CXCR4-Transgene Expression Significantly Improves Marrow Engraftment of Cultured - 《stemcells》2004-2005文献干细胞之家



免疫细胞治疗专区	欢迎关注干细胞微信公众号

12 3 4 5 6 7 8 9 10 ... 26 下一页

返回列表

查看: 403966\|回复: 258	go CXCR4-Transgene Expression Significantly Improves Marrow Engraftment of Cultured [复制链接]

江边孤钓

版主

Rank: 7 Rank: 7 Rank: 7

积分: 0
威望: 0
包包: 483

楼主

发表于 2009-3-5 10:35 |只看该作者 |倒序浏览 |打印

a Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA;8 M2 t5 U$ b4 W" I1 ^! J# V

b Department of Pediatrics, University Clinic Carl Gustav Carus, Dresden, Germany;
$ N5 H6 f( x2 I7 H
c Department of Pediatrics, The Jikei University School of Medicine, Tokyo, Japan

Key Words. Engraftment ? CXCR4 ? Ex vivo gene transfer ? Hematopoietic stem cells (HSCs)$ ?: T' W! F2 s; I
4 P4 [% X$ \4 ?% O
Correspondence: Sebastian Brenner, M.D., Department of Pediatrics, University Clinic Carl Gustav Carus, Fetscherstr. 74, 01307 Dresden, Germany. Telephone: 49-351-458-6872; Fax: 49-351-458-6333; e-mail: Sebastian.Brenner@uniklinikum-dresden.de
; n- {! o/ P  G- z2 S7 ~
ABSTRACT0 ?* G) }7 Q: c0 Z

Hematopoietic stem cell (HSC) egress to the circulation and homing to the bone marrow (BM) are regulated in part by interactions between CXC chemokine receptor 4 (CXCR4) and stromal cell–derived factor (SDF)-1 . Mobilization of CD34  peripheral blood stem cells (CD34  PBSCs) from BM occurs following administration of either granulocyte colony-stimulating factor (G-CSF; inactivation of SDF-1 and cleavage of CXCR4) or AMD3100 (partial agonist of CXCR4) . SDF-1 binding to CXCR4 on HSCs mediates chemotaxis, expression of adhesion molecules, proliferation, and survival .
# a  N- ^. f  W' j5 y( _' p
Experimental approaches under development for transplantation or gene therapy with HSCs may involve ex vivo manipulation that decreases available HSCs or affects BM homing and engraftment. The level of CXCR4 expression on CD34  HSCs decreases during prolonged ex vivo culture . Ex vivo cultured human HSCs progressively lose nonobese diabetic/severe combined immunodeficient (NOD/ SCID) mouse reconstitution potential that is improved by SDF-1 treatment . In the clinic, donor CD34  HSCs that express higher levels of CXCR4 are statistically correlated with earlier reconstitution of hematopoiesis after transplantation . All these observations suggest that loss of CXCR4 expression on CD34  HSCs during culture might precede other processes that diminish the potential to participate in long-term hematopoiesis. In the study reported here we show that transduction-mediated overexpression of CXCR4 during culture of G-CSF–mobilized CD34  PBSCs significantly improves BM repopulating potential over that of similarly cultured normal PBSCs, as measured by transplantation into sublethally irradiated NOD/SCID mice.: P& X% y7 O  k! X; t

STUDY DESIGN/ S; b" c9 a- C* ^. B! R

We first investigated the expression of CD34 and CXCR4 on G-CSF–mobilized normal CD34  PBSCs in ex vivo culture. We found that during prolonged culture, CD34 expression is progressively lost on PBSCs, such that by day 6, only 10%–38% (n = 4) of cells remained CD34 . Initial CXCR4 expression on CD34  PBSCs varied depending on the donor (20%–52%, n = 4) and was upregulated during the first 48 hours by cytokine stimulation, as was reported before . During prolonged culture, however, CXCR4 expression was progressively lost on cells that remained CD34  such that by day 6, only 1.1%–8% continued to coexpress CXCR4 (Fig. 1). Based on the loss of CXCR4 expression on CD34  cells under prolonged culture conditions, we overexpressed the CXCR4 transgene in CD34  cells.

