Background Most immune thrombocytopenia (ITP) is sporadic but a positive family history of ITP in some patients suggests that hereditary forms exist. Because of the rarity of familial ITP families available for study and the heterogeneity of sporadic ITP, family linkage analysis or genome-wide association studies are limited. Objectives Based on one ITP pedigree, we try to identify the predisposing gene in familial or sporadic ITP and reveal the way in which it causes thrombocytopenia. Methods Gene expression profiling analysis and whole-exome sequencing were performed on samples from family members with ITP, sporadic ITP cases and healthy individuals. We also evaluated the influence of potential pathogenic mutation on immune function and megakaryocyte apoptosis. Results Whole-exome sequencing identified a potential pathologic p.G76S heterozygous mutation on the TNFRSF13B gene in familial ITP patients. ITP patients harboring the G76S mutation displayed an upregulated cytokine-cytokine receptor interaction' signal, increased serum TNF, IL-17, IFN and BAFF levels, and enhanced binding capacity of APRIL ligand to B cells. Additionally, Epstein-Barr virus (EBV)-transformed B cells with the G76S mutation could induce human megakaryocyte line (Meg-01) apoptosis significantly. Conclusion p.G76S mutation on the TNFRSF13B gene is responsible for ITP, and is a genetic predisposing factor for familial or sporadic ITP.

Purpose To assess the genetic and epigenetic status of parthenogenetic human embryonic stem cells (phESCs). Methods Cytogenetics, X chromosome inactivation (XCI) and gene expression patterns were analyzed in one phESC line (FY-phES-018) that was derived from our laboratory. Results FY-phES-018 cells displayed the classical characteristics of normal hESCs. These cells had a 46, XX karyotype, and no inactive X chromosomes were observed before passage 20. After being cultured long term in vitro, some cells lost one X, and the proportion of cells with only one X gradually increased. At passage 35, almost all the cells displayed a 45, XO karyotype. Interestingly, at passage 45, the recovery of the X-chromosome was observed, and XCI became detectable; the mosaic ratio of 46, XX to 45, XO was 67:33. After passage 60, most cells displayed the 46, XX karyotype again with a mosaic ratio of 97:3. Some aberrant genomic imprinting was also observed in these cells. Conclusions The phESCs line FY-phES-018 is both genetically and epigenetically unstable; therefore, further research is needed before using these cells.

Derivation of embryonic stem cells from patient-specific cloned blastocysts by somatic cell nuclear transfer (SCNT) holds promise for both regenerative medicine and cell-based drug discovery. However, the efficiency of blastocyst formation after human SCNT is very low. The developmental competence of SCNT embryos has been previously demonstrated in several species to be enhanced by treatment with histone deacetylase inhibitors, such as trichostatin A (TSA), to increase histone acetylation. In this study, we report that treatment of SCNT embryos with 5  nM TSA for 10  h following activation incubation increased the developmental competence of human SCNT embryos constructed from β-thalassemia fibroblast cells. The efficiency of blastocyst formation from SCNT human embryos treated with TSA was approximately 2 times greater than that from untreated embryos. Cloned blastocysts were confirmed to be generated through SCNT by DNA and mitochondrial DNA fingerprinting analyses. Further, treatment of SCNT embryos with TSA improved the acetylation of histone H3 at lysine 9 in a manner similar to that observed in in vitro fertilized embryos.

Objective:To investigate the difference of X chromosome inactivation and imprinted gene expression status between the parthenogenetic human embryonic stem cells (phESC) and hESC derived from fertilized embryos. Methods: ①Relative quantitative real-time PCR was used to detect the paternally imprinted gene IGF2R and maternally imprinted gene SNRPN, IGF2 expression levels ② XIST methylation and histone H3 lysine 27 trimethylation (H3K27me3) were used to detected the X chromosome inactivation (XCI) status between the phESC and hESC. Results: ①maternally imprinted gene SNRPN, IGF2 were not expressed in phESC while the paternally imprinted gene IGF2R was expressed with more than 2 fold levels compared to the normal hESCs. ② No expression of XIST gene was detected at passage 35, which indicated that XCI was not initiated in phESC at early passages. After long-term culture, epigenetic status of these cells changed, XIST gene expression could be detected at passage 55 and the expression level was increased after differentiation. ③ DNA methylation status of promoter of XIST and positive H3K27me3 results also confirmed that phESC was initiated XCI. Conclusion:Variation XCI status suggested further investigation is necessary to clarify the epigenetic in phESC in order to ensure the safety of those cells for regenerative medicine.

