facilitating-cerebral-organoid-culture-via-lateral-soft-light

Facilitating Cerebral Organoid Culture <em>via</em> Lateral Soft Light Illumination

At present, cerebral organoid culture technology is still complicated to operate and difficult to apply on a large scale. It is necessary to find a simple ...

The Third Affiliated Hospital of Guangzhou Medical University

Key Laboratory of Reproduction and Genetics of Guangdong Higher Education Institutes

Haemoglobin (Hb) H-constant spring (CS) alpha thalassaemia (- -/-α) is the most common type of nondeletional Hb H disease in southern China. The CRISPR/Cas9-based gene correction of patient-specific induced pluripotent stem cells (iPSCs) and cell transplantation now represent a therapeutic solution for this genetic disease. We designed primers for the target sites using CRISPR/Cas9 to specifically edit the HBA2 gene with an Hb-CS mutation. After applying a correction-specific PCR assay to purify the corrected clones followed by sequencing to confirm the mutation correction, we verified that the purified clones retained full pluripotency and exhibited a normal karyotype. This strategy may be promising in the future, although it is far from representing a solution for the treatment of HbH-CS thalassemia now.

CRISPR/Cas9 gene correction of HbH-CS thalassemia-induced pluripotent stem cells.

β-thalassaemia is a prevalent hereditary haematological disease caused by mutations in the human haemoglobin β (HBB) gene. Among them, the HBB IVS2-654 (C > T) mutation, which is in the intron, creates an aberrant splicing site. Bone marrow transplantation for curing β-thalassaemia is limited due to the lack of matched donors. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), as a widely used tool for gene editing, is able to target specific sequence and create double-strand break (DSB), which can be combined with the single-stranded oligodeoxynucleotide (ssODN) to correct mutations. In this study, according to two different strategies, the HBB IVS2-654 mutation was seamlessly corrected in iPSCs by CRISPR/Cas9 system and ssODN. To reduce the occurrence of secondary cleavage, a more efficient strategy was adopted. The corrected iPSCs kept pluripotency and genome stability. Moreover, they could differentiate normally. Through CRISPR/Cas9 system and ssODN, our study provides improved strategies for gene correction of β-Thalassaemia, and the expression of the HBB gene can be restored, which can be used for gene therapy in the future.

Efficient gene correction of an aberrant splice site in β-thalassaemia iPSCs by CRISPR/Cas9 and single-strand oligodeoxynucleotides.

Myoclonus Epilepsy and Ataxia due to Potassium channel mutation (MEAK) is a rare epilepsy caused by changes in the structure and function of potassium channels due to mutations in the potassium voltage-gated channel subfamily C member 1 (KCNC1) gene. MEAK is one of the progressive myoclonus epilepsy (PME), and there are few studies on MEAK pathogenesis and targeted drugs. Here, we used peripheral blood from MEAK patients with KCNC1 (c.959G > A) gene mutation to establish induced pluripotent stem cells (iPSC). The iPSC of KCNC1 mutation established by us is a powerful tool for related research.

Generation of induced pluripotent stem cell GZHMCi001-A and GZHMCi001-B derived from peripheral blood mononuclear cells of epileptic patients with KCNC1 mutation.

Apolipoprotein (apo) B is a large, amphipathic glycoprotein which plays an important role in human lipoprotein metabolism. The 43-kb APOB gene located on the short arm of human chromosome 2 and consisted of 29 exons, mutations in the APOB gene can give rise to either hypo- or hypercholesterolemia. We used peripheral blood mononuclear cells (PBMCs) from a volunteer carrying the APOB mutation (c.10579C>T, p.Arg3527Trp) located in exon 9 to establish induced pluripotent stem cells (iPSC), which will be an effective means to reveal the key biologically relevant metabolic mechanisms, a powerful tool for medicine selection and related research.

Generation of induced pluripotent stem cell GZHMCi002-A from peripheral blood mononuclear cells with APOB mutation.

β-thalassemia is a type of inherited hemolytic anemia caused by decreased globin production due to defect of the HBB gene. The pathogenesis of the disease is imbalance of α/β globin chains. The excess of α-globin chains will form hemichromes which can damage red blood cell membranes and lead to hemolysis, ineffective erythropoiesis, and secondary iron overload. Iron overload in turn can cause complications such as growth retardation, liver cirrhosis, cardiac insufficiency, and aggravate the disease phenotype. In recent decades, genes participating in iron metabolism have been discovered, and the mechanism of iron metabolism in the development of thalassemia has gradually been elucidated. Subsequently, by manipulating the expression of key genes in iron metabolism such as hepcidin and transferrin receptor, researchers have revealed that iron restriction can improve ineffective hematopoiesis and iron overload, which may provide a potential approach for the treatment of thalassemia. This article reviews the progress of research on iron metabolism-related genes and related pathways in β-thalassemia.

[Research progress of iron metabolism in phenotype modification of β-thalassemia].

The AU-rich element (ARE)-binding factor 1 (AUF1) acts as a switch for septic shock, although its underlying mechanisms remain largely unknown. In this study, we examined the biological significance and potential molecular mechanism of AUF1 in regulating ferroptosis in sepsis-induced acute lung injury (ALI).

Article	Year
CRISPR/Cas9 gene correction of HbH-CS thalassemia-induced pluripotent stem cells. Annals of hematology\| PubMed ID: 31495903	2019
Efficient gene correction of an aberrant splice site in β-thalassaemia iPSCs by CRISPR/Cas9 and single-strand oligodeoxynucleotides. Journal of cellular and molecular medicine\| PubMed ID: 31631510	2019
Generation of induced pluripotent stem cell GZHMCi001-A and GZHMCi001-B derived from peripheral blood mononuclear cells of epileptic patients with KCNC1 mutation. Stem cell research\| PubMed ID: 32712483	2020
Generation of induced pluripotent stem cell GZHMCi002-A from peripheral blood mononuclear cells with APOB mutation. Stem cell research\| PubMed ID: 33128957	2020
[Research progress of iron metabolism in phenotype modification of β-thalassemia]. Zhonghua yi xue yi chuan xue za zhi = Zhonghua yixue yichuanxue zazhi = Chinese journal of medical genetics\| PubMed ID: 33423253	2021
AUF1 protects against ferroptosis to alleviate sepsis-induced acute lung injury by regulating NRF2 and ATF3. Cellular and molecular life sciences : CMLS\| PubMed ID: 35391558	2022

Diyu Chen

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