Supplemental Data

Files in this Data Supplement:

• Supplemental Figure 1 - (SupplementalFig1.jpg; 176 KB)
  Analysis of major histocompatibility complex antigen expression. Flow cytometry studies were performed using a Coulter Cytomics analyzer running CXP software. Analyses were performed on C57Bl/6-(β-actin)-EGFP ES cells and differentiation day 19 embryoid bodies (EBs). IFNγ (Peprotech) treatments (100ng/ml) were performed for 48 hours before sample collection. Cell suspensions of 1 million cells/sample were prepared in FACS buffer (2%FBS in Versene) and stained with PE-conjugated MHC I (eBioscience cat.#12-5999), MHC II (eBioscience cat.#12-5321), mouse IgG2a (eBioscience cat.#12-4724, or rat IgG2b (eBioscience cat.#12-4031). MHC I and MHC II expression were not detected above background levels in undifferentiated ES cells without IFNγ (A) or with IFNγ (B). In the absence of IFNγ MHC I and MHC II were not detected following 19 days of differentiation in vitro (C). However, addition of IFNγ resulted in an increase in MHC I and MHC II levels (D).
• Supplemental Figure 2 - (SupplementalFig2.jpg; 318 KB)
  Detection of ES cell-cardiomyocyte fusion using Cre-Lox recombination. Undifferentiated ES cells were transduced with an adenovirus encoding a Cre-dependent LacZ reporter gene (CMV-loxP-stop-loxP-LacZ) and were transplanted into hearts of transgenic mice expressing Cre recombinase exclusively in cardiomyocytes (αMHC-Cre). LacZ histochemistry indicates 2 cells with blue staining (arrows), indicating fusion of transplanted ES cells with host cardiomyocytes. Fusion was detected in 3 out of 3 hearts analyzed but was an extremely rare event. (Scale bar =100μm)
• Supplemental Figure 3 - (SupplementalFig3.jpg; 728 KB)
  Identification of autofluorescence mimicking EGFP expression in infarct cardiomyocytes and non-myocytes. C57 ES cells expressing EGFP were transplanted into the infarcted hearts of syngeneic hosts. Mice were killed at 3 weeks, when teratomas were well established. Sections were stained with DAPI-only to evaluate intrinsic tissue fluorescence (EGFP + autofluorescence; panel A) or with an antibody to EGFP (red; panel B) to identify ES cell descendants. (A) Analysis of intrinsic fluorescence shows green cells present in the teratomas and also in the subendocardial region of the infarct (arrows). These cells show no fluorescence on the red channel. (B) A serial section immunostained for EGFP (red) shows the epitope to be present within the teratomas, but the subendocardial cells in the infarct are EGFP-negative (arrows). This indicates the green cells in the infarct are not ES cell-derived but rather are autofluorescent host cells. (Scale bar=100μm)