85. Modeling the Genomic Integration Site Distribution of Genotoxic and Non Genotoxic Retroviral Vectors in Hematopoietic Stem/Progenitor Cells In Vivo

Abstract Diverse human cancers with poor prognosis, including many lymphoid and myeloid malignancies, exhibit high levels of Mcl-1. To explore the impact of Mcl-1 overexpression on the hematopoietic compartment, we have generated vavP-Mcl-1 transgenic mice. Their lymphoid and myeloid cells displayed increased resistance to a variety of cytotoxic agents. Myelopoiesis was relatively normal, but lymphopoiesis was clearly perturbed, with excess mature B and T cells accumulating. Rather than the follicular lymphomas typical of vavP-BCL-2 mice, aging vavP-Mcl-1 mice were primarily susceptible to lymphomas having the phenotype of a stem/progenitor cell (11 of 30 tumors) or pre-B cell (12 of 30 tumors). Mcl-1 overexpression dramatically accelerated Myc-driven lymphomagenesis. Most vavP-Mcl-1/ Eμ-Myc mice died around birth, and transplantation of blood from bitransgenic E18 embryos into unirradiated mice resulted in stem/progenitor cell tumors. Furthermore, lethally irradiated mice transplanted with E13 fetal liver cells from Mcl-1/Myc bitransgenic mice uniformly died of stem/progenitor cell tumors. When treated in vivo with cyclophosphamide, tumors coexpressing Mcl-1 and Myc transgenes were significantly more resistant than conventional Eμ-Myc lymphomas. Collectively, these results demonstrate that Mcl-1 overexpression renders hematopoietic cells refractory to many cytotoxic insults, perturbs lymphopoiesis and promotes malignant transformation of hematopoietic stem and progenitor cells.

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Functional Investigation of the Argonaute Proteins in Human Hematopoietic Stem and Progenitor Cells

Blood ◽

10.1182/blood-2020-142963 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 32-32

Author(s):

Gordon G. L. Wong ◽

Gabriela Krivdova ◽

Olga I. Gan ◽

Jessica L. McLeod ◽

John E. Dick ◽

...

Keyword(s):

Progenitor Cells ◽

Erythroid Differentiation ◽

Lineage Commitment ◽

Myeloid Differentiation ◽

Hematopoietic Stem ◽

Erythroid Precursor ◽

Lineage Differentiation ◽

Erythroid Precursors

Micro RNA (miRNA)-mediated gene silencing, largely mediated by the Argonaute (AGO) family proteins, is a post-transcriptional gene expression control mechanism that has been shown to regulate hematopoietic stem and progenitor cells (HSPCs) quiescence, self-renewal, proliferation, and differentiation. Interestingly, only the function of AGO2 in hematopoiesis has been investigated. O'Carroll et al. (2007) showed that AGO2 knockout in mice bone marrow cells interferes with B220low CD43- IgM-pre-B cells and peripheral B cell differentiation and impairs Ter119high, CD71high erythroid precursors maturation. However, the functional significance of other AGO proteins in the regulation of stemness and lineage commitment remains unclear. AGO submembers, AGO1-4 in humans, are traditionally believed to act redundantly in their function. However, our previous proteomic analysis from sorted populations of the human hematopoietic hierarchy shows each sub-member is differentially expressed during HSPCs development, suggesting each sub-member may have a specialized function in hematopoiesis. Here, we conducted CRISPR-Cas9 mediated knockout of AGO1-4 in human cord blood derived long-term (LT-) and short-term hematopoietic stem cells (ST-HSCs) and investigated the impact of the loss of function of individual AGOs in vitro and in vivo in xenograft assays. From the in vitro experiment, we cultured CRISPR-edited LT- or ST-HSCs in a single cell manner on 96-well plates pre-cultured with murine MS5 stroma cells in erythro-myeloid differentiation condition. The colony-forming capacity and lineage commitment of each individual HSC is assessed on day 17 of the culture. Initial data showed that AGO1, AGO2 and AGO3 knockout decreased the colony formation efficacy of both LT- and ST-HSCs, suggesting AGO1, AGO2 and AGO3 are involved in LT- and ST-HSCs proliferation or survival. As for lineage output, AGO1 knockout increases CD56+ natural killer cell commitment in LT-HSCs and erythroid differentiation in ST-HSCs; AGO2 knockout increases erythroid differentiation in both LT- and ST-HSCs and decreases myeloid differentiation in ST-HSCs; while AGO4 knockout seems to decrease erythroid output. For the in vivo experiment, we xenotransplanted AGO1 and AGO2 knockout LT-HSCs in irradiated immunodeficient NSG mice and assessed the change in LT-HSCs engraftment level and lineage differentiation profile at 12- and 24-week time points. We found that AGO2 knockout increased CD45+ engraftment at both 12- and 24-weeks. Aligning with our in vitro data, AGO2 knockout increases GlyA+ erythroid cells at 12- and 24-weeks. The increase in GlyA+ erythroid cells is a consequence of the 2-fold increase in GlyA+ CD71+ erythroid precursor cells, recapitulating previous findings that AGO2 knockout in mice impairs CD71high erythroid precursor maturation leading to the accumulation of undifferentiated CD71+ erythroid precursors (O'Carroll et al., 2007). Accumulation of early progenitors of the erythroid lineage, including the common myeloid progenitors (CMPs) and myelo-erythroid progenitor (MEPs) were observed, as well as their progeny including CD33+ myeloid and CD41+ megakaryocytes. For the myeloid lineage, AGO2 knockout shifts myeloid differentiation toward CD66b+ granulocytes from CD14+ monocytes. For lymphoid, AGO2 knockout decreases CD19+ CD10- CD20+ mature B-lymphoid cells, which again aligns with previous AGO2 knockout mice results. On the other hand, AGO1 knockout LT-HSCs share some similar phenotype with AGO2 knockout LT-HSCs, where AGO1 knockout increases CD71+ erythroid precursors. However, AGO1 knockout in LT-HSCs also results in unique phenotypes, with a decrease in neutrophil formation and an increase in CD4+ CD8+ T progenitor cells are observed. AGO3 and AGO4 knockout experiments are in progress. In summary, our AGO2 knockout experiments recapitulate the reported results from murine studies but also illustrate a more complete role of AGO2 in hematopoietic lineage differentiation. Moreover, AGO knockout experiments of individual submembers are revealing novel insights into their role in the regulation of stemness and lineage commitment of LT-HSCs and ST-HSCs. These data point to a unique role of different AGO isoforms in lineage commitment in human HSCs and argue against redundant functioning. Disclosures Dick: Bristol-Myers Squibb/Celgene: Research Funding.

