HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints/Permissions Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Drouet, M. Articles by Mayol, J.F. Search for Related Content PubMed PubMed Citation Articles by Drouet, M. Articles by Mayol, J.F.

Cell Cycle Activation of Peripheral Blood Stem and Progenitor Cells Expanded Ex Vivo with SCF, FLT-3 Ligand, TPO, and IL-3 Results in Accelerated Granulocyte Recovery in a Baboon Model of Autologous Transplantation but G₀/G₁ and S/G₂/M Graft Cell Content Does Not Correlate with Tranplantability

^a Centre de Recherches du Service de Santé des Armées, La Tronche, France;
^b Unité de Thérapie cellulaire, Service d'Hématologie Clinique, Groupe Hospitalier Pitié Salpétrière, Paris, France

Key Words. CD34⁺ cells • Bone marrow aplasia • Cell cycle • Nonhuman primate • Cytokines • Ex vivo expansion

M. Drouet, M.D., Ph.D., Experimental Radiohematology Unit, Centre de Recherches du Service de Santé des Armées, 24 avenue des Maquis du Grésivaudan BP 87-38702 La Tronche Cedex, France; Telephone 33-4-76-63-69-28.

	ABSTRACT

Top Abstract Introduction Materials and Methods Results Discussion References

 
Ex vivo expansion is a new strategy for hematopoietic stem and progenitor cell transplantation based on cytokine-induced amplification to produce grafts of controlled maturity. If the cell cycle position of CD34⁺ cells has been reported to govern their engraftment potential, the respective role of stem and progenitor cells in short- and long-term hematopoietic recovery remains debated. Studies focused on long-term engraftment potential suggest impairment when using cultured grafts, but the capacity to sustain short-term recovery is still controverted. The aim of this study was: A) to evaluate the consequences of cell cycle activation on short and long-term engraftment capacity, and B) to determine if cell cycle status of grafts could predict hematopoietic recovery. We showed in a nonhuman primate model of autologous peripheral blood stem and progenitor cell transplantation that cell cycle activation of CD34⁺ cells in the presence of stem cell factor + FLT3-ligand + thrombopoietin + interleukin 3 (six days of culture) which induced G₁ and S/G₂/M cell amplification (G₀: 6.1% ± 2.8%; G₀/G₁: 64.2% ± 7.2%; S/G₂/M: 30.4% ± 7.3% respectively of expanded CD34⁺ cells on average) resulted in the acceleration of short-term granulocyte recovery. By contrast, G₀/G₁ and S/G₂/M cell content of expanded grafts did not correlate with short- or long-term engraftment.

	INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion References

 
Ex vivo expansion of hematopoietic stem and progenitor cells (HSPCs) has been proposed as a new therapeutic strategy in bone marrow transplantation settings with the aim of amplifying stem cells or committed progenitor cell compartments [1, 2]. One goal of ex vivo expansion may be to increase the number of transplantable hematopoietic stem cells in a small graft to make possible a therapy which would otherwise be impossible [3]. On the other hand, the production of mature progenitors may reduce or abrogate the initial neutrothrombopenic phase of bone marrow aplasia, thus counteracting the high level of hemorrhagic and infectious morbidity during this crucial period. Meanwhile, the respective participation of mature and immature cells in short- and long-term reconstitution remains unclear. Actually, no clinical study has demonstrated thus far a clear benefit for thrombopenic patients but neutropenia can be counteracted by ex vivo expanded cell infusion associated or not with growth factor administration [4, 5]. Meanwhile, documented expanded grafts were usually performed with large numbers of cells issued from cultures of more than the 2 x 10⁶ CD34⁺ cells/kg cell threshold.

In fact, several studies demonstrated that ex vivo HSPC expansion could result in the loss of long-term hematopoietic repopulating potential [6-10]. This effect depends on culture duration and cytokine combination which govern the mitotic cycles' number after cytokine stimulation of HSPCs [11, 12]. Srour's team demonstrated that this impairment could be mainly attributed to a loss of quiescence and G₁ phase entry. Alternatively, Eaves's team recently reported the G₁ status of expanded transplantable cells [10], and a recent study suggests cooperation between G₀ and G₁ cell populations for HSPC engraftment [13]. Moreover, short-term repopulating potential could also be impaired by culture as suggested by Tisdale's and Brandt's studies [9, 14]. In fact, the cell population responsible for short- and long-term hematopoietic reconstitution as well as their cell cycle status actually remains debated. Moreover HSPC's cell cycle status could be characterized by its plasticity during culture with cyclic oscillations between proliferation and quiescence [15].

