ABSTRACT
The mammalian circadian clock, which coordinates various physiological functions, develops gradually during ontogeny. Recently, we have reported the posttranscriptional suppression of CLOCK protein expression as a key mechanism of the emergence of the circadian clock during mouse development. However, whether a common mechanism regulates the development of the human circadian clock remains unclear. In the present study, we show that human induced pluripotent stem cells (iPSCs) have no discernible circadian molecular oscillation. In addition, in vitro differentiation culture of human iPSCs required a longer duration than that required in mouse for the emergence of circadian oscillations. The expression of CLOCK protein in undifferentiated human iPSCs was posttranscriptionally suppressed despite the expression of CLOCK mRNA, which is consistent with our previous observations in mouse embryonic stem cells, iPSCs, and early mouse embryos. These results suggest that CLOCK protein expressions could be posttranscriptionally suppressed in the early developmental stage not only in mice but also in humans.
Subject(s)
CLOCK Proteins/genetics , Cell Differentiation , Circadian Clocks/genetics , Circadian Rhythm , Induced Pluripotent Stem Cells/physiology , Protein Processing, Post-Translational , CLOCK Proteins/physiology , Cells, Cultured , Circadian Clocks/physiology , Gene Expression Regulation , Humans , RNA, Messenger/geneticsABSTRACT
Target-specific genome editing using engineered nucleases has become widespread in various fields. Long gene knock-in and single-base substitutions can be performed by homologous recombination (HR), but the efficiency is usually very low. To improve the efficiency of knock-in with single-stranded oligo DNA nucleotides (ssODNs), we have investigated optimal design of ssODNs in terms of the blocking mutation, orientation, size, and length of homology arms to explore the optimal parameters of ssODN design using reporter systems for the detection of single-base substitutions. We have also investigated the difference in knock-in efficiency among the delivery forms and methods of Cas9 and sgRNA. The knock-in efficiencies for optimized ssODNs were much higher than those for ssODNs with no blocking mutation. We have also demonstrated that Cas9 protein/sgRNA ribonucleoprotein complexes (Cas9-RNPs) can dramatically reduce the re-cutting of the edited sites.
Subject(s)
CRISPR-Cas Systems/genetics , Gene Editing/methods , Gene Knock-In Techniques/methods , Ribonucleoproteins/genetics , Base Sequence/genetics , Cell Culture Techniques/methods , DNA, Single-Stranded/genetics , Feasibility Studies , HEK293 Cells , Humans , Induced Pluripotent Stem Cells , Oligonucleotides/genetics , RNA, Guide, Kinetoplastida/genetics , Transfection/methodsABSTRACT
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.
