Mesenchymal progenitor cells derived from traumatized muscle enhance neurite growth

Abstract

The success of peripheral nerve regeneration is governed by the rate and quality of axon bridging and myelination that occurs across the damaged region. Neurite growth and the migration of Schwann cells is regulated by neurotrophic factors produced as the nerve regenerates, and these processes can be enhanced by mesenchymal stem cells (MSCs), which also produce neurotrophic factors and other factors that improve functional tissue regeneration. Our laboratory has recently identified a population of mesenchymal progenitor cells (MPCs) that can be harvested from traumatized muscle tissue debrided and collected during orthopaedic reconstructive surgery. The objective of this study was to determine whether the traumatized muscle-derived MPCs exhibit neurotrophic function equivalent to that of bone marrow-derived MSCs. Similar gene- and protein-level expression of specific neurotrophic factors was observed for both cell types, and we localized neurogenic intracellular cell markers (brain-derived neurotrophic factor and nestin) to a subpopulation of both MPCs and MSCs. Furthermore, we demonstrated that the MPC-secreted factors were sufficient to enhance in vitro axon growth and cell migration in a chick embryonic dorsal root ganglia (DRG) model. Finally, DRGs in co-culture with the MPCs appeared to increase their neurotrophic function via soluble factor communication. Our findings suggest that the neurotrophic function of traumatized muscle-derived MPCs is substantially equivalent to that of the well-characterized population of bone marrow-derived MPCs, and suggest that the MPCs may be further developed as a cellular therapy to promote peripheral nerve regeneration.

Figure 1

Morphology of MSCs and MPCs cultured in either growth medium or neurotrophic induction medium: (A) phase-contrast microscopy; (B) immunocytochemistry; both × 10 magnification. Cells were stained for nestin (green, top row), BNDF (red, middle row) and with Hoechst 33342 (blue). Red and green merged panels (bottom row) indicate cells with co-localized nestin and BDNF. All scale bars=100 µm

Figure 2

Histomorphometric analysis of MPCs and MSCs cultured in growth medium (GM) and neurotrophic induction medium (NM). (A) Cell densities after 14 days of culture;*p < 0.05, Student’s t-test with n = 3. (B) Percentage of cells that stained positively for BNDF or nestin; ^a,bp < 0.05, ^b,cp < 0.05, ^a,cp < 0.01, one-way ANOVA with SNK post hoc comparisons and n = 3

Figure 3

Neurotrophic factor gene expression: (A) traumatized muscle-derived MPCs and (B) bone marrow-derived MSCs were cultured in either growth medium (GM) or neurotrophic induction medium (NM) for 14 days, and gene expression was assayed using real-time RT-PCR; *p < 0.01, Student’s t-tests with n = 4. NGF, nerve growth factor; BDNF, brain-derived neurotrophic factor; CNTF, ciliary neurotrophic factor; NT-3, neurotrophin-3

Figure 4

Neurotrophic factor production: (A) traumatized muscle-derived MPCs and (B) bone marrow-derived MSCs were cultured in either growth medium (GM) or neurotrophic induction medium (NM) for 14 days, and the concentration of neurotrophic factors secreted in the cell supernatants during the final 3 days was measured using ELISA; *p < 0.05, Student’s t-tests with n = 4

Figure 5

Neurotrophic activity assay of MPC and MSC conditioned media using cultured DRGs. (A) Neurite density was imaged using ×4 interference microscopy after culture for 3 days with growth medium or neurotrophic induction media that had been conditioned with factors secreted by MPCs or MSCs; scale bar = 250 µm. (B) The number of neurites that extended beyond the average neurite length in control DRG medium (1.75 mm) as a result of culture with growth medium or neurotrophic induction medium conditioned by MPCs or MSCs; ^a,bp < 0.05, ^b,cp < 0.05, ^a,cp < 0.01, one-way ANOVA with SNK post hoc comparisons and n = 4

Figure 6

Neurotrophic activity of MPCs and MSCs in DRG co-culture assays. (A) The DRGs were imaged using ×4 phase-contrast microscopy after co-culture with MPCs or MSCs for 3 days in growth medium or neurotrophic induction medium; scale bar = 250 µm. (B) The number of neurites that extended beyond the average neurite length in DRG medium (1.75 mm) as a result of co-culture with MPCs or MSCs in growth medium or neurotrophic induction media; ^a,b,cp < 0.05, one-way ANOVA with SNK post hoc comparisons and n = 4