Ex Vivo Neurogenesis within Enteric Ganglia Occurs in a PTEN Dependent Manner

A population of multipotent stem cells capable of differentiating into neurons and glia has been isolated from adult intestine in humans and rodents. While these cells may provide a pool of stem cells for neurogenesis in the enteric nervous system (ENS), such a function has been difficult to demonstrate in vivo. An extensive study by Joseph et al. involving 108 rats and 51 mice submitted to various insults demonstrated neuronal uptake of thymidine analog BrdU in only 1 rat. Here we introduce a novel approach to study neurogenesis in the ENS using an ex vivo organotypic tissue culturing system. Culturing longitudinal muscle and myenteric plexus tissue, we show that the enteric nervous system has tremendous replicative capacity with the majority of neural crest cells demonstrating EdU uptake by 48 hours. EdU+ cells express both neuronal and glial markers. Proliferation appears dependent on the PTEN/PI3K/Akt pathway with decreased PTEN mRNA expression and increased PTEN phosphorylation (inactivation) corresponding to increased Akt activity and proliferation. Inhibition of PTEN with bpV(phen) augments proliferation while LY294002, a PI3K inhibitor, blocks it. These data suggest that the ENS is capable of neurogenesis in a PTEN dependent manner.


Introduction
The recognition that neurogenesis occurs in defined regions of the adult central nervous system (CNS) via a population of neural stem cells (NSCs) [1,2,3,4] has provoked speculation that a similar phenomenon occurs in the enteric nervous system (ENS) [5]. The ENS is a complex network of neural crest-derived neurons and glia that span the gastrointestinal tract and control complex gut behaviors including peristalsis, secretion, and blood flow. Given their location in the gastrointestinal tract, enteric neurons face a variety of insults including the mechanical stress of gut motility, toxins absorbed from the bowel, and inflammatory/infectious processes from enteric pathogens. Thus, it seems logical to expect neurogenesis in the ENS. Indeed, so-called enteric neural stem cells (ENSCs) have been isolated from embryonic and adult intestine using a variety of techniques including growth selection and selective sorting either by cell surface markers for Ret, p75, and CD49b (alpha 2 -integrin) or fluorescent proteins [6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21]. In culture, these cells demonstrate properties of stemness including self-renewal and multipotency (differentiating into neurons, glia and myofibroblasts) [8,12]. They are also capable of colonizing gut tissue ex vivo and imparting functional changes to the neuromuscular activity of these explants [14].
However, despite the propensity of ENSCs for neurogenesis in vitro, a similar function has proven challenging to demonstrate in vivo [11]. Several ENS injury models have demonstrated significant neural plasticity, which suggests neurogenesis, but these studies failed to differentiate between new neurons and existing neurons that migrated from bordering regions [22,23,24,25]. Studies using the thymidine analog bromodeoxyuridine (BrdU) to label dividing cells have had variable success demonstrating neurogenesis in vivo [11,15,26]. While BrdU uptake was not observed in neurons under steady-state conditions in post-natal mice, it was seen following treatment with a 5-hydroxytryptamine receptor 4 (5-HT 4 ) agonist [15]. However, Joseph et al. performed an exhaustive study involving 108 rats and 51 mice under a variety of conditions including steady-state, aging, pregnancy, diabetes, dietary modifications, exercise, inflammation, and injury, yet only saw BrdU uptake in a single rat following benzalkonium chloride (BAC) treatment [11]. Some groups have utilized genetic fate mapping with loxp/cre recombinase systems under an inducible promoter to evaluate for neurogenesis in vivo [11,13]. Lineage tracing with Sox10-creERT2 mice failed to show neurogenesis under steadystate conditions (after post-natal day 84). However, using an ENS injury model with BAC, a neurotoxic detergent, 9% of neurons surrounding the site of injury were of Sox10-origin, suggesting neurogenesis [13]. A similar lineage tracing system using GFAP-creERT2 mice failed to show appreciable GFAPderived neurons following BAC treatment, however this difference may be explained by the low efficiency of cre recombination (3%-5%) in these mice [11].
In light of the apparent discrepancy regarding the neurogenic potential of ENSCs in vitro and in vivo, there is a need to better understand the pathways that control this process. Phosphatase and tensin homolog deleted on chromosome ten (PTEN) is a lipid and protein phosphatase that has a key regulatory role in cell proliferation and survival by suppressing phosphatidylinositol-3-OH kinase (PI3K)/Akt signaling [27,28]. PTEN appears to maintain stem cells in a quiescent state, with PTEN deletions causing increased proliferation in neural stem cells, haematopoietic stem cells and embryonic stem cells [29,30,31,32,33,34,35]. Tissue specific deletion of PTEN in the ENS resulted in hyperplasia of enteric ganglia [36], suggesting that PTEN may have a similar role in ENSCs. Using mice in which Cre recombinase under the Wnt1 neural crest specific promoter drives tdTomato expression in cells of neural crest origin, we employed an ex vivo organotypic tissue culture system with the thymidine analog ethynyldeoxyuridine (EdU) to study proliferation and neurogenesis in the ENS. After an initial lag phase, we find abundant EdU uptake within neurons and glia in the myenteric plexus. We find that the switch from quiescence to proliferation corresponds to a decrease in PTEN mRNA expression and an increase in phospho-PTEN (inactive form [37]). We also observe that inhibiting PTEN with potassium bisperoxo(1,10-phenanthroline)oxovanadate [bpV(phen)], augments proliferation in cells of neural crest origin.

