American Society of Hematology

Figure 6

$Figure 6. LTR capacity and mRNA expression variation in LSK/EPCR+ cells after short culture. (A) LSK/EPCR+ and LSK/EPCR− cells underwent short-term culture for 2 days in SF-O3 supplemented with SCF and Tpo, and were analyzed with flow cytometer for remaining Lin−, LSK, and LSK/EPCR+ fractions. A significantly greater population remained in Lin− fraction in the cultured LSK/EPCR+ cells than in the LSK/EPCR− cells. A limited proportion of cells remained in LSK and LSK/EPCR+ fractions. The LSK/EPCR+ () and LSK/EPCR− () cells are shown. Error bars represent SD (n = 3). *P < .05, **P < .01. (B) Apoptotic status of LSK/EPCR+ fraction remaining after short culture was analyzed. Majority of the remaining LSK/EPCR+ fraction survived from apoptosis. Error bars represent SD (n = 3). (C) Cell-cycle status of remaining LSK/EPCR+ fraction was analyzed after short culture. G1 fraction decreased and S/G2/M fraction increased significantly, compared with those as harvested. LSK/EPCR+ cells () represent as harvested. Remaining LSK/EPCR+ cells (□) after short culture are also represented. Error bars represent SD (n = 3). *P < .05, **P < .01. (D) LSK/EPCR+ cells were cultured for 2 days in SF-O3 supplemented with SCF and Tpo, and were subsequently transplanted into lethally irradiated recipient mice with BM-derived unfractionated competitor cells. After short culture, cells failed to reconstitute recipient BM. LSK/EPCR+ cells () represent as harvested. Cultured LSK/EPCR+ cells (□) are also represented. Error bars represent SD (n = 5). **P < .01. (E) EPCR (Procr) mRNA levels in LSK/EPCR+ cells decreased significantly during short culture. LSK/EPCR+ cells () represent as harvested. Cultured LSK/EPCR+ cells (□) are also represented. Error bars represent SD (n = 3). *P < .05. (F-H) Among the 91 genes tested using high-throughput quantitative RT-PCR, Cdk6, Cdh5, and Sox17 were screened based on their significantly different expression levels between LSK/EPCR+ and LSK/EPCR− cells, as well as on the significant changes in the expression of these genes in EPCR+ cells during short culture. LSK/EPCR+ () and LSK/EPCR− () cells are shown. Error bars represent SD (n = 2). ●P < .1, *P < .05, **P < .01.$

LTR capacity and mRNA expression variation in LSK/EPCR⁺ cells after short culture. (A) LSK/EPCR⁺ and LSK/EPCR⁻ cells underwent short-term culture for 2 days in SF-O3 supplemented with SCF and Tpo, and were analyzed with flow cytometer for remaining Lin⁻, LSK, and LSK/EPCR⁺ fractions. A significantly greater population remained in Lin⁻ fraction in the cultured LSK/EPCR⁺ cells than in the LSK/EPCR⁻ cells. A limited proportion of cells remained in LSK and LSK/EPCR⁺ fractions. The LSK/EPCR⁺ () and LSK/EPCR⁻ () cells are shown. Error bars represent SD (n = 3). *P < .05, **P < .01. (B) Apoptotic status of LSK/EPCR⁺ fraction remaining after short culture was analyzed. Majority of the remaining LSK/EPCR⁺ fraction survived from apoptosis. Error bars represent SD (n = 3). (C) Cell-cycle status of remaining LSK/EPCR⁺ fraction was analyzed after short culture. G1 fraction decreased and S/G2/M fraction increased significantly, compared with those as harvested. LSK/EPCR⁺ cells () represent as harvested. Remaining LSK/EPCR⁺ cells (□) after short culture are also represented. Error bars represent SD (n = 3). *P < .05, **P < .01. (D) LSK/EPCR⁺ cells were cultured for 2 days in SF-O3 supplemented with SCF and Tpo, and were subsequently transplanted into lethally irradiated recipient mice with BM-derived unfractionated competitor cells. After short culture, cells failed to reconstitute recipient BM. LSK/EPCR⁺ cells () represent as harvested. Cultured LSK/EPCR⁺ cells (□) are also represented. Error bars represent SD (n = 5). **P < .01. (E) EPCR (Procr) mRNA levels in LSK/EPCR⁺ cells decreased significantly during short culture. LSK/EPCR⁺ cells () represent as harvested. Cultured LSK/EPCR⁺ cells (□) are also represented. Error bars represent SD (n = 3). *P < .05. (F-H) Among the 91 genes tested using high-throughput quantitative RT-PCR, Cdk6, Cdh5, and Sox17 were screened based on their significantly different expression levels between LSK/EPCR⁺ and LSK/EPCR⁻ cells, as well as on the significant changes in the expression of these genes in EPCR⁺ cells during short culture. LSK/EPCR⁺ () and LSK/EPCR⁻ () cells are shown. Error bars represent SD (n = 2). ●P < .1, *P < .05, **P < .01.

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