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a Laboratory for Studies on Hematopoiesis: Molecular and Functional Aspects, Bordeaux 2 University, Bordeaux, France;
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b Establishment Aquitaine-Limousin Regional Center, Bordeaux, France;; W6 [: S9 q. J) l
& A+ d+ _" P4 W6 m- T9 uc Laboratory of Hematology, Haut L谷v那que Hospital, Pessac, France0 b A" y! u( I7 i$ i
# C: Z- `; [5 c% D2 s- fKey Words. Severe combined immunodeficiency–repopulating cells ? NOD/SCID Stem cells ? IL-3 ? Hypoxia ? Cord blood ? Ex vivo expansion8 t% ]' _3 s4 e9 m( m
0 |. W0 g* w' D* e( \ s( D+ ACorrespondence: Zoran Ivanovic, M.D., Ph.D., Laboratoire H谷matopo?豕se Normale et Pathologique FRE CNRS 2617, Universit谷 Victor Segalen Bordeaux 2, Carreire Nord–Bat. 1B–RDC, 146, rue L谷o Saignat–BP 50, 33076 Bordeaux Cedex, France. Telephone: 05-56-90-75-50; Fax: 05-56-90-75-51; e-mail: zoran.ivanovic@efs.sante.fr; Q/ w( L5 `! x' T* P* V
- D; M/ p8 z7 X. ]! r1 zABSTRACT
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. h3 H/ P# p, e9 q4 [6 e6 pThe transplantation of unmanipulated cord blood (CB) cells has two major disadvantages: (a) the low number of hematopoietic stem and progenitor cells (colony-forming cells ) in each harvest limits its application to children, and (b) there is a long period (30 days) of post-transplantation cytopenia . Simultaneous ex vivo amplification of the CFCs and primitive stem cells could resolve both problems. Extensive expansion of nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice–repopulating cells (SRCs) in long-term (4- to 12-week) cultures is not suitable for clinical application for several reasons. On the other hand, short-term (7- to 10-day) ex vivo amplification of CFCs usually leads to loss of primitive stem cells that impairs the long-term engraftment capacity of expanded cells in animals and humans . Our short-term cultures of murine bone marrow (BM) and human blood cells at 1% oxygen (O2; a concentration probably present in stem cell areas of BM ) demonstrated a better preservation of primitive stem cells than at 20% O2, but with a reduced CFC expansion . These results have been recently confirmed and strengthened by Danet et al. , who demonstrated that a 4-day culture of human BM CD34 cells at 1.5% O2 concentration ensured a transient ex vivo expansion of human BM SRCs without substantial amplification of CFCs. This positive effect of low O2 concentration on stem cell maintenance in vitro was not limited to cells issued in the marrow environment, because we found culture at 1% O2 for pre-CFCs mobilized in blood , and Koller et al. found an increased progenitor production in long-term suspension CB cultures at 5% O2. Therefore, we tried to improve the expansion of CB CD34 cells by searching for an O2 concentration that still allows full CFC amplification and has a positive effect on stem cells maintenance. In the present work, both goals were achieved at 3% O2 by using serum-free cytokine-supplemented cultures similar to those already used in our Cell Therapy Unit for clinical expansion of mobilized blood CD34 cells . These results open new perspectives for the use of CB grafts in adults.
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8 R/ Q L ~. A A: r) @MATERIALS AND METHODS$ u5 ?+ L* ]- T6 x2 ]' Z* {0 q5 Y
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CFC Expansion Is Not Affected at 3% O2 Concentration
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Preliminary experiments showed that 3% was the lowest O2 concentration, maintaining similar total cell and CFC amplification to that at 20% O2. Indeed, mean amplification of total cells (45- to 60-fold; n = 11) and of CFCs (CFU-GM BFU-E CFU-mix; 35- to 50-fold; n = 11) was similar in LC1 at 3% and 20% O2, whatever the IL-3 concentration (0, 0.5, 5, and 50 ng/ml; Fig. 1). There was no consistent increase of the number of BFU-E, as described at 1% O2 , but the size of BFU-E–derived colonies issued from 3% O2 cultures was larger (not shown).1 s5 v0 N# _ a2 }" D7 {9 y: A3 e
) v2 o( L. x7 oFigure 1. Expansion of total cells and of colony-forming cells in 7-day expansion cultures at 20% and 3% O2. White bars, cultures at 20% O2; black bars, cultures at 3% O2. Abbreviations: CFC, colony-forming cell; IL, interleukin./ c7 x: [$ h9 a T' c) I
5 a) t) h' j e. F* zBetter Pre-CFC Maintenance at 3% O2 Is IL-3 Dose Dependent
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{& ^( a _9 T8 M$ B- r gPre-CFCs were better preserved at 3% than at 20% O2, as evidenced after 28 (Fig. 2B; p $ ?) u4 N, B9 y) R
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Figure 2. Maintenance of pre-CFC activity in 7-day expansion cultures at 20% and 3% O2. Pre-CFCs present at the end of expansion (7-day primary cultures) were detected on the basis of their capacity to produce committed progenitors (CFCs) after 14 days (A) and 28 days (B) in secondary liquid cultures. White bars, primary cultures at 20% O2; black bars, primary cultures at 3% O2. Abbreviations: CFC, colony-forming cell; IL, interleukin.
