Alan J. Burns

Mutation of the proto-oncogene c-kit blocks development of interstitial cells and electrical rhythmicity in murine intestine.

1. Interstitial cells of Cajal (ICs) have been proposed as pacemakers in the gastrointestinal tract. We studied the characteristics and distribution of ICs and electrical activity of small intestinal muscles from mice with mutations at the dominant‐white spotting/c‐kit (W) locus because the tyrosine kinase function of c‐kit may be important in the development of the IC network. 2. W/WV mutants (days 3‐30 postpartum) had few ICs in the myenteric plexus region compared with wild type (+/+) siblings. The few ICs present were associated with neural elements and lay between myenteric ganglia and the longitudinal muscle layer. 3. Electrical recordings from intestinal muscle strips showed that electrical slow waves were always present in muscles of +/+ siblings, but were absent in W/WV mice. 4. Muscles from W/WV mice responded to stimulation of intrinsic nerves. Neural responses, attributed to the release of acetylcholine, nitric oxide and other unidentified transmitters, were recorded. 5. These findings are consistent with the hypothesis that ICs are a critical element in the generation of electrical rhythmicity in intestinal muscles. The data also show that neural regulation of gastrointestinal muscles can develop independently of the IC network. 6. W locus mutants provide a powerful new model for studies of the physiological role of ICs and the significance of electrical rhythmicity to normal gastrointestinal motility.

Interstitial cells of Cajal in the guinea-pig gastrointestinal tract as revealed by c-Kit immunohistochemistry

Abstract.Interstitial cells of Cajal (ICC) of various morphologies have been described in the gastrointestinal (GI) tracts of mammals. Different classes of ICC are likely to have different functional roles. ICC of the mouse GI tract have been shown to express c-kit, a proto-oncogene that codes for a receptor tyrosine kinase. We have studied the distribution of ICC within the guinea pig GI tract using antibodies to c-Kit protein and immunohistochemical techniques. c-Kit-like immunoreactivity revealed at least 6 types of ICC: (1) intramuscular ICC (IC-IM1) that lie within the muscle layers of the esophagus, stomach, and cecum, (2) ICC within the myenteric plexus region (IC-MY1) in the corpus, antrum, small intestine, and colon,(3) ICC that populate the deep muscular plexus of the small intestine (IC-DMP), (4) ICC at the submucosal surface of the circular muscle layer in the colon (IC-SM), (5) stellate ICC that are closely associated with the myenteric plexus (IC-MY2) and orientated toward the longitudinal muscle layer in the colon, and (6) branching intramuscular ICC (IC-IM2) in the proximal colon within the circular and longitudinal muscle layers. c-Kit immunohistochemistry appears to be an excellent and selective technique for labeling ICC of the guinea-pig GI tract. Labeling of these cells at the light-microscopic level provides an opportunity for characterizing the distribution, density, organization, and relationship between ICC and other cell types.

Tissue engineered human tracheas for in vivo implantation

Two years ago we performed the first clinical successful transplantation of a fully tissue engineered trachea. Despite the clinically positive outcome, the graft production took almost 3 months, a not feasible period of time for patients with the need of an urgent transplantation. We have then improved decellularization process and herein, for the first time, we completely describe and characterize the obtainment of human tracheal bioactive supports. Histological and molecular biology analysis demonstrated that all cellular components and nuclear material were removed and quantitative PCR confirmed it. SEM analysis revealed that the decellularized matrices retained the hierarchical structures of native trachea, and biomechanical tests showed that decellularization approach did not led to any influence on tracheal morphological and mechanical properties. Moreover immunohistological staining showed the preservation of angiogenic factors and angiogenic assays demonstrated that acellular human tracheal scaffolds exert an in vitro chemo-active action and induce strong in vivo angiogenic response (CAM analysis). We are now able to obtained, in a short and clinically useful time (approximately 3 weeks), a bioengineered trachea that is structurally and mechanically similar to native trachea, which exert chemotactive and pro-angiogenic properties and which could be successfully used for clinical tissue engineered airway clinical replacements.

The enteric nervous system.

