Human and Mouse Hematopoietic Colony-Forming Cell Assays

Miller, Cindy; Lai, Becky

doi:10.1385/1-59259-838-2:071

Cited by 29 publications

(33 citation statements)

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“…19 Interestingly, MPP efficiently generated myeloid colonies (59%), whereas neither CLP nor DN1 had substantial colonyforming ability (both Ͻ 2%; Figure 1D). Because both methylcellulose and OP9:OP9DL1 cultures contained the same concentrations of myeloid-promoting cytokines, the discrepant readouts suggest that other factors drive myeloid development in the OP9:OP9DL1 culture.…”

Section: Dn1 and Clp Display Robust Myeloid Potential On Op9:op9dl1 Cmentioning

confidence: 98%

In vitro assays misrepresent in vivo lineage potentials of murine lymphoid progenitors

Ehrlich

Serwold

Weissman

2011

Blood

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The identity of T-cell progenitors that seed the thymus has remained controversial, largely because many studies differ over whether these progenitors retain myeloid potential. Contradictory reports diverge in their use of various in vitro and in vivo assays. To consolidate these discordant findings, we compared the myeloid potential of 2 putative thymus seeding populations, common lymphoid progenitors (CLPs) and multipotent progenitors (MPPs), and the earliest intrathymic progenitor (DN1), using 2 in vitro assays and in vivo readouts. These assays gave contradictory results: CLP and DN1 displayed surprisingly robust myeloid potential on OP9-DL1 in vitro stromal cocultures but displayed little myeloid potential in vivo, as well as in methylcellulose cultures. MPP, on the other hand, displayed robust myeloid potential in all settings. We conclude that stromal cocultures reveal cryptic, but nonphysiologic, myeloid potentials of lymphoid progenitors, providing an explanation for contradictory findings in the field and underscoring the importance of using in vivo assays for the determination of physiologic lineage potentials. (Blood. 2011;117(9):2618-2624) IntroductionThe generation of mature hematopoietic lineages from stem cells entails the progressive loss of self-renewal capacity along with an increase in lineage restriction. Lineage restriction requires upregulation of lineage-specifying genes and down-regulation of genes that promote alternate fates, 1 probably involving progressive modulation of both transcription factor activity and chromatin structure. Accordingly, reinitiation of alternate transcriptional profiles should result in restoration of suppressed lineage potentials in cells that are not yet fully lineage-committed. Consistent with this model, signaling through ectopically expressed human interleukin-2R␣ (IL-2R␣) or granulocyte-macrophage colony-stimulating factor receptor (GM-CSFR) in common lymphoid progenitors (CLPs) allows them to mature down a myeloid fate at the expense of lymphoid development. 2,3 The observation that signaling through the same ectopic cytokine receptor is unable to reprogram fully T cell-committed thymic progenitors to a myeloid fate indicates that as lineage commitment occurs, fate decisions are indeed progressively locked in 4 : however, even these commitment decisions are not irreversible. Ectopic expression of PU.1 or C/EBP␣, which compete with Pax5 to determine myeloid versus lymphoid fates in CLP, restores myeloid potential in T-cell "committed" progenitors. 5,6 Mature B cells can also be reprogrammed to become T cells by conditional deletion of Pax5. 7 Strikingly, ectopic expression of 4 transcription factors reprograms differentiated fibroblasts to a pluripotent state. 8,9 Thus, lineage potentials revealed by experimental modifications may not reflect physiologic lineage decisions.Strikingly, culturing cells outside of their in vivo environments can also promote nonphysiologic lineage outcomes. For example, VCAM-1 Ϫ multipotent progenitors (MPPs) are highly lymphoi...

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Section: Dn1 and Clp Display Robust Myeloid Potential On Op9:op9dl1 Cmentioning

confidence: 98%

In vitro assays misrepresent in vivo lineage potentials of murine lymphoid progenitors

Ehrlich

Serwold

Weissman

2011

Blood

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“…Colony-forming cell (CFC) assays were performed by plating blood in methocellulose-containing Iscove MDM supplemented with IL-3, IL-6, and stem cell factor (MethoCult 3534; Stem Cell Technologies, Vancouver, BC) according to standard techniques. 23 …”

Section: Mobilization Protocol and Cfc Assaysmentioning

confidence: 99%

Chemosensitization of acute myeloid leukemia (AML) following mobilization by the CXCR4 antagonist AMD3100

