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Tu1448

Identification and Localization of Enteric Neural Precursors Within the SmallIntestinal Nestin NetworkSubhash Kulkarni, Ya-Yuan Fu, Laren Becker, Gunjan Tiwari, Johann Peterson, Maria-Adelaide Micci, Tatyana V. Michurina, Grigori Enikolopov, Shiue-Cheng Tang, Pankaj J.Pasricha

Background and Aim: Nestin has been proposed as a marker for neural stem cells and hasbeen also reported to be expressed by enteric neural stem cells (ENSC), along with a varietyof other proteins such as p75, α2 integrin, Sox2 and Sox10. However, little is known aboutthe nature and distribution of nestin expressing cells in the gut wall. We hypothesized thatnestin expression could be used as a method for isolating and localizing putative ENSC thatwould enable better molecular characterization of these cells, facilitate their harvesting fortherapeutic transplantation efforts as well provide greater insight into their participation, ifany, in intrinsic regenerative events. Methods: We studied a transgenic mouse that expressedgreen fluorescent protein (GFP) under the control of the nestin promoter and using wholetissue confocal microscopy of adult small intestine. Immunohistochemistry with a varietyof cell surface markers was used to further subclassify Nestin+ cells in various regions..Multicolor FACS sorting with a combination of Nestin and cell surface markers was usedto sort out different populations of cells, on which In Vitro clonal propagation assays as wellas multi-potent differentiation assays were performed. Results: Nestin+ cells form an extensivetransmural network in the small intestine, which consists of perivascular cells expressingthe surface proteoglycan NG2, as well as others both within and outside of the myentericganglia. Within the ganglia, some nestin+ cells express the surface neurotrophin receptorp75, thus identifying them as potential ENSC. When isolated from myenteric plexus andgrown in augmented Neurobasal media, we found that only Nestin+ cells propagated inculture and differentiated to produce neurons and glia. In Vitro clonal propagation anddifferentiation of nestin+ cells sub-sorted for NG2 and p75 showed that only cells expressingboth p75 and nestin (and not p75 alone) are capable of proliferating (Mean ± S.E. of percentageproliferative cells in Nestin+p75+ fraction: 1.68 ± 0.52) and differentiating to form neuronsand glia In Vitro (Mean ± S.E. of percentage of neurospheres that differentiate to form neuronsand glia: 78.26 ± 4.35; only neurons: 0 ± 0; only glia: 2.22 ± 1.96). Conclusions: Nestin andp75 together mark a population of cells that contain ENSC. These cells appear to be localizedwithin the ganglia and are distinct from other nestin+ cells such as those in the perivascularnetwork. Our results suggest that it is possible to obtain enriched populations of ENSC formolecular characterization as well as study their involvement in regenerative events.

Tu1449

3-D Microscopy of the Enteric Glial Network in the Transparent Human ColonMucosaYuan-An Liu, Yuan-Chiang Chung, Shien-Tung Pan, Yung-Chi Hou, Shih-Jung Peng,Pankaj J. Pasricha, Shiue-Cheng Tang

Background & Aims: Enteric glia form a network in the intestinal mucosa and engage inmultidirectional interactions with the epithelium, blood vessels, nerves, and immune cells.Although enteric glia have been shown to participate with various inflammatory processes,the standard microtome-based 2-D tissue analysis cannot provide a global view of the glialnetwork and its association with the adjacent tissue components in space. In this research,we prepared transparent human colon mucosa by optical clearing for 3-D imaging of theglial network to illustrate their architecture for morphological and quantitative analyses.Methods: Tissues of human colon were obtained from colectomies performed for carcinoma.Transverse sections of colon mucosa were prepared away from the tumor site. S100B andnuclear fluorescent staining were used to identify the glial cell bodies and processes andthe intestinal microstructure. Optical clearing (Liu et al., Neurogastroenterol Motil 23:e446-e457, 2011; Fu & Tang, Gastroenterology, 139:p1100-1105, 2010) was used to promotephoton penetration in the tissue for 3-D confocal microscopy and in-depth projection ofthe glial network. Results: Transparent specimens led to in-depth imaging of the fluorescence-labeled mucosal structure and glial network with high definition. Significantly, the 3-Dimage data provided a continuous flow of the anatomic information for panoramic projection.Three features of the projected glial network are seen: (1) the condensed glial cell bodiesand processes encircle the crypt bottoms, (2) the glial network elongates from the cryptbottom to the opening, and (3) the glial processes sprout at the crypt opening. Specifically,the orderly aligned glial cells at the middle segment of the colonic crypts provide anappropriate target for quantitation: counting the cell bodies with at least two S100B-labeledprocesses extending from the nuclear signals. Conclusion: The enteric glia in the humancolonic mucosa possesses location-dependent network morphology as revealed by 3-D

