Isolation and Characterization of Mesoangioblasts from Mouse, Dog, and Human Tissues

Abstract

Mesoangioblasts are recently identified stem/progenitor cells, associated with small vessels of the mesoderm in mammals. Originally described in the mouse embryonic dorsal aorta, similar though not identical cells have been later identified and characterized from postnatal small vessels of skeletal muscle and heart (not described in this unit). They have in common the anatomical location, the expression of endothelial and/or pericyte markers, the ability to proliferate in culture, and the ability to undergo differentiation into various types of mesoderm cells upon proper culture conditions. Currently, the developmental origin of mesoangioblasts, their phenotypic heterogeneity, and the relationship with other mesoderm stem cells are not understood in detail and are the subject of active research. However, from a practical point of view, these cells have been successfully used in cell transplantation protocols that have yielded a significant rescue of structure and function in skeletal muscle of dystrophic mice and dogs. Since the corresponding human cells have been recently isolated and characterized, a clinical trial with these cells is planned in the near future. This unit provides detailed methods for isolation, culture, and characterization of mesoangioblasts. Curr. Protoc. Stem Cell Biol. 3:2B.1.1-2B.1.29. C 2007 by John Wiley & Sons, Inc.Keywords: mesoangioblasts r pericytes r mesoderm progenitor cells r cell culture NOTE: Mesoangioblasts and pericyte-derived cells must be cultured under physiological O 2 conditions (5% O 2 , 5% CO 2 , 90% N 2 ; see Critical Parameters).

Necdin enhances muscle reconstitution of dystrophic muscle by vessel-associated progenitors, by promoting cell survival and myogenic differentiation

“…We isolated MABs from biopsies of muscles of adult (6-month old) C57 mice exactly according to Tonlorenzi et al 14 ( Figure 1a). Proliferating MABs (wt MABs) express very low level of necdin, therefore to study its role in these cells we generated a cell line that overexpresses necdin constitutively: we produced a lentiviral vector expressing necdin under the constitutive PGK promoter and eGFP under the CMV promoter in opposite orientation, to infect wt MABs (NDNMABs) (Figure 1b).…”

“…Unlike their embryonic counterpart, MABs derived from adult muscle are able to differentiate in skeletal muscle cells spontaneously, without the need of co-culture with myoblasts and yet they differ from satellite cells because do not express Pax7 14,16 (Figure 1g; Supplementary Figure 1B). Skeletal muscle differentiation was induced by culturing NDNMABs and GFPMABs in muscle-differentiation medium ( Figure 3a) and fusion index was calculated to compare their differentiation potential: NDNMABs showed a higher fusion index (NDNMABs: 54%; GFPMABs: 26%), with the progressive appearance of an increased number of myotubes with more nuclei over time, and in particular with 410 nuclei at 6 days (Figures 3b and c).…”

“…Stem cell populations with myogenic potential can be isolated from multiple regions of the body. 18,19 Until now, MABs appear to fulfil most of the features required for a clinical use in human patients 3,14,16 and have shown to significantly contribute to the restoration of the muscle structure and function in mouse and dog models of muscular dystrophy. 4,5,20 Still protocol optimization for cell therapy is ongoing, and different drug and gene therapy approaches have been investigated to improve their clinical outcome.…”

“…MABs were isolated and cultured exactly as previously described in Tonlorenzi et al 14 Specifically, adultderived murine MABs were isolated from muscular biopsies of a 6-month-old C57BL/6 mouse hind legs. After dissecting both the TA and rinse them in PBS with Ca 2 þ -Mg 2 þ to remove residual blood, they were carefully cleaned from adipose and connective tissues, trying to identify portions of interstitial tissue containing small vessels.…”

Characterization of in vitro cultured myoblasts isolated from duck (Anas platyrhynchos) embryo

“…Techniques to study the gene regulation mechanism on a molecular level, like RNA interference (RNAi) and gene transfection, could be performed for a better understanding of the myoblasts growth characteristics. Myoblasts have been isolated and cultured in many species including mouse, dog, chicken, and so on (Linkhart et al 1980;Tonlorenzi et al 2007), except for duck. The absent information of duck's myoblasts is the main obstacles for further investigating the mechanisms of muscle development in duck.…”