Nervi¹,

Ramírez²,

Rettig³

et al. 2009

Blood

446

370

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The CXCR4-SDF-1 axis plays a central role in the trafficking and retention of normal and malignant stem cells in the bone marrow (BM) microenvironment. Here, we used a mouse model of acute promyelocytic leukemia (APL) and a small molecule competitive antagonist of CXCR4, AMD3100, to examine the interaction of mouse APL cells with the BM microenvironment. APL cells from a murine cathepsin G-PML-RAR␣ knockin mouse were genetically modified with firefly luciferase (APL luc ) to allow tracking by bioluminescence imaging. Coculture of APL luc cells with M2-10B4 stromal cells protected the leukemia cells from chemotherapy-induced apoptosis in vitro. Upon injection into syngeneic recipients, APL luc cells rapidly migrated to the BM followed by egress to the spleen then to the peripheral blood with death due to leukostasis by day 15. Administration of AMD3100 to leukemic mice induced a 1.6-fold increase in total leukocytes and a 9-fold increase of circulating APL blast counts, which peak at 3 hours and return to baseline by 12 hours. Treatment of leukemic mice with chemotherapy plus AMD3100 resulted in decreased tumor burden and improved overall survival compared with mice treated with chemotherapy alone. These studies provide a proof-of-principle for directing therapy to the critical tethers that promote AMLniche interactions. (Blood. 2009;113: 6206-6214) IntroductionHematopoietic stem cells (HSCs) reside in the bone marrow (BM) and interact with a highly organized microenvironment composed of a diverse population of stromal cells and an extracellular matrix rich in fibronectin, collagens, and various proteoglycans. The interaction between HSCs and the BM microenvironment is critical in regulating HSC processes such as trafficking, self-renewal, proliferation, and differentiation.Egress (mobilization) of stem cells from the BM to the peripheral blood can be induced by cytokines (G-CSF and GM-CSF), chemokines (Gro-␤ and IL-8), or by small molecule inhibitors of both CXCR4 and VLA-4. 1 Interaction between the chemokine, SDF-1, and its cognate receptor CXCR4 functions as a key regulator of stem cell mobilization and trafficking. 2,3 Constitutive secretion of SDF-1 by marrow stromal cells creates a gradient by which HSCs expressing the receptor CXCR4 home to and interact with its marrow niche. 4 In response to factors such as G-CSF, SDF-1 production is down-regulated by stromal cells that release HSCs into the peripheral circulation. 5 AMD3100 is a bicyclam molecule that potently, selectively, and reversibly antagonizes the binding of SDF-1 to CXCR4. 6 In multiple clinical studies, AMD3100 rapidly and effectively mobilizes HSCs into the peripheral circulation and is currently under development as a stem cell mobilization agent prior to high-dose chemotherapy for multiple myeloma, non-Hodgkin lymphoma, and other hematologic malignancies. [7][8][9] In acute myeloid leukemia (AML), the bone marrow microenvironment provides the primary site of minimal residual disease after chemotherapy. [10][11][12] Similar to normal HSCs, AM...

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“…Total numbers of colonies were, however, in the range of normality according to the assay utilized. 25 …”

Section: In Vitro Colony-forming Capacity Of Normal Hematopoietic Promentioning

confidence: 99%

Cytokine-induced killer cells for cell therapy of acute myeloid leukemia: improvement of their immune activity by expression of CD33-specific chimeric receptors

Marin¹,

Pizzitola²,

Agostoni³

et al. 2010

Haematologica

105

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The online version of this article has a Supplementary Appendix. BackgroundCytokine-induced killer cells are ex vivo-expanded cells with potent antitumor activity. The infusion of cytokine-induced killer cells in patients with acute myeloid leukemia relapsing after allogeneic hematopoietic stem cell transplant is well tolerated, but limited clinical responses have been observed. To improve their effector functions against acute myeloid leukemia, we genetically modified cytokine-induced killer cells with chimeric receptors specific for the CD33 myeloid antigen. Design and MethodsSFG-retroviral vectors coding for anti-CD33-ζ and anti-CD33-CD28-OX40-ζ chimeric receptors were used to transduce cytokine-induced killer cells. Transduced cells were characterized in vitro for their ability to lyse leukemic targets (4-hour 51 chromium-release and 6-day co-cultures assays on human stromal mesenchymal cells), to proliferate ( 3 H-thymidine-incorporation assay) and to secrete cytokines (flow cytomix assay) after contact with acute myeloid leukemia cells. Their activity against normal CD34 + hematopoietic progenitor cells was evaluated by analyzing the colony-forming unit capacity after co-incubation. ResultsCytokine-induced killer cells were efficiently transduced with the anti-CD33 chimeric receptors, maintaining their native phenotype and functions and acquiring potent cytotoxicity (up to 80% lysis after 4-hour incubation) against different acute myeloid leukemia targets, as also confirmed in long-term killing experiments. Moreover, introduction of the anti-CD33 chimeric receptors was accompanied by prominent CD33-specific proliferative activity, with the release of high levels of immunostimulatory cytokines. The presence of CD28-OX40 in chimeric receptor endodomain was associated with a significant amelioration of the anti-leukemic activity of cytokine-induced killer cells. Importantly, even though the cytokine-induced killer cells transduced with anti-CD33 chimeric receptors showed toxicity against normal hematopoietic CD34 + progenitor cells, residual clonogenic activity was preserved. ConclusionsOur results indicate that anti-CD33 chimeric receptors strongly enhance anti-leukemic cytokine-induced killer cell functions, suggesting that cytokine-induced killer cells transduced with these molecules might represent a promising optimized tool for acute myeloid leukemia immunotherapy.Key words: chimeric T-cell receptor, acute myeloid leukemia, CD33, cell therapy, gene therapy. Haematologica 2010;95(12):2144-2152. doi:10.3324/haematol.2010 This is an open-access paper. © F e r r a t a S t o r t i F o u n d a t i o n

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Human and Mouse Hematopoietic Colony-Forming Cell Assays

Cited by 29 publications

References 1 publication

In vitro assays misrepresent in vivo lineage potentials of murine lymphoid progenitors

In vitro assays misrepresent in vivo lineage potentials of murine lymphoid progenitors

Chemosensitization of acute myeloid leukemia (AML) following mobilization by the CXCR4 antagonist AMD3100

Cytokine-induced killer cells for cell therapy of acute myeloid leukemia: improvement of their immune activity by expression of CD33-specific chimeric receptors

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