scholarly journals STAT5 is required for long-term maintenance of normal and leukemic human stem/progenitor cells

Blood ◽  
2007 ◽  
Vol 110 (8) ◽  
pp. 2880-2888 ◽  
Author(s):  
Hein Schepers ◽  
Djoke van Gosliga ◽  
Albertus T. J. Wierenga ◽  
Bart J. L. Eggen ◽  
Jan Jacob Schuringa ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Myeloid Leukemia ◽  
Cell Number ◽  
Hematopoietic Stem ◽  
Fold Reduction ◽  
Cd34 Cells ◽  
Stem And Progenitor Cells ◽  
Precise Role ◽  
Term Maintenance

Abstract The transcription factor STAT5 fulfills a distinct role in the hematopoietic system, but its precise role in primitive human hematopoietic cells remains to be elucidated. Therefore, we performed STAT5 RNAi in sorted cord blood (CB) and acute myeloid leukemia (AML) CD34+ cells by lentiviral transduction and investigated effects of STAT5 downmodulation on the normal stem/progenitor cell compartment and the leukemic counterpart. STAT5 RNAi cells displayed growth impairment, without affecting their differentiation in CB and AML cultures on MS5 stroma. In CB, limiting-dilution assays demonstrated a 3.9-fold reduction in progenitor numbers. Stem cells were enumerated in long-term culture-initiating cell (LTC-IC) assays, and the average LTC-IC frequency was 3.25-fold reduced from 0.13% to 0.04% by STAT5 down-regulation. Single-cell sorting experiments of CB CD34+/CD38− cells demonstrated a 2-fold reduced cytokine-driven expansion, with a subsequent 2.3-fold reduction of progenitors. In sorted CD34+ AML cells with constitutive STAT5 phosphorylation (5/8), STAT5 RNAi demonstrated a reduction in cell number (72% ± 17%) and a decreased expansion (17 ± 15 vs 80 ± 58 in control cultures) at week 6 on MS5 stroma. Together, our data indicate that STAT5 expression is required for the maintenance and expansion of primitive hematopoietic stem and progenitor cells, both in normal as well as leukemic hematopoiesis.

scholarly journals Long-Term Hydroxyurea Use Is Associated with Lower Levels of Hematopoietic Stem and Progenitor Cells in Patients with Sickle Cell Disease

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 985-985
Author(s):  
Seda S. Tolu ◽  
Kai Wang ◽  
Zi Yan ◽  
Andrew Crouch ◽  
Gracy Sebastian ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Progenitor Cells ◽  
Research Funding ◽  
Cell Disease ◽  
Transfusion Therapy ◽  
Hematopoietic Stem ◽  
Hematopoietic System ◽  
Cd34 Cells ◽  
Stem And Progenitor Cells

