Stem Cells http://www.stemcellsportal.com/
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Reprints/Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Denning-Kendall, P.
Right arrow Articles by Hows, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Denning-Kendall, P.
Right arrow Articles by Hows, J.

Stem Cells 2003;21:61-70 www.StemCells.com
© 2003 AlphaMed Press

Cytokine Expansion Culture of Cord Blood CD34+ Cells Induces Marked and Sustained Changes in Adhesion Receptor and CXCR4 Expressions

Patricia Denning-Kendall, Sakon Singha, Ben Bradley, Jill Hows

University of Bristol Division of Transplantation Sciences, Bristol, United Kingdom

Key Words. Stem cell expansion • Cord blood • Adhesion receptors • CXCR4 • CD34+ cells

Patricia Denning-Kendall, Ph.D., The Paul O’Gorman Lifeline Centre, University of Bristol Division of Transplantation Sciences, Southmead Hospital, Westbury-on-Trym, Bristol, BS10 5NB, United Kingdom. Telephone: 44 117 9596238; Fax: 44 117 9595342; e-mail: P.A.Denning-Kendall{at}bristol.ac.uk


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Recent studies have demonstrated defective bone marrow homing of hematopoietic stem cells after cytokine expansion culture. Adhesion receptors (ARs) are essential to the homing process, and it is possible that cytokine culture modulates AR expression. We studied changes in expression of very late antigen-4 (VLA-4), VLA-5, L-selectin, leukocyte function-associated antigen-1 (LFA-1), CD44, and the stromal cell-derived factor-1 (SDF-1) receptor, CXCR4, during cytokine culture of cord blood (CB) CD34+ cells.

Expression of ARs was studied by flow cytometry on CB CD34+ cells in whole blood, after purification and during culture for up to 10 days. Cells were cultured with stem cell factor (SCF), thrombopoietin (TPO), Flt3-ligand (Flt3), and G-CSF. Results showed that 80% or more of uncultured CD34+ cells were positive for VLA-4, L-selectin, LFA-1, CD44, and CXCR4 while 50% were positive for VLA-5. Purification of CD34+ cells did not affect AR expression, but cytokines increased expression three- to nine-fold throughout the 10-day culture period. In contrast, expression of CXCR4 decreased. Expression changes of ARs and CXCR4 on CD34+/CD38- cells mirrored those of the total CD34+ population. The results indicate that cytokine culture significantly increases AR expression on CB CD34+ cells, which may be related to the decrease in homing of cytokine-cultured hematopoietic stem cells.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Hematopoietic reconstitution after stem cell transplantation is dependent on the successful homing and proliferation of sufficient primitive repopulating cells within the bone marrow. Homing and retention of hematopoietic stem cells within the bone marrow matrix are dependent on a variety of adhesion receptors (ARs), being a similar process to transendothelial migration of leukocytes to sites of inflammation [1]. Initially, cells are attracted to the marrow capillary endothelium by the chemokine, stromal cell-derived factor-1 (SDF-1), produced by bone marrow stromal cells [2]. Interactions between stem cells and endothelial cell ligands then facilitate their transmigration from the circulation into the marrow. Migration experiments [3–5] and in vivo studies, [4, 6, 7] have implicated very late antigen-4 (VLA-4), VLA-5, P-selectin, E-selectin, platelet endothelial cell adhesion molecule-1, and CD44 as being crucial for homing and engraftment of stem cells.

In vitro expansion culture of CD34+ cells with cytokines is clinically important in cord blood (CB) transplantation, where the stem cell dose is limited, and for retroviral gene therapy. However, cytokines not only induce cell division but also drive stem cells to differentiate into more mature cells lacking the ability to repopulate [8–10]. In phase I clinical studies, in vitro-expanded peripheral blood stem cells infused into myeloablated patients resulted in long-term engraftment failure [11]. However, further research has indicated that the correct choice of cytokine combination is crucial for the maintenance of marrow repopulating cells. The use of stem cell factor (SCF), Flt-3-ligand (Flt-3), and thrombopoietin (TPO) has produced robust in vitro expansion of cytokine cultured NOD/SCID mouse repopulating cells [12, 13]. Even if repopulating cells are maintained in culture, engraftment defects will still occur if they cannot home correctly to the bone marrow or have reduced proliferative potential after homing. There is evidence of a dramatic reduction in the homing of colony-forming cells from cytokine-expanded cultures in animal models [14–16], together with altered adhesive characteristics [17, 18]. These data point to an alteration in AR function in cultured stem cells. This paper describes sustained increases in AR expression together with a decrease in CXCR4 during cytokine culture of CB CD34+ cells that could contribute to the homing defects documented in the literature.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
CB Collection
Umbilical CB samples from full-term deliveries were collected by gravity into sterile 50-ml tubes containing 1,000 IU heparin after the umbilical cord had been clamped and cut by the midwife. All collections were performed with informed consent and with local hospital ethics committee approval.