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CD34 Cell Proliferation Is Not Altered by 3% O2 Concentration9 L( I7 f5 }. J# g* K% j
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Whereas CD34 cells seeded at day 0 at 3% and 20% O2 (PKH26 proliferation test; Fig. 3) all divided at least once and showed similar 7-day proliferative history profile, those issued of 3% O2 kept a better pre-CFC potential, as evidenced by their day-28 CFC production in LC2 (Fig. 3). Thus, as already shown in mouse cell cultures at 1% O2 , the maintenance of pre-CFC at 3% O2 was not abolished by cell divisions.8 h% |3 P% w/ N( s$ g( O
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Figure 3. Proliferative history of cells cultured for 7 days at 20% and 3% O2. The day-0 fluorescence intensity of PKH2-labeled CD34 cells has been used to distinguish population of undivided cells. All cells divided at least once during the 7 days of culture; the cell proliferation is coupled with the diminution and loss of CD34 antigen expression, which was similar at 20% and 3% O2.0 R* D: c2 [: T) P4 l: f
# }4 T s( ?0 U" L% l1 vCD34 Cell Phenotype After LC1 at 3% and 20% O2
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+ H9 i- _ [/ e) O1 GAfter 7 days of culture with 0.5 ng/ml of IL-3, the percentage of cells still expressing CD34 was lower at 3% (8.7 ± 2.9) than at 20% (13.0 ± 5.0%) O2 (p
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; _; `) A# Y8 {) ^. X9 VFigure 4. Phenotypical characteristics of nucleated cells (A) and CD34 cells (B) in 7-day expansion cultures at 20% (white bars) and 3% (black bars) oxygen. Mean ± standard error of nine (CD34), six (CD38, CD41, CD133), or three (other markers) independent experiments.
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% H- b2 \0 ~- u+ GFigure 5. Relation between expression of CD34, CD38, and CD133 on the cells cultured at 20% and 3% O2. CD34 /CD38 cells disappeared in both conditions; note a lower percentage of CD133 cells at 3% O2 on CD34 and CD34– cells.. Y0 S4 G5 ]; B# V5 i2 l4 j
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SRCs Are Better Maintained in 3% O2 LC1. E2 y" [. v2 d& t. J K( m. U. t
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SRC activity in expansion products is presently the best predictive test of long-term engraftment, as shown in baboons . After a 7-day expansion of CD34 cells with 0.5 ng/ml of IL-3 in 3% and 20% O2 LC1, we transplanted NOD/SCID mice with three doses of cells representing the progeny of 20,000, 40,000, and 120,000 CD34 cells seeded at day 0. We evidenced higher engraftment capacity of cells issued from 3% O2 LC1 at three cell doses injected (Table 1). Although these results do not allow a precise calculation of SRC frequencies, they showed a much better maintenance of SRC activity at 3% (a similar level of engraftment was achieved with progeny of 20,000 CD34 cells expanded at 3% O2 and of 120,000 cells expanded at 20% O2). Even for the lowest cell dose injected (at which 33% and 62.5% of mice were not engrafted), most mice positive for CD45 were also positive for CD33 and CD19 human antigens, showing that both O2 concentrations preserve the individual multilineage capacity of SRC (Fig. 6).& _/ @/ x6 ^1 X/ s; N2 h
' j# v6 E: e& c' n$ x9 K! ]9 A6 HTable 1. Comparison of SRC maintenance after 7 days of expansion at 20% and 3% O2
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Figure 6. Engraftment of NOD/SCID mice by cells expanded at 20% or 3% O2. The quantity of expanded cells injected was calculated to represent the progeny of 20,000, 40,000, and 120,000 CD34 cells plated at day 0 (X axis) in two conditions. Analysis of human chimerism on the basis of percentages of human CD45, CD33, and CD19 cells in NOD/SCID mice bone marrow. Abbreviation: NOD/SCID, nonobese diabetic/severe combined immunodeficiency.
