Abstract
Induced pluripotent stem (iPS) cells are generated from adult somatic cells by transduction of defined factors. Given their unlimited proliferation and differentiation potential, iPS cells represent promising sources for cell therapy and tools for research and drug discovery. However, systems for the directional differentiation of iPS cells toward paraxial mesodermal lineages have not been reported. In the present study, we established a protocol for the differentiation of mouse iPS cells into paraxial mesodermal lineages in serum-free culture. The protocol was dependent on Activin signaling in addition to BMP and Wnt signaling which were previously shown to be effective for mouse ES cell differentiation. Independently of the cell origin, the number of transgenes, or the type of vectors used to generate iPS cells, the use of serum-free monolayer culture stimulated with a combination of BMP4, Activin A, and LiCl enabled preferential promotion of mouse iPS cells to a PDGFR-α+/Flk-1− population, which represents a paraxial mesodermal lineage. The mouse iPS cell-derived paraxial mesodermal cells exhibited differentiation potential into osteogenic, chondrogenic, and myogenic cells both in vitro and in vivo and contributed to muscle regeneration. Moreover, purification of the PDGFR-α+/KDR− population after differentiation allowed enrichment of human iPS cell populations with paraxial mesodermal characteristics. The resultant PDGFR-α+/KDR− population derived from human iPS cells specifically exhibited osteogenic, chondrogenic, and myogenic differentiation potential in vitro, implying generation of paraxial mesodermal progenitors similar to mouse iPS cell-derived progenitors. These findings highlight the potential of protocols based on the serum-free, stepwise induction and purification of paraxial mesodermal cell lineages for use in stem cell therapies to treat diseased bone, cartilage, and muscle.
Highlights
Embryonic stem (ES) cells have been investigated both as an experimental tool for developmental biology and as a source of cell-based therapies due to their potential for self-renewal and differentiation into all cell lineages
Since Yamanaka and Takahashi first reported the generation of pluripotent ES cell-like cells–induced pluripotent stem cells– from mouse fibroblasts by unique gene transfer-based nuclear reprogramming technology, many researchers have compared the similarities and differences between ES and iPS cells with respect to their pluripotency, undifferentiated states, genetic or epigenetic regulations, and differentiation potentials
We demonstrated a potential of mouse ES and iPS cells for paraxial mesodermal differentiation, with the exception of the requirement of Activin A during iPS cell differentiation under serum-free conditions
Summary
Embryonic stem (ES) cells have been investigated both as an experimental tool for developmental biology and as a source of cell-based therapies due to their potential for self-renewal and differentiation into all cell lineages. The use of nonintegrative vectors for gene transfer [5] or replacement of the cMyc oncogene with other safer genes such as L-Myc [6] or Glis-1 [7] permits generation of iPS cells that lack transgenes or oncogenes. Such technical advancements reduce the risk of tumorigenesis that results from reactivation of c-Myc transgenes. Suitable differentiation markers should be used to determine the signaling mechanisms that govern pluripotent stem cell differentiation toward specific lineages, so that recombinant proteins and small molecules can be used to direct differentiation
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