Spinal muscular atrophy is a genetic disorder caused by deletion of the survival motor neuron 1 (SMN1) gene that leads to loss of motor neurons in the spinal cord. Though motor neurons are selectively lost during SMA pathology, selective replacement of SMN in motor neurons does not lead to full rescue in mouse models. Due to the ubiquitous expression of SMN, it is likely that other cell types besides motor neurons are affected by its disruption and therefore may contribute to disease pathology. Here we show that astrocytes in SMAΔ7 mouse spinal cord and from SMA induced pluripotent stem cells (iPSCs) exhibit morphological and cellular changes indicative of activation prior to overt motor neuron loss. Furthermore, our in vitro studies show mis regulation of basal calcium and decreased response to ATP stimulation indicating abnormal astrocyte function. Together, these data show for the first time early disruptions in astrocytes that may contribute to SMA disease pathology.
We have developed a simple method to generate and expand multipotent, self-renewing pre-rosette neural stem cells from both human embryonic stem cells (hESCs) and human induced pluripotent stem cells (iPSCs) without utilizing embryoid body formation, manual selection techniques, or complex combinations of small molecules. Human ESC and iPSC colonies were lifted and placed in a neural stem cell medium containing high concentrations of EGF and FGF-2. Cell aggregates (termed EZ spheres) could be expanded for long periods using a chopping method that maintained cell-cell contact. Early passage EZ spheres rapidly down-regulated OCT4 and up-regulated SOX2 and nestin expression. They retained the potential to form neural rosettes and consistently differentiated into a range of central and peripheral neural lineages. Thus, they represent a very early neural stem cell with greater differentiation flexibility than other previously described methods. As such, they will be useful for the rapidly expanding field of neurological development and disease modeling, high-content screening, and regenerative therapies based on pluripotent stem cell technology.
Using stem cells to replace degenerating muscle cells and restore lost skeletal muscle function is an attractive therapeutic strategy for treating neuromuscular diseases. Myogenic progenitors are a valuable cell type for cell-based therapy and also provide a platform for studying normal muscle development and disease mechanisms in vitro. Human pluripotent stem cells represent a valuable source of tissue for generating myogenic progenitors. Here, we present a novel protocol for deriving myogenic progenitors from human embryonic stem (hES) and induced pluripotent stem (iPS) cells using free-floating spherical culture (EZ spheres) in a defined culture medium. hES cell colonies and human iPS cell colonies were expanded in medium supplemented with high concentrations (100 ng/ml) of fibroblast growth factor-2 (FGF-2) and epidermal growth factor in which they formed EZ spheres and were passaged using a mechanical chopping method. We found myogenic progenitors in the spheres after 6 weeks of culture and multinucleated myotubes following sphere dissociation and 2 weeks of terminal differentiation. A high concentration of FGF-2 plays a critical role for myogenic differentiation and is necessary for generating myogenic progenitors from pluripotent cells cultured as EZ spheres. Importantly, EZ sphere culture produced myogenic progenitors from human iPS cells generated from both healthy donors and patients with neuromuscular disorders (including Becker's muscular dystrophy, spinal muscular atrophy, and familial amyotrophic lateral sclerosis). Taken together, this study demonstrates a simple method for generating myogenic cells from pluripotent sources under defined conditions for potential use in disease modeling or cell-based therapies targeting skeletal muscle. STEM CELLS TRANSLATIONAL MEDICINE 2014;3:564-574
Spinal muscular atrophy (SMA) is a genetic disorder caused by a deletion of the survival motor neuron 1 gene leading to motor neuron loss, muscle atrophy, paralysis, and death. We show here that induced pluripotent stem cell (iPSC) lines generated from two Type I SMA subjects–one produced with lentiviral constructs and the second using a virus-free plasmid–based approach–recapitulate the disease phenotype and generate significantly fewer motor neurons at later developmental time periods in culture compared to two separate control subject iPSC lines. During motor neuron development, both SMA lines showed an increase in Fas ligand-mediated apoptosis and increased caspase-8 and-3 activation. Importantly, this could be mitigated by addition of either a Fas blocking antibody or a caspase-3 inhibitor. Together, these data further validate this human stem cell model of SMA, suggesting that specific inhibitors of apoptotic pathways may be beneficial for patients.
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