Division of Genetic Medicine, Department of Medicine, Vanderbilt University School of Medicine
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Buehler, D. P., Wiese, C. B., Skelton, S. B., Southard-Smith, E. M. An Optimized Procedure for Fluorescence-activated Cell Sorting (FACS) Isolation of Autonomic Neural Progenitors from Visceral Organs of Fetal Mice. J. Vis. Exp. (66), e4188, doi:10.3791/4188 (2012).
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 intestine1 and TH is also present in a subset of adult pelvic ganglia neurons2-4 . 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/Mmnc1. 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 intestine2-6. 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.
1. Preparation of Media (All steps done in tissue culture hood)
3. Dissociation of Subdissected Tissues
4. Filtering Cell Suspension
5. Preparing Samples for FACS
|Tissue||Sample Pool Size||Volume 7-AAD to be added to tube*|
|15.5 dpc Intestine||1-5||200 μl|
|15.5 dpc LUT||1-5||150 μl|
6. Flow Cytometry
7. Representative Results
Tissue dissociation to produce a cell suspensions for flow sorting is a delicate balance between adequate enzymatic digestion and avoiding over-digestion that can result in low cell viabilities. An example of desired level of tissue dissociation is shown in Figure 2. In appropriately digested tissue before manual trituration pieces of sub-dissected organs are still clearly evident (Figure 2b, 2f). In tissues that are enzymatically treated for too long a period of time or at too high a concentration of enzyme, the resulting suspension lacks any residual large pieces of tissue (Figure 2d, 2h).
Appropriate dissociation and manual filtering produce sort profiles at flow cytometry that typically exhibit greater than 90% viable cells and show high levels of EGFP expression (Figure 4). Cell populations obtained by this method illustrate good viability and can be captured for subsequent culture or analysis of gene expression by gating for capture of EGFP+ neuronal progenitors.
Figure 1. Distribution of TH-EGFP+ neuronal progenitors in fetal mouse LUT. Whole-mount urogenital tract at 15.5 dpc viewed ventrally under bright field illumination (a) compared to distribution of EGFP+ cells labeled by TH-EGFP transgene expression identified under fluorescence illumination (b). TH-EGFP expression is present in adrenals (a) and medially located celiac ganglia (cg). Lateral (c) view of 15.5 dpc TH-EGFP sub-dissected bladder exhibits fluorescence from transgene expression in pelvic ganglia (pg), the bladder wall (bla) and urethra (u). In dorsal view (d) EGFP+ cells are evident in the anterior dorsal urethra. Other labels: kidneys (k), testis (t), bladder (bla) and genital tubercle (gt).
Figure 2. Brightfield images of 15 dpc fetal LUT (a) and intestine (e), respectively, imaged halfway through the dissociation incubation period, at the end of the dissociation incubation before disruption (b, f), after manual disruption (c, g), and in a sample that has been overly dissociated (d, h).
Figure 3. Schematic Diagram illustrates compensation controls needed to establish rigorous FACS gating parameters.
Figure 4. Representative image of flow sort profiles at 14.5 dpc (a) and 15.5 dpc (b). Black population is comprised of single cells based on forward and side scatter that are dead and labeled by 7-AAD fluorescence. Gray population is comprised of singlet cells based on forward and side scatter that have excluded 7-AAD and are thus viable. Green gated population is indicated by boxed "GFP+" area and is comprised of single cells that have excluded 7-AAD (viable) and exhibit EGFP fluorescence.
Mouse reporter lines expressing fluorescent reporters are becoming widely available through multiple efforts in the murine genetics community1,8,9. As a result the dissociation method illustrated here can be widely applied for isolation of discrete neuronal subtypes based on neurotransmitter or receptor expression patterns from either fetal or adult tissues. While we have optimized this method based on expression of a fluorescent transgene reporter, it can also be applied to samples expressing cell surface receptors labeled by live cell immuno-labeling methods3-5,10.
