Expression of c-kit mRNA and protein during the differentiation of human hematopoietic progenitor cells.
To investigate how c-kit and c-kit ligand play a role in the function of hematopoietic stem cells, we determined the expression of c-kit in sorted human hematopoietic stem cells, CD34+CD33- cells and CD34+CD33+ cells. CD34+ cells constituted approximately 1% of the population of gated bone marrow cells and contained colony-forming cells. Two-color analysis by a fluorescence-activated cell sorter (FACS) revealed that about one-third to one-half of the total CD34+ cell population were positive for the CD33 antigen. To analyze the relative accumulation of c-kit mRNA in sorted cells, we used the reverse transcription-polymerase chain reaction (RT-PCR) method, followed by Southern blot analysis. There was a linear relationship between the amount of input RNA and products amplified in the range of 10(3) to 10(5) cells. Using this procedure, we carried out an analysis of c-kit mRNA expression in CD34+CD33-, CD34+CD33+, CD34-CD33+, and CD34-CD33- cells. Enhanced expression for c-kit mRNA was observed solely in CD34+CD33- cells. In contrast, flow cytometry shows that c-kit protein was expressed most abundantly in CD34+CD33+ cells. Colony-forming cells were generated on a human stromal cell layer for 5 weeks initiated with CD34+CD33- cells but not with CD34+CD33+ cells. During co-culture with stromal cells, CD34+CD33- cells differentiated into CD34+CD33+ cells. From these findings, it is concluded that CD34+CD33+ cells are direct progenies of CD34+CD33- cells. In this differentiation pathway, the expression of c-kit mRNA decreased and the c-kit protein increased.
Yamaguchi
Y
Y
Department of Medical Biology and Parasitology, Jichi Medical School, Tochigi-ken, Japan.
Gunji
Y
Y
Nakamura
M
M
Hayakawa
K
K
Maeda
M
M
Osawa
H
H
Nagayoshi
K
K
Kasahara
T
T
Suda
T
T
D000818:Animals; D000911:Antibodies, Monoclonal; D015703:Antigens, CD / Q000032:analysis / Q000502:physiology; D018952:Antigens, CD34; D015214:Antigens, Differentiation, Myelomonocytic / Q000032:analysis / Q000502:physiology; D002454:Cell Differentiation / Q000235:genetics; D002478:Cells, Cultured; D015870:Gene Expression; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000276:immunology; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D016133:Polymerase Chain Reaction / Q000379:methods; D011518:Proto-Oncogene Proteins / Q000235:genetics* / Q000276:immunology; D019009:Proto-Oncogene Proteins c-kit; D012333:RNA, Messenger / Q000032:analysis*; D063268:Sialic Acid Binding Ig-like Lectin 3
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D018952:Antigens, CD34; D015214:Antigens, Differentiation, Myelomonocytic; C571163:CD33 protein, human; C571165:Cd33 protein, mouse; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D063268:Sialic Acid Binding Ig-like Lectin 3; D019009:Proto-Oncogene Proteins c-kit