Hematopoietic contribution to skeletal muscle regeneration by myelomonocytic precursors

Abstract

Adult bone marrow-derived cells can participate in muscle regeneration after bone marrow transplantation. In recent studies a single hematopoietic stem cell (HSC) was shown to give rise to cells that not only reconstituted all of the lineages of the blood, but also contributed to mature muscle fibers. However, the relevant HSC derivative with this potential has not yet been definitively identified. Here we use fluorescence-activated cell sorter-based protocols to test distinct hematopoietic fractions and show that only fractions containing c-kit(+) immature myelomonocytic precursors are capable of contributing to muscle fibers after i.m. injection. Although these cells belong to the myeloid lineage, they do not include mature CD11b(+) myelomonocytic cells, such as macrophages. Of the four sources of mature macrophages tested that were derived either from monocytic culture, bone marrow, peripheral blood after granulocyte colony-stimulating factor mobilization, or injured muscle, none contributed to muscle. In addition, after transplantation of bone marrow isolated from CD11b-Cre-transgenic mice into the Cre-reporter strain (Z/EG), no GFP myofibers were detected, demonstrating that macrophages expressing CD11b do not fuse with myofibers. Irrespective of the underlying mechanisms, these data suggest that the HSC derivatives that integrate into regenerating muscle fibers exist in the pool of hematopoietic cells known as myelomonocytic progenitors.

Fig. 1.

HSC (Lin^–c-kit⁺Sca-1⁺) and common myeloid progenitors (Lin^–c-kit⁺Sca-1^–), but not Lin⁺ and c-kit^– mature hematopoietic cells, contribute to muscle regeneration. (A–D) Example of fractionation from the BM of GFP transgenic mice: Total BM cells were Lin-depleted by using purification with magnetic beads, and the depleted fraction (Lin^–) was then fractionated on the basis of c-kit and Sca-1 expression by flow cytometry. All separated fractions were injected i.m. into the TAs of mice that had just received a NTX injection. (A′–D′) Transverse sections of TA muscles 4 weeks after i.m. fraction/NTX injection are shown stained for GFP (green) and laminin (red) expression; nuclei are counterstained with Hoechst 33342 (blue). Representative TA sections of mice injected with 10⁵ Lin⁺ cells (A′), 10⁵ Lin^–c-kit^– cells (B′), 10⁵ Lin^–c-kit⁺Sca-1^– cells (C′), or 5.10⁴ Lin^–c-kit⁺Sca-1⁺ cells (D′) are shown. The total number of GFP⁺ muscle fibers observed 4 weeks after injection in several experiments and the percentage of injected TA presenting GFP⁺ fibers for each fractionation is provided in Table 1. (Scale bars, 50 μm.)

Fig. 2.

Injected BMDC contribute to intact myofibers or give rise to CD11b/Mac1⁺ cells that invade degenerating fibers. (A and A′) Representative confocal microscopic analyses of transverse sections of skeletal muscle 4 weeks after i.m. injection of cells fractionated from the BM of GFP transgenic mice detect GFP (green in A only), laminin (A and A′, red) and nuclei (A and A′, blue). Both GFP⁺ myofibers surrounded by basal lamina (arrow) and GFP⁺ clusters of cells not surrounded by basal laminal membranes (arrow heads) were observed in TA cross sections (note number of nuclei in GFP⁺ cluster in A′). (B and B′) GFP⁺ clusters (arrows) (green) were positive for CD11b (B′, red) and therefore identified as donor-derived macrophages that have invaded degenerating myofibers. Endogenous macrophages (arrowheads) are not GFP⁺.(C and C′) True GFP⁺ myofibers (arrows) (C′, green) were found to lack CD11b (red), whereas infiltrating endogenous macrophages CD11b⁺ (arrowheads) were observed. (Scale bars, 50 μm.)

Fig. 3.

Mature CD11b⁺ myelomonocytic cells from BM, PB after G-CSF mobilization, NTX-damaged muscle, or monocytic cultures do not participate in myofiber regeneration. (A and B) Flow-cytometric analysis of BM and sorting gates for isolation of Gr1⁺ (A) or CD11b⁺ (B) cells. (C) Sorting gates for CD11b⁺ and CD11b^–c-kit⁺ cells from PB after G-CSF mobilization. (D) Sorting gates for CD45⁺CD11b⁺ and CD45^–CD11b^– cells from muscle damaged by NTX. All cells were analyzed by flow cytometry and isolated from GFP⁺ transgenic mice before i.m. injection into skeletal muscles that had previously received an NTX injection. The number of TA muscles with GFP⁺ myofibers and the total number of GFP⁺ myofibers observed for each fraction analyzed are shown in Table 2. (E) Representative transverse section of TA muscle CD11b-Cre Z/EG BMT 4 weeks after NTX damage. No GFP⁺ myofibers were detected, whereas GFP⁺ macrophages (arrows) resulting from fusion between Z/EG endogenous and CD11b-Cre donor macrophages were observed. (F) TA muscle of Z/EG transgenic mice injected with myoblasts from muscle extract of CMV-Cre transgenic mice was used as positive control. (G) Lin^–c-kit⁺ cells expanded in methylcellulose culture for 1 or 2 weeks and the phenotypes of the cells tested for contribution to muscle regeneration are shown. The number of TA muscles with GFP⁺ myofibers and total number of GFP⁺ myofibers observed for each culture time point are shown in Table 2.