标题: Simultaneous Maintenance of Human Cord Blood SCID-Repopulating Cells and Expansi [打印本页] 作者: 江边孤钓 时间: 2009-3-5 10:36 标题: Simultaneous Maintenance of Human Cord Blood SCID-Repopulating Cells and Expansi
a Laboratory for Studies on Hematopoiesis: Molecular and Functional Aspects, Bordeaux 2 University, Bordeaux, France; * Z) V7 T5 F+ v# n) {0 b! \5 E8 I5 i) O- C( J
b Establishment Aquitaine-Limousin Regional Center, Bordeaux, France;/ j" c" I: h9 H. l
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c Laboratory of Hematology, Haut L谷v那que Hospital, Pessac, France% m. B& L- A1 L1 J
7 Z3 [) B! C# }/ J$ u' }& I- xKey Words. Severe combined immunodeficiency–repopulating cells ? NOD/SCID Stem cells ? IL-3 ? Hypoxia ? Cord blood ? Ex vivo expansion v% X' N0 Y- y4 \1 Z
# ^0 i9 d; q7 @. \% K. A. S$ h. i o3 sCorrespondence: 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 , ~; O2 m! s1 G! K V7 `$ b2 w. s) U0 S5 }
ABSTRACT 3 g+ G7 r. E, [) @+ M$ x1 A, \% r% U, u' x. M4 N A/ @' w
The 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. / m6 Z3 K1 U" \& T1 j2 O3 _8 H % {, L0 _* y+ HMATERIALS AND METHODS$ u: g1 ^' [4 A0 v) [
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CFC Expansion Is Not Affected at 3% O2 Concentration ( e) K, T+ ]% [' t 5 P( V3 j) p8 wPreliminary 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). ) T( `1 ^/ e5 f& J4 _ + i% i1 W0 U1 GFigure 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.( V3 s1 B0 L+ B3 O
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Better Pre-CFC Maintenance at 3% O2 Is IL-3 Dose Dependent : q' C& ~' `6 b6 }) \( ? 4 u& L4 C/ E1 g* V. x8 RPre-CFCs were better preserved at 3% than at 20% O2, as evidenced after 28 (Fig. 2B; p ; ]! j4 t$ E" Y; j- T* K! ~ 6 @; q- T2 d# XFigure 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.; v9 @. m+ N4 b. W. L
4 z0 R8 ?- V! cCD34 Cell Proliferation Is Not Altered by 3% O2 Concentration # a: y5 j1 }+ ~: e1 v ! D1 z" h. v4 ` S7 Z# tWhereas 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., z6 I2 l) h1 z$ k! ^3 m
8 K8 l2 X$ x8 z+ x8 TFigure 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% O2.8 W. R; p$ ~. U% J
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CD34 Cell Phenotype After LC1 at 3% and 20% O29 R f! o8 u" J! q0 }/ {2 {
9 S( T! o7 d5 q1 l5 iAfter 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 8 \7 q% F5 Q; G1 G) k6 l" M- E # o" q: x8 V: `+ {7 d7 |Figure 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. ' L4 O5 }* }& ]! v) O9 i8 H$ H: U. H6 r9 d* J) I3 B% i4 `
Figure 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. a2 a" f) b" Q0 V; I5 s; X
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SRCs Are Better Maintained in 3% O2 LC1 , B* U) J$ m- G# s( b7 ?8 H! f: S1 [1 W0 O9 i
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).4 w n5 y Q" M' o; J1 l# A) V( N
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Table 1. Comparison of SRC maintenance after 7 days of expansion at 20% and 3% O2 8 F2 N: i5 N! l% g- s2 Q5 {7 Y. o& o
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.0 {2 L2 |% D2 O1 A& G8 e* x+ e8 M1 j$ i
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Our 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.! L( p# U; ~9 g% @/ L) T
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Neildez-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)作者: txxxtyq 时间: 2015-6-13 12:30