Andrea C. M. Sinanan

Schwann Cell Development in Embryonic Mouse Nerves

Previously we proposed that Schwann cell development from the neural crest is a two-step process that involves the generation of one main intermediate cell type, the Schwann cell precursor. Until now Schwann cell precursors have only been identified in the rat, and much remains to be learned about these cells and how they generate Schwann cells. Here we identify this cell in the mouse and analyze its transition to form Schwann cells in terms of timing, molecular expression, and extracellular signals and intracellular pathways involved in survival, proliferation, and differentiation. In the mouse, the transition from precursors to Schwann cells takes place 2 days earlier than in the rat, i.e., between embryo days 12/13 and 15/16, and is accompanied by the appearance of the 04 antigen and the establishment of an autocrine survival circuit. Beta neuregulins block precursor apoptosis and support Schwann cell generation in vitro, a process that is accelerated by basic fibroblast growth factor 2. The development of Schwann cells from precursors also involves a change in the intracellular survival signals utilized by neuregulins: To block precursor death neuregulins need to signal through both the mitogen-activated protein kinase and the phosphoinositide-3-kinase pathways although neuregulins support Schwann cell survival by signaling through the phosphoinositide-3-kinase pathway alone. Last, we describe the generation of precursor cultures from single 12-day-old embryos, a prerequisite for culture studies of genetically altered precursors when embryos are non-identical with respect to the transgene in question.

Human adult craniofacial muscle‐derived cells: neural‐cell adhesion‐molecule (NCAM; CD56)‐expressing cells appear to contain multipotential stem cells

Skeletal muscle has been well characterized as a reservoir of myogenic precursors or satellite cells with the potential to participate in cellular repopulation therapies for muscle dysfunction. Recent evidence, however, suggests that the postnatal muscle compartment can be considered an alternative to bone marrow as a source of multipotent cells or muscle‐derived stem cells (MDSCs). MDSCs, when primed with appropriate environmental cues, can differentiate into a variety of non‐muscle cells. The present study describes the application of a new technique for the isolation of adult human myoblasts and putative MDSCs, based on microbead–immunomagnetic selection of CD56+ cells, derived from craniofacial skeletal muscle, and details changes in morphological/molecular phenotype of the purified cells when maintained in either a myogenic or a non‐myogenic milieu. Multiple immunofluorescence microscopy and two‐colour flow‐cytometric analysis of proliferating CD56+ cultures revealed positive staining for myogenic markers (CD56, desmin and M‐cadherin) as well as putative stem‐cell markers [the antigens CD34, CD90 and CD106, and Flk‐1 (fetal liver kinase‐1)/VEGFR‐2 (vascular‐endothelial‐growth‐factor receptor)]. Confluent cultures subjected to cycles of adipogenic or osteogenic induction contained either adipocytes or osteoblasts and myotubes. In conclusion, the CD56+ subpopulation within adult human skeletal muscle is heterogeneous and is composed of both lineage‐committed myogenic cells and multipotent cells (the candidate MDSCs), which are able to form non‐muscle tissue such as fat and bone.

Oct-6 (SCIP/Tst-1) is expressed in Schwann cell precursors, embryonic Schwann cells, and postnatal myelinating Schwann cells: comparison with Oct-1, Krox-20, and Pax-3.

