Please find enclosed 2 articles, which follow certain protocols to isolate cancer stem cells (CSCs)from cell lines and the commentary from PNAS (2016) can give you additional ideas. Private companies are also selling kits to specific identify, characterize and enrich CSCs-Here is one link: https://www.rndsystems.com/products/magcellect-cd24-cd44-breast-cancer-stem-cell-isolation-kit_magh111

Which specific breast cancer cell line(s) you plan to work on?

Good luck and best wishes for your project!

Please find enclosed 2 articles, which follow certain protocols to isolate cancer stem cells (CSCs)from cell lines and the commentary from PNAS (2016) can give you additional ideas. Private companies are also selling kits to specific identify, characterize and enrich CSCs-Here is one link: https://www.rndsystems.com/products/magcellect-cd24-cd44-breast-cancer-stem-cell-isolation-kit_magh111

Which specific breast cancer cell line(s) you plan to work on?

Good luck and best wishes for your project!

FACS sorting helps.

The above listed papers all assume that using cell surface markers that can be enriched for CSCs select CSCs. Using 3 such markers, as did Kuperwasser's lab, we showed that this permits selection of a CSC enriched population that comprises 1% (magnetic bead selection) to 2% (flow cytometry selection) of breast tumor cells. Of this 1 - 2 %, only 1% were actually CSCs. Thus, 1% of 2% = 0.02% of the tumor were CSCs.  Besides, CSCS will differentiate without a niche to maintain them. This is provided in our Hybrid Spheroid Assay (patented). See our abstracts.

Hi, there

First, you must select appropriate cell line to isolate cancer stem cells. The total number of CSCs are so variable depending on cell line type. According to my experience, the number of CSCs is so trivial in SKOV3 cell line but you could isolate the large amount of CSCs from HT-29 cells line. The isolated cells from HT-29 are so resistant for EMT or MET. CSCs colony and spheriod are so typical in this cell line also. therefore, be careful to select more appropirate cell line.   

Second, you must monitor the phenotype shifting of isolated CSCs by MACS or FACS and define appropriate time for your experiment. 

Reza,

You ignore the problem of how one defines CSCs. The CDxx cell surface markers are invalid definitions, as noted above. You need a functional definition like tumor initiating cell (as I noted above using Kuperwasser's limiting dilution data) or my Hybrid Spheroid Assay. This is not the same as tumor spheres, because the latter do not estimate the number oif divisions done by the single CSC.

Ines,

I agree that cell surface markers, as in Al-Hajj enrich for CSCs, but they reported that as many as 11-35% of the tumor cells were selected by their first 2 markers (CD26- CD44+). Kuperwasser added ESA/EpCam/CD326 to the selection and reduced that fraction to 2%. However, her limiting dilution assay data showed that 100 of the "2% cells" yielded 60-62.5% of the mice growing a breast tumor in their "humanized" breast fat pads. Since these cells were injected as a suspension, subject to the Poisson distribution, that means that only 1 cell was the basis of what grew (63% got >= 1 CSC, 37% got no CSC). 1% of 2% = 2/10,000. Not a very useful enrichment in you want to learn about CSCs from the 2% population selected by the markers. This result is consistent with the 4/10,000 we got as the breast cancer CSC fraction using our functional assay, the Hybrid Spheroid Assay (patented). We presented these results in 2012 (In measuring the radiosensitivity of breast cancer stem cells (CSCs), cell surface biomarkers purporting to designate a CSC “phenotype” are an insufficient definition of CSCs, since > 98% are not CSCs. Christopher S. Lange, Talal Syed, Paul K. Valestra, Erica Lai, Anna Groysman, Shy’Ann Jie, Manuj Agarwal, Elliot Navo, Robert Rotman, Lisa Dresner, Bozidar Djordjevic. Radiat. Res. 58th Annual Meeting Program Book: pg 126-127, Abstract PS5-48, September 30 – October 3, 2012, San Juan, PR). 

Dear Christopher,

I will appreciated too if you could kindly send to me your publication mentioned above.

Christopher Lange I would be grateful if I can get the copy of the said article. It is not found on that page.

Mai Ibrahim Barakat , I. Velázquez Quesada , Christopher Lange and Reza Rahbarghazi those CSCs available through vendors for sales, how reliable are they, talking about CSCs enrichment/purity?