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; k* V- J6 x4 v) o' e6 i) fDISCUSSION$ N5 U Y( b5 [+ j5 V
( {- h4 I5 t9 m2 A2 s( i! YOur results demonstrate that low O2 concentration (3%) ensures simultaneously the maintenance of primitive CB stem cells (SRCs) and expansion of committed progenitors (CFCs) ex vivo in the presence of SCF, G-CSF, MGDF (100 ng/ml each), and IL-3 (0.5 ng/ml). The positive impact of IL-3 on proliferating stem cells (pre-CFC) in serum-free medium is enhanced at low O2 tension (3%) and maximal at low concentration of IL-3 (0.5 ng/ml). Low O2 tension seems to increase the dissociation between phenotype and function of cultured cells. Nevertheless, as shown recently for adult BM cells , we establish that human CB stem cells respond to hypoxia by self-renewing divisions.; R' g4 Q2 r$ j% t; |+ w7 h( k
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ACKNOWLEDGMENTS
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7 Y# ~- Y+ y- J/ aGluckman E. Current status of umbilical cord blood hematopoietic stem cell transplantation. Exp Hematol 2000;28:1197–1205.
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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.
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Peters SO, Kittler ELW, Ramshaw S et al. Ex vivo expansion of murine marrow cells with interleukin-3 (IL-3), IL-6, IL-11, and stem cell factor leads to impaired engraftment in irradiated hosts. Blood 1996;87:30–37.2 Q2 L, l9 q& Z
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Abkovitz JL, Tabada MR, Sabo KM et al. The ex vivo expansion of feline marrow cells leads to increased numbers of BFU-E and CFU-GM but a loss of reconstituting ability. STEM CELLS 1998;16:288–293." C3 H/ V" |8 K) f: J9 o' b
& T) Q1 l6 S4 H( g, B
Tisdale JF, Hanazono Y, Sellers SE et al. Ex vivo expansion of genetically marked rhesus peripheral blood progenitor cells resulted in diminished long-term repopulating ability. Blood 1998;92:1131–1141.7 b/ R% e/ v% n! s3 ]
1 T+ L" {/ J' T3 P+ N( |( p' ~
Holyoake TL,Alcom MZ, Richmond L et al. CD34 positive PBC expanded ex vivo may not provide durable engraftment following myeloablative chemotherapy regimens. Bone Marrow Transplant 1997;19:1095–1101.
) O8 Q, E4 s8 p% k1 V$ u$ A5 @! x* v& T) e
Chow DC, Wenning LA, Miller WM et al. Modeling pO2 distributions in the bone marrow hematopoietic compartment, II: modified Kroghian models. Biophys J 2001;81:685–696.
1 A% Y% I6 v* W6 ], d( \, `, l, S/ @" W! h; ~0 d9 M
Cipolleschi MG, Dello Sbarba P, Olivotto M. The role of hypoxia in the maintenance of hemopoietic stem cells. Blood 1993;82:2031–2037.
( h1 Q6 h d! u" H0 E6 m2 i3 ~9 r1 s3 n
Cipoleschi MG, Rovida E, Ivanovic Z et al. The maintenance of hematopoietic progenitors in severe hypoxic cultures, an in vitro indicator of marrow-repopulating ability. Leukemia 2000;14:735–739.
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Ivanovic Z, Bartolozzi B, Bernabei PA et al. Incubation of murine bone marrow cells in hypoxia ensures the maintenance of marrow-repopulating activity together with the expansion of committed progenitors. Br J Haematol 2000;108:424–429.
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. S" v; H* q, N3 QIvanovic Z, Dello Sbarba P, Trimoreau F et al. Primitive human HPCs are better maintained and expanded in vitro at 1 percent oxygen than at 20 percent. Transfusion 2000; 40:1482–1488.- o' n! S0 p, _+ i& ?
9 ?, q+ j) i% W/ X/ k" a" ?Ivanovic Z, Belloc F, Faucher JL et al. Hypoxia maintains and IL3 reduces the pre-CFC potential of dividing CD34 murine bone marrow cells. Exp Hematol 2002;30:67–73.+ _0 M Z5 K! K7 Y
- H7 _2 s, ]' e/ D& P! y% KDanet G, Pan Y, Luongo JL et al. Expansion of human SCID-repopulating cells under hypoxic conditions. J Clin Invest 2003;112:126–135.$ W# K. c) x2 _6 m4 V8 p
% ?" V( |0 b( M1 E% T9 l+ S
Koller MR, Bender JG, Papoutsakis et al. Effects of synergistic cytokine combinations, low oxygen, and irradiated stroma on the expansion of human cord blood progenitors. Blood 1992:80:403–411.* [) A5 \* w2 M3 O+ ^9 a" k9 p
- _/ P" o9 F; C* E
Reiffers J, Cailliot C, Dazey B et al. Abrogation of post-myeloablative chemotherapy neutropenia by ex vivo-expanded autologous CD34 positive cells. Lancet 1999; 354:1092–1093.