Mononuclear Cell and CD34+ Cell Preparation from CB
Cord blood mononuclear cells (MNCs) were prepared from 40-50 ml CB using Ficoll-Hypaque (Lymphoprep density = 1.077 g/ml; Nycomed; Birmingham, UK; http://www.nycomed-amersham.com) density gradient separation. Red cells were removed with ammonium chloride lysing solution. CD34+ cells were prepared as previously described [19] using MiniMACS (Miltenyi Biotec Ltd; Bisley, UK; http://www.miltenyibiotec.com).

Expansion Cultures
Enriched CD34+ cells (5 x 103/ml, mean purity 90.0% ± 8.4%) were cultured at 37°C for up to 10 days. For serum replete conditions, Iscove’s modified Dulbecco’s medium ([IMDM] 300 mOsmol/kg H2O, Sigma I 7633) supplemented with 20% fetal calf serum ([FCS] HCC-6450; Stem Cell Technologies, Vancouver, Canada; http://www.stemcell.com) was used. X-VIVO 10 (BioWhittaker; Wokingham, Berkshire, UK; http://www.cambrex.com/redirect) was used for serum-free conditions. Both media were supplemented with SCF, Flt3, and TPO at 100 ng/ml plus G-CSF at 10 ng/ml (PeproTech; London, UK; http://www.peprotech.com). Cells were fed on day 7 by demi-depletion.

Flow Cytometric Analysis of CB Cells

Analysis of AR and CXCR4 Expressions on the White Cell Population of CB   Aliquots (1 ml) of CB were treated with 10 ml lysing solution to remove red cells prior to staining with AR antibodies (Abs) as red cells sequestered anti-CD44 and anti-L-selectin. After lysis, white cells from 100 µl of blood were incubated with the recommended volume (10 µl or 20 µl) of anti-human specific AR Abs coupled to phycoerythrin (PE)- and fluorescein isothiocyanate (FITC)-conjugated anti-CD34 for 30 minutes at 4°C in 100 µl Hanks’ balanced salt solution. The panel of Abs consisted of VLA-4 (clone 9F10) and L-selectin (SK11) from Becton Dickinson (Oxford, UK; http://www.bd.com) and VLA-5 (VC5), leukocyte function-associated antigen-1 (LFA-1, G43-25B), CD44 (G44-26), and CXCR4 (12G5) from Pharmingen (Oxford, UK; http://www.bdbiosciences.com/pharmingen). CD34 (HPCA-2) Ab was obtained from Beckton Dickinson. Background levels of staining were measured using isotypic controls. Cells were examined on a Coulter Epics XL flow cytometer, and data were analyzed using the Coulter EXPO32 software. Results were recorded as the median staining intensity on the total leukocyte population even if the staining on some cells fell below that of the isotype control.

Analysis of AR Expression on CD34+ Cells after Ficoll Separation, MiniMACS CD34+ Purification and Expansion Culture   For analysis of AR expression on MNCs or CD34+ cells, either fresh or after expansion culture, a maximum of 106 cells was incubated with the appropriate AR PE-conjugated Ab and FITC-conjugated anti-CD34. When required, anti-CD38-phycoerythrin cyanin 5.1(PE-Cy5) clone PNIM 2651 (Beckman Coulter; High Wycombe; http://www.beckman.com) was also added.

Statistics
All results are reported as mean ± standard deviation (SD). Differences between populations of cells were analysed using a two-tailed Student’s t test.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of Adhesion Receptors and CXCR4 on Whole CB, MNCs, and Purified CD34+ Cells
Figure 1Go demonstrates that the expression of ARs and CXCR4 by CD34+ cells was within the range expressed by other nucleated cells. The plots show lymphocytes, monocytes, and granulocytes as distinct clouds, separated by their side scatter properties.