In a previous study, we showed in a nonhuman primate model that stem cell factor (SCF) + FLT3-ligand (FLT3-L) + thrombopoietin (TPO) + interleukin 3 (IL-3) (i.e., 4F; 50 ng/ml each) expanded grafts issued from 2 x 10⁶/kg CD34⁺ cells significantly reduced both neutropenia and thrombocytopenia as compared with unmanipulated grafts [16]. Then we chose to evaluate the potential benefit of such a culture condition in the context of small numbers of initial CD34⁺ cells, which corresponds to a significant problem in clinical settings (i.e., poorly mobilizing patients). Moreover, we sought to determine whether the cell cycle phenotype of the grafts could be a prognostic factor of transplantability. We showed here in a 6-day culture system using baboon mobilized peripheral blood (MPB) CD34⁺ cells that the previously described SCF + FLT3-L + TPO + IL-3 cytokine combination [16, 17] activated CD34⁺ cells resulting in the amplification of G₀/G₁ and S/G₂/M cells. In our nonhuman primate model of autologous peripheral blood stem progenitor cell (PBSPC) transplantation, cell-cycle-activated CD34⁺ cell grafts accelerated short-term neutrophil recovery without impairment of mid/long-term reconstitution. By contrast, G₀/G₁ and S/G₂/M graft cell content did not correlate with short- or long-term recovery which suggests that cell cyle phenotype would not represent a valuable predictive factor of engraftment.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

 
Animals
Adult male baboons (Papio anubis) (26 ± 3 kg, n = 15) were housed in a CRSSA-accredited animal facility in holding rooms equipped with a reverse-filtered air barrier. The present protocol was approved by the Army Ethics Committee.

CD34⁺ Cell Source
Cytoxan + G-CSF-MPB hematopoietic cells were collected by leukapheresis on a Spectra cell separator (Cobe; Villepinte, France; http://www.cobebct.com): Cytoxan 50 mg/kg on two consecutive days (a gift from Asta-Medica; Nanterre, France; http://www.astamedica.de/index.cfm) and Neupogen 40 µg/kg/day (a gift from Amgen-Roche; Neuilly, France; http://www.amgen.com). CD34⁺ cells were isolated on immunoaffinity columns using 12.8 Class I anti-CD34 monoclonal antibody (mAb) (Cell Pro; Bothell, WA). Mean CD34⁺ cell purity evaluated using 566 Class III anti-CD 34 phycoerythrin (PE)-conjugate (a gift from T. Egeland; Oslo, Norway) was 84.2 % ± 9.4 %.

Cell Cultures
CD34⁺ cells were grown in Iscove's modified Dulbecco's medium supplemented with 1.5% bovine serum albumin (Cohn fraction V; Sigma; St Louis, MO; http://www.sigma-aldrich.com), iron saturated human transferrin (300 mg/ml; Sigma), and sonicated lipids (20 µg/ml), penicillin/streptomycin/glutamine, -thioglycerol (11.5 µmol/l; Sigma). Cells were seeded at 1 x 10⁵ cells/ml and cultured in 75-cm² vented flasks, in a humidified incubator with 5% C0₂ in air for 6 days with a combination of 100 ng/ml recombinant human (rHu) Flt3-L (a gift from Immunex; Seattle, WA; http://www.immunex.com), 100 ng/ml rHuSCF (a gift from Amgen Corp; Thousands Oaks, CA), 50 ng/ml rhuTPO (a gift from Genentech; South San Francisco, CA; http://www.gene.com), and 30 ng/ml rhuIL-3 (a gift from Novartis; Basel, Switzerland; http://www.pharma.novartis.com). Cell density was readjusted once by adding fresh medium and splitting the culture into two flasks.

