS-5 stromal experiments revealed that expression of the fusion protein perturbed hematopoietic differentiation. In all stem and progenitor populations, cells expressing TCF3-ZNF384 lost the ability to differentiate into erythroid colonies. HSC, MPP, and CMP cells expressing the fusion most commonly form undifferentiated, CD45+, CD33+ colonies. Additionally, GMP and MEP cells expressing the fusion lost their ability to form colonies. Human CD34+ cells expressing TCF3-ZNF384 successfully initiate leukemia in NSG-SGM3 mice with a median latency of 123 days. Mice presented with anemia and pathological analysis using hematoxylin and eosin staining showed infiltration of leukemic cells into the bone marrow, spleen, liver, central nervous system, and ovary. Additionally, CD33, myeloperoxidase, and major basic protein staining confirmed myeloid leukemia with a subset of eosinophil differentiation. Conclusion: Our results demonstrate that hematopoietic lineage determination is altered by the expression of TCF3-ZNF384 in human stem and progenitor cell populations. Additionally, we have created the first model of TCF3-ZNF384 leukemia which mimics the complexity of lineage deregulation in ZNF384-rearranged leukemia. Disclosures Mullighan: Cancer Prevention and Research Institute of Texas: Consultancy; Amgen: Honoraria, Speakers Bureau; Loxo Oncology: Research Funding; Pfizer: Honoraria, Research Funding, Speakers Bureau; Abbvie: Research Funding.

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The human CD34 hematopoietic stem cell antigen promoter and a 3' enhancer direct hematopoietic expression in tissue culture

Blood ◽

10.1182/blood.v80.12.3051.bloodjournal80123051 ◽

1992 ◽

Vol 80 (12) ◽

pp. 3051-3059 ◽

Cited By ~ 1

Author(s):

TC Burn ◽

AB Satterthwaite ◽

DG Tenen

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Tissue Culture ◽

Stem Cell ◽

Transcription Factors ◽

Progenitor Cell ◽

Hematopoietic Stem ◽

Culture Cells ◽

Human Cd34 ◽

Tissue Culture Cells

The human CD34 hematopoietic stem cell antigen is a highly glycosylated type 1 membrane protein of unknown function. CD34 is expressed on 1% to 4% of bone marrow cells, including pluripotent stem cells and committed progenitors of each hematopoietic lineage. CD34 has also been shown to be expressed on the small vessel endothelium of a variety of tissues and on a subset of bone marrow stromal cells. We have chosen to use the human CD34 gene as model to examine the transcription factors and cis-elements required for stem cell/progenitor cell-specific gene regulation. We show here that the CD34 gene is transcriptionally regulated in tissue culture cells. Using a luciferase reporter gene, we have isolated and characterized an active CD34 promoter. A CD34- luciferase construct, containing 4.5 kb of 5′ flanking DNA from a CD34 genomic clone, was 30-fold more active in CD34+ tissue culture cells than in HeLa cells. Sequences from the 3′ end of the CD34 gene were shown to have enhancing activity in CD34+ T-lymphoblastic RPMI-8402 cells and not in CD34- U937 cells or in nonhematopoietic HeLa cells. We also show that a cytidine-guanosine island in the 5′ end of the CD34 gene is heavily methylated in two CD34- hematopoietic cell lines and demethylated in two CD34+ cell lines. Analysis of the CD34 promoter should result in the identification of stem cell/progenitor cell- specific transcription factors and should provide a means to direct the expression of heterologous genes in hematopoietic stem cells and progenitors.

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