Establishment of human induced pluripotent stem cell line MURAi001-A from skin fibroblasts of a patient carrying a c.4404A > G mutation in the TET1 gene.

Ten-Eleven Translocation methylcytosine dioxygenase 1 (TET1) is known to play a broad tumor suppressor role through demethylating and activating tumor suppressor genes. TET1 missense mutations are previously reported in many types of leukemia. Here, the human induced pluripotent stem cell line MURAi001-A was generated from skin fibroblasts derived from a 56-year-old female patient carrying the TET1 gene mutation c.4404A > G (p.I1468M), who had a history of ovarian germ cell tumor. The MURAi001-A cell line demonstrated embryonic-like characteristics as it expressed specific stemness markers, differentiated into the three germ layers, and retained normal karyotyping.

Effects of a short abstinence period on sperm quality in oligozoospermic men.

OBJECTIVE: The aim of this study was to compare semen parameters and sperm DNA fragmentation (SDF) and explore the relationship between semen parameters and SDF between 2 and 7 days of abstinence and a short abstinence period (within 4 hours) in oligozoospermic infertile patients. METHODS: Two semen samples were collected from infertile oligozoospermic men (n=34) after an abstinence period of 2 to 7 days and within 4 hours, respectively. Sperm parameters were compared between the two abstinence duration groups, including semen volume, sperm concentration, total sperm count, sperm motility, total motile sperm count (TMSC), morphology, and SDF. RESULTS: The semen volume, concentration, and total sperm count were significantly decreased after 4 hours of abstinence than after 2 to 7 days of abstinence, with median differences of 1.2 mL (p<0.001), 2×106/mL (p=0.011), and 9.6×106/ejaculation (p<0.001), respectively. TMSC was significantly lower after a short abstinence, with a median difference of 4.24×106/ejaculate (p<0.001). However, there were no significance differences in the percentage of motility, the SDF, and the percentage of sperm with normal morphology. Interestingly, volume, concentration, total sperm count, sperm motility, and SDF, but not TMSC, exhibited significant linear correlations between the two abstinence groups in univariate regression analysis, except for TMSC. CONCLUSION: In oligozoospermic men, the volume, concentration, and total sperm count were significantly lower after a short abstinence period, but without adverse effects on sperm motility and SDF.

Induction of fetal hemoglobin: Lentiviral shRNA knockdown of HBS1L in ß0-thalassemia/HbE erythroid cells.

Imbalanced globin chain output contributes to thalassemia pathophysiology. Hence, induction of fetal hemoglobin in ß-thalassemia and other ß-hemoglobinopathies are of continuing interest for therapeutic approaches. Genome-wide association studies have identified three common genetic loci: namely ß-globin (HBB), an intergenic region between MYB and HBS1L, and BCL11A underlying quantitative fetal hemoglobin production. Here, we report that knockdown of HBS1L (all known variants) using shRNA in early erythroblast obtained from ß0-thalassemia/HbE patients triggers an upregulation of γ-globin mRNA 1.69 folds. There is modest perturbation of red cell differentiation assessed by flow cytometry and morphology studies. The levels of α- and ß-globin mRNAs are relatively unaltered. Knockdown of HBS1L also increases the percentage of fetal hemoglobin around 16.7 folds when compared to non-targeting shRNA. Targeting HBS1L is attractive because of the potent induction of fetal hemoglobin and the modest effect on cell differentiation.

In Vitro Study of Ineffective Erythropoiesis in Thalassemia: Diverse Intrinsic Pathophysiological Features of Erythroid Cells Derived from Various Thalassemia Syndromes.

Defective hemoglobin production and ineffective erythropoiesis contribute to the pathophysiology of thalassemia syndromes. Previous studies in the field of erythropoiesis mainly focused on the severe forms of thalassemia, such as ß-thalassemia major, while mechanisms underlying the pathogenesis of other thalassemia syndromes remain largely unexplored. The current study aimed to investigate the intrinsic pathophysiological properties of erythroid cells derived from the most common forms of thalassemia diseases, including α-thalassemia (hemoglobin H and hemoglobin H-Constant Spring diseases) and ß-thalassemia (homozygous ß0-thalassemia and ß0-thalassemia/hemoglobin E diseases), under an identical in vitro erythroid culture system. Cell proliferation capacity, differentiation velocity, cell death, as well as globin synthesis and the expression levels of erythropoiesis modifying factors were determined. Accelerated expansion was found in erythroblast cells derived from all types of thalassemia, with the highest degree in ß0-thalassemia/hemoglobin E. Likewise, all types of thalassemia showed limited erythroid cell differentiation, but each of them manifested varying degrees of erythroid maturation arrest corresponding with the clinical severity. Robust induction of HSP70 transcripts, an erythroid maturation-related factor, was found in both α- and ß-thalassemia erythroid cells. Increased cell death was distinctly present only in homozygous ß0-thalassemia erythroblasts and associated with the up-regulation of pro-apoptotic (Caspase 9, BAD, and MTCH1) genes and down-regulation of the anti-apoptotic BCL-XL gene.