The enteric nervous system (ENS), the intrinsic innervation of the gastrointestinal tract, consists of numerous types of neurons, and glial cells, that are distributed in two intramuscular plexuses that extend along the entire length of the gut and control co-ordinated smooth muscle contractile activity and other gut functions. All enteric neurons and glia are derived from neural crest cells (NCC). Vagal (hindbrain) level NCC provide the majority of enteric precursors along the entire length of the gut, while a lesser contribution, that is restricted to the hindgut, arises from the sacral region of the neuraxis. After leaving the dorsal neural tube NCC undergo extensive migration, proliferation, survival and differentiation in order to form a functional ENS. This article reviews the molecular mechanisms underlying these key developmental processes and highlights the major groups of molecules that affect enteric NCC proliferation and survival (Ret/Gdnf and EdnrB/Et-3 pathways, Sox10 and Phox2b transcription factors), cell migration (Ret and EdnrB signalling, semaphorin 3A, cell adhesion molecules, Rho GTPases), and the development of enteric neuronal subtypes and morphologies (Mash1, Gdnf/neurturin, BMPs, Hand2, retinoic acid). Finally, looking to the future, we discuss the need to translate the wealth of data gleaned from animal studies to the clinical area and thus better understand, and develop treatments for, congenital human diseases affecting the ENS.

Enteric nervous system stem cells derived from human gut mucosa for the treatment of aganglionic gut disorders.

BACKGROUND & AIMS Enteric nervous system stem cells (ENSSCs) provide potential therapeutic tools to replenish absent ganglia in Hirschsprungs disease. Although full-thickness human postnatal gut tissue can be used to generate ENSSCs, reliance on its harvesting from surgical resection poses significant practical limitations. This study aimed to explore whether gut tissue obtained utilizing minimally invasive routine endoscopy techniques could be used to generate ENSSCs and whether such cells retain the potential to generate an ENS upon transplantation into aganglionic gut. METHODS Postnatal human gut mucosal tissue obtained from children undergoing gastrointestinal endoscopy was used to generate cell cultures in which ENSSCs were contained within neurosphere-like bodies (NLBs). These NLBs were characterized by immunostaining, and their potential to generate components of the ENS, in vitro and upon transplantation into models of aganglionic gut, was examined. RESULTS Gut mucosal biopsy specimens were obtained from 75 children (age, 9 months-17 years). The biopsy specimens contained neural cells and ENSSCs and, on culturing, generated characteristic NLBs at all ages examined. Postnatal mucosa-derived NLBs contained cells that, akin to their embryonic counterparts, were proliferating, expressed ENSSC markers, were bipotent, and capable of generating large colonies in clonogenic cultures and multiple ENS neuronal subtypes. Upon transplantation, cells from NLBs colonized cultured recipient aganglionic chick and human hindgut to generate ganglia-like structures and enteric neurons and glia. CONCLUSIONS The results represent a significant practical advance toward the development of definitive cell replenishment therapies for ENS disorders such as Hirschsprungs disease.

Inhibition of neural crest migration underlies craniofacial dysmorphology and Hirschsprung's disease in Bardet–Biedl syndrome

Facial recognition is central to the diagnosis of many syndromes, and craniofacial patterns may reflect common etiologies. In the pleiotropic Bardet–Biedl syndrome (BBS), a primary ciliopathy with intraflagellar transport dysfunction, patients have a characteristic facial “gestalt” that dysmorphologists have found difficult to characterize. Here, we use dense surface modeling (DSM) to reveal that BBS patients and mouse mutants have mid-facial defects involving homologous neural crest-derived structures shared by zebrafish morphants. These defects of the craniofacial (CF) skeleton arise from aberrant cranial neural crest cell (NCC) migration. These effects are not confined to the craniofacial region, but vagal-derived NCCs fail to populate the enteric nervous system, culminating in disordered gut motility. Furthermore, morphants display hallmarks of disrupted Sonic Hedgehog (Shh) signaling from which NCCs take positional cues. We propose a model whereby Bbs proteins modulate NCC migration, contributing to craniofacial morphogenesis and development of the enteric nervous system. These migration defects also explain the association of Hirschsprungs disease (HD) with BBS. Moreover, this is a previously undescribed method of using characterization of facial dysmorphology as a basis for investigating the pathomechanism of CF development in dysmorphic syndromes.

