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Delineating the effects of 5-fluorouracil and follicle-stimulating hormone on mouse bone marrow stem/progenitor cells.

Shaikh A, Bhartiya D, Kapoor S, Nimkar H - Stem Cell Res Ther (2016)

Bottom Line: Nuclear OCT-4, SCA-1, and SSEA-1 coexpressing LIN(-)/CD45(-) VSELs and slightly larger LIN(-)/CD45(+) HSCs expressing cytoplasmic OCT-4 were identified and comprised 0.022 ± 0.002 % and 0.081 ± 0.004 % respectively of the total cells in BM. 5-FU treatment resulted in depletion of cells with a 7-fold reduction by D4 and normal hematopoiesis was re-established by D10.VSELs remained unaffected by 5-FU on D2 and increased on D4, whereas HSCs showed a marked reduction in numbers on D2 and later increased along with the corresponding increase in BrdU uptake and upregulation of specific transcripts (Oct-4A, Oct-4, Sca-1, Nanog, Stella, Fragilis, Pcna).The study provides a novel insight into basic hematopoiesis and has clinical relevance.

View Article: PubMed Central - PubMed

Affiliation: Stem Cell Biology Department, National Institute for Research in Reproductive Health (ICMR), Jehangir Merwanji Street, Parel, Mumbai, 400 012, India.

ABSTRACT

Background: Pluripotent, Lin(-)/CD45(-)/Sca-1(+) very small embryonic-like stem cells (VSELs) in mouse bone marrow (BM) are resistant to total body radiation because of their quiescent nature, whereas Lin(-)/CD45(+)/Sca-1(+) hematopoietic stem cells (HSCs) get eliminated. In the present study, we provide further evidence for the existence of VSELs in mouse BM and have also examined the effects of a chemotherapeutic agent (5-fluorouracil (5-FU)) and gonadotropin hormone (follicle-stimulating hormone (FSH)) on BM stem/progenitor cells.

Methods: VSELs and HSCs were characterized in intact BM. Swiss mice were injected with 5-FU (150 mg/kg) and sacrificed on 2, 4, and 10 days (D2, D4, and D10) post treatment to examine changes in BM histology and effects on VSELs and HSCs by a multiparametric approach. The effect of FSH (5 IU) administered 48 h after 5-FU treatment was also studied. Bromodeoxyuridine (BrdU) incorporation, cell cycle analysis, and colony-forming unit (CFU) assay were carried out to understand the functional potential of stem/progenitor cells towards regeneration of chemoablated marrow.

Results: Nuclear OCT-4, SCA-1, and SSEA-1 coexpressing LIN(-)/CD45(-) VSELs and slightly larger LIN(-)/CD45(+) HSCs expressing cytoplasmic OCT-4 were identified and comprised 0.022 ± 0.002 % and 0.081 ± 0.004 % respectively of the total cells in BM. 5-FU treatment resulted in depletion of cells with a 7-fold reduction by D4 and normal hematopoiesis was re-established by D10. Nuclear OCT-4 and PCNA-positive VSELs were detected in chemoablated bone sections near the endosteal region. VSELs remained unaffected by 5-FU on D2 and increased on D4, whereas HSCs showed a marked reduction in numbers on D2 and later increased along with the corresponding increase in BrdU uptake and upregulation of specific transcripts (Oct-4A, Oct-4, Sca-1, Nanog, Stella, Fragilis, Pcna). Cells that survived 5-FU formed colonies in vitro. Both VSELs and HSCs expressed FSH receptors and FSH treatment enhanced hematopoietic recovery by 72 h.

Conclusion: Both VSELs and HSCs were activated in response to the stress created by 5-FU and FSH enhanced hematopoietic recovery by at least 72 h in 5-FU-treated mice. VSELs are the most primitive pluripotent stem cells in BM that self-renew and give rise to HSCs under stress, and HSCs further divide rapidly and differentiate to maintain homeostasis. The study provides a novel insight into basic hematopoiesis and has clinical relevance.

No MeSH data available.


