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.

Sexual dimorphism of human sternum in a contemporary Spanish population.

Sex estimation is one of the first steps in forensic anthropology to identify human remains. In absence of the skull or the pelvis, any skeletal remain becomes fundamental for identification, especially in mass-disaster cases. The sternum is a potentially useful element in anthropological analysis with a high recovery rate in both forensic-and archaeological context. This study aims to develop classification functions for use in Spanish population. For this, sternum sexual dimorphism is studied in a sample of 105 individuals, known age-at-death, ancestry and sex, from San José Municipal Cemetery of Granada (Spain). Lin's concordance correlation coefficient was used to estimate intra-and inter-observer error. In discriminant analysis for estimating sex, cross-validation shows accuracy rates exceeds 90% for sternum body length and maximum width (91.8%), or total length with maximum width (90.7%). Isolated variables with higher accuracy rates are total sternum length (89.1%), and sternum body length (87%). Although there is compliance with Hyrtl's law it is not useful for estimating sex in Spanish population. These discriminant functions have also been validated successfully in two samples from Portugal (Coimbra identified skeletal collection--CISC, and 21st century identified ckeletal collection--Santarém XXI): the variables with higher accuracy rates sternum total length with its maximum width (92.3% the correctly classified individual in the sample CISC; and 83.5% in the sample of Santarém XXI) and the sternum total length (92.1% and 78.5%, respectively). The discriminant functions achieved with the collection of the San Jose cemetery of Granada can be applied to current remains, provided that study populations present a similar sexual dimorphism, like the two samples from Portuguese population presented in this study.

Morphometric sex estimation from 3D computed tomography os coxae model and its validation in skeletal remains.

In the last decade, the advances in imaging technologies have intensified the use of multislice computed tomography (MSCT) for anthropological purposes. The published literature has proved it to be a suitable tool for establishing sexually dimorphic characteristics in different anatomical areas. In this context, the main purpose of the present study was to explore the accuracy of traditional morphometric method when applied to data acquired in three-dimensional (3D) reconstructed os coxae of living Spanish population, to develop a series of statistically robust patterns for sex assessment and to test their validity in innominate remains. For this purpose, 150 volume-rendered innominate CT scans were selected to examine nine interlandmark linear distances by means of descriptive statistics and discriminant function analyses (DFA) employing the jackknife procedure for cross-validations. Five measurements were sexually dimorphic. Acetabular diameter was the single most accurate predictor. This, combined with innominate height and innominate breadth, contributed very significantly to sex estimation. Overall, classification accuracies were 89.3-95.3 % (89.3-94.7 % after cross-validation), with a sex-bias lower than 5 %. The second validation approach performed on a sample of 96 individuals from another contemporary Spanish reference collection comprising dry bones showed the high percentage of accurate classification (83.3-95.8 %). In conclusion, the assessment of sex using cross-sectional MSCT images of the os coxae is possible and the discriminant functions obtained on Spanish living individuals can also be effective for estimating sex from skeletal remains.

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.

A neural extracellular matrix-based method for in vitro hippocampal neuron culture and dopaminergic differentiation of neural stem cells.

BACKGROUND: The ability to recreate an optimal cellular microenvironment is critical to understand neuronal behavior and functionality in vitro. An organized neural extracellular matrix (nECM) promotes neural cell adhesion, proliferation and differentiation. Here, we expanded previous observations on the ability of nECM to support in vitro neuronal differentiation, with the following goals: (i) to recreate complex neuronal networks of embryonic rat hippocampal cells, and (ii) to achieve improved levels of dopaminergic differentiation of subventricular zone (SVZ) neural progenitor cells. METHODS: Hippocampal cells from E18 rat embryos were seeded on PLL- and nECM-coated substrates. Neurosphere cultures were prepared from the SVZ of P4-P7 rat pups, and differentiation of neurospheres assayed on PLL- and nECM-coated substrates. RESULTS: When seeded on nECM-coated substrates, both hippocampal cells and SVZ progenitor cells showed neural expression patterns that were similar to their poly-L-lysine-seeded counterparts. However, nECM-based cultures of both hippocampal neurons and SVZ progenitor cells could be maintained for longer times as compared to poly-L-lysine-based cultures. As a result, nECM-based cultures gave rise to a more branched neurite arborization of hippocampal neurons. Interestingly, the prolonged differentiation time of SVZ progenitor cells in nECM allowed us to obtain a purer population of dopaminergic neurons. CONCLUSIONS: We conclude that nECM-based coating is an efficient substrate to culture neural cells at different stages of differentiation. In addition, neural ECM-coated substrates increased neuronal survival and neuronal differentiation efficiency as compared to cationic polymers such as poly-L-lysine.

Modeling neural differentiation on micropatterned substrates coated with neural matrix components.

Topographical and biochemical characteristics of the substrate are critical for neuronal differentiation including axonal outgrowth and regeneration of neural circuits in vivo. Contact stimuli and signaling molecules allow neurons to develop and stabilize synaptic contacts. Here we present the development, characterization and functional validation of a new polymeric support able to induce neuronal differentiation in both PC12 cell line and adult primary skin-derived precursor cells (SKPs) in vitro. By combining a photolithographic technique with use of neural extracellular matrix (ECM) as a substrate, a biocompatible and efficient microenvironment for neuronal differentiation was developed.