Myeloerythroid-restricted progenitors are sufficient to confer radioprotection and provide the majority of day 8 CFU-S

Abstract

Whole-body irradiation at the minimal lethal dose causes bone marrow failure and death within 12-18 days. To identify the principal components of the hematopoietic system that are radioprotective, we transplanted lethally irradiated mice with purified progenitors: common myeloid progenitors (CMPs), megakaryocyte/erythrocyte-restricted progenitors (MEPs), or granulocyte/monocyte-restricted progenitors (GMPs). Transplanted CMPs gave rise to cells both of the granulocyte/monocyte (GM) series and the megakaryocyte/erythrocyte series, whereas GMPs or MEPs showed reconstitution of only GM or ME cells, respectively. CMPs and MEPs but not GMPs protected mice in a dose-dependent manner, suggesting that erythrocytes, platelets, or both are the critical effectors of radioprotection. Accordingly, CMPs and MEPs formed robust colonies in recipient bone marrow and spleen, whereas GMPs formed small colonies that rapidly disappeared. Direct comparisons of spleen CFU (CFU-S) potentials among each progenitor subset showed that MEPs contain the vast majority of day 8 CFU-S activity, suggesting that day 8 CFU-S are the precursors of radioprotective cell subsets. All animals radioprotected for 30 days subsequently survived for at least 6 months post-transplant, and showed only host-derived hematopoiesis after 30 days. These findings suggest that rare hematopoietic stem cells survive myeloablation that can eventually repopulate irradiated hosts if myeloerythroid-restricted progenitors transiently rescue ablated animals through the critical window of bone marrow failure.

Figure 1

(a) Myeloid progenitor profiles in mouse bone marrow. Live Lin^–IL-7Rα^–Thy1.1^– cells were gated and analyzed for expression of the c-Kit and Sca-1 surface markers. The IL-7Rα^–Thy1.1^–Lin^–Sca-1^–c-Kit⁺ fraction was subdivided into FcγR^loCD34⁺(CMP), FcγR^loCD34^– (MEP), and FcγR^hiCD34⁺ (GMP) populations. (b) In vivo differentiation potential of myeloid progenitors. Splenocytes from lethally irradiated congenic recipient mice were analyzed 6 days after intravenous injection of 10,000 CMPs, MEPs, or GMPs. Top row shows Gr-1/Mac-1 profiles of donor-derived (Ly5.2) cells in recipient mice (Ly5.1). Bottom row shows Ly5.2/TER119 profiles from the Gr-1^–Mac-1^– fractions shown above. FSC, forward scatter.

Figure 2

Morphology of colonies derived from sorted CMPs, MEPs, and GMPs. Tibias were sectioned at day 7 after transplantation of (a) no cells, (b) 1 × 10⁴ CMPs, (c) 6 × 10³ MEPs, and (d) 2 × 10⁴ GMPs into lethally irradiated recipients. Controls confirmed the absence of host-derived colonies. Left panels show field views at ×40 magnification, whereas right panels are close-up views at ×100 magnification. Erythroid colonies were generally larger and more compact (left close-up view in b) than were GM colonies (right close-up in b). Also note the distinct nuclear morphologies of granulocytes and monocytes in close-up colony views in b and d.

Figure 3

Radioprotection of mice transplanted with myeloid progenitors. (a–c) Kaplan-Meier survival curves of lethally irradiated mice transplanted with no cells (control, rectangles), GMPs (filled squares), MEPs (open squares), or CMPs (triangles). All mice receiving no cells died by day 14 after irradiation, consistent with hematopoietic failure. Radioprotection was defined as survival beyond day 30. (a) No radioprotection was detected at the dose of 10,000 cells. (b) While 30,000 GMPs did not confer radioprotection, the same number of CMPs and MEPs protected 41.17% (7/17) and 35.29% (6/17) of mice from postradiation death, respectively. (c) More than 60% of mice can be saved by either 50,000 CMPs or 50,000 MEPs. (d) A representative flow cytometric analysis of peripheral blood from a recipient of 50,000 MEPs at day 42 after transplantation. All Gr-1⁺Mac-1⁺ cells are host-derived (Ly5.1).

Figure 4

Proposed model of murine hematopoiesis based on prospectively isolatable bone marrow populations. HSCs are long-term self-renewing cells that generate all blood cell types. IL-7Rα expression defines the commitment of MPPs to downstream CLPs that generate the lymphoid lineages. CMPs are clonogenic precursors of both GMPs and MEPs that respectively generate myelomonocytes and erythrocytes/platelets. Radioprotection capacity is present only in the highlighted populations that can generate erythrocytes and/or platelets in vivo.