Poor quality embryos discarded from in vitro fertilization (IVF) laboratories are good sources for deriving human embryonic stem cell (hESC) lines. In this study, 166 poor quality embryos donated from IVF centers on day 3 were cultured in a blastocyst medium for 2 days, and 32 early blastocysts were further cultured in a blastocyst optimum culture medium for additional 2 days so that the inner cell masses (ICMs) could be identified and isolated easily. The ICMs of 17 blastocysts were isolated by a mechanical method, while those of the other 15 blastocysts were isolated by immunosurgery. All isolated ICMs were inoculated onto a feeder layer for subcultivation. The rates of ICM attachment, primary ICM colony formation and the efficiency of hESC derivation were similar between the ICMs isolated by the two methods (P>0.05). As a result, four new hESC lines were established. Three cell lines had normal karyotypes and one had an unbalanced Robertsonian translocation. All cell lines showed normal hESC characteristics and had the differentiation ability. In conclusion, we established a stable and effective method for hESC isolation and culture, and it was confirmed that the mechanical isolation was an effective method to isolate ICMs from poor embryos. These results further indicate that hESC lines can be derived from poor quality embryos discarded by IVF laboratories.

Objective: To investigate different genes expression in human embryonic stem cell (hESC) lines with normal and abnormal karyotype after differentiation. Methods: Relative quantitative real-time PCR was used to detect the temporal expression of gene PGK1, X chromosome tumor suppressor gene RBBP7, oncogene GPC4 as well as paternally imprinted gene H19, IGF2R and maternally imprinted gene SNRPN at different times after spontaneous differentiation in two normal and two abnormal karyotype hESC lines. The expression of OCT4, NANOG was also investigated. Results: PGK1 showed up-regulated expression in all hESCs. ②Compared to the normal hESCs, RBBP7 and GPC4 showed significantly up-regulated expression in the abnormal hESCs.③Similar imprinted gene expression patterns were found in both normal and abnormal hESCs. H19, IGF2R showed up-regulated expression following the time after differentiation while SNRPN showed down-regulated. ④ The expression of OCT4, NANOG was significantly decreased in the normal hESCs after differentiation compared to the abnormal hESCs. Conclusion: The expression patterns of PGK1 and imprinted genes in normal and abnormal hESCs were similar, which indicates that an appropriate order of those genes in hESCs. The stronger self-renewal and weaker differentiation ability and the up-regulated expression levels in oncogene and tumor suppressor gene in abnormal hESCs may be a great hurdle to the development of those cells.

BACKGROUND Human embryonic stem cell (hESC) lines derived from poor quality embryos usually have either normal or abnormal karyotypes. However, it is still unclear whether their biological characteristics are similar. METHODS Seven new hESC lines were established using discarded embryos. Five cell lines had normal karyotype, one was with an unbalanced Robertsonian translocation and one had a triploid karyotype. Their biological characteristics, short tandem repeat loci, HLA typing, differentiation capability and imprinted gene, DNA methylation and X chromosome inactivation status were compared between different cell lines. RESULTS All seven hESC lines had similar biological characteristics regardless of karyotype (five normal and two abnormal), such as expression of stage-specific embryonic antigen (SSEA)-4, tumor-rejection antigen (TRA)-1-81 and TRA-1-60 proteins, transcription factor octamer binding protein 4 mRNA, no detectable expression of SSEA-1 protein and high levels of alkaline phosphatase activity. All cell lines were able to undergo differentiation. Imprinted gene expression and DNA methylation were also similar among these cell lines. Non-random X chromosome inactivation patterns were found in XX cell lines. CONCLUSIONS The present results suggest that hESC lines with abnormal karyotype are also useful experimental materials for cell therapy, developmental biology and genetic research.

