A synthetic mRNA cell reprogramming method using CYCLIN D1 promotes DNA repair, generating improved genetically stable human induced pluripotent stem cells.

A key challenge for clinical application of induced pluripotent stem cells (iPSC) to accurately model and treat human pathologies depends on developing a method to generate genetically stable cells to reduce long-term risks of cell transplant therapy. Here, we hypothesized that CYCLIN D1 repairs DNA by highly efficient homologous recombination (HR) during reprogramming to iPSC that reduces genetic instability and threat of neoplastic growth. We adopted a synthetic mRNA transfection method using clinically compatible conditions with CYCLIN D1 plus base factors (OCT3/4, SOX2, KLF4, LIN28) and compared with methods that use C-MYC. We demonstrate that CYCLIN D1 made iPSC have (a) lower multitelomeric signal, (b) reduced double-strand DNA breaks, (c) correct nuclear localization of RAD51 protein expression, and (d) reduced single-nucleotide polymorphism (SNP) changes per chromosome, compared with the classical reprogramming method using C-MYC. CYCLIN D1 iPSC have reduced teratoma Ki67 cell growth kinetics and derived neural stem cells successfully engraft in a hostile spinal cord injury (SCI) microenvironment with efficient survival, differentiation. We demonstrate that CYCLIN D1 promotes double-stranded DNA damage repair predominantly through HR during cell reprogramming to efficiently produce iPSC. CYCLIN D1 reduces general cell stress associated with significantly lower SIRT1 gene expression and can rescue Sirt1 null mouse cell reprogramming. In conclusion, we show synthetic mRNA transfection of CYCLIN D1 repairs DNA during reprogramming resulting in significantly improved genetically stable footprint in human iPSC, enabling a new cell reprogramming method for more accurate and reliable generation of human iPSC for disease modeling and future clinical applications.

Two novel ligand-independent variants of the VEGFR-1 receptor are expressed in human testis and spermatozoa, one of them with the ability to activate SRC proto-oncogene tyrosine kinases.

The vascular endothelial growth factor receptor 1 (VEGFR-1) family of receptors is preferentially expressed in endothelial cells, with the full-length and mostly the soluble (sVEGFR-1) isoforms being the most expressed ones. Surprisingly, cancer cells (MDA-MB-231) express, instead, alternative intracellular VEGFR-1 variants. We wondered if these variants, that are no longer dependent on ligands for activation, were expressed in a physiological context, specifically in spermatogenic cells, and whether their expression was maintained in spermatozoa and required for human fertility. By interrogating a human library of mature testis cDNA, we characterized two new truncated intracellular variants different from the ones previously described in cancer cells. The new isoforms were transcribed from alternative transcription start sites (aTSS) located respectively in intron-19 (i19VEGFR-1) and intron-28 (i28VEGFR-1) of the VEGFR-1 gene (GenBank accession numbers JF509744 and JF509745) and expressed in mature testis and spermatozoa. In this paper, we describe the characterization of these isoforms by RT-PCR, northern blot, and western blot, their preferential expression in human mature testis and spermatozoa, and the elements that punctuate their proximal promoters and suggest cues for their expression in spermatogenic cells. Mechanistically, we show that i19VEGFR-1 has a strong ability to phosphorylate and activate SRC proto-oncogene non-receptor tyrosine kinases and a significant bias toward a decrease in expression in patients considered infertile by WHO criteria.

Global Proteomic and Methylome Analysis in Human Induced Pluripotent Stem Cells Reveals Overexpression of a Human TLR3 Affecting Proper Innate Immune Response Signaling.

