Simultaneous Injection of Bone Marrow Cells and Stromal Cells into Bone Marrow A - 《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 ... 28 下一页

返回列表

查看: 402114\|回复: 274	go Simultaneous Injection of Bone Marrow Cells and Stromal Cells into Bone Marrow A [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 First Department of Pathology,2 N8 w: V* G, A0 H
4 C) q  L  F; G* u7 X
b Regeneration Research Center for Intractable Diseases,- U6 \) x$ S' W% P

c Center for Cancer Therapy,

d Department of Transplantation for Regeneration Therapy, and% |' @% T: @5 \3 ~7 L. Y2 t
! X8 n6 K. ]: J3 _3 X
e Department of Orthopedics, Kansai Medical University, Moriguchi, Japan;, Q* D8 k6 _4 v! I- j7 m3 W* c; \

f Department of Pediatrics, Nanfang Hospital, Guangzhou, China4 H7 w5 B! `" ?% d4 N
" g6 ^/ H3 F! K1 F* |! h
Key Words. Intra-bone marrow bone marrow transplantation ? Stromal cell ? Major histocompatibility complex ? Colony-forming unit of spleen
& g/ V$ o& G( ]/ Y& U2 W3 i
Correspondence: Susumu Ikehara, M.D., First Department of Pathology, Kansai Medical University, 10-15 Fumizono-cho, Moriguchi City, Osaka 570-8506, Japan. Telephone: 81-66-993-8283; Fax: 81-66-884-8283; e-mail: ikehara@takii.kmu.ac.jp

ABSTRACT* b, O( H8 X- S
. X5 ?4 ~8 d9 z) ^
It has been reported that bone marrow stromal cells (BMSCs) play a crucial role in hematopoiesis and that BMSCs support hematopoietic cells by cognate interaction and production of humoral factors . Even in vitro, Dexter culture using BMSCs can maintain hematopoiesis for longer than 6 months . Moreover, several cell lines derived from the bone marrow have been reported to support hematopoiesis . MC3T3-G2/PA6 (PA6), a mouse bone marrow stromal cell line established from newborn mouse calvaria, can also support hematopoiesis in vitro . We have previously reported that the transplantation of bone marrow cells (BMCs) plus BMSCs successfully ameliorates autoimmune diseases in MRL/Mp-lpr/lpr (MRL/lpr) mice, which possess radioresistant abnormal hemopoietic stem cells . In the experiment, we injected allogeneic BMCs into a vein and transplanted a flushed bone under the renal capsule as a source of BMSCs. These could then migrate from the transplanted bone to the host bone marrow, thereby supporting hematopoiesis of the donor’s BMCs. We have also found that a major histocompatibility complex (MHC) restriction exists between bone marrow hematopoietic stem cells and BMSCs . Recently we have found that the intra–bone marrow (IBM) injection of allogeneic BMCs ameliorates intractable autoimmune diseases in MRL/lpr mice ." A# R5 z9 ~2 n" O! r# _: k3 w
# f0 r: y& |+ h* J, v# W
In this study, we demonstrate that the IBM injection of BMCs plus BMSCs promotes the hematopoiesis of donor BMCs, resulting in prolongation of survival.
& S0 j, Q* D9 C0 |
MATERIALS AND METHODS5 U+ {# l/ z1 V+ u( r
: z3 b: v/ b2 h" n. _( l; Z
Increase in Colony-Forming Unit of Spleen Counts on Day 12 of Mice That Received IBM-BMT of BMCs Plus Stromal Cells6 `7 L( _$ K) n  U7 p
& i& C! q, N- Q9 g6 `/ [; o# S0 f( D1 X
To examine whether MHC-matched stromal cells accelerate hematopoiesis in vivo, we injected eGFP  B6 BMCs and PA6 into a bone marrow cavity or a tail vein of lethally irradiated C3H mice, followed by determination of colony-forming unit of spleen (CFU-S) on day 12. As shown in Figure 1, simultaneous IBM injection of BMCs from eGFP  B6 mice plus PA6 promoted a significant increase of day-12 CFU-S compared with IBM-BMT of only BMCs or IV-BMT of BMCs plus PA6. However, day-12 CFU-S counts in the IBM-BMT of BMCs plus PA6 group still did not attain the counts of the syngeneic IBM-BMT (IBM-BMT of eGFP  B6 mice into B6 mice) group. We obtained similar data when we used BMCs from B6 mice instead of BMCs from eGFP  B6 mice.& Z& T) n; O" |, X- W- r

