In a first step, myeloma cells will be isolated using magnetic beads. Thereafter, we would like to isolate endothelial cells from the negative fraction.
Leider nein, wir benutzen ausschließlich immortalisierte humane Zelllinien - natrülich mit allen Nachteilen!
Herzliche Grüße,
Reinhard.
I have attempted to culture the so-called late-outgrowth endothelial progenitor cells (EPCs) successfully from mouse bone marrow, and they should equally work for human bone marrow. These cells are effectively endothelial cells (look at Nick Mill's paper that compares EPCs to HUVECs: http://onlinelibrary.wiley.com/doi/10.1002/stem.1280/abstract)
Here is the detailed protocol on how to do it: http://www.ncbi.nlm.nih.gov/pubmed/18770637
It works perfectly and I have been getting 100% success rate with 2 attempts. You just need to be mindful of other mesenchymal-like colony, and only picks the one that is EC-like (i.e. cobblestone-shaped cells in a circular colony) as soon as you see them in an appropriate size.
The statement in Olga Tura's paper that EPC's may not be found in human bone marrow (but instead in human circulation may derive from vasculature) was a reflection of our findings, and designed to stimulate an alternative hypothesis, for further investigation. While Ka Hou Lao may have cultivated EPC (how are these validated by Ka?) from mouse bone marrow, I will be interested to know (as principal investigator on our study) when they successfully cultivate them from human bone marrow.
Good point George. The ECFC (alternative name for late outgrowth EPC) from mouse bone marrow were validated by the usual criteria such as EC markers (CD31, CD144, VEGFR2) and excluded the hematopoietic markers (such as CD14 and CD144) by flow cytometry, but I have not validated them further with in vitro matrigel and in vivo Martrigel plug as that work was not carried further since it was not the focus of my project. ECFCs were indeed said to be found in human bone marrow in a JCI paper published back in 2000 by Lin et al. where they found these cells from a bone marrow donor was present in the recipient circulation (they termed it as EOCs, probably identical to ECFCs from their identifications). But whether that is conclusive enough is a question as the characterisation back then was not as stringent as now.
For OP, it's a method worth trying to obtain ECs from bone marrow, but others might have better suggestions in isolating ECs?
I think a functional assay trumps a phenotypic assay since as far as I know there has not yet been a widely accepted definitive unique phenotype agreed for EOC/EPC (and numerous contentious phenotypes have been proposed), and ultimately a functional assay would still be required for acceptance that any isolated cells are indeed endothelial progenitors. We were pleased with the functional assay we devised for human EPC (can be found at Barclay et al., Stem Cell Research & Therapy 2012, 3:23 - open access at http://stemcellres.com/content/3/4/23 or on Research Gate ) but other less complex assays are available.
http://stemcellres.com/content/3/4/23
Article Systematic assessment in an animal model of the angiogenic p...
I haven't isolated ECs from bone marrow, but as suggested, the CD31 coated dynabeads may work. If contamination with other cells types is a concern and that the presence of ECFCs is not an issue, then you can cultivate the isolated cells and enrich the ECs population with the CD31 (or another surface EC marker) coated dynabeads again at passage 1 to obtain a purer population.
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