Prolonged expression of the CRISPR-Cas9 nuclease and gRNA from viral vectors may cause off-target mutagenesis and immunogenicity. Thus, a transient delivery system is needed for therapeutic genome ...editing applications. Here, we develop an extracellular nanovesicle-based ribonucleoprotein delivery system named NanoMEDIC by utilizing two distinct homing mechanisms. Chemical induced dimerization recruits Cas9 protein into extracellular nanovesicles, and then a viral RNA packaging signal and two self-cleaving riboswitches tether and release sgRNA into nanovesicles. We demonstrate efficient genome editing in various hard-to-transfect cell types, including human induced pluripotent stem (iPS) cells, neurons, and myoblasts. NanoMEDIC also achieves over 90% exon skipping efficiencies in skeletal muscle cells derived from Duchenne muscular dystrophy (DMD) patient iPS cells. Finally, single intramuscular injection of NanoMEDIC induces permanent genomic exon skipping in a luciferase reporter mouse and in mdx mice, indicating its utility for in vivo genome editing therapy of DMD and beyond.
Abstract
Tendon self-renewal is a rare occurrence because of the poor vascularization of this tissue; therefore, reconstructive surgery using autologous tendon is often performed in severe injury ...cases. However, the post-surgery re-injury rate is relatively high, and the collection of autologous tendons leads to muscle weakness, resulting in prolonged rehabilitation. Here, we introduce an induced pluripotent stem cell (iPSC)-based technology to develop a therapeutic option for tendon injury. First, we derived tenocytes from human iPSCs by recapitulating the normal progression of step-wise narrowing fate decisions in vertebrate embryos. We used single-cell RNA sequencing to analyze the developmental trajectory of iPSC-derived tenocytes. We demonstrated that iPSC-tenocyte grafting contributed to motor function recovery after Achilles tendon injury in rats via engraftment and paracrine effects. The biomechanical strength of regenerated tendons was comparable to that of healthy tendons. We suggest that iPSC-tenocytes will provide a therapeutic option for tendon injury.
Rapid progress in mass spectrometry (MS) has made comprehensive analyses of the proteome possible, but accurate quantification remains challenging. Isobaric tags for relative and absolute ...quantification (iTRAQ) is widely used as a tool to quantify proteins expressed in different cell types and various cellular conditions. The quantification precision of iTRAQ is quite high, but the accuracy dramatically decreases in the presence of interference peptides that are coeluted and coisolated with the target peptide. Here, we developed “removal of interference mixture MS/MS spectra (RiMS)” to improve the quantification accuracy of isobaric tag approaches. The presence of spectrum interference is judged by examining the overlap in the elution time of all scanned precursor ions. Removal of this interference decreased protein identification (11% loss) but improved quantification accuracy. Further, RiMS does not require any specialized equipment, such as MS3 instruments or an additional ion separation mode. Finally, we demonstrated that RiMS can be used to quantitatively compare human-induced pluripotent stem cells and human dermal fibroblasts, as it revealed differential protein expressions that reflect the biological characteristics of the cells.
Human induced pluripotent stem cells (hiPSCs) can differentiate into cells of the three germ layers and are promising cell sources for regenerative medicine therapies. However, current protocols ...generate hiPSCs with low efficiency, and the generated iPSCs have variable differentiation capacity among different clones. Our previous study reported that MYC proteins (c-MYC and MYCL) are essential for reprogramming and germline transmission but that MYCL can generate hiPSC colonies more efficiently than c-MYC. The molecular underpinnings for the different reprogramming efficiencies between c-MYC and MYCL, however, are unknown. In this study, we found that MYC Box 0 (MB0) and MB2, two functional domains conserved in the MYC protein family, contribute to the phenotypic differences and promote hiPSC generation in MYCL-induced reprogramming. Proteome analyses suggested that in MYCL-induced reprogramming, cell adhesion-related cytoskeletal proteins are regulated by the MB0 domain, while the MB2 domain regulates RNA processes. These findings provide a molecular explanation for why MYCL has higher reprogramming efficiency than c-MYC.
The incomplete differentiation of human induced pluripotent stem cells (iPSCs) poses a serious safety risk owing to their potential tumorigenicity, hindering their clinical application. Here, we ...explored the potential of phospho-D-peptides as novel iPSC-eliminating agents. Alkaline phosphatases overexpressed on iPSCs dephosphorylate phospho-D-peptides into hydrophobic peptides that aggregate and induce cell death. We isolated a peptide candidate, D-3, that selectively and rapidly induced toxicity in iPSCs within 1 hr but had little influence on various non-iPSCs, including primary hepatocytes and iPSC-derived cardiomyocytes. Two hours of D-3 treatment efficiently eliminated iPSCs from both single cultures and co-cultures spiked with increasing ratios of iPSCs. In addition, D-3 prevented residual iPSC-induced teratoma formation in a mouse tumorigenicity assay. These results suggest the enormous potential of D-3 as a low-cost and effective anti-iPSC agent for both laboratory use and for the safe clinical application of iPSC-derived cells in regenerative medicine.
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•D-3 induces toxicity in iPSCs and ESCs within 1 hr of incubation•D-3 has little influence on various non-iPSCs, including hepatocytes and neurons•D-3 prevents residual iPSC-induced teratoma formation in an animal model•Alkaline phosphatase activity on the cell surface is required for D-3's toxicity
Yi Kuang et al. discovered a phosphor-peptide D-3 that responds to the activity of alkaline phosphatase on the cell surface to selectively and effectively remove iPSCs and ESCs within 1–2 hr. D-3 is a promising low-cost iPSC-eliminating agent and is simple to use.
