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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

Mature and actively dividing cells in BM are affected by 5-FU whereas primitive and relatively quiescent stem cells are spared. a The total BM nucleated cells isolated from two femurs and two tibias of the mice were counted on D2, D4, and D10 after 5-FU treatment and compared with control. Similar to histological data (Fig. 2); the cell count decreased up to D4 (7-fold) and is increased on D10 (n = 10, ***p ≤ 0.001). b The percentage of various bone marrow subtypes based on Lin and CD45 expression were evaluated on D4 and D10 after 5-FU treatment. Note that while the percentage of Lin+/CD45+ subtypes decreased on D4, its percentage increased with hematopoietic recovery. Percentage of Lin–/CD45– (enriched in VSELs) increased on D4 and slightly reduced by D10. The Lin–/CD45+ (enriched in HSCs) percentage also increased by D4, and remained high on D10 (n = 10, ***p ≤ 0.001). c Upregulation in the pluripotent, primordial germ cell and proliferation specific transcripts is observed in BM cells that survive 5-FU on D4. The upregulation of these markers confirms the presence and involvement of primitive stem cells in regeneration. The transcript levels on D4 are shown compared with control (n = 6, *p ≤ 0.05; **p ≤ 0.01). Bars in a, b represent average ± SD and in c average ± SEM. d–h Characterization of cells on D4 that survived 5-FU treatment. d H & E staining shows VSELs which are spherical in shape and small in size with high nucleo-cytoplasmic ratio located near the endosteal region. Inset (e) shows cells at higher magnification. f Nuclear OCT-4+ VSELs are clearly visualized in bone marrow sections cells in clusters (*) or singly (arrow) along the endosteal region; these cells also express (g) cell-surface SSEA-1and (h) nuclear SCA-1. Presence of nuclear OCT-4, SSEA-1, and SCA-1 confirms that VSELs survive 5-FU treatment. Nuclei are counterstained with DAPI. Scale bar = 20 μm for H & E and immunohistochemistry images, scale bar = 10 μm for immunofluorescence images. Day 2-FU, Day 4-FU, Day 10-FU days post 5-FU treatment, BM bone marrow, 5-FU 5-fluorouracil, H & E Hematoxylin and Eosin, OCT-4 octamer binding transforming factor-4, SSEA-1 stage-specific embryonic antigen-1, SCA-1, stem cell antigen-1, DAPI 4′,6-diamidino-2-phenylindole
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Fig3: Mature and actively dividing cells in BM are affected by 5-FU whereas primitive and relatively quiescent stem cells are spared. a The total BM nucleated cells isolated from two femurs and two tibias of the mice were counted on D2, D4, and D10 after 5-FU treatment and compared with control. Similar to histological data (Fig. 2); the cell count decreased up to D4 (7-fold) and is increased on D10 (n = 10, ***p ≤ 0.001). b The percentage of various bone marrow subtypes based on Lin and CD45 expression were evaluated on D4 and D10 after 5-FU treatment. Note that while the percentage of Lin+/CD45+ subtypes decreased on D4, its percentage increased with hematopoietic recovery. Percentage of Lin–/CD45– (enriched in VSELs) increased on D4 and slightly reduced by D10. The Lin–/CD45+ (enriched in HSCs) percentage also increased by D4, and remained high on D10 (n = 10, ***p ≤ 0.001). c Upregulation in the pluripotent, primordial germ cell and proliferation specific transcripts is observed in BM cells that survive 5-FU on D4. The upregulation of these markers confirms the presence and involvement of primitive stem cells in regeneration. The transcript levels on D4 are shown compared with control (n = 6, *p ≤ 0.05; **p ≤ 0.01). Bars in a, b represent average ± SD and in c average ± SEM. d–h Characterization of cells on D4 that survived 5-FU treatment. d H & E staining shows VSELs which are spherical in shape and small in size with high nucleo-cytoplasmic ratio located near the endosteal region. Inset (e) shows cells at higher magnification. f Nuclear OCT-4+ VSELs are clearly visualized in bone marrow sections cells in clusters (*) or singly (arrow) along the endosteal region; these cells also express (g) cell-surface SSEA-1and (h) nuclear SCA-1. Presence of nuclear OCT-4, SSEA-1, and SCA-1 confirms that VSELs survive 5-FU treatment. Nuclei are counterstained with DAPI. Scale bar = 20 μm for H & E and immunohistochemistry images, scale bar = 10 μm for immunofluorescence images. Day 2-FU, Day 4-FU, Day 10-FU days post 5-FU treatment, BM bone marrow, 5-FU 5-fluorouracil, H & E Hematoxylin and Eosin, OCT-4 octamer binding transforming factor-4, SSEA-1 stage-specific embryonic antigen-1, SCA-1, stem cell antigen-1, DAPI 4′,6-diamidino-2-phenylindole

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)

