Hematopoietic potential of stem cells isolated from murine skeletal muscle - PubMed (original) (raw)

Hematopoietic potential of stem cells isolated from murine skeletal muscle

K A Jackson et al. Proc Natl Acad Sci U S A. 1999.

Abstract

We have discovered that cells derived from the skeletal muscle of adult mice contain a remarkable capacity for hematopoietic differentiation. Cells prepared from muscle by enzymatic digestion and 5-day in vitro culture were harvested, and 18 x 10(3) cells were introduced into each of six lethally irradiated recipients together with 200 x 10(3) distinguishable whole bone marrow cells. After 6 or 12 weeks, all recipients showed high-level engraftment of muscle-derived cells representing all major adult blood lineages. The mean total contribution of muscle cell progeny to peripheral blood was 56 +/- 20% (SD), indicating that the cultured muscle cells generated approximately 10- to 14-fold more hematopoietic activity than whole bone marrow. When bone marrow from one mouse was harvested and transplanted into secondary recipients, all recipients showed high-level multilineage engraftment (mean 40%), establishing the extremely primitive nature of these stem cells. We also show that muscle contains a population of cells with several characteristics of bone marrow-derived hematopoietic stem cells, including high efflux of the fluorescent dye Hoechst 33342 and expression of the stem cell antigens Sca-1 and c-Kit, although the cells lack the hematopoietic marker CD45. We propose that this population accounts for the hematopoietic activity generated by cultured skeletal muscle. These putative stem cells may be identical to muscle satellite cells, some of which lack myogenic regulators and could be expected to respond to hematopoietic signals.

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Figures

Figure 1

Figure 1

Flow cytometric analysis of peripheral blood from a representative mouse transplanted with muscle cells. Peripheral blood was drawn from the transplant recipient 6 weeks after it received 18 × 103 Ly-5.1+ mononuclear muscle cells and 200 × 103 Ly-5.2+ whole bone marrow cells and then was stained with anti-Ly-5.1-biotin followed by Str-PE and antibodies to specific lineage markers. (A–C) Controls. (B–F) Peripheral blood from transplant recipient. (A) C57BL/6-Ly-5.2 peripheral blood stained with anti-Ly-5.1-biotin followed by Str-PE. (B) C57BL/6-Ly-5.1 peripheral blood stained with anti-Ly-5.1-biotin + Str-PE. (C) C57BL/6-Ly-5.1 peripheral blood stained with isotype controls for lineage markers (a mixture of rat-IgG2a-FITC and rat-IgG2b-FITC). (D) Anti-B220-FITC (B cells) with anti-Ly-5.1-biotin + Str-PE. (E) Anti-Thy-1-FITC (T cells) with anti-Ly-5.1 biotin + Str-PE. (F) Anti-Gr-1-FITC + anti-Mac-1-FITC (granulocytes and macrophages, respectively) with anti-Ly-5.1-biotin + Str-PE. The percentage of cells in each quadrant is indicated in the Upper Right corner. Bracketed numbers (D–F) are the percentages of lineage-positive cells derived from muscle. This calculation was made by dividing the number of cells in the Upper Right quadrant by the total number of lineage-positive cells (the sum of both Right quadrants).

Figure 2

Figure 2

Multilineage engraftment in all mice transplanted with muscle cells. Peripheral blood was drawn 6 and 12 weeks after transplantation and stained with antibodies against the Ly-5.1 marker, B220 (B cells), Thy-1 (T cells), and Gr-1 and Mac-1 (granulocytes and macrophages, respectively). Stained blood samples were analyzed by flow cytometry, as described in Experimental Procedures. The percentages of positive cells in each lineage are reported. Mouse 1 (M1) was killed after the 6-week analysis for subsequent study (Fig. 3).

Figure 3

Figure 3

Repopulation of lymphoid and myeloid cell compartments in secondary recipients of muscle cells. Bone marrow from mouse 1 (M1; Fig. 2) was transplanted at 8 × 105 nucleated cells per mouse into each of five lethally irradiated C57BL/6-Ly-5.2 recipients. After 5 weeks, peripheral blood was drawn and stained with antibodies against Ly-5.1, B220 (B cells), Thy-1 (T cells), and Gr-1/Mac-1 (granulocytes and macrophages) and then analyzed by flow cytometry. The findings are displayed as the percentages of cells positive for Ly-5.1 and specific lineage markers.

Figure 4

Figure 4

Flow cytometric analysis of HSC from secondary recipients of muscle cells. Bone marrow prepared from the secondary transplant recipient mouse 5 (Fig. 3) was stained with Hoechst 33342, anti-Ly-5.1-biotin, and Str-PE. (A and B) Whole bone marrow. (C and D) HSC. Whole bone marrow contained 0.03% HSC (determined by Hoechst dye efflux; refs. and 22) and contained 63% Ly-5.1+ cells (B). The HSC population (C) contained 65% Ly-5.1+ cells (D).

Figure 5

Figure 5

Flow cytometric analysis of muscle cell cultures. (A, C, and E) Whole culture. (B, D, and F) Hoechst low SP cells. The percentage of cells in each quadrant is indicated in the Upper Right corner. (A) Whole muscle cell culture stained with Hoechst 33342. The indicated region is analogous to the SP fraction of whole bone marrow and represents about 1% of the culture. (B) SP fraction as gated in A. (C) Whole culture stained with anti-Sca-1-FITC and anti-CD45-biotin + Str-PE. (D) Anti-Sca-1-FITC and anti-CD45 staining of the SP fraction. (E) Whole culture stained with anti-c-Kit-PE alone. (F) c-Kit staining of the SP fraction.

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