Previous studies have suggested that the loss of the translation initiation factor eIF4G1 homolog NAT1 induces excessive self-renewability of naive pluripotent stem cells (PSCs); yet the role of NAT1 ...in the self-renewal and differentiation of primed PSCs is still unclear. Here, we generate a conditional knockout of NAT1 in primed PSCs and use the cells for the functional analyses of NAT1. Our results show that NAT1 is required for the self-renewal and neural differentiation of primed PSCs. In contrast, NAT1 deficiency in naive pluripotency attenuates the differentiation to all cell types. We also find that NAT1 is involved in efficient protein expression of an RNA uridyltransferase, TUT7. TUT7 is involved in the neural differentiation of primed PSCs via the regulation of human endogenous retrovirus accumulation. These data demonstrate the essential roles of NAT1 and TUT7 in the precise transition of stem cell fate.
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•NAT1 is required for the self-renewal and neural differentiation of primed PSCs•Loss of NAT1 induced aberrant HERV-H expression in human PSCs•TUT7 contributes to regulate precise expression of HERV-Hs in human PSCs•NAT1 promotes the translation of TUT7
Takahashi et al. show that NAT1 is crucial for the self-renewal and neural differentiation of primed PSCs. Loss of NAT1 induced significant increase of HERV-H transcripts. NAT1 promotes the translation of TUT7, which controls HERV-H expression. Taken together, the NAT1/TUT7/HERV-H axis plays important roles in neural differentiation of PSCs.
The molecular mechanisms of cell reprogramming and differentiation involve various signaling factors. Small molecule compounds have been identified to artificially influence these factors through ...interacting cellular proteins. Although such small molecule compounds are useful to enhance reprogramming and differentiation and to show the mechanisms that underlie these events, the screening usually requires a large number of compounds to identify only a very small number of hits (e.g., one hit among several tens of thousands of compounds). Here, we show a proof of concept that xenospecific gene products can affect the efficiency of cell reprogramming to pluripotency. Thirty genes specific for the bacterium Wolbachia pipientis were forcibly expressed individually along with reprogramming factors (Oct4, Sox2, Klf4 and c‐Myc) that can generate induced pluripotent stem cells in mammalian cells, and eight were found to affect the reprogramming efficiency either positively or negatively (hit rate 26.7%). Mechanistic analysis suggested one of these proteins interacted with cytoskeleton to promote reprogramming. Our results raise the possibility that xenospecific gene products provide an alternative way to study the regulatory mechanism of cell identity.
Screening of Wolbachia‐specific proteins identified several proteins that affected mammalian cell reprogramming to pluripotency. Our results suggest that screenings of xenospecific gene products could be useful for studies on mechanisms of cell identity regulation as well as development of technologies controlling it.
The blood-brain barrier (BBB) is composed of four cell populations, brain endothelial cells (BECs), pericytes, neurons, and astrocytes. Its role is to precisely regulate the microenvironment of the ...brain through selective substance crossing. Here we generated an in vitro model of the BBB by differentiating human induced pluripotent stem cells (hiPSCs) into all four populations. When the four hiPSC-derived populations were co-cultured, endothelial cells (ECs) were endowed with features consistent with BECs, including a high expression of nutrient transporters (CAT3, MFSD2A) and efflux transporters (ABCA1, BCRP, PGP, MRP5), and strong barrier function based on tight junctions. Neuron-derived Dll1, which activates Notch signaling in ECs, was essential for the BEC specification. We performed in vitro BBB permeability tests and assessed ten clinical drugs by nanoLC-MS/MS, finding a good correlation with the BBB permeability reported in previous cases. This technology should be useful for research on human BBB physiology, pathology, and drug development.
The effects of transcription factors on the maintenance and differentiation of human-induced or embryonic pluripotent stem cells (iPSCs/ESCs) have been well studied. However, the importance of ...posttranscriptional regulatory mechanisms, which cause the quantitative dissociation of mRNA and protein expression, has not been explored in detail. Here, by combining transcriptome and proteome profiling, we identified 228 posttranscriptionally regulated genes with strict upregulation of the protein level in iPSCs/ESCs. Among them, we found 84 genes were vital for the survival of iPSCs and HDFs, including 20 genes that were specifically necessary for iPSC survival. These 20 proteins were upregulated only in iPSCs/ESCs and not in differentiated cells derived from the three germ layers. Although there are still unknown mechanisms that downregulate protein levels in HDFs, these results reveal that posttranscriptionally regulated genes have a crucial role in iPSC survival.
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•The posttranscriptionally regulated 20 genes are necessary for iPSC survival•The proteins of HSPA8, EIF3D, and NCBP2 are quickly degraded in HDFs•mRNA localization affects the protein amounts in most of the 20 genes•Translation is repressed in HDFs despite mRNA binding to ribosomes
Biological sciences; Stem cells research; Omics
Current proteome analysis method is an effective method to comprehensively analyze the proteins, but it is still difficult to identify and quantify the proteins of a specific cell in a single ...analysis due to the complexity of the proteome samples. We have been working on the development of basic technologies to solve this problem and improve the identification and quantification efficiency. We have focused on the use of monolithic columns to improve separation efficiency and have developed the RiMS (removal of interference mixture spectra) method, which improves the accuracy of isobaric labelling quantification. We applied this method to the analysis of pluripotent stem cells, resulting improved quantification accuracy of cell type-specific proteins. In this paper, we will introduce the RiMS method with an overview of the methods used for quantitative proteome analysis.
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