The existence of mammary stem cells (MaSCs) has been postulated from evidence that the mammary gland can be regenerated by transplantation of epithelial fragments in mice. Interest in MaSCs has been further stimulated by their potential role in breast tumorigenesis. However, the identity and purification of MaSCs has proved elusive owing to the lack of defined markers. We isolated discrete populations of mouse mammary cells on the basis of cell-surface markers and identified a subpopulation (Lin-CD29hiCD24+) that is highly enriched for MaSCs by transplantation. Here we show that a single cell, marked with a LacZ transgene, can reconstitute a complete mammary gland in vivo. The transplanted cell contributed to both the luminal and myoepithelial lineages and generated functional lobuloalveolar units during pregnancy. The self-renewing capacity of these cells was demonstrated by serial transplantation of clonal outgrowths. In support of a potential role for MaSCs in breast cancer, the stem-cell-enriched subpopulation was expanded in premalignant mammary tissue from MMTV-wnt-1 mice and contained a higher number of MaSCs. Our data establish that single cells within the Lin-CD29hiCD24+ population are multipotent and self-renewing, properties that define them as MaSCs.

The dendritic cells (DC) of mouse spleen and thymus were examined for expression of CD4 and CD8. Provided care was taken to avoid selective extraction or selective depletion of DC subpopulations, three main types of DC were detected in mouse spleen: a major new population of CD4+8− DEC-205low CD11bhigh DC, together with the previously described CD4−8− DEC-205low CD11bhigh DC and CD4−8αα+ DEC-205high CD11blow DC. The CD4 on the surface of the CD4+ splenic DC subpopulation was produced by the DC themselves, and CD4 RNA transcripts were present. Likewise, the CD8α on the surface of the splenic CD8+ DC was shown to be a product of the DC themselves, in agreement with earlier evidence. All three spleen DC types would be considered as mature, based on expression of CD80, CD86, and CD40 as well as on T cell stimulating function. Mouse thymuses appeared to contain two DC types; both were DEC-205highCD11blow, but they differed in the level of CD8αα expression. However, as well as this authenticated marker expression, immunofluorescent staining was also found to reflect a series of artifacts, due to the autofluorescence of contaminating cells and due to pickup of CD4 and CD8αβ. By constructing mice chimeric for the hemopoietic lineages using mixtures of wild-type bone marrow with CD4null or CD8αnull bone marrow, a marked pickup by thymic DC of Ags derived from thymocytes was demonstrated.

The development of functionally specialized subtypes of dendritic cells (DCs) can be modeled through the culture of bone marrow with the ligand for the cytokine receptor Flt3. Such cultures produce DCs resembling spleen plasmacytoid DCs (pDCs), CD8(+) conventional DCs (cDCs) and CD8(-) cDCs. Here we isolated two sequential DC-committed precursor cells from such cultures: dividing 'pro-DCs', which gave rise to transitional 'pre-DCs' en route to differentiating into the three distinct DC subtypes (pDCs, CD8(+) cDCs and CD8(-) cDCs). We also isolated an in vivo equivalent of the DC-committed pro-DC precursor cell, which also gave rise to the three DC subtypes. Clonal analysis of the progeny of individual pro-DC precursors demonstrated that some pro-DC precursors gave rise to all three DC subtypes, some produced cDCs but not pDCs, and some were fully committed to a single DC subtype. Thus, commitment to particular DC subtypes begins mainly at this pro-DC stage.

Immediate precursors of the many subtypes of dendritic cells (DCs) remain obscure. Here we purified a splenic precursor population that produced all splenic CD8+ and CD8- conventional DCs (cDCs) but not plasmacytoid DCs or other lineages. This 'pre-cDC' population included cells 'precommitted' to form either CD8+ or CD8- cDCs. The pre-cDCs, which comprised 0.05% of splenocytes, expressed a CD11c(int) CD45RA(lo) CD43(int) SIRP-alpha(int) CD4- CD8- major histocompatibility complex class II-negative surface phenotype. The pre-cDCs were not monocytes. Monocytes generated few cDCs in steady-state recipient mice. However, when transferred into mice with an inflammatory milieu dependent on granulocyte-macrophage colony-stimulating factor, monocytes produced a distinct type of splenic DC. Thus, the inflammatory status of the host influences the developmental origin and type of DC present in lymphoid tissues.

Mice homozygous for an Ikaros null mutation display distinct defects in the development of fetal and adult lymphocytes. Fetal T lymphocytes, and fetal and adult B lymphocytes and their earliest progenitors are absent. Postnatally, hematopoietic stem cells give rise to thymocyte precursors that undergo aberrant differentiation into the CD4 lineage and clonal expansion. The lack of NK cells and some gamma delta T cell subsets and a large reduction in thymic dendritic APCs suggest that Ikaros is essential for establishing early branch points in the postnatal T cell pathway. The lymphoid defects detected in Ikaros null mice reveal critical molecular differences between fetal and postnatal hematopoietic progenitors that dictate their ability to give rise to T cells. These studies also establish Ikaros as a tumor suppressor gene acting during thymocyte differentiation. Phenotypic comparison of this null mutation with a severe dominant-negative Ikaros mutation identifies molecular redundancy in the postnatal hemolymphoid system.