ABSTRACT
Epigenetic memory in induced pluripotent stem cells, which is related to the somatic cell type of origin of the stem cells, might lead to variations in the differentiation capacities of the pluripotent stem cells. In this context, induced pluripotent stem cells from human CD34(+) hematopoietic stem cells might be more suitable for hematopoietic differentiation than the commonly used fibroblast-derived induced pluripotent stem cells. To investigate the influence of an epigenetic memory on the ex vivo expansion of induced pluripotent stem cells into erythroid cells, we compared induced pluripotent stem cells from human neural stem cells and human cord blood-derived CD34(+) hematopoietic stem cells and evaluated their potential for differentiation into hematopoietic progenitor and mature red blood cells. Although genome-wide DNA methylation profiling at all promoter regions demonstrates that the epigenetic memory of induced pluripotent stem cells is influenced by the somatic cell type of origin of the stem cells, we found a similar hematopoietic induction potential and erythroid differentiation pattern of induced pluripotent stem cells of different somatic cell origin. All human induced pluripotent stem cell lines showed terminal maturation into normoblasts and enucleated reticulocytes, producing predominantly fetal hemoglobin. Differences were only observed in the growth rate of erythroid cells, which was slightly higher in the induced pluripotent stem cells derived from CD34(+) hematopoietic stem cells. More detailed methylation analysis of the hematopoietic and erythroid promoters identified similar CpG methylation levels in the induced pluripotent stem cell lines derived from CD34(+) cells and those derived from neural stem cells, which confirms their comparable erythroid differentiation potential.
Subject(s)
Cell Differentiation , Erythroid Cells/cytology , Fetal Blood/cytology , Hematopoietic Stem Cells/cytology , Induced Pluripotent Stem Cells/cytology , Neural Stem Cells/cytology , Biomarkers/metabolism , DNA Methylation , Epigenomics , Erythroid Cells/metabolism , Fetal Blood/metabolism , Fibroblasts/cytology , Fibroblasts/metabolism , Gene Expression Profiling , Hematopoietic Stem Cells/metabolism , Humans , Induced Pluripotent Stem Cells/metabolism , Neural Stem Cells/metabolism , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Though expression of the homeobox transcription factor Nanog is generally restricted to pluripotent cells and early germ cells, many contradictory reports about Nanog's involvement in tumorigenesis exist. To address this, a modified Tet-On system was utilized to generate Nanog-inducible mice. Following prolonged Nanog expression, phenotypic alterations were found to be restricted to the intestinal tract, leaving other major organs unaffected. Intestinal and colonic epithelium hyperplasia was observed-intestinal villi had doubled in length and hyperplastic epithelium outgrowths were seen after 7days. Increased proliferation of crypt cells and downregulation of the tumor suppressors Cdx2 and Klf4 was detected. ChIP analysis showed physical interaction of Nanog with the Cdx2 and Klf4 promoters, indicating a regulatory conservation from embryonic development. Despite downregulation of tumor suppressors and increased proliferation, ectopic Nanog expression did not lead to tumor formation. We conclude that unlike other pluripotency-related transcription factors, Nanog cannot be considered an oncogene.
Subject(s)
Cell Proliferation , Colon/metabolism , Epithelial Cells/metabolism , Homeodomain Proteins/metabolism , Intestinal Mucosa/metabolism , Intestine, Small/metabolism , Animals , CDX2 Transcription Factor , Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/metabolism , Cell Transformation, Neoplastic/pathology , Colon/pathology , Colonic Neoplasms/genetics , Colonic Neoplasms/metabolism , Colonic Neoplasms/pathology , Epithelial Cells/pathology , Gene Expression Regulation , Gene Knockdown Techniques , Genotype , Homeodomain Proteins/genetics , Hyperplasia , Intestinal Mucosa/pathology , Intestine, Small/pathology , Kruppel-Like Factor 4 , Kruppel-Like Transcription Factors/genetics , Kruppel-Like Transcription Factors/metabolism , Mice , Mice, Inbred C3H , Mice, Inbred C57BL , Mice, Transgenic , NIH 3T3 Cells , Nanog Homeobox Protein , Phenotype , Signal Transduction , Time Factors , Transcription Factors/genetics , Transcription Factors/metabolism , Transfection , Tumor Suppressor Proteins/genetics , Tumor Suppressor Proteins/metabolismABSTRACT
Oct4A is a core component of the regulatory network of pluripotent cells, and by itself can reprogram neural stem cells into pluripotent cells in mice and humans. However, its role in defining totipotency and inducing pluripotency during embryonic development is still unclear. We genetically eliminated maternal Oct4A using a Cre/loxP approach in mouse and found that the establishment of totipotency was not affected, as shown by the generation of live pups. After complete inactivation of both maternal and zygotic Oct4A expression, the embryos still formed Oct4-GFP- and Nanog-expressing inner cell masses, albeit non-pluripotent, indicating that Oct4A is not a determinant for the pluripotent cell lineage separation. Interestingly, Oct4A-deficient oocytes were able to reprogram fibroblasts into pluripotent cells. Our results clearly demonstrate that, in contrast to its role in the maintenance of pluripotency, maternal Oct4A is not crucial for either the establishment of totipotency in embryos, or the induction of pluripotency in somatic cells using oocytes.
