When my lab isolated and characterized cells from bone marrow, we found about 30 different cell types, i.e., totipotent stem cells, pluripotent stem cells, germ layer lineage stem cells, ectodermal stem cells, mesodermal stem cells, endodermal stem cells, hematopoietic stem cells, hematopoietic progenitor cells, mesenchymal stem cells (Caplan’s), stromal vascular cells, endothelial progenitor cells, endothelial cells, endosteal cells, osteogenic progenitor cells, osteoblasts, chondrogenic progenitor cells, chondroblasts, adipoblasts, unilocular adipocytes, etc. Which cell population are you using for your studies?
Hi Dr. Young,
I am using MSCs from rat bone marrow extracted from the hind limb. Unfortunately, we didn't have FACS or immunofluorescence, we were not able to sort/isolate MSCs accurately. So, basically, we isolate MSCs from suspended cells by adherent selection. Hypothetically, any cells that adhere and grow on a substrate will be considered as MSCs.
For this, I may have overlooked some progenitor, fibroblast, and even osteoblasts which may adhere and grow together with MSCs as well.
Long.
Dear Long,
Basically, any tissue-committed progenitor cell will adhere to culture dish. These cells make their own matrix, so they are not dependent on you to provide the matrix for them. So you might want to expand the number of your potential cell types you are isolating and plating. By the way, you do not need immunofluorescence for identification. We use phase contrast and bright field microscopy with our ELICA procedure. Using this assay you can quantify, visualize, and standardize to DNA content, all within the same well of a 96-well plate. Your specificity depends on the primary antibodies that you use for phenotypic expression markers. We routinely get our antibodies from the Developmental Studies Hybridoma Bank at the University of Iowa. I am attaching two papers that will help with the protocols.
You can also use differential cryopreservation to separate progenitor cells from stem cells. We use 7.5% v/v ultra-pure DMSO as the cryoprotectant. Progenitor cells and differentiated cells prefer flash freezing and storage in liquid nitrogen. You will get about a 90% recovery of these cells and kill off the majority of the stem cells. Conversely, stem cells prefer slow freezing and storage at minus -70C to minus -80C. You will get about 95% viability, while killing off the majority of the progenitor and differentiated cells. I will attach our cryopreservation paper for you.
One culture method to distinguish progenitor cells from stem cells is to incubate the cells with 2 micrograms/ml insulin in addition to what is already in your medium. Insulin is a progression agent. Meaning it accelerates phenotypic expression in progenitor cells but has no effect on stem cells. Lineage-committed progenitor cells should express their differentiation program within 1-4 weeks in culture.
Precursor cells (stem and progenitor) have different states of activity, i.e., quiescence, proliferation, and phenotypic expression. And these phases do not overlap. So, if you force phenotypic expression in progenitor cells with insulin, the progenitor cells usually will not proliferate (however, stem cells might continue proliferating). This is important if you are dealing with progenitor cells that conform to Hayflick's limit, and after a set number of doublings will senesce and die. It is difficult to perform experiments if the cells are at the upper range of Hayflick’s limit.
If you use a serum that is not heat inactivated it probably contains inductive factor(s) that will cause induced lineage-commitment in lineage-uncommitted stem cells. This induced phenotype usually does not show up until after you passage your cells. You can force the issue, again, by treating cultures with serum and insulin, same concentration as above. In this respect you will see phenotypic change within 1-4 weeks.
I would suggest that you screen your cultures visually to try and discern phenotypes. Then run ELICA for confirmation of differentiated cells. Next, incubate your cultures with insulin to see which progenitor cells are present and run ELICA fort conformation. Then incubate cultures with dexamethasone at 10^-6, 10^-7, 10^-8, 10^-9, and 10^-10 M concentration in your culture medium + insulin. You could use purified recombinant proteins for induction but they are way too expensive. Dexamethasone, is relative inexpensive and is a general inductive agent which will induce multiple different cell types in stem cells. If you get only mesodermal cell types the cells are mesodermal stem cells; ectodermal cell types, then ectodermal stem cells; and endodermal cell types, then endodermal cell types. If you see multiple types of two or more germ layer lineages you are probably looking at pluripotent stem cells or totipotent stem cells.
If you need anything all you need do is ask. Good luck.
Hi Henry, we are growing human mesenchymal 'stem' adherent stromal cells (Caplan).
We regard them as being a heterogeneous mix rather thna a pure cell type or differentiation stage.
Regards
Jim F
Dear Jim F,
Thank you. You understand what you are dealing with. Would you mind sending.me a link to your publications?
Thank you
This may not be an answer to your question about the nature of the cells but it is an important point in development of cell cultuer media using human albumin as a constituent and carrier molecule. I understand that American companies that factionate human plasma use a mix of tryptophan and octanoic acid to stabilise albumin during the 60C heat pasteurisation step of manufacture. CSL Behring in Australia uses just octanoic acid but at double the concentration and this results in a much higher level of this excipient that adversely affects mesenchymal cells in culture. The link below should take you to the paper.
https://www.hindawi.com/journals/sci/2015/192576/