A new stem cell biology: the continuum and microvesicles

Abstract

The hierarchical models of stem cell biology have been based on work first demonstrating pluripotental spleen-colony-forming units, then showing progenitors with many differentiation fates assayed in in vitro culture; there followed the definition and separation of "stem cells" using monoclonal antibodies to surface epitopes and fluorescent-activated cell characterization and sorting (FACS). These studies led to an elegant model of stem cell biology in which primitive dormant G0 stem cells with tremendous proliferative and differentiative potential gave rise to progressively more restricted and differentiated classes of stem/progenitor cells, and finally differentiated marrow hematopoietic cells. The holy grail of hematopoietic stem cell biology became the purification of the stem cell and the clonal definition of this cell. Most recently, the long-term repopulating hematopoietic stem cell (LT-HSC) has been believed to be a lineage negative sca-1+C-kit+ Flk3- and CD150+ cell. However, a series of studies over the past 10 years has indicated that murine marrow stem cells continuously change phenotype with cell cycle passage. We present here studies using tritiated thymidine suicide and pyronin-Hoechst FACS separations indicating that the murine hematopoietic stem cell is a cycling cell. This would indicate that the hematopoietic stem cell must be continuously changing in phenotype and, thus, could not be purified. The extant data indicate that murine marrow stem cells are continually transiting cell cycle and that the purification has discarded these cycling cells. Further in vivo BrdU studies indicate that the "quiescent" LT-HSC in G0 rapidly transits cycle. Further complexity of the marrow stem cell system is indicated by studies on cell-derived microvesicles showing that they enter marrow cells and transcriptionally alter their cell fate and phenotype. Thus, the stem cell model is a model of continuing changing potential tied to cell cycle and microvesicle exposure. The challenge of the future is to define the stem cell population, not purify the stem cell. We are at the beginning of elucidation of quantum stemomics.

Fig. 1

Spleen colony-forming assay. Colony-forming unit spleen (CFU-S) were represented by colonies or “lumps” on the surface of the spleen after infusion of marrow cells into lethally irradiated mice. Spleen colony counting was also performed at earlier time spans, 9 days or more. (Abbreviation: cGy, centigray.)

Fig. 2

Progenitor assay in semisolid matrices. (Abbreviations: CSFs, colony stimulation factors; GM-CFC, granulocyte-macrophage colony-forming cell.)

Fig. 3

Sequence of separative steps for isolation of LT-HSC.

Fig. 4

Hierarchical model of hematopoiesis.

Fig. 5

LT-HSC separated into into G0, G1, and SG2M phases by pyronin and Hoechst staining and fluorescent-activated cell sorting. These fractions were then engrafted competitively into lethally irradiated mice and engraftment outcomes determined.

Fig. 6

The phenotype of the “stem” cell continually and reversibly changes with passage through cell cycle. (Abbreviations: CLP, common lymphoid progenitor; CMP, common multipotent progenitor; LT-HSC, long-term hematopoetic stem cell; ST-HSC, short-term hematopeotic stem cell; GMP, granulocyte-macrophage progenitor; ERMegPP, megakaryocye erythroid progenitor; MP, multipotent progenitor.)

Fig. 7

Cell cycle continuum model. Each numbered circle is the same cell followed through all cycles. They show reversible changes in phenotype of marrow cells.

Fig. 8

Mouse marrow cells exposed to lung microvesicles were evaluated immediately after exposure and after 13 weeks in cytokine supported culture only mouse surfactant B&C was demonstrable, indicating a transcriptional mechanism underlying the genetic changes.

Fig. 9

Lung-derived microvesicles.

Fig. 10

Tissue specificity of microvesicle cell fate modulation. Microvesicles from liver, lung, brain and heart all modulated target marrow cells to express mRNA specific for the originator tissues.

Fig. 11

Microvesicle modulation of marrow cells on a continuum of cell cycle– related change. A modification of Figure 7.