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

Takano H, Ema H, Sudo K, Nakauchi H - J. Exp. Med. (2004)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Stem Cell Therapy, Center for Experimental Medicine, Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, 108-8639, Japan.

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.

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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.
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fig1: 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.

Mentions: After single cells underwent cell division in the presence of SCF, SCF + IL-3, SCF + TPO, or SCF + IL-3 + TPO by day 5 of culture, one member of the pair of daughter cells was transferred into another well using a micromanipulator (Fig. 1 A). Individual members of daughter cell pairs, in different wells, were cultured in parallel in the presence of SCF + IL-3 + TPO + EPO for 10–14 d. For serial micromanipulation, after single cells gave rise to daughter cells in the presence of either SCF + IL-3 or SCF + TPO, one of the two daughter cells remained in the same well where culture had been initiated, and the other was transferred into a new well containing the same combination of cytokines. After daughter cells underwent cell division, the two cells derived from each daughter cell (granddaughter cells) were separated and continuously cultured in the presence of SCF + IL-3 + TPO + EPO for 10–14 d (Fig. 1 B).


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

Takano H, Ema H, Sudo K, Nakauchi H - J. Exp. Med. (2004)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2211802&req=5

fig1: 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.
Mentions: After single cells underwent cell division in the presence of SCF, SCF + IL-3, SCF + TPO, or SCF + IL-3 + TPO by day 5 of culture, one member of the pair of daughter cells was transferred into another well using a micromanipulator (Fig. 1 A). Individual members of daughter cell pairs, in different wells, were cultured in parallel in the presence of SCF + IL-3 + TPO + EPO for 10–14 d. For serial micromanipulation, after single cells gave rise to daughter cells in the presence of either SCF + IL-3 or SCF + TPO, one of the two daughter cells remained in the same well where culture had been initiated, and the other was transferred into a new well containing the same combination of cytokines. After daughter cells underwent cell division, the two cells derived from each daughter cell (granddaughter cells) were separated and continuously cultured in the presence of SCF + IL-3 + TPO + EPO for 10–14 d (Fig. 1 B).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Stem Cell Therapy, Center for Experimental Medicine, Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, 108-8639, Japan.

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.

Show MeSH