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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

Hematopoiesis recovery is augmented after FSH treatment. a, b Cell surface expression of FSHR (green) on both small VSELs (top panel) and slightly bigger HSCs (bottom panel) which survive 5-FU treatment in mouse BM on D4. c Co-expression of SCA-1 (red) and FSHR (green) on the same cells. The nuclei are counterstained with DAPI. Scale bar = 20 μm. d, e H & E sections on D4 showed increased BM cellularity after FSH treatment on D4 (d) compared with FSH minus control. e In 5-FU + FSH-treated sections, the endogenous bone marrow regeneration was almost complete by D7 after 5-FU treatment compared with untreated controls. f Flow cytometry data showed an increase in number of VSELs and HSCs on FSH treatment. g BrdU uptake also increased on FSH treatment slightly in the primitive Lin–/CD45– (enriched in VSELs) while a significant increase (p < 0.001) was seen in Lin–/CD45+ (HSC-enriched) cells. h Upregulation of transcripts specific for pluripotent, primordial germ cells, and proliferation markers on FSH treatment compared with untreated controls. Significant increase in Oct-4 and Stella transcripts was observed. Please note that Oct-4 transcripts (which reflect HSCs) were more than Oct-4A (which reflects VSELs). Bars in f, g represent average ± SD and in h average ± SEM. Representative of five experiments in f, g and in h data obtained from three experiments. *p ≤ 0.05. ***p ≤ 0.001. D4-5-FU, D7-5-FU days post 5-FU treatment, FSH follicle-stimulating hormone, BM bone marrow, 5-FU 5-fluorouracil, H & E hematoxylin and eosin, VSEL very small embryonic-like stem cell, FSHR follicle stimulating hormone receptor, SCA-1 stem cell antigen-1, HSC hematopoietic stem cell, OCT-4 octamer binding transforming factor-4, BrdU bromodeoxyuridine (Color figure online)
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Fig6: Hematopoiesis recovery is augmented after FSH treatment. a, b Cell surface expression of FSHR (green) on both small VSELs (top panel) and slightly bigger HSCs (bottom panel) which survive 5-FU treatment in mouse BM on D4. c Co-expression of SCA-1 (red) and FSHR (green) on the same cells. The nuclei are counterstained with DAPI. Scale bar = 20 μm. d, e H & E sections on D4 showed increased BM cellularity after FSH treatment on D4 (d) compared with FSH minus control. e In 5-FU + FSH-treated sections, the endogenous bone marrow regeneration was almost complete by D7 after 5-FU treatment compared with untreated controls. f Flow cytometry data showed an increase in number of VSELs and HSCs on FSH treatment. g BrdU uptake also increased on FSH treatment slightly in the primitive Lin–/CD45– (enriched in VSELs) while a significant increase (p < 0.001) was seen in Lin–/CD45+ (HSC-enriched) cells. h Upregulation of transcripts specific for pluripotent, primordial germ cells, and proliferation markers on FSH treatment compared with untreated controls. Significant increase in Oct-4 and Stella transcripts was observed. Please note that Oct-4 transcripts (which reflect HSCs) were more than Oct-4A (which reflects VSELs). Bars in f, g represent average ± SD and in h average ± SEM. Representative of five experiments in f, g and in h data obtained from three experiments. *p ≤ 0.05. ***p ≤ 0.001. D4-5-FU, D7-5-FU days post 5-FU treatment, FSH follicle-stimulating hormone, BM bone marrow, 5-FU 5-fluorouracil, H & E hematoxylin and eosin, VSEL very small embryonic-like stem cell, FSHR follicle stimulating hormone receptor, SCA-1 stem cell antigen-1, HSC hematopoietic stem cell, OCT-4 octamer binding transforming factor-4, BrdU bromodeoxyuridine (Color figure online)

Mentions: To outline the effect of FSH on stem/progenitor cells in the mouse BM, initially the expression of FSHR on the bone marrow cells was studied. Immunofluorescence studies on BM cells that survived 5-FU treatment on D4 showed cell surface expression of FSHR on cells of two distinct sizes (Fig. 6a, b; Additional file 1: Figure S3). Co-expression of SCA-1 and FSHR was also observed, confirming that indeed stem/progenitors express FSHR (Fig. 6c). In addition to FSHR, cells that survived 5-FU treatment also expressed the primordial germ cell (PGC) marker STELLA (Additional file 1: Figure S2) and showed increase in PGC transcripts (Fig. 3c). Immunophenotyping results (Additional file 1: Table S4, Figure S3) showed that only 6 % of LIN–/CD45– VSELs and 10 % of LIN–/CD45+ HSCs expressed FSHR.Fig. 6


