Epimorphic regeneration approach to tissue replacement in adult mammals

Abstract

Urodeles and fetal mammals are capable of impressive epimorphic regeneration in a variety of tissues, whereas the typical default response to injury in adult mammals consists of inflammation and scar tissue formation. One component of epimorphic regeneration is the recruitment of resident progenitor and stem cells to a site of injury. Bioactive molecules resulting from degradation of extracellular matrix (ECM) have been shown to recruit a variety of progenitor and stem cells in vitro in adult mammals. The ability to recruit multipotential cells to the site of injury by in vivo administration of chemotactic ECM degradation products in a mammalian model of digit amputation was investigated in the present study. Adult, 6- to 8-week-old C57/BL6 mice were subjected to midsecond phalanx amputation of the third digit of the right hind foot and either treated with chemotactic ECM degradation products or left untreated. At 14 days after amputation, mice treated with ECM degradation products showed an accumulation of heterogeneous cells that expressed markers of multipotency, including Sox2, Sca1, and Rex1 (Zfp42). Cells isolated from the site of amputation were capable of differentiation along neuroectodermal and mesodermal lineages, whereas cells isolated from control mice were capable of differentiation along only mesodermal lineages. The present findings demonstrate the recruitment of endogenous stem cells to a site of injury, and/or their generation/proliferation therein, in response to ECM degradation products.

Fig. 1.

The in vitro migration of perivascular stem cells from the upper chamber of a Boyden assay to the lower chamber only in the presence of an ECM gradient confirms chemotactic and not chemokinetic activity of the degradation products. Error bars are SD among three wells, with a similar trend seen on three separate occasions (A). Gross appearance of digits in mice on day 14 after amputation and injection of ECM degradation products (B) or no injection (C). Arrows denote the amputated digit. Trichrome staining of digits in mice on day 14 postamputation and injection of ECM degradation products (D) or no injection (E). Histologic Trichrome-stained sections are at a magnification of ×40 and ×200.

Fig. 2.

Histologic sections of cell accumulations distal to the site of amputation in mice 14 days after amputation and injection of ECM degradation products or no treatment after staining for markers of multipotency. All images were taken at a magnification of ×400. Cell counts are displayed in units of number of cells. *P < 0.05; **P < 0.005 (between treatment and no treatment, or treatment and uninjured controls). Error bars are SEM.

Fig. 3.

In vitro lineage differentiation potential of cell isolated distal to the site of amputation in digits of mice treated with ECM degradation products and untreated. Neuroectodermal differentiation was confirmed via expression for neuroectodermal markers (β-tubulin-III, NeuN, and GFAP). Adipogenic differentiation was confirmed via Oil Red O staining for the presence of lipid vacuoles. Osteogenic differentiation was confirmed via Alizarin Red staining for calcium deposition.