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Hematopoietic stem/progenitor cells, generation of induced pluripotent stem cells, and isolation of endothelial progenitors from 21- to 23.5-year cryopreserved cord blood

Blood ◽

10.1182/blood-2011-01-330514 ◽

2011 ◽

Vol 117 (18) ◽

pp. 4773-4777 ◽

Cited By ~ 115

Author(s):

Hal E. Broxmeyer ◽

Man-Ryul Lee ◽

Giao Hangoc ◽

Scott Cooper ◽

Nutan Prasain ◽

...

Keyword(s):

Stem Cells ◽

Cord Blood ◽

Progenitor Cells ◽

Proliferative Potential ◽

Hematopoietic Stem ◽

Immunodeficient Mice ◽

Induced Pluripotent

Abstract Cryopreservation of hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) is crucial for cord blood (CB) banking and transplantation. We evaluated recovery of functional HPC cryopreserved as mononuclear or unseparated cells for up to 23.5 years compared with prefreeze values of the same CB units. Highly efficient recovery (80%-100%) was apparent for granulocyte-macrophage and multipotential hematopoietic progenitors, although some collections had reproducible low recovery. Proliferative potential, response to multiple cytokines, and replating of HPC colonies was extensive. CD34+ cells isolated from CB cryopreserved for up to 21 years had long-term (≥ 6 month) engrafting capability in primary and secondary immunodeficient mice reflecting recovery of long-term repopulating, self-renewing HSCs. We recovered functionally responsive CD4+ and CD8+ T lymphocytes, generated induced pluripotent stem (iPS) cells with differentiation representing all 3 germ cell lineages in vitro and in vivo, and detected high proliferative endothelial colony forming cells, results of relevance to CB biology and banking.

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Dectin-1 Stimulation of Hematopoietic Stem and Progenitor Cells Occurs In Vivo and Promotes Differentiation Toward Trained Macrophages via an Indirect Cell-Autonomous Mechanism

mBio ◽

10.1128/mbio.00781-20 ◽

2020 ◽

Vol 11 (3) ◽

Cited By ~ 1

Author(s):

Cristina Bono ◽

Alba Martínez ◽

Javier Megías ◽

Daniel Gozalbo ◽

Alberto Yáñez ◽

...

