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Fate of undifferentiated mouse embryonic stem cells within the rat heart: role of myocardial infarction and immune suppression.

He Q, Trindade PT, Stumm M, Li J, Zammaretti P, Bettiol E, Dubois-Dauphin M, Herrmann F, Kalangos A, Morel D, Jaconi ME - J. Cell. Mol. Med. (2008)

Bottom Line: We found that mESC survival depended on immunosuppression both in normal and infarcted hearts.However, upon Cyclosporin A treatment, both normal and infarcted rat hearts failed to induce selective cardiac differentiation of implanted mESC.Instead, teratomas developed in normal and infarcted rat hearts 1 week and 4 weeks (50% and 100%, respectively) after cell injection.

View Article: PubMed Central - PubMed

Affiliation: Department of Rehabilitation and Geriatrics, Laboratory of Biology of Aging, Geneva University Hospitals, Geneva, Switzerland.

ABSTRACT
Abstract It has recently been suggested that the infarcted rat heart microenvironment could direct pluripotent mouse embryonic stem cells to differentiate into cardiomyocytes through an in situ paracrine action. To investigate whether the heart can function as a cardiogenic niche and confer an immune privilege to embryonic stem cells, we assessed the cardiac differentiation potential of undifferentiated mouse embryonic stem cells (mESC) injected into normal, acutely or chronically infarcted rat hearts. We found that mESC survival depended on immunosuppression both in normal and infarcted hearts. However, upon Cyclosporin A treatment, both normal and infarcted rat hearts failed to induce selective cardiac differentiation of implanted mESC. Instead, teratomas developed in normal and infarcted rat hearts 1 week and 4 weeks (50% and 100%, respectively) after cell injection. Tight control of ESC commitment into a specific cardiac lineage is mandatory to avoid the risk of uncontrolled growth and tumourigenesis following transplantation of highly plastic cells into a diseased myocardium.

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Characterization of the ESC lines used for the transplantation experiments. (A) Expression of eGFP or CD63 transgene by CGR8 stable clones. Panel a: undifferentiated colony positive for eGFP fluorescence localizing to membranes and endosomes. (B) Phase contrast picture of the CGR8 colony. (C) Immunostaining of a CGR8 cell clone expressing the hBcl2 gene. (D) Untransfected cells do not react with the antihBcl2 antibody. (E) Comparison of the beating activity expressed as percentage of beating EBs measured in wt or hBcl2-CGR8 EBs at day 8 and day 20 of culture. In contrast to wild-type EBs, 20-day-old EBs derived from human Bcl-2 expressing cells maintained a high percentage of beating. (F) DNA laddering performed on wild-type (wt) or hBcl2-CGR8 cells (lane 1 and 2, respectively) treated with 10 μM hydrogen peroxide for 24 hrs. (G, H) Immunoreactivity of mESC to antibodies against MHC class I or class II. Undifferentiated ESC colonies were stained with anti-MHC-I (G), or anti-MHC-II (H). Images correspond to the middle slice of a deconvoluted stack of confocal images acquired by a Zeiss Meta confocal microscope.
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fig01: Characterization of the ESC lines used for the transplantation experiments. (A) Expression of eGFP or CD63 transgene by CGR8 stable clones. Panel a: undifferentiated colony positive for eGFP fluorescence localizing to membranes and endosomes. (B) Phase contrast picture of the CGR8 colony. (C) Immunostaining of a CGR8 cell clone expressing the hBcl2 gene. (D) Untransfected cells do not react with the antihBcl2 antibody. (E) Comparison of the beating activity expressed as percentage of beating EBs measured in wt or hBcl2-CGR8 EBs at day 8 and day 20 of culture. In contrast to wild-type EBs, 20-day-old EBs derived from human Bcl-2 expressing cells maintained a high percentage of beating. (F) DNA laddering performed on wild-type (wt) or hBcl2-CGR8 cells (lane 1 and 2, respectively) treated with 10 μM hydrogen peroxide for 24 hrs. (G, H) Immunoreactivity of mESC to antibodies against MHC class I or class II. Undifferentiated ESC colonies were stained with anti-MHC-I (G), or anti-MHC-II (H). Images correspond to the middle slice of a deconvoluted stack of confocal images acquired by a Zeiss Meta confocal microscope.