Background Sickle cell disease (SCD) is curable by transplantation and potentially by gene therapy, and is generally treated by a combination of blood transfusion and Hydroxyurea (HU). Characterizing the hematopoietic system in SCD patients is important because the long-term effect of HU treatment are not known, and because of lower than expected efficacy of transduction and transplantation in Hematopoietic Stem and Progenitor Cells (HSPCs) in recent gene therapy trials. Previous studies have shown that the number of bone marrow (BM) CD34+ cells is elevated in SCD patients and that HU treatment is associated with decreased level of CD34+ cells in the peripheral blood (PB) and BM relative to steady state patients. However, hematopoiesis in SCD patients naive or treated with HU or transfusion remains poorly understood. Here, we report on the characterization of the HSPC compartment in patients with SCD by prospective isolation of 49f+ long-term Hematopoietic Stem Cells (49f+LT-HSCs), Multipotent Progenitors (MPPs), Common Myeloid Progenitors (CMPs), Megakaryocyte-Erythroid Progenitors (MEPs), and Granulocyte-Monocyte Progenitors (GMPs). Methods After obtaining consent, PB and/or BM were collected from 69 patients with HBSS/SB0, aged 12 to 45years, and 25 healthy adult African American controls. Patients were divided into chronic transfusion therapy (n=19), HU (n=31) and naïve (n=19) groups. Frozen mono-nuclear cells were analyzed by flow cytometry on a BD LSRII using CD 49f, 90 45Ra, 123, 235a, 38, 34, 33 and lineage antibodies. Results FACS analysis revealed that the number PB CD34+ cells was 2.5 CD34+/uL of blood in the HU group as compared to 19 CD34+/uL in the exchange and naive groups, and 7.3 CD34+/uL in the control group (q-value <0.05 in all cases). Analysis with additional markers revealed that the decrease in circulating HSPCs in the HU group affected the entire hematopoietic system since the number of 49f+LT-HSCs, MPPs, CMPs, MEPs were all significantly lower in the HU group. The decrease in cell number in the HU group, however, was not homogeneous. The proportion of LT-HSCs was higher in the HU and transfusion groups when compared to the naive and control groups. The HU group also had the lowest proportion of MPPs and GMPs, as well as the highest proportion of MEPs. We then investigated hematopoiesis as a function of the length of HU treatment to elucidate the long-term treatment effect of this cytotoxic agent. Patients > 18 years of age that had been treated on HU for at least three years exhibited a strong statistically significant negative correlation between years on HU and CD34+/uL (R2 = 0.41), LT-HSC/uL (R2 = 0.35), MPP/uL (R2 =0.43), CMP/uL (R2 = 0.37), MEP/uL (R2 = 0.25) and GMP/uL (R2 0.39, p<0.01 in all cases). Importantly there was no correlation between WBC counts, age, HU dose, or serum erythropoietin level versus the numbers of any HSPC/uL. Lastly, we compared the number of HSPCs in paired PB and BM samples 10 controls and 4 SCD patients. This revealed that the numbers of CD34+, HSCs, MPPs, CMPs, GMPs and MEPs in the PB and BM were well correlated (r2 in 0.6-0.8 range) suggesting that in first approximation, results obtained in the PB reflect changes in the BM rather than changes in egress of HSPCs from the BM. Discussion We have observed lower numbers of circulating CD34+,49f+LT- HSCs, MPPs, CMP, GMP and MEPs in individuals with HBSS on HU therapy when compared to naive, chronic transfusion and, to a lesser extent, controls. Furthermore we observed subtle differences in the proportion of various circulating stem and progenitor cells (HSPCs) suggesting that the various treatments affects hematopoiesis in complex ways. The strong negative correlations between the length of HU treatment and the numbers of HSPCs can be explained either by decreased cell mobilization to the periphery, or by a depletion of the HSPC numbers in the BM overtime. Most patients undergoing gene therapy trials are currently taken off HU and placed on transfusion therapy for several months to increase CD34+ cell collection and LT-HSC transduction efficiency. We observed a greater number of circulating 49f+LT-HSC/uL of blood in the transfusion group than in the HU group, but the proportion of 49f+LT-HSC relative to the number of CD34+ were similar in both groups. Functional studies may help determine whether 49f+LT-HSCs from the transfusion group are qualitatively different from of the HU group and more amenable to gene therapy. Figure Disclosures Manwani: Novartis: Consultancy; Pfizer: Consultancy; GBT: Consultancy, Research Funding. Minniti:Doris Duke Foundation: Research Funding.

CITED2 Modulates Differentiation and Proliferation of Normal and Leukemic Hematopoietic Stem and Progenitor Cells Downstream of PU.1

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 922-922
Author(s):  
Hein Schepers ◽  
Patrick M. Korthuis ◽  
Jan J. Schuringa ◽  
Gerald de Haan ◽  
Edo Vellenga
Keyword(s):  
Stem Cells ◽  
Progenitor Cells ◽  
Binding Sites ◽  
Myeloid Differentiation ◽  
Leukemic Stem Cells ◽  
Hematopoietic Stem ◽  
Cell Numbers ◽  
Cd34 Cells ◽  
Stem And Progenitor Cells

