Molecular and Biological Analysis of Human Hematopoietic Stem Cells at Single-Cell Resolution

Elucidating the properties and processes regulating primitive hematopoietic cell behavior constitutes a clinically important but still elusive goal. A new challenge in addressing these questions has emerged from experiments in mice that have revealed multiple sources of intrinsically determined heterogeneity in the self renewal potential, lineage competence and proliferation state of these cells. In addition, it is has been shown that these key properties are not coordinately regulated when the cells are stimulated with different external cues. These findings have galvanized interest on the power of using index sorting to link functional responses with high-dimensional molecular profiles obtained on single cells with bioinformatically matched phenotypes. We have now used this approach to analyze the CD34+CD38−CD45RA−CD90+CD49f+ subset of normal human cord blood cells (“CD49f cells”, 10% pure LT-HSCs) before and after their exposure to different growth factor combinations in vitro. Our initial molecular analyses included measurements of several signaling intermediates, transcription factors, cell cycle regulators and surface markers measured simultaneously by mass cytometry. The results revealed new features of intrinsic heterogeneity amongst these very rare human hematopoietic cells and the differential control of their survival, proliferation and regenerative potential. These analyses also led to the discovery of the identity and hence additional molecular properties of a subset within this CD49f population that exclusively possess durable blood cell output capacity in serially transplanted immunodeficient mice. These findings set the stage for future examination of how the properties of these critical normal human hematopoietic cells are affected by development, aging and perturbations that cause disease.