Hi! I am producing forebrain brain organoids using the Lancaster protocol (Lancaster, 2014, Nature protocols). I successfully produced brain organoids with forebrain identity the first time I tried the protocol (expression of PAX6, presence of subventricular zones TBR2+, presence of cortical layers, etc...). However, when I tried to produce organoids with more iPSCs lines, most organoid batches lack forebrain identity (see attached file, I.E. no PAX6 expression in ventricular zones, no TBR2+ subventricular zones, no expression of cortical marker like TBR1). Morphologically, the organoids seem perfectly normal and meet all the quality-control requirements described by your paper (i.e. bright radial neuroepithelium following neural induction, formation of neuroepithelial buds and ventricular zones etc…), but they don't have a forebrain identity.

I was wondering if someone experimented similar issues and identified potential factors contributing to this problem.

I have rigorously checked the quality and the pluripotency of iPSCSs, their karyotype and the presence of mycoplasma before performing the experiments. I used the iPSCs between passage 15 and 20. I also performed rigorous troubleshooting (i.e. change the lot of products, add patterning molecules to the media etc…). 

Thanks a lot and have a nice day!