Identification of endoglin as a functional marker that defines long-term repopulating hematopoietic stem cells.
We describe a strategy to obtain highly enriched long-term repopulating (LTR) hematopoietic stem cells (HSCs) from bone marrow side-population (SP) cells by using a transgenic reporter gene driven by a stem cell enhancer. To analyze the gene-expression profile of the rare HSC population, we developed an amplification protocol termed "constant-ratio PCR," in which sample and control cDNAs are amplified in the same PCR. This protocol allowed us to identify genes differentially expressed in the enriched LTR-HSC population by oligonucleotide microarray analysis using as little as 1 ng of total RNA. Endoglin, an ancillary transforming growth factor beta receptor, was differentially expressed by the enriched HSCs. Importantly, endoglin-positive cells, which account for 20% of total SP cells, contain all the LTR-HSC activity within bone marrow SP. Our results demonstrate that endoglin, which plays important roles in angiogenesis and hematopoiesis, is a functional marker that defines LTR HSCs. Our overall strategy may be applicable for the identification of markers for other tissue-specific stem cells.
Chen
Chang-Zheng
CZ
Whitehead Institute for Biomedical Research, Nine Cambridge Center, Cambridge, MA 02142, USA.
Li
Min
M
de Graaf
David
D
Monti
Stefano
S
Göttgens
Berthold
B
Sanchez
Maria-Jose
MJ
Lander
Eric S
ES
Golub
Todd R
TR
Green
Anthony R
AR
Lodish
Harvey F
HF
D000818:Animals; D020296:Animals, Congenic; D015703:Antigens, CD; D015415:Biomarkers; D001854:Bone Marrow Cells / Q000166:cytology; D019070:Cell Lineage; D003114:Colony-Forming Units Assay; D018076:DNA, Complementary / Q000235:genetics; D000071063:Endoglin; D005260:Female; D005434:Flow Cytometry; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D018380:Hematopoietic Stem Cell Transplantation; D006412:Hematopoietic Stem Cells / Q000737:chemistry / Q000166:cytology*; D051379:Mice; D008810:Mice, Inbred C57BL; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D008822:Mice, Transgenic; D020411:Oligonucleotide Array Sequence Analysis; D016133:Polymerase Chain Reaction / Q000379:methods; D011828:Radiation Chimera; D011956:Receptors, Cell Surface; D013382:Subtraction Technique; D019010:Vascular Cell Adhesion Molecule-1 / Q000235:genetics / Q000502:physiology*
D015703:Antigens, CD; D015415:Biomarkers; D018076:DNA, Complementary; C064206:ENG protein, human; D000071063:Endoglin; D011956:Receptors, Cell Surface; D019010:Vascular Cell Adhesion Molecule-1