title: Maintenance of human embryonic stem cells in mesenchymal stem cell-conditioned media augments hematopoietic specification.
abstract: The realization of human embryonic stem cells (hESC) as a model for human developmental hematopoiesis and in potential cell replacement strategies relies on an improved understanding of the extrinsic and intrinsic factors regulating hematopoietic-specific hESC differentiation. Human mesenchymal stem cells (hMSCs) are multipotent cells of mesodermal origin that form a part of hematopoietic stem cell niches and have an important role in the regulation of hematopoiesis through production of secreted factors and/or cell-to-cell interactions. We have previously shown that hESCs may be successfully maintained feeder free using hMSC-conditioned media (MSC-CM). Here, we hypothesized that hESCs maintained in MSC-CM may be more prone to differentiation toward hematopoietic lineage than hESCs grown in standard human foreskin fibroblast-conditioned media. We report that specification into hemogenic progenitors and subsequent hematopoietic differentiation and clonogenic progenitor capacity is robustly enhanced in hESC lines maintained in MSC-CM. Interestingly, co-culture of hESCs on hMSCs fully abrogates hematopoietic specification of hESCs, thus suggesting that the improved hematopoietic differentiation is mediated by MSC-secreted factors rather than by MSC-hESC physical interactions. To investigate the molecular mechanism involved in this process, we analyzed global (LINE-1) methylation and genome-wide promoter DNA methylation. hESCs grown in MSC-CM showed a decrease of 17% in global DNA methylation and a promoter DNA methylation signature consisting of 45 genes commonly hypomethylated and 102 genes frequently hypermethylated. Our data indicate that maintenance of hESCs in MSC-CM robustly augments hematopoietic specification and that the process seems mediated by MSC-secreted factors conferring a DNA methylation signature to undifferentiated hESCs which may influence further predisposition toward hematopoietic specification.
authors.affiliation: Stem Cells, Development, and Cancer Laboratory, GENYO: Centro de Genómica e Investigación Oncológica Pfizer-Universidad de Granada-Junta de Andalucía, Granada, Spain. veronica.ramos@juntadeandalucia.es
authors.forename: Verónica
authors.initials: V
authors.lastname: Ramos-Mejía
authors.forename: Agustín F
authors.initials: AF
authors.lastname: Fernández
authors.forename: Verónica
authors.initials: V
authors.lastname: Ayllón
authors.forename: Pedro J
authors.initials: PJ
authors.lastname: Real
authors.forename: Clara
authors.initials: C
authors.lastname: Bueno
authors.forename: Per
authors.initials: P
authors.lastname: Anderson
authors.forename: Francisco
authors.initials: F
authors.lastname: Martín
authors.forename: Mario F
authors.initials: MF
authors.lastname: Fraga
authors.forename: Pablo
authors.initials: P
authors.lastname: Menendez
mesh_terms: D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology*; D002460:Cell Line; D018920:Coculture Techniques; D017077:Culture Media, Conditioned / Q000494:pharmacology; D019175:DNA Methylation / Q000187:drug effects / Q000502:physiology; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism*; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D059630:Mesenchymal Stem Cells
chemical_list: D017077:Culture Media, Conditioned