Mature and actively dividing cells in BM are affected by 5-FU whereas primitive and relatively quiescent stem cells are spared. a The total BM nucleated cells isolated from two femurs and two tibias of the mice were counted on D2, D4, and D10 after 5-FU treatment and compared with control. Similar to histological data (Fig. 2); the cell count decreased up to D4 (7-fold) and is increased on D10 (n = 10, ***p ≤ 0.001). b The percentage of various bone marrow subtypes based on Lin and CD45 expression were evaluated on D4 and D10 after 5-FU treatment. Note that while the percentage of Lin+/CD45+ subtypes decreased on D4, its percentage increased with hematopoietic recovery. Percentage of Lin–/CD45– (enriched in VSELs) increased on D4 and slightly reduced by D10. The Lin–/CD45+ (enriched in HSCs) percentage also increased by D4, and remained high on D10 (n = 10, ***p ≤ 0.001). c Upregulation in the pluripotent, primordial germ cell and proliferation specific transcripts is observed in BM cells that survive 5-FU on D4. The upregulation of these markers confirms the presence and involvement of primitive stem cells in regeneration. The transcript levels on D4 are shown compared with control (n = 6, *p ≤ 0.05; **p ≤ 0.01). Bars in a, b represent average ± SD and in c average ± SEM. d–h Characterization of cells on D4 that survived 5-FU treatment. d H & E staining shows VSELs which are spherical in shape and small in size with high nucleo-cytoplasmic ratio located near the endosteal region. Inset (e) shows cells at higher magnification. f Nuclear OCT-4+ VSELs are clearly visualized in bone marrow sections cells in clusters (*) or singly (arrow) along the endosteal region; these cells also express (g) cell-surface SSEA-1and (h) nuclear SCA-1. Presence of nuclear OCT-4, SSEA-1, and SCA-1 confirms that VSELs survive 5-FU treatment. Nuclei are counterstained with DAPI. Scale bar = 20 μm for H & E and immunohistochemistry images, scale bar = 10 μm for immunofluorescence images. Day 2-FU, Day 4-FU, Day 10-FU days post 5-FU treatment, BM bone marrow, 5-FU 5-fluorouracil, H & E Hematoxylin and Eosin, OCT-4 octamer binding transforming factor-4, SSEA-1 stage-specific embryonic antigen-1, SCA-1, stem cell antigen-1, DAPI 4′,6-diamidino-2-phenylindole
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Fig3: Mature and actively dividing cells in BM are affected by 5-FU whereas primitive and relatively quiescent stem cells are spared. a The total BM nucleated cells isolated from two femurs and two tibias of the mice were counted on D2, D4, and D10 after 5-FU treatment and compared with control. Similar to histological data (Fig. 2); the cell count decreased up to D4 (7-fold) and is increased on D10 (n = 10, ***p ≤ 0.001). b The percentage of various bone marrow subtypes based on Lin and CD45 expression were evaluated on D4 and D10 after 5-FU treatment. Note that while the percentage of Lin+/CD45+ subtypes decreased on D4, its percentage increased with hematopoietic recovery. Percentage of Lin–/CD45– (enriched in VSELs) increased on D4 and slightly reduced by D10. The Lin–/CD45+ (enriched in HSCs) percentage also increased by D4, and remained high on D10 (n = 10, ***p ≤ 0.001). c Upregulation in the pluripotent, primordial germ cell and proliferation specific transcripts is observed in BM cells that survive 5-FU on D4. The upregulation of these markers confirms the presence and involvement of primitive stem cells in regeneration. The transcript levels on D4 are shown compared with control (n = 6, *p ≤ 0.05; **p ≤ 0.01). Bars in a, b represent average ± SD and in c average ± SEM. d–h Characterization of cells on D4 that survived 5-FU treatment. d H & E staining shows VSELs which are spherical in shape and small in size with high nucleo-cytoplasmic ratio located near the endosteal region. Inset (e) shows cells at higher magnification. f Nuclear OCT-4+ VSELs are clearly visualized in bone marrow sections cells in clusters (*) or singly (arrow) along the endosteal region; these cells also express (g) cell-surface SSEA-1and (h) nuclear SCA-1. Presence of nuclear OCT-4, SSEA-1, and SCA-1 confirms that VSELs survive 5-FU treatment. Nuclei are counterstained with DAPI. Scale bar = 20 μm for H & E and immunohistochemistry images, scale bar = 10 μm for immunofluorescence images. Day 2-FU, Day 4-FU, Day 10-FU days post 5-FU treatment, BM bone marrow, 5-FU 5-fluorouracil, H & E Hematoxylin and Eosin, OCT-4 octamer binding transforming factor-4, SSEA-1 stage-specific embryonic antigen-1, SCA-1, stem cell antigen-1, DAPI 4′,6-diamidino-2-phenylindole
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.


Related in: MedlinePlus