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a First Department of Pathology,
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& A8 t" ?' [' Mb Regeneration Research Center for Intractable Diseases,
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! t, s1 E4 W6 X9 a+ }c Center for Cancer Therapy,1 K# q6 w: t0 B3 i9 q4 J
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d Department of Transplantation for Regeneration Therapy, and
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e Department of Orthopedics, Kansai Medical University, Moriguchi, Japan;
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Key Words. Intra-bone marrow bone marrow transplantation ? Stromal cell ? Major histocompatibility complex ? Colony-forming unit of spleen! h5 o& T+ H! F" \( y
5 \% R4 O3 |# ~/ [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
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! ^' P7 ~0 u1 f4 mABSTRACT
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8 U2 t. m9 S4 H. |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 .4 C X8 ^ X- r" | I: P# ~
" \# @4 C2 o* o/ q) P; fIn this study, we demonstrate that the IBM injection of BMCs plus BMSCs promotes the hematopoiesis of donor BMCs, resulting in prolongation of survival.
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MATERIALS AND METHODS
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6 \6 J$ K' u1 R& T+ S/ t4 V, ^Increase in Colony-Forming Unit of Spleen Counts on Day 12 of Mice That Received IBM-BMT of BMCs Plus Stromal Cells
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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.5 t. v, S9 Z/ H# R9 y _( B
( y0 r8 G. g. Y- E$ l# g7 j9 YFigure 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
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Early Recovery of Donor Hemopoietic Cells in Mice That Received IBM-BMT of BMCs Plus Stromal Cells/ d# ?; C2 ]; Z, {
. A2 Y) h6 s$ K+ N0 M& w& \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 PA6.2 E. s. J/ p& ]. [6 K2 T# K
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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
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1 e! B9 P! n( S6 }0 j8 oNext, 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.) C2 [) J+ Z7 S) T, a" J5 ]
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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., X- H! v- J7 d: y# E$ E: m
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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
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6 d; K; T$ O! o# u* GProlonged Survival in Mice That Received IBM-BMT of BMCs Plus Stromal Cells
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* c/ J) y" K7 G- J0 c* t2 A1 y8 o- KWe 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 .
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) z3 X8 X6 |( c y5 {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.
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Simultaneously Injecting Cultured BMSCs Plus BMCs into Same Bone Accelerates Hematopoiesis
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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.0 V8 L- A6 A; `- A9 M& x
1 H% l. a; n& g) ], e( x1 [, J/ ~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 ) C2 v7 H% N: e. l
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DISCUSSION4 Y8 f+ m# m# a7 X9 m6 X( S
& s8 k. k. w# v$ K8 WWe 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.7 }2 \# `8 O J7 {. g
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Yuming Zhang and Yasushi Adachi contributed equally to this work.
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