标题: Early Fetal Liver Readily Repopulates B Lymphopoiesis in Adult Bone Marrow [打印本页] 作者: 江边孤钓 时间: 2009-3-5 10:52 标题: Early Fetal Liver Readily Repopulates B Lymphopoiesis in Adult Bone Marrow
Institute of Pathological Physiology, First Medical Faculty, Charles University, Prague, Czech Republic+ a3 W2 }3 b: x
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Key Words. Fetal liver ? Hematopoietic stem cell ? B lymphopoiesis ? Gene expression 8 ?, R" V5 B: R- S# v# j% S $ c( |9 F( `) q) D, B9 S' g6 b: JCorrespondence: Ko-Tung Chang, Ph.D., Institute of Pathological Physiology, First Medical Faculty, Charles University, U Nemocnice 5, 128 53 Prague, Czech Republic. Telephone: 420-2-2496-5934; Fax: 420-2-2491-2834; e-mail: kotungc@bcm.tmc.edu - S1 Q* V2 v2 A( z6 d8 P5 x0 ^+ ?& J+ V3 @, B
ABSTRACT+ T/ U! o6 m# R% N3 B/ `
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Hematopoiesis is a time- and a site-dependent event during ontogeny of vertebrates . During gastrulation in mice, ventral mesodermal cells segregate to the two sites where blood cells form independently. The first wave of hematopoietic activity, known as primitive hematopoiesis, appears in the ventral blood islands of the yolk sac (YS) at embryonic day (E) 7.5 . This transient extraembryonic hematopoiesis gives rise to primitive nucleated erythrocytes between E7 and E11 to cope with the oxygen demand of a rapidly growing embryo. Adult-type definitive hematopoiesis begins as the second wave of hematopoiesis in the dorsolateral plate of aorta-gonads-mesonephros (AGM) region at E10.5 . This intraembryonic hematopoiesis thereafter shifts to the fetal liver (FL) at E11 and E12, where production of all hematopoietic cells is initiated. Finally, from near birth until the end of life, hematopoiesis resides in the bone marrow (BM), spleen, thymus, and lymphatic tissues.# N3 [% q Y. j
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Multipotent progenitors are detected readily by in vitro culture of YS cells. However, long-term repopulating hematopoietic stem cell (LTR-HSC) activity is not present in the YS before E11 , because cells from the YS are unable to reconstitute the entire hematopoietic system in lethally irradiated adult mice for more than several months . LTR-HSCs emerge in the AGM region just before the establishment of the hematopoietic liver activity and subsequently colonize the hepatic tissue. FL becomes a main hematopoietic organ during the fetal period, and HSCs expand dramatically there between E12 and E16 . However, the microenvironment of FL undergoes continuous changes during this time, and the metabolic function of hepatocytes gradually becomes the major function of the liver .! w0 ]" q* g1 ]' E- }3 t, k% Y7 H: q! B
2 b* J! N: N% M- n2 L4 l, z. RDefinitive erythropoiesis and fetal T lymphopoiesis peak in the FL at day 12, whereas B lineage–restricted progenitors are still rare to be detected . Although B-lymphoid activity can be found beginning at E8 or E9 in the YS by fetal thymic organ culture , B-lineage precursors were detected in FL from E11 and pre-B cells in E13 FL . A population of fully B-committed progenitors that can be differentiated in vitro into B-lineage cells emerges in the FL at E10 or E11 . This shows that the B-cell differentiation program is not delayed during hematopoietic ontogeny. However, it does not address the question of whether the B-lineage progenitors/ stem cells from early FLs are ready to function in the environment of adult BM. The question is pertinent, because LTR-HSC activity of the cells derived from E11.5 AGM is enhanced when cultivated in the presence of FL nonhematopoietic cells . These reports prompted us to study whether HSCs of the AGM origin developing additionally in the FL may be specifically imprinted by the interaction with the FL microenvironment to convert into fully functional adult hematopoietic tissue. To test the tentative instructive role of FL regarding B-lymphopoiesis ontogeny, we studied the gene expression associated with normal hematopoiesis in FL from E12.5, E14.5, and E17.5. We also compared the short- and long-term repopulating potential of FL HSCs from E12.5 and E17.5 by measuring competitive reconstitution of B- and T-lymphoid and granulocyte/macrophage lineages from unfractioned FL cells in sublethally irradiated adult mice.% g1 M/ S+ W1 R: R
