Isolation of CD34+ Hematopoietic Stem Cells

Bone marrow samples were harvested from human subjects from the posterior iliac crest using a Yamshidi needle, with aspirations at 5–7 different levels of approximately 10 ml at each level^{29 (link)}. The study has been approved by the Ethics Committee for Human Subjects at the University Heidelberg and written informed consent was obtained from each individual. We recruited 17 subjects with the age ranging from 21 to 71 years for the different experiments within this study. The bone marrow aspirates were processed by FICOLL density fractionation for isolation of mononuclear cells (MNCs). After staining with CD34-APC and CD14-PE (both from BD Biosciences, San Jose, CA) the CD34+ cell population was isolated using a FACSAria II flow cytometry cell sorter (BD Biosciences). After Fluorescence Activated Cell Sorting (FACS), the sorted cells were analyzed for their purity by reanalyzing an aliquot of the respective cell population^{10 (link)}.