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First published online August 25, 2005
Stem Cells Vol. 24 No. 1 January 2006, pp. 65 -73
doi:10.1634/stemcells.2004-0351; www.StemCells.com
© 2006 AlphaMed Press

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TISSUE-SPECIFIC STEM CELLS

Very Low O2 Concentration (0.1%) Favors G0 Return of Dividing CD34+ Cells

Francis Hermittea, Philippe Brunet de la Grangea, Francis Bellocb, Vincent Pralorana,b, Zoran Ivanovicc

a CNRS UMR 5164: University of Bordeaux 2, Bordeaux, France;
b University Hospital, Hematology Laboratory, Bordeaux, France;
c French Blood Transfusion Organisation in Aquitaine-Limousin, Bordeaux, France

Key Words. CD34 • Cell cycle • Hematopoietic stem cell • Hypoxia • Oxygen

Correspondence: Vincent Praloran, M.D., Ph.D., CNRS UMR 5164, Université de Bordeaux 2, 33076 Bordeaux CEDEX, France. Telephone: 33-5-57–57-16-11; Fax: 33-5-56-51-42-18; e-mail: Vincent.Praloran{at}hemato.u-bordeaux2.fr


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 References
 
Physiological bone marrow oxygen concentrations are everywhere lower than 4% and almost null in some areas. We compared the effects of 20%, 3%, and 0.1% O2 concentrations on cord blood CD34+ cell survival, cycle, and functionality in serum-free cultures for 72 hours with or without interleukin-3 (IL-3). As from 24 hours, IL-3 improved cell survival and proliferation in all conditions. After 72 hours, cells were 1.5 and 2.5 times more in quiescence (G0) at 3% and 0.1% O2, respectively, than at 20%; transforming growth factor-ß signaling seemed not to be involved. To explore cell cycle further, fresh CD34+ cells were stained with PKH26 and cultured for 72 hours, and then undivided and divided cells were sorted. At 0.1% O2, 46.5% ± 19.1% of divided cells returned to G0 compared with 7.9% ± 0.3% at 20%. Colony formation and nonobese diabetic/severe combined immunodeficient mice engraftment efficiency were similar after 3 days at 20% and 0.1% O2 concentrations but lower than at T0. In conclusion, a low O2 concentration, close to those found in bone marrow stem cell niches, induces the G0 return of CD34+ cells without impairing their functional capacity.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Conclusion
 References
 
Hematopoiesis allows the permanent and regulated production of mature blood cells with limited life duration. Pluripotent hematopoietic stem cells (HSCs) can self-renew or give rise to myeloid and lymphoid progenitors that proliferate and differentiate into precursors whose amplification and maturation produce blood cells [1, 2]. Most human HSCs and hematopoietic progenitors can be identified and purified due to their cell surface expression of the CD34 antigen [3]. The majority of HSCs are quiescent in vivo [1, 4], and all are characterized by their self-renewal and long-term engraftment capacities in irradiated recipients [2, 5]. Survival and proliferation of HSCs are regulated in vitro by interactions with stromal cells and/or growth factors. Among growth factors protecting CD34+ cells from apoptosis, interleukin-3 (IL-3) in association with megakaryocyte growth and development factor (MGDF) and stem cell factor (SCF) improved the maintenance and self-renewal of HSCs in serum-free cultures at 3% O2 concentration [6].

Numerous in vitro studies analyzed the complex processes involved in the maintenance of HSCs [2], but few emphasized the role of O2 concentration, an environmental factor that could play an important role in the regulation of hematopoiesis in vivo. Indeed, most cells undergo low-oxygen tensions because O2 concentration ranges from 14% to less than 0.4% in mammalian organs under normal atmosphere (20% O2) [7]. Particularly, O2 concentration in the bone marrow ranges from 2% to 4% in arteries [8, 9], and a recent biophysical model supports the concept of anoxic bone marrow areas [10] where HSCs could be localized [11].

