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miR-17 promotes expansion and adhesion of human cord blood CD34(+) cells in vitro.

Yang Y, Wang S, Miao Z, Ma W, Zhang Y, Su L, Hu M, Zou J, Yin Y, Luo J - Stem Cell Res Ther (2015)

Bottom Line: However, the overexpression of miR-17 in vivo reduced the hematopoietic reconstitution potential of CB CD34(+) cells compared to that of control cells.The increased expression of major adhesion molecules in miR-17 overexpressed CB CD34(+) cells suggests that the adhesion between miR-17 overexpressed CB CD34(+) cells and their niche in vivo is regulated abnormally, which may further lead to the reduced hematopoietic reconstitution capability of 17/OE cells in engrafted mice.We conclude that the proper expression of miR-17 is required, at least partly, for normal hematopoietic stem cell-niche interaction and for the regulation of adult hematopoiesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical & Research Technology, School of Medicine, University of Maryland, Baltimore, MD, 21201, USA. yangyx@bjmu.edu.cn.

ABSTRACT

Introduction: We have recently found that miR-17 is necessary in the cell-extrinsic control of cord blood (CB) CD34(+) cell function. Here, we demonstrated that the proper level of miR-17 is also necessary in the cell-intrinsic control of the hematopoietic properties of CB CD34(+) cells.

Methods: The miR-17 overexpression and knockdown models were created using primary CB CD34(+) cells transfected by the indicated vectors. Long-term culture, colony forming, adhesion and trans-well migration assays were carried out to investigate the function of miR-17 on CB CD34(+) cells in vitro. NOD prkdc (scid) Il2rg () mice were used in a SCID repopulating cell assay to investigate the function of miR-17 on CB CD34(+) cells in vivo. A two-tailed Student's t-test was used for statistical comparisons.

Results: In vitro assays revealed that ectopic expression of miR-17 promoted long-term expansion, especially in the colony-forming of CB CD34(+) cells and CD34(+)CD38(-) cells. Conversely, downregulation of miR-17 inhibited the expansion of CB CD34(+) cells. However, the overexpression of miR-17 in vivo reduced the hematopoietic reconstitution potential of CB CD34(+) cells compared to that of control cells. The increased expression of major adhesion molecules in miR-17 overexpressed CB CD34(+) cells suggests that the adhesion between miR-17 overexpressed CB CD34(+) cells and their niche in vivo is regulated abnormally, which may further lead to the reduced hematopoietic reconstitution capability of 17/OE cells in engrafted mice.

Conclusion: We conclude that the proper expression of miR-17 is required, at least partly, for normal hematopoietic stem cell-niche interaction and for the regulation of adult hematopoiesis.

No MeSH data available.


Related in: MedlinePlus

The effect of miR-17 modulation on the expansion of CB CD34+CD38−/CD38+ cells. a The expansion of CB CD34+ cells upon miR-17 modulation after culturing for 20 days. CB CD34+ cells were transfected with vectors for miR-17 overexpression (17/OE), miR-17 knockdown (17/KD), or control (CTRL) and cultured in cytokine-driven serum-free medium. Fold expansion is shown as mean + SD (n = 6). *p < 0.05, between 17/OE or 17/KD, and CTRL group (Student’s t-test). b CB CD34+ cells, transfected with ectopic miR-17 vector (17/OE or 17/KD), were further separated through the analysis of CD38 expression. Cells were cultured in cytokine-driven serum-free medium and the cell numbers were monitored. The data are presented as the fold expansion (n = 6, mean + SD). *p < 0.05; **p < 0.01, between 17/OE or 17/KD and CTRL group (Student’s t-test). c Colony forming assays of CB CD34+ cells upon miR-17 modulation. After 7 or 14 days of culture, the indicated cell numbers were plated for colony forming assays. The colonies, including BFU-Es, CFU-GMs, and CFU-Mix’s, with greater than 50 cells were counted after 14–16 days. The results are expressed as mean + SD (n = 6). *p < 0.05, between 17/OE or 17/KD, and CTRL group (Student’s t-test). CFU colony forming unit
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Fig2: The effect of miR-17 modulation on the expansion of CB CD34+CD38−/CD38+ cells. a The expansion of CB CD34+ cells upon miR-17 modulation after culturing for 20 days. CB CD34+ cells were transfected with vectors for miR-17 overexpression (17/OE), miR-17 knockdown (17/KD), or control (CTRL) and cultured in cytokine-driven serum-free medium. Fold expansion is shown as mean + SD (n = 6). *p < 0.05, between 17/OE or 17/KD, and CTRL group (Student’s t-test). b CB CD34+ cells, transfected with ectopic miR-17 vector (17/OE or 17/KD), were further separated through the analysis of CD38 expression. Cells were cultured in cytokine-driven serum-free medium and the cell numbers were monitored. The data are presented as the fold expansion (n = 6, mean + SD). *p < 0.05; **p < 0.01, between 17/OE or 17/KD and CTRL group (Student’s t-test). c Colony forming assays of CB CD34+ cells upon miR-17 modulation. After 7 or 14 days of culture, the indicated cell numbers were plated for colony forming assays. The colonies, including BFU-Es, CFU-GMs, and CFU-Mix’s, with greater than 50 cells were counted after 14–16 days. The results are expressed as mean + SD (n = 6). *p < 0.05, between 17/OE or 17/KD, and CTRL group (Student’s t-test). CFU colony forming unit