The POU domain transcription factor Oct‐6 (SCIP/ Tst‐1) is likely to control important stages of Schwann cell development, including the initiation of myelination around birth. Here, we use immunocytochemical and reverse transcriptase‐polymerase chain reaction techniques to examine Oct‐6 earlier in nerve development, to test the idea that Oct‐6 has an additional role in Schwann cell precursors or early embryonic Schwann cells, a possibility raised by previous studies on transgenic mice. Consistent with this, we find low but unambiguous levels of Oct‐6 mRNA and protein in Schwann cell precursors of mouse and rat (nerves from 12‐ and 14‐day‐old embryos, respectively), with expression levels gradually increasing during early Schwann cell development and towards birth. Unexpectedly, Oct‐6 immunoreactivity is clearly present in nuclei of most myelinating cells at least as late as postnatal day 12. Furthermore, many nonmyelinating Schwann cells express Oct‐6 in adult life. A comparison of Oct‐6 mRNA with other Schwann cell transcription factors—namely, Oct‐1, Krox‐20, and Pax‐3—reveals that each factor exhibits strong developmental regulation and a unique expression pattern in embryonic nerves. Therefore, they are likely to play distinct regulatory roles in early development of the Schwann cell lineage.

Gelatinase-B (matrix metalloproteinase-9; MMP-9) secretion is involved in the migratory phase of human and murine muscle cell cultures

The remodelling of connective tissue components is a fundamental requirement for a number of pivotal processes in cell biology. These may include myoblast migration and fusion during development and regeneration. In other systems, similar biological processes are facilitated by secretion of the matrix metalloproteinases (MMPs), especially the gelatinases. This study investigated the activity of the gelatinases MMP-2 and 9 by zymography on cell conditioned media in cultures of cells derived from explants of the human masseter muscle and in the murine myoblast cell-line C2C12. Expression of MMP-9 by western blotting and TIMP-1, the major inhibitor of MMPs, by northern blotting, during all phases of myoblast proliferation, migration, alignment and fusion, was also measured. Irrespective of the origin of the cultures, MMP-9 activity was secreted only by single cell and pre-fusion cultures whilst MMP-2 activity was secreted at all stages as well as by myotubes. The loss of MMP-9 activity was due to the loss of MMP-9 protein expression. TIMP-1 mRNA was not detectable at the single cell stage but its expression increased as cells progressed through the pre-fusion and post-fusion stages to reach a maximal in myotube containing cultures. Migration of cells derived from human masseter muscle was inhibited, using a specific anti-MMP-9 blocking monoclonal antibody (6-6B). These data are consistent with the concept that regulation of matrix turnover via MMP-9 may be involved in the events leading to myotube formation, including migration. Loss of expression of this enzyme and expression of TIMP-1 mRNA is associated with myotube containing cultures. Consequently, the ratio between MMPs and TIMPs maybe important in determining myoblast migration and differentiation.

The IGF-I splice variant MGF increases progenitor cells in ALS, dystrophic, and normal muscle

The effects of muscle splice variants of insulin‐like growth factor I (IGF‐I) on proliferation and differentiation were studied in human primary muscle cell cultures from healthy subjects as well as from muscular dystrophy and ALS patients. Although the initial numbers of mononucleated progenitor cells expressing desmin were lower in diseased muscle, the E domain peptide of IGF‐IEc (MGF) significantly increased the numbers of progenitor cells in healthy and diseased muscle. IGF‐I significantly enhances myogenic differentiation whereas MGF E peptide blocks this pathway, resulting in an increased progenitor (stem) cell pool and thus potentially facilitating repair and maintenance of this postmitotic tissue.

Muscling in on stem cells

Skeletal muscle is one of the few adult tissues that possesses the capacity for regeneration (restoration of lost functional tissue) as opposed to repair. This capacity is due to the presence of ‘muscle stem cells’ known as satellite cells. Detailed investigation of these cells over the past 50 years has revealed that both these and other cells within the skeletal muscle complex are capable of regenerating both muscle and other cell types as well. Here, we review this information, and suggest that skeletal muscle is an exciting reservoir of cells for regenerating skeletal muscle itself, as well as other cell types.

Northcroft Memorial Lecture 2005: Muscling in on malocclusions: Current concepts on the role of muscles in the aetiology and treatment of malocclusion

The British Orthodontic Society invites outstanding contributors from the field of Orthodontics to give the guest lecture in memory of George Northcroft. In 2005 the guest lecturer was Professor Nigel Hunt. The article that follows was presented as the Northcroft Memorial Lecture 2005 at the World Orthodontic Congress, Paris.

