I am interested in looking at various aspects of patient specific neuronal lines from an iPSC differentiation angle. The iPSCs will be generated from EBV-lymphocytes of patients. Any tricks or caution before proceeding further and what is the time line to arrive at a neuronal lineage from EBV step. The EBV cells are available but iPSC and lineage specification has to be done.
Hi Kalpita.
We are very experienced in making human iPSCs from many different cell types... but we've been having no success making them from EBV-immortalized B-cells. We've tried the published methods with no luck from several different donors... there were a couple of papers in Blood 3-4 years ago describing methods to do this - Choi and Rajesh were the first authors of these 2 papers - I'm sure you've already found them. But we've only tried with the episomal plasmid sets and methods described in these papers (as well as the episomes + "generic" iPSC media conditions). No attempts with other reprogramming methods such as mRNA or Sendai virus. These might be worth a try??? My understanding is that lenti/retroviruses don't transduce these cells very well.
If you or anyone else has any thoughts or suggestions on how to get this to work I'd love to hear them!
If you are able to make the iPSCs, there are many good neural differentiation methods out there. Check out the work of Lorenz Studer.
Good luck!
Neuronal/Glial Differentiation: Primitive EBV cultures should be treated with RA (100 nM) from day 10 and SHH (100 ng/mL) from day 14. On day 25, neural progenitors will be differentiated on a laminin substrate in the differentiation medium consisting of neurobasal medium, N2 supplement, and cAMP (1 μM). For motoneuron differentiation, the iPSC will be adhered to laminin substrate and culture in the presence of a mixture of BDNF, glial cell-derived neurotrophic factor (GDNF), and IGF1 (10 ng/mL). For glia differentiation, iPSC will be expanded in suspension for another 2 months in a medium consisting of DMEM/F12, N1 supplement (100 ng/mL), and cAMP (1 μM), and for oligodendorcytes, T3 (60 ng/mL), platelet-derived growth factor-AA (PDGF-AA), insulin-like growth factor 1 (IGF1), and neurotrophin 3 (NT3), all at 10 ng/mL (6), will be added. The cell will then adhered to plastic (for astrocytes) or ornithine substrate (for oligodendrocytes) and cultured for 7 days before immunocytochemical analysis. motoneuron clusters willbe then plated onto the myocyte co-cultures, differentiation for 2 days in DMEM containing 2% FBS.
Hi Christopher,
Glad you shared your experience. I did read couple of papers and I realize that fibroblast gives way better result than B cells. Also we dont work on iPSC at all. we are going to take help from labs who work on it.
Hi Kalpita - once you are able to obtain iPSCs, if you are interested in receiving hands-on training in iPSC culture, our institution offers a training course in basic techniques for culturing human pluripotent stem cells: https://research.cchmc.org/stemcell/training. Good luck with everything.
Hi Christopher,
Since I am a finishing grad student, I don't have lot of time to invest in learning and setting up experiments but its a complementary work to my thesis work. i can only take up projects like these with an angle of neurogenetic diseases in my post doc in coming summer. Thanks for the encouragement.
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