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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

Pluripotent very small embryonic-like stem cells (VSELs) in adult mouse bone marrow. a Expression of pluripotent stem cell makers including nuclear OCT-4 (red) and SOX-2 (red) and cell surface SSEA-1 (green) and SCA-1 (green) were detected on small-sized cells in bone marrow smears. Also note presence of cytoplasmic OCT-4 in slightly bigger cells. These cells were more abundant and please refer to Additional file 1: Figure S1 to see additional images of cytoplasmic OCT-4-expressing cells. Scale bar = 20 μm. b Flow cytometry analysis of VSELs and HSCs in bone marrow. Cells of size 2–8 μm were gated using size calibration beads as reference, followed by sequential selection of LIN–/SCA-1+ cells. This population was evaluated for expression of CD45. The LIN–/SCA-1+/CD45– cells were VSELs, while LIN–/SCA+/CD45+ cells were HSCs. The average percentage of VSELs and HSCs with SD from 10 experiments is reported. Representative image is shown. c Dual immunofluorescence was performed using anti-SSEA1 antibody (green) with nuclear OCT-4 (red, upper) and SCA-1 (red, lower). Cells co-expressing nuclear OCT-4 and SSEA-1 and SSEA-1 and SCA-1 were observed. Scale bar = 20 μm. In all images, nuclei are counterstained with DAPI. HSC Hematopoietic stem cell, OCT-4 octamer binding transforming factor-4, SSEA-1 stage-specific embryonic antigen-1, Sca-1 stem cell antigen-1, Sox 2 sex-determining region (box 2), DAPI 4′,6-diamidino-2-phenylindole (Color figure online)
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Fig1: Pluripotent very small embryonic-like stem cells (VSELs) in adult mouse bone marrow. a Expression of pluripotent stem cell makers including nuclear OCT-4 (red) and SOX-2 (red) and cell surface SSEA-1 (green) and SCA-1 (green) were detected on small-sized cells in bone marrow smears. Also note presence of cytoplasmic OCT-4 in slightly bigger cells. These cells were more abundant and please refer to Additional file 1: Figure S1 to see additional images of cytoplasmic OCT-4-expressing cells. Scale bar = 20 μm. b Flow cytometry analysis of VSELs and HSCs in bone marrow. Cells of size 2–8 μm were gated using size calibration beads as reference, followed by sequential selection of LIN–/SCA-1+ cells. This population was evaluated for expression of CD45. The LIN–/SCA-1+/CD45– cells were VSELs, while LIN–/SCA+/CD45+ cells were HSCs. The average percentage of VSELs and HSCs with SD from 10 experiments is reported. Representative image is shown. c Dual immunofluorescence was performed using anti-SSEA1 antibody (green) with nuclear OCT-4 (red, upper) and SCA-1 (red, lower). Cells co-expressing nuclear OCT-4 and SSEA-1 and SSEA-1 and SCA-1 were observed. Scale bar = 20 μm. In all images, nuclei are counterstained with DAPI. HSC Hematopoietic stem cell, OCT-4 octamer binding transforming factor-4, SSEA-1 stage-specific embryonic antigen-1, Sca-1 stem cell antigen-1, Sox 2 sex-determining region (box 2), DAPI 4′,6-diamidino-2-phenylindole (Color figure online)

Mentions: Earlier reports [6, 53] and our initial immunofluorescence studies on cell smears of mice bone marrow confirmed the presence of rare, small, spherical cells with high nucleo-cytoplasmic ratio expressing pluripotent stem cell markers including nuclear OCT-4A and SOX-2 and cell-surface SCA-1 and SSEA-1 (Fig. 1a). Interestingly, few larger cells with low nucleo-cytoplasmic ratio and prominent cytoplasm were also observed which expressed cytoplasmic OCT-4 (Fig. 1a; Additional file 1: Figure S1). These cells with cytoplasmic OCT-4 were present in more numbers and were possibly the HSCs. Based on these initial results, the BM cells were investigated further by flow cytometry (Fig. 1b) using the protocol and unique gating strategy reported by Kucia et al. [6]. Small cells (2–8 μm) were gated and analyzed for expression of Lineage (LIN) markers and pluripotent stem cell marker (SCA-1). Next, the LIN–/SCA-1+ cells were gated and analyzed for the expression of hematopoietic marker CD45. The LIN– and CD45– non-hematopoietic cells expressing pluripotent marker SCA-1 were the VSELs (LIN–/CD45–/SCA-1+), while the LIN–CD45+ hematopoietic cells with SCA-1 expression were the HSCs (LIN–/CD45+/SCA-1+). As shown in Fig. 1b, the percentage of VSELs (0.022 ± 0.002 %) was almost 3-fold lower than the HSCs (0.081 ± 0.004 %) (n = 10). A similar strategy was used to determine the percentage of small cells expressing OCT-4A and SSEA-1 and it was observed that the percentage of LIN–/CD45– cells co-expressing these markers was almost similar to SCA-1+ VSELs (Additional file 1: Table S3). This co-expression of markers in the small cells suggested that SCA-1+ VSELs also express OCT-4A and SSEA-1. Dual immunostaining of the bone marrow smears confirmed that cells co-expressed SCA-1 with OCT-4A and SCA-1 with SSEA-1 (Fig. 1c).Fig. 1


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)