View larger version (43K):
[in this window]
[in a new window]
  		Figure 1. Typical flow cytometry plots showing staining for ARs on CB using PE-labeled antibodies against side scatter (SS). Red cells were removed from samples of whole blood prior to staining. The plots show AR expression on all leukocytes (gray) with staining on the CD34+ cells shown in black. This was achieved by "backgating" CD34+ cells stained with a FITC-conjugated CD34 antibody.

 
To assess the impact of cell processing, the levels of AR expression on CD34+ cells in whole blood, MNCs, and purified CD34+ cells from the same samples were compared for AR expression. There was a trend toward greater AR expression on CD34+ cells after Ficoll and MiniMACS CD34+ purification (Fig. 2Go), although after MiniMACS purification only LFA-1 remained significantly higher than that on whole blood. Therefore, it was considered valid to use the expression of ARs on purified CD34+ cells as a baseline to study changes in AR expression during cytokine culture of CD34+ cells.



View larger version (18K):
[in this window]
[in a new window]
  		Figure 2. Median staining intensity (arbitrary units + standard deviation) for AR expression on CB CD34+ cells in whole lysed CB and CD34+ cells in MNC populations and in MiniMACS-purified CD34+ cell populations (from the same five samples). Asterisks indicate where staining intensity was significantly higher (p < 0.05) than that on fresh whole blood.

 
Expansion Culture of CB CD34+ Cells
The combination of SCF, Flt3, TPO, and G-CSF in either IMDM (with serum) or in serum-free conditions, produced a significant and similar expansion of nucleated cells, CD34+ cells, and colony-forming units (CFUs) (Table 1Go). CFUs were measured as described previously and include CFU-granulocyte-macrophage (CFU-GM), BFU-E, CFU-erythroid (CFU-E), and CFU-mixture (CFU-MIX) [20]. Fresh CD34+ cells contained 9.2% ± 4.5% CD38- cells. The proportion of CD34+ cells that were CD38- was lower on day 3 (D3) of culture, at 1.7% ± 0.6%, but was then maintained at between 5% and 8% for up to 10 days of culture. These conditions were chosen to provide optimal preservation of primitive repopulating cells [21].


View this table:
[in this window]
[in a new window]
  		Table 1. In vitro expansion of CD34+ CB cells
 
Changes in AR and CXCR4 Expressions During Expansion Culture of CB CD34+ Cells
The percentages of CD34+ cells that were positive for AR expression before and after 7 days of culture are shown in Table 2Go. VLA-4, L-selectin, and CD44 were expressed on >80% of uncultured CD34+ cells, and there was no significant difference after 7 days of culture. The percentages of cells positive for VLA-5 and LFA-1 were significantly greater. However, the median staining intensity, also shown in Table 2Go, which relates to the number of molecules expressed on each cell, showed significant and sustained greater expression after culture for all ARs studied. Figures 3 and 4GoGo show the staining profiles for uncultured CD34+ cells (day 0) and for cells after 5 days of culture. It can be seen that the greater staining intensity for VLA-4, VLA-5, LFA-1, and CD44 was due to greater expression on the vast majority of cells. For example, over 90% of CD34+ cells on day 5 had a higher VLA-4 staining intensity than any of the uncultured cells. Although some cells expressed higher levels of L-selectin after culture, there was also a higher proportion of cells that were negative. Lower overall levels of CXCR4 expression after culture were due to a markedly greater proportion of cells that did not express CXCR4 on their surface. Throughout the culture, the staining intensity of the isotype control on CD34+ cells remained below 0.5. Results were identical with or without serum.


View this table:
[in this window]
[in a new window]
  		Table 2. Effect of cell culture AR expression on CD34+ cells
 


View larger version (32K):
[in this window]
[in a new window]
  		Figure 3. Flow cytometric analysis of AR expression on fresh uncultured MiniMACs-purified CB CD34+ cells (gray) and after culture for 5 days with 100 ng/ml SCF, Flt3, and TPO plus 10 ng/ml G-CSF in IMDM/10% FCS (black). The plots show fluoresence intensity (log) versus relative cell number. The expression of ARs on the CD34+CD38- subpopulation after 5 days culture is shown in separate plots.

