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A Do-It-Yourself and 3D-Printed Time-Lapse Imaging Platform for In-Incubator Live Cell Observation

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The Third Affiliated Hospital of Guangzhou Medical University

Key Laboratory of Reproduction and Genetics of Guangdong Higher Education Institutes

Guangzhou Medical University

Southern Medical University

Guangzhou Weike Zexing Instrument Technology Co., Ltd.

Targeted disruption of Cripto-1 in mice caused embryonic lethality at E7.5, whereas we unexpectedly found that ectopic Cripto-1 expression in mouse embryos also led to embryonic lethality, which prompted us to characterize the causes and mechanisms underlying embryonic death due to ectopic Cripto-1 expression. RCLG/EIIa-Cre embryos displayed complex phenotypes between embryonic day 14.5 (E14.5) and E17.5, including fatal hemorrhages (E14.5-E15.5), embryo resorption (E14.5-E17.5), pale body surface (E14.5-E16.5) and no abnormal appearance (E14.5-E16.5). Macroscopic and histological examination revealed that ectopic expression of Cripto-1 transgene in RCLG/EIIa-Cre embryos resulted in lethal cardiac defects, as evidenced by cardiac malformations, myocardial thinning, failed assembly of striated myofibrils and lack of heartbeat. In addition, Cripto-1 transgene activation beginning after E8.5 also caused the aforementioned lethal cardiac defects in mouse embryos. Furthermore, ectopic Cripto-1 expression in embryonic hearts reduced the expression of cardiac transcription factors, which is at least partially responsible for the aforementioned lethal cardiac defects. Our results suggest that hemorrhages and cardiac abnormalities are two important lethal factors in Cripto-1 transgenic mice. Taken together, these findings are the first to demonstrate that sustained Cripto-1 transgene expression after E11.5 causes fatal hemorrhages and lethal cardiac defects, leading to embryonic death at E14.5-17.5.

Ectopic expression of Cripto-1 in transgenic mouse embryos causes hemorrhages, fatal cardiac defects and embryonic lethality.

Hemophilia A (HA), is a X-linked recessive congenital bleeding disorder, caused by deficiency of the coagulation factorVIII (FVIII) which is encoded by coagulation factor 8 (F8). HA affects 1 of every 5,000 males worldwide. The intron 22 inversion (Inv22) mutation of F8 causes about 45% of severeHA cases.Here, we generated induced pluripotent stem cells (iPSCs) from a HA patient with Inv22 mutation by electroporation of urine-derived cells (UCs) with episomal plasmids under feeder-free, virus-free, serum-free condition and without oncogene c-MYC. This iPSCs line could facilitate future applications of human iPSCs by provide a valuable cell model.

Generation of induced pluripotent stem cell GZLSL-i001-A derived from urine-derived cells of Hemophilia A patient with Inv22 mutation.

β-thalassemia, an autosomal recessive blood disorder that reduces the production of hemoglobin, is majorly caused by the point mutation of the HBB gene resulting in reduced or absent β-globin chains of the hemoglobin tetramer. Animal models recapitulating both the phenotype and genotype of human disease are valuable in the exploration of pathophysiology and for in vivo evaluation of novel therapeutic treatments. The docile temperament, short vital cycles, and low cost of rabbits make them an attractive animal model. However, β-thalassemia rabbit models are currently unavailable. Here, using CRISPR/Cas9-mediated genome editing, we point mutated the rabbit β-globin gene HBB2 with high efficiency and generated a β-thalassemia rabbit model. Hematological and histological analyses demonstrated that the genotypic mosaic F0 displayed a mild phenotype of anemia, and the heterozygous F1 exhibited typical characteristics of β-thalassemia. Whole-blood transcriptome analysis revealed that the gene expression was altered in HBB2-targeted when compared with WT rabbits. And the highly expressed genes in HBB2-targeted rabbits were enriched in lipid and iron metabolism, innate immunity, and hematopoietic processes. In conclusion, using CRISPR-mediated HBB2 knockout, we have created a β-thalassemia rabbit model that accurately recapitulates the human disease phenotype. We believe this tool will be valuable in advancing the investigation of pathogenesis and novel therapeutic targets of β-thalassemia and associated complications.

CRISPR/Cas9-mediated β-globin gene knockout in rabbits recapitulates human β-thalassemia.

