Intravenous SPION-labeled adipocyte-derived stem cells targeted to the brain by magnetic attraction in a rat stroke model: An ultrastructural insight into cell fate within the brain.

Mesenchymal stem cell therapy after stroke is a promising option investigated in animal models and clinical trials. The intravenous route is commonly used in clinical settings guaranteeing an adequate safety profile although low yields of engraftment. In this report, rats subjected to ischemic stroke were injected with adipose-derived stem cells (ADSCs) labeled with superparamagnetic iron oxide nanoparticles (SPIONs) applying an external magnetic field in the skull to retain the cells. Although most published studies demonstrate viability of ADSCs, only a few have used ultrastructural techniques. In our study, the application of a local magnetic force resulted in a tendency for higher yields of SPION-ADSCs targeting the brain. However, grafted cells displayed morphological signs of death, one day after administration, and correlative microscopy showed active microglia and astrocytes associated in the process of scavenging. Thus, we conclude that, although successfully targeted within the brain, SPION-ADSCs viability was rapidly compromised.

Inflammatory demyelination induces ependymal modifications concomitant to activation of adult (SVZ) stem cell proliferation.

Ependymal cells (E1/E2) and ciliated B1cells confer a unique pinwheel architecture to the ventricular surface of the subventricular zone (SVZ), and their cilia act as sensors to ventricular changes during development and aging. While several studies showed that forebrain demyelination reactivates the SVZ triggering proliferation, ectopic migration, and oligodendrogenesis for myelin repair, the potential role of ciliated cells in this process was not investigated. Using conventional and lateral wall whole mount preparation immunohistochemistry in addition to electron microscopy in a forebrain-targeted model of experimental autoimmune encephalomyelitis (tEAE), we show an early decrease in numbers of pinwheels, B1 cells, and E2 cells. These changes were transient and simultaneous to tEAE-induced SVZ stem cell proliferation. The early drop in B1/E2 cell numbers was followed by B1/E2 cell recovery. While E1 cell division and ependymal ribbon disruption were never observed, E1 cells showed important morphological modifications reflected by their enlargement, extended cytoskeleton, and reinforced cell-cell junction complexes overtime, possibly reflecting protective mechanisms against ventricular insults. Finally, tEAE disrupted motile cilia planar cell polarity and cilia orientation in ependymal cells. Therefore, significant ventricular modifications in ciliated cells occur early in response to tEAE suggesting a role for these cells in SVZ stem cell signalling not only during development/aging but also during inflammatory demyelination. These observations may have major implications for understanding pathophysiology of and designing therapeutic approaches for inflammatory demyelinating diseases such as MS.

Identification and Characterization of the Dermal Panniculus Carnosus Muscle Stem Cells.

The dermal Panniculus carnosus (PC) muscle is important for wound contraction in lower mammals and represents an interesting model of muscle regeneration due to its high cell turnover. The resident satellite cells (the bona fide muscle stem cells) remain poorly characterized. Here we analyzed PC satellite cells with regard to developmental origin and purported function. Lineage tracing shows that they originate in Myf5(+), Pax3/Pax7(+) cell populations. Skin and muscle wounding increased PC myofiber turnover, with the satellite cell progeny being involved in muscle regeneration but with no detectable contribution to the wound-bed myofibroblasts. Since hematopoietic stem cells fuse to PC myofibers in the absence of injury, we also studied the contribution of bone marrow-derived cells to the PC satellite cell compartment, demonstrating that cells of donor origin are capable of repopulating the PC muscle stem cell niche after irradiation and bone marrow transplantation but may not fully acquire the relevant myogenic commitment.

Murine muscle engineered from dermal precursors: an in vitro model for skeletal muscle generation, degeneration, and fatty infiltration.

Skeletal muscle can be engineered by converting dermal precursors into muscle progenitors and differentiated myocytes. However, the efficiency of muscle development remains relatively low and it is currently unclear if this is due to poor characterization of the myogenic precursors, the protocols used for cell differentiation, or a combination of both. In this study, we characterized myogenic precursors present in murine dermospheres, and evaluated mature myotubes grown in a novel three-dimensional culture system. After 5-7 days of differentiation, we observed isolated, twitching myotubes followed by spontaneous contractions of the entire tissue-engineered muscle construct on an extracellular matrix (ECM). In vitro engineered myofibers expressed canonical muscle markers and exhibited a skeletal (not cardiac) muscle ultrastructure, with numerous striations and the presence of aligned, enlarged mitochondria, intertwined with sarcoplasmic reticula (SR). Engineered myofibers exhibited Na(+)- and Ca(2+)-dependent inward currents upon acetylcholine (ACh) stimulation and tetrodotoxin-sensitive spontaneous action potentials. Moreover, ACh, nicotine, and caffeine elicited cytosolic Ca(2+) transients; fiber contractions coupled to these Ca(2+) transients suggest that Ca(2+) entry is activating calcium-induced calcium release from the SR. Blockade by d-tubocurarine of ACh-elicited inward currents and Ca(2+) transients suggests nicotinic receptor involvement. Interestingly, after 1 month, engineered muscle constructs showed progressive degradation of the myofibers concomitant with fatty infiltration, paralleling the natural course of muscular degeneration. We conclude that mature myofibers may be differentiated on the ECM from myogenic precursor cells present in murine dermospheres, in an in vitro system that mimics some characteristics found in aging and muscular degeneration.

Adult neural stem cells from the subventricular zone: a review of the neurosphere assay.

The possibility of obtaining large numbers of cells with potential to become functional neurons implies a great advance in regenerative medicine. A source of cells for therapy is the subventricular zone (SVZ) where adult neural stem cells (NSCs) retain the ability to proliferate, self-renew, and differentiate into several mature cell types. The neurosphere assay, a method to isolate, maintain, and expand these cells has been extensively utilized by research groups to analyze the biological properties of aNSCs and to graft into injured brains from animal models. In this review we briefly describe the neurosphere assay and its limitations, the methods to optimize culture conditions, the identity and the morphology of aNSC-derived neurospheres (including new ultrastructural data). The controversy regarding the identity and "stemness" of cells within the neurosphere is revised. The fine morphology of neurospheres, described thoroughly, allows for phenotypical characterization of cells in the neurospheres and may reveal slight changes that indirectly inform about cell integrity, cell damage, or oncogenic transformation. Along this review we largely highlight the critical points that researchers have to keep in mind before extrapolating results or translating experimental transplantation of neurosphere-derived cells to the clinical setting.