: f7 \ o1 k1 d" {- W5 A" V! N& q V$ @* q+ V; {7 Y/ d
Desplat V, Faucher JL, Mahon FX et al. Hypoxia modifies proliferation and differentiation of CD34 CML cells. STEM CELLS 2002;20:347–354.; m4 k. N9 F# \4 [7 |
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Denning-Kendall PA, Evely R, Singha S et al. In vitro expansion of cord blood does not prevent engraftment of severe combined immunodeficient repopulating cells. Br J Haematol 2002;116:218–228.
5 @9 z" E0 [ @1 F8 E/ R7 z2 V/ ?- G( {. F1 [- C7 ?, {
Cipolleschi MG, D’Ippolito G, Bernabei PA et al. Severe hypoxia enhances the formation of erythroid bursts from human cord blood cells and the maintenance of BFU-E in vitro. Exp Hematol 1997;25:1187–1194.: a$ g. I6 @( S) p
0 O$ T: g8 O$ _" G2 C' m
Breider D, Jacobson S. Interleukin-3 supports expansion of long-term multilineage repopulating activity after multiple stem cell division in vitro. Blood 2000;96:1748–1755.
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0 t3 m* I8 t& wPiacibello 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 1998;12:718–727.% o' n8 P" R+ C1 L" q" K7 E
) C* r: ?% L e# DRottem M, Okada T, Goff JP et al. Mast cells cultured from the peripheral blood of normal donors and patients with mastocytosis originate from a CD34 /Fc RI-cell population. Blood 1994;84:2489–2496." [( K, D- T l5 {
5 m( v3 z7 E+ H- `" }Norol F, Drouet M, Pflumio F et al. Ex vivo expansion marginally amplifies repopulating cells from baboon peripheral blood mobilized CD34 cells. Br J Haematol 2002;117: 924–934.0 j. `4 ^1 s# ^- T) z; ~
) o8 w# G- W+ s. r4 ^; @
McNiece I, Jones R, Bearman SI 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.
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+ z3 Y7 I) D1 a, kIvanovic Z. Interleukin-3 (IL-3) and ex-vivo maintenance of hematopoietic stem cells: facts and controversies. Eur Cytokine Netw 2004;15:6–13.' L1 [. F' Y( K( p4 Y& k
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Dexter T, Whetton AD, Basil GW. Haemopoietic cell growth and glucose transport: its role in cell survival and the relevance of this in normal haematopoiesis and leukemia. Differentiation 1984;27:163–167.- u9 y0 b! t' D% i) r$ s
; s' m, y, `7 RZhang JZ, Behroz A, Ismail-Beigi F. Regulation of glucose transport by hypoxia. Am J Kidney Dis 1999;34:189–202.% s' k- O( U# l" d
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McCoy KDN, Ahmed N, Tan AS et al. The hematopoietic growth factor interleukin-3, promotes glucose transport by increasing the specific activity and maintaining the affinity for glucose of plasma membrane glucose transporters. J Biol Chem 1997;272:17276–17282.
\7 P4 e4 x* W: o! g9 U0 j' ^4 d. u2 }' @3 e6 A
Ahmed N, Berridge MV. Regulation of glucose transport by interleukin-3 in growth factor-dependent and oncogene-transformed bone marrow-derived cell lines. Leuk Res 1997;21:609–618.
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0 d' k8 n3 n, M1 W; NDorrell C, Gan OI, Pereira DS et al. Expansion of human cord blood CD34 CD38– cells in ex vivo culture during retroviral transduction without a corresponding increase in SCID repopulating cells (SRC) frequency: dissociation of SRC phenotype and function. Blood 2000;95:102–110.
/ C1 m6 a" b4 w; X g$ |5 C0 q K+ G, `- ?/ e9 C
Xu R, Reems JA. Umbilical cord blood progeny cells that retain a CD34 phenotype after ex vivo expansion have less engraftment potential than unexpanded CD34 cells. Transfusion 2001;41:213–218.
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* n" m) b* T7 O& b5 PDonaldson C, Denning-Kendal P, Bradley B et al. The CD34 CD38– 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.
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Danet GH, Lee HW, Luongo JL et al. Dissociation between stem cell phenotype and NOD/SCID repopulating activity in human peripheral blood CD34 cells after ex vivo expansion. Exp Hematol 2001;12:1465–1473.
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% w( _- c* M7 g' J4 XNeildez-Nguyen TM, Wajcman H, Marden MC et al. Human erythroid cells produced ex vivo at large scale differentiate into red blood cells in vivo. Nat Biotech 2002;20:467–472.(Zoran Ivanovica,b, Franci) |
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发表于 2009-3-5 10:36
发表于 2015-6-13 12:30