Spinal cerebellar ataxia type 3 (SCA3), also known as Machado-Joseph disease, is the result of abnormal repeat amplification of CAG of the ATXN3 gene. It is one of the main types of autosomal dominant ataxia, with motor symptoms of cerebellar ataxia, mainly accompanied by non-motor symptoms, such as ocular symptoms, psychiatric symptoms, and nutritional disorders. Currently, no effective treatment is available for patients with SCA3. The construction of induced pluripotent stem cells (iPSCs) from two SCA3 patients (14/74 CAG repeats) will be an excellent tool for studying SCA3 disease mechanisms and for drug screening.

Generation of two induced pluripotent stem cell lines, GZHMCi009-A and GZHMCi010-A, derived from peripheral blood mononuclear cells of two SCA3 patients with 14/74 CAG repeats of the ATXN3 mutation.

Reactivation of γ-globin expression is a promising therapeutic approach for β-hemoglobinopathies. Here, we propose a novel Cas9/AAV6-mediated genome editing strategy for the treatment of β-thalassemia: Natural HPFH mutations -113A > G, -114C > T, -117G>A, -175T > C, -195C > G, and -198T > C were introduced by homologous recombination following disruption of BCL11A binding sites in  promoters. Precise on-target editing and significantly increased γ-globin expression during erythroid differentiation were observed in both HUDEP-2 cells and primary HSPCs from β-thalassemia major patients. Moreover, edited HSPCs maintained the capacity for long-term hematopoietic reconstitution in B-NDG hTHPO mice. This study provides evidence of the effectiveness of introducing naturally occurring HPFH mutations as a genetic therapy for β-thalassemia.

Induction of Fetal Hemoglobin by Introducing Natural Hereditary Persistence of Fetal Hemoglobin Mutations in the γ-Globin Gene Promoters for Genome Editing Therapies for β-Thalassemia.

Bisphenol A (BPA) is a well-known environmental endocrine disruptor that has detrimental effects on reproduction. This study aimed to investigate whether BPA exposure could disrupt the function of cumulus cells and influence oocyte maturation and development. Porcine oocytes at the germinal vesicle stage were exposed to BPA for 44 h. The results revealed that BPA exposure led to dysfunction in cumulus cells by inhibiting meiotic division, inducing endoplasmic reticulum stress, and disrupting steroid synthesis. Furthermore, BPA exposure significantly increased reactive oxygen species and caused abnormal distribution of mitochondria in the oocytes. Notably, matured oocytes in the MII stage from the BPA-exposed groups showed significantly reduced development to the blastocyst stage, along with increased autophagy and apoptosis. These findings suggest that cumulus-oocyte complexes are sensitive to BPA exposure during the germinal vesicle stage, and the toxic effects of BPA on cumulus cells can severely inhibit oocyte and parthenogenetic embryos development.

ArticleTotal : 3	Year
Publication title	2022
Publication title	2024
Publication title	2026

Article	Year
Ectopic expression of Cripto-1 in transgenic mouse embryos causes hemorrhages, fatal cardiac defects and embryonic lethality. Scientific reports\| PubMed ID: 27687577	2016
Generation of induced pluripotent stem cell GZLSL-i001-A derived from urine-derived cells of Hemophilia A patient with Inv22 mutation. Stem cell research\| PubMed ID: 33212352	2020
CRISPR/Cas9-mediated β-globin gene knockout in rabbits recapitulates human β-thalassemia. The Journal of biological chemistry\| PubMed ID: 33639162	2021
Generation of two induced pluripotent stem cell lines, GZHMCi009-A and GZHMCi010-A, derived from peripheral blood mononuclear cells of two SCA3 patients with 14/74 CAG repeats of the ATXN3 m... Stem cell research\| PubMed ID: 35421843	2022
Induction of Fetal Hemoglobin by Introducing Natural Hereditary Persistence of Fetal Hemoglobin Mutations in the γ-Globin Gene Promoters for Genome Editing Therapies for β-Thalassemia. Frontiers in genetics\| PubMed ID: 35656314	2022
Bisphenol A impairs oocyte maturation by dysfunction of cumulus cells. Theriogenology\| PubMed ID: 39615448	2025

Bangzhu Chen

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