PHOTOCLEAVABLE REAGENTS FOR ISOLATION OF STEM CELLS

干细胞的分离是治疗多种疾病的关键技术。由于干细胞可以增殖和分化，它们的分离有利于移植和基因治疗。虽然在通过基于生物素-抗生物素蛋白的免疫吸附分离干细胞方面已经取得了进展，但是需要改进以提高干细胞的产量、纯度和处理速度。在第一阶段，开发了一种基于专有光裂解试剂的细胞分离新方法。已经产生了与可光裂解的生物素缀合的抗体，其与链霉亲和素包被的表面紧密结合，然后通过UV-A光释放。一种先导化合物在红移波长处表现出显著的光致裂解。使用这种方法，CD34+骨髓性白血病KG-1细胞已被成功捕获和光释放。与细胞内自由基清除剂组合，生长和克隆形成测定显示与未照射的细胞相比没有差异。第二阶段的研究将针对这种新技术的优化，用于捕获和光释放细胞，用于移植，快速细胞培养和鉴定。将在波士顿大学医学院使用含人造血干细胞的标本进行临床前CD34+细胞分离和小鼠移植研究。拟定商业应用：基于光可裂解生物素（PC-生物素）、光可裂解标记物（PC-标记物）和PC-抗体（PC-抗体）的新型光诱导细胞选择（PICS）技术的开发将具有用于分离和检测各种细胞（包括干细胞、内皮细胞和病原体）的商业应用。潜在产品包括PC-生物素和PC-标记化合物、PC-抗体缀合物、光可裂解的FACS标记、用于细胞分离的试剂盒、检测和用于细胞分离的自动化装置。

The isolation of stem cells is a critical technology for the treatment of a wide range of diseases. Since stem cells can proliferate and differentiate, their isolation facilitates transplantation and gene therapies. While progress has been made in the isolation of stem cells through biotin-avidin based immunoadsorption, improvements are needed in order to increase yields, purity and rapidity of processing of stem cells. During Phase I, a new method for the isolation of cells has been developed which is based on proprietary photocleavable reagents. Antibodies conjugated with photocleavable biotins have been produced which bind tightly to streptavidin coated surfaces and are then released by UV-A light. One lead compound developed exhibits significant photocleavage at red-shifted wavelengths. Using this approach, CD34+ myelogenous leukemia KG-1 cells have been successfully captured and photoreleased. In combination with an intracellular free radical scavenger, growth and clonogenic assays reveal no differences compared to non-irradiated cells. Phase II research will be directed at the optimization of this new technology for the capture and photorelease of cells for use in transplantation, rapid cell culturing and identification. Preclinical CD34+ cell isolation and mouse engraftment studies will be conducted at Boston University Medical School using human hematopoietic stem cell-containing specimens. PROPOSED COMMERCIAL APPLICATION: Development of new PhotoInduced Cell Selection, PICS, technology based on photocleavable biotins (PC-Biotin), photocleavable markers (PC- Marker) and PC-antibodies (PC-antibodies) will have commercial applications for the isolation and detection of a wide variety of cells including stem cells, endothelial and pathogens. Potential products include PC-Biotin and PC-Marker compounds, PC-antibody conjugates, photocleavable FACS labels, kits for cell isolation, detection and automated devices for cell separation.