Abstract Abstract 922 Recently, it was demonstrated that the transcriptional co-activator CITED2 has a conserved role in the maintenance of normal adult hematopoiesis. Little is known regarding the regulation of CITED2, its expression levels in leukemic stem cells (LSCs) and whether CITED2 expression contributes to leukemogenesis. Using microarray data, we found variable CITED2 expression in ∼90% of sorted CD34+ AML cells (n=46). Q-PCR on 12 of these samples indicated that 50% of the leukemic samples displayed higher expression of CITED2 as compared to mobilized peripheral blood (PB) or cord blood (CB)-derived CD34+ hematopoietic stem and progenitor cells (HSCPs). To verify whether this expression of CITED2 in leukemic cells is functional, we performed RNAinterference on primary CD34+ cells from acute myeloid leukemia (AML) patients (n=9). AML samples with high CITED2 expression were susceptible to lentiviral downregulation of CITED2 as evidenced by the loss of transduced cells from long-term leukemic cultures. To investigate a potential role of CITED2 in leukemogenesis, lentiviral gain-of-function experiments were performed with CB-derived CD34+ HSCPs. These experiments indicate that CITED2 expression increases cell numbers up to 5-fold in long-term culture-initiating cell (LTC-iC) experiments. This cell expansion on MS5-stromal cultures was paralleled by a short-term maintenance of progenitors. Colony-forming-cell (CFC) assays demonstrated a 3.5 fold higher output of CFC colonies compared to control cultures up to 2 weeks of MS-5 co-culture. CITED2-expressing colonies were larger than control colonies, verifying the increased cell numbers in LTC-iC experiments. To further dissect the effects of CITED2, transduced CD34+CD38− HSCs and CD34+CD38+ progenitors were sorted for single-cell liquid cultures and cell-divisions were followed for 6 days. Analysis of 360 single CD34+CD38− HSCs indicated that CITED2 overexpression leads to an enhanced quiescence (cells with no division) and decreased proliferation (cells that divide). However, tracking the divisions of 360 single CD34+CD38+ progenitor cells demonstrated the opposite: Cells overexpressing CITED2 divided more than control cells. These data are consistent with a role for CITED2 in leukemic stem cells (LSCs), where LSCs are thought to be more quiescent than leukemic progenitors. Since leukemias are also characterized by a differentiation block, we subsequently analyzed the differentiation of CB-derived CD34+ HSCPs upon overexpression of CITED2. In LTC-iC experiments, a shift in myelo-monocytic differentiation was observed. Enhanced CITED2 expression led to an increased percentage of CD15-positive cells (64%) at the expense of CD14-positive cells (9%) compared to control cultures (35% and 32% respectively). This bias towards granulocytic differentiation was also observed on May-Grunwald-Giemsa (MGG) stains and was furthermore confirmed in CFC assays. Furthermore, when analyzing erythroid differentiation, a clear reduction in CD71bright GPA+ cells could be observed in CITED2 expressing cells, compared to control cells (2% vs. 12% respectively), which was confirmed by CFC assays and MGG stains. Towards clarifying the regulation of CITED2, we scanned the CITED2 promoter for transcription factor binding sites and identified several PU.1 binding sites. Gene expression comparison between PU.1 and CITED2 in a panel of primary AML samples indicated that, apart from FAB M2 AMLs, PU.1 expression is inversely correlated with CITED2 expression. To functionally investigate this inverse correlation, we performed chromatin immunoprecipitations (ChIP) and demonstrated that PU.1 is indeed able to bind to the PU.1 binding sites in the CITED2 promoter of CB CD34+ cells. Subsequent overexpression of PU.1 in CB CD34+ HSPCs led to a 2-fold reduction in CITED2 expression. Taken together, we propose a model in which PU.1 tightly regulates CITED2 expression during normal myeloid differentiation. In certain AML subsets, this model would predict that low PU.1 expression results in failure to lower CITED2 expression below a certain threshold, which subsequently results in maintenance of LSC quiescence. Furthermore, the enhanced CITED2 levels result in an increased proliferation of downstream leukemic progenitors, where together with low PU.1 levels the normal myeloid differentiation program is perturbed contributing to leukemic development. Disclosures: No relevant conflicts of interest to declare.