When considering the clinical applications of autologous cell replacement therapy of human induced pluripotent stem cells (iPSC)-derived cells, there is a clear need to better understand what the immune response will be before we embark on extensive clinical trials to treat or model human disease. We performed a detailed assessment comparing human fibroblast cell lines (termed F1) reprogrammed into human iPSC and subsequently differentiated back to fibroblast cells (termed F2) or other human iPSC-derived cells including neural stem cells (NSC) made from either retroviral, episomal, or synthetic mRNA cell reprogramming methods. Global proteomic analysis reveals the main differences in signal transduction and immune cell protein expression between F1 and F2 cells, implicating wild type (WT) toll like receptor protein 3 (TLR3). Furthermore, global methylome analysis identified an isoform of the human TLR3 gene that is not epigenetically reset correctly upon differentiation to F2 cells resulting in a hypomethylated transcription start site in the TLR3 isoform promoter and overexpression in most human iPSC-derived cells not seen in normal human tissue. The human TLR3 isoform in human iPSC-NSC functions to suppress NF-KB p65 signaling pathway in response to virus (Poly IC), suggesting suppressed immunity of iPSC-derived cells to viral infection. The sustained WT TLR3 and TLR3 isoform overexpression is central to understanding the altered immunogenicity of human iPSC-derived cells calling for screening of human iPSC-derived cells for TLR3 expression levels before applications. Stem Cells 2019;37:476-488.

Gordon H. Dixon's trace in my personal career and the quantic jump experienced in regulatory information.

Even before Rosalin Franklin had discovered the DNA double helix, in her impressive X-ray diffraction image pattern, Erwin Schröedinger, described, in his excellent book, What is Life, how the finding of aperiodic crystals in biological systems surprised him (an aperiodic crystal, which, in my opinion is the material carrier of life). In the 21st century and still far from being able to define life, we are attending to a quick acceleration of knowledge on regulatory information. With the discovery of new codes and punctuation marks, we will greatly increase our understanding in front of an impressive avalanche of genomic sequences. Trifonov et al. defined a genetic code as a widespread DNA sequence pattern that carries a message with an impact on biology. These patterns are largely captured in transcribed messages that give meaning and identity to the particular cells. In this review, I will go through my personal career in and after my years of work in the laboratory of Gordon H. Dixon, extending toward the impressive acquisition of new knowledge on regulatory information and genetic codes provided by remarkable scientists in the field. Abbreviations: CA II: carbonic anhydridase II (chicken); Car2: carbonic anhydridase 2 (mouse); CpG islands: short (>0.5 kb) stretches of DNA with a G+C content ≥55%; DNMT1: DNA methyltransferases 1; DNMT3b: DNA methyltransferases 3B; DSB: double-strand DNA breaks; ERT: endogenous retrotransposon; ERV: endogenous retroviruses; ES cells: embryonic stem cells; GAPDH: glyceraldehide phosphate dehydrogenase; H1: histone H1; HATs: histone acetyltransferases; HDACs: histone deacetylases; H3K4me3: histone 3 trimethylated at lys 4; H3K79me2: histone 3 dimethylated at lys 79; HMG: high mobility group proteins; HMT: histone methyltransferase; HP1: heterochromatin protein 1; HR: homologous recombination; HSE: heat-shock element; ICRs: imprinted control regions; IRF: interferon regulatory factor; LDH-A/-B: lactate dehydrogenase A/B; LTR: long terminal repeats; MeCP2: methyl CpG binding protein 2; OCT4: octamer-binding transcription factor 4; PAF1: RNA Polymerase II associated factor 1; piRNA: PIWI-interacting RNA; poly(A) tails: poly-adenine tails; PRC2: polycomb repressive complex 2; PTMs: post-translational modifications; SIRT 1: sirtuin 1, silent information regulator; STAT3: signal transducer and activator of transcription; tRNAs: transfer RNA; tRFs: tRNA-derived fragments; TSS: transcription start site; TE: transposable elements; UB I: polyubiquitin I; UB II: polyubiquitin II; UBE 2N: ubiquitin conjugating enzyme E2N; 5'-UTR: 5'-untranslated sequences; 3'-UTR: 3'-untranslated sequences.