Recruitment of progenitor cells by an extracellular matrix cryptic peptide in a mouse model of digit amputation

Abstract

Biologic scaffolds composed of extracellular matrix (ECM) have been used successfully in preclinical models and humans for constructive remodeling of functional, site-appropriate tissue after injury. The mechanisms underlying ECM-mediated constructive remodeling are not completely understood, but scaffold degradation and site-directed recruitment of both differentiated and progenitor cells are thought to play critical roles. Previous studies have shown that degradation products of ECM scaffolds can recruit a population of progenitor cells both in vitro and in vivo. The present study identified a single cryptic peptide derived from the α subunit of the collagen III molecule that is chemotactic for a well-characterized perivascular stem cell in vitro and causes the site-directed accumulation of progenitor cells in vivo. The oligopeptide was additionally chemotactic for human cortical neural stem cells, rat adipocyte stem cells, C2C12 myoblast cells, and rat Schwann cells in vitro. In an adult murine model of digit amputation, treatment with this peptide after mid-second phalanx amputation resulted in a greater number of Sox2+ and Sca1+,Lin- cells at the site of injury compared to controls. Since progenitor cell activation and recruitment are key prerequisites for epimorphic regeneration in adult mammalian tissues, endogenous site-directed recruitment of such cells has the potential to alter the default wound healing response from scar tissue toward regeneration.

FIG. 1.

Identification of chemotactic peptide. Urinary bladder matrix digest was fractionated and chemotactic ability quantified against perivascular stem cells by ammonium sulfate (A, B), size exclusion (C), ion exchange (D, E), and reverse phase (F, G) chromatography. Peptide was identified via mass spectroscopy and synthesized to assay for chemotactic potential for human perivascular stem cells (H). A BLAST search for the isolated peptide sequence showed over 75% homology with the Collagen IIIα molecule over eight separate species (I). Error bars are mean±SD. *p<0.05 as compared to negative control. SD, standard deviation.

FIG. 2.

Peptide promotes migration of multiple cell types in vitro. The chemotactic ability of the peptide was tested over six orders of magnitude concentration against human neuroepithelial cortical (CTX) stem cells (A), human adipose stem cells (B), mouse myoblast (C2C12) cells (C), rat Schwann (RT4-D6P2T) cells (D), human microvascular endothelial (HMEC) cells (E), and rat intestinal epithelial (IEC6) cells (F). Error bars are Mean±SD. *p<0.05 as compared to negative control.

FIG. 3.

Peptide treatment results in greater number of cells in vivo. Adult mouse hind foot digits were amputated at the mid-second phalanx and treated with 15μL of either PBS or peptide. Histologic examination by hematoxylin and eosin staining showed a thinner, invaginating epithelium concomitant with a denser cellular response after peptide treatment (A) as compared to PBS carrier control treatment (B). Histologic sections showed that peptide treatment led a greater number of Sox2, Sca1, and Ki67-positive cells at the site of amputation (C). Flow cytometric analysis confirmed that Sca1+ cells did not express markers of differentiated blood lineage (D). Co-expression of Sca1 and Sox2 was observed in subsets of cells after cytospin and co-immunolabeling (arrow) (E). Images were taken at 40×magnification (A, B), 630×magnification (A, B), or 400×magnification (C, E). Error bars are Mean±SD. *p<0.05. **p<0.01. PBS, phosphate-buffered saline. Color images available online at www.liebertonline.com/tea

FIG. 4.

To confirm that Sca1+ and Sox2+ cells were actively proliferating, isolated cells were cytospun and co-immunolabeled for either Sca1 or Sox2 and a marker of cells in the M phase of the cell cycle, phosphorylated Histone H3. Subsets of Sca1+ and Sox2+ co-expressed nuclear Histone H3 (arrows). Images were taken at 400×magnification. Color images available online at www.liebertonline.com/tea