Characterization and Quantification of Unmanipulated and Expanded Cells
Viable cells were counted in a hemocytometer using trypan blue exclusion assay and analyzed on May-Grünwald-Giemsa stained cytospins. As reported [3], we performed clonogenic assays (colony-forming unit-granulocyte/ macrophage [CFU-GM], colony-forming unit-megakaryocyte [CFU-MK]), long-term culture-initiating cell (LTC-IC), and nonobese diabetic-severe combined immunodeficient (NOD-SCID) assays, and flow cytometric analysis (EPICS XL, Beckman Coulter; Miami, FL; http://www.coulter.com) before and after culture (anti-CD90 [BD Biosciences; San Jose, CA], anti-CD38 [Orthoclinical; Levallois, France; http://www.orthoclinical.com], antimyeloperoxydase [Caltag; San Francisco, CA; http://www.caltag.com], anti-CD41 [Coultronics; Margency, France], anti CXCR-4 [BD Biosciences] mAbs). Cells which expressed CD34 antigen after 6 days of culture at the same level as initial CD34⁺ cells in term of mean fluorescence intensity (MFI) were defined as CD34^+high cells; cells which exhibited a reduced CD34 expression, mainly labeled as CD34/CD41 cells or CD34/MPO cells, were defined as CD34^+low cells. Cell cycle analysis was also performed using flow cytometry. The cells were first incubated with biotinylated anti-CD34⁺ mAb (566 clone) for 30 minutes on ice, washed and reincubated with streptavidin fluorescein isothiocyanate (FITC [Dako; Trappes, France; http://www.dako.dk]) and then fixed for 1 hour. After washing, the cell pellet was resuspended in 70% ethanol and kept overnight at 4°C in the dark. Cells were then washed and incubated with propidium iodide (50 µg/ml, Sigma) in phosphate buffer solution for 30 minutes and immediately analyzed. For G₀/G₁ cell quantification, DNA and nuclear protein Ki67 (only present during G₁ and S/G₂/M phases of cell cycle) cell content was evaluated [18]. Briefly, after CD34⁺ labeling (566 PE) and cell fixation in phosphate buffered saline (PBS)-formaldehyde 1%, cells were permeabilized in PBS-Triton 0;1% (30 minutes on ice). After being washed twice, cells were incubated with anti-Ki-67 FITC conjugated MIB-1 mAb (Beckman Coulter) for 1 hour at 4°C. Finally, cells were washed and resuspended in PBS-7 aminoactinomycin (7AAD) (Sigma, 0.5 µg/ml). Flow cytometric analysis was performed within 1 hour and G₀, G₁, S/G₂/M cell count determined (FCS Express, De novo Software; Toronto, Canada; http://www.denovosoftware.com). Figure 1, quadrant 1 shows cells negative for nuclear Ag Ki67, which showed a diploid amount of DNA (7AAD) = G₀ cells; quadrant 2 shows active cells (Ki67 positive cells) which have not begun to replicate DNA (diploid cells) = G₁ cells; quadrant 3 shows active cells (Ki67 positive cells) which replicate DNA = S/G₂/M.

View larger version (41K): [in this window] [in a new window]   Figure 1. CD34+ expression and cell cycle phenotype of fresh and ex vivo expanded baboon cells. CD34+ expression and cell cycle phenotype were determined by flow cytometric analysis as indicated in Materials and Methods (triple labelings with 566-PE mAb, anti Ki67 cell-FITC mAb, 7AAD). A: CD34+ expression shown here as monoparametric analysis conducted concomitantly with triple labelings. B: cell cycle phenotype of total CD34+ cells shown as a biparametric dot plot from triple labeling. x axis = DNA / 7AAD cell content; y axis = nuclear protein Ki67 cell content. 1: G0 CD34+ cells; 2: G1 CD34+ cells; 3: S/G2/M CD34+ cells. Results from one representative baboon cell sample.

Statistical Analysis
In the short-term recovery study, the times to neutrophil and platelet reconstitution after grafting were compared for unmanipulated and expanded cells using the Mann and Whitney nonparametric test; values of p of less than 0.01 were considered to be statistically significant. Global evaluation was performed between unmanipulated (n = 5) and ex vivo expanded groups (n = 10). With respect to the animals which did not exhibit any reconstitution, we attributed the highest time to recovery observed, (i.e., the last rank) in order to perform the statistical analysis.

Correlation Study
Spearman's correlation coefficients were determined between CD34⁺ and CD34^+high G₀/G₁-S/G₂/M cells, on the one hand, and (A) day to PMN/PLT recovery and (B) AUC of PMN/PLT counts from day 60 to day 360 post-transplantation, on the other hand.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cell Cycle Analysis of 4F Cultured CD34⁺ Cells
Cell cycle analysis was performed for five unmanipulated grafts and for 10 expanded grafts as follows: unmanipulated CD34⁺ cells 0.3, 0.5, 0.75 x 10⁶/kg (n = 2, n = 2, n = 1, respectively); expanded grafts from initial CD34⁺ cell number 0.3/0.5/0.75 x 10⁶ (n = 5, n = 3, n = 2, respectively).