In IVF clinics, many poor quality human embryos have been discarded because they showed no survival characteristics at the end of culture and the implantation rate is very low if poor quality embryos are transferred.This study was designed to investigate an effective method for the derivation of human embryonic stem cells (hESC) from poor quality embryos discarded from in vitro fertilization (IVF) laboratories,maintain the hES cell without feeder and serum, minimize exposure to animal cells and proteins.blastocysts used in this study from IVF discarded embryo, ICM isolated by immunosurgery, then placed on mitomycin C-treated MEF feeder layers, after 9 passage, hES cell maintained in conditioned media without Serum,collect conditon medium from human forskins fibroblasts feeder, add 8ng/ml bFGF befoure use.human embryonic stem cell colonies were subsequently characterized by cell surface marker staining, karyotyping and teratoma formation.We derive two new hESC lines form discarded blastocysts,the two cell line can maintained in conditioned medium from human foreskin fibroblasts feeder cell with 20% knockout serum replacement.express several pluripotency markers including alkaline phosphatase, SSEA-4, Tra-1-60, Tra 1-60,Tra 1-81, Oct-4,GAPDH-R, and SOX2-F, remain in undifferentiated state with normal karyotype after prolonged passages and can form embryoid bodies in vitro and teratoma in vivo.

Parthenogenetic embryonic stem cells (PESC) play important roles in the study of gene function and may also be important in the cell therapy.In the present study, one pluripotent human embryonic stem cell (hPESC) line was isolated from parthenogenetic blastocysts.These cells remained undifferentiated state after more than 38 passages and still maintained a normal 46, XX karyotype.The hPESC display a similar morphology as compared with traditional human embryonic stem cells (hESC) derived from fertilized eggs, such as forming colonies with tightly packed cells, prominent nucleoli, and a small cytoplasm to nucleus ratio.The hPESC line expressed traditional hESC markers, such as SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, and did not express SSEA-1.These cells also showed positive expression of transcription factor OCT-4, Nanog, Sox2 and a high level of alkaline phosphatase activity.DNA profiling of hPESC line indicated that it is MHC matched with the oocyte donor.In order to identify the methylation status in undifferentiated hPESC line, DNA methylation pattern of the imprint control (IC) region of the SNRPN-gene was examined, and only methylated maternal SNRPN -gene was detected in this study.Examination of X chromosome inactivation status showed that both X chromosomes are active, indicating that DNA methylation on both X chromosomes is globally reduced in hPESC line.Furthermore, the monoallelic expression of parentally expressed imprinted gene IGF2 and maternally expressed imprinted gene H19 were also analyzed , the results provided the further evidence of the parthenogenetic origin of the hPESC line.In conclusion, we have generated a phESC line and this cell line can be differentiated into various types of cells, which may be of great value in the future application on the treatment of human diseases and for further study of epigenetic mechanisms.

Derivation and characterization of new Chinese human embryonic stem cell lines with normal and abnormal karyotypes

Objective:To establish the optimum method of deriving mouse parthenogenetic embryonic stem cells.Meth- ods:The B6D2F1 hybrid mouse oocytes were parthenogenetic activated with strontium chloride(SrCI2)and cytochalasin B(CB). The parthenogenetic blastocysts and morulas were seeded on MEF feeder respectively to examine the primary ICM appearance and compare the derivation rate.Results:Twelve batches of mouse parthenogenetic embryonic stem cells were established.The estab- lished cells were positive for SSEA-1 and negative for SSEA-4,TRA-1-81,TRA-1-60 markers.They showed a high level of AKP activity,kept the normal Karyotypes,formed both embryoid bodies in vitro and teratomas in vivo.Conclusions:Mouse parthenogenetic embryonic stem cell lines were successfully derived from blastocysts and morulas without zona pellucida.This tech- nology should provide a plateform for deriving human homozygous stem cells that will have a potential value in autologous cell therapy.