Ethics Statement
Experimental protocols were approved by the Administrative Panel on Laboratory Animal Care (APLAC) at the Stanford University, California in accordance with the guidelines provided by the National Institutes of Health.

Tissue Preparation and ex vivo Organotypic Culturing
Mice were anesthetized with isofluorane and euthanized by cervical dislocation. A laparotomy was performed and the small intestine was removed and lavaged with PBS containing penicillinstreptomycin (PS; Invitrogen, Carlsbad CA). Small intestine was cut into segments of 2 cm in length and placed over a sterile plastic rod. A superficial longitudinal incision was made along the serosal surface and the longitudinal muscle with the adherent myenteric plexus (LMMP) was peeled off from the underlying tissue using a wet sterile cotton swab and placed in Opti-MEM containing PS. LMMP was cultured in stem cell medium (SCM) consisting of Neurobasal medium containing B27, 2 mM L-glutamine and 100 U/ml PS. EdU (Invitrogen, Carlsbad CA) was added to media at a final concentration of 25 mM. BpV(phen) (BioVision, Milpitas CA) was added to culture media at a final concentration of 200 or 400 nM, and LY294002 (Cell Signaling, Danvers MA) was added at a final concentration of 10 or 50 mM. The following growth factors were added to SCM for all experiments except those involving inhibitors or differentiation: fibroblast growth factor (bFGF, 10 ng/ml), epidermal growth factor (EGF, 10 ng/ ml) and glial cell-derived neurotrophic factor (GDNF, 10 ng/ml) (Invitrogen, Carlsbad CA). Tissue was cultured at 37uC and 5% CO 2 , and medium was replaced every day. For differentiation experiments, LMMP was cultured in SCM containing EdU for 48 h before chasing in differentiating media (SCM without growth factors bFGF, EGF and GDNF) for an additional 5 days. For flow cytometry and microwell experiment, LMMP was digested in Digestion Buffer consisting of M199 media (Invitrogen, Carlsbad CA) containing 0.1% BSA, 1 mM CaCl 2 , 20 mM HEPES, 150 mM P188, 50 U/ml DNAse I (Worthington, Lakewood NJ), 1.1 mg/ml collagenase (Sigma, St. Louis MO), and dispase 1 mg/ ml (Sigma, St. Louis MO) for 40 minutes at 37uC and 5% CO 2 . Tissue was washed in PBS with 1% BSA then passed through a 40 mm nylon mesh cell strainer before fixation.