Mentions: We genetically modified the mouse ESC line CGR8 [16] to express as reporter gene either the enhanced green fluorescent protein (eGFP) fused with CD63 or the human anti-apoptotic gene Bcl2 (hBcl2), inserted into pcDNA3.1(-) vectors at EcoRI and HindIII restriction sites. The constructs were electroporated into CGR8 cells according to standard protocols in a Gene Pulser (BioRad Laboratories, Reinach, Switzerland) at 240 V, 500 μF. Expressed eGFP-CD63 fusion protein targets eGFP to the plasma membrane and intracellular endosomes (Fig. 1A) and prevents the dilution of the marker, thus enabling a better visualization of cells by immunohistochemistry, as opposed to diffuse cytoplasmic eGFP detection. The hBcl2- CGR8 clone confers a resistance to apoptosis when exposed to an unfavourable environment such as in the infarcted heart. Transfected EGFP-CD63 positive mESC were sorted by flow cytometry (FACStar+, Becton Dickinson, Allschwil, Switzerland), while stable clones carrying hBcl2 were selected by neomycin resistance after 10 days treatment with G418 (250 μg/ml, Gibco, Invitrogen, Basel, Switzerland). Wild-type (wt) D3 mESC were propagated on mitomycin-treated mouse embryonic fibroblasts, while stable CGR8 ESC clones were propagated in the presence of leukaemia inhibitory factor (LIF). EGFP and hBlc2 were identified by immunohistochemistry using anti-eGFP and anti-Blc2 antibodies, respectively (see below).


Fate of undifferentiated mouse embryonic stem cells within the rat heart: role of myocardial infarction and immune suppression.

He Q, Trindade PT, Stumm M, Li J, Zammaretti P, Bettiol E, Dubois-Dauphin M, Herrmann F, Kalangos A, Morel D, Jaconi ME - J. Cell. Mol. Med. (2008)

Characterization of the ESC lines used for the transplantation experiments. (A) Expression of eGFP or CD63 transgene by CGR8 stable clones. Panel a: undifferentiated colony positive for eGFP fluorescence localizing to membranes and endosomes. (B) Phase contrast picture of the CGR8 colony. (C) Immunostaining of a CGR8 cell clone expressing the hBcl2 gene. (D) Untransfected cells do not react with the antihBcl2 antibody. (E) Comparison of the beating activity expressed as percentage of beating EBs measured in wt or hBcl2-CGR8 EBs at day 8 and day 20 of culture. In contrast to wild-type EBs, 20-day-old EBs derived from human Bcl-2 expressing cells maintained a high percentage of beating. (F) DNA laddering performed on wild-type (wt) or hBcl2-CGR8 cells (lane 1 and 2, respectively) treated with 10 μM hydrogen peroxide for 24 hrs. (G, H) Immunoreactivity of mESC to antibodies against MHC class I or class II. Undifferentiated ESC colonies were stained with anti-MHC-I (G), or anti-MHC-II (H). Images correspond to the middle slice of a deconvoluted stack of confocal images acquired by a Zeiss Meta confocal microscope.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3823046&req=5