In our hands we observed several factors that affected the percentage of cells that survived the dissociation procedure. These include elapsed time from initial dissection of fetal tissue to isolation of cells by flow cytometry, gentle handling of cell suspensions both during filtering and at the flow sorter, as well as the enzyme type used in dissociation. The survival of both enteric and LUT progenitors benefited from rapid dissection/dissociation and use of gentle conditions during cell sorting (17psi, 100 micron nozzle, 3,000 events/sec). We found that prior to isolation at the flow sorter, the total population of material observed by the instrument (all events including debris and multiplets) generally exhibited about 80% survival for cell dissociates generated from either fetal intestine or LUT. To exclude debris and clumps of cells, initial gates for forward scatter (FSC) and side scatter (SSC) were imposed on this total population. Then the total population is further refined to a "daughter" population by voltage pulse geometry gating for width and height measurement of FSC and SSC to exclude doublets. Within this refined "daughter" population non-viable cells that are 7-AAD+ were excluded so that only viable cells exhibiting the fluorescent EGFP reporter were collected. We observed that the frequency of survival for neuronal progenitors in this daughter population was relatively low when dissociation was performed in a blend of trypsin+collagenase that is routinely used in many cases for this purpose (0.5% Trypsin, Gibco; 10 mg/ml Collagenase IV, Worthington)2,5-7. To obtain higher percentages of surviving neuronal progenitors from LUT tissue isolates, we tested alternate enzyme treatments for dissociation of tissue to derive cell suspensions for flow sorting (Table 2). Neuronal progenitors exhibited improved viability upon dissociation in dispase (5 mg/ml) a gentle dissociation method that is often used for isolation of neural tubes or other neural progenitor populations11. However, the greatest viability observed was obtained with Accumax, a proprietary blend of protease, collagenolytic activity and DNase of unknown proportions, (Table 2). Dissociation in Accumax yielded the best survival for both enteric neuronal progenitors and LUT neuronal progenitors with modest differences in survival between the two sources. Our results suggest that distinct neuronal populations may differ in their sensitivity to dissociation conditions. Thus, investigators seeking to isolate neuronal progenitors or mature neurons should evaluate dissociation conditions to identify those that produce the greatest cell survival and yield for distinct tissue sources. The method we describe serves as a starting point for such procedures in the peripheral nervous system.
|Enzyme in dissociation solution||% Survival Enteric NP||% Survival LUT NP|
|Tryp + Collag||29.5 +/- 8.2%, n=11||46.2 +/- 8.6%, n=4|
|Dispase||35.1 +/- 9.8%, n=10||46.3 +/- 6.6%, n=9|
|Accumax||66.5 +/- 7.8%, n=8||58.1 +/- 5.2%, n=10|
Table 2. Viability of neuronal progenitors (NP) in different dissociation conditions.
No conflicts of interest declared.
The authors would like to thank Catherine Alford for suggestions on cell dissociation methods and Kevin Weller, David Flaherty and Brittany Matlock for support in the Flow Cytometry Shared Resource at Vanderbilt University Medical Center and Melissa A. Musser for artistic assistance with illustrations. We thank Drs. Jack Mosher and Sean Morrison for advice on implementing isolation of neuronal progenitors. The VMC Flow Cytometry Shared Resource is supported by the Vanderbilt Ingram Cancer Center (P30 CA68485) and the Vanderbilt Digestive Disease Research Center (P30 DK058404). This work was supported by funding from US National Institutes of Health grants DK064251, DK086594, and DK070219.
|Accumax||Sigma(mfr: Innovative Cell Technologies)||A7089-100ML||Store frozen in 1 ml aliquots|
|DNase I||Sigma||D-4527||Stored frozen at -20 °C 5 mg/ml in 1xHBSS,
(Used in Quench, Quench 1:5)
|10X PBS pH 7.4||Gibco||70011-044||Make up to 1x with tissue culture grade water then sterile filter|
|10X HBSS w/o Ca or Mg||Gibco||14185-052||Make up to 1x with tissue culture grade water then sterile filter|
|Leibovitz’s L-15 medium||Gibco||21083027|
|Penicillin /Streptomycin 100X||Gibco||15140-133||Store aliquoted at -20 °C|
|BSA||Sigma||A3912-100G||Store aliquoted at -20 °C, 100 mg/ml in water|
|Biowhittaker 1M HEPES in 0.85% NaCl||Lonza||17-737E|
|38 μm NITEX Nylon Mesh Membrane||Sefar America||3-38/22||Cut into ~3 cm squares. UV treat overnight to sterilize in the tissue culture hood.|
|5 ml polystyrene tubes||Falcon||352058|
|15 ml conical tubes||Corning||430790|
|Fine Dissecting Forceps||Fine Science Instruments||11251-30||Dumont#5 forcep, Dumoxel, standard tip 0.1x0.06mm|
|Dissecting Spoon||Fine Science Instruments||10370-18|
Formal Correction: Erratum: An Optimized Procedure for Fluorescence-activated Cell Sorting (FACS) Isolation of Autonomic Neural Progenitors from Visceral Organs of Fetal Mice
Posted by JoVE Editors on 10/01/2012. Citeable Link.
The authors middle initials were omitted from the publication of An Optimized Procedure for Fluorescence-activated Cell Sorting (FACS) Isolation of Autonomic Neural Progenitors from Visceral Organs of Fetal Mice. The author's names have been updated to:
Dennis P. Buehler, Carrie B. Wiese, S. B. Skelton, E. Michelle Southard-Smith
Dennis Buehler, Carrie Wiese, S. B. Skelton, Michelle Southard-Smith