Macrophages improve survival, proliferation and migration of engrafted myogenic precursor cells into MDX skeletal muscle

Abstract

Transplantation of muscle precursor cells is of therapeutic interest for focal skeletal muscular diseases. However, major limitations of cell transplantation are the poor survival, expansion and migration of the injected cells. The massive and early death of transplanted myoblasts is not fully understood although several mechanisms have been suggested. Various attempts have been made to improve their survival or migration. Taking into account that muscle regeneration is associated with the presence of macrophages, which are helpful in repairing the muscle by both cleansing the debris and deliver trophic cues to myoblasts in a sequential way, we attempted in the present work to improve myoblast transplantation by coinjecting macrophages. The present data showed that in the 5 days following the transplantation, macrophages efficiently improved: i) myoblast survival by limiting their massive death, ii) myoblast expansion within the tissue and iii) myoblast migration in the dystrophic muscle. This was confirmed by in vitro analyses showing that macrophages stimulated myoblast adhesion and migration. As a result, myoblast contribution to regenerating host myofibres was increased by macrophages one month after transplantation. Altogether, these data demonstrate that macrophages are beneficial during the early steps of myoblast transplantation into skeletal muscle, showing that coinjecting these stromal cells may be used as a helper to improve the efficiency of parenchymal cell engraftment.

Figure 1. MPCs and MPs used in the study.

(A) MPCs issued from Tg:CAG-GFP muscle were cultured and expanded (A, left) before their in vitro or in vivo use. These cells are capable of forming differentiated myotubes (Aright) (blue = Hoechst). Bar = 10 µm. (B) MPs were differentiated from bone marrow precursors. Differentiation into MPs is assessed by the positive labelling of more than 99% of the cells for the pan-macrophage marker F4/80. (C) Expression of iNOS, TNFα and COX2 was assessed by RT-qPCR on resting (NT) pro-inflammatory (Pro) and anti-inflammatory (Anti) MPs. Results represent mean ± sem of 4 independent RT-qPCR experiments. *P<0.05.

Figure 2. Effects of MPs on MPC engraftment in skeletal muscle.

(A) GFP-MPCs were injected in mdx TA muscle with or without MPs at 1∶0 and 1∶5 ratios. At different time points after transplantation, the amount of GFP contained in injected muscles was evaluated by immunoblotting and expressed in % of the signal evaluated at time 0.5/1 h (results represent mean ± sem of 3 independent experiments). *: versus time 0.5/1 h (** = P<0.01, *** = P<0.001). #: 1∶5 versus 1∶0 ratio at each time point (# = P<0.05). (B) Radiolabelled MPCs were injected in mdx TA muscle with or without MPs at 1∶5 ratio. At 0 (2/4 h), 2 and 5 days after transplantation, radioactivity contained in injected muscles was measured and expressed in cpm (results represent mean ± sem of 4 independent experiments). *: versus d0(2/4 h) P<0.05. (C) Sections of muscles injected with GFP-MPCs with or without MPs were labelled for caspase 3. Arrows show double stained apoptotic MPCs. Bar = 10 µm. (D) Sections of muscles injected with GFP-MPCs with or without MPs were labelled for ki67. Arrows show double stained cycling MPCs. Bar = 10 µm.

Figure 3. Localisation of MPCs and MPs at sites of transplantation.

GFP-MPCs were injected with or without MPs which were labelled with PKH26 before the cotransplantation into mdx muscle. Immunolabellings of the pan-macrophage marker F4/80 (magenta) were performed on sections at day 1 and 5 after transplantation. Green = MPCs, cyan = PKH-labelled MPS, blue = Hoechst). Arrows show colocalization of cyan and magenta stainings (white) indicating the expression of F4/80 by transplanted MPs. Two examples from different muscles are given for day 5. Bar = 10 µm. Inserts on the right panel represent higher magnification of the fields delimited by rectangles. Bar = 20 µm.

Figure 4. Expression of the M2 marker CD206 by MPs at site of transplantation.

GFP-MPCs were injected with or without MPs which were labelled with PKH26 before the cotransplantation into mdx muscle. Immunolabellings of CD206 (magenta) were performed on sections at day 5 after transplantation. Green = MPCs, cyan = PKH-labelled MPS, blue = Hoechst). Arrows show colocalization of cyan and magenta stainings (white) indicating the expression of CD206 by transplanted MPs. Two examples from different muscles are given. Bar = 10 µm. Inserts on the right panel represent higher magnification of the fields delimited by rectangles. Bar = 20 µm.

Figure 5. Effects of MPs on MPC adhesion in vitro.

MPCs were seeded with or without MP-conditioned medium at various concentrations or with NIH-3T3 fibroblast-conditioned medium of B lymphocyte-conditioned medium for 5 h. Adherent cells were counted and adhesion was expressed in % of adherent cells counted in control medium (results represent mean ± sem of 3 independent experiments). * = P<0.05.

Figure 6. Effects of MPs on MPC migration in vitro and in vivo.

(A) MPCs were seeded in the upper chamber of chemotactic inserts with or without MP-conditioned medium at various concentrations or with NIH-3T3 fibroblast-conditioned medium of B lymphocyte-conditioned medium. In (B) MPs were seeded in the lower chamber at various densities. The number of migrating cells was counted 24 h later and migration was expressed in % of migrating cells counted in control medium. (results represent mean ± sem of 3 independent experiments). * = P<0.05. (C) GFP-MPCs were injected with or without MPs at various ratios through a microtube migrating device in mdx TA muscle as described in Materials and Methods. After 48 h, migration of cells is evaluated by calculating the distance of cells from the microtube and expressed in µm. (results represent mean ± sem of 3 independent experiments). * = P<0.05. On bottom, representative micrographs show migration of MPCs from the microtube, when injected alone (1∶0) or with MPs (1∶5).

Figure 7. Effect of MPs on MPC participation to regenerating myofibres.

GFP-MPCs were injected in mdx TA muscle with or without MPs at 1∶0 and 1∶5 ratios. 30 days after transplantation, muscle sections were analyzed for the expression of dystrophin. The number of dystrophin positive myofibres is given for one field and represents mean ± sem of 3 independent experiments). ** = P<0.01. Representative micrographs show dystrophin positive myofibres in the 1∶0 and 1∶5 conditions (two examples per condition). Bar = 10 µm.