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。虽然在通过生物素-亲和素免疫吸附分离干细胞方面取得了进展，但还需要改进，以提高干细胞的产量、纯度和加工速度。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。与光可切割生物素结合的抗体已被生产出来，这种抗体与链霉亲和素包被的表面紧密结合，然后被UV-A光释放。一种先导化合物在红移波长处表现出明显的光解理作用。利用这种方法，CD34+骨髓性白血病KG-1细胞已被成功捕获并光释放。结合细胞内自由基清除剂，生长和克隆测定显示与未辐照细胞相比没有差异。第二阶段的研究将针对这种新技术的优化，用于细胞的捕获和光释放，用于移植、快速细胞培养和鉴定。临床前CD34+细胞分离和小鼠植入研究将在波士顿大学医学院进行，使用含有人类造血干细胞的标本。拟议的商业应用：基于光可切割生物素（PC- biotin）、光可切割标记物（PC- Marker）和PC-抗体（PC- antibody）的新型光诱导细胞选择（PICS）技术的开发将具有商业应用，可用于分离和检测多种细胞，包括干细胞、内皮细胞和病原体。潜在产品包括pc -生物素和pc -标记化合物，pc -抗体偶联物，光可切割的FACS标签，细胞分离试剂盒，检测和细胞分离自动化设备。

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.