Genetic enhancement in cultured human adult stem cells conferred by a single nucleotide recoding.
Ontology highlight
ABSTRACT:
SUBMITTER: Yang J
PROVIDER: S-EPMC5587854 | biostudies-literature | 2017 Sep
REPOSITORIES: biostudies-literature
Publications
Genetic enhancement in cultured human adult stem cells conferred by a single nucleotide recoding.
Yang Jiping J Li Jingyi J Suzuki Keiichiro K Liu Xiaomeng X Wu Jun J Zhang Weiqi W Ren Ruotong R Zhang Weizhou W Chan Piu P Izpisua Belmonte Juan Carlos JC Qu Jing J Tang Fuchou F Liu Guang-Hui GH
Cell research 20170707 9
Similar Datasets
Project description:Premature senescence-associated functional decay and neoplastic transformation of transplanted human stem cells or their derivatives represent two major roadblocks for regenerative medicine. Cellular senescence acts as a major mechanism antagonizing neoplastic transformation, and stem cells evading senescence are prone to oncogenic transformation. So far it is unknown whether there is any genetic code, rewriting of which can simultaneously brake cellular senescence and oncogenic transformation programs. Here, we identify a single nucleotide in human genome(target on the transcription factor NRF2) as a candidate code, switch of which blocks both senescence and transformation pathways. Genetic modification stabilizes the wild type human mesenchymal stem cells (hMSCs) at a “sustainably younger” state. More importantly, the same genetic manipulation endows hMSCs with the ability of counteracting oncogenic transformation. Our study thus provides the first proof-of-concept of genetic enhancement of human stem cells, a strategy holding potential to generate superior and safer stem cell materials for cell replacement therapy.
2017-07-21 | GSE84694 | GEO
Project description:Adipose-derived mesenchymal stem cells (ADSCs) show considerable promise for clinical applications in regenerative medicine. We performed a large-scale single-cell transcriptomic sequencing of 24,358 cultured human ADSCs from three donors. We provide a high-quality dataset, which would be a valuable resource for dissecting the intrapopulation heterogeneity of cultured ADSCs as well as interrogating lineage priming patterns for any interested lineages at single-cell resolution.
| S-EPMC6390702 | biostudies-literature
Project description:We assessed single nucleotide variations (SNVs) between individual cells in two cancer cell lines; DU145, from brain metastasis of prostate tumor with deficient mismatch repair; and HT1080, a fibrosarcoma cell line. Clones of individual cells were isolated, and sequenced using Ion Ampliseq comprehensive cancer panel that covered the exomes of 409 oncogenes and tumor suppressor genes. Five clones of DU145 and four clones of HT1080 cells were analyzed. We found from 7 to 12 unique SNVs between DU145 clones, while HT1080 clones showed no more than one unique SNV. We then sub-cloned individual cells from some of these isolated clones of DU145 and HT1080 cells. The sub-clones were expanded from a single cell to approximately one million cells after about 20 cell divisions. The sub-clones of DU145 cells had from one to four new unique SNVs within the sequenced regions. No unique SNVs were found between sub-clones of HT1080 cells. Our data demonstrate that the extent of genetic variation at the single nucleotide level in cultured cancer cells is significantly affected by the status of the DNA mismatch repair system.
| S-EPMC5623640 | biostudies-literature
Project description:Selenocysteine (Sec) is naturally incorporated into proteins by recoding the stop codon UGA. Sec is not hardwired to UGA, as the Sec insertion machinery was found to be able to site-specifically incorporate Sec directed by 58 of the 64 codons. For 15 sense codons, complete conversion of the codon meaning from canonical amino acid (AA) to Sec was observed along with a tenfold increase in selenoprotein yield compared to Sec insertion at the three stop codons. This high-fidelity sense-codon recoding mechanism was demonstrated for Escherichia coli formate dehydrogenase and recombinant human thioredoxin reductase and confirmed by independent biochemical and biophysical methods. Although Sec insertion at UGA is known to compete against protein termination, it is surprising that the Sec machinery has the ability to outcompete abundant aminoacyl-tRNAs in decoding sense codons. The findings have implications for the process of translation and the information storage capacity of the biological cell.
