Mouse regenerating myofibers detected as false-positive donor myofibers with anti-human spectrin.

Abstract Stem cell transplantation is being tested as a potential therapy for a number of diseases. Stem cells isolated directly from tissue specimens or generated via reprogramming of differentiated cells require rigorous testing for both safety and efficacy in preclinical models. The availability of mice with immune-deficient background that carry additional mutations in specific genes facilitates testing the efficacy of cell transplantation in disease models. The muscular dystrophies are a heterogeneous group of disorders, of which Duchenne muscular dystrophy is the most severe and common type. Cell-based therapy for muscular dystrophy has been under investigation for several decades, with a wide selection of cell types being studied, including tissue-specific stem cells and reprogrammed stem cells. Several immune-deficient mouse models of muscular dystrophy have been generated, in which human cells obtained from various sources are injected to assess their preclinical potential. After transplantation, the presence of engrafted human cells is detected via immunofluorescence staining, using antibodies that recognize human, but not mouse, proteins. Here we show that one antibody specific to human spectrin, which is commonly used to evaluate the efficacy of transplanted human cells in mouse muscle, detects myofibers in muscles of NOD/Rag1(null)mdx(5cv), NOD/LtSz-scid IL2Rγ(null) mice, or mdx nude mice, irrespective of whether they were injected with human cells. These "reactive" clusters are regenerating myofibers, which are normally present in dystrophic tissue and the spectrin antibody is likely recognizing utrophin, which contains spectrin-like repeats. Therefore, caution should be used in interpreting data based on detection of single human-specific proteins, and evaluation of human stem cell engraftment should be performed using multiple human-specific labeling strategies.

Human fetal skeletal muscle contains a myogenic side population that expresses the melanoma cell-adhesion molecule.

Muscle side population (SP) cells are rare myogenic progenitors distinct from satellite cells, the known tissue-specific stem cells of skeletal muscle. Studies in mice demonstrated that muscle SP cells give rise to satellite cells in vivo. Given that muscle SP cells are heterogeneous, it has been difficult to prospectively enrich for myogenic progenitors within the SP fraction, particularly from human tissue. Further, conditions that favor the expansion of human muscle SP cells while retaining their myogenic potential have yet to be reported. In this study, human fetal muscle SP and main population (MP) cells were purified based on the expression of melanoma cell adhesion molecule (MCAM), a marker we previously reported to enrich for cells with myogenic potential. To define the relationship between MCAM expression and the degree of myogenic commitment, single cells were analyzed for the expression of myogenic-specific markers. Myogenic factors strongly associated with MCAM expression in single cells, particularly Myf5. Different MCAM+ populations, including SP cells, were expanded and assayed for fusion potential in vitro and engraftment potential in vivo. All MCAM+ subpopulations fused robustly into myotubes in vitro, whereas the MCAM- subpopulations did not. Further, MCAM+ SP cells exhibited the highest fusion potential in vitro and were the only fraction to engraft in vivo, although at low levels, following propagation. Thus, MCAM can be used to prospectively enrich for myogenic muscle SP cells in human fetal muscle. Moreover, we provide evidence that human MCAM+ SP cells have intrinsic myogenic activity that is retained after propagation.

Isolation and characterization of human fetal myoblasts.

Dissociated human fetal skeletal muscle contains myogenic cells, as well as non-myogenic cells such as adipocytes, fibroblasts, and lymphocytes. It is therefore important to determine an efficient and reliable isolation method to obtain a purer population of myoblasts. Toward this end, fluorescence-activated cell sorting in conjunction with robust myogenic cell surface markers can be utilized to enrich for myoblasts in dissociated muscle. In this chapter, we describe a method to significantly enrich for myoblasts using -melanoma cell adhesion molecule (MCAM), which we have determined to be an excellent marker of human fetal myoblasts. The myoblasts resulting from this isolation method can then be expanded in vitro and still retain significant myogenic activity as shown by an in vitro fusion assay. The ability to isolate a highly myogenic population from dissociated muscle facilitates the in vitro study of skeletal muscle development and muscle diseases. Furthermore, robust expansion of these cells will lead to new insights in the development of cell-based therapies for human muscle disorders.

Activity-dependent regulation of inhibitory synapse development by Npas4.

Neuronal activity regulates the development and maturation of excitatory and inhibitory synapses in the mammalian brain. Several recent studies have identified signalling networks within neurons that control excitatory synapse development. However, less is known about the molecular mechanisms that regulate the activity-dependent development of GABA (gamma-aminobutyric acid)-releasing inhibitory synapses. Here we report the identification of a transcription factor, Npas4, that plays a role in the development of inhibitory synapses by regulating the expression of activity-dependent genes, which in turn control the number of GABA-releasing synapses that form on excitatory neurons. These findings demonstrate that the activity-dependent gene program regulates inhibitory synapse development, and suggest a new role for this program in controlling the homeostatic balance between synaptic excitation and inhibition.

Mouse Eri1 interacts with the ribosome and catalyzes 5.8S rRNA processing.

Eri1 is a 3'-to-5' exoribonuclease conserved from fission yeast to humans. Here we show that Eri1 associates with ribosomes and ribosomal RNA (rRNA). Ribosomes from Eri1-deficient mice contain 5.8S rRNA that is aberrantly extended at its 3' end, and Eri1, but not a catalytically inactive mutant, converts this abnormal 5.8S rRNA to the wild-type form in vitro and in cells. In human and murine cells, Eri1 localizes to the cytoplasm and nucleus, with enrichment in the nucleolus, the site of preribosome biogenesis. RNA binding residues in the Eri1 SAP and linker domains promote stable association with rRNA and thereby facilitate 5.8S rRNA 3' end processing. Taken together, our findings indicate that Eri1 catalyzes the final trimming step in 5.8S rRNA processing, functionally and spatially connecting this regulator of RNAi with the basal translation machinery.

FOXP3 controls regulatory T cell function through cooperation with NFAT.

Antigen stimulation of immune cells activates the transcription factor NFAT, a key regulator of T cell activation and anergy. NFAT forms cooperative complexes with the AP-1 family of transcription factors and regulates T cell activation-associated genes. Here we show that regulatory T cell (Treg) function is mediated by an analogous cooperative complex of NFAT with the forkhead transcription factor FOXP3, a lineage specification factor for Tregs. The crystal structure of an NFAT:FOXP2:DNA complex reveals an extensive protein-protein interaction interface between NFAT and FOXP2. Structure-guided mutations of FOXP3, predicted to progressively disrupt its interaction with NFAT, interfere in a graded manner with the ability of FOXP3 to repress expression of the cytokine IL2, upregulate expression of the Treg markers CTLA4 and CD25, and confer suppressor function in a murine model of autoimmune diabetes. Thus by switching transcriptional partners, NFAT converts the acute T cell activation program into the suppressor program of Tregs.