Hi Emre,

Like Daniel sed, your sorted cancer stem like cells are just differenciating. You need to create the optimal conditions for its maintenance and expantion in vitro.

check our paper to see how we did to maintain and amplify sorted stem cells.

https://www.researchgate.net/publication/228068563_Bronchioloalveolar_stem_cells_from_the_lamb_lungs_of_can_differentiate_into_Clara_cells_or_alveolar_type_II_cells

good luck!

Article Bronchioloalveolar stem cells from the lamb lungs of can dif...

Hi Emre,

The good news is that you appear to be isolating the correct cells!!  CSC will always differentiate as they expand in culture.  It something that I look for as a quality control check for conformation that I am getting real CSC.  SO, you need to culture them as spheres in low attachment plates.  Use 6 well dishes (Nunc Sphera is my favorite brand) and seed 50,000 cells per well into DMEM/F12 blend basal medis supplemented with 20ng/ml EGF and 20ng/ml bFGF.  Those are the only 2 supplements you need to add (some kind of antibiotic is needed after sorting!).  The CSC will start to form tight spheres in a few days.  You don't want to let the spheres get too big, so you can passage them ever 4-5 days, using trypsin to break them back into single cells and reseed new wells.  EGF and bFGF are not stable in culture so you have to add new every day.  There are hundreds of papers in PubMed that describe this process it has been used for the last 30 years.  It is probably the only way to expand CSC while maintaing their "stemness".

Feel free to contact me if you have further questions,

Steve

Dear Emre,

as I googled CD133+CD15+, the following paper came up:

http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0005498

Citing conclusions: "The finding that a significant fraction of clonogenic neural stem cells lack the established markers CD133 and CD15, and that some of these cells may be dormant or slow-cycling, has implications for approaches to identify and isolate neural stem cells and brain cancer stem cells. Our data also suggest the possibility that CD133 may be specifically down-regulated during G0/G1, and this should be considered when this marker is used to identify and isolate other tissue and cancer stem cells."

Could this also be your case?

Zeynep brings up a good point, Emre what kind of cancer are you working with, and are these cells from an established cell line or fresh tumor tissue?

Steve

Hi,

I am sorting CD133+ cells from melanoma by FACS Aria III and in my experience when i have cultured these cells as spheroid in stem cell specific medium then the expression of CD133 reduce drastically in comparison when i have cultured as monalyer in DMEM containing 10% FBS. 

Hello

I agree with researchers at several points

1- that you appear to be isolating the correct cells

2 - must define for us the type of cancer stem cells, which are dealing with so that we can absorb the decrease in the expression of the cells , which probably tha cells differentiated into other types of cancer cells , is well known the cancer cells have phenotypic diversity , Which may be due the decrease expressing of your cells

3- Consider in components of medium you use or receptors for growth and chemotactic factors , involved in cancer stem cells maintenance or differentiation .

All these points will help you to understanding the reason of the decrease expression your cells .

Best wishes

Firstly, thanks for the all of your answers.I will culture them as a sphere. I am waiting growing of my cells. I study with established cancer cell lines. Not fresh tumor tissue. I will be careful about all of your suggestions.

The biomarkers that you are using may enrich for cancer stem cells (CSCs) but the selected biomarker based "phenotype" almost certainly is a mixed population in which the CSCs are a very small fraction. The clearest evidence for this is that the sorted population is a relatively high fraction of the total tumor population (for fresh from the patient samples). For breast cancer the flow sorted or magnetic bead sorted CD26-CD44+CD326+(ESA/EPCAM) cells are only 2 or 1% of the tumor (respectively), but in limited dilution assays of these cells directly into the NOD/SCID mouse "humanized" breast fat pad, 100 such cells yield 6/10 or 5/8 (60-62.5%) of the animals get the tumor (see Kuperwasser lab papers). Since a single CSC will produce a tumor (e.g., Hewitt & Wilson 1958 mouse leukemia study) and a Poisson distribution calculation yields for an average of 1 CSC injected, 37% of animals get no CSC and hence no tumor, and thus 63% have 1 or more CSCs and get the tumor, therefore the fraction of original tumor cells that are CSCs is 1-2% times 1/100 = 1-2x10^-4 as the CSC fraction in the tumor. This agrees with the 4x10^-4 CSC fraction measured without enrichment in the Hybrid Spheroid Assay (pat. pending).  For cervical cancer we get 0.5% as the functional CSC fraction. In established cell lines, these markers are almost meaningless except as a means of relative enrichment.

See: 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. 

See also papers by Leoni Kunz-Schugart (Technische Universitat Dresden) who reported in the same meeting that the CD133 supposed marker for colon cancer stem cells taken fresh from patient samples disappeared very quickly in culture and then came back again with no stable expression.

I very much doubt that the bulk of the cells of any established cell line meet the functional requirements of McCulloch & Till (1962-4) for a stem cell, i.e., unlimited proliferation, ability to differentiate, and ability to make more of self (i.e., more stem cells). Cell lines have immortal cells (express telomerase), but these are not necessarily stem cells. Note that the majority of CSC descendants are amplifying transit cells with limited proliferation future.