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. 2015 Apr 11;6(1):66.
doi: 10.1186/s13287-015-0037-x.

Microencapsulated equine mesenchymal stromal cells promote cutaneous wound healing in vitro

Leen Bussche  1 , Rebecca M Harman  2 , Bethany A Syracuse  3 , Eric L Plante  4 , Yen-Chun Lu  5 , Theresa M Curtis  6 , Minglin Ma  7 , Gerlinde R Van de Walle  8
Affiliations

Microencapsulated equine mesenchymal stromal cells promote cutaneous wound healing in vitro

Leen Bussche et al. Stem Cell Res Ther. .

Abstract

Introduction: The prevalence of impaired cutaneous wound healing is high and treatment is difficult and often ineffective, leading to negative social and economic impacts for our society. Innovative treatments to improve cutaneous wound healing by promoting complete tissue regeneration are therefore urgently needed. Mesenchymal stromal cells (MSCs) have been reported to provide paracrine signals that promote wound healing, but (i) how they exert their effects on target cells is unclear and (ii) a suitable delivery system to supply these MSC-derived secreted factors in a controlled and safe way is unavailable. The present study was designed to provide answers to these questions by using the horse as a translational model. Specifically, we aimed to (i) evaluate the in vitro effects of equine MSC-derived conditioned medium (CM), containing all factors secreted by MSCs, on equine dermal fibroblasts, a cell type critical for successful wound healing, and (ii) explore the potential of microencapsulated equine MSCs to deliver CM to wounded cells in vitro.

Methods: MSCs were isolated from the peripheral blood of healthy horses. Equine dermal fibroblasts from the NBL-6 (horse dermal fibroblast cell) line were wounded in vitro, and cell migration and expression levels of genes involved in wound healing were evaluated after treatment with MSC-CM or NBL-6-CM. These assays were repeated by using the CM collected from MSCs encapsulated in core-shell hydrogel microcapsules.

Results: Our salient findings were that equine MSC-derived CM stimulated the migration of equine dermal fibroblasts and increased their expression level of genes that positively contribute to wound healing. In addition, we found that equine MSCs packaged in core-shell hydrogel microcapsules had similar effects on equine dermal fibroblast migration and gene expression, indicating that microencapsulation of MSCs does not interfere with the release of bioactive factors.

Conclusions: Our results demonstrate that the use of CM from MSCs might be a promising new therapy for impaired cutaneous wounds and that encapsulation may be a suitable way to effectively deliver CM to wounded cells in vivo.

