In Situ Tissue Regeneration: Chemoattractants for Endogenous Stem Cell Recruitment

Introduction

The goal of tissue engineering is to orchestrate regeneration of injured or diseased tissue through the use of cells, signaling molecules, and/or biomaterials.^1–3 The rational design of regenerative therapies is guided by lessons learned from embryonic development, adult homeostasis, and stem cell biology.^1,3–7 Stem cell migration is required for morphogenesis and organogenesis during development and for tissue maintenance and injury repair in adults. Recruitment of mesenchymal stem cells (MSC) to an injury site is the first stage of a biomimetic therapy for tissue regeneration.

MSC are a cornerstone of tissue engineering therapies because they have multilineage potential. MSC have been isolated from bone marrow ⁸ and many other tissues, including fat, periosteum, synovium, and dental pulp.^9–11 Evidence suggests that MSC reside in perivascular niches throughout the body.^12,13 MSC differentiation has been demonstrated along mesodermal, ¹⁴ ectodermal,^15,16 and endodermal ¹⁷ lineages. ¹¹ MSC also support regeneration through trophic actions.^18,19

Regenerative therapies have typically been based on ex vivo expanded bone marrow MSC (bMSC), delivered systemically^20,21 or as part of a cell-scaffold construct.^22–24 However, there are drawbacks associated with MSC harvest and expansion, including cost, logistics, potential donor site morbidity, and regulatory hurdles. Ex vivo culture may also alter MSC signaling responses, ²⁵ reduce homing capacity,^25–27 and alter immunogenicity. ²⁸ Recruitment of endogenous MSC avoids these drawbacks, and there is growing recognition that in situ regeneration by endogenous MSC recruitment is a viable alternative or adjunctive to MSC transplantation.^29–32 Approaches under development use chemoattactants to enhance migration of endogenous MSC to accelerate wound healing and/or colonize biomaterial scaffolds.^33–35 Recent proof of concept studies demonstrating tissue regeneration by endogenous MSC recruitment have motivated further development of this approach.^36–38 The most effective chemoattractants for recruiting endogenous MSC may vary with tissue type and remain to be determined.

MSC are recruited to locations in adult tissues by homing through the vascular network and by interstitial migration within tissue. ³⁹ The mode of recruitment exploited in in situ tissue regeneration is chemotaxis, which is directional migration in response to a gradient of soluble chemoattractants, such as growth factors or chemokines. A key requirement for therapy success is the recruitment of a sufficient number of MSC to the injury site to achieve regeneration. ⁴⁰ The focus of this review is chemoattractants for MSC recruitment. The mechanisms that govern MSC migration to injury sites have not been fully elucidated^27,41 and are beyond the scope of this review. Biomaterials and strategies for chemoattractant delivery are described elsewhere.^31,42 In recent years, a substantial body of work has been published in which various chemoattractants have been tested for MSC recruitment in vitro and in several in vivo models. The objective of this review is to synthesize and evaluate evidence of recruitment efficacy by reported MSC chemoattractants.

Growth Factors

Growth factors are polypeptide extracellular signaling molecules that stimulate cell survival, migration, proliferation, and differentiation. Growth factors play critical regulatory roles in embryogenesis ⁴³ and in guiding wound repair. ⁴⁴ bMSC express a variety of growth factor receptors (Table 1).

Chemokines

Chemotactic cytokines, or chemokines, are a family of small molecules of approximately 8-10 kDa in size that induce leukocyte migration. ¹⁰² They are classified into four groups based on the number and spacing of their cystein residues: CC, CXC, C, and CX₃C. ¹⁰³ Chemokines are key regulators of cell migration for tissue homeostasis, immune responses, and wound healing. bMSC express a variety of chemokine receptors (Table 2).

Other Chemoattractants

In addition to the better known growth factors and chemokines, other molecules have also been identified as MSC chemoattractants. Four examples of these other chemoattractants are reviewed. bMSC express receptors for these molecules (Table 3).

Discussion

A biomimetic engineering approach to in situ tissue regeneration by endogenous MSC requires the orchestration of three main stages: MSC recruitment, MSC differentiation, and neotissue maturation. The first stage must result in the recruitment of a sufficient number of MSC that are capable of effecting regeneration of the injured or diseased tissue. One of the challenges for engineering endogenous MSC recruitment is the selection of effective chemoattractant(s).

Reported chemotactic responses vary among MSC isolated from the same tissue type and among MSC isolated from different tissue types. Differences in culture conditions and cell passage numbers contribute to the variation. Both cell density and passage number correspond to changes in MSC morphologies and gene expression levels of some phenotype markers.^25,89,160 Donor-related differences also contribute to the variance, ¹⁶¹ as do species-related, and even strain-related, differences.^89,162 Differences among MSC isolated from different tissues^161,163 may reflect the diversity among MSC niches. ¹⁶⁴

Exposure of MSCs to a chemoattractant may stimulate collateral responses in addition to the chemotaxis desired. Treatment with TGFα, ⁸¹ TNF-α, ¹⁵⁵ or overexpression of IGF-1 ⁵⁹ each stimulated the expression of other growth factors by the MSC. HGF inhibited proliferation and resulted in the loss of the MSC phenotype.^93,95 HMGB-1 inhibited proliferation and promoted osteogenic differentiation. ¹⁴⁹ TLR priming changed MSC cytokine and chemokine expression patterns and differentiation responses; the changes were dependent on the TLR primed. ¹⁵⁷

The in vitro and in vivo environments differ considerably; in vitro MSC chemotaxis may not translate to in vivo chemotaxis. In vitro, MSC are typically exposed to one chemoattractant, while in vivo, MSC are exposed to a multiplicity of signals that are influenced by the inflammatory state. In vivo, MSC are recruited from a tissue niche, while in vitro, MSC are recruited from a cell suspension or from a monolayer. In vivo, chemoattractants are subject to diffusion away from the regeneration site, enzymatic degradation, and/or deactivation. ³¹ In vivo, chemoattractants interact with the extracellular matrix. Proteoglycans bind, store, and release growth factors and chemokines. ¹⁶⁵ Matrix binding has been shown to enhance growth factor function over that of the soluble form. ⁴² In vivo, other cells, such as fibroblasts, may also respond to a chemoattractant by migrating and/or by producing growth factors and chemokines. ⁵⁸ Testing in animal models showed that lower doses of growth factor resulted in a greater chemotactic response ⁵³ and that chemotaxis in vitro may not correspond to chemotaxis in vivo. ¹⁵

Much remains to be discovered about mechanisms that govern MSC chemotaxis; however, proof-of-concept studies have demonstrated the promise of endogenous MSC recruitment for tissue regeneration. Each chemoattractant reviewed has been reported to induce MSC chemotaxis. The key will be to determine which chemoattractant(s) (1) recruit a sufficient number of MSC capable of effecting regeneration, (2) do not recruit cells that interfere with regeneration, and (3) do not interfere with subsequent stages of tissue regeneration. The rational design of MSC recruitment strategies will require a synergistic combination of in vitro and in vivo experiments. Novel cell labeling techniques ⁵³ will be required to monitor the spatiotemporal dynamics of MSC chemotaxis in vivo. Successful endogenous MSC recruitment is the first stage of successful tissue regeneration.