 


View larger version (32K):
[in this window]
[in a new window]
  		Figure 4. Flow cytometric analysis of AR expression on fresh uncultured MiniMACs-purified CB CD34+ cells (gray) and after culture for 5 days with 100 ng/ml SCF, Flt3, and TPO plus 10 ng/ml G-CSF in IMDM/10% FCS (black). The plots show fluoresence intensity (log) versus relative cell number. The expression of ARs on the CD34+CD38- subpopulation after 5 days culture is shown in separate plots.

 
Changes on CD34+38- cells
If changes in AR expression are to have an effect on homing and engraftment of hematopoietic cells, it is likely that they would have to exert their effects on the primitive cells of the engrafting phenotype. It is known that NOD/SCID repopulating cells reside within the CD34+ CD38- population [8]. Therefore, we tested whether similar increases in AR expression to those in the total CD34+ population, also occurred on CD34+CD38- cells. It is not possible to monitor this in serum-free conditions as CD38 is underexpressed [22], so for these experiments, CD34+ cells were cultured in IMDM and serum. We compared the median staining intensity of ARs in the total CD34+ population with the staining on the 3% of CD34+ cells with the lowest CD38 staining (Fig. 5Go). Figure 6Go shows that changes in AR expression on CD34+CD38- cells were very similar to those in the total CD34+ population. Even after 5 days of culture, AR expression on CD34+CD38- cells was at the same level, if not greater than the total CD34+ population. The figure also shows the time course of changes in AR and CXCR4 expression with several ARs being upregulated after only 24 hours of culture, before CD34+CD38- cells had entered the cell cycle (data not shown). By day 7 of culture, expression of CD44 and L-selectin on CD34+ and CD34+CD38- cells was greater by around threefold compared with fresh cells, and VLA-4, VLA-5, and LFA-1 expressions were greater by seven- to ninefold.



View larger version (56K):
[in this window]
[in a new window]
  		Figure 5. Flow cytometry plot showing the distribution of CD34+CD38+ cells after cytokine culture. The plot shows cells stained after 2 days of culture. Gate A contains all the CD34+ cells, and gate B shows the 3% of CD34+ cells with the lowest CD38 expression. These gates were used to analyze AR expression and cell cycle characteristics of these cell populations.

 


View larger version (22K):
[in this window]
[in a new window]
  		Figure 6. Expression of ARs on CB CD34+ and CD34+CD38- cells during cytokine culture for 1, 2, or 5 days compared with MiniMACS-purified uncultured cells (D0). The first bar of each pair represents median AR staining intensity on the total CD34+ population, and the second bar represents median staining intensity on the CD34+CD38- population in the same samples (n = 3).

 
The staining profiles of the individual ARs for the total CD34+ population and the CD34+CD38- population mirrored each other closely after 5 days of culture (Figs. 3 and 4GoGo). A substantial proportion (12%-14%), however, were low or negative for VLA-4 or LFA-1 staining even though the majority of cells had much higher staining than fresh uncultured CD34+ cells. VLA-5 staining on CD34+CD38- cells, in contrast, showed a second population of cells (34% of cells in the example shown, Fig. 3Go) with very high staining, which was not apparent in the total CD34+ population. L-selectin in both fresh and cultured cells showed two distinct peaks, with up to 30% of cells being negative.


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have shown that several ARs are expressed on CB CD34+ cells. The levels of expression are within the ranges seen on other leukocytes in that 80% or more CB CD34+ cells were positive for VLA-4, L-selectin, LFA-1, CD44, and the SDF-1 receptor, CXCR4, and nearly 50% were positive for VLA-5. One other study of AR expression on CD34+ cells within whole blood showed similar percentages of positive cells, but low levels of CD44 expression (median staining intensity <5) were reported as red cells were not removed before staining [23]. There are several references to AR expression either on MNCs or purified CD34+ cells [24–27]. However, these studies did not compare AR expression on fractionated cells with unprocessed cells. Our data show that processing and purification of CB did not affect the expressions of ARs on CD34+ cells, and that reported levels of AR expression on purified CD34+ cells were representative of those in whole blood.

