Mouse embryonic stem cells. Line D3. Differentiated through embryoid body formation (hanging droplet technique). Dissociated on noted day of differentiation and FACS sorted for the Green fluorescent protein. Transgene: Nkx2-5 enhancer, Hsp68 minimal promoter, GFP.
Biomaterial provider
N/A
Treatment protocol
See description
Growth protocol
See description
Extracted molecule
total RNA
Extraction protocol
Qiagen mini shredder, Qiagen Rneasy kit.
Label
Biotin
Label protocol
Two rounds amplification protocol for total RNA following Affymetrix� specifications using T7 promoter oligo dT primer for cDNA synthesis, and ENZO IVT kit (www.affymetrix.com, GeneChip Expression Analysis, Technical Manual, 2003).
Hybridization protocol
16hrs at 45 C with rotation (60rpm) as described by Affymetrix in their GeneChip Expression Analysis Technical Manual, 2004.
Scan protocol
Using Affymetrix� GeneChip Scanner 3000 and default parameters described by the manufacturer in their GeneChip Expression Analysis Technical Manual, 2004.
Description
Cell culture and cell differentiation Mouse embryonic stem cells (mESCs) were maintained undifferentiated by culturing on a feeder layer of mitomycin C pretreated primary mouse embryonic fibroblasts (PMEFs, Specialty Media, cat.# PMEF-H) at 37oC, 90% humidity and 5% CO2. The cell line used was the mESC D3 (passage #12-18). The basic culture medium was comprised of DMEM (Invitrogen, cat.# 11965-092), heat inactivated (56oC for 30 minutes) fetal bovine serum (Hyclone, cat#. S11550, 15%), Glutamax (Invitrogen, cat.# 35050-065, 1%), non-essential amino acids (Invitrogen, cat.# 11140-050, 1%), sodium pyruvate (Invitrogen, cat.# 11360-070, 1%), ?-mercaptoethanol (Sigma, cat.# M7522, 0.1mM), gentamicin (Cambrex, cat.# 17-518, 25?g/ml) and ESGRO leukemia inhibitory factor (Chemicon, cat.# ESG1106, 1000units/ml). Prior to initiation of differentiation The PMEF feeder layer was subtracted and the mESCs were maintained on a 0.01% gelatin pretreated plate for 24-48 hours. Differentiation through embryoid body (EB) formation was initiated by dissociating the mESCs using trypsin (Invitrogen, cat#. 25300-054, 0.05%) and resuspending them in basic culture medium (without the leukemia inhibitory factor) at a final concentration of 5x104 cells/ml. The hanging droplet (HD) technique was used for EB formation. HDs were plated on the lid of H2O containing tissue culture dishes at a volume of 20?L/droplet (approximately 1000 cells/ droplet). Two days post initiation of differentiation the EBs were transferred in suspension on poly-HEMA (Sigma, cat#. P3932) treated tissue culture dishes. The plates were pretreated with poly-HEMA in order to avoid any cell attachment on the plastic. Basic culture medium was supplemented with ascorbic acid(Takahashi et al., 2003) (Sigma, cat#. A4403, 0.1mg/ml) and it was exchanged with fresh medium every 2 days during differentiation. Transfection of mouse embryonic stem cells The mouse Nkx2.5 enhancer fragment #5(Lien et al., 1999) and the hsp68 minimal promoter was excised from the provided vector (XhoI/ NcoI) and inserted in front of the EGFP gene in the Bluescript vector. A vector containing the hygromycin resistance gene (hygromycin phosphotransferase) under the control of the mouse polymerase II promoter was also used in order to select for the transfected mESCs. Prior to transfection the two vectors were linearized. Undifferentiated mESCs were grown to confluency on a layer of primary embryonic fibroblasts and dissociated with trypsin. The cells were combined with the linearized DNA and electroporated (Biorad Gene Pulser Xcell, 240V/ 500?F). The cells were then replated on a fresh layer of PMEFs. Hygromycin B (Invitrogen, cat.# 10687-010, 50?g/ml) was used for 7 days after transfection for selection. Resistant colonies were picked and grown on layers of PMEFs in the presence of antibiotic. Once sufficient cells were present the clones were differentiated in order to check for the correct expression of the GFP in the cardiac areas (spontaneously contracting) of the differentiating cultures. Successful clones were further amplified and used for characterization of the GFP expressing cardiac progenitor cells. In order to select mESC derived cardiomyocytes for the microarray analysis experiment, a stable transgenic clone of mESCs was made that allowed the expression of the neomycin resistance gene under the control of the alpha myosin heavy chain promoter (U71441) as described by Klug et al., (Klug et al., 1996). The transgenic clone was prepared as described above. FACS Analysis For determination of the CPC yield in the differentiating cells, EBs were harvested at the indicated timepoints (between days 6 and 10 of differentiation). Each sample contained approximately 100 hanging droplets on day 0 of differentiation. The EBs were washed in PBS and resuspended in 0.05% trypsin at 4oC (1/2 hour) and 37oC (10 minutes). The