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.神经母细胞瘤（Neuroblastoma）是最常见的起源于交感肾上腺细胞（sympathoadrenal, SA）的颅外实体瘤，可分为肾上腺素能型与间质型。本研究比较了四种不同的分化方案，用以将诱导多能干细胞（induced pluripotent stem cells, iPSC）定向诱导为SA细胞以及两类中间细胞状态——神经中胚层祖细胞（neuromesodermal progenitors, NMP）与躯干神经嵴细胞（trunk neural crest cells, tNCC），同时该方案亦可用于生成MYCN驱动的肿瘤。有趣的是，可诱导出最高水平NMP标志物的方案，并未同步产生最高水平的tNCC或SA细胞标志物。我们筛选出一套可通过两株不同性别的iPSC系稳定产出高表达SA细胞标志物的细胞的方案；该方案同样可生成高表达神经母细胞瘤特异性标志物PHOX2B的肿瘤。但从转录组层面分析，所有方案生成的肿瘤均与神经母细胞瘤特征相似。其中两种方案可稳定生成肾上腺素能型神经母细胞瘤，另外两种方案的肿瘤分型则较为模糊。综上，我们成功筛选出一套可稳定诱导生成肾上腺素能型神经母细胞瘤的方案。总体实验设计：我们首先在体外通过逆转录定量聚合酶链反应（RT-qPCR）检测tNCC/SA相关标志物，对四种不同的tNCC/SA分化方案进行初步比较。随后将野生型多能干细胞（WT PSCs）转导MYCN基因，经分化后原位移植至免疫缺陷小鼠体内。我们还通过RNA测序（RNA-seq）对生成的肿瘤进行转录组分析，并将其与神经嵴来源肿瘤的转录组进行比对。