Introduction: Utilizing single-cell cloning of the COMMA-D cell line engineered to express β-galactosidase (CDβ) cell line, which exhibits normal in vivo morphogenesis, distinct multipotent, ductal-limited, alveolar-limited and luminal-restricted progenitors, have been isolated and characterized.
Methods: A single-cell suspension of CDβ cells was stained using Hoechst dye 33342, followed by analysis and sorting. Cells that effluxed the dye appeared on the left side of a FACS analysis panel and were referred to as side population (SP) cells. Cells that retained the dye appeared on the right side and were referred to as non-SP (NSP) cells. Cells from both SP and NSP regions were sorted and analyzed for outgrowth potential. Additionally, individual clones were derived from single cells sorted from each region.
Results: There was no difference in the outgrowth potential of the SP vs. NSP cells when 5,000 cells per fat pad were transplanted. However, individual clones derived from single cells sorted from either SP or NSP regions had varying growth potential. A total of nine clones were identified, four of which possessed in vivo mammary outgrowth potential and five of which lacked in vivo outgrowth potential. Two of the clones formed mammary lobuloalveolar structures that contained both ducts and alveoli and were termed multipotent. Two of the clones generated either ductal-only or alveolar-only structures and were referred to as ductal-limited or alveolar-limited progenitor clones, respectively. The ability to expand the clones in vitro allowed for the characterization of their unique molecular phenotypes. Among the mammary-specific markers tested, high cytokeratin 5 (CK5) expression was the only marker that correlated with the clones' outgrowth potential. Among the clones that did not show any in vivo outgrowth potential when transplanted alone, one clone showed in vivo growth and incorporated into the mammary lumen when mixed with normal mammary epithelial cells. This clone also showed the highest in vitro expression of CK8 and Elf5and may represent a luminal-restricted progenitor clone. In addition, six "biclones," each made from an SP cell plus an NSP cell, were analyzed. Of these six, three exhibited lobuloalveolar growth.
Conclusions: Distinct immortalized mammary progenitors have been isolated and characterized. Importantly, the results of this study provide further evidence for the existence of distinct ductal and alveolar mammary progenitors.