INTRODUCTION: The human enteric nervous system (ENS) is a complex network of neurons and glia that extends throughout the length of the bowel. There are many neuron and glia types, but very little is known about the human ENS compared to other species. There are two main ENS layers. The myenteric plexus neurons are clustered into ganglia with thick nerve fiber bundles running between ganglia. Submucosal neuron ganglia are scattered throughout the region between circular muscle and the epithelial lining of the bowel. One key problem with studying human ENS is that these cells are a very minor component of the bowel wall (< 1: 10,000 cells) and they are surrounded by other tissue. The bowel wall is not easily dissociated making it challenging to isolate cells of the human ENS for any type of analsysis.OBJECTIVES: To obtain a suspension of single nuclei from human colon myenteric plexus that could be used for RNA-seq.METHODS: 1) Human colon "myenteric plexus" is dissected from the bowel wall after labeling with a live cell dye and then frozen in OCT. 2) Frozen "myenteric plexus" is sectioned on a cryostat to generate small fragments and disrupt muscle and connective tissue. 3) RNA quality is assessed from a few sections using an Agilent Bioanalyzer. 4) Nuclei are obtained from frozen sections using Dounce homogenization and labeled with Hoechst 33342 dye. 5). Nuclei are separated from other cell debris by FACS. 6) RNA-seq data were obtained using the 10X Genomic single cell sequencing platform.RESULTS: Live dye labeling works well on living tissue to visualize the ENS in human colon. Careful dissection is required to micro-dissect "myenteric plexus" from from within bowel wall muscle layers. A suspension of single nuclei is efficiently isolated by freezing isolated tissue in OCT, frozen sectioning, Dounce homogenizing, and FACS sorting Hoechst 33342 stained nuclei. The isolated nuclei are suitable for sequencing on the 10X Genomics single cell sequencing platform. This procedure dramatically enriched for cells in the region of the myenteric plexus, but unfortunately only a small percentage of the cells isolated are myenteric neurons. In addition to myenteric neurons, we obtained data from enteric glia, smooth muscle, muscularis macrophage, interstitial cells of Cajal, and PDGFRalpha+ cells that are known to be closely associated with myenteric plexus.CONCLUSION: We tried many approaches to optimize this procedure including additional steps to enrich for myenteric neuron nuclei. These additional procedures invariably reduced total cell yield without enriching significantly for neuronal nuclei. We do not know yet why neuronal nuclei remain a small component of our total nuclei population, but suspect that neuronal nuclei are more fragile than other nuclei isolated. This approach generated valuable data at a single nucleus RNA-seq level for all cell types within the muscle wall known to control bowel motility.