Pluripotent stem cells (PSCs) have the potential to produce any types of cells from all three basic germ layers and the capacity to self-renew and proliferate indefinitely in vitro. The two main types of PSCs, embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), share common features such as colony morphology, high expression of Oct4 and Nanog, and strong alkaline phosphatase activity. In recent years, increasing evidences suggest that telomere length represents another important internal factor in maintaining stem cell pluripotency. Telomere length homeostasis and its structural integrity help to protect chromosome ends from recombination, end fusion, and DNA damage responses, ensuring the divisional ability of mammalian cells. PSCs generally exhibit high telomerase activity to maintain their extremely long and stable telomeres, and emerging data indicate the alternative lengthening of telomeres (ALT) pathway may play an important role in telomere functions too. Such characteristics are likely key to their abilities to differentiate into diverse cell types in vivo. In this review, we will focus on the function and regulation of telomeres in ESCs and iPSCs, thereby shedding light on the importance of telomere length to pluripotency and the mechanisms that regulate telomeres in PSCs.
Animals ; Humans ; Models, Biological ; Pluripotent Stem Cells ; metabolism ; Telomerase ; metabolism ; Telomere ; metabolism ; Telomere Homeostasis

Objective:To investigate the clinical features and therapeutic efficacy of patients with hereditary leiomyomatosis and renal cell carcinoma(RCC) syndrome-associated RCC (HLRCC-RCC) in China.Methods:The clinical data of 119 HLRCC-RCC patients with fumarate hydratase (FH) germline mutation confirmed by genetic diagnosis from 15 medical centers nationwide from January 2008 to December 2021 were retrospectively analyzed. Among them, 73 were male and 46 were female. The median age was 38(13, 74) years. The median tumor diameter was 6.5 (1.0, 20.5) cm. There were 38 cases (31.9%) in stage Ⅰ-Ⅱand 81 cases (68.1%) in stage Ⅲ-Ⅳ. In this group, only 11 of 119 HLRCC-RCC patients presented with skin smooth muscle tumors, and 44 of 46 female HLRCC-RCC patients had a history of uterine fibroids. The pathological characteristics, treatment methods, prognosis and survival of the patients were summarized.Results:A total of 86 patients underwent surgical treatment, including 70 cases of radical nephrectomy, 5 cases of partial nephrectomy, and 11 cases of reductive nephrectomy. The other 33 patients with newly diagnosed metastasis underwent renal puncture biopsy. The results of genetic testing showed that 94 patients had FH gene point mutation, 18 had FH gene insertion/deletion mutation, 4 had FH gene splicing mutation, 2 had FH gene large fragment deletion and 1 had FH gene copy number mutation. Immunohistochemical staining showed strong 2-succinocysteine (2-SC) positive and FH negative in 113 patients. A total of 102 patients received systematic treatment, including 44 newly diagnosed patients with metastasis and 58 patients with postoperative metastasis. Among them, 33 patients were treated with tyrosine kinase inhibitor (TKI) combined with immune checkpoint inhibitor (ICI), 8 patients were treated with bevacizumab combined with erlotinib, and 61 patients were treated with TKI monotherapy. Survival analysis showed that the median progression-free survival (PFS) of TKI combined with ICI was 18 (5, 38) months, and the median overall survival (OS) was not reached. The median PFS and OS were 12 (5, 14) months and 30 (10, 32) months in the bevacizumab combined with erlotinib treatment group, respectively. The median PFS and OS were 10 (3, 64) months and 44 (10, 74) months in the TKI monotherapy group, respectively. PFS ( P=0.009) and OS ( P=0.006) in TKI combined with ICI group were better than those in bevacizumab combined with erlotinib group. The median PFS ( P=0.003) and median OS ( P=0.028) in TKI combined with ICI group were better than those in TKI monotherapy group. Conclusions:HLRCC-RCC is rare but has a high degree of malignancy, poor prognosis and familial genetic characteristics. Immunohistochemical staining with strong positive 2-SC and negative FH can provide an important basis for clinical diagnosis. Genetic detection of FH gene germ line mutation can confirm the diagnosis. The preliminary study results confirmed that TKI combined with ICI had a good clinical effect, but it needs to be confirmed by the results of a large sample multi-center randomized controlled clinical study.