There are two reports suggesting that AR expression on CD34+ cells during cytokine culture is unchanged [28, 29], while Chute et al. reported that culture of bone marrow CD34+ cells on porcine microvascular endothelial cells (PMVECs) with cytokines for 7 days produced a significant increase in the expression of VLA-4, CD58(LFA-3), and L-selectin but not of LFA-1 or CD44 [25]. In contrast, we have shown a consistent and sustained increase in expression of all ARs investigated on CD34+ cells in cytokine culture. It is possible that different microenvironments and cytokine mixtures affect expression of individual ARs to a greater or lesser degree. However, our experiments used culture conditions that are most likely to be used clinically with a growth factor combination that sustains repopulating cells [21].

The lower L-selectin expression we observed after 24 hours of culture followed by rapid recovery may be a similar phenomenon to the temporary shedding of L-selectin molecules by activation of a matrix metalloproteinase (MMP) seen during cryopreservation of CD34+ cells [26], as MMPs can also be activated by cytokines.

This report demonstrates that changes in AR expression also occur on primitive CD34+CD38- cells and is the first to show that not all CD34+CD38- cells are affected to the same extent. Although there was a dramatically higher expression of ARs, up to 30% of the cells were low or even negative for VLA-4, LFA-1, and L-selectin after cytokine culture. In addition, a subpopulation of CD34+CD38- cells expressed very high levels of VLA-5. We do not know the AR phenotype of the human engrafting cell or if this changes during cytokine culture. Homing capabilities of human CD34+ cells are inhibited by pretreatment with antibodies to VLA-4, VLA-5, and LFA-1 [30], suggesting they require expression of these antigens. However, Gunji et al. [31] reported that cells capable of colony-forming-cell production on stromal layers are LFA-1 negative, and murine repopulating cells are both LFA-1 and L-selectin negative [32]. The significance of CD34+ cells that have high or low AR expression after culture can be investigated by cell sorting followed by transplantation into NOD/SCID mice.

Recent studies have shown that engraftment of primitive human CD34+ cells in NOD/SCID mice was dependent on the SDF-1 receptor, CXCR4, regardless of the initial level of surface expression [33, 34]. Cells that are initially CXCR4 negative contain internal CXCR4 that can migrate to the surface under the right environment. After cytokine culture, we observed a much greater proportion of CXCR4 negative cells. This may be due to a downregulation of CXCR4 production, in which case, there could also be fewer internal CXCR4, or an oscillation of CXCR4 from the surface to the interior. Given the reliance upon migration toward SDF-1 of engrafting cells, an absolute decrease in CXCR4 expression could have severe consequences on the engrafting capability of CD34+ cells.

Specific homing and engraftment of hematopoietic stem cells within the bone marrow are reliant on a multistep process involving selectin receptors, integrins, LFA-1, and finally, CD44. Locally produced cytokines may also be required for activation of integrins [35], upregulation of the CXCR4 receptor [36], and activation of matrix metalloproteinases [37]. This orchestrated series of events may be compromised if hematopoietic cells express abnormally high levels of ARs, low levels of CXCR4, and are also in the cell cycle. It is possible that cytokine culture prematurely activates hematopoietic cells so that they bind nonspecifically to extramedullary endothelial surfaces. This would account for the dramatic decrease in homing of cytokine-cultured hematopoietic cells in the NOD/SCID mouse model [14–16]. Our data clearly show changes in CD34+ cell AR expression induced by cytokine culture. We propose that this may be related to decreases in homing and engraftment of cultured hematopoietic cells. The impact of altered CD34+ AR expression on marrow homing and engraftment requires investigation in vivo in the NOD/SCID mouse model.


   ACKNOWLEDGMENT
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
This work was supported by The Leukemia Research Fund, Children With Leukemia, and the Bud Flanagan Leukemia Fund. We thank the midwives in the Delivery Suite Southmead Hospital and all mothers who donated cord blood for the project.