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MATERIALS AND METHODS 2 {2 k# }1 W0 F" K! Z5 W; }$ L! P+ B$ h- E' a! v; |
mRNA Levels in Early-, Mid-, and Late-Gestational FL and in Adult Liver Compared with Adult BM (Table 2)% n8 B: f7 |# g$ A& L% E @2 p
- W& b, a c& lLiver Developmental Genes ? The mRNA levels of albumin, transferrin receptor 2, and X-box binding protein-1 increased during liver development. They were significantly different from adult BM in the case of albumin and transferrin receptor 2.1 z( d; r8 P4 F% I9 D
0 k0 Q% p2 Q/ g: C' \, CTable 2. Comparison of the levels of mRNA for selected genes in fetal liver from E12.5, E14.5, and E17.5 and in adult liver with that of adult BM taken as 1.0 % {; D# t5 X& A5 ?0 {- ~) n9 s( s 1 t% m6 l0 P. Z6 r1 dLineage-Specific Genes ? The transcription factor Pax5 and pre-B lineage antigen CD19 mRNAs levels were low in FL from the early and middle gestational ages as well as in adult liver but were slightly higher in E17.5 FL than in adult BM. + c! g$ I# N$ J) R. \1 [% |' o7 h$ n z9 g2 y' d$ G5 F# t }The expression of -globin gene was high throughout the whole FL development. Surprisingly, the mRNA level in the adult liver was shown to be comparable to that in BM. It might have been caused by contamination with reticulocytes from blood contained in the liver, which was not perfused before sample collection. / M' V* O$ d" \: Q7 d : {: P/ d" D) X4 f' IIntegrin ? Very late antigen 4 (VLA-4) mRNA tended to be higher in FL at days 14.5 and days 17.5 of gestation compared with adult BM, but the difference was not significant. ! ~. ?/ E' B6 d! x6 ~ 2 K4 O" r8 T, ]: E+ ]$ E* yCytokines and Their Receptors ? SCF and c-kit * X- i3 u* R8 P" F0 Y8 a' g & t( C1 {& m8 F, B% J: zThe expression of both kinds of mRNA had a tendency to be higher in FL from all stages studied compared with adult BM. ( D: q5 D8 M5 u! B$ O6 [2 G' X9 Q, `! [% Y G9 ^8 q4 G" P& V
SDF-1 (CXCL12) and CXCR47 ]# A$ }. \+ W8 l
' l7 ?" r3 I# e& Z$ }( HThe highest expression of both stromal-derived factor-1(SDF-1) and CXCR4 was in FL from E17.5, but SDF-1 mRNA was also high in adult liver. CXCR4 mRNA expression was significantly lower in the early and middle stages of FL compared with that of E17.5 FL (p " i# f9 t, F' }0 e ! K. {; W' t0 R8 m: PErythropoietin and Erythropoietin Receptor 0 N1 X' H: ?1 t7 U- y" V- Z K6 W . W5 p: k% n$ W( I' j8 _Erythropoietin (Epo) mRNA was only marginally expressed in BM, whereas all stages of FL significantly expressed the Epo gene. Epo receptor (EpoR) mRNA was higher in FL compared with BM./ f+ b9 x P* W. h: s6 _6 l% ^
; f+ ^0 V% q0 p DOncostatin M and Oncostatin M Receptor' l" a, G3 C$ U, p2 |
" H4 Z1 O) n" b0 @7 m COncostatin M mRNA was highly expressed in all stages of FL. Oncostatin M receptor mRNA peaked in adult liver.5 Z- S! D7 m. E+ m8 f7 w' S* J
% o. M) l' L2 W6 q$ r6 cThe vascular endothelial growth factor (VEGF) mRNA, as well as the mRNA for its receptor (KDR/flk-1), was more expressed in FL, as well as in adult liver, compared with adult BM.$ w W r, y, B# [
+ l- I9 `9 a; f- @" a& ]Flt3 Ligand$ m" v/ j" w) ~" W
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Flt3 ligand mRNA was significantly more expressed in all FL and adult liver samples compared with adult BM. ' E$ Q# K, X% N( |% b6 o 4 A- e" t6 c$ B( g+ _Interleukin-7 Receptor( w. b2 t7 W) m3 e* t
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The receptor mRNA was expressed in middle and late FL comparably to its expression in adult BM. " a8 N; q+ @7 m4 ]+ ?) Y6 \ 7 Y& {1 c) R' e7 q0 bFlow Cytometry Analysis of B-Cell Lineage Antigenic Surface Markers B220 and CD19 in Early and Late Gestational FL and Adult BM/ |! c y0 h3 C; U+ C! O% k( _
