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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.


Effect of 5-FU on bone marrow cellularity. Hematoxylin and eosin-stained sections of decalcified femur (a) and affected histology on D2, D4, and D10 (b–d) after 5-FU treatment. Gradual decrease in bone marrow cells is observed on D2 (b) and D4 (c) with lowest cellularity on D4 (c) compared with control (a). By D10, endogenous recovery of hematopoiesis is seen (d). Note an influx of RBCs on D2 and D4. Scale bar = 20 μm. All images are taken on the Nikon 90i microscope. D2-5-FU, D4-5-FU, D10-5-FU days post 5-fluorouracil treatment
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Fig2: Effect of 5-FU on bone marrow cellularity. Hematoxylin and eosin-stained sections of decalcified femur (a) and affected histology on D2, D4, and D10 (b–d) after 5-FU treatment. Gradual decrease in bone marrow cells is observed on D2 (b) and D4 (c) with lowest cellularity on D4 (c) compared with control (a). By D10, endogenous recovery of hematopoiesis is seen (d). Note an influx of RBCs on D2 and D4. Scale bar = 20 μm. All images are taken on the Nikon 90i microscope. D2-5-FU, D4-5-FU, D10-5-FU days post 5-fluorouracil treatment

Mentions: The effect of 5-FU was studied on mouse BM cellularity by examining the histological sections of decalcified femur post treatment on D2, D4, and D10 along with untreated controls. 5-FU treatment caused an apparent reduction in cell numbers (Fig. 2b) compared with control (Fig. 2a), with the lowest bone marrow cellularity observed on D4 (Fig. 2c); however by D10, BM sections showed similar histoarchitecture to that of the control sections, confirming endogenous regeneration (Fig. 2d). Also, 5-FU treatment resulted in a marked influx of erythrocytes in the bone marrow on D4 (Fig. 2c). Histological observations were further confirmed by BM total cell count taken on the same days. As expected, the cell count steadily decreased by 3-fold on D2 and by 7-fold on D4 (p < 0.001) compared with the control (Fig. 3a). The cell count on D4 (3.04 ± 0.79 × 106) was at least 2-fold lower (p < 0.001) than on D2 (7.65 ± 0.74 × 106). On D10 post treatment, the cell numbers (10.42 ± 2.3 × 106) were increased, suggestive of endogenous bone marrow regeneration.Fig. 2


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)

Effect of 5-FU on bone marrow cellularity. Hematoxylin and eosin-stained sections of decalcified femur (a) and affected histology on D2, D4, and D10 (b–d) after 5-FU treatment. Gradual decrease in bone marrow cells is observed on D2 (b) and D4 (c) with lowest cellularity on D4 (c) compared with control (a). By D10, endogenous recovery of hematopoiesis is seen (d). Note an influx of RBCs on D2 and D4. Scale bar = 20 μm. All images are taken on the Nikon 90i microscope. D2-5-FU, D4-5-FU, D10-5-FU days post 5-fluorouracil treatment
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4837595&req=5

Fig2: Effect of 5-FU on bone marrow cellularity. Hematoxylin and eosin-stained sections of decalcified femur (a) and affected histology on D2, D4, and D10 (b–d) after 5-FU treatment. Gradual decrease in bone marrow cells is observed on D2 (b) and D4 (c) with lowest cellularity on D4 (c) compared with control (a). By D10, endogenous recovery of hematopoiesis is seen (d). Note an influx of RBCs on D2 and D4. Scale bar = 20 μm. All images are taken on the Nikon 90i microscope. D2-5-FU, D4-5-FU, D10-5-FU days post 5-fluorouracil treatment
Mentions: The effect of 5-FU was studied on mouse BM cellularity by examining the histological sections of decalcified femur post treatment on D2, D4, and D10 along with untreated controls. 5-FU treatment caused an apparent reduction in cell numbers (Fig. 2b) compared with control (Fig. 2a), with the lowest bone marrow cellularity observed on D4 (Fig. 2c); however by D10, BM sections showed similar histoarchitecture to that of the control sections, confirming endogenous regeneration (Fig. 2d). Also, 5-FU treatment resulted in a marked influx of erythrocytes in the bone marrow on D4 (Fig. 2c). Histological observations were further confirmed by BM total cell count taken on the same days. As expected, the cell count steadily decreased by 3-fold on D2 and by 7-fold on D4 (p < 0.001) compared with the control (Fig. 3a). The cell count on D4 (3.04 ± 0.79 × 106) was at least 2-fold lower (p < 0.001) than on D2 (7.65 ± 0.74 × 106). On D10 post treatment, the cell numbers (10.42 ± 2.3 × 106) were increased, suggestive of endogenous bone marrow regeneration.Fig. 2

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.