Expansion of Normal and Leukemic Human Hematopoietic Stem/Progenitor Cells Requires Rac-Mediated Interaction with Stromal Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1398-1398
Author(s):  
Jan Jacob Schuringa ◽  
Marjan Rozenveld-Geugien ◽  
Inge Baas ◽  
Djoke van Gosliga ◽  
Edo Vellenga
Keyword(s):  
Signal Transduction ◽  
Progenitor Cells ◽  
Stromal Cells ◽  
Myeloid Leukemia ◽  
Dominant Negative ◽  
Dramatic Decrease ◽  
Human Cord Blood ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract We have studied Rac signal transduction in human cord-blood (CB) and acute myeloid leukemia (AML) CD34+ cells and determined that Rac proteins are critically involved in the interaction between human stem/progenitor cells and stroma. Constitutive activation of Rac signaling was achieved by retroviral introduction of Rac1-V12 into CB-derived CD34+ cells, while inhibition of Rac signaling was established by retroviral introduction of dominant negative Rac1-N17 or by utilizing the Rac inhibitor NSC23766. Inhibition of Rac signaling resulted in a proliferative disadvantage of CB-CD34+ cells when cultured on MS5 stromal cells. Cells were severely disturbed in their migration towards and direct association with MS5 stroma when Rac signaling was inhibited. The Long Term Culture-Initiating Cells (LTC-ICs) migrated underneath the stromal MS5 layer within 24 hrs after plating, and similar results were obtained for about 50% of the Colony Forming Cells (CFCs). However, transient inhibition of Rac signaling for 24–72 hrs resulted in a shift of LTC-ICs and CFCs to the suspension phase as determined by colony assays and CAFC week 5 enumeration. When Rac signaling signal transduction was inhibited during the 5 week coculture period a dramatic decrease in LTC-IC frequency from 0.6% to 0.15% was observed. Many of these phenotypes were reversed in the presence of activated Rac1-V12, including improved migration towards and association with MS5 cells and elevated LTC-IC frequencies. Importantly, in CAFC assays using AML cells (n=8) that were enriched for leukemic stem cells on the basis of a CD34+/CD38low phenotype we observed a dramatic decrease in leukemic CAFC formation as well as strongly diminished clonal expansion in the presence of the Rac inhibitor NSC23766. Taken together, our data indicate that Rac signal transduction is required for the maintenance and expansion of both normal as well as leukemic stem/progenitor cells by mediating their interaction with stromal cells.

Repression of BMI1 in normal and leukemic human CD34+ cells impairs self-renewal and induces apoptosis

Blood ◽  
2009 ◽  
Vol 114 (8) ◽  
pp. 1498-1505 ◽  
Author(s):  
Aleksandra Rizo ◽  
Sandra Olthof ◽  
Lina Han ◽  
Edo Vellenga ◽  
Gerald de Haan ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Myeloid Leukemia ◽  
Oxygen Species ◽  
Self Renewal ◽  
Human Cd34 ◽  
Bmi1 Expression ◽  
Cd34 Cells ◽  
Stem And Progenitor Cells ◽  
Cord Blood Cd34

Abstract High expression of BMI1 in acute myeloid leukemia (AML) cells is associated with an unfavorable prognosis. Therefore, the effects of down-modulation of BMI1 in normal and leukemic CD34+ AML cells were studied using a lentiviral RNA interference approach. We demonstrate that down-modulation of BMI1 in cord blood CD34+ cells impaired long-term expansion and progenitor-forming capacity, both in cytokine-driven liquid cultures as well as in bone marrow stromal cocultures. In addition, long-term culture-initiating cell frequencies were dramatically decreased upon knockdown of BMI1, indicating an impaired maintenance of stem and progenitor cells. The reduced progenitor and stem cell frequencies were associated with increased expression of p14ARF and p16INK4A and enhanced apoptosis, which coincided with increased levels of intracellular reactive oxygen species and reduced FOXO3A expression. In AML CD34+ cells, down-modulation of BMI1 impaired long-term expansion, whereby self-renewal capacity was lost, as determined by the loss of replating capacity of the cultures. These phenotypes were also associated with increased expression levels of p14ARF and p16INK4A. Together our data indicate that BMI1 expression is required for maintenance and self-renewal of normal and leukemic stem and progenitor cells, and that expression of BMI1 protects cells against oxidative stress.

Long-term maintenance of human hematopoietic stem/progenitor cells by expression of BMI1

Blood ◽  
2008 ◽  
Vol 111 (5) ◽  
pp. 2621-2630 ◽  
Author(s):  
Aleksandra Rizo ◽  
Bert Dontje ◽  
Edo Vellenga ◽  
Gerald de Haan ◽  
Jan Jacob Schuringa
Keyword(s):  
Progenitor Cells ◽  
Ex Vivo ◽  
Scid Mice ◽  
Polycomb Group ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Colony Forming Capacity ◽  
Term Maintenance