Control unmanipulated baboon MPB CD34⁺ cells are mainly in G₀/G₁ phase of cell cycle, and very few cells are in S/G₂/M phase. As previously reported, after 6 days of culture, only a fraction of ex vivo expanded CD34⁺ cells expressed CD34 antigen in a similar way to initial CD34⁺ cells (CD34⁺⁺ = CD34^high) in terms of MFI, and another fraction exhibited a reduced expression (CD34^low) (Fig. 1). Whatever the CD34 antigen expression level, cultured cells were activated. A strong minority of CD34⁺ cultured cells exhibited an S/G₂/M pattern (33.4% ± 12.3% and 30.4% ± 7.3% for CD34^+high and CD34⁺, respectively) and the majority were in G₀/G₁ phase (64.6% ± 11.3% and 64.2% ± 7.2%) with only a reduced fraction of G₀ cells (6.1% ± 2.8% for CD34⁺ cells) (Fig. 1).

Cell Cycle Activation Induced by Ex Vivo Expansion Improves Short-Term Neutrophil Recovery
Unmanipulated grafts contained 0.28 to 0.66 x 10⁶ G₀/G₁ CD34⁺ cells/kg and 3.1 x 10⁴/kg S/G₂/M cells on average (Table 1). Cultured grafts always exhibited cell cycle activation as G₀/G₁ (from 1.4 to 7.8 10⁶/kg depending on the CD34⁺ cell dose) and S/G₂/M (from 0.77 to 3.47 x 10⁶/kg) cell contents were increased; in fact, G₀/G₁ cell amplification appeared to be related to G₁ cell amplification with no increase of the fraction of the quiescent cells. Animals grafted with activated ex vivo expanded cells received many more G₀/G₁ and S/G₂/M cells than controls (p = 0.002).

G₀/G₁ and S/G₂/M Cell Content Did Not Correlate with Mid/Long-Term Engraftment for Expanded Group
No correlation was established between G₀/G₁ and S/G₂/M cells and PLT AUC. S/G₂/M cells did not significantly correlate with PMN AUC (r²: 0.009).

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

 
Conflicting results about the capacity of expanded cells to sustain long-term but also short-term engraftment have been reported in animal models, which appeared mainly related to cytokine combination and culture duration used. For example, in a nonhuman primate model, Tisdale et al. reported in an elegant model of PB CD34⁺ transduction that ex vivo expansion in the presence of IL-6, IL-3, SCF, and FLT-3L significantly impaired long- and short-term hematopoietic recovery as compared with unmanipulated cells [9]. Brandt et al. showed in their baboon model of HSPC transplantation a delayed hematopoietic recovery when using ex vivo expanded grafts cultured on porcine microvascular endothelial cells when compared with unmanipulated grafts [14]. In fact, a lack of knowledge remains about the respective role of immature and mature cells in short- and long-term recovery [19] as well as about their cell cycle status [20]. More information is required about the cell compartment to be selectively amplified in order to improve ex vivo expanded graft efficacy.

Actually, ex vivo cell culture has been suspected or demonstrated in animal models to be unable to sustain immature cell survival or growth. For instance, Srour's studies demonstrated that LTC-IC potential can be retained in vitro during only three mitotic cycles in SCF, FLT-3L, TPO, IL-3, and IL-6 cultures and that NOD-SCID engraftment capacity was impaired after a 36-hour incubation in the presence of a combination of IL-3, SCF, and TPO or SCF, TPO, and FLT-3L, mainly due to the G₀/G₁ cell transit [11]. The engrafting properties of the HSPCs in different cell cycle phases could be related to adhesion molecule expression [21, 22]. Alternatively, Eaves's team showed that expanded transplanted cells were not quiescent but rather G₁ cells. Meanwhile, in contrast with the previous studies, G₀ and G₁ umbilical cord blood CD34⁺ cells have been reported to exhibit similar repopulating capacity as described in the NOD/SCID mouse model [23]. In their animal model, Tisdale and Brandt showed that the CD34⁺ cells' massive exit from quiescence and differentiation (99.8% in Brandt's study), a consequence of prolonged culture (10-14 days), impaired short-term reconstitution.

