Simultaneous Maintenance of Human Cord Blood SCID‐Repopulating Cells and Expansion of Committed Progenitors at Low O ₂ Concentration (3%)

Abstract

The transplantation of unmanipulated cord blood (CB) cells has two major disadvantages: (a) the low number of hematopoietic stem and progenitor cells (colony-forming cells [CFCs]) in each harvest limits its application to children, and (b) there is a long period (30 days) of post-transplantation cytopenia [1]. Simultaneous ex vivo amplification of the CFCs and primitive stem cells could resolve both problems. Extensive ex-pansion of nonobese diabetic/severe combined immuno-deficiency (NOD/SCID) mice-repopulating cells (SRCs) in long-term (4-to 12-week) cultures [2] is not suitable for clinical application for several reasons. On the other hand, short-term (7-to 10-day) ex vivo amplification of CFCs usually leads to loss of primitive stem cells that impairs the long-term engraftment capacity of expanded cells in animals and humans [3][4][5][6] AbstractIn the present work, we tested the hypothesis that liquid cultures (LCs) of cord blood CD34 + cells at an appropriate low O 2 concentration could simultaneously allow colony-forming cell (CFC) expansion and nonobese diabetic/severe combined immunodeficiency mice-repopulating cell (SRC) maintenance. We first found that 3% was the minimal O 2 concentration, still allowing the same rate of CFC expansion as at 20% O 2 . We report here that 7-day LCs of cord blood CD34 + cells at 3% O 2 maintain SRC better than at 20% O 2 and allow a similar amplification of CFCs (35-to 50-fold) without modifying the CD34 + cell proliferation. Their phenotypic profile (antigens: HLA-DR, CD117, CD33, CD13, CD11b, CD14, CD15, and CD38) was not modified, with exception of CD133, whose expression was lower at 3% O 2 . These results suggest that low O 2 concentrations similar to those found in bone marrow participates in the regulation of hematopoiesis by favoring stem cell-renewing divisions. This expansion method that avoids stem cell exhaustion could be of paramount interest in hematopoietic transplantation by allowing the use of small-size grafts in adults. Stem Cells 2004;22:716-724

Severe Hypoxia Defines Heterogeneity and Selects Highly Immature Progenitors Within Clonal Erythroleukemia Cells

“…Indeed, severe hypoxia enhances maintenance in vitro of normal stem cells capable of short-term hematopoietic reconstitution (STR-HSC). This finding was later confirmed for human long-term repopulating (LTR) stem cells reconstituting NOD/ SCID mice [6,7]. We also showed that hypoxia protects stem cells from stimuli that induce lineage commitment to differentiation [5].…”

“…This study was based on the adaptation to leukemia cells [11,12] of the CRA assay developed in our laboratory [2] and successfully applied [3,5,7,13] for the quantification in vitro of the stem cell potential of normal hematopoietic cell populations. The CRA assay, in combination with incubation under severe hypoxia as a selection procedure, showed that the MEL cell population includes highly hypoxia-sensitive CFC as well as their hypoxia-resistant, CRC-type progenitors.…”

“…It is worth pointing out that adaptation to hypoxia is a property of stem cells that is expressed independently of their actual location in areas where oxygen tension is markedly reduced and, therefore, does not require LSC to be confined in those areas. That LSC may remain hypoxia-adapted independently of their actual tissue location is supported by the fact that normal circulating HSC exhibit the same resistance to hypoxia as bone marrow HSC [3,7].…”

“…In the study reported here, targeted to deepen the characterization of the effects of severe hypoxia on leukemia progenitor and stem cells, we used the murine Friend's erythroleukemia (MEL) cell line to address the question of whether cell subsets characterized by different sensitivity to hypoxia are detectable within a clonal leukemia cell line and whether these differences define the level within the progenitor/ stem cell hierarchy. To identify LSC, we used the Culture Repopulating Ability (CRA) assay, developed in our laboratory [2] and extensively applied [3,5,7] to quantify in vitro normal STR-HSC and later adapted to study their leukemic counterpart [11,12]. We found that the MEL cell line is functionally and metabolically heterogeneous in that it comprises hypoxia-sensitive CFC as well as hypoxia-resistant culture repopulating cells (CRC), which correspond to STR-type LSC.…”

The Soluble Form of LR11 Protein Is a Regulator of Hypoxia-induced, Urokinase-type Plasminogen Activator Receptor (uPAR)-mediated Adhesion of Immature Hematological Cells

“…Several studies have suggested that long term HSCs reside mainly in the endosteal sites of the BM, in which the pO 2 is very low (3,6). Cell biological studies using HS(P)Cs indicated that their functions as well as their quiescence state are maintained most effectively under hypoxic conditions (10,(47)(48)(49)(50)(51)(52). Hypoxia stabilizes the HIF-1␣ protein, a master regulator of oxygen homeostasis, and activates HIF-1␣-mediated signals in HSCs (48,53,54).…”