Androgens affect myogenesis In Vitro and increase local IGF-1 expression

PURPOSE The mechanism whereby anabolic androgens are associated with hypertrophy of skeletal muscle is incompletely understood but may involve an interaction with locally generated insulin-like growth factor (IGF) 1. The present investigation utilized a cell culture model of human skeletal muscle-derived cell maturation to test the hypothesis that androgens increase differentiation of human muscle precursor cells in vitro and to assess effects of androgen with or without IGF-1 on IGF-1 messenger RNA (mRNA) expression in human muscle precursor cells. METHODS Differentiation of muscle-derived cells was induced under standard low-serum conditions. Cultures were then exposed to androgen (testosterone (T)) at 50, 100, and 500 nM or IGF-1 (10-50 ng·mL⁻¹). Immunocytochemistry and real-time polymerase chain reaction (RT-PCR) were used to assess effects of androgens and IGF-1 after 3- (early) or 7-d (late) muscle differentiation, respectively; RT-PCR was used to quantify the effects on androgen receptor expression. RESULTS Under low-serum conditions, 3-d exposure to androgens or IGF-1 or both resulted in no significant increase in cellular myogenic commitment. After 7-d exposure, however, T and IGF-1 were both found to increase fusion index with no observable synergistic effect. T also increased IGF-1 mRNA generation (P < 0.0001), whereas exogenous IGF-1 (P < 0.001) reduced IGF-1 mRNA transcription relative to control. The T effect was reversible after treatment with flutamide, an androgen receptor antagonist. CONCLUSIONS Both T and IGF-1 increase myogenic commitment after 7-d exposure to a differentiation medium. With T causing a concomitant increase in IGF-1 mRNA underpinning IGF-1 as a central mediator in the cellular pathways associated with muscle hypertrophy, including those affected by androgens. The novel system described has the potential for elucidating the pattern of growth factor effects associated with androgens in skeletal muscle.

Alpha v beta 3 and alpha v beta 5 integrins and their role in muscle precursor cell adhesion.

Background information. Functional adaptation of skeletal muscle is a requirement for different muscle groups (e.g. craniofacial, ocular and limb) to undergo site‐specific changes. Such tissue remodelling depends on dynamic interactions between muscle cells and their extracellular matrix, via participation of multifunctional molecules such as integrins. In view of data suggesting a role in fundamental muscle biology and muscle development in other systems, the present study has focused on expression and function of αv integrins, in cultured adult human craniofacial muscle (masseter) precursor cells and myotubes, and the predominantly fibroblastic IC (interstitial cells) population.

Myosin proteins identified from masseter muscle using quantitative reverse transcriptase–polymerase chain reaction—a pilot study of the relevance to orthodontics

There is a clearly established relationship between masticatory muscle structure and facial form. Human studies in this area, however, have been limited, especially in consideration of the myosin heavy chain (MyHC) family of contractile proteins. The aim of this pilot study was to assess if differences were detectable between genotype with respect to MyHC isoforms and the vertical facial phenotype in a sample of nine Caucasian female patients, age range 18-49 years, using a novel rapid technique. Masseter muscle biopsies were taken from patients with a range of vertical facial form. The levels of expression of the MyHC isoform genes MYH 1, 2, 3, 6, 7, and 8 were compared with the expression in a female calibrator patient aged 23 years with normal vertical facial form, using quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. Statistical analysis was undertaken using Pearson correlation coefficient. The results showed that there were distinct differences in gene expression between patients with a wide range of variation although changes in MYH1 were consistent with one cephalometric variable, the maxillo-mandibular angle. The full procedure, from start to finish, can be completed within half a day. Rapid genotyping of patients in this way could reveal important information of relevance to treatment. This technology has potential as a diagnostic and prognostic aid when considering correction of certain malocclusions.