标题: Murine Bone Marrow Stromal Cells Sustain In Vivo the Survival of Hematopoietic S [打印本页] 作者: 江边孤钓 时间: 2009-3-5 10:47 标题: Murine Bone Marrow Stromal Cells Sustain In Vivo the Survival of Hematopoietic S
a Department of Cytology and Histology,- m$ c$ s, u* Q# ^
! E! b/ `. b; L& {! s- gb Department of Pathological Anatomy, and$ S" J3 c7 Y% ~. M' r x
& ^$ x- ?& p9 w. t' Z* ic Laboratory of Connective Tissues Biology, University of Li豕ge, Li豕ge, Belgium, }' z3 x% M9 Z+ f8 \2 p5 {4 `, E0 C
k5 O# j: { R; m* XKey Words. Hematopoiesis ? Microenvironment ? Stromal cells ? MS-5 cell line ? In vivo model M9 k! l& O6 v i5 Q3 R
" J) m$ Z3 G0 e( c1 n* V- MCorrespondence: Fr谷d谷rique Hubin, Department of Cytology and Histology, University of Li豕ge, Li豕ge, 4000, Belgium. Telephone: 003-24-366-2403; Fax: 003-24-366-2919; e-mail: F.Hubin@ulg.ac.be% F; g& S4 Z2 x/ N( f
% c0 f4 R! F- `' D" XABSTRACT. q P8 V b( ^, r1 O7 ~ f: o) w' \
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Bone marrow stroma is made of several cell types and an extracellular matrix, which together form a suitable microenvironment modulating quiescence, self-renewal, and commitment of hematopoietic stem cells and the proliferation, maturation, and apoptosis of more mature hematopoietic cells . However, it has not been possible so far to correlate these functions with particular cell types of the microenvironment, which consists mainly of macrophages, endothelial cells, fibroblasts, and adipocytes . Hence, the establishment of stromal cell clones has gained importance in the dissection of microenvironmental functions. However, it must be kept in mind that the in vivo situation is much more complex than the in vitro model and that the cultures lack the organized three-dimensional structure of reticular network in the bone marrow. Hence, the development of in vivo models in which the interactions between hematopoietic and microenvironmental cells can be studied is absolutely necessary. Hematopoietic cells can be grown in immunodeficient mice, in which their development can be sustained either by blood-forming tissues of the mouse host itself or by surgically implanted human hematopoietic organs . Yet the overall level of hematopoiesis achieved is still highly variable. It is therefore likely that stem cell/stroma interactions are not optimally reproduced. Furthermore, the cellular complexity of the stroma in these models prevents the understanding of the relationship between stromal cells and hematopoietic cells./ ?$ [% v8 n: Z/ b, ]
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To establish a simplified in vivo situation, we have explored the possibility to graft a bone marrow fibroblastic cell line (MS-5) with hematopoietic cells into the kidney capsule of syngenic mice. MS-5 cell line was derived from the irradiated adherent layer of a Dexter-type long-term culture . Depending on the culture conditions, this cell line provides a permissive environment in vitro for B-cell differentiation and for generation of granulocytes but also supports murine colony-forming unit-spleen (CFU-S), granulocyte macrophage-colony-forming unit (CFU-GM), burst-forming unit-erythroid (BFU-E), and bone marrow cells with reconstituting ability . We tried with this model to determine whether hematopoiesis was induced only by a single stromal cell line in vivo without the influence of medium culture and, if so, to clarify putative roles of bone marrow fibroblastic cells in vivo. Furthermore, the potential to produce active bone marrow outside the medullary space could be useful in certain clinical conditions in individuals with irreversible stromal injury. : r! Q" T- h8 v* a 7 y3 t- G5 X& c, f. D" m ?MATERIALS AND METHODS( e, a/ T4 j$ S8 ?' I# m0 H
?0 h8 x3 D$ g/ Z, d7 s, l/ |Differentiation of Murine Stromal Cells MS-5 into Adipocytes0 X7 C' ?9 n: H! O" u. U
. }: Z* ~. m- c4 q' F: XIn vitro, MS-5 stromal cells present a fibroblastic morphology with long spindle-shaped processes. They are alkaline phosphatase–positive and acid phosphatase–negative and contain discrete lipidic droplets stained by oil red O. When 5 x 106 cells are transplanted into kidney capsule, most of them adopt an adipocytic morphology as soon as 5 days after injection, and some of them keep a fibroblastic morphology. These cells are intensely stained by oil red O (Fig. 1A) but lack alkaline phosphatase activity (Fig. 1B). Hematopoietic cells are not detected in the graft. The experiment was performed three times on a total of 22 mice; the results were identical at each tested delay (5, 10, 15, and 30 days). They indicate that MS-5 stromal cells in vivo differentiate into adipocytes and suggest that they do not exert any chemoattractive activity upon hematopoietic stem cells. * a9 g& f* Z8 ]& K. ~0 s2 u0 W' H9 y4 Q+ H
