Barbara Gawronska-Kozak

Scarless skin repair in immunodeficient mice

Scarring, the end result of the wound healing process in adult mammals, is a problem of significant clinical importance. We observed that athymic nude‐nu mice, similar to mammalian fetuses, are able to restore the structure and integrity of injured skin through a process resembling regeneration, where scar formation is absent. Among the postinjured skin tissues collected from athymic nude‐nu, wild‐type controls (C57BL/6J), severe‐combined immunodeficient, Rag (lack of B and T cells), athymic (thymectomized neonates and adult C57BL/6J), and mice treated with an immunosuppressant (cyclosporin A), only athymic nude‐nu mice showed: a lack of scar by histological examination (hematoxylin & eosin and Massons trichrome staining), low levels of collagen (as determined by hydroxyproline content), high levels of hyaluronic acid, a statistically significant increase in elastic modulus for injured samples over unwounded (biomechanical testing) and low levels of the pro‐scarring cytokines platelet‐derived growth factor‐B and transforming growth factor β1. Additionally, immunohistochemical and Western blot analyses of postinjured tissues as well as flow cytometry analysis of blood samples showed the presence of CD8‐positive cells in all studied animals except nude‐nu mice. We conclude that scarless skin healing in athymic nude‐nu mice provides a new model to study the influence of the immune system on tissue regeneration.

Mesenchymal stem cells from the outer ear: a novel adult stem cell model system for the study of adipogenesis

Adipocytes arise from multipotent stem cells of mesodermal origin, which also give rise to the muscle, bone, and cartilage lineages. However, signals and early molecular events that commit multipotent stem cells into the adipocyte lineage are not well established mainly due to lack of an adequate model system. We have identified a novel source of adult stem cells from the external murine ears referred to here as an ear mesenchymal stem cells (EMSC). EMSC have been isolated from several standard and mutant strains of mice. They are self‐renewing, clonogenic, and multipotent, since they give rise to osteocytes, chondrocytes, and adipocytes. The in vitro characterization of EMSC indicates very facile adipogenic differentiation. Morphological, histochemical, and molecular analysis after the induction of differentiation showed that EMSC maintain adipogenic potentials up to fifth passage. A comparison of EMSC to the stromal‐vascular (S‐V) fraction of fat depots, under identical culture conditions (isobutyl‐methylxanthine, dexamethasone, and insulin), revealed much more robust and consistent adipogenesis in EMSC than in the S‐V fraction. In summary, we show that EMSC can provide a novel, easily obtainable, primary culture model for the study of adipogenesis.

Scarless skin wound healing in FOXN1 deficient (nude) mice is associated with distinctive matrix metalloproteinase expression.

Similar to mammalian fetuses FOXN1 deficient (nude) mice are able to restore the structure and integrity of injured skin in a scarless healing process by mechanisms independent of the genetic background. Matrix metalloproteinases (MMPs) are required for regular skin wound healing and the distinctive pattern of their expression has been implicated to promote scarless healing. In this study, we analyzed the temporal and spatial expression patterns of these molecules during the incisional skin wounds in adult nude mice. Macroscopic and histological analyses of skin wounds revealed an accelerated wound healing process, minimal granulation tissue formation and markedly diminished scarring in nude mice. Quantitative RT-PCR (Mmp-2, -3, -8, -9, -10, -12, -13, -14 and Timp-1, -2, -3), Western blots (MMP-13) and gelatin zymography (MMP-9) revealed that MMP-9 and MMP-13 showed a unique, bimodal pattern of up-regulation during the early and late phases of wound healing in nude mice. Immunohistochemically MMP-9 and MMP-13 were generally detected in epidermis during the early phase and in dermis during the late (remodeling) phase. Consistent with these in vivo observations, dermal fibroblasts cultured from nude mice expressed higher levels of types I and III collagen, MMP-9 and MMP-13 mRNA levels and higher MMP enzyme activity than wild type controls. Collectively, these finding suggest that the bimodal pattern of MMP-9 and MMP-13 expression during skin repair process in nude mice could be a major component of their ability for scarless healing.

Role of Adiponectin and Inflammation in Insulin Resistance of Mc3r and Mc4r Knockout Mice

Objective: To investigate the involvement of hypoadiponectinemia and inflammation in coupling obesity to insulin resistance in melanocortin‐3 receptor and melanocortin‐4 receptor knockout (KO) mice (Mc3/4rKO).

Ear mesenchymal stem cells (EMSC) can differentiate into spontaneously contracting muscle cells

We have previously shown that cells isolated from the outer ears of adult mice are a source of mesenchymal stem cells that can be induced to differentiate into adipo‐, osteo‐, and chondrocytes. In this study, we demonstrate that ear mesenchymal stem cells (EMSC) express stromal cell‐associated markers (CD44, CD73) and stem cell marker Sca‐1 and can be differentiated into spontaneously contracting muscle cells. Treatment of cells with epidermal growth factor (EGF) change their morphology from fibroblast shapes into stick‐like structures that show repeated spontaneous contractions. Under conditions that promote myogenic differentiation, EMSC expressed mRNA for myoD and ventricular specific myosin light chain (MLC‐2v) and protein for connexin 43, sarcomeric α‐actinin, myocyte enhancer factor 2c (MEF2c), myosin heavy chain (MyHC), myogenin, and sarco‐endoplasmic reticulum Ca2+ATPase (SERCA) 1. However, the cells were negative for Nkx2.5, GATA4, and ANP. Intracellular Ca2+ transients in spontaneously beating EMSC, visualized by Fluo‐3AM, showed a frequency of Ca2+ oscillations ranging over 28–59/min (mean 41.17 ± SEM 1.54). We also demonstrated that small pieces of ear tissues (ear punches) collected from live mice provide sufficient numbers of EMSC to isolate, culture and differentiate them into myocytes. Due to the ease of acquiring an expanding repertoire of differentiated EMSC cell types by a noninvasive surgical procedure, we conclude that the ear may prove to be a potential source of autologous cells for regenerative medicine, as supported by the fact that ears are one of the best sources of cells for somatic cell nuclear transfer (SCNT). J. Cell. Biochem. 102: 122–135, 2007.

Flow cytometric and immunohistochemical detection of in vivo BrdU-labeled cells in mouse fat depots.

This study has determined the natural frequency and localization of progenitor/stem cells within fat depots in situ based on their ability to retain DNA nucleotide label (BrdU). Neonate and mature male C57BL6/J mice were injected intraperitoneally with BrdU- and label-retaining cells (LRC) were quantified in fat depots by immunohistochemical, immunofluorescent, and flow cytometric methods. In neonates, LRC constituted 27% of the cells in inguinal fat (iWAT) and 65% in interscapular brown fat (BAT) after Day 10 and 26% of the cells in epididymal fat (eWAT) after Day 28. After 52 days, the LRC accounted for 0.72% of iWAT, 0.53% of eWAT and 1.05% of BAT, respectively. The BrdU-labeled cells localized to two areas: single cells distributed among adipocytes or those adjacent to the blood vessels wall. In mature C57BL6/J mice, flow cytometric analysis determined that a majority of the LRC were also positive for stem cell antigen-1 (Sca-1).

Recruitment of fat cell precursors during high fat diet in C57BL/6J mice is fat depot specific

The study aims to explore long‐term dietary effects on increases in body mass and fat depot enlargement through the recruitment of early in life labeled progenitor cells to the adipolineage.