- m9 W0 {0 B" X4 x! N) R: gFlow cytometry analysis of unfractioned FL from E12.5 and E17.5 and adult BM was performed using antibodies against the pan-B-cell markers (Fig. 1). The mean percentages of B220 cells in FL from E12.5 and E17.5 and in adult BM were 1.42%, 5.72%, and 13.64%, and those of CD19 cells were 0.89%, 5.69%, and 14.04%, respectively. All of these differences were highly significant (p / m9 k, }! i3 A6 D, M4 a
% t" }3 A/ f' W# ~. eFigure 1. Flow cytometry analysis of B-cell antigenic surface markers B220 and CD19 in FL at days 12.5 and 17.5 of gestation and in adult BM. A representative result from three independent experiments is shown. FL cells were obtained, on average, from six embryos of the same mother. Adult BM was obtained from both femur and tibia pairs of a normal mouse. Relative proportions of B-lineage cells: B220 (A) and CD19 (B). Abbreviations: BM, bone marrow; FL, fetal liver.& j( v, Y8 }0 N) w5 A2 N
$ W* z, m m* @/ c" cComparison of Short- and Long-Term Repopulating Capacity of Progenitor/Stem Cells from Early- and Late-Gestation FL and from Adult BM1 S* Q+ P( k6 q) \
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Eight mice irradiated with 6 Gy were transplanted with 5 x 106 of FL cells from E12.5 or E17.5 or with 5 x 106 cells from adult BM. The percentage of donor cells at different times after the transplantations is presented in Figure 2A. At up to 16 weeks, the chimerism resulting from transplantation of E12.5 FL cells was significantly lower than that achieved by transplantation of FL cells from E17.5. This difference was much more pronounced during the first 2 months after transplantation, corresponding with short-term repopulation. The proportions of B- and T-lymphoid cells in total donor-derived nucleated cells were not different among recipients of either E12.5 or E17.5 FL cells or adult BM cells (Figs. 2B–2D). : ?% B4 R; }% i' b, v: v) G( d" b. N, |* \! S1 D: K
Figure 2. Competitive repopulating capacity of E12.5 and E17.5 FL cells against adult BM. Five million cells collected from FL at day 12.5 or 17.5 of gestation and from adult BM were transplanted into sublethally irradiated recipients (6 Gy). (A): Total contribution to peripheral blood nucleated cells from donor cells. (B–D): Percentage of B and T lymphocytes and granulocytes/macrophages in donor-derived nucleated cells (total donor nucleated cells = 100%). n = 8; means ± standard error of the mean are shown. Significant difference between E12.5 and E17.5 of FL: **p 6 W( i( z: \# i& c
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Inhibition of Donor-Derived Granulocyte/ Macrophage Formation in Recipients with a Low Level of Engraftment 4 T) R0 e% o1 r. R1 R+ S" ~- s4 K: y0 W1 G4 O" M, M5 `" n
A total of 106 FL cells from day 17.5 was transplanted into mice that received either 3 or 6 Gy of irradiation. There was a very low engraftment in 3-Gy irradiated mice (9 b9 v2 Y* ~; A
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Figure 3. Inhibition of donor-derived granulocyte/macrophage formation in recipients with a low level of engraftment. A total of 106 cells collected from fetal liver at day 17.5 of gestation was transplanted into 3- or 6-Gy irradiated recipients (n = 8). Data show the percentage (means ± standard error of the mean) of B and T lymphocytes and granulocytes/macrophages in donor-derived peripheral blood after 12 weeks. There was a significant difference of granulocytes/macrophages fraction derived from transplanted cells (6.06 ± 1.6% versus 22.83 ± 1.8%, *p - w1 j. l" n9 c5 g1 n& M+ X( W) A. w
DISCUSSION $ z* `3 f( C" r q2 J/ T& J4 ?* P: F' G4 I7 H. |
This work was supported by research grant MSM 111100003 from the Ministry of Education of the Czech Republic. We thank D. Dyrov芍 and D. Singerov芍 for excellent technical assistance and Z. Volk芍nov芍 and J. Jobbikov芍 for the animal maintenance. We also thank Drs. Max Cooper, Paul W. Kincade, and Josef T. Prchal for their critical review of the manuscript. " \" s c2 s# G4 i" g3 o+ t0 Y! U* }4 L
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