Optical imaging of PKH-labeled hematopoietic cells in recipient bone marrow in vivo
Optical imaging of PKH-labeled hematopoietic cells in recipient bone marrow in vivo
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DOI：
10.1634/stemcells.20-6-501
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发表时间：
2002-01-01
期刊：
影响因子：
5.2
通讯作者：
Farkas, DL
中科院分区：
文献类型：
--
作者：
Askenasy, N;Farkas, DL
这项工作描述了一种光学技术，用于表征造血干细胞（HSC）在生理条件下植入的早期阶段，并在真实的时间。将PKH膜连接体标记的骨髓细胞（BMC）注射到预先手术的条件受体（B10->4B10.BR小鼠）中，以在股骨骨骺上放置光学窗。通过荧光显微镜在体内追踪标记的细胞。用激光镊子测试细胞与BM基质的粘附，并通过碘化丙啶（PI）排除试验测定活力，如从原位新鲜切除的股骨中PKH 67-PI对的能量传递测量所确定的。在体内追踪的最佳浓度下，1-4 μ M PKH染料既不损害BMC的活力，也不损害同种异体HSC在骨髓清除受体中重建造血的能力。光学窗口允许股骨中23%-26%的PKH标记的BMC的体内可视化。移植后16小时，同基因和异基因BMCs的归巢效率分别为1.77% ± 0.15%和0.21% ± 0.02%。在移植后16小时切除的股骨中，70% +/-9%的细胞粘附于BM基质，并且三分之二的细胞为PI阴性（活的）。受体骨髓中标记的HSC的体内跟踪和原位评估为植入的早期阶段提供了重要的定量和定性见解。早期事件的相关性和持久植活的效率作为优化移植条件的系统方法的基础。
This work describes an optical technique for characterization of the early stages of hematopoietic stem cell (HSC) engraftment under physiological conditions and in real time. Bone marrow cells (BMCs) labeled with PKH membrane linkers were injected into conditioned recipients (B10-->4B10.BR mice) preoperated for placement of optical windows over femoral epiphyses. Labeled cells were tracked in vivo by fluorescence microscopy. Cellular adhesion to the BM stroma was tested with laser tweezers, and viability was assayed by the propidium iodide (PI) exclusion test, as determined from energy-transfer measurements of the pair PKH67-PI in freshly excised femurs in situ. At optimal concentrations for in vivo tracking, 1-4 muM PKH dyes neither impaired the viability of BMCs nor the capacity of allogeneic HSCs to reconstitute hematopoiesis in myeloablated recipients. The optical window allowed in vivo visualization of 23%-26% of the PKH-labeled BMCs in the femur. The homing efficiencies at 16 hours posttransplantation were quantified as 1.77% 0.15% and 0.21% +/- 0.02% for syngeneic and allogeneic BMCs, respectively. In femurs excised 16 hours after transplantation, 70% +/- 9% of the cells were adherent to the BM stroma, and two-thirds of the cells were PI negative (viable). In vivo tracking and in situ assessment of labeled HSCs in recipient BM provide important quantitative and qualitative insights into the early stages of engraftment. Correlation of early events and the efficiency of durable engraftment serve as the basis for a systematic approach toward optimization of the conditions for transplantation.