Keyword(s):

Bone Marrow ◽

Candida Albicans ◽

Progenitor Cells ◽

Recipient Mouse ◽

Dependent Manner ◽

Toll Like Receptor ◽

Hematopoietic Stem ◽

Content Type ◽

Stem And Progenitor Cells

ABSTRACT Toll-like receptor (TLR) agonists drive hematopoietic stem and progenitor cells (HSPCs) to differentiate along the myeloid lineage. In this study, we used an HSPC transplantation model to investigate the possible direct interaction of β-glucan and its receptor (dectin-1) on HSPCs in vivo. Purified HSPCs from bone marrow of B6Ly5.1 mice (CD45.1 alloantigen) were transplanted into dectin-1−/− mice (CD45.2 alloantigen), which were then injected with β-glucan (depleted zymosan). As recipient mouse cells do not recognize the dectin-1 agonist injected, interference by soluble mediators secreted by recipient cells is negligible. Transplanted HSPCs differentiated into macrophages in response to depleted zymosan in the spleens and bone marrow of recipient mice. Functionally, macrophages derived from HSPCs exposed to depleted zymosan in vivo produced higher levels of inflammatory cytokines (tumor necrosis factor alpha [TNF-α] and interleukin 6 [IL-6]). These results demonstrate that trained immune responses, already described for monocytes and macrophages, also take place in HSPCs. Using a similar in vivo model of HSPC transplantation, we demonstrated that inactivated yeasts of Candida albicans induce differentiation of HSPCs through a dectin-1- and MyD88-dependent pathway. Soluble factors produced following exposure of HSPCs to dectin-1 agonists acted in a paracrine manner to induce myeloid differentiation and to influence the function of macrophages derived from dectin-1-unresponsive or β-glucan-unexposed HSPCs. Finally, we demonstrated that an in vitro transient exposure of HSPCs to live C. albicans cells, prior to differentiation, is sufficient to induce a trained phenotype of the macrophages they produce in a dectin-1- and Toll-like receptor 2 (TLR2)-dependent manner. IMPORTANCE Invasive candidiasis is an increasingly frequent cause of serious and often fatal infections. Understanding host defense is essential to design novel therapeutic strategies to boost immune protection against Candida albicans. In this article, we delve into two new concepts that have arisen over the last years: (i) the delivery of myelopoiesis-inducing signals by microbial components directly sensed by hematopoietic stem and progenitor cells (HSPCs) and (ii) the concept of “trained innate immunity” that may also apply to HSPCs. We demonstrate that dectin-1 ligation in vivo activates HSPCs and induces their differentiation to trained macrophages by a cell-autonomous indirect mechanism. This points to new mechanisms by which pathogen detection by HSPCs may modulate hematopoiesis in real time to generate myeloid cells better prepared to deal with the infection. Manipulation of this process may help to boost the innate immune response during candidiasis.

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Phenotypic correction of Fanconi anemia group C knockout mice

Blood ◽

10.1182/blood.v95.2.700 ◽

2000 ◽

Vol 95 (2) ◽

pp. 700-704 ◽

Cited By ~ 40

Author(s):

Kimberly A. Gush ◽

Kai-Ling Fu ◽

Markus Grompe ◽

Christopher E. Walsh

Keyword(s):

Bone Marrow ◽

Progenitor Cells ◽

Mitomycin C ◽

Fanconi Anemia ◽

Bone Marrow Cells ◽

Bone Marrow Failure ◽

Genetic Disorder ◽

Hematopoietic Stem ◽

Marrow Cells

Fanconi anemia (FA) is a genetic disorder characterized by bone marrow failure, congenital anomalies, and a predisposition to malignancy. FA cells demonstrate hypersensitivity to DNA cross-linking agents, such as mitomycin C (MMC). Mice with a targeted disruption of the FANCC gene (fancc −/− nullizygous mice) exhibit many of the characteristic features of FA and provide a valuable tool for testing novel therapeutic strategies. We have exploited the inherent hypersensitivity offancc −/− hematopoietic cells to assay for phenotypic correction following transfer of the FANCC complementary DNA (cDNA) into bone marrow cells. Murine fancc −/− bone marrow cells were transduced with the use of retrovirus carrying the humanfancc cDNA and injected into lethally irradiated recipients. Mitomycin C (MMC) dosing, known to induce pancytopenia, was used to challenge the transplanted animals. Phenotypic correction was determined by assessment of peripheral blood counts. Mice that received cells transduced with virus carrying the wild-type gene maintained normal blood counts following MMC administration. All nullizygous control animals receiving MMC exhibited pancytopenia shortly before death. Clonogenic assay and polymerase chain reaction analysis confirmed gene transfer of progenitor cells. These results indicate that selective pressure promotes in vivo enrichment offancc-transduced hematopoietic stem/progenitor cells. In addition, MMC resistance coupled with detection of the transgene in secondary recipients suggests transduction and phenotypic correction of long-term repopulating stem cells.

