High frequency of long-term culture-initiating cells retain in vivo repopulation and self-renewal capacity

Objective We wished to test if the long-term culture initiating cell (LTC-IC) assay measures primitive hematopoietic stem cells. An LTC-IC is defined by its ability to repopulate a stromal layer by forming colonies of myeloid cells. A negative well should never have received a stem cell, whereas a positive well should have been initiated by a stem cell. If these colonies were derived from stem cells, then a subset of the positive wells should retain stem cell activity. Materials and Methods Limiting dilution cultures were initiated on the stromal cell line S17. Individual clonal cultures from LTC-IC assays were assessed for repopulation capacity in W14W41 mice. Results In long-term repopulation experiments, little activity was found in the negative wells, whereas 50% of the positive wells contained repopulating stem cells. The diverse in vivo repopulation patterns of the clonally derived stem cells suggest that this assay detects the full spectrum of stem cell types. Secondary transfers show that the clonally derived stem cells have self-renewal capacity. Experiments with mixtures of genetically distinguished cells showed that most (>90%) of the cultures were clonal. Conclusions Our data present the first formal link between LTC-IC and repopulating stem cells. Moreover, the culture system presents a new way of generating a high frequency of clonally repopulating stem cells.