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Springer TA. Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm. Cell 1994;76:301–314.[CrossRef][Medline]

  2. Aiuti A, Webb IJ, Bleul C et al. The chemokine SDF-1 is a chemoattractant for human CD34+ hematopoietic progenitor cells and provides a new mechanism to explain the mobilization of CD34+ progenitors to peripheral blood. J Exp Med 1997;185:111–120.[Abstract/Free Full Text]

  3. Naiyer AJ, Jo DY, Ahn J et al. Stromal derived factor-1-induced chemokinesis of cord blood CD34(+) cells (long-term culture-initiating cells) through endothelial cells is mediated by E-selectin. Blood 1999;94:4011–4019.[Abstract/Free Full Text]

  4. Peled A, Kollet O, Ponomaryov T et al. The chemokine SDF-1 activates the integrins LFA-1, VLA-4, and VLA-5 on immature human CD34(+) cells: role in transendothelial/stromal migration and engraftment of NOD/SCID mice. Blood 2000;95:3289–3296.[Abstract/Free Full Text]

  5. Yong KL, Watts M, Shaun-Thomas N et al. Transmigration of CD34+ cells across specialized and nonspecialized endothelium requires prior activation by growth factors and is mediated by PECAM-1 (CD31). Blood 1998;91:1196–1205.[Abstract/Free Full Text]

  6. van der Loo JC, Xiao X, McMillin D et al. VLA-5 is expressed by mouse and human long-term repopulating hematopoietic cells and mediates adhesion to extracellular matrix protein fibronectin. J Clin Invest 1998;102:1051–1061.[Medline]

  7. Frenette PS, Subbarao S, Mazo IB et al. Endothelial selectins and vascular cell adhesion molecule-1 promote hematopoietic progenitor homing to bone marrow. Proc Natl Acad Sci USA 1998;95:14423–14428.[Abstract/Free Full Text]

  8. Bhatia M, Wang JC, Kapp U et al. Purification of primitive human hematopoietic cells capable of repopulating immune-deficient mice. Proc Natl Acad Sci USA 1997;94:5320–5325.[Abstract/Free Full Text]

  9. Bryder D, Jacobsen SE. Interleukin-3 supports expansion of long-term multilineage repopulating activity after multiple stem cell divisions in vitro. Blood 2000;96:1748–1755.[Abstract/Free Full Text]

  10. Piacibello W, Gammaitoni L, Bruno S et al. Negative influence of IL3 on the expansion of human cord blood in vivo long-term repopulating stem cells. J Hematother Stem Cell Res 2000;9:945–956.[CrossRef][Medline]

  11. Holyoake TL, Alcorn MJ, Richmond L et al. CD34 positive PBPC expanded ex vivo may not provide durable engraftment following myeloablative chemoradiotherapy regimens. Bone Marrow Transplant 1997;19:1095–1101.[CrossRef][Medline]

  12. Piacibello W, Sanavio F, Severino A et al. Engraftment in nonobese diabetic severe combined immunodeficient mice of human CD34(+) cord blood cells after ex vivo expansion: evidence for the amplification and self-renewal of repopulating stem cells. Blood 1999;93:3736–3749.[Abstract/Free Full Text]

  13. Ueda T, Tsuji K, Yoshino H et al. Expansion of human NOD/SCID-repopulating cells by stem cell factor, Flk2/Flt3 ligand, thrombopoietin, IL-6, and soluble IL-6 receptor. J Clin Invest 2000;105:1013–1021.[Medline]

  14. Szilvassy SJ, Bass MJ, Van Zant G et al. Organ-selective homing defines engraftment kinetics of murine hematopoietic stem cells and is compromised by ex vivo expansion. Blood 1999;93:1557–1566.[Abstract/Free Full Text]

  15. Spiegel A, Kollet O, Peled A et al. Both human leukemic pre-B ALL and normal cord blood CD34+ cells home into NOD/SCID mice in a SDF-1/CXCR4-dependent manner but differ in their signaling and activation. Blood 2001;98:313a.

  16. Kerre TC, Vandekerckhove B, Offner F et al. The behavior of long-term cultured human hematopoietic stem cells early after transplantation into NOD/SCID mice provides explanations for their impaired long-term exgraftment in this model. Blood 2001;98:645a.