Thus, we decided to evaluate the engraftment potential of short-term (6 days) CD34⁺ cell cultures involving early-acting cytokines in order to amplify the CD34⁺ cell compartment. As the 2 x 10⁶/kg CD34⁺ cell threshold cannot be reached in numerous cancer patients, especially after multiple chemotherapies, we focused this work on the efficacy of grafts expanded from a reduced initial number of CD34⁺ cells. In our study, cell cycle analysis showed that after 6 days of incubation with 4F, CD34⁺ cells were mainly in G₀/G₁ phase (64.2% ± 7.2%), with a minority of them in S/G₂/M phase (30.4% ± 7.3%). Whether G₀ cells observed over the duration of the culture were the same population of initial quiescent cells or whether they were issued from the reentry in G₀ phase according to the cell cycle plasticity concept remains to be established. However, our results are in agreement with recent works based on early-acting cytokine cultures [24]. In our study, injection of low doses of activated expanded cells improved short-term WBC and PMN recovery [3] but had no effect on short-term PLT recovery and mid/long-term recovery of the two cell lines (Table 2). As G₀ cell percentage was on average 60% in fresh CD34⁺ cells and 6.1% ± 2.8% after six days of cultures (Fig. 1), the increased number of transplanted cells as compared to those grafted to the control animals consisted of nonquiescent G₁ and S/G₂/M cells.

Our results on short-term hematopoietic recovery incited us to evaluate whether G₀/G₁ and S/G₂/M graft cell content may represent predictive factor(s) of engraftment for expanded grafts, especially for WBC and PMN reconstitution. Unfortunately, our data did not show any significant correlation between cell cycle parameters and short- or long-term PMN/PLT recovery (Table 3). In their murine model, Szlivassy et al. recently demonstrated similar short-term reconstitution after grafting G₀/G₁ or S/G₂/M expanded Sca-1⁺c-kit⁺-Lin^– cells [25]. Thus, we tested whether G₀/G₁ + S/G₂/M graft cell content (i.e., total CD34⁺ cell population) correlates with early reconstitution. Again, no significant correlation could be established (r²: 0.3568), in agreement with McNiece's study which reported the nonpertinent predictive value of expanded peripheral blood CD34⁺ cell graft content.

	REFERENCES

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Emerson SG. Ex vivo expansion of hematopoietic precursors, progenitors, and stem cells: the next generation of cellular therapeutics. Blood 1996;87:3082–3088.[Free Full Text]

2. Brugger W, Heimfeld S, Berenson RJ et al. Reconstitution of hematopoiesis after high-dose chemotherapy by autologous progenitor cells generated ex vivo. N Engl J Med 1995;333:283–287.[Abstract/Free Full Text]

3. Norol F, Drouet M, Pflumio F et al. Ex vivo expansion marginally amplifies pluripotent repopulating cells from baboon peripheral blood mobilized CD34⁺ cells. Blood 2000;96:765a.

4. Reiffers J, Caillot C, Dazey B et al. Abrogation of post-myeloablative chemotherapy neutropenia by ex-vivo expanded CD34-positive cells. Lancet 1999;354:1092–1093.[CrossRef][Medline]

5. McNiece I, Jones R, Scott I et al. Ex vivo expanded peripheral blood progenitor cells provide rapid neutrophil recovery after high dose chemotherapy in patients with breast cancer. Blood 2000;96:3001–3007.[Abstract/Free Full Text]

6. Gothot A, Pyatt R, McMahel J et al. Functional heterogeneity of human CD34⁺ cells isolated in subcompartments of the G₀/G₁ phase of the cell cycle. Blood 1997;90:4384–4393.[Abstract/Free Full Text]

7. Gothot A, van der Loo JCM, Wade Clapp D et al. Cell cycle-related changes in repopulating capacity of human mobilized peripheral blood CD34⁺ cells in non-obese diabetic/severe combined immune-deficient mice. Blood 1998;92:2641–2649.[Abstract/Free Full Text]

8. Traycoff CM, Orazi A, Ladd AC et al. Proliferation-induced decline of primitive hematopoietic progenitor cell activity is coupled with an increase in apoptosis of ex vivo expanded CD34⁺ cells. Exp Hematol 1998;26:53–62.[Medline]

9. Tisdale JF, Hanazono Y, Sellers SE et al. Ex vivo expansion of genetically marked rhesus peripheral blood progenitor cells results in diminished long-term repopulating activity. Blood 1998;92:1131–1141.[Abstract/Free Full Text]

10. Glimm H, Oh IH, and Eaves CJ. Human hematopoietic stem cells stimulated to proliferate in vitro lose engraftment potential during their S/G₂/M transit and do not reenter G₀. Blood 2000;96:4185–4193.[Abstract/Free Full Text]

11. Srour EF, Tong X, Heilman D et al. Cytokine modulation of the primitive hematopoietic potential of individual CD34⁺CD38^–/lo cells in G₀ during successive in vitro cell divisions. Exp Hematol 1999;27(suppl 1):108.

