Karkala Faidra, de Bosscher Indy, Windster Jonathan D, Stroebel Savio, van Zanten Lars, Alves Maria M, Sacchetti Andrea
Department of Pediatric Surgery, Erasmus University Medical Center, Sophia Children's Hospital, 3015 GD Rotterdam, The Netherlands.
Department of Clinical Genetics, Erasmus University Medical Center, Sophia Children's Hospital, 3015 GD Rotterdam, The Netherlands.
Int J Mol Sci. 2025 May 18;26(10):4824. doi: 10.3390/ijms26104824.
Isolation of neurons and glia from the enteric nervous system (ENS) enables ex vivo studies, including the analysis of genomic and transcriptomic profiles. While we previously reported a fluorescence-activated cell sorting (FACS)-based isolation protocol for human ENS cells, no equivalent exists for mice. As directly applying the human protocol to mouse tissue resulted in low recovery of live ENS cells, we optimized tissue dissociation using mouse colons. A 30 min Liberase-based digestion showed optimal recovery of viable ENS cells, with CD56 and CD24 emerging as the most reliable markers to select and subdivide these cells. ENS' identity was further validated by FACS, using neuronal (TUBB3) and glial (SOX10) markers and reverse transcriptase quantitative PCR on sorted fractions. Overall, the mouse ENS expression profile significantly overlapped with the human one, showing that current dissociation protocols yield a mixed population of enteric neurons and glia. Nonetheless, using the imaging flow cytometer BD S8 FACS Discover and ELAVL4 as a neuronal soma-associated marker, we observed enrichment of neurons in a CD56/CD24 population. In conclusion, we present here a protocol for high-purity FACS-based isolation of viable mouse ENS cells, suitable for downstream applications.
从肠神经系统（ENS）中分离神经元和神经胶质细胞能够进行体外研究，包括对基因组和转录组图谱的分析。虽然我们之前报道了一种基于荧光激活细胞分选（FACS）的人ENS细胞分离方案，但小鼠却没有类似的方案。由于将人类方案直接应用于小鼠组织会导致活的ENS细胞回收率较低，我们使用小鼠结肠优化了组织解离方法。基于 Liberase 的 30 分钟消化显示出可行的 ENS 细胞的最佳回收率，CD56 和 CD24 成为选择和细分这些细胞的最可靠标记。通过 FACS，使用神经元（TUBB3）和神经胶质（SOX10）标记以及对分选组分进行逆转录酶定量 PCR，进一步验证了 ENS 的特性。总体而言，小鼠 ENS 表达谱与人类表达谱有显著重叠，表明当前的解离方案产生了肠神经元和神经胶质细胞的混合群体。尽管如此，使用成像流式细胞仪 BD S8 FACS Discover 和 ELAVL4 作为神经元胞体相关标记，我们在 CD56/CD24 群体中观察到神经元的富集。总之，我们在此提出一种基于 FACS 的高纯度分离可行小鼠 ENS 细胞的方案，适用于下游应用。