  17. Giet O, Huygen S, Beguin Y et al. Cell cycle activation of hematopoietic progenitor cells increases very late antigen-5-mediated adhesion to fibronectin. Exp Hematol 2001;29:515–524.[CrossRef][Medline]

  18. Yamaguchi M, Ikebuchi K, Hirayama F et al. Different adhesive characteristics and VLA-4 expression of CD34(+) progenitors in G0/G1 versus S+G2/M phases of the cell cycle. Blood 1998;92:842–848.[Abstract/Free Full Text]

  19. Denning-Kendall PA, Horsley H, Donaldson C et al. Different behaviour of fresh and cultured CD34+ cells during immunomagnetic separation. Br J Haematol 1999;105:780–785.[CrossRef][Medline]

  20. Denning-Kendall P, Donaldson C, Nicol A et al. Optimal processing of human umbilical cord blood for clinical banking. Exp Hematol 1996;24:1394–1401.[Medline]

  21. Kobari L, Pflumio F, Giarratana M-C et al. In vitro and in vivo evidence for the long-term multilineage (myeloid, B, NK, and T) reconstitution capacity of ex vivo expanded human CD34+ cord blood cells. Exp Hematol 2000;28:1470–1480.[CrossRef][Medline]

  22. Donaldson C, Denning-Kendall P, Bradley B et al. The CD34(+)CD38(neg) population is significantly increased in haemopoietic cell expansion cultures in serum-free compared to serum-replete conditions: dissociation of phenotype and function. Bone Marrow Transplant 2001;27:365–371.[CrossRef][Medline]

  23. Timeus F, Crescenzio N, Basso G et al. Cell adhesion molecule expression in cord blood CD34+ cells. STEM CELLS 1998;16:120–126.[Abstract/Free Full Text]

  24. Asosingh K, Renmans W, Van der Gucht K et al. Circulating CD34+ cells in cord blood and mobilized blood have a different profile of adhesion molecules than bone marrow CD34+ cells. Eur J Haematol 1998;60:153–160.[Medline]

  25. 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]

  26. Hattori Y, Kato H, Nitta M et al. Decrease of L-selectin expression on human CD34+ cells on freeze-thawing and rapid recovery with short-term incubation. Exp Hematol 2001;29:114–122.[CrossRef][Medline]

  27. Roy V, Verfaillie CM. Expression and function of cell adhesion molecules on fetal liver, cord blood and bone marrow hematopoietic progenitors: implications for anatomical localization and developmental stage specific regulation of hematopoiesis. Exp Hematol 1999;27:302–312.[CrossRef][Medline]

  28. Dravid G, Rao SG. Ex vivo expansion of stem cells from umbilical cord blood: expression of cell adhesion molecules. STEM CELLS 2002;20:183–189.[Abstract/Free Full Text]

  29. Reems JA, Mielcarek M, Torok-Storb B. Differential modulation of adhesion markers with ex vivo expansion of human umbilical CD34+ progenitor cells. Biol Blood Marrow Transplant 1997;3:133–141.[Medline]

  30. Kollet O, Spiegel A, Peled A et al. Rapid and efficient homing of human CD34(+)CD38(-/low)CXCR4(+) stem and progenitor cells to the bone marrow and spleen of NOD/SCID and NOD/SCID/B2m(null) mice. Blood 2001;97:3283–3291.[Abstract/Free Full Text]

  31. Gunji Y, Nakamura M, Hagiwara T et al. Expression and function of adhesion molecules on human hematopoietic stem cells: CD34+ LFA-1- cells are more primitive than CD34+ LFA-1+ cells. Blood 1992;80:429–436.[Abstract/Free Full Text]

  32. Orschell-Traycoff CM, Hiatt K, Dagher RN et al. Homing and engraftment potential of Sca-1(+)lin(-) cells fractionated on the basis of adhesion molecule expression and position in cell cycle. Blood 2000;96:1380–1387.[Abstract/Free Full Text]

  33. Rosu-Myles M, Gallacher L, Murdoch B et al. The human hematopoietic stem cell compartment is heterogeneous for CXCR4 expression. Proc Natl Acad Sci USA 2000;97:14626–14631.[Abstract/Free Full Text]

  34. Kollet O, Petit I, Khan J et al. Sorted human cord blood CD34+CXCR4- cells contain internal CXCR4 which can oscillate in vivo and rescue low SDF-1 dependent repopulation in NOD/SCID mice. Blood 2001;98:71a.

