I'm going to work with human hematopoietic and leukemic stem cells derived from bone marrow. Can anyone suggest me the some protocols regarding cell isolation, culture and differentiation? And also, is CD34+CD38- feature enough to be a hematopoietic stem cell?
Most of the peripheral blood leukemic cells are not stem cells (unless the leukemia progressed to blast crisis/acute stage). Most leukemic stem cells settle in bone marrow by homing. So you need to mobilize these leukemic stem cells into peripheral blood before collecting blood samples. Mobilize the CD34+ leukemic stem cells using CXCR4 Antagonist AMD3100 (Plerixafor) OR filgrastim (also it mobilizes lymphoma stem cells..so, it depends on the patients disease you're working with. Since you'll be focusing on leukemic patients, lymphoma mobilization will be of less by chance) and then collect blood for MACS purification to enrich CD34+ and subsequent MACS to deplete CD38+ (note: deplete CD38). Alternatively, after mobilization use FACS sorting to get CD34+ CD38- population. Good Luck...!!
Thank you very much for your precious advice. I will certainly follow that. However, I was wondering which culture media and cytokine/growth factor cocktail would you suggest for expansion and differentiation of these stem cells?
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