Reprogramming of Parental Genomes in Embryonic Stem Cell – Fibroblast Heterokaryons and Synkaryons

Gridina M.M *, Serov O.L ,


Objective: Up to date it was generally known that the hybrids, obtained after fusion of somatic cells with pluripotent cells, acquire various features of pluripotency from the embryonic stem (ES) cell fusion partner, including prolonged self-renewal ability, expression of pluripotency-specific genes, lack of expression of tissue-specific genes, developmental potential to contribute to the development of all three germ layers of the soma as well as to the germ cell lineage, and an undifferentiated epigenetic cell state. Heterokaryon formation (fused cells with two nuclei and a common cytoplasm) offers a unique tool to study the reprogramming process. Materials and Methods: We developed design of experiment allowed us to analyze heterokaryons and synkaryons (hybrid cells) in a few hour's time after cell fusion. We fused the ES cells marked by GFP and fibroblasts labeled with fluorescent microspheres. Heterokaryons or hybrid cells were considered if they had both markers. Its reliability was confirmed using sequencing by presence of mitochondrial DNA derived from both parental cells. Results: At 4-8 hours after fusion most heterokaryons had the fibroblasts-like morphology and were negative for alkaline phosphatase. The first hybrid cells appeared at 20 hours after fusion and were presented by both ES-like and fibroblasts-like cells which were prevalent. After a time about half of hybrid cells had ES-like morphology and formed colonies. In contrast to this, the fibroblasts-like hybrid cells didn’t show self-renewal ability: they were unable to form colonies and grew as disconnected single cells. Immunofluorescent analysis demonstrated that all the hybrid cells with ES-like morphology were positive for expression of “pluripotent” genes: Oct4 and Nanog; negative for expression of genes typical for differentiated cells: lamin A/C, I-type Collagen and Fibronectin and contained active all parental Х-chromosomes. However, in population of hybrid cells with fibroblast-like morphology and positive for fibroblast-specific genes (I-type Collagen and Fibronectin) there were a lot of hybrid cells which had intermediate phenotype: they did not express of Oct4, Nanog as well as I-type Collagen and Fibronectin and all Х-chromosomes were active. Conclusion: It is suggested that some cells underwent only partial reprogramming. Thus analysis of early events, which occurred directly after the fusion, showed that reprogramming process was more complex and it could not be described by “all or nothing” model.