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)

Hematopoiesis recovery is augmented after FSH treatment. a, b Cell surface expression of FSHR (green) on both small VSELs (top panel) and slightly bigger HSCs (bottom panel) which survive 5-FU treatment in mouse BM on D4. c Co-expression of SCA-1 (red) and FSHR (green) on the same cells. The nuclei are counterstained with DAPI. Scale bar = 20 μm. d, e H & E sections on D4 showed increased BM cellularity after FSH treatment on D4 (d) compared with FSH minus control. e In 5-FU + FSH-treated sections, the endogenous bone marrow regeneration was almost complete by D7 after 5-FU treatment compared with untreated controls. f Flow cytometry data showed an increase in number of VSELs and HSCs on FSH treatment. g BrdU uptake also increased on FSH treatment slightly in the primitive Lin–/CD45– (enriched in VSELs) while a significant increase (p < 0.001) was seen in Lin–/CD45+ (HSC-enriched) cells. h Upregulation of transcripts specific for pluripotent, primordial germ cells, and proliferation markers on FSH treatment compared with untreated controls. Significant increase in Oct-4 and Stella transcripts was observed. Please note that Oct-4 transcripts (which reflect HSCs) were more than Oct-4A (which reflects VSELs). Bars in f, g represent average ± SD and in h average ± SEM. Representative of five experiments in f, g and in h data obtained from three experiments. *p ≤ 0.05. ***p ≤ 0.001. D4-5-FU, D7-5-FU days post 5-FU treatment, FSH follicle-stimulating hormone, BM bone marrow, 5-FU 5-fluorouracil, H & E hematoxylin and eosin, VSEL very small embryonic-like stem cell, FSHR follicle stimulating hormone receptor, SCA-1 stem cell antigen-1, HSC hematopoietic stem cell, OCT-4 octamer binding transforming factor-4, BrdU bromodeoxyuridine (Color figure online)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig6: Hematopoiesis recovery is augmented after FSH treatment. a, b Cell surface expression of FSHR (green) on both small VSELs (top panel) and slightly bigger HSCs (bottom panel) which survive 5-FU treatment in mouse BM on D4. c Co-expression of SCA-1 (red) and FSHR (green) on the same cells. The nuclei are counterstained with DAPI. Scale bar = 20 μm. d, e H & E sections on D4 showed increased BM cellularity after FSH treatment on D4 (d) compared with FSH minus control. e In 5-FU + FSH-treated sections, the endogenous bone marrow regeneration was almost complete by D7 after 5-FU treatment compared with untreated controls. f Flow cytometry data showed an increase in number of VSELs and HSCs on FSH treatment. g BrdU uptake also increased on FSH treatment slightly in the primitive Lin–/CD45– (enriched in VSELs) while a significant increase (p < 0.001) was seen in Lin–/CD45+ (HSC-enriched) cells. h Upregulation of transcripts specific for pluripotent, primordial germ cells, and proliferation markers on FSH treatment compared with untreated controls. Significant increase in Oct-4 and Stella transcripts was observed. Please note that Oct-4 transcripts (which reflect HSCs) were more than Oct-4A (which reflects VSELs). Bars in f, g represent average ± SD and in h average ± SEM. Representative of five experiments in f, g and in h data obtained from three experiments. *p ≤ 0.05. ***p ≤ 0.001. D4-5-FU, D7-5-FU days post 5-FU treatment, FSH follicle-stimulating hormone, BM bone marrow, 5-FU 5-fluorouracil, H & E hematoxylin and eosin, VSEL very small embryonic-like stem cell, FSHR follicle stimulating hormone receptor, SCA-1 stem cell antigen-1, HSC hematopoietic stem cell, OCT-4 octamer binding transforming factor-4, BrdU bromodeoxyuridine (Color figure online)
Mentions: To outline the effect of FSH on stem/progenitor cells in the mouse BM, initially the expression of FSHR on the bone marrow cells was studied. Immunofluorescence studies on BM cells that survived 5-FU treatment on D4 showed cell surface expression of FSHR on cells of two distinct sizes (Fig. 6a, b; Additional file 1: Figure S3). Co-expression of SCA-1 and FSHR was also observed, confirming that indeed stem/progenitors express FSHR (Fig. 6c). In addition to FSHR, cells that survived 5-FU treatment also expressed the primordial germ cell (PGC) marker STELLA (Additional file 1: Figure S2) and showed increase in PGC transcripts (Fig. 3c). Immunophenotyping results (Additional file 1: Table S4, Figure S3) showed that only 6 % of LIN–/CD45– VSELs and 10 % of LIN–/CD45+ HSCs expressed FSHR.Fig. 6

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