Lentiviral labeling of mouse and human enteric nervous system stem cells for regenerative medicine studies.

BACKGROUND: Reliable methods of labeling human enteric nervous system (ENS) stem cells for use in novel cell replacement therapies for enteric neuropathies are lacking. Here, we explore the possibility of using lentiviral vectors expressing fluorescent reporter genes to transduce, label, and trace mouse and human ENS stem cells following transplantation into mouse gut. METHODS: Enteric nervous system precursors, including ENS stem cells, were isolated from enzymatically dissociated mouse and human gut tissues. Lentivirus containing eGFP or mCherry fluorescent reporter genes was added to gut cell cultures at a multiplicity of infection of 2-5. After fluorescence activated cell sorting for eGFP and subsequent analysis with markers of proliferation and cell phenotype, transduced mouse and human cells were transplanted into the gut of C57BL/6 and immune deficient Rag2-/gamma chain-/C5 mice, respectively and analyzed up to 60 days later. KEY RESULTS: Mouse and human transduced cells survived in vitro, maintained intense eGFP expression, proliferated as shown by BrdU incorporation, and formed characteristic neurospheres. When transplanted into mouse gut in vivo and analyzed up to 2 months later, transduced mouse and human cells survived, strongly expressed eGFP and integrated into endogenous ENS networks. CONCLUSIONS & INFERENCES: Lentiviral vectors expressing fluorescent reporter genes enable efficient, stable, long-term labeling of ENS stem cells when transplanted into in vivo mouse gut. This lentiviral approach not only addresses the need for a reliable fluorescent marker of human ENS stem cells for preclinical studies, but also raises the possibility of using lentiviruses for other applications, such as gene therapy.

Inhibition of cell death results in hyperganglionosis: implications for enteric nervous system development.

The enteric nervous system (ENS) is derived from vagal and sacral neural crest cells (NCC) that delaminate from the neural tube and undergo extensive migration and proliferation in order to colonize the entire length of the gut and differentiate into many millions of neurons and glial cells. Although apoptotic programmed cell death is an essential physiological process during development of the majority of the vertebrate nervous system, apoptosis within early ENS development has not been comprehensively investigated. The aim of this study was to determine the presence and extent of apoptosis within the vagal NCC population that gives rise to most of the ENS in the chick embryo. We demonstrated that apoptotic cells, as shown by terminal deoxynucleotidyl transferase biotin-dUTP nick end labelling and active caspase-3 immunoreactivity, are present within an electroporated green fluorescent protein (GFP) and human natural killer-1 (HNK-1) immunopositive NCC population migrating from the vagal region of the neural tube to the developing foregut. Inhibition of caspase activity in vagal NCC, by electroporation with a dominant-negative form of caspase-9, increased the number of vagal NCC available for ENS formation, as shown by 3-dimensional reconstruction of serial GFP or HNK-1 labelled sections, and resulted in hyperganglionosis within the proximal foregut, as shown by NADPH-diaphorase whole gut staining. These findings suggest that apoptotic cell death may be a normal process within the precursor pool of pre-enteric NCC that migrates to the gut, and as such it may play a role in the control of ENS formation.

Red and white muscle development in the trout (Oncorhynchus mykiss) as shown by in situ hybridisation of fast and slow myosin heavy chain transcripts.

The axial muscle of most teleost species consists of a deep bulk of fast-contracting white fibres and a superficial strip of slow-contracting red fibres. To investigate the embryological development of fast and slow muscle in trout embryos, we carried out single and double in situ hybridisation with fast and slow myosin heavy chain (MyHC)-isoform-specific riboprobes. This showed that the slow-MyHC-positive cells originate in a region of the somite close to the notochord. As the somite matures in a rostrocaudal progression, the slow-MyHC-positive cells appear to migrate radially away from the notochord to the lateral surface of the myotome, where they form the superficial strip of slow muscle. Surprisingly, the expression pattern of the fast MyHC showed that the differentiation of fast muscle commences in the medial domain of the somite before the differentiation and migration of the slow muscle precursors. Later, as the differentiation of fast muscle progressively spreads from the inside to the outside of the myotome, slow-MyHC-expressing cells become visible medially. Our observations that the initial differentiation of fast muscle takes place in proximity to axial structures and occurs before the differentiation and migration of slow muscle progenitors are not in accord with the pattern of muscle formation in teleosts previously described in the zebrafish Danio rerio, which is often used as the model organism in fishes.

Differential expression of two nonallelic MyoD genes in developing and adult myotomal musculature of the trout (Oncorhynchus mykiss).

Previously we identified two nonallelic MyoD encoding genes in the rainbow trout. These two MyoD genes (TMyoD and TMyoD2) were duplicated during the tetraploidization of the salmonid genome. In this study we show that TMyoD and TMyoD2 exhibit a distinct spatiotemporal pattern of expression that defines discrete cell populations in the developing somite. TMyoD expression is first detected in the mid-gastrula on either side of the elongating embryonic shield. During the anterior-to-posterior wave of somite formation the TMyoD transcript is initially present in adaxial cells of both the presomitic mesoderm and the forming somites. A lateral extension of TMyoD expression occurs only when the myotomes acquire their characteristic chevron shape pointing rostrally. By contrast, the initial expression of TMyoD2 occurs in somites that have already formed and is limited to the posterior compartment of somites. Further, in postlarval trout we observed a differential expression of TMyoD and TMyoD2 genes in muscle fibers with differing phenotype. Collectively, these data provide evidence that the two trout MyoD encoding genes have evolved to become functionally different. A comparison of the expression patterns of the two trout MyoD genes with that of myogenin allowed us to position them in the regulatory pathway leading to muscle differentiation.

Expression of a cysteine-rich protein (CRP) encoding gene during early development of the trout.

Members of the cysteine-rich protein (CRP) define a subclass of LIM-only proteins implicated mainly in muscle differentiation. Until now, very little is known concerning the expression of CRP encoding genes during vertebrate development. We describe here the isolation of a trout (Oncorhynchus mykiss) gene encoding a cysteine-rich protein (TCRP) and the pattern of its mRNA accumulation during embryogenesis, focusing on somitogenesis. TCRP encodes a putative protein with two LIM domains linked to a short glycine-rich region that displays 86%, 76%, 67% identity with chicken CRP2, CRP1 and MLP/CRP3 proteins, respectively. Whole-mount in situ hybridisation showed that TCRP transcript is first detected just before somitogenesis in the paraxial mesoderm, while it is absent in the axial structures. During somitogenesis, the expression of TCRP was observed caudally in the elongating presomitic mesoderm and in the last formed somites. The labelling for TCRP was found to fade as the somites mature. At the end of the somitogenesis, TCRP transcripts accumulation was restricted to pronephros and bronchial arches.