Hence, different levels of reduced oxygenation induce different cellular responses in vitro. Previous studies showed enhanced colony formation from bone marrow cells cultured at 10% O2 [12] and 5% O2 [13–15] as well as from cord blood (CB) cells [15, 16]. Moreover, culture of CD34+ cells at 5% O2 enhances the production of megakaryocytes [17], whereas at 20% O2 it promotes their maturation with platelet formation [17, 18]. This low O2 concentration (5%) also modulates cytokine effects [19] and cytokine receptor and lineage-specific marker expressions in cultures [18]. A lower O2 concentration (1%) influences in vitro erythropoiesis [20] and preserves primitive HSCs better than 20% O2 but limits colony-forming cell (CFC) amplification in cultures of murine [21–23] and human cells [24]. Recent results show that culture at 1.5%–3% O2 preserves pre-CFCs and severe combined immunodeficient (SCID) repopulating cells (SRCs) better than at 20% O2, without affecting CFC expansion [25, 26]. Lastly, CD34+ CB cells survive 24 hours at 0%–0.5% O2 in serum-free media with only thrombopoietin [27].

These data led us to explore the combined in vitro effects of IL-3 and low O2 concentrations (3% and 0.1%) on survival, cell cycle progression, colony-forming ability, and pre-CFC and SRC maintenance of cultured CB CD34+ cells.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Conclusion
 References
 
Full-Term Delivery Placental CB Samples
Samples collected (with the mother’s informed consent) in sterile bags containing anticoagulant were obtained from the French National Blood Service (Regional Center of Bordeaux). Only samples unsuitable for CB bank storage in view of allogenic transplantation (<100 g) were used for our experiments.

CD34+ Purification and Primary Liquid Culture
Mononuclear cells were separated on Ficoll (d = 1.077; Laboratoires EUROBIO, Courtabeouf, France, http://www.eurobio.fr) and CD34+ cells isolated using the Mini-MACS kit (Miltenyi Biotec, Bergisch Gladbach, Germany, http://www.miltenyibiotec.com) according to the manufacturer’s instructions. Cell purity assessed by flow cytometry (FC) on an Epics XL cytometer (Beckman Coulter, Fullerton, CA, http://www.beckmancoulter.com) using an anti-CD34 antibody (HPCA2) (Becton, Dickinson and Company, Franklin Lakes, NJ, http://www.bd.com) always exceeded 95%.

Purified CD34+ cells were seeded in 24-well plates (5 x 104 cells/ml per well) for 24, 48, and 72 hours in Stem {alpha} A (STEM ALPHA, St. Clement les Places, France, http://www.stemalpha.fr) with streptomycin (100 µg/ml) and penicillin (100 U/ml), with or without IL-3 (0.1, 0.5, and 20 ng/ml) (Tebu-bio, Le Perray en Yvelines, France, http://www.tebu-bio.com), and with or without transforming growth factor (TGF)-ß Receptor II blocking antibody (TGF-ß RII) (5 µg/ml) (R&D Systems, Minneapolis, http://www.rndsystems.com). All cultures were maintained at 37°C, in humidified atmosphere containing atmospheric air (20% O2) (NuAire incubator, NuAire, Plymouth, MN, http://www.nuaire.com), 3% O2 (Jouan incubator with an O2 control device PRO:OX110 [BioSpherix, Ltd., Redfield, NY, http://www.biospherix.com] or air-tight chamber filled with gas mixture), or 0.1% O2 (air-tight chamber filled with gas mixture) with 5% CO2 in all cases. At each time point of primary liquid culture (LC1), viable cells were counted on a Malassez Cell with Trypan Blue exclusion before further processing. To avoid variations of O2 concentrations during culture, samples for each time point were incubated in individual chambers.

Flow Cytometry
Apoptosis was assessed by annexin-V labeling after 24, 48, and 72 hours of LC1 with the APOPTESTTM–FITC (fluorescein isothiocyanate) kit (DakoCytomation, Carpinteria, CA, http://www.dakocytomation.us) according to manufacturer protocol.