Figure 1. Flow cytometric analysis demonstrates the change in expression of CXCR4 by CD34  PBSCs during ex vivo culture. (A): Twenty percent of CD34 PBSCs express CXCR4 on day 0 (cross lines set at 99th percentile of isotype antibody controls, right column). (B): At 6 days of culture only 8% of those cells continuing to express CD34 also express CXCR4. (C): In contrast, 87.2% of CD34  cells transduced with RD114-MFGS-CXCR4 on days 2–6 of culture express CXCR4 transgene on day 6. Abbreviation: PBSC, peripheral blood stem cell.
% S, @9 Q) Y) r
Cultured CD34  PBSCs transduced overnight on days 2–6 with murine retrovirus vector RD114-pseudotyped MFGS-CXCR4, expressed high levels of CXCR4 (Fig. 1). Either normal (nontransduced) CD34  PBSCs or RD114-MFGS-gp91phox (phagocyte oxidase transmembrane subunit)–transduced CD34  PBSCs were used as control (92% of cells transduced; not shown). CXCR4 transgene–positive hematopoietic progenitor cells expressed 11- to 18-fold more CXCR4 per cell (measured by mean fluorescence intensity) than normal or control-transduced cells (n = 5). CXCR4-transduced hematopoietic progenitors (five independent tests with CD34  PBSCs from five different donors) sampled on culture day 6 demonstrated increased calcium flux SDF-1 response that was 160%–220% of naive cultured CD34  PBSCs (Fig. 2, inset). Respective CXCR4-transduced PBSC migration over 30 minutes on day 6 in response to SDF-1 was on average 3.4-fold greater than migration of cultured control cell migration in response to SDF-1 (Fig. 2). These ex vivo results suggest CXCR4 transgene–enhanced migration and calcium-flux response to SDF-1. Since cells were not split into CD34  and CD34– populations for the calcium flux and migration assays, it should be noted that upregulated naive CXCR4 on more differentiated cells in culture might have influenced the results.- S% f* m9 Z* Q

Figure 2. Enhanced ionized intracellular calcium flux and cellular migration of CXCR4-transduced CD34  PBSCs in response to SDF-1. The inset shows one representative run of the fura2-AM response (fluorescence emission ratio at 510 nm of 340 nm versus 380 nm excitation) of control-cultured (open diamonds) and CXCR4-transduced PBSCs (dark-filled circles) to sequential stimulation with buffer, 10 nM SDF-1, and 10 nM ATP. The bar graph shows the number of CXCR4-transduced PBSCs that migrated through 5 μm polycarbonate transwells within 30 minutes (open bar in response to buffer; dark-filled bar in response to 10 nM SDF-1). The middle light cross-hatched bar shows the migration response of control-cultured PBSCs to 10 nM SDF-1. Given are mean values and standard errors of five separate runs with PBSCs from five different donors. Abbreviations: PBSC, peripheral blood stem cell; SDF-1, stromal cell–derived factor-1.

In a NOD/SCID mouse study, we compared repopulation potential of CXCR4-transduced or normal (nontransduced) cells at day 6 of culture, in which each mouse was transplanted with the progeny from 4 x 106 CD34  PBSCs seeded at culture initiation. At the day of transplantation, the CXCR4-transduced CD34  cells expressed CXCR4 in >87%, compared with 8% naive CXCR4 expression in the normal (non-transduced) CD34  population (Fig. 1; Table 1, column 5). After 6 weeks, BM of 10 mice transplanted with the CXCR4-transduced 6-day cultured CD34  PBSCs demonstrated 19.9% ± 3.4% engraftment of human CD45  cells, while BM of eight mice transplanted with the normal 6-day cultured CD34  PBSCs demonstrated significantly lower (p

Table 1. Human cell engraftment in NOD/SCID mice, CXCR4-transgene expression and cell migration ex vivo

Figure 3. Significantly higher human cell engraftment for MFGS-CXCR4–transduced PBSCs in nonobese diabetic/ severe combined immunodeficient mice. At 6 weeks after transplantation, human cell engraftment (measured by anti-human CD45) was 19.9% ± 3.4% for 6-day cultured MFGS-CXCR4–transduced PBSCs (dark filled bar) compared with 9.3% ± 1.3% engraftment for similarly cultured nontransduced (normal) PBSCs (hatched bar) from the same donor (see Table 1). Abbreviation: PBSC, peripheral blood stem cell.
9 G" b2 c" H/ L" A7 }, P3 Q% i
We previously reported accelerated loss of the NOD/ SCID mouse marrow repopulating potential of human CD34  PBSCs between the 4th and 6th days of ex vivo culture . With CD34  PBSCs from a different healthy volunteer, we compared NOD/SCID repopulation potential of CXCR4 and gp91phox control-transduced cells at day 6 of culture, in which each mouse was transplanted with the progeny from only 1 x 106 CD34  PBSCs seeded at culture initiation. Under similar conditions, no engraftment of normal CD34  PBSCs was seen in NOD/SCID mice in previous experiments (unpublished data). At the day of transplantation, the CXCR4-transduced PBSCs expressed CXCR4 in 95%, compared with : y( _& c/ y, q2 N8 Y
7 y7 a3 }4 ~- B; H' a
Figure 4. Enhanced NOD/SCID engraftment potential of 6-day cultured CXCR4-transduced CD34  PBSCs. (A) and (C) shows a representative flow cytometry analysis of bone marrow harvested at 9 weeks after transplantation of a NOD/SCID mouse with 6-day cultured control-transduced CD34  PBSCs demonstrating no engraftment, while (B) and (D) show similar analyses of mice transplanted with CXCR4-transduced PBSCs, demonstrating significant engraftment of human CD45  cells, of which most also express high levels of CXCR4 transgene (D, upper right quadrant). Abbreviations: NOD/SCID, nonobese diabetic/severe combined immunodeficient; PBSC, peripheral blood stem cell.% Z; q4 {$ i/ i" p. C
! V1 h# ]% a& a* V# O5 _% P
The studies demonstrate that, under the prolonged ex vivo culture conditions, the CXCR4 transduction of CD34  PBSCs results in significantly improved long-term NOD/ SCID engraftment conferred by transduction-mediated forced overexpression of CXCR4, and this is consistent with recently published data by Kahn et al. . Our results indicate that loss of marrow reconstitution potential of cultured HSCs not only is due to differentiation (and thus loss of self-renewal potential) but also may be preceded by loss of CXCR4 expression crucial for homing. This represents direct evidence that CXCR4 mediates HSC homing and engraftment to the BM, and it provides incentive for development of clinically applicable methods for increasing levels of expression of CXCR4 to enhance engraftment of ex vivo manipulated HSCs.

