Transcriptome transfer produces a predictable cellular phenotype

Abstract

Cellular phenotype is the conglomerate of multiple cellular processes involving gene and protein expression that result in the elaboration of a cell's particular morphology and function. It has been thought that differentiated postmitotic cells have their genomes hard wired, with little ability for phenotypic plasticity. Here we show that transfer of the transcriptome from differentiated rat astrocytes into a nondividing differentiated rat neuron resulted in the conversion of the neuron into a functional astrocyte-like cell in a time-dependent manner. This single-cell study permits high resolution of molecular and functional components that underlie phenotype identity. The RNA population from astrocytes contains RNAs in the appropriate relative abundances that give rise to regulatory RNAs and translated proteins that enable astrocyte identity. When transferred into the postmitotic neuron, the astrocyte RNA population converts 44% of the neuronal host cells into the destination astrocyte-like phenotype. In support of this observation, quantitative measures of cellular morphology, single-cell PCR, single-cell microarray, and single-cell functional analyses have been performed. The host-cell phenotypic changes develop over many weeks and are persistent. We call this process of RNA-induced phenotype changes, transcriptome-induced phenotype remodeling.neuron ͉ transcriptome-induced phenotype remodeling ͉ single cell ͉ Waddington I n multicellular organisms, all cells contain nearly identical copies of the genome but exhibit drastically different phenotypes. Even a single neuron has a set of phenotypic characteristics that distinguish it from other neurons as well as other cell types, such as the nearby astrocytes. Indeed, as Waddington proposed in his classical epigenetic landscape model, genetically predetermined cells can follow any specific permitted trajectories that eventually lead to different cellular phenotypes (1). From this point of view, the genome serves as a repository of dynamic control information whose state can be reprogrammed to match the stable phenotypic states.Emerging evidence has demonstrated the reversibility and flexibility of the cellular phenotype. Gurdon et al. first showed that the ability to obtain fertile adult male and female frogs by injecting endoderm nuclei into enucleated eggs (2). This result not only forms the foundation of the field in nuclear transplantation but also provides evidence that the cytoplasmic components of a differentiated cell can support nuclear reprograming. Generation of induced pluripotent stem (iPS) cells by transfection of transcription factors into dividing fibroblasts (3), followed by cell selection, represents a new strategy to globally revert a mature cell into a different cell type (4-9). The need for redifferentiation of these embryonic stem cell-like-iPS cells into desired cell types adds a layer of complexity that is difficult to control (10, 11). Nevertheless, studies of nuclear reprogramming from genomic and epigenetic modification, as seen from so...

Transcriptome transfer provides a model for understanding the phenotype of cardiomyocytes

“…We have previously shown that the transfer of transcriptome (TIPeR) from a rat astrocyte into a rat neuron converts the electrically active neuron into an electrically quiescent tAstrocyte cell (10). We asked whether the change in expression profile (i.e., modulation of relative abundance of gene products) could transdifferentiate electrically quiescent cells into electrically excitable cells.…”

Single cell optical transfection

“…During irradiation, a tiny, submicrometre-sized pore, lasting fractions of a second, is generated on the plasma membrane. Cell viabilities greater than or equal to 90 per cent are often reported in the literature (Tirlapur & Konig 2002;Kohli et al 2005a;Peng et al 2007;Baumgart et al 2008;Lei et al 2008;Uchugonova et al 2008b Baumgart et al 2008;Uchugonova et al 2008b), and mRNA (Barrett et al 2006;Sul et al 2009). Recently, our own laboratory has even shown that a single 100 nm gold particle can be optically tweezed and subsequently injected into a cell (McDougall et al 2009; as discussed in more detail later).…”

“…The field is at an exciting juncture with the publication of two seminal papers from the Eberwine group (Barrett et al 2006;Sul et al 2009). Here, the focus of study was not the technique of optical injection itself, but the application of the technique to biological questions difficult to answer using other methods.…”

Transcriptome Analysis of Single Cells

“…The amplified aRNA can also be used to confirm cell phenotype conversion studies in which a full set of mRNAs from one cell type are transfected into a different cell type in order to induce transition of the phenotype of the latter cell type into that of the former, a procedure developed by the lab and known as TIPeR. 17 These studies are particularly useful for studying disease states and cell phenotypes and such studies are currently ongoing in the lab. RT-PCR or qPCR can be performed on the amplified material to confirm the expression of cell-specific genes.…”