Expansion of human SCID-repopulating cells under hypoxic conditions

Abstract

It has been proposed that bone marrow (BM) hematopoietic stem and progenitor cells are distributed along an oxygen (O2) gradient, where stem cells reside in the most hypoxic areas and proliferating progenitors are found in O2-rich areas. However, the effects of hypoxia on human hematopoietic stem cells (HSCs) have not been characterized. Our objective was to evaluate the functional and molecular responses of human BM progenitors and stem cells to hypoxic conditions. BM lineage–negative (Lin–) CD34+CD38– cells were cultured in serum-free medium under 1.5% O2 (hypoxia) or 20% O2 (normoxia) for 4 days. Using limiting dilution analysis, we demonstrate that the absolute number of SCID-repopulating cells (SRCs) increased by 5.8-fold in hypoxic cultures compared with normoxia, and by 4.2-fold compared with freshly isolated Lin–CD34+CD38– cells. The observed increase in BM-repopulating activity was associated with a preferential expansion of Lin–CD34+CD38– cells. We also demonstrate that, in response to hypoxia, hypoxia-inducible factor-1α protein was stabilized, surface expression of angiogenic receptors was upregulated, and VEGF secretion increased in BM Lin–CD34+ cultures. The use of low O2 levels to enhance the survival and/or self-renewal of human BM HSCs in vitro represents an important advance and could have valuable clinical implications

Hypoxia mediates low cell-cycle activity and increases the proportion of long-term–reconstituting hematopoietic stem cells during in vitro culture

“…These results are in agreement with a recent study on CD34 + 38 -cells from human cord blood [18,21] Because quiescent cells in the BM are most abundant in the hypoxic region close to the endosteal surface, and in association with sinusoids and distant to capillaries [12], it seems that hypoxia could be a critical regulator to maintain HSCs.…”

“…While concordant studies have shown distinct effects of hypoxia on some HSCs and progenitors [17][18][19][20][21], no study has been performed with phenotypically defined mouse HSCs, neither has the molecular programme of HIF-1 been investigated in highly enriched HSCs. In the present study, we cultured FACS-sorted long-term and short-term reconstituting FLT3 -CD34 -/+ Lin -Sca-1 + c-Kit + (LSK) cells in 20% or 1% O 2 for 2-4 days and analyzed the effects on cell proliferation and ex vivo expansion as well as engraftment potential.…”

The role of oxygen availability in embryonic development and stem cell function

“…It has been proposed that HSCs and their proliferating progenitors are naturally distributed along this gradient, with the HSCs occupying the most hypoxic niches 54,55 . Furthermore, Danet et al demonstrated that bone marrow HSCs cultured at 1.5% O 2 promoted their ability to engraft and repopulate the haematopoietic organ of immunocompromised recipient mice 56 . Intriguingly, antibodies against markers that substantially enriched for HSCs during purification identify cells that are localized to the sinusoidal endothelium, as opposed to the more hypoxic endosteal lining 57 .…”

HIF-2α regulates Oct-4: effects of hypoxia on stem cell function, embryonic development, and tumor growth

“…Stromal cell contacts, extracellular matrix proteins, temperature, and oxygen (O 2 ) levels in the immediate microenvironment can influence stem cell function and differentiation. In particular, low O 2 levels (hypoxia) promote the survival of neural crest stem cells, hematopoietic stem cells, and human ES cells (Morrison et al 2000;Studer et al 2000;Danet et al 2003;Ezashi et al 2005). Hypoxia also regulates the number of ICM cells in bovine blastocysts and enhances hemangioblast and hematopoietic progenitor development.…”