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CD70/CD27 signaling promotes blast stemness and is a viable therapeutic target in acute myeloid leukemia

Journal of Experimental Medicine ◽

10.1084/jem.20152008 ◽

2016 ◽

Vol 214 (2) ◽

pp. 359-380 ◽

Cited By ~ 40

Author(s):

Carsten Riether ◽

Christian M. Schürch ◽

Elias D. Bührer ◽

Magdalena Hinterbrandner ◽

Anne-Laure Huguenin ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Stem Cell ◽

Progenitor Cells ◽

Myeloid Leukemia ◽

Hematopoietic Stem ◽

Cell Divisions ◽

Cell Gene ◽

Stem Cell Gene ◽

Acute Myeloid

Aberrant proliferation, symmetric self-renewal, increased survival, and defective differentiation of malignant blasts are key oncogenic drivers in acute myeloid leukemia (AML). Stem cell gene signatures predict poor prognosis in AML patients; however, with few exceptions, these deregulated molecular pathways cannot be targeted therapeutically. In this study, we demonstrate that the TNF superfamily ligand–receptor pair CD70/CD27 is expressed on AML blasts and AML stem/progenitor cells. CD70/CD27 signaling in AML cells activates stem cell gene expression programs, including the Wnt pathway, and promotes symmetric cell divisions and proliferation. Soluble CD27, reflecting the extent of CD70/CD27 interactions in vivo, was significantly elevated in the sera of newly diagnosed AML patients and is a strong independent negative prognostic biomarker for overall survival. Blocking the CD70/CD27 interaction by mAb induced asymmetric cell divisions and differentiation in AML blasts and AML stem/progenitor cells, inhibited cell growth and colony formation, and significantly prolonged survival in murine AML xenografts. Importantly, hematopoietic stem/progenitor cells from healthy BM donors express neither CD70 nor CD27 and were unaffected by blocking mAb treatment. Therefore, targeting CD70/CD27 signaling represents a promising therapeutic strategy for AML.

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

Blood ◽

10.1182/blood.v92.12.4612 ◽

1998 ◽

Vol 92 (12) ◽

pp. 4612-4621 ◽

Cited By ~ 128

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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Hematopoietic progenitors in cyclic neutropenia: effect of granulocyte colony-stimulating factor in vivo

Blood ◽

10.1182/blood.v75.10.1951.1951 ◽

1990 ◽

Vol 75 (10) ◽

pp. 1951-1959 ◽

Cited By ~ 39

Author(s):

AR Migliaccio ◽

G Migliaccio ◽

DC Dale ◽

WP Hammond

Keyword(s):

Growth Factor ◽

Progenitor Cells ◽

Blood Cells ◽

Granulocyte Colony Stimulating Factor ◽

Colony Stimulating Factor ◽

Cyclic Neutropenia ◽

Hematopoietic Stem ◽

Gm Csf ◽

Stimulating Factor

Abstract The number and growth factor requirements of committed progenitor cells (colony-forming units-granulocyte/macrophage and burst-forming units- erythroid) in three patients with cyclic neutropenia (two congenital, one acquired) were studied before and during therapy with recombinant human granulocyte colony-stimulating factor (G-CSF; 3 to 10 micrograms/kg/d). When the patients with congenital disease were treated with G-CSF, the cycling of blood cells persisted, but the cycle length was shortened from 21 days to 14 days, and the amplitude of variations in blood counts increased. There was a parallel shortening of the cycle and increase of the amplitude of variations (from two- to three-fold to 10- to 100-fold) in the number of both types of circulating progenitor cells in these two patients. In the patient with acquired cyclic neutropenia, cycling of both blood cells and progenitors could not be seen. In cultures deprived of fetal bovine serum, erythroid and myeloid bone marrow progenitor cells from untreated patients and from normals differed in growth factor responsiveness. As examples, maximal growth of granulocyte/macrophage (GM) colonies was induced by granulocyte/macrophage (GM)-CSF plus G-CSF in the patients, whereas a combination of GM-CSF, G-CSF and interleukin- 3 (IL-3) was required in the normals, and erythropoietin alone induced fourfold more erythroid bursts from cyclic neutropenic patients than from normal donors (46% versus 11% of the maximal colony number, respectively). The growth factor responsiveness of marrow progenitor cells slightly changed during the treatment toward the values observed with normal progenitors. These results indicate that treatment with G- CSF not only ameliorated the neutropenia, but also increased the amplitude and the frequency of oscillation of circulating progenitor cell numbers. These data are consistent with the hypothesis that G-CSF therapy affects the proliferation of the hematopoietic stem cell.

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