Objective  To isolate and culture breast cancer stem cells (BCSCs) from breast cancer tissues after different cycles of neoadjuvant chemotherapy

and investigate their proliferation and differentiation biological characteristics in vitro in order to obtain the impact of neoadjuvant chemotherapy on the isolation and culture of BCSCs.    Methods  Breast tissue samples from 20 patients receiving TAC neoadjuvant chem

otherapy in our department from October 2009 to April 2010 were collected. There were 5 cases with 1 cycle of chemotherapy

6 cases with 2 cycles

7 cases with 3 cycles

and 2 cases with 4 cycles. The tissue samples (1 cm×1 cm×1 cm) were obtained through operation and cut into pieces for culture. The suspended cells were cultured in serum-free media (SFM) supplemented with growth factors. Cells grown in these conditions as nonadherent spherical clusters of cells (usually named “mammospheres”

MSs) were enriched in BCSCs. The self-renewal capacity of mammospheres-derived cells (MSDCs) was evaluated by colony formation assay. Conversely

the differentiation of MSDCs was induced by culturing them in media supplemented with 5% fetal bovine serum without growth factors. Expressions of epithelial (ESA) and Nestin in MSDCs was detected by immunocytochemistry. ALDH1

+

cells of CMSDCs were detected by flow cytometry (FCM).    Results  The 5 specimens with 1 cycle of chemotherapy cultured in SFM had no MS formation

but the 3 of 6 specimens with 2 cycles formed MSs in the media described above. Interestingly

MSs were directly isolated from the 5 of 9 specimens with more than 3 cycles of chemotherapy

but the remaining 4 cases obtained only a small amount of necrotic cells and tissue fragments. MSDCs adhered and differentiated in media under differentiating conditions

and which contains ALDH1

+

cells.    Conclusion  Isolation of BCSCs from breast cancer tissues after adjuvant chemotherapy should select samples after 2 cycles of chemotherapy. BCSCs are enriched by new adjuvant chemotherapy. ALDH1

+

BCSCs distribute more widely in breast cancer tissues than those of the CD44

+

CD24

-

ones.