Sphere Formation Assay
At the end of 48 h incubation in SCM, LMMP were pulsed with EdU for 12 h then dissociated in Digestion Buffer. Cells were washed in PBS with 10% FBS, passed through a sterile 40 mm nylon mesh cell strainer, and cultured in SCM for 3 days until neurosphere-like bodies (NLBs) were easily visualized. These primary NLBs were transferred to fibronectin-coated 60-mm plates (BD Biosciences) and cultured as an adherent monolayer.
Cells were dissociated into single cells with Accutase (Invitrogen) and either layered on microwells (,5000 cells/microwell grid) or cultured on fibronectin-coated plates for additional passage. Images of cells in microwells were captured on inverted fluorescence microscope at 46 objective. Cells were cultured in microwells for 6-8 days to allow sphere formation. Microwells were fixed, permeabilized and the Click-IT EdU reaction performed before imaging with a CCD cooled camera on a Nikon C1 confocal microscope at 606 objective. For sphere cell counts, cross-sectional images of NLBs were captured using confocal microscopy and spheres were counted for total number of cells . EdU + cells of neural crest origin were found in anaphase (inset) confirming that EdU uptake reflects cell division. Remarkably the rate of EdU uptake increased dramatically after 24 h reaching close to 45% of tdT-expressing cells by 72 h (C). The increase in EdU uptake after 24 h corresponded to increased total area of tdT expression per field (D) and increased total number of tdT-expressing cells per field (E) suggesting that EdU uptake reflects proliferation of cells. Area measurements (D) were made by performing automatic threshold determination on the red (tdT) channel in individual high-resolution images and measuring the area in percentage of each image with intensity above the threshold using ImageJ software. Area percentages were then converted to absolute units of mm 2 . (C) Statistical significance testing by exact binomial test; scale bar = 50 mm. (using DAPI) and number of EdU + cells. Only those spheres that could be traced to a single cell at day 1 were included in analysis.
The experiment was performed in biologic replicates using 3 animals and data was pooled for analysis. Figure 2. EdU + cells have sphere-forming potential and express ENSC markers. Following a ''pulse'' with EdU, LMMP was digested and cells were cultured in media without EdU for a prolonged ''chase'' (up to 25 days). After either 1 or 2 passages, dissociated cells were seeded into microwells allowing tracking of single cells. Using fluorescence microscopy, single tdT-expressing cells (A, arrow) were observed that formed NLBs (B, arrow). Upon fixation and labeling of EdU, subsets of NLBs were found that contained EdU + cells (C, arrow). The majority of NLBs remained small (#3 cells/sphere cross-section) and none of these contained EdU+ cells (D). However, EdU was detected in a substantial subset of large NLBs (.3 cells/ sphere cross-section) (E). The average sphere size was larger in NLBs that EdU was detected, a difference that was statistically significant for both secondary and tertiary NLBs (F). Flow cytometric analysis was performed on LMMP that was pulsed with EdU at 48 h and gated for tdT + /EdU + single cells with debris and doublets excluded. The .97% of these cells co-expressed ENSC markers (white histogram) including p75, CD49b and Nestin (D, gray histogram is no primary control). * = Semi-quantative measurements of band intensity using pixel intensity were performed using ImageJ (NIH) and expressed in densitometric units normalized to the loading control (GAPDH).

RNA Isolation and qRT-PCR
RNA from tissue was extracted using Qiagen RNeasy mini kit (Qiagen, Valencia CA), quantified using Nanodrop 2000c and converted to cDNA using High Capacity RNA-to-cDNA kit (ABI, Foster City CA). Quantification of PTEN expression was carried Figure 3. EdU uptake occurs in neurons and glia ex vivo. Flow cytometric analysis was performed on LMMP that was cultured with EdU for 72 h and gated for single cells that co-expressed tdT and the specified neuronal or glial marker (white histogram). EdU uptake was detected in 54% of Tuj/tdT + cells (A), 64.7% of PGP9.5/tdT + cells (B), 62.1% of S100ß/tdT + cells (C), and 73.1% of GFAP/tdT + cells. The gray histogram represents LMMP cultured with EdU for only 24 h was used as a negative control. After culturing LMMP for 48 h in media containing EdU then chasing for 5 days in differentiating media without EdU, immunofluorescence revealed cells with EdU uptake (green) that immunostained for neuronal and glial markers (red) including Tuj (E), PGP9.5 (F), S100ß (G), GFAP (H), nNOS (I), and VIP (J). Scale bars: 50 mm in E-J. doi:10.1371/journal.pone.0059452.g003 out using TaqMan Gene expression Assays and ABI StepOne plus real time instrument with the Taqman probes (ABI, Foster City CA) to PTEN (Mm00477208_m1) and HPRT (Mm01545399_m1). All experiments were performed in biological triplicates. Expression of each gene was normalized to the housekeeping gene HPRT. Fold change in gene expression between groups was calculated using the Pfaffl method.

