Dear Pham,

Have you characterized the MSCs from both sources. In both the cases you get the progenitor cells first and later by growing them in specified conditions you can get MSCs. As for as your doubt, size of the cells doesn't matter much and what matters is its multipotency. MSCs from younger sources will give you more stable and long lasting MSCs provided when you maintain them proper. So first characterize and use MSCs from younger sources for better results..all the best       

Hi,

 I do not have any experience with rat MSCs, however our experience with human MSCs, show more stem/progenitor like properties in MSCs derived from infants compared to adults. Which I guess you can extrapolate to your rat MSCs.

 Hope that helps.

hello Long,

Definitely u need to characterize cells and size is of least significance in this case. But if you get MSCs from young animals, you will get a long culture with more proliferations and less chromosomal aberrations, which are accumulated over time. Technically if you compare, then quanttatively u will get more stemness in young MSCs as I have done the same in case of human samples. So if u want to consider the differences then u can take these things into account. If your work is not dependent on these differences then you can assume zero interindiviual differences in stem cells. Hope it helps.!

Old MSCs exhibited decreased proliferation and differentiation potential. Dubbling time and differentiation potential is better in MSCs isolated from young rats.

http://m.tej.sagepub.com/content/2/1/2011_741213.full

http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0045052

It is abvious younger rat will have more stemless than old one but very important point is, from where it is harvested which makes big change. 

Hello

There are many studies that provided evidence of that there are age-related changes in MSCs derived from the bone marrow of old rodents with regard to plasticity and basic stem cell biology.

see the article in attachment

Good Luck 

Thank you a lot for all the answers which I found of much help for me. I think my question has been answered now. 

Dear Long Pham,

Try to find information's in the following articles:

1. Asumda FZ, Chase PB. Age-related changes in rat bone-marrow mesenchymal stem cell plasticity. BMC Cell Biol. 2012; 12:44.

2. Zheng H, Martin JA, Duwayri Y, Falcon G, Buckwalter JA. Impact of aging on rat bone marrow-derived stem cell chondrogenesis. J. Gerontol. Biol. Sci. Med. Sci. 2007; 62(2):136-48.

3. Zhang DZ, Gai LY, Liu HW. Differences between adipose-derived stem cells and mesenchymal stem cells in differentiation into cardiomyocytes. Shen Li Xue Bao 2008; 60(3).341-7.

Greetings from Serbia,

Jasmin

Dear

MSC also getting aging.

Morphology in culture may dependent on cell seeding condition.

Difference of capability regarding stemness between young and old one seems to be one of main reasons.

Hope it helps you 

Dear Long,

You need to characterize cells to know what you have. There are both multipotent mesenchymal progenitor cells and multipotent mesenchymal stem cells in tissues. The progenitor cells have a defined 8-10 cell proliferation limit and have SH2, SH3, &SH4 cell surface markers. The mesenchymal stem cells have unlimited proliferation potential and have Thy-1 cell surface marker. Additional points you need to consider are as follows. As an animal increases in age the ECM gets more dense trapping the cells within their connective tissue niches. Young animals can be digested with collagenase in 15 minutes, in contrast older animals need a longer digestion time to extract similar numbers of cells. Then the rule of thumb is that progenitor cells decrease with age of the animal due to Hayflick's limit, whereas stem cell numbers stay constant due to unlimited proliferation potential.

Thank you so much for enlightening me with your answers. I appreciate your kind supports. I have another typical question to ask:

I am trying to extract MSCs from rat bone marrow (yellow marrow) using substrate adhesion method. The principle of the method is that MSCs will be stick on to the culture dish surface while other cells (red blood and hematopoeitic) are floating. Just by changing media and washing with Phosphate Buffer Saline (PBS), the cellular mixture will be purified with more MSCs.

The method is simple and seems to me the most common way to extract MSCs. However, I have some doubt about the homogeneity of the MSCs. Like you said, there are progenitor and stem cells coexist, and the progenitor may also stick to the surface. Thus, MSCs will never be 100% homogenous using this method, right? Or do we have any other method to purify MSCs more?

Thank you. Look forward to your answers.

Dear Dr Long,

You can set up a "panning" method where you first bind antibodies to MSCs to the surface of a plate. Then add a suspension of bone marrow, allow to incubate, rinse off suspension with buffer, then knock off attached cells with 2.0M NaCl in buffer. That will give you the most purified population of cells at a fraction of the cost of magenetic bead separation or FACS. This technique also allows you to reuse the "panning" culture plate by "rejuvenating" the surface of these binding by removing the 2M salt solution with 5-10 washings with buffer. You can store plate, covered with buffer, at 4 degrees centigrade between uses. 

Hi Dr. Henry,

Thanks again for your answer. 

I wonder how long for each step you mentioned earlier. For example, how long I incubate the bone marrow to have MSCs bind to antibodies?

When knocking off attached cells with salt solution, how much quantity (mL) you use? Do you have to incubate in this stage?

Please go to PubMed and search for "Wiberg M". You'll find two or more papers on this matter. Prof Mikael Wiberg is a Hand Surgeon and anatomist in Umea University, Sweden

Dear Long Pham,

Usual incubation time varies between 30-60 minutes dependent on conditions, i.e., temperature (at 4C run for 60 min, at ambient temp run at 30 min), solutions (DPSB run for 60 min, optimum binding solution run for 30 min), etc. Actually, I need to know exactly what you intend to do before I can help to design a system for you.

With respect to knocking off cells with salt solution, it depends on the surface of the vessel you will be using. For instance, if you are using a Miltenyi column or 35 mm culture dish, then 0.5 ml is sufficient; if using T25 flask then 1.0 ml is sufficient; if using T-75 flask then 5.0 ml is sufficient. You would need to "incubate" salt solution on cells for 5 min.