The polycomb group (PcG) gene BMI1 has been identified as one of the key epigenetic regulators of cell fates during different stages of development in multiple murine tissues. In a clinically relevant model, we demonstrate that enforced expression of BMI1 in cord blood CD34+ cells results in long-term maintenance and self-renewal of human hematopoietic stem and progenitor cells. Long-term culture-initiating cell frequencies were increased upon stable expression of BMI1 and these cells engrafted more efficiently in NOD-SCID mice. Week 5 cobblestone area-forming cells (CAFCs) were replated to give rise to secondary CAFCs. Serial transplantation studies in NOD-SCID mice revealed that secondary engraftment was only achieved with cells overexpressing BMI1. Importantly, BMI1-transduced cells proliferated in stroma-free cytokine-dependent cultures for more than 20 weeks, while a stable population of approximately 1% to 5% of CD34+ cells was preserved that retained colony-forming capacity. Whereas control cells lost most of their NOD-SCID engraftment potential after 10 days of ex vivo culturing in absence of stroma, NOD-SCID multilineage engraftment was retained by overexpression of BMI1. Thus, our data indicate that self-renewal of human hematopoietic stem cells is enhanced by BMI1, and we classify BMI1 as an intrinsic regulator of human stem/progenitor cell self-renewal.

scholarly journals Multicolor Flowcytometry Analysis of Hematopoietic Stem and Progenitor Cells Subsets Among Basal and Mobilized Peripheral CD34+ Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5117-5117
Author(s):  
Valentina Giai ◽  
Elona Saraci ◽  
Eleonora Marzanati ◽  
Christian Scharenberg ◽  
Monica De Stefanis ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Healthy Volunteers ◽  
Progenitor Cells ◽  
Hematopoietic Stem ◽  
Multipotent Progenitors ◽  
Cd34 Cells ◽  
Stem And Progenitor Cells ◽  
Progenitor Compartment

Abstract BACKGROUND: In the recent years, numerous studies based on multicolor flowcytometry have analyzed the different subpopulations of bone marrow (BM) hematopoietic stem and progenitor cells (HSPCs) (Manz MG et al, PNAS 2002; Majeti R et al, Cell Stem Cell 2007): the common myeloid progenitors (CMPs: Lin-CD34+CD38+CD45RA-CD123+), the granulocyte-macrophage progenitors (GMPs: Lin-CD34+CD38+CD45RA+CD123+) and the megakaryocyte-erythroid progenitors (MEPs: Lin-CD34+CD38+CD45RA-CD123-) constitute the progenitor compartment, while the hematopoietic stem cells (HSCs: Lin-CD34+CD38- CD45RA-CD90+), the multipotent progenitors (MPPs: Lin-CD34+CD38- CD45RA-CD90-) and the lymphoid-myeloid multipotent progenitors (LMPPs: Lin-CD34+CD38- CD45RA+CD90-) represent the more immature HSPCs. In animal models, the progenitor compartment includes short-term repopulating cells, leading to the hematological recovery in the first 5 weeks after transplantation, whereas the stem cell compartment comprehends the long-term repopulation cells, responsible for the long-term hematological recovery. However, very little is known about the different subpopulations of HSPCs among peripheral blood (PB) CD34+ in basal state and after mobilization for harvest and transplantation. Our study was conducted to analyze PB CD34+ cells from healthy volunteers and from hematological patients during CD34+ cells