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Fetal hematopoietic stem cells express MFG-E8 during mouse embryogenesis.

Lee J, Choi BI, Park SY, An SY, Han J, Kim JH - Exp. Mol. Med. (2015)

Bottom Line: Using immunohistochemistry, we showed that MFG-E8 was specifically expressed in CD34(+) HSCs at all hematopoietic sites, including the yolk sac, aorta-gonad-mesonephros region, placenta and fetal liver, during embryogenesis.Fluorescence-activated cell sorting and polymerase chain reaction analyses demonstrated that CD34(+) cells, purified from the fetal liver, expressed additional HSC markers, c-Kit and Sca-1, and that these CD34(+) cells, but not CD34(-) cells, highly expressed MFG-E8.Together, this study demonstrates, for the first time, that MFG-8 is expressed in fetal HSC populations, and that MFG-E8 may have a role in embryonic hematopoiesis.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Stem Cells and Tissue Regeneration, Division of Biotechnology, College of Life Sciences and Biotechnology, Korea University, Seoul, Korea.

ABSTRACT
The milk fat globule-EGF-factor 8 protein (MFG-E8) has been identified in various tissues, where it has an important role in intercellular interactions, cellular migration, and neovascularization. Previous studies showed that MFG-E8 is expressed in different cell types under normal and pathophysiological conditions, but its expression in hematopoietic stem cells (HSCs) during hematopoiesis has not been reported. In the present study, we investigated MFG-E8 expression in multiple hematopoietic tissues at different stages of mouse embryogenesis. Using immunohistochemistry, we showed that MFG-E8 was specifically expressed in CD34(+) HSCs at all hematopoietic sites, including the yolk sac, aorta-gonad-mesonephros region, placenta and fetal liver, during embryogenesis. Fluorescence-activated cell sorting and polymerase chain reaction analyses demonstrated that CD34(+) cells, purified from the fetal liver, expressed additional HSC markers, c-Kit and Sca-1, and that these CD34(+) cells, but not CD34(-) cells, highly expressed MFG-E8. We also found that MFG-E8 was not expressed in HSCs in adult mouse bone marrow, and that its expression was confined to F4/80(+) macrophages. Together, this study demonstrates, for the first time, that MFG-8 is expressed in fetal HSC populations, and that MFG-E8 may have a role in embryonic hematopoiesis.

No MeSH data available.


Related in: MedlinePlus

Expression of milk fat globule-EGF-factor 8 (MFG-E8) in CD34+ cells in the fetal liver. (a and b) Flow cytometric analysis of CD34+ cells in the developing liver before (a) and after (b) purification with anti-CD34 antibody. (c) quantitative PCR (qPCR) analysis of sorted cells for the expression of Cd34. Cd34 expression in the CD34+ fraction was presented as relative expressions (fold changes) over CD34− fraction after normalization to β-actin expression. *P<0.05. (d and e) real-time reverse transcription-polymerase chain reaction (RT-PCR) (d) and qPCR (e) analyses of sorted CD34+ and CD34− fractions for the expression of Mfg-e8. Note that only a CD34+ fraction expresses Mfg-e8. P<0.05. Mfg-e8 expression in the CD34+ fraction was presented as relative expressions (fold changes) over CD34− fraction after normalization to β-actin expression.
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fig4: Expression of milk fat globule-EGF-factor 8 (MFG-E8) in CD34+ cells in the fetal liver. (a and b) Flow cytometric analysis of CD34+ cells in the developing liver before (a) and after (b) purification with anti-CD34 antibody. (c) quantitative PCR (qPCR) analysis of sorted cells for the expression of Cd34. Cd34 expression in the CD34+ fraction was presented as relative expressions (fold changes) over CD34− fraction after normalization to β-actin expression. *P<0.05. (d and e) real-time reverse transcription-polymerase chain reaction (RT-PCR) (d) and qPCR (e) analyses of sorted CD34+ and CD34− fractions for the expression of Mfg-e8. Note that only a CD34+ fraction expresses Mfg-e8. P<0.05. Mfg-e8 expression in the CD34+ fraction was presented as relative expressions (fold changes) over CD34− fraction after normalization to β-actin expression.

Mentions: The fetal liver is colonized by HSCs at days 10 and 11 of gestation, and HSC expansion initiates on E13. The fetal liver therefore serves as a main organ for HSC expansion and differentiation, until hematopoiesis in the bone marrow is established. Fetal liver HSCs have a similar surface phenotype to placental HSCs, including the hematopoietic marker CD34.22, 23 We purified CD34+ cells from fetal livers at E12.5 (n=55 from 6 pregnant mice) to determine the selective expression of MFG-E8 in CD34+ HSCs. The CD34 antigen was expressed by 21.6% of whole-fetal liver cells after depletion of RBCs by lysis. In addition, the percentage increased up to 99% after purification by fluorescence-activated cell sorting, using anti-CD34 antibody (Figures 4a and b). The purification of CD34+ cells was confirmed by qPCR, showing that Cd34 was expressed only in the CD34+ fraction, but not in the CD34− fraction (Figure 4c). Importantly, RT-PCR analysis revealed that although c-Kit was expressed in both CD34+ and CD34− fractions, Mfg-e8 expression was detected only in cells that expressed c-Kit, Sca-1 and Cd34, whereas the CD34− fraction expressed very low levels, if any, of Mfg-e8 (Figure 4d). The high level of Mfg-e8 expression in CD34+ cells was further validated by qPCR analysis (Figure 4e).


