Comprehensive analysis of prime editing outcomes in human embryonic stem cells.

Prime editing is a versatile and precise genome editing technique that can directly copy desired genetic modifications into target DNA sites without the need for donor DNA. This technique holds great promise for the analysis of gene function, disease modeling, and the correction of pathogenic mutations in clinically relevant cells such as human pluripotent stem cells (hPSCs). Here, we comprehensively tested prime editing in hPSCs by generating a doxycycline-inducible prime editing platform. Prime editing successfully induced all types of nucleotide substitutions and small insertions and deletions, similar to observations in other human cell types. Moreover, we compared prime editing and base editing for correcting a disease-related mutation in induced pluripotent stem cells derived form a patient with α 1-antitrypsin (A1AT) deficiency. Finally, whole-genome sequencing showed that, unlike the cytidine deaminase domain of cytosine base editors, the reverse transcriptase domain of a prime editor does not lead to guide RNA-independent off-target mutations in the genome. Our results demonstrate that prime editing in hPSCs has great potential for complementing previously developed CRISPR genome editing tools.

An improved method for the derivation of high quality iPSCs in the absence of c-Myc.

Induced pluripotent stem cells (iPSCs) hold tremendous potential for the development of new regenerative medicine therapies and the study of molecular mechanisms of pluripotency and development. However, reactivation of c-Myc, which results in tumor formation in chimeric mice, is a major roadblock in the translation of iPSCs into therapies. Although ectopic expression of c-Myc is not absolutely required for somatic reprogramming, in the absence of c-Myc, the overall efficiency of reprogramming is drastically reduced and the reprogramming time is increased. Subtle, abnormal epigenetic modifications in iPSCs derived in the absence of c-Myc have also been documented. Therefore, we developed a reprogramming method without c-Myc to generate high-quality iPSCs, a prerequisite to harnessing the full potential of iPSCs. In this study, we determined that serum replacement (SR)-based culture conditions dramatically increased the transcription factor-mediated reprogramming of mouse embryonic fibroblast cells (MEFs). The process was shortened to approximately 8 days when Oct4/Sox2/Klf4 (3F)-transduced MEFs were first cultured for 3 days under low serum conditions (LS protocol). The 3F-derived iPSCs that were generated by this method resembled mouse ES cells (mESCs) in morphology, gene expression, and in vitro differentiation. Finally, we observed that 3F-derived iPSC colonies were able to reach definite pluripotency in terms of molecular signatures when the catalytic function of c-Myc was tolerated. The 3F induction of pluripotency described here should facilitate the use of iPSCs and may also facilitate the mechanistic dissection of somatic reprogramming.

Self-assembling antimicrobial peptides on nanotubular titanium surfaces coated with calcium phosphate for local therapy.

Bacterial infection is a serious medical problem leading to implant failure. The current antibiotic based therapies rise concerns due to bacterial resistance. The family of antimicrobial peptides (AMP) is one of the promising candidates as local therapy agents due to their broad-spectrum activity. Despite AMPs receive increasing attention to treat infection, their effective delivery to the implantation site has been limited. Here, we developed an engineered dual functional peptide which delivers AMP as a biomolecular therapeutic agent onto calcium phosphate (Ca-P) deposited nanotubular titanium surfaces. Dual functionality of the peptide was achieved by combining a hydroxyapatite binding peptide-1 (HABP1) with an AMP using a flexible linker. HABP functionality of the peptide provided a self-coating property onto the nano-topographies that are designed to improve osteointegration capability, while AMP offered an antimicrobial protection onto the implant surface. We successfully deposited calcium phosphate minerals on nanotubular titanium oxide surface using pulse electrochemical deposition (PECD) and characterized the minerals by XRD, FT-IR, FE-SEM. Antimicrobial activity of the engineered peptide was tested against S. mutans (gram- positive) and E. coli (gram-negative) both in solution and on the Ca-P coated nanotubular titanium surface. In solution activity of AMP and dual functional peptide have the same Minimum Inhibitory Concentration (MIC) (32 mg/mL). The peptide also resulted in the reduction of the number of bacteria both for E.coli and S. mutans compare to control groups on the surface. Antimicrobial features of dual functional peptides are strongly correlated with their structures suggesting tunability in design through linkers regions. The dual-function peptide offers single-step solution for implant surface functionalization that could be applicable to any implant surface having different topographies.

Activation-induced deaminase-coupled DNA demethylation is not crucial for the generation of induced pluripotent stem cells.

DNA methylation constitutes a major obstacle in the reprogramming of cells to pluripotency. Although little is known regarding the molecular mechanisms of DNA demethylation, activation-induced deaminase (AID), which is known to function in antibody diversification, has been implicated in DNA demethylation through a base excision repair (BER)-mediated pathway. Here we comprehensively examine the plausibility of coupled AID-BER demethylation in the generation of induced pluripotent stem cells (iPSCs) and show that AID is dispensable for reprogramming cells into iPSCs. Additionally, the overexpression of AID and other factors involved in AID-coupled DNA demethylation does not increase the efficiency of reprogramming. Moreover, BER is not likely to play a role in this process. Our results indicate that the reactivation of key genes governing the pluripotency circuitry occurs through a mechanism that is independent of deamination-coupled demethylation.

Ground-state conditions promote robust Prdm14 reactivation and maintain an active Dlk1-Dio3 region during reprogramming.

Induced pluripotent stem cells (iPSCs) are capable of unlimited self-renewal and can give rise to all three germ layers, thereby providing a new platform with which to study mammalian development and epigenetic reprogramming. However, iPSC generation may result in subtle epigenetic variations, such as the aberrant methylation of the Dlk1-Dio3 locus, among the clones, and this heterogeneity constitutes a major drawback to harnessing the full potential of iPSCs. Vitamin C has recently emerged as a safeguard to ensure the normal imprinting of the Dlk1-Dio3 locus during reprogramming. Here, we show that vitamin C exerts its effect in a manner that is independent of the reprogramming kinetics. Moreover, we demonstrate that reprogramming cells under 2i conditions leads to the early upregulation of Prdm14, which in turn results in a highly homogeneous population of authentic pluripotent colonies and prevents the abnormal silencing of the Dlk1-Dio3 locus.