Distinct mammary stem cells orchestrate long-term homeostasis of adult mammary gland.

The murine mammary gland is sustained by distinct pools of stem cells that are limited in space and time, exhibiting both unipotency and bipotency. However, the specific identities of the bipotent and unipotent mammary stem cells remain unclear. In this study, we investigated spatial heterogeneity of the mammary gland at the single-cell transcriptional level. We found that mammary basal cells exhibited spatially distinct populations and characteristics, which can be further divided based on the expression of CD34 and CD200 markers. Notably, CD34^-CD200⁺ basal cells enriched at the nipple region demonstrated strong long-term self-renewal ability and possessed the highest stem cell frequency, while CD34⁺CD200^- basal cells enriched in the terminal end buds (TEBs) showed reduced stem cell potency. Through lineage tracing experiments based on their signature genes, we discovered that Bcl11b⁺ cells were enriched in the CD34^-CD200⁺ population and exhibited bipotency even in the postnatal mammary gland, with an increasing contribution to mammary epithelia observed during long-term tracing and after multiple rounds of pregnancies. Conversely, lineage tracing of Sema3a⁺ cells, enriched in the CD34⁺CD200^- population, predominantly revealed their unipotent nature and significant contribution during alveologenesis. Notably, the Bcl11b⁺ cells displayed a slow response to pregnancy but contributed to long-term mammary homeostasis, in contrast to the rapid response observed in Sema3a⁺ cells. In addition, Bcl11b progenies survived much better than Sema3a progenies during involution stage, thereby exhibiting increased coverage in the mammary gland after multiple rounds of pregnancies. Importantly, depletion of Bcl11b in Krt14⁺ mammary basal cells resulted in reduced bipotency of mammary stem cells and impaired their long-term contribution to the mammary gland. Overall, our study identifies distinct bipotent and unipotent populations of mammary basal cells with different dynamic properties that play critical roles in maintaining postnatal mammary homeostasis. These findings are crucial for advancing our understanding of breast health and breast cancer research.