A whole genome transcriptome analysis was carried

A whole genome transcriptome analysis was carried out to clarify the mechanisms of the cell type specific cytotoxicity of HDAC inhibition on the abnormal cells. The transcriptome analysis revealed the HDAC inhibitor responsive genes in the normal and abnormal cells. The genes with altered levels in abnormal hESCs and reciprocal regulation in response to HDAC inhibition were identified as putative factors explaining selective sensitivity of abnormal hESCs to HDAC inhibition. Previous studies with mouse PD 0332991 have shown that many of these genes are direct targets of Hdac1, including Tnfrsf19, Id2, Arid5B and Epha4 in mouse ESCs and in Lrp8 and Pmp22 in mouse trophoblast cells (Kidder and Palmer, 2012) and SPP1 in human fibroblasts (Pazolli et al., 2012). We have further validated that LRP8 and SPP1 are regulated in HDAC1 dependent manner in human embryonal carcinoma cells (NT2D1) correlating with the reduced growth of the cells after HDAC1 knockdown. The genes with altered expression and selective sensitivity to HDAC inhibition in abnormal cells are linked to development and age associated to severe developmental and neurological diseases and cancers, such as Alzheimer\'s disease, Parkinson\'s disease, gliomas or leukemias. This indicates that the genes showing altered expression and selective sensitivity in HDAC inhibition in abnormal cells are important for the normal cellular differentiation, development and aging. On the other hand these genes link functionally to neuronal and hematological development and seem to be vulnerable to culture induced changes. Consistently, including HDAC1, most of the putative upstream regulators of these genes have previously known function in hematological development. In addition, many of them, such as TP53 and YY1, link to transformation and oncogenesis. These observations may partly explain why HDAC inhibitors are promising or effective in the treatment in leukemias. These candidate genes with altered regulation in abnormal cells and their upstream regulators are likely to be important for the improved survival and proliferation of the abnormal hESCs and are highly valuable as potential targets for prevention of abnormal or malignant growth. Whether the activity of HDACs is increased before or after genomic alterations occur in stem cells remains to be elucidated. Interestingly, a recent study describes a key function of HDAC1 and HDAC2 in DNA damage response to promote DNA nonhomologous end-joining. Moreover, hypersensitization of HDAC1/2 deficient cells to DNA damage inducing agents was demonstrated (Miller et al., 2010). Thus, it is possible that accumulation of abnormalities in the HDAC1/2 proteins themselves may pronounce the cells to DNA damage and genetic instability. This is further supported by a recent study showing that HDAC1 and Nurd complex are the key modulators of aging related chromatin defects in primary fibroblasts and activity of HDAC1 is decreased during aging (Pegoraro et al., 2009). Thus, defective function of HDACs and accumulation of genomic abnormalities in the stem cells during extended culture may mimic senescence of the cells and in particular long-lived stem cells residing in aging somatic tissues. Similar to ESCs, changes occurring in the chromatin of long-lived somatic stem cells are likely to affect the growth, differentiation and regenerative potential of these cells exposing the tissues to malignant growth and degenerative diseases. Future studies determining the importance of HDAC mediated protein complexes in the maintenance of somatic stem cells in aging tissues and their contribution to malignant and age related degenerative diseases will be of great interest. The following are the supplementary data related to this article.
Acknowledgments We thank staff at the CSCB, particularly to Gemma Bray, Katie Amps, Heather Spink, Greg Bingham and Christine Piggot for the technical support provided for the study. We acknowledge Dr. Neil Harrison for the feedback and critical review of the manuscript. We also thank the staff at Turku Centre for Biotechnology, Päivi Junni and Marjo Hakkarainen, for maintenance of the stock stem cell cultures and Jianliang Li, Stuart Avery and Miro Viitala for technical assistance. Furthermore, Sarita Heinonen and Finnish Microarray and Sequencing Centre are acknowledged for technical support with the Illumina arrays. This study was funded by the Academy of Finland (Project 116713), Helsingin Sanomat Foundation, Finnish Cancer Foundation, JDRF and ESTOOLS.