12. Bhatia M, Bonnet D, Kapp U et al. Quantitative analysis reveals expansion of human hematopoietic repopulating cells after short-term ex vivo culture. J Exp Med 1997;186:619–624.[Abstract/Free Full Text]

13. Byk T, Kahn J, Kollet O et al. G₁ CD34⁺/CD38+ cells potentiate the motility and engraftment of quiescent G₀ CD34⁺/CD38^–/low SCID repopulating cells. Blood 2000;96:288a.

14. Brandt JE, Bartholomew AM, Fortman JD et al. Ex vivo expansion of autologous bone marrow CD34⁺ cells with porcine microvascular endothelial cells results in a graft capable of rescuing lethally irradiated baboons. Blood 1999;94:106–113.[Abstract/Free Full Text]

15. Habibian HK, Peters SO, Hsieh CC et al. The fluctuating phenotype of the lymphohematopoietic stem cell with cell cycle transit. J Exp Med 1998;188:393–398.[Abstract/Free Full Text]

16. Norol F, Drouet M, Mathieu J et al. Ex vivo expanded mobilized peripheral blood CD34⁺ cells accelerate haematological recovery in a baboon model of aulogous transplantation. Br J Haematol 2000;109:162–172.[CrossRef][Medline]

17. Drouet M, Mathieu J, Grenier N et al. The reduction of in vitro radiation-induced Fas-related apoptosis in CD34⁺ progenitor cells by SCF, FLT-3 ligand, TPO, and IL-3 in combination resulted in CD34⁺ cell proliferation and differentiation. STEM CELLS 1999:17;273-285.[Abstract/Free Full Text]

18. Jordan CT, Yamasaki G, Minamoto D. High-resolution cell cycle analysis of defined phenotypic subsets within primitive human hematopoietic cell populations. Exp Hematol 1996;24:1347–1355.[Medline]

19. Ziljmans JM, Visser JWM, Laterveer L et al. The early phase of engraftment after murine blood cell transplantation is mediated by hematopoietic stem cells. Proc Natl Acad Sci USA 1998;95:725–729.[Abstract/Free Full Text]

20. Quesenberry P, Becker P, Nielson S et al. Stem cell engraftment and cell cycle phenotype. Leukemia 1999;13(suppl 1):S92–S93.

21. Srour EF, Wilpshaar J, Wolbert FM et al. Homing and cell cycle activation of human hematopoietic progenitor cells in NOD/SCID mice. Blood 1999;94(suppl 1):654a.

22. Chute JP, Saini AA, Kampen RL et al. A comparative study of the cell cycle status and primitive cell adhesion molecule profile of human CD34⁺ cells cultured in stroma-free versus porcine microvascular endothelial cell cultures. Exp Hematol 1999;27:370–379.[CrossRef][Medline]

23. Wilpshaar J, Falkenburg JHF, Tong X et al. Similar repopulating capacity of mitotically active and resting umbilical cord blood CD34⁺ cells in NOD/SCID mice. Blood 2000;96:2100–2107.[Abstract/Free Full Text]

24. Murray LJ, Young JC, Osborne LJ et al. Thrombopoietin, flt3, and kit ligands together suppress apoptosis of human mobilized CD34⁺ cells and recruit primitive CD34⁺Thy-1⁺ cells into rapid division. Exp Hematol 1999;27:1019–1028.[CrossRef][Medline]

25. Szilvassy SJ, Meyerose TE, Grimes B. Effects of cell cycle activation on the short-term engraftment properties of ex vivo expanded murine hematopoietic cells. Blood 2000;95:2829–2837.[Abstract/Free Full Text]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints/Permissions Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Drouet, M. Articles by Mayol, J.F. Search for Related Content PubMed PubMed Citation Articles by Drouet, M. Articles by Mayol, J.F.