mobilization. Our main aim was to understand if the proportions of different HSPCs among PB CD34+ cells were similar to those found in BM and whether the mobilizing regimens employed in chemo treated patients differently affected CD34+ cells subfractions in PB. METHODS: multicolor flowcytometry was used to analyze CD34+ cells from 4 BM samples and 9 PB samples from healthy volunteers and 32 PB samples from hematological patients prior CD34+ cells harvesting. RESULTS: Percentages of CD34+ cells subpopulations were different in basal PB compared to the BM: indeed, CMPs, GMPs and MEPs constituted respectively 27.6% ± 9.5, 23.8% ± 7.2 and 27.6% ± 16.2 of BM CD34+ cells and 47.8% ± 9.5, 10.3% ± 6.9 and 16.1% ± 7.6 of the total PB CD34+ cells. HSCs constituted 2.1% of BM and 1.5% of PB CD34+ cells. The differences between BM and circulating CMPs and GMPs were significant (p<0.005 and p<0.01). No differences in subpopulations proportions were shown comparing G-CSF mobilized and basal PB CD34+ cells. Interestingly, the 2 patients mobilized with AMD3100 (the inhibitory molecule for CXCR4) showed a higher percentage of GMPs (33.8% and 37.8% versus the average 16.3% ± 9.8 in G-CSF mobilized samples) and a lower fraction of CMPs (29.5% and 41.6% versus the average 58% ± 12 in G-CSF mobilized samples). In order to understand this result, we looked then at the CXCR4 mean fluorescence intensity among the progenitor subsets: GMPs showed significantly higher levels of this molecule compared to CMPs and MEPs. Regarding the mobilizing chemotherapy regimens, CMPs percentages were higher (61.1% versus 49.1%, p: 0.038) and GMPs’ were significantly lower (11.1% versus 27.6%, p<0.0001) in cyclophosphamide treated patients, compared to patients mobilized with other chemotherapy regimens. The percentage of HSCs did not significantly differ among bone marrow, unmobilized and mobilized PB CD34+ cells. Therefore, since an average collection of mobilized PB cells contains approximately one log more CD34+ cells than a BM harvest, a similarly higher amount of HSC are infused with mobilized CD34+ cell transplantation. A linear positive correlation between the number of mobilized CD34+ cells and the number of mobilized CMPs, GMPs, and MEPs was observed indicating that the proportions of different HSPCs did not significantly change among high- and low-mobilizers. There were no correlations between the number of mobilized subpopulations and leucocytes, hemoglobin and platelets levels. CONCLUSIONS: Our data displayed the heterogeneity of HSPC compartment between PB and BM. Many factors could contribute to this variegated scenario. These mechanisms comprehension can help us to choose the most suitable chemotherapy and cytokine administrations in order to improve clinical outcomes as infections complications, length of aplasia and transfusion requirements during an hematopoietic stem cell transplantation. Disclosures Palumbo: Bristol-Myers Squibb: Consultancy, Honoraria; Genmab A/S: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Janssen-Cilag: Consultancy, Honoraria; Millennium Pharmaceuticals, Inc.: Consultancy, Honoraria; Onyx Pharmaceuticals: Consultancy, Honoraria; Array BioPharma: Honoraria; Amgen: Consultancy, Honoraria; Sanofi: Honoraria. Boccadoro:Celgene: Honoraria; Janssen: Honoraria; Onyx: Honoraria.

