小鼠造血细胞移植后早期造血重建的动力学变化
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摘要
目的:揭示移植后早期(4 w)供体来源的造血干/祖细胞(hematopoietic stem/progenitor cell,HSPC)以及各类分化成熟血细胞的动态变化特点。方法:利用小鼠同种同基因造血干细胞移植模型分别移植供体(B6-Ly5.1)5×10~6全骨髓单个核细胞(wBMMNC)和富集的3×10~5c-Kit~+HSPC到致死剂量照射的同品系受体小鼠(B6-Ly 5.2),然后动态监测移植后早期(4 w内)受体内供体来源细胞的造血重建和谱系分化情况。结果:wBMMNC移植后1 w(5-7 d)供体来源细胞开始出现增殖,在移植后2-3 w达到增殖高峰;c-Kit~+HSPC移植后第1-2 w供体细胞即出现增殖高峰并一直维持到移植后4 w。wBMMNC移植后1 w,骨髓中供体来源细胞主要向髓系细胞分化,伴随有少量B细胞生成,而几乎没有T细胞的生成;在移植后2-4 w,供体来源细胞几乎全部分化为髓系细胞。c-Kit~+HSPC移植后1-3w供体来源细胞几乎全部分化为髓系细胞,移植后4 w时出现B细胞的重建。移植后1-2 w,c-Kit~+细胞移植组供体来源LKS~-和LKS~+细胞的绝对数显著高于wBMMNC移植组(P<0.001)。双光子显微镜观察受体小鼠颅骨发现,cKit~+细胞移植后4-5 d,10~5及10~6细胞移植组即可观察到簇状分布的供体来源细胞的增殖。结论:c-Kit~+细胞移植后供体来源HSPC(LKS~-和LKS~+)重建动力学速率显著快于wBMMNC移植组,c-Kit~+细胞移植后1-3 w主要向髓系细胞分化,4 w才开始出现部分淋系分化。c-Kit~+细胞移植后1 w之内供体来源HSPC迫于造血压力迅速增殖分化,从而维持机体对于应激造血的迫切需求。
Objective: To observe the dynamic changes of hematopoietic reconstitution and multiple lineages differentiation at early phase after transplantation. Methods: Whole bone marrow mononuclear cells(wBMMNC,5 × 106) and enriched c-Kit~+ hematopoietic stem/progenitor cells(HSPC, 3 × 10~5) from the BM of B6-Ly5. 1 mice were transplanted into lethally irradiated B6-Ly5. 2 mice, the frequencies and absolute numbers of donor-derived cells(including LKS~-and LKS~+) were detected by flow cytometry. The multiple lineages differentiation of do nor-derived cells was also monitored by flow cytometry. The homing and early phase proliferations of donor-derived cells were observed by two-photon microscope.Results: The donor-derived cells started to proliferation from 5-7 days after transplantation and reached the peak value at 2-3 weeks after wBMMNC transplantation. The donor-derived cells proliferated from 1-2 weeks and maintained until 4 weeks after c-kit~+ HSPC transplantation. At 1 week after transplantation, the donor-derived cells mainly differentiated into myeloid cells with a few lymphoid cells production(B cells) but the production of T cells was not observed at most in wBMMNC transplanted group, while myeloid cells occupied the majority of donor-derived cells at 2-4 weeks; donor-derived cells almost totally differentiated into myeloid cells at 1-3 weeks after transplantation in c-Kit~+ transplanted group and donor-derived B cells appeared at 4 weeks. The absolute number of donor-derived LKS~-and LKS~+ cells in the B M of c-Kit~+ transplanted group were much higher than that of wBMMNC group(P < 0. 001) at 2 weeks respectively.The clustering proliferation of c-Kit~+ cells at 4-5 days after transplantation was observed by twophoton microscope. Conclusion: The dynamical rate of proliferation and reconstitution of donor-derived cells are much earlier and quicker in c-Kit~+ group than those of wBMMNC group. c-Kit~+ cells mainly differentiate into myeloid cells within1-3 weeks and the lymphoid cell differentiation starts at 4 weeks after transplantation. The immediate proliferation and differentiation of c-Kit~+ cells within 1 week maybe due to the urgent needs of hematopoietic regeneration under the myeloablated hosts.
Objective: To observe the dynamic changes of hematopoietic reconstitution and multiple lineages differentiation at early phase after transplantation. Methods: Whole bone marrow mononuclear cells(wBMMNC,5 × 106) and enriched c-Kit~+ hematopoietic stem/progenitor cells(HSPC, 3 × 10~5) from the BM of B6-Ly5. 1 mice were transplanted into lethally irradiated B6-Ly5. 2 mice, the frequencies and absolute numbers of donor-derived cells(including LKS~-and LKS~+) were detected by flow cytometry. The multiple lineages differentiation of do nor-derived cells was also monitored by flow cytometry. The homing and early phase proliferations of donor-derived cells were observed by two-photon microscope.Results: The donor-derived cells started to proliferation from 5-7 days after transplantation and reached the peak value at 2-3 weeks after wBMMNC transplantation. The donor-derived cells proliferated from 1-2 weeks and maintained until 4 weeks after c-kit~+ HSPC transplantation. At 1 week after transplantation, the donor-derived cells mainly differentiated into myeloid cells with a few lymphoid cells production(B cells) but the production of T cells was not observed at most in wBMMNC transplanted group, while myeloid cells occupied the majority of donor-derived cells at 2-4 weeks; donor-derived cells almost totally differentiated into myeloid cells at 1-3 weeks after transplantation in c-Kit~+ transplanted group and donor-derived B cells appeared at 4 weeks. The absolute number of donor-derived LKS~-and LKS~+ cells in the B M of c-Kit~+ transplanted group were much higher than that of wBMMNC group(P < 0. 001) at 2 weeks respectively.The clustering proliferation of c-Kit~+ cells at 4-5 days after transplantation was observed by twophoton microscope. Conclusion: The dynamical rate of proliferation and reconstitution of donor-derived cells are much earlier and quicker in c-Kit~+ group than those of wBMMNC group. c-Kit~+ cells mainly differentiate into myeloid cells within1-3 weeks and the lymphoid cell differentiation starts at 4 weeks after transplantation. The immediate proliferation and differentiation of c-Kit~+ cells within 1 week maybe due to the urgent needs of hematopoietic regeneration under the myeloablated hosts.
