Neural crest progenitors and stem cells

In the vertebrate embryo, multiple cell types originate from a common structure, the neural crest (NC), which forms at the dorsal tips of the neural epithelium. The NC gives rise to migratory cells that colonise a wide range of embryonic tissues and later differentiate into neurones and glial cells of the peripheral nervous system (PNS), pigment cells (melanocytes) in the skin and endocrine cells in the adrenal and thyroid glands. In the head and the neck, the NC also yields mesenchymal cells that form craniofacial cartilages, bones, dermis, adipose tissue, and vascular smooth muscle cells. The NC is therefore a model system to study cell diversification during embryogenesis and phenotype maintenance in the adult. By analysing the developmental potentials of quail NC cells in clonal cultures, we have shown that the migratory NC is a collection of heterogeneous progenitors, including various types of intermediate precursors and highly multipotent cells, some of which being endowed of self-renewal capacity. We also have identified common progenitors for mesenchymal derivatives and neural/melanocytic cells in the cephalic NC. These results are consistent with a hierarchical model of lineage segregation wherein environmental cytokines control the fate of progenitors and stem cells. One of these cytokines, the endothelin3 peptide, promotes the survival, proliferation, and self-renewal capacity of common progenitors for glial cells and melanocytes. At post-migratory stages, when they have already differentiated, NC-derived cells exhibit phenotypic plasticity. Epidermal pigment cells and Schwann cells from peripheral nerves in single-cell culture are able to reverse into multipotent NC-like progenitors endowed with self-renewal. Therefore, stem cell properties are expressed by a variety of NC progenitors and can be re-acquired by differentiated cells of NC origin, suggesting potential function for repair

Dermal Substitutes Support the Growth of Human Skin-Derived Mesenchymal Stromal Cells: Potential Tool for Skin Regeneration

<div><p>New strategies for skin regeneration are needed in order to provide effective treatment for cutaneous wounds and disease. Mesenchymal stem cells (MSCs) are an attractive source of cells for tissue engineering because of their prolonged self-renewal capacity, multipotentiality, and ability to release active molecules important for tissue repair. In this paper, we show that human skin-derived mesenchymal stromal cells (SD-MSCs) display similar characteristics to the multipotent MSCs. We also evaluate their growth in a three-dimensional (3D) culture system with dermal substitutes (Integra and Pelnac). When cultured in monolayers, SD-MSCs expressed mesenchymal markers, such as CD105, Fibronectin, and α-SMA; and neural markers, such as Nestin and βIII-Tubulin; at transcriptional and/or protein level. Integra and Pelnac equally supported the adhesion, spread and growth of human SD-MSCs in 3D culture, maintaining the MSC characteristics and the expression of multilineage markers. Therefore, dermal substitutes support the growth of mesenchymal stromal cells from human skin, promising an effective tool for tissue engineering and regenerative technology.</p></div

Comparative Experimental Study of Wound Healing in Mice: Pelnac versus Integra

<div><p>Strategies for skin regeneration have been developed to provide effective treatment for cutaneous wounds and disease. Dermal substitutes have been used to cover the lesion to facilitate cell colonization, thereby promoting dermal regeneration. However, very little is known about Pelnac matrix especially at histological level. Therefore, the present work carried out an experimental <i>in vivo</i> comparative analysis between Pelnac and Integra, the most used dermal templates, in a mouse model of full-thickness skin wounds. Histological sections performed at the 3^rd, 6^th and 9^th days after surgery were analyzed with regard to inflammatory response and vascularization. Both templates were completely incorporated in all animals at the end of the analyzed period. Pelnac-treated animals displayed reduced granulation tissue during the first 6 days of treatment compared to the animals treated with Integra at the same time period. The number of inflammatory cells (neutrophils) was similar in both groups during the period, significantly reducing at the end of inflammatory phase (9^th day of treatment) consistent with the progression of healing process. In addition, the density of blood vessels was also statistically similar in both matrices. Therefore, the two dermal templates displayed comparable biological behavior in tissue repair. It is noteworthy that this is the first experimental study comparing Pelnac and Integra dermal templates with focus on full-thickness skin wounds.</p></div

Fibroblast growth factor-2 bound to specific dermal fibroblast-derived extracellular vesicles is protected from degradation