Figure 1. Significant increase of CFU-S counts on day 12 in the IBM-BMT of BMCs plus PA6 group. BMCs (1 x 105) of eGFP  mice or PA6 (1 x 105) were transplanted to C3H or B6 mice that had been irradiated at 9.5 Gy, as described in Materials and Methods. Twelve days after BMT, the mice were euthanized to determine CFU-S counts and spleen weights. (A): Weight of spleens on day 12 after BMT. *p 9 ^3 y6 D) l4 }" q) a' \

Early Recovery of Donor Hemopoietic Cells in Mice That Received IBM-BMT of BMCs Plus Stromal Cells* N4 M. B. k7 P
9 R4 F0 m: T/ k5 |4 f4 f
We counted the number of WBCs, RBCs, and platelets in the PB of the recipients to examine the recovery of hematopoiesis 12 days after transplantation. As shown in Figures 2A–2C, the BMCs plus PA6 group treated with IBM-BMT showed good recovery of hematopoiesis compared with the groups treated with IV-BMT of BMCs plus PA6 or IBM-BMT of only BMCs. Although there were no significant differences between these groups because of their large standard deviations, we repeatedly noted good recovery in the group treated with IBM-BMT of BMCs plus PA6.

Figure 2. Better recovery of hematopoiesis in PB after IBM-BMT of BMCs plus PA6 than IBM-BMT of BMCs. BMCs (1 x 105) of eGFP  mice or PA6 (1 x 105) were transplanted to C3H mice or B6 mice that had been irradiated at 9.5 Gy, as described in Materials and Methods. Twelve days after BMT, the mice were euthanized to examine the numbers of WBCs (A), RBCs (B), and platelets (C) in PB and also to analyze the percentage of eGFP  nuclear cells in the WBCs (D). Each group consisted of more than four mice. *p
7 o) F- f8 {/ j& ^! \  P
Next, to examine whether transplanted BMCs differentiate into mature hemopoietic cells, we determined the percentage of eGFP  nuclear cells in the PB of the host mice. As shown in Figure 2D, a significantly high percentage of eGFP  WBCs was found in the PB of mice that received IBM-BMT of eGFP  BMCs plus PA6 compared with mice that received IBM-BMT of only BMCs or IV-BMT of BMCs plus PA6. The mean percentage of eGFP  WBCs in the PB of mice in the IBM-BMT of BMCs plus PA6 group was approximately 50%, whereas the PB of mice in the group that received IBM-BMT of only BMCs or IV-BMT of BMCs plus PA6 showed only approximately 20% of donor cells. There were also significant differences between the groups treated with IBM-BMT of BMCs plus PA6 and IV-BMT of BMCs plus PA6 or IBM-BMT of BMCs. These results suggest that IBM-BMT of BMCs plus MHC-matched stromal cells is effective in promoting the hematopoiesis of donor BMCs.+ k  Z6 x# }* c; t

Next, we also analyzed the recovery of donor hematopoietic cells in the bone marrow. We examined the BMC-injected bone (tibia) of the IBM-BMT groups and randomly selected tibias in other groups. As shown in Figure 3, total number of bone marrow cells, percentage of eGFP  cells, number of eGFP  cells, number of CD11b  cells, number of B220  cells, and number of Ter119  cells increased to a greater extent in the group treated with IBM-BMT of BMCs plus PA6 compared with other groups. In fact, there was no significant difference between some groups, but we observed a clear tendency toward better hematopoietic recovery in the group treated with IBM-BMT of BMCs plus PA6.1 q" T) G/ O; C" E) O) w. r/ U$ U) ~

