Molecular detection of stem cell plasticity

成体干细胞可塑性的明确证据将对理解器官发育具有重要意义，并有望提供新的治疗性细胞替代疗法。骨髓干细胞可塑性的严格证明需要检测造血和器官再生细胞之间的分子同一性。逆转录病毒和慢病毒载体整合产生独特、稳定的整合位点，是克隆衍生的理想标记。我们已经开发了一种基于pcr的技术，用于在单细胞水平上对独特的逆转录病毒和慢病毒整合位点进行直接基因组测序。本课题旨在研究MGMT/ gfp标记的寡克隆骨髓干细胞移植到人乙型肝炎病毒大抗原转基因小鼠和组织蛋白酶L敲除小鼠模型后的迁移和器官特异性分化。将在这些有和没有辐射损伤的转基因模型中研究肝再生和分化为心肌细胞。gfp阳性细胞将与特异性分化标记一起通过双色或三色免疫荧光进行鉴定。对gfp标记的器官特异性细胞进行非接触激光光学显微解剖，然后进行单细胞整合位点分析，将最终证明或反驳这类细胞具有造血功能的共同克隆起源。小鼠骨髓中多能干细胞的数量和频率的量化将定义可塑性的程度，并有助于建立对多能干细胞生理学的更好理解。

Definitive evidence of adult stem cell plasticity would be of outstanding significance for understanding organ development and would promise novel curative cell replacement therapies. Rigorous proof of bone marrow stem cell plasticity requires the detection of molecular identity between cells in hematopoiesis and organ regeneration. Retro- and lentivirus vector integration generates unique, stable integration sites and is an ideal marker for clonal derivation. We have developed a PCR-based technique for the direct genomic sequencing of unique retro- and lentiviral integration sites at the single cell level. This proposal aims to study the immigration and organ-specific differentiation of MGMT/GFP-marked oligoclonal bone marrow stem cells after transplantation into murine models of human hepatitis B virus large antigen transgenic mice and cathepsin L knockout mice. Hepatoregeneration and differentiation into cardiomyocytes will be studied in these transgenic models with and without radiation damage. GFP-positive cells will be identified together with specific markers of differentiation by double or triple color immunofluorescence. Non-contact laser optical microdissection of GFP-marked, organ-specific cells followed by single cell integration site analysis will definitively prove or disprove a common clonal origin of such cells with hematopoiesis. The quantification of the number and frequency of pluripotent stem cells presents in murine marrow will define the extent of plasticity and help to establish a better understanding of multipotent stem cell physiology.