Asymmetric division and lineage commitment at the level of hematopoietic stem cells: inference from differentiation in daughter cell and granddaughter cell pairs

Abstract

How hematopoietic stem cells (HSCs) commit to a particular lineage is unclear. A high degree of HSC purification enabled us to address this issue at the clonal level. Single-cell transplantation studies revealed that 40% of the CD34-/low, c-Kit+, Sca-1+, and lineage marker- (CD34-KSL) cells in adult mouse bone marrow were able, as individual cells, to reconstitute myeloid and B- and T-lymphoid lineages over the long-term. Single-cell culture showed that >40% of CD34-KSL cells could form neutrophil (n)/macrophage (m)/erythroblast (E)/megakaryocyte (M) (nmEM) colonies. Assuming that a substantial portion of long-term repopulating cells can be detected as nmEM cells within this population, we compared differentiation potentials between individual pairs of daughter and granddaughter cells derived in vitro from single nmEM cells. One of the two daughter or granddaughter cells remained an nmEM cell. The other showed a variety of combinations of differentiation potential. In particular, an nmEM cell directly gave rise, after one cell division, to progenitor cells committed to nm, EM, or M lineages. The probability of asymmetric division of nmEM cells depended on the cytokines used. These data strongly suggest that lineage commitment takes place asymmetrically at the level of HSCs under the influence of external factors.

Figure 1.

Micromanipulation of daughter cell pairs and granddaughter cell pairs derived from single CD34⁻KSL cells in vitro. (A) After single CD34⁻KSL cells underwent first divisions in the presence of SCF, SCF + IL-3, SCF + TPO, or SCF + IL-3 + TPO, members of daughter cell pairs were separated by micromanipulation and further cultured in the presence of SCF + IL-3 + TPO + EPO to permit full differentiation along myeloid lineage. (B) After single CD34⁻KSL cells underwent first divisions in the presence of SCF + IL-3 or SCF + TPO, members of daughter cell pairs were separated into wells containing SCF + IL-3 or SCF + TPO. After each daughter cell underwent second division, granddaughter cells were separated and individually cultured in the presence of SCF + IL-3 + TPO + EPO.

Figure 2.

Colony-forming ability of single CD34⁻KSL cells. CD34⁻ KSL cells were individually cultured in the presence of SCF, IL-3, TPO, and EPO for 2 wk. Percentages of CFCs with different differentiation potentials are shown based on three independent experiments. Colony cells were morphologically identified as neutrophils (n), macrophages (m), erythroblasts (E), or megakaryocytes (M). Otherwise, unidentified immature cells were designated as blastlike cells (bl). The nmEM cells constituted 43.2 ± 3.2% (mean ± SD; n = 3) of the colony-forming CD34⁻KSL cells.

Figure 3.

Distribution of lineage-committed daughter and granddaughter cells. The numbers of daughter and granddaughter cells that lost the capacity to differentiate along one or more lineages are graphed, using data in Tables II and III. All possible combinations of differentiation potential are shown in the same order as that presented in Fig. 2.

Figure 4.

Myeloid lineage restriction model. The mode of lineage commitment at the level of HSCs may differ from that at the level of progenitor cells. Our model for HSCs is presented in combination with the model proposed by Weissman's group (34). An HSC can directly give rise to lineage-committed progenitor cells such as nm, EM, or M progenitor cells through initial HSC division in asymmetric manner. It may give rise to a common myeloid progenitor (CMP) after a certain number of divisions. The CMP gives rise to a megakaryocyte/erythrocyte lineage–restricted progenitor (MEP) and to a granulocyte/macrophage lineage-restricted progenitor (GMP). The MEP progressively gives rise to a megakaryocyte-committed progenitor (MKP) and to an erythrocyte lineage-committed progenitor (ErP). P, probability of asymmetric division.