引文
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3 Chabannon C,Kuball J,Bondanza A,et al.Hematopoietic stem cell transplantation in its 60s:a platform for cellular therapies.Sci Transl Med,2018;10(436).pii:eaap9630.doi:10.1126/scitranslmed.aap9630.
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8 Yamamoto R,Wilkinson AC,Ooehara J,et al.Large-scale clonal analysis resolves aging of the mouse hematopoietic stem cell compartment.Cell Stem Cell,2018;22(4):600-607.
9 Ema H,Uchinomiya K,Morita Y,et al.Repopulation dynamics of single haematopoietic stem cells in mouse transplantation experiments:Importance of stem cell composition in competitor cells.JTheor Biol,2016;394:57-67.
10 Shen H,Yu H,Liang PH,et al.An acute negative bystander effect of gamma-irradiated recipients on transplanted hematopoietic stem cells.Blood,2012;119(15):3629-3637.
11师鸣,胡林萍,张孝兵,等.过氧化氢酶对人造血干细胞在免疫缺陷小鼠中植入的作用.中国实验血液学杂志,2015;23(1):183-189.
12王钊,施圆圆,杨欣,等.移植前血清细胞因子水平对重型再生障碍性贫血患者异基因造血干细胞移植疗效的影响研究.中国实验血液学杂志,2018;26(2):502-507.
13 Yamamoto R,Morita Y,Ooehara J,et al.Clonal analysis unveils self-renewing lineage-restricted progenitors generated directly from hematopoietic stem cells.Cell,2013;154(5):1112-1126.
14 Tan XW,Liao H,Sun L,et al.Fetal microchimerism in the maternal mouse brain:a novel population of fetal progenitor or stem cells Able to cross the blood-brain barrier?Stem Cells,2005;23(10):1443-1452.
15 Laurenti E,Gottgens B.From haematopoietic stem cells to complex differentiation landscapes.Nature,2018;553(7689):418-426.
2 Yamamoto R,Wilkinson AC,Nakauchi H.Changing concepts in hematopoietic stem cells.Science,2018;362(6417):895-896.
3 Chabannon C,Kuball J,Bondanza A,et al.Hematopoietic stem cell transplantation in its 60s:a platform for cellular therapies.Sci Transl Med,2018;10(436).pii:eaap9630.doi:10.1126/scitranslmed.aap9630.
4 De Koning C,Plantinga M,Besseling P,et al.Immune reconstitution after allogeneic hematopoietic cell transplantation in children.Biol Blood Marrow Transplant,2016;22(2):195-206.
5 Forsberg EC,Serwold T,Kogan S,et al.New evidence supporting megakaryocyte-erythrocyte potential of flk2/flt3+multipotent hematopoietic progenitors.Cell,2006;126(2):415-426.
6 Shin JY,Hu W,Naramura M,et al.High c-Kit expression identifies hematopoietic stem cells with impaired self-renewal and megakaryocytic bias.J Exp Med,2014;211(2):217-231.
7 Oguro H,Ding L,Morrison SJ.SLAM family markers resolve functionally distinct subpopulations of hematopoietic stem cells and multipotent progenitors.Cell Stem Cell,2013;13(1):102-116.
8 Yamamoto R,Wilkinson AC,Ooehara J,et al.Large-scale clonal analysis resolves aging of the mouse hematopoietic stem cell compartment.Cell Stem Cell,2018;22(4):600-607.
9 Ema H,Uchinomiya K,Morita Y,et al.Repopulation dynamics of single haematopoietic stem cells in mouse transplantation experiments:Importance of stem cell composition in competitor cells.JTheor Biol,2016;394:57-67.
10 Shen H,Yu H,Liang PH,et al.An acute negative bystander effect of gamma-irradiated recipients on transplanted hematopoietic stem cells.Blood,2012;119(15):3629-3637.
11师鸣,胡林萍,张孝兵,等.过氧化氢酶对人造血干细胞在免疫缺陷小鼠中植入的作用.中国实验血液学杂志,2015;23(1):183-189.
12王钊,施圆圆,杨欣,等.移植前血清细胞因子水平对重型再生障碍性贫血患者异基因造血干细胞移植疗效的影响研究.中国实验血液学杂志,2018;26(2):502-507.
13 Yamamoto R,Morita Y,Ooehara J,et al.Clonal analysis unveils self-renewing lineage-restricted progenitors generated directly from hematopoietic stem cells.Cell,2013;154(5):1112-1126.
14 Tan XW,Liao H,Sun L,et al.Fetal microchimerism in the maternal mouse brain:a novel population of fetal progenitor or stem cells Able to cross the blood-brain barrier?Stem Cells,2005;23(10):1443-1452.
15 Laurenti E,Gottgens B.From haematopoietic stem cells to complex differentiation landscapes.Nature,2018;553(7689):418-426.
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