Chromatin accessibility in canine stromal cells and its implications for canine somatic cell reprogramming

Abstract

Naturally occurring disease in pet dogs is an untapped and unique resource for stem cell‐based regenerative medicine translational research, given the many similarities and complexity such disease shares with their human counterparts. Canine‐specific regulators of somatic cell reprogramming and pluripotency maintenance are poorly understood. While retroviral delivery of the four Yamanaka factors successfully reprogrammed canine embryonic fibroblasts, adult stromal cells remained resistant to reprogramming in spite of effective viral transduction and transgene expression. We hypothesized that adult stromal cells fail to reprogram due to an epigenetic barrier. Here, we performed assay for transposase‐accessible chromatin using sequencing (ATAC‐seq) on canine stromal and pluripotent stem cells, analyzing 51 samples in total, and establishing the global landscape of chromatin accessibility before and after reprogramming to induced pluripotent stem cells (iPSC). We also studied adult stromal cells that do not yield iPSC colonies to identify potential reprogramming barriers. ATAC‐seq analysis identified distinct cell type clustering patterns and chromatin remodeling during embryonic fibroblast reprogramming. Compared with embryonic fibroblasts, adult stromal cells had a chromatin accessibility landscape that reflects phenotypic differentiation and somatic cell‐fate stability. We ultimately identified 76 candidate genes and several transcription factor binding motifs that may be impeding somatic cell reprogramming to iPSC, and could be targeted for inhibition or activation, in order to improve the process in canines. These results provide a vast resource for better understanding of pluripotency regulators in dogs and provide an unbiased rationale for novel canine‐specific reprogramming approaches.

Panobinostat Effectively Increases Histone Acetylation and Alters Chromatin Accessibility Landscape in Canine Embryonic Fibroblasts but Does Not Enhance Cellular Reprogramming

“…Moreover, resulting iPSCs are often poorly characterized and are likely to represent partially reprogrammed cells, as only a few of the canine iPSC publications demonstrate spontaneous in vivo differentiation (i.e., teratoma formation), and the contribution of canine iPSC to chimera formation has not been reported. Finally, although in our hands and others, canine embryonic fibroblasts (CEFs) are reprogrammable; adult fibroblasts remain resistant to OCT4-SOX2-KLF4-MYC ( OSKM )-induced reprogramming ( 11 , 12 ). We found that adult fibroblasts have a more restrictive genomic accessibility landscape compared with CEF that “locks” adult cells in their somatic fate and prevents their reprogramming and phenotypic switch ( 11 ).…”

“…Finally, although in our hands and others, canine embryonic fibroblasts (CEFs) are reprogrammable; adult fibroblasts remain resistant to OCT4-SOX2-KLF4-MYC ( OSKM )-induced reprogramming ( 11 , 12 ). We found that adult fibroblasts have a more restrictive genomic accessibility landscape compared with CEF that “locks” adult cells in their somatic fate and prevents their reprogramming and phenotypic switch ( 11 ).…”

“…ATAC-seq data analysis further highlights the numerous pathways and transcription factors altered by our treatment and suggests a rationale for a targeted, canine-specific, reprogramming approach. Overall, although reprogramming efficiency was not increased, our data show that panobinostat and vitamin C treatment induces wide chromatin remodeling and that reprogramming may be enhanced by CEBP, a reprogramming enhancing transcription factor that was inhibited by our treatment and which its depletion was suggested as a barrier to canine somatic cell reprogramming ( 11 ).…”

A Preview of Selected Articles

“…Researchers led by Amir Kol (University of California Davis, Davis, CA) hypothesized that incomplete chromatin remodeling, which underlies the reprogramming process, 20 may represent the barrier that impedes the generation of iPSCs from adult canine cells. In a recent STEM CELLS Translational Medicine article, 6 Questa et al employed assay for transposase‐accessible chromatin using sequencing (ATAC‐seq) during the OCT4 , SOX2 , KLF4 , and MYC ‐mediated reprogramming of amenable (canine embryonic fibroblasts) and non‐amendable (canine adult dermal fibroblasts and ASCs) to explore this hypothesis. The authors hoped to compare the chromatin landscape associated with successful and unsuccessful reprogramming to identify problematic regions that interfere with the process.…”

“…Recent OCT4 ‐focused research efforts have sought to decipher how external forces acting upon pluripotent stem cells prompt the increased expression of pluripotency‐associated genes, such as OCT4 , and explore the contribution of OCT4 in the reprogramming of cells of species other than human, mouse, rat, or monkey into iPSCs 3 . In our first Featured Article published this month in STEM CELLS , Nath et al describe how the fluid shear stress generated during bioreactor culture helps to maintain the pluripotency of mouse ESCs through the upregulated expression of Oct4 , Sox2 , and Nanog ‐ a phenomenon they term “mechanopluripotency.” 5 In a Related Article published recently in STEM CELLS Translational Medicine , Questa et al identified the alterations to the global chromatin landscape that occur during the OCT4 , SOX2 , KLF4 , and MYC ‐induced reprogramming of canine cells and define candidate barriers that prevent adult canine cells from undergoing efficient reprogramming 6 …”