Asymmetrical lymphoid and myeloid lineage commitment in multipotent hematopoietic progenitors - PubMed (original) (raw)

Asymmetrical lymphoid and myeloid lineage commitment in multipotent hematopoietic progenitors

Anne Y Lai et al. J Exp Med. 2006.

Abstract

The mechanism of lineage commitment from hematopoietic stem cells (HSCs) is not well understood. Although commitment to either the lymphoid or the myeloid lineage is popularly viewed as the first step of lineage restriction from HSCs, this model of hematopoietic differentiation has recently been challenged. The previous identification of multipotent progenitors (MPPs) that can produce lymphocytes and granulocyte/macrophages (GMs) but lacks erythroid differentiation ability suggests the existence of an alternative HSC differentiation program. Contribution to different hematopoietic lineages by these MPPs under physiological conditions, however, has not been carefully examined. In this study, we performed a refined characterization of MPPs by subfractionating three distinct subsets based on Flt3 and vascular cell adhesion molecule 1 expression. These MPP subsets differ in their ability to give rise to erythroid and GM lineage cells but are equally potent in lymphoid lineage differentiation in vivo. The developmental hierarchy of these MPP subsets demonstrates the sequential loss of erythroid and then GM differentiation potential during early hematopoiesis. Our results suggest that the first step of lineage commitment from HSCs is not simply a selection between the lymphoid and the myeloid lineage.

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Figures

Figure 1.

Figure 1.

Subfractionation of MPPs by Flt3 and VCAM-1 expression. (A) Flt3 and VCAM-1 expression on HSCs and MPPs by FACS analysis. (B) FACS analysis of Flt3 and VCAM-1 expression levels on MPP subsets after two rounds of sorting. The purity of the sorted populations was >95%.

Figure 2.

Figure 2.

MegE differentiation potential is restricted to Flt3loVCAM-1+ MPPs. (A) In vitro MegE and GM differentiation potential of MPP subsets assessed by colony-forming assay on methylcellulose. Data represent the average plating efficiency of triplicate methylcellulose cultures of each MPP population. (B) Comparison of MegE-affiliated GATA1 and EpoR expression levels in HSCs, MPP subsets, CLPs, and MEPs by quantitative PCR analysis. GATA1 and EpoR expression levels in HSCs were arbitrarily defined as unit 1. Data shown represent the mean value from triplicate reactions. (C) In vivo MegE differentiation by Flt3loVCAM-1+ MPPs. Peripheral blood from recipient mice injected with Flt3loVCAM-1+ MPPs was analyzed at 2 wk after injection to detect donor-derived (GFP+) CD41+CD61+ platelets. (D) Comparison of in vivo MegE and GM differentiation potential of MPP subsets. Peripheral blood from recipient mice injected with purified MPP subsets was analyzed at 2 wk after injection to evaluate the donor chimerism (GFP+) of CD41+CD61+ platelets (○) or Mac-1+ GM cells (•). Horizontal bars in the graph denote the average values from three mice analyzed in each group. *, P < 0.05, calculated by the Student's t test compared with percentage of chimerism of Flt3loVCAM-1+ MPPs.

Figure 3.

Figure 3.

Lymphoid lineage differentiation potential and lymphoid lineage priming in MPP subsets. (A) All MPP subsets contributed to lymphoid lineage differentiation in vivo. Peripheral blood collected from reconstituted mice was analyzed for donor-derived B220+ B cells and CD3+ T cells at 3–5 wk after injection. FACS plot shown is pregated on GFP+CD45.1− population. Almost all B220+ cells are CD19+IgM+, and most CD3+ cells are TCRβ+ in the peripheral blood (Fig. S2). It should be noted that due to the setting of the FACS machine, the CD3+ population does not clearly appear in the plots. (B) Analysis of lymphoid-specific RAG1 and IL-7Rα expression levels in MPP subsets by quantitative PCR. Data shown represent the mean value from triplicate reactions. RAG1 and IL-7Rα expression levels in CLPs were arbitrarily set at 10,000 and 100, respectively.

Figure 4.

Figure 4.

Flt3loVCAM-1+ MPPs, but not Flt3hiVCAM-1+ MPPs, differentiate into CMPs in vivo. (A) Detection of CMPs, GMPs, and MEPs derived from Flt3loVCAM-1+ MPPs and Flt3hiVCAM-1+ MPPs. Equal number of cells from the two MPP populations (CD45.2) were injected into CD45.1+ recipient mice. Bone marrow cells were harvested 6 d after injection for FACS analysis, and the total cell number of donor-derived CMPs, GMPs, and MEPs in recipient mice from two independent experiments is shown. FACS plots of these donor-derived bone marrow populations are shown in Fig. S3. Although the chimerism of donor-derived cells in the bone marrow varied between experiments, the difference in the contribution of CMPs, GMPs, and MEPs from Flt3loVCAM-1+ MPPs and Flt3hiVCAM-1+ MPPs remained consistent. (B) Proposed model of hierarchy in MPP subsets and lymphoid- and myeloid lineage–restricted progenitors during hematopoiesis, based on in vivo differentiation potential of MPPs. Flt3loVCAM-1+ MPPs represent the first branching point during adult hematopoiesis, which diverge into CMPs and Flt3hiVCAM-1+ MPPs. Flt3hiVCAM-1+ MPPs represent the second branching point, diverging to lymphoid lineage and a GM differentiation pathway that is independent of CMPs. Because Flt3hiVCAM-1− MPPs do not contribute to GM lineages significantly in vivo, we consider these MPPs as lymphoid-specified progenitors before lymphoid lineage commitment in CLPs or pro–T cells in the thymus. Dotted lines represent potential migration of bone marrow progenitor candidates to the thymus for T cell development based on evidence by recent studies (reference 25).

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