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.

Asparagine synthetase (ASNS) deficiency (ASNSD; MIM #615574) is a rare neurodevelopmental disorder caused by mutations in the ASNS gene. The ASNS gene maps to cytogenetic band 7q21.3 and is 35 kb long. ASNSD is characterised by congenital microcephaly, severely delayed psychomotor development, seizures, and hyperekplexic activity. Here, we reported a family with compound heterozygous mutations in ASNS (NM_001178076:c.551C>T; c. 944A>C) and established induced pluripotent stem cells (iPSCs) from blood samples. To date, limited functional data have been reported to explain the underlying pathophysiology of ASNSD; therefore, iPSCs from these patients may be powerful tools for studying disease mechanisms.

Generation of four induced pluripotent stem cell lines, GZWWTi001-A, GZWTZi001-A, GZWXYi001-A, and GZWXDi001-A, derived from peripheral blood mononuclear cells from a family with asparagine synthetase deficiency.

MSCs are kind of cultured cells that reside in different tissues as inducers or regulators of physiological and pathological processes. Here, we derived MSCs from amniotic fluid and compared their differentiation ability and immunosuppression effect on PHA-activated PBMC with those of MSCs isolated from umbilical cords. Amniotic fluid MSCs were isolated and cultured on commercial AFC medium and classic MSC medium, and the number and size of colonies were used to evaluate differences in primary and passaged culture. Rate of proliferation, population doubling time, cell morphology, cell surface markers and mRNA expression were measured in subcultured cells. Furthermore, a comparative study was performed with umbilical cord MSCs to assess the ability of differentiation and immunosuppressive effect of PHA-stimulated PBMCs. Amniotic fluid MSCs were isolated and expanded by three methods, and exhibited nearly all the characteristics of umbilical cord MSCs. Compared with umbilical cord MSCs, amniotic fluid MSCs had an enhanced osteogenic and chrondrogenic differentiation capability, and stronger immunosuppression effect of inhibition of PHA-activated PBMC division. Culture with commercial AFCs medium yielded the highest percentage of CD105 expression and showed some advantages in primary cell isolation, cell source-specific marker retention and cell proliferation. We demonstrated that amniotic fluid MSCs exhibited some advantages over umbilical cord MSCs, and different culture media caused cell proliferation, cell surface marker and cell morphology change, but were not associated with varying differentiation capability and immune effects.

Different culture method changing CD105 expression in amniotic fluid MSCs without affecting differentiation ability or immune function.

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.

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.

Cases in which the duplication of chromosome 8p (dup 8p) is observed are characterized by facial dysmorphism, agenesis/hypoplasia of the corpus callosum, heart defects and severe mental retardation. The frequency of dup 8p cases is higher compared to other chromosomes because of the Non-allelic homologous recombination (NAHR) between two segmental duplication regions (SDs) containing olfactory receptor gene clusters, REPD (repeat-distal) and REPP (repeat-proximal), located in chromosome 8p23.1. Here we generated a human iPSC line from a patient's amniotic fluid cells with a 18 Mb duplication in 8p23.3p22, which will serve as useful tools for studying dup 8p syndrome.

Generation of induced pluripotent stem cell GZHMCi005-A from amniotic fluid-derived cells with duplication of chromosome 8p.

The deletions of the long arm of chromosome 14 involving the 14q24-q32 region have been identified as deletion 14q (del 14q) syndrome, but were rarely reported. The patients with del 14q syndrome are observed a peculiar facial appearance and neurological defects, but the molecular mechanisms were not clear. Here we generated a human iPSC line from the patient's amniotic fluid cells with 24 Mb deletion in 14q24.3q32.31 which will serve as useful tools for studying the mechanism of del 14q syndrome and the genes involved, which will provide useful basic theory of prenatal diagnosis.

Generation of induced pluripotent stem cell line GZHMCi006-A from amniotic fluid-derived cells with deletion 14q syndrome.

The quality of the early embryo is vital to embryonic development and implantation. As a highly conserved serine/threonine kinase, p21-activated kinase 2 (Pak2) participates in diverse biologic processes, especially in cytoskeleton remodeling and cell apoptosis. In mice, Pak2 knock out and endothelial depletion of Pak2 showed embryonic lethality. However, the role of Pak2 in preimplantation embryos remains unelucidated.

Article	Year
CRISPR/Cas9 gene correction of HbH-CS thalassemia-induced pluripotent stem cells. Annals of hematology\| PubMed ID: 31495903	2019
Generation of four induced pluripotent stem cell lines, GZWWTi001-A, GZWTZi001-A, GZWXYi001-A, and GZWXDi001-A, derived from peripheral blood mononuclear cells from a family with asparagine ... Stem cell research\| PubMed ID: 31698190	2019
Different culture method changing CD105 expression in amniotic fluid MSCs without affecting differentiation ability or immune function. Journal of cellular and molecular medicine\| PubMed ID: 32119193	2020
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
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
Generation of induced pluripotent stem cell GZHMCi005-A from amniotic fluid-derived cells with duplication of chromosome 8p. Stem cell research\| PubMed ID: 33588214	2021
Generation of induced pluripotent stem cell line GZHMCi006-A from amniotic fluid-derived cells with deletion 14q syndrome. Stem cell research\| PubMed ID: 33894549	2021
Pak2 reduction induces a failure of early embryonic development in mice. Reproductive biology and endocrinology : RB&E\| PubMed ID: 34879863	2021

Nengqing Liu

Biography

JoVE Journal Publications

Other Publications