Related in: MedlinePlus

Mouse BM stem/progenitor cells survive and proliferate in response to 5-FU treatment. a Flow cytometry analysis of LIN–/CD45– VSELs and LIN–/CD45+ HSCs showed an increase in percentage post treatment compared with control. Compared with D2, a significant increase was observed on D4. On D10 the percentage of both these cells is reduced (N = 12, ***p ≤ 0.001). b Propidium iodide-based cell cycle analysis of BM cells carried out on D2, D4, and D10 after 5-FU compared with the control showed a decrease in S-phase cells on D2 and an increase in the G0/G1 cell percentage. By D4 the percentage of S-phase cells increased and continued until D10. The increase in S-phase cells suggests proliferation of cells in response to 5-FU treatment (N = 6, ***p ≤ 0.001). c Proliferation of VSELs (LIN–/CD45–) and HSCs (Lin–/CD45+) was confirmed by an increase in percentage of BrdU-positive cells on D4 compared with control (N = 5, **p ≤ 0.01,***p ≤ 0.001). In (a-c) Bars represent average ± SD. Day 2-FU, Day 4-FU, Day 10-FU days post 5-fluorouracil treatment, BM bone marrow, VSEL very small embryonic-like stem cell, HSC hematopoietic stem cell, BrdU bromodeoxyuridine
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Fig4: Mouse BM stem/progenitor cells survive and proliferate in response to 5-FU treatment. a Flow cytometry analysis of LIN–/CD45– VSELs and LIN–/CD45+ HSCs showed an increase in percentage post treatment compared with control. Compared with D2, a significant increase was observed on D4. On D10 the percentage of both these cells is reduced (N = 12, ***p ≤ 0.001). b Propidium iodide-based cell cycle analysis of BM cells carried out on D2, D4, and D10 after 5-FU compared with the control showed a decrease in S-phase cells on D2 and an increase in the G0/G1 cell percentage. By D4 the percentage of S-phase cells increased and continued until D10. The increase in S-phase cells suggests proliferation of cells in response to 5-FU treatment (N = 6, ***p ≤ 0.001). c Proliferation of VSELs (LIN–/CD45–) and HSCs (Lin–/CD45+) was confirmed by an increase in percentage of BrdU-positive cells on D4 compared with control (N = 5, **p ≤ 0.01,***p ≤ 0.001). In (a-c) Bars represent average ± SD. Day 2-FU, Day 4-FU, Day 10-FU days post 5-fluorouracil treatment, BM bone marrow, VSEL very small embryonic-like stem cell, HSC hematopoietic stem cell, BrdU bromodeoxyuridine

Mentions: Flow cytometry analysis confirmed survival and an increase in the numbers of LIN–/CD45–/SCA-1+ VSELs and LIN–/CD45+/SCA-1+ HSCs after 5-FU treatment. The VSELs were resistant to 5-FU and the percentage of VSELs increased on D2 after treatment (Fig. 4a). However, to confirm that the increase in percentage was not a result of decreased bone marrow cellularity, absolute numbers were calculated (Table 1). These data showed that the VSELs remained constant after treatment whereas more than 50 % of HSCs were destroyed by 5-FU.Fig. 4


Delineating the effects of 5-fluorouracil and follicle-stimulating hormone on mouse bone marrow stem/progenitor cells.

Shaikh A, Bhartiya D, Kapoor S, Nimkar H - Stem Cell Res Ther (2016)

Mouse BM stem/progenitor cells survive and proliferate in response to 5-FU treatment. a Flow cytometry analysis of LIN–/CD45– VSELs and LIN–/CD45+ HSCs showed an increase in percentage post treatment compared with control. Compared with D2, a significant increase was observed on D4. On D10 the percentage of both these cells is reduced (N = 12, ***p ≤ 0.001). b Propidium iodide-based cell cycle analysis of BM cells carried out on D2, D4, and D10 after 5-FU compared with the control showed a decrease in S-phase cells on D2 and an increase in the G0/G1 cell percentage. By D4 the percentage of S-phase cells increased and continued until D10. The increase in S-phase cells suggests proliferation of cells in response to 5-FU treatment (N = 6, ***p ≤ 0.001). c Proliferation of VSELs (LIN–/CD45–) and HSCs (Lin–/CD45+) was confirmed by an increase in percentage of BrdU-positive cells on D4 compared with control (N = 5, **p ≤ 0.01,***p ≤ 0.001). In (a-c) Bars represent average ± SD. Day 2-FU, Day 4-FU, Day 10-FU days post 5-fluorouracil treatment, BM bone marrow, VSEL very small embryonic-like stem cell, HSC hematopoietic stem cell, BrdU bromodeoxyuridine
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Fig4: Mouse BM stem/progenitor cells survive and proliferate in response to 5-FU treatment. a Flow cytometry analysis of LIN–/CD45– VSELs and LIN–/CD45+ HSCs showed an increase in percentage post treatment compared with control. Compared with D2, a significant increase was observed on D4. On D10 the percentage of both these cells is reduced (N = 12, ***p ≤ 0.001). b Propidium iodide-based cell cycle analysis of BM cells carried out on D2, D4, and D10 after 5-FU compared with the control showed a decrease in S-phase cells on D2 and an increase in the G0/G1 cell percentage. By D4 the percentage of S-phase cells increased and continued until D10. The increase in S-phase cells suggests proliferation of cells in response to 5-FU treatment (N = 6, ***p ≤ 0.001). c Proliferation of VSELs (LIN–/CD45–) and HSCs (Lin–/CD45+) was confirmed by an increase in percentage of BrdU-positive cells on D4 compared with control (N = 5, **p ≤ 0.01,***p ≤ 0.001). In (a-c) Bars represent average ± SD. Day 2-FU, Day 4-FU, Day 10-FU days post 5-fluorouracil treatment, BM bone marrow, VSEL very small embryonic-like stem cell, HSC hematopoietic stem cell, BrdU bromodeoxyuridine
Mentions: Flow cytometry analysis confirmed survival and an increase in the numbers of LIN–/CD45–/SCA-1+ VSELs and LIN–/CD45+/SCA-1+ HSCs after 5-FU treatment. The VSELs were resistant to 5-FU and the percentage of VSELs increased on D2 after treatment (Fig. 4a). However, to confirm that the increase in percentage was not a result of decreased bone marrow cellularity, absolute numbers were calculated (Table 1). These data showed that the VSELs remained constant after treatment whereas more than 50 % of HSCs were destroyed by 5-FU.Fig. 4