Cell cycle was analyzed using propidium iodide (PI) and anti–Ki-67 antibody (DakoCytomation). PI discriminates cells in with 2n, more than 2n, and 4n DNA content. The nuclear antigen Ki-67 is expressed by cells in active phases of the cell cycle (G1 to M). The absence of Ki-67 is now considered as a reliable indicator of quiescence/G0 in most cell types (e.g., CD34+ cells) [28] because it correlates with other markers of quiescence [29, 30]. After culture, cells were washed (phosphate-buffered saline [PBS], Ca2+/Mg2+ free, EDTA [10 mM], fetal calf serum [FCS] [5%] and azide [0.05%]), fixed, and permeabilized for 30 minutes at 4°C in a solution containing formaldehyde (2%), saponine (0.02%), and Hepes 10 mM, H2O. Cells were washed twice and stained with an anti–Ki-67-FITC antibody or with the corresponding isotypic control for 30 minutes. After washing, cells were labeled with PI and analyzed by FC.

For fresh cell division tracking, the PKH26 dye (Sigma, St. Louis, http://www.sigmaaldrich.com) was used according to manufacturer instructions. Cells were separated into two populations with an Epics Elite ESP cell sorter (Beckman Coulter) after LC1 according to their PKH26 fluorescence intensity: undivided cells with fluorescence intensity similar to T0 and divided cells with decreased fluorescence intensity. Cells were then labeled with cell cycle markers as described above.

TGF-ß RII expression was analyzed using an anti–TGF-ß RII antibody (R&D Systems). After culture, cells were washed as previously described and stained with an anti–CD34-phycoerythrin (PE) (Becton, Dickinson and Company) antibody and an anti–TGF-ß RII-FITC antibody or with respective isotypic control for 30 minutes at 4°C. Then cells were washed, resuspended in PBS, and analyzed by FC.

CFC Assay
After 72 hours of LC1 with IL-3, 5 µl of cell suspension were seeded in 250 µl of methyl-cellulose (Stem{alpha}.ID, STEM ALPHA) in duplicate [26]. Culture dishes were then incubated for 14 days at 37°C in humidified atmosphere with 20% O2, 5% CO2 before counting the number of colony-forming units-granulocyte macrophage (CFU-GM), burst-forming units-erythrocyte (BFU-E), and colony-forming units-mix (CFU-Mix). All CFC assay results were normalized for 5 x 104 cells seeded in LC1 at T0.

Secondary Liquid Cultures
Secondary liquid culture (LC2) at 20% O2 allows an estimation of the number of pre-CFCs developed in LC1 by measuring their capacity to generate CFCs at different time points of LC2 [26, 31]. Briefly, after 72 hours of LC1 with IL-3 (20 ng/ml) at 20%, 3% O2, or 0.1% O2, cells were washed and replated in Stem {alpha} AG (STEM ALPHA). Cells were incubated at 37°C in humidified atmosphere (20% O2, 5% CO2) with weekly semidepopulation and addition of fresh medium. After 14, 21, and 28 days of LC2, cells were plated in methyl-cellulose to detect CFCs as described above.

SRC Analysis
After 72 hours of LC1 with IL-3 at 20% and 0.1% O2, total cell progeny of 20,000 and 40,000 CD34+ cells plated at T0 was injected intravenously with 2,000,000 irradiated (35 Gy; 60Co source; Gammatron, Siemens, France, http://www.siemens.com) human mononuclear cells as carriers to irradiated (3.5 Gy) 8- to 10-week-old nonobese diabetic (NOD)/SCID mice (Central Animal-Keeping Facility of the University of Bordeaux 2). Eight weeks after injection, mice were sacrificed and femoral bone marrow cells were harvested with 1 ml of RPMI 1640 complemented with 20% FCS. After Ficoll, mononuclear cells were incubated in PBS, EDTA 5 mM, horse serum albumin 0.5%, and rat serum 5% (StemCell Technologies, Vancouver, British Columbia, Canada, http://www.stemcell.com) at 4°C to block Fc receptors. Cells were washed (PBS, EDTA 5 mM, and human albumin 0.5%) and incubated with a PC5-coupled anti-human CD45 antibody for 20 minutes at 4°C (Immunotech, Luminy, France, http://www.immunotech.com) or with PE-coupled anti-human CD33 or CD19 antibodies (Becton, Dickinson and Company). Washed cells were then analyzed on a fluorescence-activated cell sorter (Becton, Dickinson and Company). Bone marrow cells of a noninjected mice were incubated with anti-human CD45, CD33, or CD45 antibodies as control to determine positivity thresholds (0.4% for CD45, 0.25% for CD33, and 0.5% for CD19).

