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、纯度和处理速度。在第一阶段，已经开发了一种新的细胞分离方法，该方法基于专有的光解试剂。已经产生了与可光裂解生物素结合的抗体，这些抗体紧密结合在链霉亲和素涂层的表面，然后被UV-A光释放。一种已开发的先导化合物在红移波长下显示出显著的光解作用。利用这种方法，成功地捕获了CD34髓系白血病KG-1细胞并进行了光释放。结合细胞内自由基清除剂，生长和克隆试验显示与未受辐射的细胞相比没有差异。第二阶段的研究将针对这一新技术的优化，以捕获和光释放用于移植、快速细胞培养和鉴定的细胞。波士顿大学医学院将使用含有人类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。拟议的商业应用：开发新的光诱导细胞选择，PICS，基于光可裂解生物素(PC-Biotin)、光可裂解标记(PC-Marker)和PC抗体(PC-抗体)的技术将在分离和检测包括干细胞、内皮细胞和病原体在内的各种细胞方面具有商业应用。潜在的产品包括PC-生物素和PC-Marker化合物，PC-抗体结合物，可光裂解的FACS标记，细胞分离、检测和细胞分离的自动化设备。

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.