Statistical Analysis
Data is expressed as mean +/2 standard error of mean or +/ 295% confidence interval, as noted, and was analyzed using Student's unpaired two-tailed t-test, ANOVA, or exact binomial test as appropriate. Significance was deemed when the p value was less than 0.05. For cell counts, at least 5 random fields containing myenteric plexus were counted for each analyte and each sample was performed in triplicate. Statistical analysis was performed with GraphPad Prism 5 (GraphPad Software Inc, La Jolla CA) or R (v. 2.15.1).

Proliferative Capacity of Neural Crest Cells Ex Vivo
Cre recombinase under the neural crest-specific Wnt1 promoter has been used to exclusively label or genetically alter enteric neural crest cells within the mouse gut [7,10,38,39]. We utilized a Wnt1-cre;tdTomato mouse line which allows clear visualization of cells of neural crest origin within dissected LMMP [7]. After culturing LMMP in the presence of EdU (a thymidine analog) for 48 h, we found that 78.5%(63.1) of tdTomato (tdT) expressing cells demonstrated EdU uptake. Proliferation was largely confined to cells of neural crest origin as 74.5% (66.6) of all EdU + cells expressed tdT. In order to examine the time course at which proliferation occurs, we administered 5 h pulses of EdU immediately after LMMP dissection and at intervals up to 72 hours ( Figure 1A). We detected virtually no EdU + cells at the 5 h and 24 h time points. Proliferation rapidly increased at 48 h and 72 h, ultimately reaching 45% of tdT + cells by 72 h (Figure 1B and C). EdU + neural crest cells were frequently seen in anaphase ( Figure 1B, inset) confirming that EdU uptake indicates cell division. A similar high percentage of EdU uptake in cells of neural crest origin was observed by flow cytometry ( Figure  S1A). Uptake corresponded to an increase in the total area of tdT expression per examined field ( Figure 1D) and an increase in the total number of tdT-expressing cells per field ( Figure 1E), confirming that it reflects a true expansion of neural crest cells within the tissue. Noticeably, EdU uptake first occurred primarily in cells within the ganglia ( Figure 1B, arrows) suggesting this to be the location of ENSCs.

EdU + Cells Demonstrate Sphere-forming Potential
The ability of single cells to form NLBs over multiple passages has been used as an assay for self-renewal and ''stemness'' [17,40]. We digested LMMP that had been in culture for 48 h and pulsed with EdU for 12 hours, and cultured the dissociated cells in media without EdU for up to 25 days. Cells were seeded into microwells after 1 or 2 passages to allow clonal analysis of sphere-forming potential. Single tdT-expressing cells (Figure 2A, arrow) were found that formed both secondary and tertiary NLBs ( Figure 2B, arrow) after multiple days in culture. Of the NLBs that could be traced to single cells on day 1, only a small fraction (10.1% (7/69) of secondary and 14.3% (18/126) of tertiary NLBs) became large NLBs (.3 cells/cross-section) after 6-7 days in culture. Upon EdU labeling, cells with EdU uptake could be detected in a subset of NLBs suggesting that the clonal cell from which the sphere was derived contained EdU ( Figure 2C, arrow). EdU was not found in any of the secondary (0/62) or tertiary (0/108) NLBs that remained small (#3 cells/cross-section) ( Figure 2D). However, it was detected in 57.4% (4/7) of secondary and 27.8% (5/18) of tertiary NLBs that were large (.3 cells/cross-section) suggesting a correlation with EdU positivity and sphere-forming potential ( Figure 2E). Overall, secondary and tertiary NLBs in which EdU was detected contained a dramatically larger number of cells per cross-section compared to their EdU negative counterparts ( Figure 2F, p,.0001).
Certain types of stem cells including NSCs have been shown to partition chromosomes asymmetrically during cell division [41,42,43], a stem cell property known as the 'immortal strand hypothesis' [44]. Unlike embryonic stem cells or fibroblasts in which BrdU signal became undetectable after seven cell divisions, NSCs retained BrdU signal in 8.7% of cells after 10 days (,10 population doublings) [42]. Similar to NSCs, we detected EdU signal in a small fraction of cells within the sphere (figure 2C and 2E). Of the tertiary NLBs in which EdU was detected, signal was found in 14.9% (62.9) of cells. Given that these cells had been passaged twice and cultured without EdU for 25 days it suggests that asymmetric segregation of DNA during cell division is also a property of ENSCs. EdU + Cells Express Markers of ENSCs A characteristic of ENSCs is their expression of certain markers including Nestin, p75, and CD49b [11]. We digested LMMP following 48 h in organotypic culture, including a 12 h EdU pulse, and performed flow cytometry on single cells that were fixed, labeled for EdU, and stained for ENSC markers. Greater than 90% of tdT-expressing EdU + cells also expressed p75, CD49b, Nestin ( Figure 2G), and GFAP (data not shown) suggesting that they represent ENSCs. This contrasted with the non-neural crest EdU + fraction (tdT 2/ EdU + ) where the majority of cells did not express p75, Nestin or GFAP (although did express CD49b) ( Figure S1B).

