Abstract
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 103 to 105 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.
| Original language | English |
|---|---|
| Pages (from-to) | 1233-1238 |
| Number of pages | 6 |
| Journal | Experimental Hematology |
| Volume | 21 |
| Issue number | 9 |
| Publication status | Published - 1993 |
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Keywords
- c-kit
- CD34
- FACS
- RT-PCR
ASJC Scopus subject areas
- Cancer Research
- Cell Biology
- Genetics
- Hematology
- Oncology
- Transplantation
Cite this
Expression of c-kit mRNA and protein during the differentiation of human hematopoietic progenitor cells. / Yamaguchi, Y.; Gunji, Y.; Nakamura, M.; Hayakawa, K.; Maeda, M.; Osawa, H.; Nagayoshi, K.; Kasahara, T.; Suda, T.
In: Experimental Hematology, Vol. 21, No. 9, 1993, p. 1233-1238.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Expression of c-kit mRNA and protein during the differentiation of human hematopoietic progenitor cells
AU - Yamaguchi, Y.
AU - Gunji, Y.
AU - Nakamura, M.
AU - Hayakawa, K.
AU - Maeda, M.
AU - Osawa, H.
AU - Nagayoshi, K.
AU - Kasahara, T.
AU - Suda, T.
PY - 1993
Y1 - 1993
N2 - 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 103 to 105 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.
AB - 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 103 to 105 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.
KW - c-kit
KW - CD34
KW - FACS
KW - RT-PCR
UR - http://www.scopus.com/inward/record.url?scp=0027738401&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0027738401&partnerID=8YFLogxK
M3 - Article
C2 - 7687219
AN - SCOPUS:0027738401
VL - 21
SP - 1233
EP - 1238
JO - Experimental Hematology
JF - Experimental Hematology
SN - 0301-472X
IS - 9
ER -