Down-regulation of the transcriptional repressor ZNF802 (JAZF1) reactivates fetal hemoglobin in ß0-thalassemia/HbE.

Reactivating of fetal hemoglobin (HbF; α2γ2) can ameliorate the severity of ß-thalassemia disease by compensating for adult hemoglobin deficiency in patients. Previously, microarray analysis revealed that zinc finger protein (ZNF)802 (also known as Juxta-posed with another zinc finger gene-1 (JAZF1)) was upregulated in human erythroblasts derived from adult peripheral blood compared with fetal liver-derived cells, implying a potential role as a HbF repressor. However, deficiency in ZNF802 induced by lentiviral shRNA in ß0-thalassemia/hemoglobinE erythroblasts had no effect on erythroblast proliferation and differentiation. Remarkably, the induction of HBG expression was observed at the transcriptional and translational levels resulting in an increase of HbF to 35.0 ± 3.5%. Interestingly, the embryonic globin transcripts were also upregulated but the translation of embryonic globin was not detected. These results suggest ZNF802 might be a transcriptional repressor of the γ-globin gene in adult erythroid cells.

Establishment of human induced pluripotent stem cell line MUi028-A from normal fetal skin fibroblasts.

MUi028-A human induced pluripotent stem cell (hiPSC) line was generated from normal fetal skin fibroblasts using a non-integrative reprogramming method. Reprogramming factors OCT4, SOX2, KLF4, L-MYC, and LIN28, and TP53 shRNA in three episomal vectors were delivered by electroporation. The MUi028-A cell line had embryonic stem cell characteristics with consistent expression of specific pluripotency markers and the capability of in vitro differentiation into the three germ layers. This iPSC line can be used as a control to study disease mechanisms.

Association of the Degree of Erythroid Expansion and Maturation Arrest with the Clinical Severity of ß0-Thalassemia/Hemoglobin E Patients.

INTRODUCTION: ß-Thalassemia/hemoglobin E represents one-half of all the clinically severe ß-thalassemias worldwide. Despite similar genetic backgrounds, patients show clinical heterogeneity ranging from nearly asymptomatic to transfusion-dependent thalassemia. The underlying disease modifying factors remain largely obscure. METHODS: To elucidate the correlation between ineffective erythropoiesis and ß0-thalassemia/hemoglobin E (HbE) disease severity, in vitro culture of erythroid cells derived from patients with different clinical symptoms was established. Cell proliferation, viability, and differentiation were investigated. To identify potential molecular mechanisms leading to the arrested erythroid maturation, the expression levels of erythropoiesis modifying factors were measured. RESULTS: The ß0-thalassemia/HbE cells exhibited enhanced proliferation, limited differentiation, and impaired erythroid terminal maturation but did not show accelerated erythroblast differentiation and increased cell death. Erythroblasts derived from mild patients showed the highest proliferation rate with a faster cell division time, while erythroblasts derived from severe patients displayed extremely delayed erythroid maturation. Downregulation of growth differentiation factor 11 and FOXO3a was observed in mild ß0-thalassemia/HbE erythroblasts, while upregulation of heat shock protein 70 and activin receptor 2A was revealed in severe erythroblasts. DISCUSSION/CONCLUSION: The degree of erythroid expansion and maturation arrest contributes to the severity of ß0-thalassemia/HbE patients, accounting for the disease heterogeneity. The findings suggest a restoration of erythroid maturation as a promising targeted therapy for severe patients.

Generation of a human induced pluripotent stem cells (MUi015-A) from skin fibroblast of retinoblastoma patient with 13q deletion syndrome.

The 13q deletion syndrome is a rare chromosomal disorder caused by loss of the long arm of chromosome 13, and usually entails developmental delay, intellectual disability, behavioral problems and distinctive facial features. In this study, we successfully generated a human iPSC line (MUi015-A) from skin fibroblasts of a patient who had large deletion of chromosome 13, del(13)(q14q22). The MUi015-A line exhibited embryonic stem cell characteristics with consistent pluripotency marker expression and the capability of differentiating into three germ layers. The cell line provides a good tool in studying pathophysiology of the tumors, drug testing and gene therapy.