Pluripotent very small embryonic-like stem cells (VSELs) in adult mouse bone marrow. a Expression of pluripotent stem cell makers including nuclear OCT-4 (red) and SOX-2 (red) and cell surface SSEA-1 (green) and SCA-1 (green) were detected on small-sized cells in bone marrow smears. Also note presence of cytoplasmic OCT-4 in slightly bigger cells. These cells were more abundant and please refer to Additional file 1: Figure S1 to see additional images of cytoplasmic OCT-4-expressing cells. Scale bar = 20 μm. b Flow cytometry analysis of VSELs and HSCs in bone marrow. Cells of size 2–8 μm were gated using size calibration beads as reference, followed by sequential selection of LIN–/SCA-1+ cells. This population was evaluated for expression of CD45. The LIN–/SCA-1+/CD45– cells were VSELs, while LIN–/SCA+/CD45+ cells were HSCs. The average percentage of VSELs and HSCs with SD from 10 experiments is reported. Representative image is shown. c Dual immunofluorescence was performed using anti-SSEA1 antibody (green) with nuclear OCT-4 (red, upper) and SCA-1 (red, lower). Cells co-expressing nuclear OCT-4 and SSEA-1 and SSEA-1 and SCA-1 were observed. Scale bar = 20 μm. In all images, nuclei are counterstained with DAPI. HSC Hematopoietic stem cell, OCT-4 octamer binding transforming factor-4, SSEA-1 stage-specific embryonic antigen-1, Sca-1 stem cell antigen-1, Sox 2 sex-determining region (box 2), DAPI 4′,6-diamidino-2-phenylindole (Color figure online)
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Related In: Results  -  Collection

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Fig1: Pluripotent very small embryonic-like stem cells (VSELs) in adult mouse bone marrow. a Expression of pluripotent stem cell makers including nuclear OCT-4 (red) and SOX-2 (red) and cell surface SSEA-1 (green) and SCA-1 (green) were detected on small-sized cells in bone marrow smears. Also note presence of cytoplasmic OCT-4 in slightly bigger cells. These cells were more abundant and please refer to Additional file 1: Figure S1 to see additional images of cytoplasmic OCT-4-expressing cells. Scale bar = 20 μm. b Flow cytometry analysis of VSELs and HSCs in bone marrow. Cells of size 2–8 μm were gated using size calibration beads as reference, followed by sequential selection of LIN–/SCA-1+ cells. This population was evaluated for expression of CD45. The LIN–/SCA-1+/CD45– cells were VSELs, while LIN–/SCA+/CD45+ cells were HSCs. The average percentage of VSELs and HSCs with SD from 10 experiments is reported. Representative image is shown. c Dual immunofluorescence was performed using anti-SSEA1 antibody (green) with nuclear OCT-4 (red, upper) and SCA-1 (red, lower). Cells co-expressing nuclear OCT-4 and SSEA-1 and SSEA-1 and SCA-1 were observed. Scale bar = 20 μm. In all images, nuclei are counterstained with DAPI. HSC Hematopoietic stem cell, OCT-4 octamer binding transforming factor-4, SSEA-1 stage-specific embryonic antigen-1, Sca-1 stem cell antigen-1, Sox 2 sex-determining region (box 2), DAPI 4′,6-diamidino-2-phenylindole (Color figure online)
Mentions: Earlier reports [6, 53] and our initial immunofluorescence studies on cell smears of mice bone marrow confirmed the presence of rare, small, spherical cells with high nucleo-cytoplasmic ratio expressing pluripotent stem cell markers including nuclear OCT-4A and SOX-2 and cell-surface SCA-1 and SSEA-1 (Fig. 1a). Interestingly, few larger cells with low nucleo-cytoplasmic ratio and prominent cytoplasm were also observed which expressed cytoplasmic OCT-4 (Fig. 1a; Additional file 1: Figure S1). These cells with cytoplasmic OCT-4 were present in more numbers and were possibly the HSCs. Based on these initial results, the BM cells were investigated further by flow cytometry (Fig. 1b) using the protocol and unique gating strategy reported by Kucia et al. [6]. Small cells (2–8 μm) were gated and analyzed for expression of Lineage (LIN) markers and pluripotent stem cell marker (SCA-1). Next, the LIN–/SCA-1+ cells were gated and analyzed for the expression of hematopoietic marker CD45. The LIN– and CD45– non-hematopoietic cells expressing pluripotent marker SCA-1 were the VSELs (LIN–/CD45–/SCA-1+), while the LIN–CD45+ hematopoietic cells with SCA-1 expression were the HSCs (LIN–/CD45+/SCA-1+). As shown in Fig. 1b, the percentage of VSELs (0.022 ± 0.002 %) was almost 3-fold lower than the HSCs (0.081 ± 0.004 %) (n = 10). A similar strategy was used to determine the percentage of small cells expressing OCT-4A and SSEA-1 and it was observed that the percentage of LIN–/CD45– cells co-expressing these markers was almost similar to SCA-1+ VSELs (Additional file 1: Table S3). This co-expression of markers in the small cells suggested that SCA-1+ VSELs also express OCT-4A and SSEA-1. Dual immunostaining of the bone marrow smears confirmed that cells co-expressed SCA-1 with OCT-4A and SCA-1 with SSEA-1 (Fig. 1c).Fig. 1

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