S-836AGA Abstracts

imaging with optical clearing. Our findings suggest a practical approach for in-depth imagingand quantitative analysis of glial cells in the human gut mucosal specimen.

In-depth projections of the enteric glial network at the crypt bottom (A and B) and opening(C and D). Gray: S100B-labeled enteric glia. Green: nuclei. Projection depth: 80 μm.

Tu1450

Platelet-Derived Growth Factor Receptor α (Pdgfrα)-Expressing “Fibroblast-Like Cells” in Human Diabetic and Idiopathic GastroparesisMadhusudan Grover, Cheryl E. Bernard, Pankaj J. Pasricha, Henry P. Parkman, ThomasL. Abell, Linda Anh B. Nguyen, William J. Snape, K.Robert Shen, Michael G. Sarr, JamesM. Swain, Michael L. Kendrick, Simon J. Gibbons, Tamas Ordog, Gianrico Farrugia

Background: Emerging evidence suggests that “fibroblast-like cells” (FLC) may play a rolein the regulation of gastrointestinal (GI) motor function, especially, inhibitory neurotransmis-sion. FLC are ultrastructurally distinct from other interstitial cell types including interstitialcells of Cajal (ICC). FLC also express small-conductance Ca2+-activated K+ channels (SK3).In mice, platelet-derived growth factor receptor α (PDGFRα) antibody has been shown tolabel FLC. The distribution of PDGFRα-immunoreactive (ir) FLC in human GI smoothmuscle is not well known. Aims: The aims of this study were to determine the morphologyand distribution of PDGFRα-ir FLC in human gastric muscle and to determine if FLC arealtered in gastroparesis, a condition associated with loss of key cell types, including ICC.Methods: Full thickness gastric body biopsies from 10 diabetic, 10 idiopathic gastroparesispatients, and 5 healthy subjects were immunolabeled using SK3 and PDGFRα staining forFLC and Kit staining for ICC. Intramuscular FLC and ICC bodies were quantified. Results:Human intramuscular PDGFRα-ir cells had slender cell bodies and long, thin processes andwere more abundant in the longitudinal compared to the circular muscle. In the region ofthe myenteric plexus, FLC had smaller, rounder cell bodies, compared with intramuscularFLC, and had 3-4 processes and formed networks, often around the myenteric ganglia. AllSK3-ir cell structures showed complete overlap with PDGFRα-ir in the muscle layer andthe myenteric plexus region. In the submucosa, however, only PDGFRα-ir cells were foundand SK3-ir was absent in that region. Both intramuscular and myenteric FLC were in closeproximity to ICC, but their cell bodies did not overlap. No differences were seen in thedistribution or morphology of FLC in diabetic or idiopathic gastroparesis patients. On overallquantification, FLC numbers did not differ between the three groups (FLC/hpf:11.0±1.0 fordiabetic gastroparesis, 11.7±1.0 for idiopathic gastroparesis and 10.8±0.9 for controls,p>0.05). When FLC numbers in gastroparetics with and without ICC loss were compared,still no significant differences were found. Conclusions: PDGFRα identifies FLC in humangastric muscle. The morphologic features of FLC, such as long processes and formation ofnetworks with other FLC and presence of gap junctions with smooth muscle cells, and theirtopographic localization in close proximity to ICC, nerve fibers and ganglia raise the possibilitythat this cell type may also participate in the regulation of normal human gastrointestinalmotility. However, FLC were not altered in morphology, distribution or overall numbers ingastroparesis patients. Their role in gastric function in health and disease states needs to befurther determined. Supported by P01DK68055 and the Gastroparesis Clinical ResearchConsortium U01DK074008