Fibroblast growth factor-2 (FGF2) has multiple roles in cutaneous wound healing but its natural low stability prevents the development of its use in skin repair therapies. Here we show that FGF2 binds the outer surface of dermal fibroblast (DF)-derived extracellular vesicles (EVs) and this association protects FGF2 from fast degradation. EVs isolated from DF cultured in the presence of FGF2 harbor FGF2 on their surface and FGF2 can bind purified EVs in absence of cells. Remarkably, FGF2 binding to EVs is restricted to a specific subpopulation of EVs, which do not express CD63 and CD81 markers. Treatment of DF with FGF2-EVs activated ERK and STAT signaling pathways and increased cell proliferation and migration. Local injection of FGF2-EVs improved wound healing in mice. We further demonstrated that binding to EVs protects FGF2 from both thermal and proteolytic degradation, thus maintaining FGF2 function. This suggests that EVs protect soluble factors from degradation and increase their stability and half-life. These results reveal a novel aspect of EV function and suggest EVs as a potential tool for delivering FGF2 in skin healing therapies

MSC phenotypic characterization of human skin-derived cells.

<p>(A) Morphological analysis of skin-derived cells by phase contrast microscopy. (B) MTS cell proliferation/viability assay. (C) Osteogenic and (D) adipogenic differentiation. (C) Cells cultured in inductive medium formed Alizarin Red S-stained mineralized nodules and (D) Oil red O-stained lipid clusters. (E) Flow cytometry analysis of hematopoietic (CD34, CD45) and MSC (CD90, CD73, CD105) markers. Specific markers are shown by black curves and controls by gray curves. ***p<0.001. Scale bar: (C–F): 50 µm. Other pictures: 200 µm.</p

Multilineage potential of SD-MSCs.

<p>(A) Gene expression profile by RT-PCR of SD-MSCs cultivated in standard medium. (B–E) Immunofluorescence staining of (B) CD105 and (C) Fibronectin. (D–F) Double-staining of α-SMA and βIII-Tubulin, (G–I) α-SMA and Nestin and (J–L) βIII-Tubulin and Nestin co-expression. (F, I and L): Merged pictures of D–E, G–H, J–K, respectively. Cell nuclei were stained with DAPI (blue). Arrows: Nestin nuclear staining. Scale bar: 50 µm.</p

Immunophenotypic profile of human SD-MSCs cultured in Integra and Pelnac.

<p>Flow cytometry analysis of SD-MSCs (CD90, CD73, CD105) and hematopoietic (CD45) markers in SD-MSCs after 3 days of culture in Integra (upper panel) and Pelnac (lower panel). Curves in black show the specific markers, and gray curves correspond to controls.</p

3D cultures of human SD-MSCs in (A–D) Integra and (E–H) Pelnac.

<p>Confocal microscopy of SD-MSCs cultured in (A–C) Integra and (D–F) Pelnac. (A and E) DAPI nuclear staining of SD-MSCs. (B and F) Integra and (F) Pelnac dermal substitutes, respectively (green autofluorescence). (C and G) merged images of (A and B) and (E and F), respectively. (D and H) MTS cell viability assay of SD-MSCs cultivated in Integra and Pelnac, respectively. ***p<0.001, **p<0.01. Scale bar: 100 um.</p

Scanning electron microscopy (SEM) images of SD-MSCs cultured in (A–D) Integra and (E–H) Pelnac.

<p>(A) Cross-sectional view of Integra dermal substitute alone showing the silicone (*) and the inner layer. (B–D) 3D culture of SD-MSCs in Integra 48 hours after seeding. (E) Surface and (F) cross-sectional views of Pelnac showing the collagen layer. (G–H) 3D culture of SD-MSCs in Pelnac 48 hours after seeding. Insets in B: different magnifications of a SD-MSC.</p

Histological aspects of wounds treated with dermal substitutes.

<p>HE-stained transversal sections of a full-thickness skin wound treated with (A-C) Integra or (D-F) Pelnac collected at (A, D) 3, (B, E) 6 and (C, F) 9 days after the procedure show a clear distinction between the dermal substitute (DM) and the wound bed (WB). Original magnification 100X.</p