Clustered Regularly Interspaced Short Palindromic Repeats/genetics* ; CRISPR-Cas Systems/genetics* ; Gene Editing

Animals ; Archaeal Proteins ; genetics ; metabolism ; Deoxyribonuclease I ; genetics ; metabolism ; Gene Editing ; methods ; Humans ; Natronobacterium ; enzymology ; genetics

In mammalian cells, long noncoding RNAs (lncRNAs) form complexes with proteins to execute various biological functions such as gene transcription, RNA processing and other signaling activities. However, methods to track endogenous lncRNA dynamics in live cells and screen for lncRNA interacting proteins are limited. Here, we report the development of CERTIS (CRISPR-mediated Endogenous lncRNA Tracking and Immunoprecipitation System) to visualize and isolate endogenous lncRNA, by precisely inserting a 24-repeat MS2 tag into the distal end of lncRNA locus through the CRISPR/Cas9 technology. In this study, we show that CERTIS effectively labeled the paraspeckle lncRNA NEAT1 without disturbing its physiological properties and could monitor the endogenous expression variation of NEAT1. In addition, CERTIS displayed superior performance on both short- and long-term tracking of NEAT1 dynamics in live cells. We found that NEAT1 and paraspeckles were sensitive to topoisomerase I specific inhibitors. Moreover, RNA Immunoprecipitation (RIP) of the MS2-tagged NEAT1 lncRNA successfully revealed several new protein components of paraspeckle. Our results support CERTIS as a tool suitable to track both spatial and temporal lncRNA regulation in live cells as well as study the lncRNA-protein interactomes.

The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system has been widely used for genome engineering and transcriptional regulation in many different organisms. Current CRISPR-activation (CRISPRa) platforms often require multiple components because of inefficient transcriptional activation. Here, we fused different phase-separation proteins to dCas9-VPR (dCas9-VP64-P65-RTA) and observed robust increases in transcriptional activation efficiency. Notably, human NUP98 (nucleoporin 98) and FUS (fused in sarcoma) IDR domains were best at enhancing dCas9-VPR activity, with dCas9-VPR-FUS IDR (VPRF) outperforming the other CRISPRa systems tested in this study in both activation efficiency and system simplicity. dCas9-VPRF overcomes the target strand bias and widens gRNA designing windows without affecting the off-target effect of dCas9-VPR. These findings demonstrate the feasibility of using phase-separation proteins to assist in the regulation of gene expression and support the broad appeal of the dCas9-VPRF system in basic and clinical applications.
Humans ; Transcriptional Activation ; RNA, Guide, CRISPR-Cas Systems ; Gene Expression Regulation ; CRISPR-Cas Systems/genetics*

β-Thalassemia is a global health issue, caused by mutations in the HBB gene. Among these mutations, HBB -28 (A>G) mutations is one of the three most common mutations in China and Southeast Asia patients with β-thalassemia. Correcting this mutation in human embryos may prevent the disease being passed onto future generations and cure anemia. Here we report the first study using base editor (BE) system to correct disease mutant in human embryos. Firstly, we produced a 293T cell line with an exogenous HBB -28 (A>G) mutant fragment for gRNAs and targeting efficiency evaluation. Then we collected primary skin fibroblast cells from a β-thalassemia patient with HBB -28 (A>G) homozygous mutation. Data showed that base editor could precisely correct HBB -28 (A>G) mutation in the patient's primary cells. To model homozygous mutation disease embryos, we constructed nuclear transfer embryos by fusing the lymphocyte or skin fibroblast cells with enucleated in vitro matured (IVM) oocytes. Notably, the gene correction efficiency was over 23.0% in these embryos by base editor. Although these embryos were still mosaic, the percentage of repaired blastomeres was over 20.0%. In addition, we found that base editor variants, with narrowed deamination window, could promote G-to-A conversion at HBB -28 site precisely in human embryos. Collectively, this study demonstrated the feasibility of curing genetic disease in human somatic cells and embryos by base editor system.
APOBEC-1 Deaminase ; genetics ; metabolism ; Base Sequence ; Blastomeres ; cytology ; metabolism ; CRISPR-Cas Systems ; Embryo, Mammalian ; metabolism ; pathology ; Female ; Fibroblasts ; metabolism ; pathology ; Gene Editing ; methods ; Gene Expression ; HEK293 Cells ; Heterozygote ; Homozygote ; Humans ; Point Mutation ; Primary Cell Culture ; Promoter Regions, Genetic ; Sequence Analysis, DNA ; beta-Globins ; genetics ; metabolism ; beta-Thalassemia ; genetics ; metabolism ; pathology ; therapy