EdU + Cells Express Neuronal Markers
In order to evaluate for neurogenesis in our ex vivo system, LMMP incubated with EdU was digested after 72 hours of culture and stained for glial (GFAP and S100ß) and neuronal (PGP9.5 and Tuj) markers. By flow cytometry, EdU uptake was detected in over 50% of neural crest cells that expressed neuronal markers (54% of tdT + /Tuj + , Figure 3A white, and 64.7% of tdT + /PGP9.5 + , figure 3B white). The percentage of EdU uptake was even higher for tdT + cells expressing glial markers (62.1% of tdT + /S100ß + , Figure 3C white, and 73.1% of tdT + /GFAP + , Figure 3D white). To confirm neuronal EdU uptake, LMMP was cultured for 48 h with a 12 h EdU pulse, then chased in differentiating media for 5 days. Upon EdU labeling and immunostaining, EdU uptake was detected in cells that stained for pan-neuronal markers Tuj ( Figure 3E) and PGP9.5 ( Figure 3F), and neuronal subtypes nNOS ( Figure 3I) and VIP ( Figure 3J). EdU + cells that stained for glial markers S100ß ( Figure 3G) and GFAP ( Figure 3H) were also found.

Proliferation of Cells of Neural Crest Origin is Dependent on the PTEN/PI3K/Akt Pathway
We evaluated whether loss of inhibition by PTEN may account for the increase in proliferation over time. On qRT-PCR analysis, PTEN mRNA expression declined by over 50% by 24 h ( Figure 4A). A similar decline in PTEN protein expression was not observed by western blot analysis ( Figure 4B). One possible explanation for this discrepancy was the dramatic increase in PTEN phosphorylated on the C-terminus (Ser380) ( Figure 4B), which has been associated with increased PTEN stability but decreased activity [37,45]. Based on pixel intensity, the phospho-PTEN:total PTEN ratio was 80-fold higher at 24 h and 48 h compared with steady state levels at isolation ( Figure 4B). Loss of PTEN inhibition corresponded with a rise in Akt activity. On western blot analysis, the phospho-Akt:total Akt ratio increased by over 3-4 fold at 24 h and 48 h compared with steady state at 0 h ( Figure 4C). To determine the influence of growth factors (FGF, EGF and GDNF) in the media, we performed proliferation assays in the presence and absence of growth factors. While growth factors did augment proliferation, substantial EdU uptake was observed even in their absence ( Figure 4D). In order to confirm that the PI3K/Akt pathway is required for proliferation, we performed a dose response experiment with the PI3K inhibitor LY294002 in the absence of growth factors. Compared with untreated control, 10 mM and 50 mM of LY294002 caused a 37% and 99% reduction in EdU uptake at 30 h ( Figure 4E).
Next, we assessed the effect the PTEN inhibitor bpV(phen) on proliferation. Evaluating the dose response to bpV(phen) over 7 h (also in the absence of growth factors), we found that inhibitor concentrations of 200 nM and 400 nM resulted in over 15-fold and 7-fold elevations in the ratio of phospho-Akt:total Akt respectively, relative to control ( Figure 5A). Organotypic cultures incubated with EdU in the presence or absence of bpV(phen) (200 nM) for 25 h revealed that the PTEN inhibitor resulted in a 3fold increase in proliferation compared to control ( Figure 5B and C). The percent of EdU uptake increased from 3.9% (60.37) to 12.2% (60.83) of tdT + cells with bpV(phen) (figure 5C).

