The problems of chemical differentiation could be circumvented, at least in theory, by sequentially transfecting huES cells with the genes encoding for the key transcription factors whose activation is known to choreograph pancreatic development. With this idea in mind, many groups around the world have used a number of vectors to deliver active cassettes to stem cells of all origins, including Pdx1, Pax4, Foxa2, Ngn3, NeuroD and many others. Among the vectors, adenoviruses have been highly favored due to the fact that they infect both dividing and nondiving cells, and usually do not integrate in a permanent manner into the genome. Retroviruses, in contrast, have a preference for dividing cells and integrate permanently. While the latter feature has proven of great help for permanent labeling and proof-of-principle reprogramming studies, concerns about insertional mutagenesis and heterochronic reactivation are still considered almost insurmountable hurdles in the road toward clinical applications. Direct transfection of plasmid DNA in virusfree settings has been proposed as a middle way, particularly for reprogramming experiments.
If conceived as a sequential approach, however, the notion of adding several genes to progressively specify stem cells in vitro would pose daunting technical difficulties. It would be very difficult to replicate the native expression pattern of most of these genes, which is either transient or fluctuating during development . Stepwise clonal derivation of cells transfected with one inducible gene at a time would be utterly impractical. Moreover, uncertainty about the copy number and site of integration of these cassettes would raise the aforementioned safety concerns. Strategies based on homologous recombination for targeting the integration of reprogramming genes into specific loci would be safer, but quite unworkable due to the number of genes involved. Also, it might not solve the problem of subsequent reactivation of these genes. Still, the in vitro behavior of cells typically defies conventional wisdom about natural development. Ngn3, for instance, is thought to act only transiently at the time of endocrine specification, and its down-regulation is supposed to be necessary for differentiation to proceed. However, ectopic constitutive expression of Ngn3 seems to be permissive for differentiation in other settings. Similar arguments can be made about ES cell reprogramming factors Oct3/4 and Sox2, whose permanent expression would be theoretically incompatible with the activation of differentiation pathways – yet differentiation is not impeded when iPS cells constitutively expressing these two factors are allowed to form teratomas. A plausible explanation for these observations is that the forced expression of some of these genes will trigger endogenous networks that will irreversibly take over the process from that point on, perhaps even deregulating the ectopic gene.Other forms of genetic manipulation are not intended to induce phenotypic changes, but to favor the in vitro identification (and possible sorting/selection) of desired differentiation outcomes, as well as posttransplantation tracing. These include plain tagging, the generation of cell lines expressing fluorescent reporter genes under the control of tissue-specific regulatory sequences and gene-trap strategies for the specific ablation of cells that do not have the desired characteristics.
Tags: clonal derivation of cells, Direct transfection of plasmid DNA, dividing and nondiving cells, gene-trap strategies, genes encoding, heterochronic reactivation, homologous recombination, insertional mutagenesis, iPS cells, posttransplantation tracing, problems of chemical differentiation, Retroviruses, transfecting huES cells