fig01: Characterization of the ESC lines used for the transplantation experiments. (A) Expression of eGFP or CD63 transgene by CGR8 stable clones. Panel a: undifferentiated colony positive for eGFP fluorescence localizing to membranes and endosomes. (B) Phase contrast picture of the CGR8 colony. (C) Immunostaining of a CGR8 cell clone expressing the hBcl2 gene. (D) Untransfected cells do not react with the antihBcl2 antibody. (E) Comparison of the beating activity expressed as percentage of beating EBs measured in wt or hBcl2-CGR8 EBs at day 8 and day 20 of culture. In contrast to wild-type EBs, 20-day-old EBs derived from human Bcl-2 expressing cells maintained a high percentage of beating. (F) DNA laddering performed on wild-type (wt) or hBcl2-CGR8 cells (lane 1 and 2, respectively) treated with 10 μM hydrogen peroxide for 24 hrs. (G, H) Immunoreactivity of mESC to antibodies against MHC class I or class II. Undifferentiated ESC colonies were stained with anti-MHC-I (G), or anti-MHC-II (H). Images correspond to the middle slice of a deconvoluted stack of confocal images acquired by a Zeiss Meta confocal microscope.
Mentions: We genetically modified the mouse ESC line CGR8 [16] to express as reporter gene either the enhanced green fluorescent protein (eGFP) fused with CD63 or the human anti-apoptotic gene Bcl2 (hBcl2), inserted into pcDNA3.1(-) vectors at EcoRI and HindIII restriction sites. The constructs were electroporated into CGR8 cells according to standard protocols in a Gene Pulser (BioRad Laboratories, Reinach, Switzerland) at 240 V, 500 μF. Expressed eGFP-CD63 fusion protein targets eGFP to the plasma membrane and intracellular endosomes (Fig. 1A) and prevents the dilution of the marker, thus enabling a better visualization of cells by immunohistochemistry, as opposed to diffuse cytoplasmic eGFP detection. The hBcl2- CGR8 clone confers a resistance to apoptosis when exposed to an unfavourable environment such as in the infarcted heart. Transfected EGFP-CD63 positive mESC were sorted by flow cytometry (FACStar+, Becton Dickinson, Allschwil, Switzerland), while stable clones carrying hBcl2 were selected by neomycin resistance after 10 days treatment with G418 (250 μg/ml, Gibco, Invitrogen, Basel, Switzerland). Wild-type (wt) D3 mESC were propagated on mitomycin-treated mouse embryonic fibroblasts, while stable CGR8 ESC clones were propagated in the presence of leukaemia inhibitory factor (LIF). EGFP and hBlc2 were identified by immunohistochemistry using anti-eGFP and anti-Blc2 antibodies, respectively (see below).

Bottom Line: We found that mESC survival depended on immunosuppression both in normal and infarcted hearts.However, upon Cyclosporin A treatment, both normal and infarcted rat hearts failed to induce selective cardiac differentiation of implanted mESC.Instead, teratomas developed in normal and infarcted rat hearts 1 week and 4 weeks (50% and 100%, respectively) after cell injection.

View Article: PubMed Central - PubMed

Affiliation: Department of Rehabilitation and Geriatrics, Laboratory of Biology of Aging, Geneva University Hospitals, Geneva, Switzerland.

ABSTRACT
Abstract It has recently been suggested that the infarcted rat heart microenvironment could direct pluripotent mouse embryonic stem cells to differentiate into cardiomyocytes through an in situ paracrine action. To investigate whether the heart can function as a cardiogenic niche and confer an immune privilege to embryonic stem cells, we assessed the cardiac differentiation potential of undifferentiated mouse embryonic stem cells (mESC) injected into normal, acutely or chronically infarcted rat hearts. We found that mESC survival depended on immunosuppression both in normal and infarcted hearts. However, upon Cyclosporin A treatment, both normal and infarcted rat hearts failed to induce selective cardiac differentiation of implanted mESC. Instead, teratomas developed in normal and infarcted rat hearts 1 week and 4 weeks (50% and 100%, respectively) after cell injection. Tight control of ESC commitment into a specific cardiac lineage is mandatory to avoid the risk of uncontrolled growth and tumourigenesis following transplantation of highly plastic cells into a diseased myocardium.

Show MeSH
Related in: MedlinePlus