title: An optimized procedure for fluorescence-activated cell sorting (FACS) isolation of autonomic neural progenitors from visceral organs of fetal mice.
abstract: During development neural crest (NC)-derived neuronal progenitors migrate away from the neural tube to form autonomic ganglia in visceral organs like the intestine and lower urinary tract. Both during development and in mature tissues these cells are often widely dispersed throughout tissues so that isolation of discrete populations using methods like laser capture micro-dissection is difficult. They can however be directly visualized by expression of fluorescent reporters driven from regulatory regions of neuron-specific genes like Tyrosine hydroxylase (TH). We describe a method optimized for high yields of viable TH+ neuronal progenitors from fetal mouse visceral tissues, including intestine and lower urogenital tract (LUT), based on dissociation and fluorescence-activated cell sorting (FACS). The Th gene encodes the rate-limiting enzyme for production of catecholamines. Enteric neuronal progenitors begin to express TH during their migration in the fetal intestine and TH is also present in a subset of adult pelvic ganglia neurons . The first appearance of this lineage and the distribution of these neurons in other aspects of the LUT, and their isolation has not been described. Neuronal progenitors expressing TH can be readily visualized by expression of EGFP in mice carrying the transgene construct Tg(Th-EGFP)DJ76Gsat/Mmnc. We imaged expression of this transgene in fetal mice to document the distribution of TH+ cells in the developing LUT at 15.5 days post coitus (dpc), designating the morning of plug detection as 0.5 dpc, and observed that a subset of neuronal progenitors in the coalescing pelvic ganglia express EGFP. To isolate LUT TH+ neuronal progenitors, we optimized methods that were initially used to purify neural crest stem cells from fetal mouse intestine. Prior efforts to isolate NC-derived populations relied upon digestion with a cocktail of collagenase and trypsin to obtain cell suspensions for flow cytometry. In our hands these methods produced cell suspensions from the LUT with relatively low viability. Given the already low incidence of neuronal progenitors in fetal LUT tissues, we set out to optimize dissociation methods such that cell survival in the final dissociates would be increased. We determined that gentle dissociation in Accumax (Innovative Cell Technologies, Inc), manual filtering, and flow sorting at low pressures allowed us to achieve consistently greater survival (>70% of total cells) with subsequent yields of neuronal progenitors sufficient for downstream analysis. The method we describe can be broadly applied to isolate a variety of neuronal populations from either fetal or adult murine tissues.
authors.affiliation: Division of Genetic Medicine, Department of Medicine, Vanderbilt University School of Medicine, USA.
authors.forename: Dennis P
authors.initials: DP
authors.lastname: Buehler
authors.forename: Dennis
authors.initials: D
authors.lastname: Buehler
authors.forename: Carrie B
authors.initials: CB
authors.lastname: Wiese
authors.forename: Carrie
authors.initials: C
authors.lastname: Wiese
authors.forename: S B
authors.initials: SB
authors.lastname: Skelton
authors.forename: E Michelle
authors.initials: EM
authors.lastname: Southard-Smith
authors.forename: Michelle
authors.initials: M
authors.lastname: Southard-Smith
mesh_terms: D000818:Animals; D004622:Embryo, Mammalian; D005260:Female; D005434:Flow Cytometry / Q000379:methods*; D007422:Intestines / Q000166:cytology / Q000196:embryology; D051379:Mice; D058953:Neural Stem Cells / Q000166:cytology*; D011247:Pregnancy; D014566:Urogenital System / Q000166:cytology / Q000196:embryology