Figure 1. Murine stromal cells (MS-5) injected underneath the kidney’s capsule differentiate into adipocytes. Oil red O staining in red confirms the presence of lipid droplets (A) (x200), whereas alkaline phosphatase activity of the stromal cells has disappeared (B) (x200). ; ^9 _0 `' m9 `! |( M9 P ' Z4 \# _6 V( k2 S8 q7 r9 gStromal Cells in the Bone Marrow Are Not Sufficient for Hematopoietic Cell Survival" {/ i; `" m* n; r s: G/ A& O
, } A9 m+ L. { v+ W |A total of 5 x 106 bone marrow mononuclear cells probably containing both stromal cells and hematopoietic cells was used for transplantation. Cells within this graft adopt a fibroblastic morphology, are alkaline phosphatase–negative (Fig. 2) and oil red O–negative, and do not sustain hematopoiesis; no hematopoietic cells were detected in two experiments using 14 mice whatever the delay analyzed (5–30 days)." e: M8 k' G- G6 j
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Figure 2. Bone marrow mononuclear cells grafted underneath the kidney’s capsule display a fibroblastic morphology. These cells are alkaline phosphatase–negative (x200).' `; R( e5 K, }; a( o
4 t2 E& V0 u/ q- ?/ b3 K" QEctopic Hematopoiesis Occurred Under Kidney Capsule of Mice That Received Stromal Cells MS-5 and Bone Marrow Cells $ S6 C: s6 u3 Q/ H1 @# t5 T+ ~+ h% }- l$ R% V
When 5 x 106 MS-5 stromal cells and 1 x 106 bone marrow mononuclear cells were transplanted together, transient granulopoiesis can be observed. Twenty-three mice were used in three different experiments; a representative graft at 15 days after transplantation is shown in Figure 3.7 ~- k* m7 L f9 H) Z
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Figure 3. Coinjection of stromal and bone marrow mononuclear cells underneath the kidney’s capsule gives rise to transient granulopoiesis. (A): Chloroacetate esterase activity identifies clusters of granulopoietic cells (x200). A network of (B) alkaline phosphatase fibroblastic cells (x200) and of (C) collagen III fibers (x200) surrounds these clusters of granulopoiesis. (D): The lack of acid phosphatase activity excludes the possibility that macrophages were present in the graft (x200). (E): Three months after transplantation, granulopoiesis disappeared and graft was comprised of adipocytes (x200). 1 ]7 {$ ^6 r6 x8 B1 I+ f6 I0 O8 o" R ) r# E4 s: N- s9 }- aClusters of granulopoietic cells identified by their morphology and their chloroacetate esterase activity are surrounded by a network of alkaline phosphatase–positive fibroblastic cells, which produce collagen III fibers detected by Gomori staining (Figs. 3A–3C). In some areas devoid of hematopoietic cells, oil red O–positive adipocytes are observed. To eliminate a possible presence of macrophages into the graft, specific enzymology (acid phosphatase) was performed that reveals that no macrophages were present (Fig. 3D).- R" L0 o. |& b! ~" l. i
- G' Z& X# t/ u3 j2 B+ G& {$ E& uOne month after transplantation, granulopoietic cells had disappeared, and the graft was almost completely comprised of adipocytes; 3 months after transplantation, only adipocytes were found in the graft (Fig. 3E).6 V0 P" t/ s# w; s0 c
6 [5 ]( J1 }% O" y+ B: aIdentification of Engrafting Cells Using Stable Transfected GFP-MS-5 Cells # n& Y# M, g7 e4 }0 I9 {+ S $ }0 K& }/ @* oTo assess the origin of alkaline phosphatase–positive fibroblastic cells in the graft, MS-5 stromal cells were stably transfected with a plasmid expressing GFP. Fluorescence microscopy of the grafts (34 mice, four different experiments) revealed that 15 days after grafting, clusters of granulopoiesis are surrounded by a network of green fluorescent MS-5 cells (Figs. 4A, 4B), whereas 1 month after, only green fluorescent adipocytes are observed (five of six mice; Fig. 4C). ' a6 |( p, J' A9 R4 m! F" k 3 m6 d& k& }" G3 t3 j) f( L& TFigure 4. After coinjection of green fluorescent protein (GFP)–transfected MS-5 cells and bone marrow mononuclear cells, fluorescence microscopy reveals that (A) clusters of granulopoiesis (x200) are surrounded by (B) a network of fibroblastic cells expressing GFP (15 days, x200). (C): At 30 days, only green adipocytes are observed (x200). + \- D* \+ n$ ^8 G# j1 R" H* V6 J8 z
