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Steady-state blood CD34+ HSCs are CXCR4lowCD133+
or bone marrow collection would be of great interest, espe- cially in the context of allogeneic transplantation.
Ex vivo expansion procedures have evolved over the last few years and it is now possible to amplify committed HPCs to a great extent without losing the long-term reconstituting HSCs.4,5 Recently, we demonstrated the presence of both short- and long-term reconstituting HSCs in human SS-PB and also observed that the activity of these cells increases dramatically after ex vivo expansion.6,7 In this manner, we can safely source substantial numbers of SS-PB HPCs and HSCs, thus overcoming major obsta- cles to subsequent transplantation. In the light of this, SS- PB HPCs and HSCs should be reconsidered in the context of hematopoietic transplantation.
Based on previous literature regarding HSC activity,6 it was not possible to specify whether the increase in activ- ity of HSCs capable of reconstituting in vivo hematopoiesis of severe combined immune-deficient mice (SCID) repop- ulating cells (SRCs) after ex vivo expansion is: (i) due to amplification of these cells during ex vivo culture; or (ii) corresponds to pre-existing SRCs before ex vivo expansion (at time 0), which during expansion (until day 7), gained the ability to engraft after transplantation; or (iii) a combi- nation of the above.
In order to address this issue, we investigated both HSC functional capacity in in vivo assays and the expression of membrane markers known to be associated with cell adhesion and homing, such as CD9, CD26, CD49d, CD49e, CD49f and especially CXCR4, as well as markers enabling the enrichment of HSCs (CD133, CD90, CD45RA). The choice of the tetraspanin CD9 was based on the fact that it is regulated by the activity of stromal cell-derived factor-1 (SDF-1; the ligand of CXCR4 recep- tor)8 and CD26, since it is known to be an inhibitor of activity of the SDF-1/CXCR4 couple,9 which plays an essential role in HSC mobilization and homing.10-12 CD49d (VLA4), CD49e (VLA5), and CD49f (VLA6) are adhesion molecules of the integrin family associated with the anchorage and adhesion of cells in different situations and are considered essential for HSC homing.13 Furthermore CD49f, CD45RA and CD90 are used as markers of cord blood (CB) and/or bone marrow (BM) HSCs.14,15 Despite the fact that it largely overlaps with CD34, CD133 was chosen since it is not expressed on some subpopulations of committed progenitors and, hence, is more likely to include the HSCs.16-18
We found that HSC activity increases due to both amplification in their number and to enhancement of their individual proliferative capacity. Furthermore, in vivo reconstituting cells (both short- and long-term reconstitut- ing cells i.e. ST-HSCs and LT-HSCs, respectively) in the fresh SS-PB CD34+ cell population belong to the subpopu- lation of CD133+ cells which are either CXCR4low or CXCR4neg, while after ex vivo expansion they are present only in the CD133+CXCR4low population.
Methods
Human steady-state peripheral blood cells
Leukocytes were recovered from leukodepletion filters (T2975, Fresenius Kabi, Louviers, France) by counterflow elution as described elsewhere6,19,20 with a slight modification, i.e. the cells were flushed directly into 50 mL tubes (Falcon, Dutscher, Brumath, France) (see Online Supplementary Methods).
Isolation and cryopreservation of CD34+ cells
CD34+ cells were isolated from the mononuclear cell fraction using Miltenyi’s (Miltenyi Biotec, Paris, France) “indirect” immuno-magnetic technique19 (“LS” columns; Vario Macs Device). The CD34+ cell purity was 85-90% and the yield was 3-5x105 CD34+ cells per leukodepletion filter.
For each sample, 20 to 24 leukodepletion filters were processed and CD34+ cells were pooled before cryopreservation (4% human serum albumin solution, 10% dimethylsulfoxide; Wak-Chemie, Steinbach, France).21 Samples were thawed in cold 4% human serum albumin and washed in selection buffer. After thawing, the CD34+ cell purity was 90-95%.
Ex vivo expansion of CD34+ cells recovered from leukodepletion filters
All tests were performed on CD34+ cells after thawing, before expansion (day 0) and after expansion (day 7). Day-0 CD34+ cells were seeded at 2x104 cells/mL, and cultured in 75 cm2 flasks (NUNC, Roskilde, Denmark) for 7 days in liquid (clinical-grade serum-free medium Macopharma HP01) cultures supplemented with granulocyte colony-stimulating factor 100 ng/mL (Neuropen, Amgen SAS, Neuilly-sur-Seine, France), stem cell factor 100 ng/mL, thrombopoietin 20 ng/mL and interleukin-3 0.5 ng/mL (all from Peproteck, Rocky Hill, NJ, USA) (see Online Supplementary Methods).
CD34+ cell detection, immunophenotypic analysis and selection of cell subfractions
The CD34+ cell concentrations/purities were determined as pre- viously described.19,22 Fluorescent monoclonal antibodies were used to analyze/isolate CXCR4neg, CXCR4low, CXCR4bright subfrac- tions, and CXCR4negCD133-, CXCR4negCD133+, CXCR4lowCD133-, CXCR4lowCD133+, CXCR4brightCD133-, and CXCR4brightCD133+ subfractions. The details are provided in the Online Supplementary Methods.
Detection of stem cells by their in vivo repopulating capacity
The only way to evaluate the activity of HSCs properly is to test their in vivo capacity of hematopoietic reconstitution.23 Hence, we employed the most widely used assay based on the repopulation, by human cells, of hematopoietic tissues of immune-deficient mice, thereby evaluating the cells usually called SRCs (Figure 1). This approach enables the detection of two SRC populations:
i) Short-term HSCs (ST-HSCs). ST-HSC activity was evaluated in vivo, following transplantation of different phenotypically defined fractions of human SS-PB CD34+ cells in immunodeficient [NOD/SCID/gamma-null (NSG)] mice. As described previously,24 the animal experiments were performed in compliance with French regulations (license n. 3306002) and with the approval of the Ethics Committee (n. 50120213-A). Either 1x105 CD34+ cells or 1x105 cells of sorted subfractions at day 0 were injected per mouse. After expansion, 2x105 of total day-7 cells or 2x105 cells of sorted subfrac- tions were transplanted per mouse. In some experiments, the post- culture (day-7) equivalent of a defined number of day-0 cells i.e. the total day-7 progeny of a defined day-0 cell number, was injected per mouse (Figure 1). For all experiments, 10- to 12-week old female NSG mice (central animal-keeping facility of Bordeaux University) were conditioned by means of intra-peritoneal injections of 25 mg/kg busulfan (Busilvex, Pierre Fabre, Boulogne, France),25,26 After 8 weeks, the animals were sacrificed and their femoral mononu- clear BM cells isolated and analyzed for human CD45, CD19 and CD33 (with anti-human antibodies coupled with, respectively, flu- orescein isothiocyanate, phycoerythrin and allophycocyanin; BD Biosciences, Le Pont de Claix, France) by flow-cytometry (FACS Canto II; BD Biosciences, Le Pont de Claix, France). To avoid false-
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