OBJECTIVE:To establish a protocol for culturing of human embryonic stem cells (HUES4) without any animal-derived feeder cells and to investigate the karyotype stabilities of HUES4 cells after long-term cultivation.METHODS:HUES4 cells were cultured on mitomycin C treated MEFs or human foreskin fibroblast feeder cells. The pluripotency of the ES cells was analyzed by immunocytochemistry staining to detect the expression of pluripotent marker, karyotype of the ES cells at passage 27, 34, 41, 44 and short tandem repeat (STR) at passage 27 were analyzed.RESULTS:The HUES4 cells cultured on human feeder cells were positive for alkaline phosphatase activity, SSEA-4, TRA-1-60 and TRA-1-81 staining, but negative for SSEA-1. Analysis of karyotype at different passages suggested an abnormal karyotype 46, XY, t(9;15)(q22;q26) mosaicism occurred in HUES4, and the ratios of abnormal increased with passage.CONCLUSION:HUES4 could be cultured without animal-derived feeder cells and the incidence of abnormal karyotype might be increased with long-term culture.

BACKGROUND:Natural antisense transcripts (NATs) are endogenous RNA molecules that exhibit partial or complete complementarity to other RNAs, and that may contribute to the regulation of molecular functions at various levels. In recent years, large-scale NAT screens in several model organisms have produced much data, but there is no database to assemble all these data. AntiCODE intends to function as an integrated NAT database for this purpose.RESULTS:This release of antiCODE contains more than 30,000 non-redundant natural sense-antisense transcript pairs from 12 eukaryotic model organisms. In order to provide an integrated NAT research platform, efficient browser, search and Blast functions have been included to enable users to easily access information through parameters such as species, accession number, overlapping patterns, coding potential etc. In addition to the collected information, antiCODE also introduces a simple classification system to facilitate the study of natural antisense transcripts.CONCLUSION:Though a few similar databases also dealing with NATs have appeared lately, antiCODE is the most comprehensive among these, comprising almost all currently detected NAT pairs.

To increase the efficiency of establishing mouse embryonic stem (ES) cell lines derived from various mouse strains. B6D2F1 (C57BL/6×DBA/2),129/SV×DBA/2,C57BL/6,BALB/C mouse were used in this report,blastocysts were obtained from 3.5 dpc (days post-coition) super ovulated mouse,or harvested morula from 2.5 dpc super ovulated mouse,cultured to blastocysts stage in vitro. Blastocysts transferred into ES culture medium with feeder cells,after 4-5 days of culture,inner cell mass (ICM) were picked up and transferred to the new culture dish with feeder cells,dissociate ICM by 0.05% trypsin after overnight culture,ES cells were passaged every 3-4 days. ES cell lines were established with obserration of phase contrast microscope and identified by karyotype,AKP staining,differentiation ability,SSEA-1,SSEA-4,TRA-1-60,TRA-1-81 immunostaining. 10 ES cell lines were established,each cell line exhibited typical mouse ES cell characteristics. 10 ES cell lines derived from B6D2F1 (C57BL/6×DBA/2),129/SV×DBA/2,C57BL/6,BALB/C mouse strains were established. Dissociating of ICM after overnight culture might be beneficial for establishment of mouse ES cell lines.

On four proteins interaction datasets, including Vazquez dataset, YP dataset, DIP‐core dataset, and SPK dataset, MFGO was tested and compared with the popular MR (majority rule) and GOM methods. Experimental results confirm MFGO's improvement on both speed and accuracy. Especially, MFGO method has a distinctive advantage in accurately predicting functions for proteins with few neighbors. Moreover, the robustness of the approach was validated both in a dataset containing a high percentage of unknown proteins and a disturbed dataset through random insertion and deletion. The analysis shows that a moderate amount of misplaced interactions do not preclude a reliable function assignment.