Resolution of pluripotential intermediates in murine hematopoietic differentiation by global complementary DNA amplification from single cells: confirmation of assignments by expression profiling of cytokine receptor transcripts.
Although hematopoiesis is known to proceed from stem cells through a graded series of multipotent, oligopotent, and unipotent precursor cells, it has been difficult to resolve these cells physically one from another. There is, therefore, corresponding uncertainty about the exact distribution and timing of the expression of genes known to be important in hematopoietic differentiation. In earlier work, the generation of a set of amplified complementary DNAs (cDNAs) from single precursor cells was described, whose biologic potential was determined by the outcome of cultured sibling cells. In this study, the new acquisition of cDNA from multipotent myeloid precursor cells is described, as is the mapping of RNA-level expression of 17 distinct cytokine receptors (c-kit, Flk-1, Flk-2/Flt-3, c-fms, gp130, erythropoietin receptor, GM-CSFRalpha, G-CSFR, TNFR1, IL-1RI, IL-1RII, IL-2Rbeta, IL-3-specific beta receptor, IL-4R, IL-6Ralpha, IL-7Ralpha, and IL-11Ralpha) to the enlarged sample set, spanning stages from pentapotent precursors through oligopotent intermediates to committed and maturing cells in the myeloid and lymphoid lineages. Although the enhanced scope and resolving power of the analysis yielded previously unreported observations, there was overall agreement with known biologic responsiveness at individual stages, and major contradictions did not arise. Moreover, each precursor category displayed a unique overall pattern of hybridization to the matrix of 17 receptor probes, supporting the notion that each sample pool indeed reflected a unique precursor stage. Collectively, the results provide supportive evidence for the validity of the cDNA assignments to particular stages, the depth of the information captured, and the unique capacity of the sample matrix to resolve individual stages in the hematopoietic hierarchy.
Billia
F
F
Ontario Cancer Institute and the Department of Medical Biophysics, University of Toronto, Ontario, Canada.
Barbara
M
M
McEwen
J
J
Trevisan
M
M
Iscove
N N
NN
D000818:Animals; D001853:Bone Marrow / Q000737:chemistry; D002454:Cell Differentiation; D019070:Cell Lineage; D018076:DNA, Complementary / Q000235:genetics; D005784:Gene Amplification; D020869:Gene Expression Profiling; D006410:Hematopoiesis / Q000235:genetics*; D006412:Hematopoietic Stem Cells / Q000166:cytology*; D051379:Mice; D008808:Mice, Inbred CBA; D009693:Nucleic Acid Hybridization; D019009:Proto-Oncogene Proteins c-kit / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics*; D016186:Receptor, Macrophage Colony-Stimulating Factor / Q000235:genetics; D018121:Receptors, Cytokine / Q000235:genetics*; D017467:Receptors, Erythropoietin / Q000235:genetics; D016188:Receptors, Granulocyte Colony-Stimulating Factor / Q000235:genetics; D018123:Receptors, Interleukin / Q000235:genetics; D019948:Receptors, Interleukin-4 / Q000235:genetics
D018076:DNA, Complementary; D012333:RNA, Messenger; D018121:Receptors, Cytokine; D017467:Receptors, Erythropoietin; D016188:Receptors, Granulocyte Colony-Stimulating Factor; D018123:Receptors, Interleukin; D019948:Receptors, Interleukin-4; D019009:Proto-Oncogene Proteins c-kit; D016186:Receptor, Macrophage Colony-Stimulating Factor