scholarly journals Efficient Nanoparticle-Mediated Delivery of Gene Editing Reagents into Human Hematopoietic Stem and Progenitor Cells

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2933-2933
Author(s):  
Rkia El Kharrag ◽  
Kurt Berckmueller ◽  
Margaret Cui ◽  
Ravishankar Madhu ◽  
Anai M Perez ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Quality Control ◽  
Progenitor Cells ◽  
Gene Editing ◽  
Ex Vivo ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Stem And Progenitor Cells

Abstract Autologous hematopoietic stem cell (HSC) gene therapy has the potential to cure millions of patients suffering from hematological diseases and disorders. Recent HSCs gene therapy trials using CRISPR/Cas9 nucleases to treat sickle cell disease (SCD) have shown promising results paving the way for gene editing approaches for other diseases. However, current applications depend on expensive and rare GMP facilities for the manipulation of HSCs ex vivo. Consequently, this promising treatment option remains inaccessible to many patients especially in low- and middle-income settings. HSC-targeted in vivo delivery of gene therapy reagents could overcome this bottleneck and thereby enhance the portability and availability of gene therapy. Various kinds of nanoparticles (lipid, gold, polymer, etc.) are currently used to develop targeted ex vivo as well as in vivo gene therapy approaches. We have previously shown that poly (β-amino ester) (PBAE)-based nanoparticle (NP) formulations can be used to efficiently deliver mRNA into human T cells and umbilical cord blood-derived CD34 + hematopoietic stem and progenitor cells (HSPCs) (Moffet et al. 2017, Nature Communications). Here, we optimized our NP formulation to deliver mRNA into GCSF-mobilized adult human CD34 + HSPCs, a more clinically relevant and frequently used cell source for ex vivo and the primary target for in vivo gene therapy. Furthermore, we specifically focused on the evaluation of NP-mediated delivery of CRISPR/Cas9 gene editing reagents. The efficiency of our NP-mediated delivery of gene editing reagents was comprehensively tested in comparison to electroporation, the current experimental, pre-clinical as well as clinical standard for gene editing. Most important for the clinical translation of this technology, we defined quality control parameters for NPs, identified standards that can predict the editing efficiency, and established protocols to lyophilize and store formulated NPs for enhanced portability and future in vivo applications. Nanoformulations were loaded with Cas9 ribonucleoprotein (RNP) complexes to knock out CD33, an established strategy in our lab to protect HSCs from anti-CD33 targeted acute myeloid leukemia (AML) immunotherapy (Humbert et al. 2019, Leukemia). RNP-loaded NPs were evaluated for size and charge to correlate physiochemical properties with the outcome as well as establish quality control standards. NPs passing the QC were incubated with human GCSF-mobilized CD34 + hematopoietic stem and progenitor cells (HSPCs). In parallel, RNPs were delivered into CD34 + cells using our established EP protocol. NP- and EP-edited CD34 + cells were evaluated phenotypically by flow cytometry and functionally in colony-forming cell (CFC) assays as well as in NSG xenograft model. The optimal characteristics for RNP-loaded NPs were determined at 150-250 nm and 25-35 mV. Physiochemical assessment of RNP-loaded NP formations provided an upfront quality control of RNP components reliably detecting degraded components. Most importantly, NP charge directly correlated with the editing efficiency (Figure A). NPs achieved more than 85% CD33 knockout using 3-fold lower dose of CRISPR nucleases compared to EP. No impact on the erythromyeloid differentiation potential of gene-edited cells in CFC assays was observed. Finally, NP-modified CD34 + cells showed efficient and sustained gene editing in vivo with improved long-term multilineage engraftment potential in the peripheral blood (PB) and bone marrow stem cell compartment of NSG mice in comparison to EP-edited cells (Figure B). Here we show that PBAE-NPs enable efficient CRISPR/Cas9 gene editing of human GCSF-mobilized CD34 + cells without compromising the viability and long-term multilineage engraftment of human HSPCs in vivo. Most importantly, we defined physiochemical properties of PBAE-NPs that enable us to not only determine the integrity of our gene-editing agents but also predict the efficiency of editing in HSPCs. The requirement of 3-fold less reagents compared to EP, the ability to lyophilize quality-controlled and ready to administer gene therapy reagents, and the opportunity to engineer the surface of PBAE-NPs with HSC-targeting molecules (e.g. antibodies) could make this also a highly attractive and portable editing platform for in vivo HSC gene therapy. Figure 1 Figure 1. Disclosures Kiem: VOR Biopharma: Consultancy; Homology Medicines: Consultancy; Ensoma Inc.: Consultancy, Current holder of individual stocks in a privately-held company. Radtke: Ensoma Inc.: Consultancy; 47 Inc.: Consultancy.

scholarly journals Endothelial Cells Promote Expansion of Long-Term Engrafting Marrow Hematopoietic Stem and Progenitor Cells in Primates

2016 ◽  
Vol 6 (3) ◽  
pp. 864-876 ◽  
Author(s):  
Jennifer L. Gori ◽  
Jason M. Butler ◽  
Balvir Kunar ◽  
Michael G. Poulos ◽  
Michael Ginsberg ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Progenitor Cells ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells

Adhesion to Fibronectin Maintains Regenerative Capacity During Ex Vivo Culture and Transduction of Human Hematopoietic Stem and Progenitor Cells

Blood ◽  
1998 ◽  
Vol 92 (12) ◽  
pp. 4612-4621 ◽  
Author(s):  
M.A. Dao ◽  
K. Hashino ◽  
I. Kato ◽  
J.A. Nolta
Keyword(s):  
Progenitor Cells ◽  
Ex Vivo ◽  
Xenograft Model ◽  
Retroviral Vectors ◽  
Current Data ◽  
Regenerative Capacity ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells ◽  
Ex Vivo Culture

Abstract Recent reports have indicated that there is poor engraftment from hematopoietic stem cells (HSC) that have traversed cell cycle ex vivo. However, inducing cells to cycle in culture is critical to the fields of ex vivo stem cell expansion and retroviral-mediated gene therapy. Through the use of a xenograft model, the current data shows that human hematopoietic stem and progenitor cells can traverse M phase ex vivo, integrate retroviral vectors, engraft, and sustain long-term hematopoiesis only if they have had the opportunity to engage their integrin receptors to fibronectin during the culture period. If cultured in suspension under the same conditions, transduction is undetectable and the long-term multilineage regenerative capacity of the primitive cells is severely diminished.

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