Expansion of human SCID-repopulating cells under hypoxic conditions

Danet, Guénahel H.; Pan, Yi; Luongo, Jennifer L.; Bonnet, Dominique; Simon, M. Celeste

doi:10.1172/jci17669

Cited by 278 publications

(72 citation statements)

References 63 publications

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“…Nonetheless, it is important to note that multiple cell types proliferate in vitro under hypoxic conditions including human hematopoietic stem cells and human fibroblasts (Bell et al, 2007; Danet et al, 2003). Hypoxia also increases pulmonary vascular smooth muscle cell proliferation through TOR signaling (Krymskaya et al, 2011).…”

Section: Resultsmentioning

confidence: 99%

TOR Signaling Couples Oxygen Sensing to Lifespan in C. elegans

Schieber

Chandel

2014

Cell Reports

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Summary Metazoans adapt to a low oxygen environment (hypoxia) through activation of stress response pathways. Here we report that transient hypoxia exposure extends lifespan in C. elegans through mitochondrial ROS-dependent regulation of the nutrient sensing kinase TOR and its upstream activator RHEB-1. The increase in lifespan during hypoxia requires the intestinal GATA-type transcription factor, ELT-2, downstream of TOR signaling. Using RNA-Sequencing, we describe an ELT-2-dependent hypoxia response that includes an intestinal glutathione S-transferase, GSTO-1, and uncover that GSTO-1 is required for lifespan under hypoxia. These results indicate mitochondrial ROS-dependent TOR signaling integrates metabolic adaptations to confer survival under hypoxia.

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Section: Resultsmentioning

confidence: 99%

TOR Signaling Couples Oxygen Sensing to Lifespan in C. elegans

Schieber

Chandel

2014

Cell Reports

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“…▲ CRITICAL STEP Cells should be cultured in a low-oxygen incubator (5% O 2 ) for maintenance and expansion of LT-HSCs 49,50 .…”

Section: Methodsmentioning

confidence: 99%

CRISPR/Cas9 genome editing in human hematopoietic stem cells

2018

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Genome editing via homologous recombination (HR) (gene targeting) in human hematopoietic stem cells (HSCs) has the power to reveal gene–function relationships and potentially transform curative hematological gene and cell therapies. However, there are no comprehensive and reproducible protocols for targeting HSCs for HR. Herein, we provide a detailed protocol for the production, enrichment, and in vitro and in vivo analyses of HR-targeted HSCs by combining CRISPR/Cas9 technology with the use of rAAV6 and flow cytometry. Using this protocol, researchers can introduce single-nucleotide changes into the genome or longer gene cassettes with the precision of genome editing. Along with our troubleshooting and optimization guidelines, researchers can use this protocol to streamline HSC genome editing at any locus of interest. The in vitro HSC-targeting protocol and analyses can be completed in 3 weeks, and the long-term in vivo HSC engraftment analyses in immunodeficient mice can be achieved in 16 weeks. This protocol enables manipulation of genes for investigation of gene functions during hematopoiesis, as well as for the correction of genetic mutations in HSC transplantation–based therapies for diseases such as sickle cell disease, β-thalassemia, and primary immunodeficiencies.

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“…Uncovering mechanisms in HSC biology can identify new strategies to enhance numbers and function of HSCs and improve engraftment efficacy. While HSCs and hematopoietic progenitor cells (HPCs) proliferate better in-vitro in hypoxia than normoxia (Bradley et al, 1978; Broxmeyer et al, 1985; Danet et al, 2003; Lu and Broxmeyer, 1985; Smith and Broxmeyer, 1986), all HSC/HPC studies are performed after cell collection and processing in ambient air (∼21% O 2 ) regardless of subsequent processing in hypoxia or air. The BM and CB environment where HSCs reside is extremely hypoxic compared to air (Morrison and Scadden, 2014, Nombela-Arrieta et al, 2013, Spencer et al, 2014).…”

Section: Introductionmentioning

confidence: 99%

Enhancing Hematopoietic Stem Cell Transplantation Efficacy by Mitigating Oxygen Shock

Mantel

O’Leary

Chitteti

et al. 2015

Cell

261

274

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Summary Hematopoietic stem cells (HSCs) reside in hypoxic niches within bone marrow and cord blood. Yet, essentially all HSC studies have been performed with cells isolated and processed in non-physiologic ambient air. By collecting and manipulating bone marrow and cord blood in native conditions of hypoxia, we demonstrate that brief exposure to ambient oxygen decreases recovery of long-term repopulating HSCs and increases progenitor cells, a phenomenon we term Extra Physiologic Oxygen Shock/Stress (EPHOSS). Thus, true numbers of HSCs in the bone marrow and cord blood are routinely underestimated. We linked ROS production and induction of the mitochondrial permeability transition pore (MPTP) via cyclophilin D and p53 as mechanisms of EPHOSS. MPTP inhibitor Cyclosporine A protects mouse bone marrow and human cord blood HSCs from EPHOSS during collection in air, resulting in increased recovery of transplantable HSCs. Mitigating EPHOSS during cell collection and processing by pharmacological means may be clinically advantageous for transplantation.

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Expansion of human SCID-repopulating cells under hypoxic conditions

Cited by 278 publications

References 63 publications

TOR Signaling Couples Oxygen Sensing to Lifespan in C. elegans

TOR Signaling Couples Oxygen Sensing to Lifespan in C. elegans

CRISPR/Cas9 genome editing in human hematopoietic stem cells

Enhancing Hematopoietic Stem Cell Transplantation Efficacy by Mitigating Oxygen Shock

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