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Apr 12, 2012

Small Molecule Inhibitors Dramatically Boost Efficiency of Fibroblast to Neuron Conversion

  • Scientists have dramatically increased the efficiency of converting human fibroblast cells directly into functional neurons by combining transcription factor-based reprogramming with the addition of small molecules that block the pathways that otherwise hinder cell transformation.

    The researchers, led by a team at the University of Bonn’s Life & Brain Center, claim that applying their technique to neonatal fibroblasts enabled them to generate cultures containing up to 80% functional neuron-like cells, which provided yields of more than 200% (accounting for further cell division during the conversion process). Oliver Brüstle and colleagues describe their technology and experimental results in Nature Methods in a paper titled “Small molecules enable highly efficient neuronal conversion of human fibroblasts.”

    A transcription factor transduction-based method for directly converting fibroblasts into neuronal cells (or induced neurons) was first reported in 2010. However, while the achievement has demonstrated that cells of one lineage can be prompted to convert into cells of a completely different lineage without the need to reprogram into induced pluripotent stem cells (iPSCs), the process is very inefficient, the Life & Brain team explained.  

    Their reasoning was that conversion might be facilitated by adding to the equation factors known to play a role in neural induction. More specifically, they devised a method to exploit the fact that inhibiting SMAD signalling and blocking glycogen synthase kinase-3β (GSK-3β) is known to facilitate highly efficient neural differentiation of human embryonic stem cells and iPSCs.

    The team took three human postnatal fibroblasts cell lines derived from donors ranging from newborn to four years old, and transduced them to express Ascl1 and Ngn2. Initial studies with the fibroblasts indicated that these were the two transcription factors that gave rise to the highest percentage of βIII-tub+ cells.

    To these AScl1/Ngn2-expressing cells the team effected SMAD pathway blockade using the activin-like kinase 5 inhibitor SB-431542 (SB) together with noggin, and also the GSK-3β inhibitor CHI99021 (CHIR). After 2 weeks of sustained transgene induction the compounds were removed from the cultures, and expression of the neuronal marker βIII-tub was analyzed at day 23.

    The results strikingly demonstrated that addition of the small molecules boosted the numbers of  βIII-tub+ cells generated 17-fold when compared with transcription factor transduction alone. In fact, for one of the cell lines used, the percentage of βIII-tub+ cells generated in cultures treated for 1 week was 65.1 ± 5.3%; and this reached a maximum 83.5 ± 4.3% after a two weeks of exposure to the small molecule inhibitors. Modifying the process further, the investigators found that the need to use high concentrations of noggin could be negated by using a low concentration of noggin and a 0.5 micromolar concentration of LDN-193189, which inhibits ALK2, -3, and -6.

    In terms of overall conversion efficiency, the combined transduction-small molecule inhibition approach resulted in yields of up to 230%, with respect to the initial number of plated cells. Notably, the generation of iNs seemed to be independent of the passage number and wasn’t linked with karyotypic abnormalities. Also, critically, further evaluation of the resulting iNs confirmed that they were active, exhibiting morphological, immunocyhtochemical, and functional properties of postmitotic neurons of different neurotransmitter phenotypes. “We were able to demonstrate how the genes typical for skin fibroblast were gradually down-regulated and nerve cell-specific genes were activated during the cell transformation,” comments co-author Julia Ladewig, Ph.D.

    The authors say their experimental approach should be suitable for the direct generation of bulk quantities of human iNs for biomedical applications.

     


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