Stress responses in the adult rat are programmed early in life by maternal care and associated with epigenomic marking of the hippocampal exon 1 7 glucocorticoid receptor (GR) promoter. To examine whether such epigenetic programming is reversible in adult life, we centrally infused the adult offspring with the essential amino acid L-methionine, a precursor to S-adenosyl-methionine that serves as the donor of methyl groups for DNA methylation. Here we report that methionine infusion reverses the effect of maternal behavior on DNA methylation, nerve growth factor-inducible protein-A binding to the exon 1 7 promoter, GR expression, and hypothalamic-pituitaryadrenal and behavioral responses to stress, suggesting a causal relationship among epigenomic state, GR expression, and stress responses in the adult offspring. These results demonstrate that, despite the inherent stability of the epigenomic marks established early in life through behavioral programming, they are potentially reversible in the adult brain.

Maternal care alters epigenetic programming of glucocorticoid receptor (GR) gene expression in the hippocampus, and increased postnatal maternal licking/grooming (LG) behavior enhances nerve growth factor-inducible protein A (NGFI-A) transcription factor binding to the exon 1 7 GR promoter within the hippocampus of the offspring. We tested the hypothesis that NGFI-A binding to the exon 1 7 GR promoter sequence marks this sequence for histone acetylation and DNA demethylation and that such epigenetic alterations subsequently influence NGFI-A binding and GR transcription. We report that (1) NGFI-A binding to its consensus sequence is inhibited by DNA methylation, (2) NGFI-A induces the activity of exon 1 7 GR promoter in a transient reporter assay, (3) DNA methylation inhibits exon 1 7 GR promoter activity, and (4) whereas NGFI-A interaction with the methylated exon 1 7 GR promoter is reduced, NGFI-A overexpression induces histone acetylation, DNA demethylation, and activation of the exon 1 7 GR promoter in transient transfection assays. Site-directed mutagenesis assays demonstrate that NGFI-A binding to the exon 1 7 GR promoter is required for such epigenetic reprogramming. In vivo, enhanced maternal LG is associated with increased NGFI-A binding to the exon 1 7 GR promoter in the hippocampus of pups, and NGFI-A-bound exon 1 7 GR promoter is unmethylated compared with unbound exon 1 7 GR promoter. Knockdown experiments of NGFI-A in hippocampal primary cell culture show that NGFI-A is required for serotonin-induced DNA demethylation and increased exon 1 7 GR promoter expression. The data are consistent with the hypothesis that NGFI-A participates in epigenetic programming of GR expression.

Human induced pluripotent stem (iPS) cells may represent the ideal cell source for research and applications in regenerative medicine. However, standard culture conditions that depend on the use of undefined substrates and xenogeneic medium components represent a significant obstacle to clinical translation. Recently, we reported a defined culture system for human embryonic stem (ES) cells using a fully defined synthetic polymer coating, poly[2-(methacryloyloxy)ethyl dimethyl-(3-sulfopropyl)ammonium hydroxide] (PMEDSAH), in conjunction with xeno-free and defined culture medium. Here we tested the hypothesis that iPS cells grown in this defined culture system can be differentiated into mesenchymal stem cells (iPS-MSCs). Human iPS cells were cultured on PMEDSAH and differentiated into functional MSCs, as confirmed by expression of characteristic MSC markers (CD166+, CD105+, CD73+, CD44+, CD34− and CD45−) and their ability to differentiate in vitro into adipogenic, chondrogenic and osteoblastic lineages. To demonstrate the potential of iPS-MSCs to regenerate bone in vivo, the newly derived cells were induced to osteoblast differentiation for 4 days and transplanted into calvaria defects in immoncompromised mice for 8 weeks. MicroCT analysis and histology demonstrated de novo bone formation in the calvaria defects for animals treated with iPS-MSCs, but not for the control group. Moreover, positive staining for human nuclear antigen and human mitochondria monoclonal antibodies unambiguously confirmed the participation of the transplanted human iPS-MSCs in the regenerated bone. These results confirmed that human iPS cells grown in a defined and xeno-free system have the capability to differentiate into functional MSCs with the ability to form bone in vivo.

To enhance the understanding of differentiation patterns and bone formation capacity of hESCs, we determined (1) the temporal pattern of osteoblastic differentiation of human embryonic stem cell-derived mesenchymal stem cells (hESCs-MSCs), (2) the influence of a three-dimensional matrix on the osteogenic differentiation of hESCs-MSCs in long term culture and (3) the bone forming capacity of osteoblast-like cells derived from hESCs-MSCs in calvarial defects. Incubation of hESCs-MSCs in osteogenic medium induced osteoblastic differentiation of hESCs-MSCs into mature osteoblasts in a similar chronological pattern to human bone marrow stromal cells and primary osteoblasts. Osteogenic differentiation was enhanced by culturing the cells on three-dimensional collagen scaffolds. Fluorescent activated cell sorting of alkaline phosphatase expressing cells was used to obtain an enriched osteogenic cell population for in vivo transplantation. The identification of green fluorescence protein and expression of human specific nuclear antigen in osteocytes in newly formed bone verified the role of transplanted human cells in the bone regeneration process. The current cell culture model and osteogenic cell enrichment method could provide large numbers of osteoprogenitor cells for analysis of differentiation patterns and cell transplantation to regenerate skeletal defects.