  35. Levesque JP, Haylock DN, Simmons PJ. Cytokine regulation of proliferation and cell adhesion are correlated events in human CD34+ hemopoietic progenitors. Blood 1996;88:1168–1176.[Abstract/Free Full Text]

  36. Peled A, Petit I, Kollet O et al. Dependence of human stem cell engraftment and repopulation of NOD/SCID mice on CXCR4. Science 1999;283:845–848.[Abstract/Free Full Text]

  37. Janowska-Wieczorek A, Marquez LA, Dobrowsky A et al. Differential MMP and TIMP production by human marrow and peripheral blood CD34(+) cells in response to chemokines. Exp Hematol 2000;28:1274–1285.[CrossRef][Medline]

Received June 24, 2002; accepted for publication August 20, 2002.



This article has been cited by other articles:


Home page
 Stem CellsHome page
K. Yamamura, K. Ohishi, M. Masuya, E. Miyata, Y. Sugimoto, S. Nakamura, A. Fujieda, H. Araki, and N. Katayama
Ex Vivo Culture of Human Cord Blood Hematopoietic Stem/Progenitor Cells Adversely Influences Their Distribution to Other Bone Marrow Compartments After Intra-Bone Marrow Transplantation
Stem Cells, February 1, 2008; 26(2): 543 - 549.
[Abstract] [Full Text] [PDF]

Home page
 Molecular Cancer TherapeuticsHome page
L. Jin, Y. Tabe, S. Konoplev, Y. Xu, C. E. Leysath, H. Lu, S. Kimura, A. Ohsaka, M.-B. Rios, L. Calvert, et al.
CXCR4 up-regulation by imatinib induces chronic myelogenous leukemia (CML) cell migration to bone marrow stroma and promotes survival of quiescent CML cells
Mol. Cancer Ther., January 1, 2008; 7(1): 48 - 58.
[Abstract] [Full Text] [PDF]

Home page
 Stem CellsHome page
K. Li, C. K. Y. Chuen, S. M. Lee, P. Law, T. F. Fok, P. C. Ng, C. K. Li, D. Wong, A. Merzouk, H. Salari, et al.
Small Peptide Analogue of SDF-1{alpha} Supports Survival of Cord Blood CD34+ Cells in Synergy with Other Cytokines and Enhances Their Ex Vivo Expansion and Engraftment into Nonobese Diabetic/Severe Combined Immunodeficient Mice
Stem Cells, January 1, 2006; 24(1): 55 - 64.
[Abstract] [Full Text] [PDF]

Home page
 Stem CellsHome page
S. Brenner, N. Whiting-Theobald, T. Kawai, G. F. Linton, A. G. Rudikoff, U. Choi, M. F. Ryser, P. M. Murphy, J. M.G. Sechler, and H. L. Malech
CXCR4-Transgene Expression Significantly Improves Marrow Engraftment of Cultured Hematopoietic Stem Cells
Stem Cells, December 1, 2004; 22(7): 1128 - 1133.
[Abstract] [Full Text] [PDF]

Home page
 Stem CellsHome page
C. Hart, D. Drewel, G. Mueller, J. Grassinger, M. Zaiss, L. A. Kunz-Schughart, R. Andreesen, A. Reichle, E. Holler, and B. Hennemann
Expression and Function of Homing-Essential Molecules and Enhanced In Vivo Homing Ability of Human Peripheral Blood-Derived Hematopoietic Progenitor Cells after Stimulation with Stem Cell Factor
Stem Cells, July 1, 2004; 22(4): 580 - 589.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
J. Kahn, T. Byk, L. Jansson-Sjostrand, I. Petit, S. Shivtiel, A. Nagler, I. Hardan, V. Deutsch, Z. Gazit, D. Gazit, et al.
Overexpression of CXCR4 on human CD34+ progenitors increases their proliferation, migration, and NOD/SCID repopulation
Blood, April 15, 2004; 103(8): 2942 - 2949.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
S. S. Perry, H. Wang, L. J. Pierce, A. M. Yang, S. Tsai, and G. J. Spangrude
L-selectin defines a bone marrow analog to the thymic early T-lineage progenitor
Blood, April 15, 2004; 103(8): 2990 - 2996.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Reprints/Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Denning-Kendall, P.
Right arrow Articles by Hows, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Denning-Kendall, P.
Right arrow Articles by Hows, J.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
STEM CELLS THE ONCOLOGIST CME ALPHAMED PRESS JOURNALS
http://www.peprotech.com/