Discussion
Discrepancies between in vivo and in vitro studies have created confusion regarding the capacity for neurogenesis in the adult ENS. We and others have found that under in vitro conditions, ENSCs isolated and propagated from adult intestine can readily differentiate into neurons [7,8,9,11,12,14,16,17,18,21]. However, neurogenesis has been more difficult to demonstrate in vivo. Neurogenesis is undetectable under steady state conditions in the adult ENS and is only seen in variable amounts under certain conditions (ie. injury, 5-HT4 agonist) [11,13,15,26]. Here we introduce a novel ex vivo organotypic culturing system that allows us to study neurogenesis in the intact myenteric plexus over time. Unlike dissociated cells, our ex vivo system has the advantage of preserving the architecture of the myenteric plexus and maintaining many of the interactions within the so-called ''stem cell niche''. Yet clearly it does not mimic in vivo conditions. In the process of preparing our ex vivo tissue cultures, ENSCs are removed from the influences of systemic factors supplied by the vasculature and by neural connections with the mucosal layers and extraintestinal tissue. Furthermore, the ''preparation'' whereby the muscle layer is stripped from the submucosa likely causes a disruption of the extracellular matrix with release of local factors. Finally, the tissue is then influenced by extrinsic factors in the culturing method including increased oxygen tension and nutrients and other factors supplied in the media. Despite these inherent differences with the in vivo state, our ex vivo system has demonstrated several important findings. First, the ENS appears to have a remarkably high capacity for proliferation with the majority of cells of neural crest origin demonstrating EdU uptake by 48 h. Next, we used our model to test the effect of compounds on promoting or delaying proliferation. We found that small molecule inhibitors of PI3K and PTEN either markedly prolonged the lag phase (PI3K inhibitor LY294002) ( Figure 4E) or shortened it (PTEN inhibitor bpV(phen) ( Figure 5D). Thus this system offers the potential for finding other compounds that regulate quiescence possibly through highthroughput screening. Such compounds may have therapeutic benefit in restoring bowel function following surgery or for intestinal neuromuscular disorders.
Finally, we were able to gain insight into the regulatory process that controls the transition from quiescence to proliferation. Proliferation appears to be driven by PTEN inhibition via phosphorylation of the C-terminal tail which leads to decreased phosphatase activity. The exact kinase(s) responsible for this phosphorylation are unknown but a recent publication has implicated Src and casein kinase 2 alpha [46]. Several lines of evidence support our finding that PTEN is an important regulator of ENSC quiescence. In studies using conditional PTEN deletions with PTEN fl/fl mice, PTEN appears to have a generalized role in suppressing proliferation in neural and haematopoietic stem cells [30,31,32,33,34,35]. PTEN activity also affects proliferation and pluripotency in embryonic stem cells [29]. Furthermore, loss of PTEN in humans has been associated with intestinal ganglioneuromatosis [47,48] and a mouse conditional knockout of PTEN in the ENS resulted in enteric ganglia hypertrophy and hyperplasia and a chronic intestinal pseudoobstruction disease phenotype [36]. Finally, local inhibition of PTEN following CNS or peripheral nerve injury has been shown to promote regeneration through neurite outgrowth [49,50,51]. Thus manipulation of PTEN may be a therapeutic strategy for promoting regeneration of the ENS. Transient reduction in PTEN activity is likely the best strategy since complete elimination of PTEN could cause ganglioneuromatosis or other functional disturbances, and may ultimately deplete the stem cell reserve [30,34,35]. Our ex vivo system likely reflects an injury state with disruption of the neural environment leading to suppression of PTEN. A possible explanation for the vast difference in proliferation seen our ex vivo system compared with in vivo injury models may be what the ex vivo system lacks. Unlike in vivo, it is not influenced by the inflammatory response to injury including systemic cytokines and the local influx of inflammatory cells. In the CNS, inflammation has been shown to impair neurogenesis following injury [52]. NSC function and neurogenesis was restored using indomethacin, a common non-steroidal anti-inflammatory [52]. Ultimately, future in vivo studies should examine whether a combined approach of inhibiting PTEN and reducing inflammation will enhance regeneration of the ENS. Figure S1 Majority of tdT + cells are EdU + , and EdU + / tdT 2 cells do not express p75 or Nestin. When flow cytometry was performed on LMMP cultured with EdU for 72 h, nearly 60% of tdT-expressing cells demonstrated EdU uptake (A). Flow cytometry of LMMP pulsed with EdU at 48 h revealed that the majority of dividing non-neural crest cells (tdT 2/ EdU + cells) did not express p75 or Nestin; however 63% expressed CD49b (B). (TIF)