Differential Effects of Culture Conditions on the Migration Pattern of Stromal C - 《stemcells》2004-2005文献干细胞之家



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发表于 2009-3-5 10:36 |只看该作者 |倒序浏览 |打印

a Institute of Immunology and. X4 f1 o7 o3 q
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b Department of Gynecology, Marienhospital, University of Witten/Herdecke, Witten, Germany
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Key Words. SDF-1 ? Ex vivo expansion ? Cell migration ? Cytokines ? Hematopoietic stem cells. u2 B/ R( _" N# \

Correspondence: Corinna Weidt, Institute of Immunology, University of Witten/Herdecke, Stockumer Str. 10, 58448 Witten, Germany. Telephone: 49-2302-669-152; Fax:49-2302-669-158; e-mail: cweidt@uni-wh.de% a' }5 U, ]5 V/ Z
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ABSTRACT
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The aptitude of hematopoietic stem cells (HSCs) to migrate is increasingly becoming of interest to researchers and oncologists, not only for the benefit of augmenting the success rate of hematopoietic system regeneration after transplantation of HSCs but also in examining microchimerism , tissue renewal, and disease . In light of technical and ethical issues concerning the usage of embryonic stem cells as a quiescent panacean source, it is all the more important to scrutinize the apparent migrational capacity of facilely available HSCs. Migration is a sine qua non characteristic that enables the homing of HSCs to bone marrow and damaged tissue . The importance of investigating cells in a three-dimensional (3D) surrounding becomes apparent when comparative 2D and 3D experiments are performed. Experiments to this effect have unambiguously shown that a 3D environment brings forth differential results  and must therefore be considered.6 V% U0 T8 f- D" O* @" n% _. y
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To investigate the locomotory behavior of HSCs in 3D, we used the highly efficacious migratory stimulant stromal cell–derived factor-1 (SDF-1). This chemokine is constitutively expressed by numerous tissues  and serves as a chemoattractant for tumor cells , lymphocytes , monocytes, neutrophil granulocytes, endothelial precursors, and HSCs and has been suggested to play a role as a homeostatic regulator in tissue renewal . SDF-1 belongs to the CXC subgroup of chemokines and binds to the CXCR4 receptor. CXCR4 is a member of the seven-helix receptor family, and signaling induces the migration of HSCs via calcium release and reorganization of actin cytoskeleton structure . The CD34  population of HSCs is comprised of CXCR4  and CXCR4– subpopulations. CXCR4 expression and subsequent migration with SDF-1 was seen to be stimulated and enhanced by the addition of stem cell factor (SCF) or SCF and interleukin 6 (IL-6) to the medium .
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IL-6 and Flt3-ligand (FL) are common supplements used for HSC expansion . We thus chose to examine cell migration characteristics of HSCs cultivated under these conditions. The study of single-cell migration in a 3D collagen matrix, using video microscopy and computer-aided analysis , enabled us to examine the migration pattern of a population of cord blood–derived cells isolated via the CD133 antigen. A change in the average migration rate can be the result of a permutation of several factors, including the recruitment of immobile cells and a change in the migratory pattern, which is comprised of time active, pauses, and speed. Results of our analysis using FL-incubated HSCs  compared with FL/IL-6-incubated HSCs in response to SDF-1 show a differential effect on the recruitment and migration pattern  of these cells induced by IL-6. This demonstrates that the inflammatory cytokine IL-6, when present in the culture media, leads to an altered migratory phenotype of these cells.: J8 w3 u- ^& V" N# {& ?6 h
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MATERIALS AND METHODS2 z3 t. [& j1 e3 O
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Cord blood–derived cells were placed in a collagen-based migration system after cultivation in either FL or a combination of FL and IL-6. Cultivation in IL-6 alone does not allow for such an experiment, because cells are no longer viable after 5 days (data not shown).
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Cells previously cultivated in FL alone showed an increase in the average migration rate of 7.4% when stimulated with SDF-1 (Fig. 2A). The average population of moving cells in the control was 63.9%, and the average population of SDF-1–stimulated cells was 78.4%. Additional analysis showed that recruitment of cells alone explained the increase in the average migration rate, because single-cell analysis disclosed no change in the migratory behavior of individual cells (data not shown). In contrast, cells cultivated in FL/IL-6 showed an increase in the average migration rate of 21.7% when stimulated with SDF-1 (Fig. 2B). Actually motile cells comprised 72.2% in the control population and 81.1% in SDF-1–induced cells, whereby recruitment could only explain a minute part of the elevated average migration rate (data not shown).
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Figure 2. Average migration of FL- and FL/IL-6–cultivated CD133 cells response to SDF-1. (A): The average migration rate of FL-cultivated CD133 cells in response to SDF-1 (1 μg/ml) showed an average increase of 7.4% due to a recruitment of nonmoving cells. (B): CD133 cells cultivated in FL/IL-6 responded to SDF-1 (1 μg/ml) stimulation, with an average increased migration rate of 21.3%. Each diagram represents the results of cells isolated from three independent cord blood donors. Abbreviations: FL, Flt3-ligand; IL-6, interleukin 6; SDF-1, stromal cell–derived factor-1.
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Based on the observation that cells do not move constantly, we calculated the percent time in which cells were active. Control cells were active on average 21.1% of the time, and SDF-1–stimulated cells were active 42.6% of the time (Fig. 3A; p 3 \) q7 O$ H! g: {9 ^1 w
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Figure 3. Migration pattern of FL/IL-6–cultivated CD133 cells in response to SDF-1. Time-lapse video microscopy combined with computer-assisted cell tracking allows for detailed analysis of the migration pattern on a single-cell level. The following parameters were analyzed in this study: time active (%) (A), velocity (excluding pauses; μm/min) (B), speed (including pauses; μm/min) (C), pause frequency (D), pause length (min) (E), and distance migrated (μm) (F). Stimulation of FL/IL-6–cultivated CD133 cells with SDF-1 resulted in a decrease in velocity. In contrast, there was a significant increase in the time active, speed, pause frequency, and distance migrated. Abbreviations: FL, Flt3-ligand; IL-6, interleukin 6; SDF-1, stromal cell–derived factor-1.
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To investigate whether the migration effect seen was caused by the differential conditions, FL and FL/IL-6 per se, we analyzed the migration pattern of these cells without SDF-1 stimulation. Upon comparing the migration pattern of FL- and FL/IL-6-incubated cells, we found no significant difference. For a summary of the results, see Table 1.
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Table 1. Culture conditions used and migration analysis results% I, ]& \" |; L- U8 y+ `- p2 [
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DISCUSSION& ^5 k* Y/ D# ]; `9 N0 n$ `
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These data are of particular relevance in light of obligatory HSC expansion before therapy, whereby SDF-1 plays a significant role in maintaining and reinstating the hematopoi-etic system and also conceivably in tissue regeneration. Parameters such as recruitment, speed, and pauses made are elementary in characterizing migration and demonstrably play a significant role in the migration of IL-6–incubated, SDF-1–stimulated HSCs. This level of qualitative and quantitative analysis bares potential to help interpret repopulation data with HSCs and gives reason to carefully consider the supplements of expansion media, which may enhance or even compromise the efficacy of therapy involving HSC migration.4 O' p8 p3 ~& @8 [  e& W

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