Bottom Line: Nuclear OCT-4, SCA-1, and SSEA-1 coexpressing LIN(-)/CD45(-) VSELs and slightly larger LIN(-)/CD45(+) HSCs expressing cytoplasmic OCT-4 were identified and comprised 0.022 ± 0.002 % and 0.081 ± 0.004 % respectively of the total cells in BM. 5-FU treatment resulted in depletion of cells with a 7-fold reduction by D4 and normal hematopoiesis was re-established by D10.VSELs remained unaffected by 5-FU on D2 and increased on D4, whereas HSCs showed a marked reduction in numbers on D2 and later increased along with the corresponding increase in BrdU uptake and upregulation of specific transcripts (Oct-4A, Oct-4, Sca-1, Nanog, Stella, Fragilis, Pcna).The study provides a novel insight into basic hematopoiesis and has clinical relevance.

View Article: PubMed Central - PubMed

Affiliation: Stem Cell Biology Department, National Institute for Research in Reproductive Health (ICMR), Jehangir Merwanji Street, Parel, Mumbai, 400 012, India.

ABSTRACT

Background: Pluripotent, Lin(-)/CD45(-)/Sca-1(+) very small embryonic-like stem cells (VSELs) in mouse bone marrow (BM) are resistant to total body radiation because of their quiescent nature, whereas Lin(-)/CD45(+)/Sca-1(+) hematopoietic stem cells (HSCs) get eliminated. In the present study, we provide further evidence for the existence of VSELs in mouse BM and have also examined the effects of a chemotherapeutic agent (5-fluorouracil (5-FU)) and gonadotropin hormone (follicle-stimulating hormone (FSH)) on BM stem/progenitor cells.

Methods: VSELs and HSCs were characterized in intact BM. Swiss mice were injected with 5-FU (150 mg/kg) and sacrificed on 2, 4, and 10 days (D2, D4, and D10) post treatment to examine changes in BM histology and effects on VSELs and HSCs by a multiparametric approach. The effect of FSH (5 IU) administered 48 h after 5-FU treatment was also studied. Bromodeoxyuridine (BrdU) incorporation, cell cycle analysis, and colony-forming unit (CFU) assay were carried out to understand the functional potential of stem/progenitor cells towards regeneration of chemoablated marrow.

Results: Nuclear OCT-4, SCA-1, and SSEA-1 coexpressing LIN(-)/CD45(-) VSELs and slightly larger LIN(-)/CD45(+) HSCs expressing cytoplasmic OCT-4 were identified and comprised 0.022 ± 0.002 % and 0.081 ± 0.004 % respectively of the total cells in BM. 5-FU treatment resulted in depletion of cells with a 7-fold reduction by D4 and normal hematopoiesis was re-established by D10. Nuclear OCT-4 and PCNA-positive VSELs were detected in chemoablated bone sections near the endosteal region. VSELs remained unaffected by 5-FU on D2 and increased on D4, whereas HSCs showed a marked reduction in numbers on D2 and later increased along with the corresponding increase in BrdU uptake and upregulation of specific transcripts (Oct-4A, Oct-4, Sca-1, Nanog, Stella, Fragilis, Pcna). Cells that survived 5-FU formed colonies in vitro. Both VSELs and HSCs expressed FSH receptors and FSH treatment enhanced hematopoietic recovery by 72 h.

Conclusion: Both VSELs and HSCs were activated in response to the stress created by 5-FU and FSH enhanced hematopoietic recovery by at least 72 h in 5-FU-treated mice. VSELs are the most primitive pluripotent stem cells in BM that self-renew and give rise to HSCs under stress, and HSCs further divide rapidly and differentiate to maintain homeostasis. The study provides a novel insight into basic hematopoiesis and has clinical relevance.

No MeSH data available.


Related in: MedlinePlus