To add:

It has been now 10 days since I have osteoinduced, I changed out half the media at Day 7 and have culture that is beginning to look like the image below. There is branching out of the fibroblast like cells, more of the other cell population and also some floating cells.

Dear Brianna,

I strongly recommend you to do the following:

Plate bone marrow in cell plate and leave it for 4 days; usually, I use alpha-MEM with 10% FBS and 1% P/S; under these circumstances, MSCs will adhere but not all other cells (like hematopoietic cells, blood cells, etc). It is important, at this stage, to leave MSCs in contact with these cells, in order to recreate a stem cell niche. After these 4 days, you will have a population of cells that are fibroblast-like cells. Wash your plate several times with cell medium (alpha-MEM alone), in order to remove non-adherent cells (3-4x – watch cells in microscope, in order to ensure that you have no more blood cells – in your pictures, it seems that you have other bone-marrow cells).

At this stage, if you perform a phenotype analysis (e.g., FACS) you should have the following markers: CD117, CD29, CD105, CD73, but not CD45 or CD14 (others may be used); Change your cell medium twice a week; leave cells till 90% confluence, and plate your cells for the assays; here, I strongly recommend the following osteogenic-medium: α-MEM plus 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin, and 2.5 μg/ml amphotericin, supplemented with 50 μg/ml ascorbic acid, 10 mM β-glycerophosphate, and 10 nM dexamethasone.

In order to confirm osteogenic differentiation, I usually measure Runx-2, Osterix transcription factors, type I Collagen, osteocalcin, bone nodule formation, ALP activity, among others.

Let me know if this was helpful. Take a look at one of our papers (e.g., PMID: 25169056). Here, we use human MSCs, but these ads work well in rodents.

Regards, good luck

Hi Jose,

Thank you for the suggestions. I will try that next time I isolate. Is there a reason you prefer aMEM over LG DMEM? I do confirm osteogenic differentiation by both staining with ALP, and ARed and running RT-qPCR for Bglap and Alpl on collected RNA. The first two times I isolated I did not see as much as a heterogenous population, but now I am for some reason. Also, I have been having more trouble now with getting good quality RNA to use for subsequent analysis. 

Thank you for the suggestion. 

Hi Brianna,

I'm also culturing mouse BM-MSCs, and I'm totally new in this field because I had used C3H10T1/2 for osteogenic differentiation before. Would you mind sharing the protocol to isolate BM-MSCs from mice with me? 

Thank you in advance.

P.S: Thanks Jose for the differentiation protocol :)

Hi Mai,

To isolate BM-MSCs we first begin by cervical dislocation as we believe CO2 euthanasia to impact our MSCs. We then aseptically take out the femur and tibia from the mouse and thoroughly clean the bones, keeping them attached at the knee joint. All of this is done in a surgery room. After this step we transfer to the hood where we flush out the bone marrow using 5 mL of LG-DMEM 10% FBS 1%P/S/ bone (20 mL/ mouse if doing left and right bones). The marrow will need to be pipetted after flushing as it comes out in clumps sometimes. Then we filter through a 70 uM filter into a new conical, spin down at 1500 rpm for 5 minutes, take off supernatant. At this step you should see a sizeable portion of red marrow on the bottom of the conical. Then we re-suspend in 10 mL-12 mL LG-DMEM and place in a 10 cm plate to grow up.

I hope this helps. Good luck!

Thank you so much for the protocol, Brianna. I'll try it for my experiments.

Good luck and hope your cell culture will work well!