Mentions: We then investigated the ability of miR-17 modulation to expand CB CD34+ cells in vitro. CD34+ cells were cultured in cytokine-driven serum-free medium. After 20 days of culturing, the number of total cells was counted. As shown in Fig. 2a, CD34+ cells were expanded significantly upon ectopic miR-17. After knockdown of miR-17, there was a trend toward a decrease in the total number of CD34+ cells, although statistical analyses of the cohort indicated that it did not meet statistical significance (p > 0.05). In order to assess whether there is a different response between CD34+CD38− populations and CD34+CD38+ populations upon miR-17 modulation, the expansion curves from the two populations from CD34+ cells were investigated. As shown in Fig. 2b, although both CD34+CD38− and CD34+CD38+ cells were expanded to different extents upon miR-17 modulation, the ectopic miR-17 tends to expand CD34+CD38− cells more, rather than CD34+CD38+ cells, especially during the first 15 days. The expansion fold of CD34+CD38− cells from the 17/OE cells after being cultured for 15 days was significantly higher than that from the CTRL cells, especially during the first 5 days. Despite the presence of the HSC-supporting cytokines, CD34+CD38− cells may undergo differentiation and become more committed along with culturing in vitro. Ectopic miR-17 in CD34+ cells preferentially supports a specific expansion of the CD34+CD38− populations, especially during the first 5 days, suggesting that the ectopic miR-17 may specifically affect the least differentiated cells that have the characteristics of stem or early progenitor cells. After knockdown of miR-17, there was a trend toward a decrease in the expansion capacity of CD34+CD38−/CD38+ cells, although statistical analyses of the cohort indicated that it did not meet statistical significance (p > 0.05) outside of the first 5 days from CD34+CD38− cells. Taken together, these data indicate that the ectopic miR-17 in CD34+ cells preferentially supports a specific expansion of the CD34+CD38− populations in vitro.Fig. 2


miR-17 promotes expansion and adhesion of human cord blood CD34(+) cells in vitro.

Yang Y, Wang S, Miao Z, Ma W, Zhang Y, Su L, Hu M, Zou J, Yin Y, Luo J - Stem Cell Res Ther (2015)