Data Analysis
Mean values ± SE of the mean were calculated from data of independent experiments. Differences between experiments were assessed using the Student’s t-test.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Conclusion
 References
 
CD34+ Cells Survive in Culture at Very Low O2 Concentration (0.1%)
The effect of O2 concentration (20% and 0.1%) on CD34+ cell survival during a 3-day LC1 with or without IL-3 was first evaluated by Trypan blue exclusion test. The absence of IL-3 at 20% or 0.1% O2 resulted in a similar 70% decrease of viable cells (Fig. 1AGo). Addition of IL-3 (0.1, 0.5, and 20 ng/ml) significantly improved cell survival in a dose-dependent manner (data not shown) to reach 60% of T0 viable cells at 20 ng/ml IL-3 at 20% (28,389 ± 8,500) and 0.1% O2 (28,368 ± 7,202) (p < .01) (Fig. 1AGo). Similar results were obtained when cells were cultured 72 hours with IL-3 at 3% O2 (27,167 ± 10,222). Further experiments were carried out with 20 ng/ml of IL-3. The percentage of apoptotic cells measured by annexin-V staining was similar at 20% and 0.1% O2 (Figs. 1B, 1CGo). Moreover, despite a significant increase in the apoptotic fraction from 24 hours to 72 hours of LC1, the number of viable cells remained stable at 20% and 0.1% O2, suggesting that IL-3 allowed a fraction of cells to divide. Direct analysis of the proliferative status with PKH26 (Fig. 2AGo) showed that 25%–30% of cells divided at least once within 72 hours of LC1 at both 20% and 0.1% O2 (Fig. 2BGo) with more than 80% of viable divided cells in both conditions.


Figure 1
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  		Figure 1. Time-dependent survival and apoptosis of purified cord blood CD34+ cells in liquid culture at 20% and 0.1% O2. (A): Cells were seeded at 50,000 cells per ml with (circles) or without (triangles) 20 ng/ml interleukin-3 (IL-3). Viable cells (trypan blue negative) were counted after 24, 48, and 72 hours of culture. Values are the mean ± SE of three to 18 independent experiments (*: p < .05; **: p <.01). (B): Representative plots of annexin V (Ann V)/propidium iodide (PI) labeling at T0 and after 24, 48, and 72 hours of culture with IL-3. (C): Percentages of apoptotic cells at 20% (white bar) and 0.1% (black bar) O2 in the presence of IL-3. Values are the mean ± SE of three to four experiments (**: p <.01).

 

Figure 2
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  		Figure 2. Cell division tracking of purified cord blood CD34+ cells in liquid culture at 20% and 0.1% O2. Cells were seeded at 50,000 cells per ml with 20 ng/ml interleukin-3. (A): Representative plots of PKH26 labeling at T0 and after 72 hours of culture. Top and bottom lines represent the percentages of undivided and divided cells at 10 and 72 hours of culture, respectively. (B): Percentage of undivided and divided cells after 72 hours of culture at 20% (white bar) and 0.1% (black bar) O2 was assessed on the basis of PKH26 fluorescence. Values are the mean ± SE of five experiments.

 
Cycling CD34+ Cells Return to G0 After 72 hours of LC1 at 0.1% O2
The maintenance of cell divisions at 0.1% O2 led us to compare the cell cycle status after 72 hours of LC1 at 20%, 3%, and 0.1% O2. Double-staining with PI and anti–Ki-67-FITC revealed that most fresh CB CD34+ cells were in G0 (47.7% ± 9.8%) and G1 (51.6% ± 9.8%) (Fig. 3AGo). After 72 hours of LC1 without IL-3, most surviving cells were in G0 and G1 at 20% (74.5% ± 16.1% and 19.7% ± 12.3%, respectively) and 0.1% O2 (66.8% ± 10.9% and 32.5% ± 10.9%, respectively). After 72 hours of LC1 with IL-3, the number of cells in G0 was 1.5 and 2.5 times greater at 3% O2 (7,923 ± 4,039, p = .05) and at 0.1% O2 (13,335 ± 4648, p < .01), respectively, than at 20% O2 (5,723 ± 2,510). On the other hand, the number of cells in G1 was lower after 3 day LC1 at 3% O2 (14,146 ± 4,619) and 0.1% O2 (9,528 ± 3,537, p < .01) than at 20% O2 (17,732 ± 6,793) (Fig. 3AGo).