Mutations in lectin complement pathway genes COLEC11 and MASP1 cause 3MC syndrome

3MC syndrome has been proposed as a unifying term encompassing the overlapping Carnevale, Mingarelli, Malpuech and Michels syndromes. These rare autosomal recessive disorders exhibit a spectrum of developmental features, including characteristic facial dysmorphism, cleft lip and/or palate, craniosynostosis, learning disability and genital, limb and vesicorenal anomalies. Here we studied 11 families with 3MC syndrome and identified two mutated genes, COLEC11 and MASP1, both of which encode proteins in the lectin complement pathway (collectin kidney 1 (CL-K1) and MASP-1 and MASP-3, respectively). CL-K1 is highly expressed in embryonic murine craniofacial cartilage, heart, bronchi, kidney and vertebral bodies. Zebrafish morphants for either gene develop pigmentary defects and severe craniofacial abnormalities. Finally, we show that CL-K1 serves as a guidance cue for neural crest cell migration. Together, these findings demonstrate a role for complement pathway factors in fundamental developmental processes and in the etiology of 3MC syndrome.

A rat decellularized small bowel scaffold that preserves villus-crypt architecture for intestinal regeneration

Management of intestinal failure remains a clinical challenge and total parenteral nutrition, intestinal elongation and/or transplantation are partial solutions. In this study, using a detergent-enzymatic treatment (DET), we optimize in rats a new protocol that creates a natural intestinal scaffold, as a base for developing functional intestinal tissue. After 1 cycle of DET, histological examination and SEM and TEM analyses showed removal of cellular elements with preservation of the native architecture and connective tissue components. Maintenance of biomechanical, adhesion and angiogenic properties were also demonstrated strengthen the idea that matrices obtained using DET may represent a valid support for intestinal regeneration.

Discarded human kidneys as a source of ECM scaffold for kidney regeneration technologies

In the United States, more than 2600 kidneys are discarded annually, from the total number of kidneys procured for transplant. We hypothesized that this organ pool may be used as a platform for renal bioengineering and regeneration research. We previously showed that decellularization of porcine kidneys yields renal extracellular matrix (ECM) scaffolds that maintain their basic components, support cell growth and welfare in vitro and in vivo, and show an intact vasculature that, when such scaffolds are implanted in vivo, is able to sustain physiological blood pressure. The purpose of the current study was to test if the same strategy can be applied to discarded human kidneys in order to obtain human renal ECM scaffolds. The results show that the sodium dodecylsulfate-based decellularization protocol completely cleared the cellular compartment in these kidneys, while the innate ECM framework retained its architecture and biochemical properties. Samples of human renal ECM scaffolds stimulated angiogenesis in a chick chorioallantoic membrane assay. Importantly, the innate vascular network in the human renal ECM scaffolds retained its compliance. Collectively, these results indicate that discarded human kidneys are a suitable source of renal scaffolds and their use for tissue engineering applications may be more clinically applicable than kidneys derived from animals.

Immunomodulatory effect of a decellularized skeletal muscle scaffold in a discordant xenotransplantation model

Decellularized (acellular) scaffolds, composed of natural extracellular matrix, form the basis of an emerging generation of tissue-engineered organ and tissue replacements capable of transforming healthcare. Prime requirements for allogeneic, or xenogeneic, decellularized scaffolds are biocompatibility and absence of rejection. The humoral immune response to decellularized scaffolds has been well documented, but there is a lack of data on the cell-mediated immune response toward them in vitro and in vivo. Skeletal muscle scaffolds were decellularized, characterized in vitro, and xenotransplanted. The cellular immune response toward scaffolds was evaluated by immunohistochemistry and quantified stereologically. T-cell proliferation and cytokines, as assessed by flow cytometry using carboxy-fluorescein diacetate succinimidyl ester dye and cytometric bead array, formed an in vitro surrogate marker and correlate of the in vivo host immune response toward the scaffold. Decellularized scaffolds were free of major histocompatibility complex class I and II antigens and were found to exert anti-inflammatory and immunosuppressive effects, as evidenced by delayed biodegradation time in vivo; reduced sensitized T-cell proliferative activity in vitro; reduced IL-2, IFN-γ, and raised IL-10 levels in cell-culture supernatants; polarization of the macrophage response in vivo toward an M2 phenotype; and improved survival of donor-derived xenogeneic cells at 2 and 4 wk in vivo. Decellularized scaffolds polarize host responses away from a classical TH1-proinflammatory profile and appear to down-regulate T-cell xeno responses and TH1 effector function by inducing a state of peripheral T-cell hyporesponsiveness. These results have substantial implications for the future clinical application of tissue-engineered therapies.