Transcriptional changes in somatic cells recovered from embryonic stem-somatic heterokaryons.
Following fusion, embryonic stem cells (ESCs) are capable of reprogramming somatic cells in cell hybrids. It has also been shown that transcriptional changes can occur in a heterokaryon, without nuclear hybridization. However, it is unclear whether these changes can be sustained after the removal of the dominant nucleus. In this study, we analyze the changes in embryonic stem (ES)-somatic heterokaryons following the removal of the ESCs nucleus. We also show that after ES-somatic cell fusion using tetraploid ESCs, a heterokaryon can be reverted to an autologous diploid state by differential enucleation of the denser tetraploid ES nucleus. To recover somatic cells from ES-somatic heterokaryons, we fused tetraploid ESCs containing the thymidine kinase (TK) suicide gene with mesenchymal stem cells containing a green fluorescent protein (GFP) transgene under the control of the OCT4 promoter. Following post-fusion enucleation (PFE), negative selection against the tetraploid ES genome was achieved using ganciclovir. The resulting GFP-positive clones were analyzed and shown to have undergone changes in growth characteristics, alkaline phosphatase activity, and gene expression using RT-PCR and microarray analysis. These results demonstrate that a change in transcriptional expression can be detected in somatic cells after the removal of the ES nucleus from ES-somatic heterokaryons.
Sumer
Huseyin
H
Monash Institute of Medical Research, Monash University, Clayton, Victoria 3168, Australia.
Jones
Karen L
KL
Liu
Jun
J
Rollo
Benjamin N
BN
van Boxtel
Antonius L
AL
Pralong
Daniele
D
Verma
Paul J
PJ
D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation; D002459:Cell Fusion / Q000379:methods*; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D016000:Cluster Analysis; D018920:Coculture Techniques; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000378:metabolism; D005434:Flow Cytometry; D020869:Gene Expression Profiling; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006822:Hybrid Cells / Q000166:cytology* / Q000378:metabolism; D051379:Mice; D008822:Mice, Transgenic; D008856:Microscopy, Fluorescence; D020411:Oligonucleotide Array Sequence Analysis; D039904:Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism; D011123:Polyploidy; D020133:Reverse Transcriptase Polymerase Chain Reaction
D049452:Green Fluorescent Proteins; D000469:Alkaline Phosphatase