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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

Proliferation of nuclear OCT-4+ VSELs. a Dual immunofluorescence on cryosections of D4 femur detected the presence of nuclear OCT-4+ cells co-expressing proliferation marker PCNA. Nuclear OCT-4/PCNA co-expressing VSELs were observed close to the endosteal region. The nuclei were counterstained with DAPI. Scale bar = 100 μm. Inset: magnified image of cluster of OCT-4/PCNA coexpressing cells. Scale bar = 10 μm. b, c Results of the CFU assay: small and large hematopoietic colonies of type CFU-GM or CFU-GEMM were observed on culture of cells that survived 5-FU on D4 in Methocult media. d GFP+ cell clusters observed after 10 days of culture of 5-FU-treated GFP cells in Methocult media. e Typical cobblestone formation representative of hematopoietic colonies seen in 5-FU-treated cell culture. f Representative image of colonies observed in CFU assay at 20× magnification, colony formed is of the type CFU-GEMM. OCT-4 octamer binding transforming factor-4, PCNA proliferating cell nuclear antigen, DAPI 4′,6-diamidino-2-phenylindole, CFU-GM colony-forming unit granulocytes/macrophage, CFU-GEMM colony-forming unit-granulocyte, erythroid, macrophage, megakaryocyte
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Fig5: Proliferation of nuclear OCT-4+ VSELs. a Dual immunofluorescence on cryosections of D4 femur detected the presence of nuclear OCT-4+ cells co-expressing proliferation marker PCNA. Nuclear OCT-4/PCNA co-expressing VSELs were observed close to the endosteal region. The nuclei were counterstained with DAPI. Scale bar = 100 μm. Inset: magnified image of cluster of OCT-4/PCNA coexpressing cells. Scale bar = 10 μm. b, c Results of the CFU assay: small and large hematopoietic colonies of type CFU-GM or CFU-GEMM were observed on culture of cells that survived 5-FU on D4 in Methocult media. d GFP+ cell clusters observed after 10 days of culture of 5-FU-treated GFP cells in Methocult media. e Typical cobblestone formation representative of hematopoietic colonies seen in 5-FU-treated cell culture. f Representative image of colonies observed in CFU assay at 20× magnification, colony formed is of the type CFU-GEMM. OCT-4 octamer binding transforming factor-4, PCNA proliferating cell nuclear antigen, DAPI 4′,6-diamidino-2-phenylindole, CFU-GM colony-forming unit granulocytes/macrophage, CFU-GEMM colony-forming unit-granulocyte, erythroid, macrophage, megakaryocyte

Mentions: An in vivo BrdU incorporation assay was then carried out to examine whether both the LIN–/CD45+ (enriched in HSCs) and LIN–/CD45– (enriched in VSELs) populations proliferated in response to 5-FU treatment. There was a significant increase in the BrdU uptake by both these cell types (p < 0.001) on D4 compared with untreated control, confirming their proliferation (Fig. 4c). BM cryosections on D4 showed the presence of VSELs which co-expressed nuclear OCT-4 and PCNA near the endosteal region of the BM (Fig. 5a). Together, the uptake of BrdU by non-hematopoietic cells and the expression proliferation marker PCNA by the pluripotent OCT4-expressing cells confirms that VSELs underwent self-renewal and thus contributed to restoring hematopoiesis by D10. We further examined the functional potential of cells that survived 5-FU treatment in vitro using a colony-forming unit (CFU) assay. Cells that survived 5-FU responded to the cytokines in the Methocult media and developed 20–25 small and large colonies (Fig. 5b–e).Fig. 5


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)

Proliferation of nuclear OCT-4+ VSELs. a Dual immunofluorescence on cryosections of D4 femur detected the presence of nuclear OCT-4+ cells co-expressing proliferation marker PCNA. Nuclear OCT-4/PCNA co-expressing VSELs were observed close to the endosteal region. The nuclei were counterstained with DAPI. Scale bar = 100 μm. Inset: magnified image of cluster of OCT-4/PCNA coexpressing cells. Scale bar = 10 μm. b, c Results of the CFU assay: small and large hematopoietic colonies of type CFU-GM or CFU-GEMM were observed on culture of cells that survived 5-FU on D4 in Methocult media. d GFP+ cell clusters observed after 10 days of culture of 5-FU-treated GFP cells in Methocult media. e Typical cobblestone formation representative of hematopoietic colonies seen in 5-FU-treated cell culture. f Representative image of colonies observed in CFU assay at 20× magnification, colony formed is of the type CFU-GEMM. OCT-4 octamer binding transforming factor-4, PCNA proliferating cell nuclear antigen, DAPI 4′,6-diamidino-2-phenylindole, CFU-GM colony-forming unit granulocytes/macrophage, CFU-GEMM colony-forming unit-granulocyte, erythroid, macrophage, megakaryocyte
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Fig5: Proliferation of nuclear OCT-4+ VSELs. a Dual immunofluorescence on cryosections of D4 femur detected the presence of nuclear OCT-4+ cells co-expressing proliferation marker PCNA. Nuclear OCT-4/PCNA co-expressing VSELs were observed close to the endosteal region. The nuclei were counterstained with DAPI. Scale bar = 100 μm. Inset: magnified image of cluster of OCT-4/PCNA coexpressing cells. Scale bar = 10 μm. b, c Results of the CFU assay: small and large hematopoietic colonies of type CFU-GM or CFU-GEMM were observed on culture of cells that survived 5-FU on D4 in Methocult media. d GFP+ cell clusters observed after 10 days of culture of 5-FU-treated GFP cells in Methocult media. e Typical cobblestone formation representative of hematopoietic colonies seen in 5-FU-treated cell culture. f Representative image of colonies observed in CFU assay at 20× magnification, colony formed is of the type CFU-GEMM. OCT-4 octamer binding transforming factor-4, PCNA proliferating cell nuclear antigen, DAPI 4′,6-diamidino-2-phenylindole, CFU-GM colony-forming unit granulocytes/macrophage, CFU-GEMM colony-forming unit-granulocyte, erythroid, macrophage, megakaryocyte
Mentions: An in vivo BrdU incorporation assay was then carried out to examine whether both the LIN–/CD45+ (enriched in HSCs) and LIN–/CD45– (enriched in VSELs) populations proliferated in response to 5-FU treatment. There was a significant increase in the BrdU uptake by both these cell types (p < 0.001) on D4 compared with untreated control, confirming their proliferation (Fig. 4c). BM cryosections on D4 showed the presence of VSELs which co-expressed nuclear OCT-4 and PCNA near the endosteal region of the BM (Fig. 5a). Together, the uptake of BrdU by non-hematopoietic cells and the expression proliferation marker PCNA by the pluripotent OCT4-expressing cells confirms that VSELs underwent self-renewal and thus contributed to restoring hematopoiesis by D10. We further examined the functional potential of cells that survived 5-FU treatment in vitro using a colony-forming unit (CFU) assay. Cells that survived 5-FU responded to the cytokines in the Methocult media and developed 20–25 small and large colonies (Fig. 5b–e).Fig. 5

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