Butyrate Greatly Enhances Derivation of Human Induced Pluripotent Stem Cells by Promoting Epigenetic Remodeling and the Expression of Pluripotency-Associated Genes

Mali et al. Stem Cells. 2010 Mar 3

Generation of inducible pluripotent stem cells (iPS) offers the possibility of creating patient-specific stem cells with embryonic stem cell therapeutic potential from adult sources.  Recently the main hurdle of iPS cell generation, the need for introduction of oncogenes in the adult cells, has been removed by use of chemical modulators as well as alternative non-cancer causing genes.  Another drawback of creating iPS cells is the need for mass screening of many transfected target cells before identification and extraction of the correct cell can be made.  In the current paper the histone deacetylase inhibitor butyrate was used to enhance potency of iPS generation in vitro.  Histone deacetylase inhibitors are a type of compounds that decrease the density of DNA in chromosomes.  By performing this function the DNA because more amenable to reprogramming, in the sense that the cells can be coaxed to de-differentiate with less effort.  Another histone deacetylase inhibitor, valproic acid, which is used clinically to treat convulsions, has been shown to increase the ability of blood making stem cells to self-replicate with higher efficiency, which is a characteristic of earlier de-differentiation.    

In a recent paper it was demonstrated that temporary treatment with butyrate increases efficacy of iPS generation by 15-51 fold using two techniques that are commonly used for generation of these cells.  It was demonstrated that in the presence of butyrate stimulation a remarkable (>100-200 fold) increase on reprogramming in the absence of either KLF4 or MYC transgene.

This suggests that butyrate may be a useful agent to incorporate in the iPS generation protocols that are currently under development.  Furthermore, butyrate treatment did not negatively affect properties of iPS cell lines established. The generated iPS cell lines, including those derived from an adult patient with sickle cell disease by two methods show normal karyotypes and pluripotency.

To mechanistically identify molecular pathways of butyrate enhancement of iPS generation, the investigators performed conducted genome-wide gene expression and promoter DNA methylation microarrays and other epigenetic analyses on established iPS cells and cells from intermediate stages of the reprogramming process.

 By day 6-12 after exposing cells to butyrate, enhanced histone 3 acetylation, promoter DNA demethylation, and the expression of endogenous pluripotency-associated genes including DPPA2, whose over-expression partially substitutes for butyrate stimulation is known.

According to Dr. Mali " Thus, butyrate as a cell permeable small molecule provides a simple tool to further investigate molecular mechanisms of cellular reprogramming. Moreover, butyrate stimulation provides an efficient method for reprogramming various human adult somatic cells, including those from patients that are more refractory to reprogramming"

Methods of increasing efficacy of iPS generation have included not only chemical manipulation but also starting from cell sources that are generally considered more immature.  For example a previous study demonstrated that mesenchymal stem cells create a much higher per-cell number of iPS cells as compared to skin fibroblasts. 

One of the interesting points of this finding is that butyrate may theoretically be useful at expanding potential of stem cells already in an organism.  Since butyrate is used clinically for treatment of urea cycle disorders and is non-toxic at pharmacological doses, it may be a good candidate for expanding stem cells in vivo.  Manipulation of the stem cell compartment by administration of therapeutic agents has already been performed for mobilization, which has been published with the neutraceutical Stem-Kine http://www.translational-medicine.com/content/pdf/1479-5876-7-106.pdf.