Figure 3
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  		Figure 3. Cell cycle progression of CD34+ cells cultured at 20%, 3%, or 0.1% O2 for 72 hours with IL-3 (20 ng/ml). Purified CB CD34+ cells were seeded at 50,000 cells per ml with IL-3 (20 ng/ml). Cell cycle was analyzed using an FITC-coupled Ki-67 antibody and PI. G0 cells were identified as Ki-67 negative and DNA 2n (PI labeling); G1 and SG2M cells were Ki-67 positive and DNA ≥ 2n. (A): Total numbers of cells per culture in the different phases of cell cycle at T0 (gray bar) and after 72 hours of culture at 20% (white bar), 3% (gray-white bar), or 0.1% (black bar). Values shown are the mean ± SE of six to 11 experiments (*: p < .05; **: p < .01). (B): Purified CB CD34+ cells labeled with PKH26 at the beginning of the culture and cultured 72 hours at 20% (white bar) or 0.1% (black bar) O2 were sorted according to their PKH26 fluorescence in undivided and divided cell population. Values shown are the mean ± SE of three experiments (*: p < .05). (C): Plots of divided and undivided cells in G0, G1, and SG2M (one representative experiment out of three). Abbreviations: CB, cord blood; FITC, fluorescein isothiocyanate; IL, interleukin; PI, propidium iodide.

 
The higher number of cells in G0 together with a lower number of cells in G1 at 0.1% O2 than at 20% and 3% O2 after 72 hours of LC1 led us to explore the cell cycle of sorted cells according to their division status assessed by measuring PKH26 fluorescence. Although it was 1.7-fold higher at 0.1% O2 (12,632 ± 3,330) than at 20% O2 (7,391 ± 3,042), the absolute numbers of undivided cells residing in G0 were not significantly different. By contrast, the absolute number of cells that returned to G0 after division was significantly higher (3.5- fold) at 0.1% O2 than at 20% O2 (3,090 ± 865 versus 878 ± 63, respectively) (Fig. 3BGo). Figure 3CGo shows the result of one representative experiment out of three experiments for each O2 concentration.

To explore the mechanisms involved in the survival and the G0 maintenance of CB CD34+ cells at 0.1% O2, we analyzed TGF-ß involvement and the expression of its type II receptor. No expression of TGF-ß RII was detected on CB CD34+ cells either fresh or after 72 hours of LC1 at 20% and 0.1% O2. Moreover, a blocking anti–TGF-ß RII antibody did not reduce the percentage of cells in G0 at 0.1% O2, suggesting that TGF-ß does not participate to the G0 maintenance of CB CD34+ cells at 0.1% O2 (data not shown).

Effect of Low O2 Concentration on CFU-GM, BFU-E, CFU-Mix, and pre-CFCs
Functionality of CD34+ cells after 72 hours LC1 at 20%, 3%, and 0.1% O2 with IL-3 was first assessed in vitro by their CFC and pre-CFC capacities. As reported in Figure 4AGo, the number of CFU-GM, but not of BFU-E and CFU-Mix, is significantly lower after 72 hours of LC1 at 0.1% O2 than at 20% O2 (p < .05). This difference in the number of CFU-GM is not significant when cells were cultured 72 hours at 3% O2. However, LC1 at 0.1% seems to better preserve pre-CFCs because the CFC production tested after 14, 21, and 28 days of LC2 was 1.5 higher on average than LC1 at 3% and 20% (Fig. 4BGo).


Figure 4
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  		Figure 4. Functional capacities of purified CB CD34+ cells (50,000 cells/ml) at T0 (gray bar) and after 72 hours of culture at 20% (white bar), 3% (gray-white bar), or 0.1% (black bar) O2 with IL-3 (20 ng/ml). (A): CFC-derived colonies were evaluated after 14 days in methyl-cellulose. Values shown are the mean ± SE of six to 15 experiments. (B): Pre-CFCs harvested from LC1 were analyzed by evaluating their production of CFCs after 14, 21, and 28 days in LC2. Values shown are the mean ± SE of three to five experiments. Abbreviations: BFU-E, burst-forming unit-erythrocyte; CB, cord blood; CFC, colony-forming cell; CFU-GM, colony-forming unit-granulocyte macrophage; CFU-Mix, colony-forming unit-mix; IL, interleukin; LC1, primary liquid culture; LC2, secondary liquid culture.

