ABSTRACT
The self-renewal and differentiation capacities of human pluripotent stem cells (hPSCs) make them a promising source of material for cell transplantation therapy, drug development, and studies of cellular differentiation and development. However, the large numbers of cells necessary for many of these applications require extensive expansion of hPSC cultures, a process that has been associated with genetic and epigenetic alterations. We have performed a combinatorial study on both hESCs and hiPSCs to compare the effects of enzymatic vs. mechanical passaging, and feeder-free vs. mouse embryonic fibroblast feeder substrate, on the genetic and epigenetic stability and the phenotypic characteristics of hPSCs. In extensive experiments involving over 100 continuous passages, we observed that both enzymatic passaging and feeder-free culture were associated with genetic instability, higher rates of cell proliferation, and persistence of OCT4/POU5F1-positive cells in teratomas, with enzymatic passaging having the stronger effect. In all combinations of culture conditions except for mechanical passaging on feeder layers, we noted recurrent deletions in the genomic region containing the tumor suppressor gene TP53, which was associated with decreased mRNA expression of TP53, as well as alterations in the expression of several downstream genes consistent with a decrease in the activity of the TP53 pathway. Among the hESC cultures, we also observed culture-associated variations in global gene expression and DNA methylation. The effects of enzymatic passaging and feeder-free conditions were also observed in hiPSC cultures. Our results highlight the need for careful assessment of the effects of culture conditions on cells intended for clinical therapies.
Subject(s)
Epigenesis, Genetic , Genome, Human , Genomic Instability , Human Embryonic Stem Cells/metabolism , Cell Culture Techniques , Cell Differentiation , Cell Line , Cell Self Renewal , Cell Transformation, Neoplastic/genetics , Cells, Cultured , Chromosome Aberrations , Chromosome Deletion , Chromosome Duplication , Chromosomes, Human, Pair 12 , Chromosomes, Human, Pair 17 , Chromosomes, Human, Pair 20 , DNA Methylation , Gene Expression Profiling , Human Embryonic Stem Cells/cytology , Human Embryonic Stem Cells/pathology , Humans , Phenotype , Pluripotent Stem Cells/metabolism , Polymorphism, Single Nucleotide , Time Factors , Tumor Suppressor Protein p53/geneticsABSTRACT
New small molecules that regulate the step-wise differentiation of human pluripotent stem cells into dopaminergic neurons have been identified. The steroid, guggulsterone, was found to be the most effective inducer of neural stem cells into dopaminergic neurons. These neurons are extensively characterized and shown to be functional. We believe this new approach offers a practical route to creating neurons of sufficient quality to be used to treat Parkinson's disease patients.
