Comparison of human pluripotent stem cell differentiation protocols to generate neuroblastoma tumors.

Neuroblastoma is the most common extra cranial solid tumor derived from sympathoadrenal (SA) cells and characterized as either adrenergic or mesenchymal. Here, we compared four different protocols to differentiate induced pluripotent stem cells (iPSC) toward SA cells and intermediate cell states (neuromesodermal progenitors [NMP], trunk neural crest cells [tNCC]) as well as generating MYCN-driven tumors. Interestingly, the protocols that created cells with the highest level of NMP markers did not produce cells with the highest tNCC or SA cell markers. We identified a protocol that consistently produced cells with the highest level of SA markers using two iPSC lines of different genders. This protocol also generated tumors with the highest level of the neuroblastoma-specific marker, PHOX2B. Transcriptomally, however, each protocol generates tumors that resemble neuroblastoma. Two of the protocols repeatedly produced adrenergic neuroblastoma whereas the other two protocols were ambiguous. Thus, we identified a protocol that reliably generates adrenergic neuroblastoma. Overall design: We first compared four different tNCC/SA differentiation protocols in vitro by RT-qPCR for tNCC/SA markers. WT PSCs were then transduced with MYCN, differentiated, and implanted orthotopically into immunocompromised mice. We also analyzed the tumors by RNA-seq and compared the transcriptomes with neural crest derived tumors.