Figure 3. Better recovery of hematopoiesis in the BM after IBM-BMT of BMCs plus PA6 than IBM-BMT of BMCs. BMCs were obtained from BMC-injected tibias in the IBM-BMT groups or randomly selected tibias from other groups. The numbers of total BMCs of one tibia were examined. BMCs were next stained with TC-labeled anti-CD45 Ab and PE-labeled anti-CD11b Ab, TC-labeled anti-CD45 Ab and PE-labeled anti-B220 Ab, or TC-labeled anti-CD45 Ab and PE-labeled anti-Ter119 Ab. Stained cells were analyzed using FACStar. The percentages of eGFP  cells in CD45  cells, eGFP  cells in CD11b  cells, eGFP  cells in B220  cells, or eGFP  cells in Ter119  cells in the BM were examined. The total cell numbers of each surface marker–positive cell were calculated with the total cell numbers of tibias and percentages of each fraction. Each group consisted of more than four mice. *p : k+ u) _/ j, g5 K
. u* f7 t1 v: \. H( M
Prolonged Survival in Mice That Received IBM-BMT of BMCs Plus Stromal Cells
' L& d( \( b2 `4 A2 p
We next examined whether IBM-BMT of BMCs plus MHC-matched stromal cells had the ability to prolong the survival of transplanted host mice. As shown in Figure 4, the survival rate of mice that received IBM-BMT of BMCs plus PA6 was approximately 60%, even on day 60, by which time mice in all groups other than syngeneic IBM-BMT had already died. These results suggest that rapidly developed donor hematopoietic cells enabled the mice to survive in the IBM-BMT of BMCs plus PA6 group and that sufficient hematopoiesis did not occur in other groups, except for syngeneic BMT, because the dead mice had shown no symptoms and no pathological findings of graft versus host disease. Even in the IBM-BMT of BMCs group, in which we previously observed more rapid hematopoiesis than in the IV-BMT BMCs group, the mice died rapidly, probably because of the very small number of BMCs transplanted .4 ~4 B6 S: G+ H. A% E" X( E7 B
: o! S9 m2 f5 P* H& T. u, o$ F% R
Figure 4. Improvement of survival rate after IBM-BMT of BMCs plus PA6. BMCs (1 x 105) of eGFP  mice or PA6 (1 x 105) were transplanted to C3H mice or B6 mice that had been irradiated at 9.5 Gy. The mice were observed for survival rate up to 60 days after BMT. There were 10 or more mice in each group. p values were examined with log-rank test. Significances were detected between IBM-BMT of BMCs plus PA6 and other groups. Abbreviations: BMC, bone marrow cell; BMT, bone marrow transplantation; IBM, intra-bone marrow; IV, intravenous.

Simultaneously Injecting Cultured BMSCs Plus BMCs into Same Bone Accelerates Hematopoiesis
) w  C, M' J' |) w
To apply this method clinically, we used BMSCs instead of PA6 in our system. Because there are very few BMSCs in freshly isolated BMCs, we cultured BMSCs to enrich and to augment the number of BMSCs . Next, we injected cultured BMSCs plus BMCs into the tibias to examine whether cultured BMSCs can accelerate hematopoiesis. As shown in Figure 5, cultured BMSCs plus BMCs showed higher CFU-S counts and heavier spleen weight, suggesting significantly enhanced hematopoiesis. We also prepared several kinds of control to exclude the possibility of nonspecific physical stimulation. Saline was used as a control for volume expansion, and latex beads were used for the control of volume expansion and physical stimulation. Neither injection of saline nor latex beads augmented the CFU-S counts or spleen weight. IBM-BMT of latex beads plus BMCs or IBM-BMT of fixed PA6 plus BMCs resulted in a smaller number of CSF-S than IBM-BMT of only BMCs, suggesting that latex beads and fixed PA6 disturbed hematopoiesis.; {8 _- C0 Q6 i, O1 M1 P! W