percentage of GFP+ cells was determined in a BD FACS Calibur (Cell Quest Pro software). Age matched EBs from untransfected mESCs were used as negative controls. Cells from the same samples were also counted. FACS sorting The BD Biosciences FACSAria Cell sorting system was used to sort the GFP+ and GFP- cells between days 5 and 10 of differentiation. EBs were harvested and dissociated as described above. The cells were suspended in sorting medium (96% DMEM without phenol red, 1% fetal bovine serum, 1% Glutamax, 1% non essential amino acids, 1% sodium pyruvate, 25?g/ml gentamicin) and kept at 4oC. The cells were sorted into a fetal bovine serum rich medium (20%) in order to increase their survival. For further culture the cells were resuspended in mESC culture medium at a concentration of 5x105 cells/ml and cultured in suspension for 48 hours. The cells were then transferred on tissue culture plates for another 48 hours. For RNA isolation the cells were spun down and washed in PBS before the RNA isolation procedure. To assay their proliferation capacity, GFP+ CPCs were FACS sorted on day 5 of differentiation. Cells were reaggregated in suspension (5x105 cells/ ml) in the presence of mouse recombinant Igf1 (Chemicon, Cat.# GF121, 100ng/ml) for 14 days. Cell aggregates were fixed and stained for the colocalization of the antigens: Nkx2-5 and Ki67 or Pcna. The same ICC reagents were used as described above. GeneChip microarray hybridization Total RNA for the microarray expression analysis was isolated from FACS sorted cells (GFP+ and GFP-) on days 5, 6, 7, and 8 of differentiation as described above. Two separate differentiation batches were prepared for the sake of data reproducibility. Total RNA was also isolated from terminally differentiated mESC derived genetically selected cardiomyocytes 3 weeks post initiation of differentiation. The GeneChip� two cycle target labeling kit (Affymetrix, cat.# 900494) was used to convert 100ng of total RNA to biotinylated labeled cRNA. This was then hybridized to the Affymetrix murine MOE430 2.0 genome GeneChip� array (Affymetrix, cat.# 900496). Fluorescence was detected using the Affymetrix GS3000 GeneArray scanner and image analysis for each chip was done through the GeneChip� operating system software from Affymetrix (GCOS1.3) using the standard default settings. Microarray data analysis To estimate the gene expression signals, data analysis was conducted on the chips� CEL file probe signal values at the Affymetrix probe pair (perfect match (PM) probe and mismatch (MM) probe) level, using the statistical technique RMA (Robust Multiarray Analysis) (Irizarry et al., 2003) with the bioconductor package Affy. The data normalization procedure utilized the quantile normalization method (Bolstad et al., 2003) to reduce the obscuring variation between microarrays, which might be introduced during the processes of sample preparation, manufacture, fluorescence labeling, hybridization and/or scanning. The Spotfire DecisionSite software package was used for the identification of uniquely upregulated or downregulated (at least 1.5 times increase or decrease in the expression value) probe sets in the CPC population when compared to the rest of the cells in the differentiating EBs. Probe sets that were considered unique for the CPC population were found to be commonly upregulated or downregulated during all four days of analysis. Probe sets that exhibited an increasing or decreasing pattern of expression with at least 1.5 times increase or decrease in the expression values of day 5 CPCs and day 8 CPCs were also reported. Probe sets of the CPC population that exhibited upregulation or downregulation by at least 1.5 times when compared to the mESC derived cardiomyocytes were also reported. The final analysis included probe sets that exhibited a different temporal pattern of expression in the CPC population when compared to the rest of the cells along the four days of differentiation. Specifically in order to identify these probe sets gene expression curve over time was modeled flexibly on a natural cubic spline basis. A q-value for each gene was computed to estimate the false discovery rate (FDR) incurred when calling the gene significant. Differentially expressed genes were obtained by setting a cut-off to the calculated q-values (Storey et al., 2005). The probe level data analysis was implemented with the bioconductor package, Affy under R environment and the time course analysis was performed with the software package EDGE. Finally, only probe sets with gene ontology information associated with them were reported. The Ingenuity Pathways Analysis software package was used to identify canonical pathways that are active in the CPC population (www.ingenuity.com).
Data processing
Image analysis using GCOS 1.4, using MAS 5.0 algorithm and manufacturer�s specifications. Global scaling of images to a target intensity of 150.