REFERENCES
9 M/ t' ]8 ~$ n+ Z  h2 M% s3 l. t$ m
Abkowitz JL, Robinson AE, Kale S et al. The mobilization of hematopoietic stem cells during homeostasis and after cytokine exposure. Blood 2003;102:1249–1253.. c" x6 o# P& y- F
8 V. _# v# E/ b
Peled A, Petit I, Kollet O et al. Dependence of human stem cell engraftment and repopulation of NOD/SCID mice on CXCR4. Science 1999;283:845–848.3 y! l7 ^* M4 g' g& i
  N' r/ ]7 T: S, z) v
Petit I, Szyper-Kravitz M, Nagler A et al. G-CSF induces stem cell mobilization by decreasing bone marrow SDF-1 and up-regulating CXCR4. Nat Immunol 2002;3:687–694./ Y. X  B; T8 \5 P
7 K- G; {+ N! l- D/ O
Levesque JP, Hendy J, Takamatsu Y et al. Disruption of the CXCR4/CXCL12 chemotactic interaction during hematopoietic stem cell mobilization induced by GCSF or cyclophosphamide. J Clin Invest 2003;111:187–196.

Liles WC, Broxmeyer HE, Rodger E et al. Mobilization of hematopoietic progenitor cells in healthy volunteers by AMD3100, a CXCR4 antagonist. Blood 2003;102:2728–2730.- {2 h% H: h* B, Z

Broxmeyer HE, Cooper S, Kohli L et al. Transgenic expression of stromal cell-derived factor-1/CXC chemokine lig-and 12 enhances myeloid progenitor cell survival/antiapoptosis in vitro in response to growth factor withdrawal and enhances myelopoiesis in vivo. J Immunol 2003;170: 421–429.7 o7 t" l6 P0 z, p

Lataillade JJ, Clay D, Dupuy C et al. Chemokine SDF-1 enhances circulating CD34( ) cell proliferation in synergy with cytokines: possible role in progenitor survival. Blood 2000;95:756–768.

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.% D" N- s8 j  N

Denning-Kendall P, Singha S, Bradley B et al. Cytokine expansion culture of cord blood CD34( ) cells induces marked and sustained changes in adhesion receptor and CXCR4 expressions. STEM CELLS 2003;21:61–70.) i3 {  [- q. U
# c+ H, u) P0 ~# b/ X( i; }
Glimm H, Tang P, Clark-Lewis I et al. Ex vivo treatment of proliferating human cord blood stem cells with stroma-derived factor-1 enhances their ability to engraft NOD/SCID mice. Blood 2002;99:3454–3457.( D. S0 K, w: `; B4 u/ d
( k8 ]5 u6 [3 L3 W% x
Spencer A, Jackson J, Baulch-Brown C. Enumeration of bone marrow "homing" haemopoietic stem cells from G-CSF-mobilised normal donors and influence on engraftment following allogeneic transplantation. Bone Marrow Transplant 2001;28:1019–1022.4 L3 X) q- |( e# [

Brenner S, Whiting-Theobald NL, Linton GF et al. Concentrated RD114-pseudotyped MFGS-gp91phox vector achieves high levels of functional correction of the chronic granulomatous disease oxidase defect in NOD/SCID/beta-microglobulin–/– repopulating mobilized human peripheral blood CD34  cells. Blood 2003;102:2789–2797.

Kahn J, Byk T, Jansson-Sjostrand L et al. Overexpression of CXCR4 on human CD34  progenitors increases their proliferation, migration, and NOD/SCID repopulation. Blood 2004;103:2942–2949.(Sebastian Brennera,b, Nar)

收藏0 分享0 顶0 踩0