We have tested the hypothesis that non-cellular extracts from normal mammary tissue can reprogram non-mammary and cancer cell in the epithelium-divested mammary fat pad in vivo. We have published evidence that mammary extracellular matrix (mECM) can redirect testicular and embryonic stem cells from the mouse to mammary epithelial cell fates in vivo. Similar preparations from other organ systems were found negative for this activity. Mammary epithelial cell lineage analysis was performed via the Lyon's Hypothesis, which states that one of the two X chromosomes is silenced in each female cell during embryogenesis. Implants from a single mammary gland of an H253 transgenic female mouse heterozygous for a lacZ-labeled X chromosome were analyzed at various time points following transplantation into the epithelium-cleared mammary fat pads of immune-compromised mice. Our results show lacZ-marked X chromosome, demonstrated by X-gal staining, are confined to a single epithelial clone that gave rise to the cap cells of growing terminal end bud (TEB) in the expanding mammary outgrowths and also the basal myoepithelial cells of the mammary ducts. Luminal cells in these ducts were uniformly negative for lacZ expression indicating that they were derived from cellular precursors that contained a silenced lac-Z marked X chromosome. This observation confirms the earlier work of Williams and Daniel, who concluded that the terminal end bud-associated cap cells of growing mammary ducts were the precursors of the basal (myoepithelial cells) of mature mammary ducts. The results of our study indicates that a single clone (marked by an X chromosome carrying a lacZ gene) is responsible for the appearance of cap cells in the growing terminal end bud (TEB) and in turn give rise to the myoepithelial lineage of the mammary ductal system. This same clone is shown among the body cells of the marked TEBs as a group of closely associated cells also marked by lacZ. We conclude that this clone found in the body of the TEB gives rise to the specialized cap cells and subsequently to the myoepithelial lineage of the subtending mammary ducts. This is a slightly different interpretation presented by Williams and Daniel in 1983, who postulated that the cap cells themselves were multipotent and gave rise to both luminal and myoepithelial progeny in the growing ductal system. It is clear from our work that none of the luminal cells in the subtending ducts bear the un-silenced lacZ-marked X chromosome. We conclude that the luminal cells arise from a different set of progenitors. These conclusions mesh very well with our earlier discovery that lobule-only and duct-only progenitors are present amongst nulliparous mammary epithelium during transplantation of limiting dilutions of epithelial cells from primary cultures of post pubertal females. Confirmation of separate ductal and lobule-limited precursors was confirmed during serial passages of mammary tissue into subsequently impregnated hosts where ductal and lobular development were shown to be lost independently from one another. Telomeres are nucleoprotein structures comprised of simple repeat sequences located at the ends of chromosomes. They shorten upon cell division and are restored by a specialized ribonucleic-protein complex called telomerase (TER), which is made up of essential protein and RNA components. To determine if this activity was essential to mouse mammary gland growth in vivo, we serially transplanted mammary fragments from TER +/-, and TER -/- mammary tissues. Homozygous TER-/- and heterozygous TER+/-, where telomerase RNA template had been deleted, were examined over 6 transplant generations (12wk.) to determine if the rate of reaching growth senescence was adversely affected by the absence of telomerase template. Individual implants from both homozygous and heterozygous TER null outgrowth showed growth senescence beginning at the second transplant generation. This result suggests that either mammary epithelial stem cells must maintain their telomere length in order to self-renew, or that the absence or reduction of telomerase template results in more frequent death/extinction of stem cells during symmetric self-renewing divisions. A third possibility results from the deletion (or reduction) of stem cell niche maintenance signals necessary to stem cell renewal. Examination of TER-/-, TER+/- and wild type (TER+/+) senescent outgrowths revealed the absence of estrogen receptor-positive (ER+) epithelium although progesterone receptor-positive (PR+) cells were abundant. It was shown earlier that ER-sensing epithelial cells are indispensable for post-pubescent mammary ductal growth. Despite their inability to establish mammary growth in vivo, TER+/- cells were able to re-direct neural stem cells to mammary cell fates. Mammotropic hormones and growth factors play a very important role in mammary growth and differentiation. Here, hormones including Estrogen, Progesterone, Prolactin, their cognate receptors, and the growth factor Amphiregulin, are tested with respect to their roles in signaling non-mammary cells to redirect to mammary epithelial cell fate(s). This was done in the context of glandular regeneration in pubertal athymic female mice. Our previous studies demonstrated that mammary stem cell niches are recapitulated during gland regeneration in vivo. During this process, cells of non-mammary origin cooperate with mammary epithelial cells to form mammary stem cell niches and thus respond to normal developmental signals. In all cases tested, hormone signaling is dispensable for non-mammary cells to undertake mammary epithelial cell fate(s), proliferate, and contribute progeny to chimeric mammary outgrowths. Importantly, redirected non-mammary cell progeny, regardless of their source, have the ability to self-renew and contribute offspring to secondary mammary outgrowths derived from transplanted chimeric mammary fragments, thus suggesting that some of these cells are capable of mammary stem cell/progenitor functions. Currently, Amphiregulin, a growth factor required for ductal elongation in the mammary gland, was found to be non-essential for reprogramming non-mammary cells to mammary cell fate(s), in vivo. In a recent paper, it was reported that polyploid cells are frequent in lactating mammary tissues. This phenomenon was observed in mammary tissue sampled from five separate mammalian species. According to that report, these binucleated cells occur late in pregnancy and early in lactation. Unfortunately, this paper did not mention a number of earlier observations and findings that remain pertinent to this day. In these classical experiments, the authors demonstrated in vivo that DNA synthesis continued without commensurate cell division during late pregnancy and lactation, and that this DNA synthesis was imperative for functional differentiation of the mammary epithelium. Older studies, from our laboratory and others, showed that DNA synthesis was indispensable to the induction of milk protein production in explant cultures of mammary tissue from unprimed, nulliparous mice. This dependence on DNA synthesis in mammary explant cultures stimulated by lactogenic hormones was found to be dispensable following a single pregnancy. The absolute requirement for DNA synthesis in nulliparous mouse mammary explants stimulated to synthesize milk protein in vitro has remained unexplained, as has the need for DNA synthesis prior to the onset of lactation. From a historical perspective, it is more likely that binuclear secretory cells in the lactating mammary gland are a consequence of the DNA synthesis requirement for lactational differentiation, rather than an essential activity essential to supporting lactation.
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