ITS is normally supplied as a 100 X solution. Just add it at 1 X viz. 1:100 dilution in your media i.e. 1 ml of 100 X ITS in 100 ml media.

Thank you very much.

I would agree with Dr. Kapoor above. However, you bring up an interesting point. We have cloned from single cells using serial-diluted single cell clonogenic analysis (ring cloning does not work to generate populations of cells derived from a single cell), mesenchymal stem cells (contains telomerase enzyme - assessd by both WYSIWYG molecular analysis and antibody ELICA assay) and mesenchymal progenitor cells (absence of telomerase enzyme by antibody ELICA assay). The clones of mesenchymal progenitor cells utilize the ITS solution perfectly fine. However, ITS is harmful to clones of true mesenchymal stem cells - the transferrin and selenium kills the cells - we tested each component individually using dose response curves with our ELICA (DNA analysis portion of the) microassay. The insulin basically has no effect on stem cells, but it will accelerate the phenotypic expression of progenitor cells (which occurs similarly with IGF-I and IGF-II. IGF-I and IGF-II work at a lower physiological concentrations than insulin, but insulin is cheaper to use, i.e., 2 micrograms per ml medium). Therefore, if your cells are growing quite well in an ITS-based medium my suspicion is that you are growing, for the most part, mesenchymal progenitor cells. They have a defined life span of 8-10 population doublings in rodents and 50-70 population doublings in humans.

A second method to tell the two populations apart is that mesenchymal progenitor cells when they reach contact inhibition will die not matter what you do to the cultures (that is the reason why you need to replate the cells before they reach confluence). In contrast, contact inhibited mesenchymal stem cells will survive perfectly fine in the contact inhibited state as a single layer of cells as long as you feed them fresh culture medium when the color of the medium changes towards yellow (a bulid up of lactic acid in the medium).

A third method to tell the two populations apart is their freezing parameters. The optimal freezing conditions for mesenchymal progenitor cells are 10% v/v DMSO (ATCC DMSO is fine) in plating medium and flash freezing and storage in liquid nitrogen - recoveries should be in the 90-95% range. The optimum freezing condition for mesenchymal stem cells is 7.5% v/v of 99.99% pure DMSO (Allied Chemical Co.) in plating medium and slow freezing and storage at -70 degress centigrade +/- 5 degrees centigrade - recoveries should be 95-98%. The key is that -70 C will KILL progenitor cells and liquid nitrogen will KILL stem cells. Check Cell Biochem Biophys 40:1-80, 2004 for more particulars. The manuscript can be down-loaded from my publication list.

Thanks Mr Young for your answer and helpful explanation. I couldn’t agree more that many papers lack prior characterizations of the cells being investigated. The assertion that the so-called MSCs could be induced to ‘any’ cell types outside their committed lineage pattern is often made without taking into account that the cells taken from their residing location, particularly bone marrow, are truly heterogeneous.

By the way, I’m doing a preliminary optimization of components to be used in developing a serum-free medium (the concentration of ITS as follows; 10 ug/ml insulin; 5,5 ug/ml transferrin and 5 ng/ml selenium) for murine bone marrow cell culture. bFGF is added as mitogenic factor. The cells manage to attach 24 hours after primary isolation, yet retain the same round morphology and display slight proliferation.

I hope you don’t mind answering my questions in regards to your explanations. Could you please explain why transferrin and selenium toxicity are different between the two cell populations considering the true MSCs develop from their progenitors? I’d also like to know whether you culture MSCs and/or MSC progenitors in serum-free or serum-containing medium. There are several papers stating that ITS supplement is required in serum-free condition (normally with DMEM/F12) to replace some vital components in serum although in one paper, Jung S et al (2011) did isolation and expansion of human MSC without selenium, yet with apo-transferrin in their defined PPRF-msc6 medium. Do their toxicity (transferrin and selenium) apply the same way in both conditions?

Last but not least, I would also like to have your perspective about the key factors (perhaps the most influential) determining cell fate from the pluripotent epiblast-like cells to their transformation into the committed (unipotent) ones; is it the niche, genes or other factors that enable the event?

Sorry again for many asking and thanks in advance for your help.

Thanks Mr Young for your answer and helpful explanation. I couldn’t agree more that many papers lack prior characterizations of the cells being investigated. The assertion that the so-called MSCs could be induced to ‘any’ cell types outside their committed lineage pattern is often made without taking into account that the cells taken from their residing location, particularly bone marrow, are truly heterogeneous.

