Myeloerythroid-restricted progenitors are sufficient to confer radioprotection and provide the majority of day 8 CFU-S (original) (raw)
In this report, we functionally characterize three myeloerythroid-restricted progenitor subsets that have recently been isolated — CMPs and their lineal descendants, MEPs and GMPs (7). CMPs are downstream of HSCs, because CMPs do not give rise to HSCs, do not have self-renewal potential, and lack T cell differentiation potential (Figure 4). The identification of the CMPs, in addition to the previously reported CLPs (6), suggests that commitment to a myeloid or lymphoid lineage is a key developmental “choice” that occurs at distinct stages downstream of HSCs. The restricted capacity in vivo of CMPs and CLPs to differentiate into myeloid or lymphoid cells also suggests that significant plasticity between these lineages is rare once commitment occurs (31).
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.
The ability to prospectively isolate the progenitors representing the major branch points of the hematopoietic tree allows the functionalities of each subset to be tested in a precise manner. In this way, we examined each stem and progenitor subset for classical in vivo colony-forming and radioprotection activity. We found that the majority of day 8 CFU-S activity resides within the MEP population and is absent in GMPs, supporting previous findings that day 8–9 CFU-S are largely erythroid (1, 15, 32). CMPs presumably form splenic colonies by first giving rise to MEPs. Our data also reveal that about half of day 12 CFU-S activity resides within lineage-committed progenitors and about half within the more primitive HSC/MPP population (1, 2, 15). The day 12 activity observed within the MEP population may be explained by previous work suggesting that some day 12 colonies derive from surviving day 8 colonies (33). CLPs do not possess either day 8 or day 12 CFU-S activity (6). Thus, this study shows directly that the classical day 8 CFU-S assay largely measures MEP activity.
Radioprotection is defined as the ability to rapidly reconstitute an ablated blood-forming system in order to maintain viability of the host for a limited period of time, generally 30 days in the mouse. The populations responsible for this activity have been the subject of controversy (reviewed in ref. 34). While we have previously shown that as few as 100 HSCs can provide radioprotection (2, 3, 30), it has been argued that HSCs do not contribute directly to radioprotection since their relative quiescence causes significant delay of effector cell production (35, 36). Recently, other groups have also reported that long-term HSCs have poor radioprotective capacity and require the assistance of additional bone marrow subsets for full radioprotection (4, 10).
We tested the capacity of myeloid-restricted CMPs, MEPs, and GMPs to radioprotect lethally irradiated congenic recipient mice without additional provision of HSCs or helper bone marrow. While transplantation of GMPs alone provided no radioprotection, both CMP and MEP transplants showed dose-dependent rescue of irradiated recipients. The fact that CMPs and MEPs but not GMPs contain radioprotective capacity, together with the results of blood cell counts at day 12, strongly indicate that deficiencies of red blood cells, platelets, or both are normally responsible for mortality from hematopoietic failure following total body irradiation in mice. The hematocrits and platelet numbers 12 days after the injection of 30,000 MEPs or CMPs alone are comparable to the levels achieved by transplantation of a saturating dose of c-Kit+Thy-1.1loLin–/loSca-1+ (KTLS) HSCs (≥ 5,000 cells) (30). Interestingly, host-type cells gradually recovered in rescued recipients, and after 30 days constituted all blood cells. This is similar to transplants of radioprotective doses of ST-HSCs or MPPs (2), indicating that transplanted CMPs, MEPs, MPPs, and ST-HSCs provide transient production of erythrocytes and platelets sufficient to maintain host viability until rare, residual HSCs present in the host can take over. Therefore, in these studies, radioprotection is determined only by the capacity of cells to provide sufficient erythropoiesis and/or megakaryopoiesis during the period of bone marrow aplasia and is not the exclusive property of the HSC/MPP compartment. The requirement for high numbers of CMPs or MEPs to obtain significant radioprotection is probably due to their lack of self-renewal capacity, relatively short life spans, and small progeny burst sizes when compared with HSCs, which can replenish the CMP and MEP pools indefinitely.
Although the spleen does not normally contribute to human hematopoiesis, there are a number of conditions in which extensive splenic hematopoiesis can occur (37). This strongly indicates that human spleens contain a microenvironment capable of supporting proliferation and differentiation of hematopoietic stem and progenitor cells in vivo. Unfortunately, very few studies have been done to examine this aspect of hematopoietic recovery after bone marrow transplantation. It is generally agreed that macroscopic spleen colonies equivalent to mouse CFU-S do not exist in humans. However, microscopic bone marrow and spleen colonies can be found in a majority of patients shortly after bone marrow transplantation (38, 39). Interestingly, their characteristics are very similar to what we describe here for CMP- and MEP-derived colonies. Whether these colonies originate from the human counterparts of mouse CMPs and MEPs remains to be determined.
Cytopenia after total body irradiation or high-dose chemotherapy is one of the most serious problems in clinical oncology and transplantation. In our murine model, CMPs and MEPs significantly increase hematocrit and platelet counts in ablated recipients, whereas neutropenia cannot be prevented by transplantation of myeloid-restricted progenitors. Using a mathematical model simulating the clinical situation of peripheral blood transplantation, Scheding et al. have predicted that even transplantation of 1 × 107 CFU-GM/kg is not sufficient to avoid neutropenia after high-dose chemotherapy (40). We have transplanted high doses of GMPs — up to 100,000 per recipient (2 × 106/kg) — and never observed significant increases in blood neutrophil counts (data not shown). However, it is likely that neutropenia alone does not cause mortality unless serious bacterial or fungal infections occur. Therefore, the importance of myeloid progenitors to providing sufficient granulopoiesis could not be clearly demonstrated in this transplantation model because all irradiated recipients were kept on antibiotics under aseptic conditions. In another experimental system, we recently found that in conjunction with HSC transplants, the addition of CMP and GMP, but not MEP populations, protects mice from lethal infection by Aspergillus or Pseudomonas during this neutropenic period (A. Bitmansour et al., unpublished observations). Additionally, these progenitor populations are efficiently expanded and mobilized following standard protocols used in clinical transplantation and are all devoid of T lymphocyte differentiation potential in vivo (T. Na Nakorn, unpublished observations). Taken together, these results suggest that high-dose myeloid-restricted progenitor therapy might be effective in ameliorating the morbidity and mortality from myelosuppression caused by high-dose irradiation or chemotherapy regimens.
In summary, we have tested the behaviors of myeloerythroid-restricted progenitor subsets in a transplantation setting to model their normal physiological roles as closely as possible. All prospectively isolated myeloerythroid progenitor populations (CMPs, GMPs, and MEPs) showed transient reconstitution of irradiated hosts, and displayed fate outcomes in accordance with those of in vitro assays. Within the limits of the in vivo assays, they are non–self-renewing cells. CMPs and MEPs rapidly formed robust colonies in recipient marrow and spleen. Most importantly, CMPs and MEPs provided dose-dependent rescue of otherwise lethal irradiation. Our results also suggest that erythrocytes, platelets, or both are the critical effectors of radioprotection. The identification of counterpart populations in humans may allow us to test whether these progenitors can be used as adjuncts in hematopoietic cell transplantation to overcome some of the major complications seen in the clinic.