| S-EPMC4004526 | biostudies-literature
Project description:One unexplored aspect of HIV-1 genetic architecture is how codon choice influences population diversity and evolvability. Here we compared the levels of development of HIV-1 resistance to protease inhibitors (PIs) between wild-type (WT) virus and a synthetic virus (MAX) carrying a codon-pair-reengineered protease sequence including 38 (13%) synonymous mutations. The WT and MAX viruses showed indistinguishable replication in MT-4 cells or peripheral blood mononuclear cells (PBMCs). Both viruses were subjected to serial passages in MT-4 cells, with selective pressure from the PIs atazanavir (ATV) and darunavir (DRV). After 32 successive passages, both the WT and MAX viruses developed phenotypic resistance to PIs (50% inhibitory concentrations [IC50s] of 14.6 ± 5.3 and 21.2 ± 9 nM, respectively, for ATV and 5.9 ± 1.0 and 9.3 ± 1.9, respectively, for DRV). Ultradeep sequence clonal analysis revealed that both viruses harbored previously described mutations conferring resistance to ATV and DRV. However, the WT and MAX virus proteases showed different resistance variant repertoires, with the G16E and V77I substitutions observed only in the WT and the L33F, S37P, G48L, Q58E/K, and L89I substitutions detected only in the MAX virus. Remarkably, the G48L and L89I substitutions are rarely found in vivo in PI-treated patients. The MAX virus showed significantly higher nucleotide and amino acid diversity of the propagated viruses with and without PIs (P < 0.0001), suggesting a higher selective pressure for change in this recoded virus. Our results indicate that the HIV-1 protease position in sequence space delineates the evolution of its mutant spectrum. Nevertheless, the investigated synonymously recoded variant showed mutational robustness and evolvability similar to those of the WT virus.IMPORTANCE Large-scale synonymous recoding of virus genomes is a new tool for exploring various aspects of virus biology. Synonymous virus genome recoding can be used to investigate how a virus's position in sequence space defines its mutant spectrum, evolutionary trajectory, and pathogenesis. In this study, we evaluated how synonymous recoding of the human immunodeficiency virus type 1 (HIV-1) protease affects the development of protease inhibitor (PI) resistance. HIV-1 protease is a main target of current antiretroviral therapies. Our present results demonstrate that the wild-type (WT) virus and a virus with recoded protease exhibited different patterns of resistance mutations after PI treatment. Nevertheless, the developed PI resistance phenotypes were indistinguishable between the recoded virus and the WT virus, suggesting that the HIV-1 strain with synonymously recoded protease and the WT virus are equally robust and evolvable.
| S-EPMC6069183 | biostudies-literature
Project description:Human dental pulp stem cells (hDPSCs) are easily obtained multipotent cells, however, their potential value in regenerative medicine is hindered by the phenotypic and functional changes after conventional monolayer expansion. Here, we employed single-cell RNA sequencing (scRNA-seq) to comprehensively study the transcriptional difference between the freshly isolated and monolayer cultured DPSCs. The cell cluster analysis based on our scRNA-seq data showed that monolayer culture resulted in a significant cellular composition switch compared to the freshly isolated DPSCs. However, one subpopulation, characterized as MCAM(+)JAG(+)PDGFRA(-), maintained the most transcriptional characteristics compared to their freshly isolated counterparts. Notably, immunofluorescent staining revealed that the MCAM(+)JAG(+)PDGFRA(-) hDPSCs uniquely located in the perivascular region of human dental pulp tissue. Flow-cytometry analysis confirmed that their proportion remained relatively stable (~2%) regardless of physiological senescence or dental caries. Consistent with the annotation of scRNA-seq data, MCAM(+)JAG(+)PDGFRA(-) hDPSCs showed higher proliferation capacity and enhanced in vitro multilineage differentiation potentials (osteogenic, chondrogenic and adipogenic) compared with their counterparts PDGFRA(+) subpopulation. Furthermore, the MCAM(+)JAG(+)PDGFRA(-) hDPSCs showed enhanced bone tissue formation and adipose tissue formation after 4-week subcutaneous implantation in nude mice. Taken together, our study for the first time revealed the cellular composition switch of monolayer cultured hDPSCs compared to the freshly isolated hDPSCs. After in vitro expansion, the MCAM(+)JAG(+)PDGFRA(-) subpopulation resembled the most transcriptional characteristics of fresh hDPSCs which may be beneficial for further tissue regeneration applications.
