BACKGROUND: This systematic review describes the most common methodologies for immortalizing human and animal mesenchymal stem cells (MSCs). This study follows the rules of PRISMA and is registered in the Institutional Review Board of PROSPERO International of systematic reviews, numbered protocol code: CRD42020202465.
METHODS: The data search systematization was based on the words \"mesenchymal stem cell\" AND \"immortalization.\" The search period for publications was between 2000 and 2022, and the databases used were SCOPUS, PUBMED, and SCIENCE DIRECT. The search strategies identified 384 articles: 229 in the SCOPUS database, 84 in PUBMED, and 71 in SCIENCE DIRECT. After screening by titles and abstracts, 285 articles remained. This review included thirty-nine articles according to the inclusion and exclusion criteria.
RESULTS: In 28 articles, MSCs were immortalized from humans and 11 animals. The most used immortalization methodology was viral transfection. The most common immortalized cell type was the MSC from bone marrow, and the most used gene for immortalizing human and animal MSCs was hTERT (39.3%) and SV40T (54.5%), respectively.
CONCLUSIONS: Also, it was observed that although less than half of the studies performed tumorigenicity assays to validate the immortalized MSCs, other assays, such as qRT-PCR, colony formation in soft agar, karyotype, FISH, and cell proliferation, were performed in most studies on distinct MSC cell passages.

IDH wild-type (wt) grade 2/3 astrocytomas are a heterogenous group of tumors with disparate clinical and molecular profiles. cIMPACT-NOW recommendations incorporated in the new 2021 World Health Organization (WHO) Classification of Central Nervous System (CNS) Tumors urge minimal molecular criteria to identify a subset that has an aggressive clinical course similar to IDH -wt glioblastomas (GBMs). This paper describes the use of a panel of molecular markers to reclassify IDH -wt grade 2/3 diffuse astrocytic gliomas (DAGs) and study median overall survival concerning for to IDH -wt GBMs in the Indian cohort. IDH -wt astrocytic gliomas (grades 2, 3, and 4) confirmed by IDHR132H immunohistochemistry and IDH1/2 gene sequencing, 1p/19q non-codeleted with no H3F3A mutations were included. TERT promoter mutation by Sanger sequencing, epidermal growth factor receptor amplification, and whole chromosome 7 gain and chromosome 10 loss by fluorescence in situ hybridization was assessed and findings correlated with clinical and demographic profiles. The molecular profile of 53 IDH -wt DAGs (grade 2: 31, grade 3: 22) was analyzed. Eleven cases (grade 2: 8, grade 3: 3) (20.75%) were reclassified as IDH -wt GBMs, WHO grade 4 ( TERT promoter mutation in 17%, epidermal growth factor receptor amplification in 5.5%, and whole chromosome 7 gain and chromosome 10 loss in 2%). Molecular GBMs were predominantly frontal (54.5%) with a mean age of 36 years and median overall survival equivalent to IDH -wt GBMs (18 vs. 19 mo; P =0.235). Among grade 2/3 DAGs not harboring these alterations, significantly better survival was observed for grade 2 versus grade 3 DAGs (25 vs. 16 mo; P =0.002). Through the incorporation of a panel of molecular markers, a subset of IDH -wt grade 2 DAGs can be stratified into molecular grade 4 tumors with prognostic and therapeutic implications. However, IDH -wt grade 3 DAGs behave like GBMs irrespective of molecular profile.

Immune checkpoint blockade antibodies are setting a new standard of care for cancer patients. It is therefore important to assess any new immune-based therapies in the context of immune checkpoint blockade. Here, we evaluate the impact of combining a synthetic consensus TERT DNA vaccine that has improved capacity to break tolerance with immune checkpoint inhibitors. We observed that blockade of CTLA-4 or, to a lesser extent, PD-1 synergized with TERT vaccine, generating more robust anti-tumor activity compared to checkpoint alone or vaccine alone. Despite this anti-tumor synergy, none of these immune checkpoint therapies showed improvement in TERT antigen-specific immune responses in tumor-bearing mice. αCTLA-4 therapy enhanced the frequency of T-bet+/CD44+ effector CD8+ T cells within the tumor and decreased the frequency of regulatory T cells within the tumor, but not in peripheral blood. CTLA-4 blockade synergized more than Treg depletion with TERT DNA vaccine, suggesting that the effect of CTLA-4 blockade is more likely due to the expansion of effector T cells in the tumor rather than a reduction in the frequency of Tregs. These results suggest that immune checkpoint inhibitors function to alter the immune regulatory environment to synergize with DNA vaccines, rather than boosting antigen-specific responses at the site of vaccination.

Mutations in the dyskerin gene (DKC1) cause X-linked dyskeratosis congenita (DC), a rare and fatal premature aging syndrome characterized by defective telomere maintenance. Dyskerin is a highly conserved nucleolar protein, and a component of the human telomerase complex that is essential for human telomerase RNA (hTR) stability. However, its regulation remains poorly understood. Here, we report that dyskerin can be modified by small ubiquitin-like modifiers (SUMOs). We find that human DC-causing mutations in highly conserved dyskerin SUMOylation consensus sites lead to impaired hTR accumulation, telomerase activity and telomere maintenance. Finally, we show that modification of dyskerin by SUMOylation is required for its stability. Our findings provide the first evidence that dyskerin stability is regulated by SUMOylation and that mutations altering dyskerin SUMOylation can lead to defects in telomere maintenance that are characteristics of DC.

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