The effect of miR-17 modulation on the expansion of CB CD34+CD38−/CD38+ cells. a The expansion of CB CD34+ cells upon miR-17 modulation after culturing for 20 days. CB CD34+ cells were transfected with vectors for miR-17 overexpression (17/OE), miR-17 knockdown (17/KD), or control (CTRL) and cultured in cytokine-driven serum-free medium. Fold expansion is shown as mean + SD (n = 6). *p < 0.05, between 17/OE or 17/KD, and CTRL group (Student’s t-test). b CB CD34+ cells, transfected with ectopic miR-17 vector (17/OE or 17/KD), were further separated through the analysis of CD38 expression. Cells were cultured in cytokine-driven serum-free medium and the cell numbers were monitored. The data are presented as the fold expansion (n = 6, mean + SD). *p < 0.05; **p < 0.01, between 17/OE or 17/KD and CTRL group (Student’s t-test). c Colony forming assays of CB CD34+ cells upon miR-17 modulation. After 7 or 14 days of culture, the indicated cell numbers were plated for colony forming assays. The colonies, including BFU-Es, CFU-GMs, and CFU-Mix’s, with greater than 50 cells were counted after 14–16 days. The results are expressed as mean + SD (n = 6). *p < 0.05, between 17/OE or 17/KD, and CTRL group (Student’s t-test). CFU colony forming unit
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Fig2: The effect of miR-17 modulation on the expansion of CB CD34+CD38−/CD38+ cells. a The expansion of CB CD34+ cells upon miR-17 modulation after culturing for 20 days. CB CD34+ cells were transfected with vectors for miR-17 overexpression (17/OE), miR-17 knockdown (17/KD), or control (CTRL) and cultured in cytokine-driven serum-free medium. Fold expansion is shown as mean + SD (n = 6). *p < 0.05, between 17/OE or 17/KD, and CTRL group (Student’s t-test). b CB CD34+ cells, transfected with ectopic miR-17 vector (17/OE or 17/KD), were further separated through the analysis of CD38 expression. Cells were cultured in cytokine-driven serum-free medium and the cell numbers were monitored. The data are presented as the fold expansion (n = 6, mean + SD). *p < 0.05; **p < 0.01, between 17/OE or 17/KD and CTRL group (Student’s t-test). c Colony forming assays of CB CD34+ cells upon miR-17 modulation. After 7 or 14 days of culture, the indicated cell numbers were plated for colony forming assays. The colonies, including BFU-Es, CFU-GMs, and CFU-Mix’s, with greater than 50 cells were counted after 14–16 days. The results are expressed as mean + SD (n = 6). *p < 0.05, between 17/OE or 17/KD, and CTRL group (Student’s t-test). CFU colony forming unit
Mentions: We then investigated the ability of miR-17 modulation to expand CB CD34+ cells in vitro. CD34+ cells were cultured in cytokine-driven serum-free medium. After 20 days of culturing, the number of total cells was counted. As shown in Fig. 2a, CD34+ cells were expanded significantly upon ectopic miR-17. After knockdown of miR-17, there was a trend toward a decrease in the total number of CD34+ cells, although statistical analyses of the cohort indicated that it did not meet statistical significance (p > 0.05). In order to assess whether there is a different response between CD34+CD38− populations and CD34+CD38+ populations upon miR-17 modulation, the expansion curves from the two populations from CD34+ cells were investigated. As shown in Fig. 2b, although both CD34+CD38− and CD34+CD38+ cells were expanded to different extents upon miR-17 modulation, the ectopic miR-17 tends to expand CD34+CD38− cells more, rather than CD34+CD38+ cells, especially during the first 15 days. The expansion fold of CD34+CD38− cells from the 17/OE cells after being cultured for 15 days was significantly higher than that from the CTRL cells, especially during the first 5 days. Despite the presence of the HSC-supporting cytokines, CD34+CD38− cells may undergo differentiation and become more committed along with culturing in vitro. Ectopic miR-17 in CD34+ cells preferentially supports a specific expansion of the CD34+CD38− populations, especially during the first 5 days, suggesting that the ectopic miR-17 may specifically affect the least differentiated cells that have the characteristics of stem or early progenitor cells. After knockdown of miR-17, there was a trend toward a decrease in the expansion capacity of CD34+CD38−/CD38+ cells, although statistical analyses of the cohort indicated that it did not meet statistical significance (p > 0.05) outside of the first 5 days from CD34+CD38− cells. Taken together, these data indicate that the ectopic miR-17 in CD34+ cells preferentially supports a specific expansion of the CD34+CD38− populations in vitro.Fig. 2

Bottom Line: However, the overexpression of miR-17 in vivo reduced the hematopoietic reconstitution potential of CB CD34(+) cells compared to that of control cells.The increased expression of major adhesion molecules in miR-17 overexpressed CB CD34(+) cells suggests that the adhesion between miR-17 overexpressed CB CD34(+) cells and their niche in vivo is regulated abnormally, which may further lead to the reduced hematopoietic reconstitution capability of 17/OE cells in engrafted mice.We conclude that the proper expression of miR-17 is required, at least partly, for normal hematopoietic stem cell-niche interaction and for the regulation of adult hematopoiesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical & Research Technology, School of Medicine, University of Maryland, Baltimore, MD, 21201, USA. yangyx@bjmu.edu.cn.

ABSTRACT

Introduction: We have recently found that miR-17 is necessary in the cell-extrinsic control of cord blood (CB) CD34(+) cell function. Here, we demonstrated that the proper level of miR-17 is also necessary in the cell-intrinsic control of the hematopoietic properties of CB CD34(+) cells.

Methods: The miR-17 overexpression and knockdown models were created using primary CB CD34(+) cells transfected by the indicated vectors. Long-term culture, colony forming, adhesion and trans-well migration assays were carried out to investigate the function of miR-17 on CB CD34(+) cells in vitro. NOD prkdc (scid) Il2rg () mice were used in a SCID repopulating cell assay to investigate the function of miR-17 on CB CD34(+) cells in vivo. A two-tailed Student's t-test was used for statistical comparisons.

Results: In vitro assays revealed that ectopic expression of miR-17 promoted long-term expansion, especially in the colony-forming of CB CD34(+) cells and CD34(+)CD38(-) cells. Conversely, downregulation of miR-17 inhibited the expansion of CB CD34(+) cells. However, the overexpression of miR-17 in vivo reduced the hematopoietic reconstitution potential of CB CD34(+) cells compared to that of control cells. The increased expression of major adhesion molecules in miR-17 overexpressed CB CD34(+) cells suggests that the adhesion between miR-17 overexpressed CB CD34(+) cells and their niche in vivo is regulated abnormally, which may further lead to the reduced hematopoietic reconstitution capability of 17/OE cells in engrafted mice.

Conclusion: We conclude that the proper expression of miR-17 is required, at least partly, for normal hematopoietic stem cell-niche interaction and for the regulation of adult hematopoiesis.

No MeSH data available.


Related in: MedlinePlus