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Figures

Figure 1
Figure 1
Mesenchymal stromal cell (MSC) conditioned medium (CM) promotes migration of dermal fibroblasts in scratch assays (n = 3). (A) Representative phase-contrast images of wounded NBL-6 cells cultured with MSC-derived CM (lower panels) as compared with control CM (upper panels) over a 48-hour period, and migration distances of NBL-6 cells are expressed as micrometer per hour in 48 hours. **P <0.01. (B) Migration of NBL-6 cells cultured with MSC-derived CM as compared with CM from mitomycin C-treated MSCs (2,000 ng/mL). (C) Viability (dashed line) and proliferation (solid line) of NBL-6 cells cultured in the presence of various concentrations of mitomycin C. NBL-6, horse dermal fibroblast cell; OD, optical density.
Figure 2
Figure 2
Mesenchymal stromal cell (MSC) conditioned medium (CM) promotes wound closure using Electric Cell-substrate Impedance Sensing (n = 3). Wound-healing rates of NBL-6 cells cultured with MSC-derived CM or control CM, as determined by electrical impedance in ohms detected from 0 to 24 hours after wounding (A) and total wound closure time expressed in hours (B). **P <0.01. (C) Phase-contrast images of wounded NBL-6 cells cultured with MSC-derived CM (upper panels) as compared with control CM (lower panels) over a 24-hour period. Scale bars = 100 μm. NBL-6, horse dermal fibroblast cell.
Figure 3
Figure 3
Mesenchymal stromal cell (MSC) conditioned medium (CM) alters gene expression levels in dermal fibroblasts (n = 3). (A) Schematic illustration of NBL-6 treatments for RNA isolation. Fold change of mRNA levels, as detected by quantitative reverse transcription-polymerase chain reaction, in (B) NBL-6 cells cultured with MSC-derived CM as compared with control CM or (C) NBL-6 cells cultured with CM from preconditioned MSCs as compared with CM from non-preconditioned MSCs. Left panel shows genes involved in reduction of hypergranulation or enhancement of wound healing or both, and right panel shows genes that stimulate hypergranulation or delay wound healing or both. *P <0.05; **P <0.01. ANXA2, annexin A2; CCL2, CC chemokine 2; CoCl2, cobalt chloride; COLI, collagen type 1A1; COLIII, collagen type 3; CTSK, cathepsin K; CxCL10, CXC chemokine 10; IFNγ, interferon-gamma; IL-8, interleukin-8; MMP1, metallopeptidase 1; MMP2, metallopeptidase 2; NBL-6, horse dermal fibroblast cell; PAI1, plasminogen activator inhibitor-1; PLAT, tissue plasminogen activator; PLAU, plasminogen activator urokinase; SDC2, syndecan 2; SDC4, syndecan 4; TIMP1, metallopeptidase inhibitor 1; TNFα, tumor necrosis factor-alpha.
Figure 4
Figure 4
Mesenchymal stromal cells (MSCs) retain stem cell characteristics after microencapsulation (n = 3). (A) Expression of surface markers, as detected by flow cytometry, on MSCs removed from capsules after 2 days post-encapsulation as compared with control MSCs that were never encapsulated. Percentage of total cells positive is shown on the left, and representative histograms are shown on the right. *P <0.05. (B) Representative images of MSCs stained with Alizarin Red, Alcian Blue, and Oil Red O to detect osteocyte, chondrocyte, and adipocyte differentiation, respectively. Left column: undifferentiated MSCs cultured in expansion medium; center column: control MSCs that were never encapsulated cultured in MSC differentiation media; and right column: MSCs removed from capsules after 2 days post-encapsulation cultured in MSC differentiation media. Scale bars = 10 μm. (C) Population doubling times of MSCs removed from capsules after 2 days post-encapsulation as compared with control MSCs that were never encapsulated. MHC, major histocompatibility complex.
Figure 5
Figure 5
Mesenchymal stromal cells (MSCs) remain viable after long-term microencapsulation (n = 3). (A) Representative images of MSCs encapsulated (left), immediately after removal of capsules (center), and after several days of culture (right). (B) Viability, as determined by Trypan blue exclusion, of MSCs removed from capsules at 5-day intervals starting at 2 days after encapsulation. (C) Numbers of viable cells removed from equal numbers of capsules at 5-day intervals starting at 2 days after encapsulation. **P <0.01. (D) Population doubling times of MSCs removed from capsules at 5-day intervals starting at 2 days post-encapsulation. **P <0.01. (E) Representative phase-contrast images of cultured MSCs removed from capsules 22 days post-encapsulation as compared with control MSCs that were never encapsulated. Scale bars = 50 μm.
Figure 6
Figure 6
Conditioned medium (CM) from microencapsulated mesenchymal stromal cells (MSCs) promotes NBL-6 migration and alters gene expression (n = 3). (A) Migration distances of NBL-6 cells cultured in CM from encapsulated MSCs as compared with MSC CM in in vitro scratch assays. Data are expressed as micrometer per hour in 48 hours. (B) Fold change of mRNA, as detected by quantitative reverse transcription-polymerase chain reaction, in NBL-6 cells cultured with encapsulated MSC CM as compared with control MSC CM. Left panel shows genes involved in reduction of hypergranulation or enhancement of wound healing or both, and right panel shows genes that stimulate hypergranulation or delay wound healing or both. *P <0.05. COLIII, collagen type 3; CxCL10, CXC chemokine 10; IL, interleukin; MMP1, metallopeptidase 1; NBL-6, horse dermal fibroblast cell.
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