By the way, I’m doing a preliminary optimization of components to be used in developing a serum-free medium (the concentration of ITS as follows; 10 ug/ml insulin; 5,5 ug/ml transferrin and 5 ng/ml selenium) for murine bone marrow cell culture.

It has taken me 20 years to devise recombinant human (rh)-based serum-free medium for germ layer lineage stem cells, pluripotent stem cells and totipotent stem cells. Best of luck on your future endeavors.

In bone marrow from mouse, rat and human we have isolated monocytes, osteoclasts, osteo-progenitor cells, osteoblasts, osteocytes, endothelial cells, smooth muscle cells, hematopoietic cells, hematopoietic stem (actually progenitor) cells, mesodermal stem cells, ectodermal stem cells, endodermal stem cells, germ layer lineage stem cells, pluripotent stem cells and totipotent stem cells. So my question to you is which population(s) do you plan on culturing in your serum-free medium? If you insist on using transferrin and selenium you will KILL off the “stem” cells in the above list and only be propagating progenitor cells.

bFGF is added as mitogenic factor.

Please do NOT use bFGF as a mitogenic factor, while it will work for progenitor cells it has a different activity on stem cells. What the people that wrote the manuscripts failed to realize (they were culturing progenitor cells) is that bFGF has multiple functions with respect to stem cells. Yes, it is a slight mitogen, increasing cellular proliferation 2-3 times above control medium values in progenitor cells, ectodermal stem cells and endodermal stem cells. But that is its secondary function. Its primary function is to induce FIBROGENESIS in mesodermal stem cells, pluripotent stem cells, and totipotent stem cells. Now, this will not occur right away, but you will begin to see it crop up in your cultures after the second or third passage. Then your cultures will be “overgrown” with fibroblasts. Actually, you will have induced them yourself using bFGF. TGF-b has the same effects as bFGF over the same time frame on the same cell populations. The reason that the others did not recognize the fibrogenic inductive effects of bFGF (or TGB-b) is that genomically committed progenitor cells (and ectodermal stem cells and endodermal stem cells) will NOT react to an inductive factor outside their respective cell lineage. What I mean to say is that osteoblasts will stay as osteoblasts regardless of whether an inductive factor for fibroblasts is present in the medium or not.

If you want a mitogen, I would suggest using PDGF-BB (R&D Syst...

Thanks Mr Young for your comment. I'm planning to culture 'true' mesenchymal stem cells in serum-free condition. I'll consider taking out transferrin & selenium from the medium. Actually, I'm also thinking about choosing PDGF as mitogen as well, but many investigations claimed that bFGF and/or TGF-beta1 are crucial in 'MSC' propagation, their self-renewal, 'survival' over multiple passages and their involvement in some signaling pathways, such as PI3K-AKT-MDM2, Notch etc. Based on your experience, is PDGF-BB only already enough as mitogen for the above purposes? I also read an article about EGF (Tamama K et al, 2005) and it said that EGF could promote ex-vivo expansion of MSC without triggering their differentiation. I intend to add some plant extracts to enhance their proliferation though.

Hi Preeti Kapoor thanks for the information. So generally we use 1X ITS ?

I am working with cancerous cell lines can I grow them in 1X ITS without any FCS?

Thank you.

I would make a small (1%) correction to Dr. Kapoor's answer: 1: 100 means to add 1 ml ITS to 99 ml medium. This yield 1 part per 100 parts or 1:100 dilution. Adding 1 ml to 100 ml medium make 1: 101 dilution.

For ease of understanding the differences between telomerase negative mesenchymal progenitor stem cells which will form three cell types and telomerase positive mesenchymal stem cells which will form at least 37 separate mesodermal cell types, I have revised the name of the latter to telomerase positive mesodermal stem cells. We have finally published a synopsis of our work comparing telomerase positive stem cells to a telomerase negative mesenchymal progenitor stem cells. The data can be found in two papaers: Characterization … published in 2020 and Donor Selection … also published in2020. Both are available from ResearchGate for free download.

Small correction to Dr. Weiss' answer. 1:100 means 1 ml in 100 ml of total volume. An aqueous solution made with a substance with a specific gravity and solution characteristics very different from water, such as ethanol, will require putting 1 ml of ethanol and the filling to 100 ml with water. Adding 1 ml of ethanol to 99 ml of water does not equal 100 ml total volume. This is v/v measurement. Probably not relevant to the original question.