We have recently described a modification of the long-term culture- initiating cell (LTC-IC) assay that allows the identification and quantitation of a subset of murine LTC-IC that are able to generate both myeloid and lymphoid progeny in vitro. These “LTC-ICML” are inseparable from either standard LTC-IC or cells with long-term in vivo lympho-myeloid repopulating potential (competitive repopulating units, or CRU) present in normal adult mouse marrow but are detected at 15- to 30-fold lower frequencies. To determine whether at least part of this discrepancy is due to a reduced sensitivity of the LTC-ICML assay, a number of strategies to try to improve the original protocol were tested. Prolongation of the second phase of the assay (when the cells are maintained under lymphoid LTC conditions) from 7 to 10 days and inclusion of Flt3 ligand (FL) in the assay used to detect the production of B-lineage cells (CFU-pre-B) increased the average yield of CFU-pre-B per LTC-ICML by a factor of 3- to 20-fold; however, neither of these manipulations increased the frequency of LTC-ICML detected. Similarly, the use of S17 fibroblasts as feeders also did not improve the sensitivity of the LTC-ICML assay. On the other hand, approximately threefold more LTC-ICML could be detected when the initial phase of the assay (when the cells are maintained under myeloid LTC conditions) was shortened from 4 weeks to 1 week. In addition, this latter modification showed the existence in adult mouse marrow of a previously unrecognized, very early B220- stage of lymphoid development characterized by a relative resistance to both 5-fluorouracil and hydrocortisone. The discovery of such cells is of significant interest; however, their presence in adult mouse marrow in numbers comparable to those of both LTC-ICML and myeloid-restricted LTC-IC imposes severe restrictions on the use of the shorter LTC-ICML assay which offset its increased sensitivity. Interestingly, the present studies also show that both LTC-IC and LTC-ICML numbers are significantly better maintained (approximately twofold to sixfold) in myeloid LTC than are CRU. This differential maintenance of LTC-IC and CRU in vitro may be related to the fact that the detection of each of these two functionally defined progenitors involves the use of different ligand receptor systems. Alternatively, it is possible that at least some LTC- IC and CRU may represent developmentally distinct cell types. Taken together, these findings suggest a model of hematopoietic stem cell regulation in which retention of totipotentiality and maintenance of responsiveness to specific regulators may not be tightly linked.

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