Fetal hematopoietic stem cells express MFG-E8 during mouse embryogenesis.

Lee J, Choi BI, Park SY, An SY, Han J, Kim JH - Exp. Mol. Med. (2015)

Expression of milk fat globule-EGF-factor 8 (MFG-E8) in CD34+ cells in the fetal liver. (a and b) Flow cytometric analysis of CD34+ cells in the developing liver before (a) and after (b) purification with anti-CD34 antibody. (c) quantitative PCR (qPCR) analysis of sorted cells for the expression of Cd34. Cd34 expression in the CD34+ fraction was presented as relative expressions (fold changes) over CD34− fraction after normalization to β-actin expression. *P<0.05. (d and e) real-time reverse transcription-polymerase chain reaction (RT-PCR) (d) and qPCR (e) analyses of sorted CD34+ and CD34− fractions for the expression of Mfg-e8. Note that only a CD34+ fraction expresses Mfg-e8. P<0.05. Mfg-e8 expression in the CD34+ fraction was presented as relative expressions (fold changes) over CD34− fraction after normalization to β-actin expression.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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fig4: Expression of milk fat globule-EGF-factor 8 (MFG-E8) in CD34+ cells in the fetal liver. (a and b) Flow cytometric analysis of CD34+ cells in the developing liver before (a) and after (b) purification with anti-CD34 antibody. (c) quantitative PCR (qPCR) analysis of sorted cells for the expression of Cd34. Cd34 expression in the CD34+ fraction was presented as relative expressions (fold changes) over CD34− fraction after normalization to β-actin expression. *P<0.05. (d and e) real-time reverse transcription-polymerase chain reaction (RT-PCR) (d) and qPCR (e) analyses of sorted CD34+ and CD34− fractions for the expression of Mfg-e8. Note that only a CD34+ fraction expresses Mfg-e8. P<0.05. Mfg-e8 expression in the CD34+ fraction was presented as relative expressions (fold changes) over CD34− fraction after normalization to β-actin expression.
Mentions: The fetal liver is colonized by HSCs at days 10 and 11 of gestation, and HSC expansion initiates on E13. The fetal liver therefore serves as a main organ for HSC expansion and differentiation, until hematopoiesis in the bone marrow is established. Fetal liver HSCs have a similar surface phenotype to placental HSCs, including the hematopoietic marker CD34.22, 23 We purified CD34+ cells from fetal livers at E12.5 (n=55 from 6 pregnant mice) to determine the selective expression of MFG-E8 in CD34+ HSCs. The CD34 antigen was expressed by 21.6% of whole-fetal liver cells after depletion of RBCs by lysis. In addition, the percentage increased up to 99% after purification by fluorescence-activated cell sorting, using anti-CD34 antibody (Figures 4a and b). The purification of CD34+ cells was confirmed by qPCR, showing that Cd34 was expressed only in the CD34+ fraction, but not in the CD34− fraction (Figure 4c). Importantly, RT-PCR analysis revealed that although c-Kit was expressed in both CD34+ and CD34− fractions, Mfg-e8 expression was detected only in cells that expressed c-Kit, Sca-1 and Cd34, whereas the CD34− fraction expressed very low levels, if any, of Mfg-e8 (Figure 4d). The high level of Mfg-e8 expression in CD34+ cells was further validated by qPCR analysis (Figure 4e).

Bottom Line: Using immunohistochemistry, we showed that MFG-E8 was specifically expressed in CD34(+) HSCs at all hematopoietic sites, including the yolk sac, aorta-gonad-mesonephros region, placenta and fetal liver, during embryogenesis.Fluorescence-activated cell sorting and polymerase chain reaction analyses demonstrated that CD34(+) cells, purified from the fetal liver, expressed additional HSC markers, c-Kit and Sca-1, and that these CD34(+) cells, but not CD34(-) cells, highly expressed MFG-E8.Together, this study demonstrates, for the first time, that MFG-8 is expressed in fetal HSC populations, and that MFG-E8 may have a role in embryonic hematopoiesis.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Stem Cells and Tissue Regeneration, Division of Biotechnology, College of Life Sciences and Biotechnology, Korea University, Seoul, Korea.

ABSTRACT
The milk fat globule-EGF-factor 8 protein (MFG-E8) has been identified in various tissues, where it has an important role in intercellular interactions, cellular migration, and neovascularization. Previous studies showed that MFG-E8 is expressed in different cell types under normal and pathophysiological conditions, but its expression in hematopoietic stem cells (HSCs) during hematopoiesis has not been reported. In the present study, we investigated MFG-E8 expression in multiple hematopoietic tissues at different stages of mouse embryogenesis. Using immunohistochemistry, we showed that MFG-E8 was specifically expressed in CD34(+) HSCs at all hematopoietic sites, including the yolk sac, aorta-gonad-mesonephros region, placenta and fetal liver, during embryogenesis. Fluorescence-activated cell sorting and polymerase chain reaction analyses demonstrated that CD34(+) cells, purified from the fetal liver, expressed additional HSC markers, c-Kit and Sca-1, and that these CD34(+) cells, but not CD34(-) cells, highly expressed MFG-E8. We also found that MFG-E8 was not expressed in HSCs in adult mouse bone marrow, and that its expression was confined to F4/80(+) macrophages. Together, this study demonstrates, for the first time, that MFG-8 is expressed in fetal HSC populations, and that MFG-E8 may have a role in embryonic hematopoiesis.

No MeSH data available.


Related in: MedlinePlus