Mammary epithelial stem cells: transplantation and self-renewal analysis

Abstract

An entire mammary epithelial outgrowth, capable of full secretory differentiation, may be comprised of the progeny of a single cellular antecedent. This conclusion is based upon the maintenance of retroviral insertion sites within the somatic DNA of successive transplant generations derived from a single mammary fragment. In addition, dissociation of these clonal dominant glands and implantation of dispersed cells at limiting dilution demonstrated that both duct-limited and lobule-limited outgrowths were developed, as well as complete, fully differentiated glands. Thus, transplantation has revealed three distinct mammary epithelial progenitors in the mouse. Similar studies have extended this observation to rat mammary tissue. Recently, using cre-lox conditional activation of reporter genes, a new epithelial progenitor, specific for mammary secretory epithelium in postlactation females has been uncovered. In situ, these cells were shown to regenerate secretory lobules upon successive pregnancies. In transplant studies, they demonstrated the capacity for self-renewal and contributed to the new generation of all of the known epithelial cell types among mammary epithelium. In limiting dilution, the parity-induced progenitors were capable of engendering lobule-limited and duct-limited outgrowths in their entirety, but not completely developed glands. Serial transplant studies indicate that these progenitors have a significant but limited capacity for self-renewal.

Figure 1

Section from a serially transplanted alveolar outgrowth induced by MMTV. Hyperplastic alveolar outgrowths possess an indefinite replicative lifespan. Consequently, in contrast to normal growth senescent implants, small light cells, (SLC), and undifferentiated large light cells (ULLC) (see Fig. 2) are routinely present. bm, basement membrane; myo, myoepithelial cell; fibro, fibroblast. Bar represents 1 µm.

Figure 2

Another portion of the hyperplastic outgrowth shown in Fig. 1, illustrating location and positioning of ULLC. bm, basement membrane; fibro, fibroblast; mfg, milk‐fat globule. Bar represents 1 µm.

Figure 3

This diagram represents our current model for the ultimate mammary stem cell (SLC), which gives rise to lobular and ductal progenitors that subsequently differentiate into all types representative of mammary epithelium. Adapted from Chepko and Smith (1997).

Figure 4

Total DNA from serially transplanted clonal dominant epithelial populations of five successive generations was subjected to digestion with EcoR1 followed by Southern Blot analysis. The subsequent blot was probed with a ³²P‐labelled MMTV‐LTR‐specific probe. EcoR1 cuts within the genome of the MMTV, producing two host–viral junction fragments of each provirus insertion. In panel A, five specific host–viral restriction fragments (arrows) were found in the original outgrowth (lane 6), and in all of the succeeding generations. Transplant generations 2 through 5 are represented by the middle four lanes (2–5). The DNA in lane 1 is from the mammary tumour that arose in a fourthth generation outgrowth. In panel B, DNA from a fully developed R12 outgrowth (lane 7) is compared with DNA from a lobule‐limited outgrowth in the contralateral gland at parturition (lane 8). In both cases, all five MMTV‐host restriction fragments are detected.

Figure 5

Diagram of the cell fate potential of WAP‐Cre‐activated cells upon subsequent pregnancies or in transplants.