 
SRC Activity of Fresh and Cultured (20% and 0.1% O2) CD34+ Cells
To explore the effect of O2 concentration on the maintenance of HSCs with engraftment capacities, we compared the SRC activity of cell suspensions after 72 hours of LC1 at 20% and 0.1% O2 by injecting them in NOD/SCID mice. Injection of freshly purified CD34+ cells in NOD/SCID mice led to an efficient engraftment with the two cell doses tested (20,000 and 40,000) as shown by analysis of human CD45 expression (Fig. 5AGo). However, whereas all mice were also positive for CD19 antigen, only 70% of those injected with 20,000 CD34+ cells were positive for CD33 (Figs. 5B, 5CGo). Injection of cell progeny of 20,000 and 40,000 cells seeded at T0 led to similar engraftment efficiencies (30% and 50%, respectively, measured by the expression of human CD45) after both 20% and 0.1% O2 LC1 (Fig. 5AGo). However, LC1 at 20% O2 seems to maintain better myeloid (human CD33) and lymphoid (human CD19) differentiation potential of stem cells as revealed by the higher human CD33 and CD19 chimerism in engrafted mice.


Figure 5
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  		Figure 5. Engraftment of nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice by cells at T0 and expanded at 20% or 0.1% O2. After 72 hours of cord blood CD34+ cell culture, the progeny of 20,000 ({blacktriangleup}) and 40,000 ({diamondsuit}) CD34+ cells plated at T0 was injected in NOD/SCID mice by the tail vein. Human CD45(A), CD33(B), and CD19(C) chimerism in NOD/SCID mice was analyzed 8 weeks after injection. The threshold of detection was determined by analyzing eight noninjected mice (bullet).

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Conclusion
 References
 
The present work brought major results concerning the effects of 0.1% O2 on CB CD34+ cells in vitro. We firstly showed that 72 hours of serum-free culture with IL-3 induced the same limited cell death rate (40%) at 20%, 3%, and 0.1% O2. These results confirm and extend those of Saxonhouse et al. on CD34+ cells cultured 24 hours at 0.1% O2 [27] by showing that 0.1% O2 does not increase apoptosis of fresh CD34+ cells. IL-3 induces cell cycle progression in quiescent normal and malignant CD34+ cells at 20% O2 [32, 33], by itself allowing G1-S transition [34]. Direct analysis of fresh CB cell proliferation with PKH26 labeling confirms these observations at 20% O2 and extends it to 0.1% O2 because 25%–30% of cells divided at least once. This result is also stressed by cell cycle analysis because only 1% of freshly purified CB CD34+ cells are in SG2M cell cycle phases against 10%–30% after 72 hours LC1 at 20%, 3%, and 0.1% O2. However, cell cycle analysis of whole CD34+ cell population revealed that 30% and 50% of cells were in G0 at 3% and 0.1% O2 cultures, respectively, compared with only 20% at 20% O2 culture. More importantly, the very low (but physiological) [10] O2 concentration (0.1% O2) in the presence of IL-3 allowed the re-entry in G0 of more than 40% of divided cells, unlike 20% O2. This phenomenon has not yet been described for CD34+ cells in vitro.

Examining molecules able to block primitive HSCs in G0 and to induce their G0 re-entry after cycling at 0.1% O2, we first considered TGF-ß because it inhibits [35] or blocks [36] hematopoietic cell proliferation in vitro. However, despite pronounced in vitro effects in cultures at 20% O2, TGF-ß signaling is required neither for cell cycle control nor for normal differentiation and maturation of murine primitive HSCs in vivo [37]. Furthermore, marrow repopulating activity is independent of TGF-ß signaling in murine [37] and human [30] hematopoietic cells. Our results with cultures at 0.1% O2, close to those found in vivo, are in agreement with the results of Larsson et al. [37]. Indeed, the expression of TGF-ß RII at the cell surface was negligible on fresh CD34+ cells as well as after culture at 20% and 0.1% O2, and the TGF-ß RII neutralization did not decrease the percentage of cells in G0 at 0.1% O2.