Figure 5. BMSCs isolated from BMCs accelerate hematopoiesis. Several kinds of control groups (IBM-BMT of only saline, only latex beads, BMCs plus latex beads, BMCs plus fixed PA6, and cultured BMSCs) were prepared as described in Materials and Methods. IBM-BMT was carried out from B6 to C3H mice using B6-cultured BMSCs plus B6 eGFP  BMCs. Twelve days after BMT, mice were euthanized, their spleens were weighed (A), and their CFU-S (B) was counted. Each group consisted of more than four mice. *p 1 j& |9 u6 z+ q- r' I$ g. M
! j, D' [" e" E; s! s. l+ D
DISCUSSION
  A) \+ ~; v& _% w! C
We thank Ms. Murakami-Shinkawa, Ms. Tokuyama, and Ms. Miura for their expert technical assistance and Mr. Eastwick-Field and Ms. Ando for the preparation of this manuscript. This study was supported by grants from Haiteku Research Center of the Ministry of Education, grant-in-aid for scientific research (B) 11470062, grant-in-aid for scientific research (Hoga) 16659107, grants-in-aid for scientific research on priority area (A) 10181225 and (A) 11162221, a grant from Millennium of Ministry of Education, Culture, Sports, Science and Technology, and a grant from the Scientific Frontier program of the Ministry of Education, Culture, Sports, Science and Technology. The Department of Transplantation for Regeneration Therapy, Kansai Medical University, was sponsored by Otsuka Pharmaceutical Company, Ltd.
2 u& I$ t2 t) l7 p6 \
Yuming Zhang and Yasushi Adachi contributed equally to this work.

REFERENCES
  N* H/ s1 k+ U+ M. E
Izumi-Hisha H, Than S, Ogata H et al. Monoclonal antibodies against a preadipose cell line (MC3T3-G2/PA6) which can support hemopoiesis. Hybridoma 1991;10:103–112.

Whitlock CA, Witte ON. Long term culture of B lymphocytes and their precursors from murine bone marrow. Proc Natl Acad Sci U S A 1982;79:3608–3612.( X+ v0 g( G1 ?) H" i4 w( \. K
  U8 V- ^1 x* l
Suda T, Ito M, Ogawa Y et al. Interleukin-7 production and function in stromal cell dependent B cell development. J Exp Med 1989;170:333–338.
+ v& r# p$ q5 p- ?- x% j
Ohkawa H, Harigaya K. The effect of direct cell to cell interaction of the clonal human marrow stromal cell line on the differentiation and proliferation of the myeloid leukemic cell line, HL-60. Cancer Res 1987;47:2879–2882.
. U, N# F+ X5 p- I
Harigaya K, Cronkite EP, Miller ME et al. Murine bone marrow cell line producing colony-stimulating factor. Proc Natl Acad Sci U S A 1981;78:6963–6966.- b" P" k1 S- H5 R6 R) Z
& Q# X) A0 c; j, n6 d8 j
Dexter TM, Allen TD, Lajtha LG. Conditions controlling the proliferation of hemopoietic stem cells in vitro. J Cell Physiol 1977;91:335–344.

Zipori D, Toledo J, von der Mark K. Phenotypic heterogeneity among stromal cell lines from mouse bone marrow disclosed in their extracellular matrix composition and interactions with normal and leukemic cells. Blood 1985;66:447–455.

Whitlock CA, Tidmarsh GF, Muller-Sieburg C et al. Bone marrow stromal cell lines with lymphopoietic activity express high levels of pre-B neoplasia-associated molecules. Cell 1987;48:1009–1021.! {3 V& ?( b7 Z* o* N" i' I$ K+ n

Ogawa M, Nishikawa S, Ikuta K et al. B cell ontogeny in murine embryo studied by a culture system with the monolayer of a stromal cell clone, ST-2: B cell progenitor develops first in the embryonal body rather than in the yolk sac. EMBO J 1988;7:1337–1343.

