Cell Fusion (Heterokaryon) technology for monitoring nuclear reprogramming

 

We are using global bi-species RNAseq to identify genes involved in reprogramming cell fate (iPS or directed reprogramming) using heterokaryons. We are discovering genes that are essential and common to diverse approaches for the reprogramming of cells from one fate to that of another. Heterokaryons are non-dividing fusion products of two different cell types, from different species and have several unique advantages for discovery of determinants of cell fate:

(1) Heterokaryons - non-dividing cell fusion products with distinct intact nuclei can be analyzed in which the genetic material of each cell remains separate

(2) Cells of different species are fused allowing a distinction of the specific changes in gene expression in each fused cell type

(3) The onset of reprogramming is synchronous, as it is initiated upon addition of the fusagen

(4) Reprogramming is fast (within hours) and efficient (> 70% of fused cells

(5) The earliest changes in gene expression that occur within hours and up to a few days post fusion can be assessed

(6) Algorithms developed together with Wing Wong (Dept. of Statistics) allow the expression of every human transcript (40 million) to be analyzed (only conserved 50bp reads are eliminated).

Using this approach, the Blau lab showed that previously silent genes could be activated in diverse differentiated human cell types revealing the plasticity of cell fate in the 1980s. Notably, after fusion with mouse muscle cells, human muscle genes were expressed by human amniotic cells, keratinocytes, hepatocytes and skin fibroblasts even in the absence of cell division or DNA replication, showing that mammalian cell differentiation is not “terminal” but readily reversed.

Gene dosage, or nuclear ratio, was crucial and determined which cell phenotype prevailed. Cells of different lineages, ectodermal keratinocytes, endodermal hepatocytes, and mesodermal fibroblasts exhibited distinct efficiencies and timing of reprogramming toward a muscle fate, indicating that the underlying molecular mechanisms differ.

Recent studies have employed the heterokaryon technology to provide mechanistic insights into the reprogramming of somatic cells to an embryonic stem cell fate. Among other discoveries, this work revealed a novel role for AID as a component of the mammalian active DNA demethylase machinery and demonstrated the essential role of this enzyme at the onset of reprogramming cells in heterokaryons and in reprogramming cells to pluripotency.


* Blau et al, 1985, Science (PDF)

* Bhutani et al., 2009, Nature (PDF)