Ectopic Hematopoiesis Occurs When Mice Receive Stromal Cells MS-5 and CD45 Hematopoietic Subpopulations $ T0 F8 J2 r5 R8 d8 s. V( W6 t+ V6 R
To exclude an eventual contribution of stromal cells present in the suspension of total bone marrow mononuclear cells, hematopoietic cells were purified on the basis of CD45 expression. The graft of MS-5 stromal cells and CD45 cells (32 mice in three different experiments) gives rise to the same results as MS-5 stromal cells and total bone marrow mononuclear cells; granulopoiesis developed also in these graft conditions (Figs. 5A, 5B).& B3 [3 p- Q% b5 ^. t
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Figure 5. Isolated CD45 cells coinjected with green fluorescent protein (GFP)–transfected MS-5 cells underneath the kidney’s capsule give rise to (A) clusters of granulopoiesis (x200) surrounded by (B) a network of fibroblastic cells expressing GFP (15 days, x200). / p! h% f8 e* M# ^0 k+ E! Y2 z: u3 v0 {4 h
Stromal MS-5 Cells Are Able to Sustain Murine Hematopoietic Stem Cell Survival4 c6 E4 B1 p) T+ j- x; x# k
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In the next step, MS-5 stromal cells were grafted with c-kit Sca-1 Lin– cells isolated from mononuclear bone marrow cells (33 mice in three different experiments). Clusters of hematopoietic cells, surrounded by a network of green fluorescent MS-5 cells, were observed in the graft. These cells do not present chloroacetate esterase activity excluding a granulopoietic nature (Figs. 6A, 6B). They are neither B nor T lymphocytes because they do not express B220 and CD3 (Figs. 7A, 7B) nor erythroid cells, do not express Ter-119 (Fig. 7C), and are quiescent (Ki-67–negative; Fig. 7D) like hematopoietic stem cells. * Q- g( Q, r8 a$ ?' l3 ]# f0 W/ G. a* I4 K. Q* l) [
Figure 6. Murine c-kit Sca-1 Lin– cells coinjected with green fluorescent protein (GFP)–transfected MS-5 cells underneath the kidney’s capsule give rise to clusters that do not belong to the granulocytic lineage as assessed by (A) the absence of chloroacetate esterase (x200) and that are surrounded by (B) a network of fibroblastic cells expressing GFP (15 days, x200). ! Q6 C% @& h( i( C- b$ @/ |: H0 j/ J. W. O7 c/ E' w$ f. R
Figure 7. Hematopoietic cells obtained after coinjection of c-kit Sca-1 Lin– cells and green fluorescent protein–transfected MS-5 cells do not express (A) B2220 (10 days, x500), (B) CD3 (10 days, x500), (C) Ter-119 (10 days, x200), and (D) Ki-67 (10 days, x500). (E–H): Positive controls, respectively, for B220 (lymph node), CD3 (lymph node), Ter-119 (bone marrow), and Ki-67 (bone marrow). ' ], w6 V. @! i8 d2 _: p) D 6 j- T/ i _- G! xOne thousand cells isolated from the graft were transplanted into 15 lethally irradiated mice, and survival of transplanted mice was monitored daily during 30 days after transplantation. For positive control, murine c-kit Sca-1 Lin– stem cells were also used for transplantation. ' ^) e, [" u. h7 {5 b7 ^" [ 4 @1 P3 P7 ]! QBoth groups of mice were still alive 30 days after transplantation. At day 15 after transplantation, the femurs of transplanted mice contained an average of 3 x 106 hematopoietic cells, and macroscopic colonies (CFU-S) were observed in their spleens. 1 M/ {2 {3 ]& o* x0 Y6 j* r' F8 K% X( }# F2 N
The ability of the cells isolated from the graft to form colonies in vitro was compared with that of a c-kit Sca-1 Lin– suspension; c-kit Sca-1 Lin– cells form CFU-Mix (3 colonies), CFU-GM (37 colonies), CFU-G (14 colonies), CFU-M (8 colonies), and BFU-E (7 colonies), whereas only one CFU-Mix, CFU-GM, and CFU-G was detected after plating the cells isolated from the graft. Z9 H) D7 U9 ?3 g* N: O! [# m
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DISCUSSION6 Z7 O7 w1 q) S( l4 v! p7 {
0 Z4 j) j. Y! L" OThis work was supported by a grant of the Fond National de la Recherche Scientifique. Fr谷d谷rique Hubin and Zakia Belaid are T谷l谷vie fellows. The technical assistance of Marie-Christine Petit and Delphine Delneuville is acknowledged. We also thank Charles Lambert and Philippe Ruggeri for their contribution in the transfection procedures. * `8 t# R. N. i6 }$ Z% h 4 P# Z! q" r$ c7 }9 fDISCLOSURES& Y t+ l* ^4 V( [5 q8 W9 b
9 e" t% l! u/ P. S( x9 w, Z$ uThe authors indicate no potential conflicts of interest.7 Z- j0 v) b6 u0 v `6 f! l: s
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