| S-EPMC8674359 | biostudies-literature
Project description:The potential use of human mesenchymal stem cells for therapeutic applications implies large scale in vitro culture, increasing the probability of genetic instability and transformation. We examine here the incidence of unbalanced and balanced chromosome rearrangements in polyclonal and single cell-derived cultures of human adipose stem cells to senescence. G-banding karyotyping of the polyclonal cultures shows a normal karyotype. In addition, high-resolution microarray-based comparative genomic hybridization analyses relative to uncultured adipose stem cells from the same donors reveal overall genomic stability in long-term (approximately 6 months) polyclonal and clonal culture. One adipose stem cell clone displayed minor deletions in gene-rich telomeric and sub-telomeric regions on three chromosomes in early passage. This however, was detected only in a sub-population of cells that was subsequently spontaneously eliminated from the culture. Apparent pericentromeric instabilities are also occasionally detected in specific chromosomes. Our results indicate that clonal chromosomal aberrations may arise transiently in early passage adipose stem cells (ASC) cultures. Nonetheless, incidence of these aberrations seems to be negligible in the majority of long-term ASC cultures, at least under the culture conditions used here.
| S-EPMC3822542 | biostudies-literature
Project description:We describe a time-resolved nascent single-cell RNA sequencing (RNA-seq) approach that measures gene-specific transcriptional noise and the fraction of active genes in S. cerevisiae. Most genes are expressed with near-constitutive behavior, while a subset of genes show high mRNA variance suggestive of transcription bursting. Transcriptional noise is highest in the cofactor/coactivator-redundant (CR) gene class (dependent on both SAGA and TFIID) and strongest in TATA-containing CR genes. Using this approach, we also find that histone gene transcription switches from a low-level, low-noise constitutive mode during M and M/G1 to an activated state in S phase that shows both an increase in the fraction of active promoters and a switch to a noisy and bursty transcription mode. Rapid depletion of cofactors SAGA and MED Tail indicates that both factors play an important role in stimulating the fraction of active promoters at CR genes, with a more modest role in transcriptional noise.
| S-EPMC11405135 | biostudies-literature
Project description:Morphological and clinical heterogeneity of advanced colorectal cancer is probably caused by genetic variability in putative cancer stem cell genes, including Lgr5. Here, we investigated 23 variants of the Lgr5 gene in normal tissue, primary tumors, lymph node metastases and distant metastases of stage III and stage IV colorectal cancer patients. These data were compared to results of immunohistochemical Lgr5 expression analysis and to prognostic clinical parameters. No differences were found comparing germline and somatic Lgr5 genotype in primary tumors, but additional Lgr5 gene alterations could be demonstrated in lymph node and distant metastases. Significant negative correlation was seen between Lgr5 allelic variation and Lgr5 protein expression (p=0.0394), which mainly can be attributed to the negative influence of non-coding Lgr5 gene variations on Lgr5 protein expression (p=0.0166). Lgr5 gene variants could be found more frequently in primary tumors of stage III patients with increased time to recurrence, in distant metastases of patients with better survival and in lymph node metastases of patients with poorer survival compared to patients with Lgr5 wild type in primary and metastatic tissues, respectively. However, the analytic power of these prognostic data was low due to small sample size in the investigated groups. In conclusion, our data indicate that Lgr5 allelic variation affect Lgr5 protein expression in colorectal carcinomas. The somatic Lgr5 genotype seems to be relatively stable in primary tumors, but becomes vulnerable during the metastatic process of colorectal cancer. This instability has possibly prognostic importance, which has to be further evaluated by large cohort studies.
| S-EPMC3426388 | biostudies-literature
Project description:The effect of the cellular reprogramming process per se on mutation load remains unclear. To address this issue, we performed whole exome sequencing analysis of induced pluripotent stem cells (iPSCs) reprogrammed from human cord blood (CB) CD34(+) cells. Cells from a single donor and improved lentiviral vectors for high-efficiency (2-14%) reprogramming were used to examine the effects of three different combinations of reprogramming factors: OCT4 and SOX2 (OS), OS and ZSCAN4 (OSZ), OS and MYC and KLF4 (OSMK). Five clones from each group were subject to whole exome sequencing analysis. We identified 14, 11, and 9 single nucleotide variations (SNVs), in exomes, including untranslated regions (UTR), in the five clones of OSMK, OS, and OSZ iPSC lines. Only 8, 7, and 4 of these, respectively, were protein-coding mutations. An average of 1.3 coding mutations per CB iPSC line is remarkably lower than previous studies using fibroblasts and low-efficiency reprogramming approaches. These data demonstrate that point nucleotide mutations during cord blood reprogramming are negligible and that the inclusion of genome stabilizers like ZSCAN4 during reprogramming may further decrease reprogramming-associated mutations. Our findings provide evidence that CB is a superior source of cells for iPSC banking.
| S-EPMC3613421 | biostudies-literature