These short-term cultures (72 hours) at 0.1% generated the same negative effect on CFU-GM previously shown in prolonged cultures of mouse bone marrow [21], human peripheral blood cells [22], and CB CD34+ cells [20] at 1% O2. This phenomenon was not observed at higher O2 concentrations (3% and 20% O2). By contrast, we did not observe variations of BFU-E and CFU-Mix. This could be partly due to the higher O2 requirement of CFU-GM [38]. However, the CFC production by pre-CFCs tested after 14, 21, and 28 days of LC2 was higher than after 72 hours LC1 at 3% and 20% O2. One can suggest that the higher number of G0 cells led to a better maintenance of more primitive stem cells, as previously described [32]. It is known that CB CD34+ cells engraft NOD/SCID mice better than CD34+ bone marrow cells [39]. However, whereas fresh CB CD34+ cells engrafted all mice even with 20,000 cells injected, 72 hours of LC1 cells did not engraft all mice at both 20% and 0.1% O2. This could be partly related to the presence of IL-3 alone in these cultures. The similar percentage of mice engrafted with cells issued from LC1 at 20% and 0.1% O2 is in some respects surprising because the percentages and absolute numbers of G0 cells are different between the two conditions. Indeed, some studies concluded that bone marrow mononuclear cells and mobilized CD34+ blood cells in G0 have a better engraftment potential than cells in other phases of the cell cycle [30, 40, 41]. By contrast, other studies showed no differences in engraftment capacity of CB and fetal cells, whatever their cell cycle phase [42, 43], and unimpaired SRC activity of HSCs cycling actively ex vivo [44–46]. Thus, our results could reflect the maintenance of engraftment capacity of actively proliferating SRCs at 20% of O2 (in line with [43–46]), the loss of homing/engraftment capacity of SRCs after re-entering in G0 at 0.1% O2, or the expression of genes independent of cell cycle status and involved in bone marrow homing of SRCs. Interestingly, cultured cycling human CD34+ cells engrafted in the bone marrow of NOD/SCID mice were quiescent 40 hours after injection [47]. Although the hypothesis of re-entering in G0 of previously cycling cells was not proposed by these authors to explain their results, our data suggest that it should be considered, particularly because of the presence of nearly anoxic areas in the bone marrow tissue [10].


   CONCLUSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
References
 
Our data demonstrate that 0.1% allows the survival of primitive HSCs among CB CD34+ cells. In addition, and as important, we provide the first evidence that culture at 0.1% O2 favors the return of cycling CD34+ cells in G0, a quiescent state that characterizes steady-state HSCs. Altogether, these data and previous studies by our group and other groups [17, 25, 26] stress the proposal that variations of bone marrow O2 concentrations in the physiological range (0.1%–5% O2) are key regulators of primitive hematopoiesis by controlling the maintenance and re-entry in quiescence of HSCs after cycling (0.1%), their differentiation potential as well as the proliferation and maturation of committed progenitors (1.5%–5% O2).


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 References
 
Francis Hermitte is the recipient of a grant from the "Ministère de la Recherche" of France. This work was supported by grants from the "Comités de la Charente et de la Gironde" of the "Ligue Nationale Française Contre le Cancer." We thank Mrs. J. Plassat, Mr. P. Chemin from the Radiotherapy Unit of "Institut Bergonié," Bordeaux, and Mrs. Orthet from the Radiotherapy Unit of "Hôpital ST André," Bordeaux, for NOD/SCID mice irradiation as well as Pierre Costet for his efforts to maintain NOD/SCID mice colony at the Central Animal Facility of Bordeaux 2 University. We thank Dr. A. Hatzfeld and Dr. J. J. Lataillade for their help in TGF-ß investigation. We are grateful to Tanesha Naiken for her valuable help in English writing and to Sylvie Hermouet for her critical reading and helpful suggestions about the manuscript.

DISCLOSURES
The authors indicate no potential conflicts of interest.


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 References
 

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Received December 10, 2004; accepted for publication June 23, 2005.




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