Kodama H, Amagai Y, Koyama H et al. Hormonal responsiveness of preadipose cell line derived from newborn mouse calvaria. J Cell Physiol 1982;112:83–88.* q. j* h9 }; W+ e9 y
3 m  y9 Z2 W; q' y- J
Kodama H, Amagai Y, Koyama H et al. A new preadipose cell line derived from newborn mouse calvaria can promote the proliferation of pluripotent hemopoietic stem cells. J Cell Physiol 1982;112:89–95.

Ishida T, Inaba M, Hisha H et al. Requirement of donor-derived stromal cells in the bone marrow for successful allogeneic bone marrow transplantation. J Immunol 1994;152: 3119–3127.: J* v* m0 \" Z7 I! h

Sugiura K, Hisha H, Ishikawa J et al. Major histocompatibility complex restriction between hematopoietic stem cells and stromal cells in vitro. STEM CELLS 2001;19:46–58.' ]; }0 Z5 w6 L9 i' x

Sugiura K, Inaba M, Hisha H et al. Requirement of major histocompatibility complex-compatible microenvironment for spleen colony formation (CFU-S on day 12 but not on day 8). STEM CELLS 1997;15:461–468.9 w+ R" ^0 U  s1 y$ O' X0 D

Hashimoto F, Sugiura K, Inoue K et al. Major histocompatibility complex restriction between hematopoietic stem cells and stromal cells in vivo. Blood 1997;89:49–54.

Kushida T, Inaba M, Hisha H et al. Intra-bone marrow injection of allogeneic bone marrow cells: a powerful new strategy for treatment of intractable autoimmune diseases in MRL/lpr mice. Blood 2001;97:3292–3299.

Okabe M, Ikawa M, Kominami K et al. "Green mice" as a source of ubiquitous green cells. FEBS Lett. 1997;407:313–319.

Fan TX, Hisha H, Jin TN et al. Successful allogeneic bone marrow transplantation (BMT) by injection of bone marrow cells via portal vein: stromal cells as BMT-facilitating cells. STEM CELLS 2001;19:144–150.+ F" T7 L. t! R- D% \0 M0 g
# q' W1 |6 N9 C$ L
Till JE, McCulloch EA. A direct measurement of the radiation sensitivity of normal bone marrow cells. Radiat Res 1961;85:213–222.) ]  X6 o9 q! B7 y4 L4 V2 l  F1 ?

Kodama H, Sudo H, Koyama H et al. In vitro hematopoiesis within a microenvironment created by MC3T3-G2/PA6 preadipocyte. J Cell Physiol 1984;118:233–240.

Kodama H, Hagiwara H, Sudo H et al. MC3T3-G2/PA6 preadipocytes support in vitro proliferation of hematopoietic stem cells through a mechanism different from that of interleukin 3. J Cell Physiol 1986;129:20–26.
2 ?: S" ^! Q" }% |" K
Satoh M, Mioh H, Shiotsu Y et al. Mouse bone marrow stromal cell line MC3T3-G2/PA6 with hematopoietic-supporting activity expresses high levels of stem cell antigen Sca-1. Exp Hematol 1997;25:972–979.& |# r5 u: H$ N7 [( A

Witte PL, Frantsve LM, Hergott M et al. Cytokine production and heterogeneity of primary stromal cells that support B lymphopoiesis. Eur J Immunol 1993;23:1809–1817.* @# W+ I4 F8 C' x9 Y
+ S4 \' s  d( u$ E% y
Zhang Y, Yasumizu R, Sugiura K et al. Fate of allogeneic or syngeneic cells in intravenous or portal vein injection: possible explanation for the mechanism of tolerance induction by portal vein. Eur J Immunol 1994;24:1558–1565.
% V- P: g. d% s! L
Naito S, Kinjo M, Tanaka K. Fate of intravenously injected cultured human cancer cells in the lungs of congenitally athymic nude mice. Acta Pathol Jpn 1980;30:541–547.
' {+ E9 j& X0 _8 \+ O  j; e
Morse MA, Coleman E, Akabani G et al. Migration of human dendritic cells after injection in patients with metastatic malignancies. Cancer Res 1999;59:56–58.(Yuming Zhanga,f, Yasushi )

收藏0 分享0 顶0 踩0