Optimization of seeding density of OP9 cells to improve hematopoietic differentiation efficiency.

BACKGROUND: OP9 mouse stromal cell line has been widely used to induce differentiation of human embryonic stem cells (hESCs) into hematopoietic stem/progenitor cells (HSPCs). However, the whole co-culture procedure usually needs 14-18 days, including preparing OP9 cells at least 4 days. Therefore, the inefficient differentiation system is not appreciated. We aimed to optimize the culture conditions to improve differentiation efficiency. METHODS: In the experimental group, we set six different densities of OP9 cells and just cultured them for 24 h before co-culture, and in the control group, OP9 cells were cultured for 4 days to reach an overgrown state before co-culture. Then we compared the hematopoietic differentiation efficiency among them. RESULTS: OP9 cells were randomly assigned into two groups. In the experimental group, six different plated numbers of OP9 cells were cultured for 1 day before co-culture with hESCs. In contrast, in the control group, OP9 cells were cultured for 4 days at a total number of 3.1 × 104 cells/cm2 in a 6-well plate to reach an overgrown state before co-culture. Hematopoietic differentiation was evaluated with CD34 immunostaining, and compared between these two groups. We could not influence the differentiation efficiency of OP9 cells with a total number of 10.4 × 104 cells/cm2 in a 6-well plate which was cultured just for 1 day, followed by co-culture with hESCs. It reached the same differentiation efficiency 5 days earlier than the control group. CONCLUSION: The peak of CD34 + cells appeared 2 days earlier compared to the control group. A total number of 1.0 × 106 cells in a 6-well plate for OP9 cells was appropriate to have high differentiation efficiency.

MIR22HG Regulates the Proliferation, Epithelial-Mesenchymal Transition, and Apoptosis in Colorectal Carcinoma.

Background: Recent investigations have suggested that long noncoding RNA (lncRNA) MIR22HG is commonly dysregulated in multiple types of malignancies. Nevertheless, the role of these MIR22HG in human colorectal carcinoma (CRC) are not well explored. Materials and Methods: Quantitative real-time polymerase chain reaction (qPCR) and in situ hybridization (ISH) assay were used to measure the expression of MIR22HG. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), flow cytometry, and migration, as well as invasion assays, were utilized to determine the roles of MIR22HG on growth, apoptosis, migration, and invasiveness of CRC cell. The expression of E-cadherin and N-cadherin was measured using Western blotting and immunohistochemistry staining assay. CRC cell growth in vivo was analyzed using nude mice xenograft. Results: The qPCR and ISH assay revealed that MIR22HG was downregulated in CRC sample compared with in normal tissue. MIR22HG was also significantly downexpressed in CRC cells compared with that in normal colonic epithelial cell line. Overexpression of MIR22HG inhibited the growth, migration ability, and invasiveness of CRC cell in vitro. In addition, MIR22HG suppressed the epithelial-mesenchymal transition (EMT) and induced the apoptosis of human CRC cell. Moreover, the authors demonstrated that MIR22HG inhibited the tumor growth of CRC cell and regulated the expression of EMT markers (E-cadherin and N-cadherin) in vivo. Conclusion: Altogether, these results imply that lncRNA MIR22HG restrained the aggressive phenotypes of CRC cell.

Clinical application of oligo array-CGH for detecting balanced translocations in preimplantation genetic diagnosis.

Array comparative genomic hybridization (array-CGH), which facilitates to detect unbalanced reciprocal translocation and allows screening aneuploidy for chromosomes, has been repeatedly verified to be valid for diagnosis of translocations in preimplantation human embryos. Currently, the main microarrays used for CGH are bacterial artificial chromosome (BAC)-based arrays. Compared with the BAC-based arrays, oligonucleotide (oligo)-based arrays have a relatively higher resolution and optimal coverage particularly in the subtelomeric regions. Herein, we described the clinical application of a newly designed oligo-based array by Agilent in preimplantation genetic diagnosis (PGD) and aneuploidy screening for balanced translocations. In the study, a total of 144 embryos from 9 couples carrying Robertsonian translocations and 5 carrying reciprocal translocations were biopsied on day 3 for array-CGH analysis. Overall, 135 (93.8%) embryos were successfully diagnosed to be free of either aneuploidies or unbalanced fragments. However, the remained 9 (6.2%) embryos failed to be amplified due to failed cell lysis, DNA damage or the absence of nuclei in the biopsied cells. Collectively, 23 embryos were identified as "euploid and balanced" and suitable to be transferred. Finally, 9 embryos of satisfactory quality were transferred to 6 women, among which 4 recipients exhibited positive hCG level. Fortunately, one recipient with positive hCG level has delivered one baby, and two pregnancies were continuing. Our study served as the first clinical application of oligo-based array CGH technology in PGD for both reciprocal and Robertsonian translocations concomitant with comprehensive aneuploidy screening.

Generation of site-specific mutant mice using the CRISPR/Cas9 system.

The CRISPR/Cas9 system is a recently developed important technology for genome editing in cellular and animal models. Here we established a CRISPR/Cas9-based system of generating site-specific mutant mice using DNA double-strand breaks (DSBs) induced homologous recombination (HR)-dependent or independent repair mechanism. Through co-microinjection of Cas9 mRNA and single-guide RNA (sgRNA) targeting genomic DNA sequence corresponding to enzyme activity of lysine (K)-specific demethylase 2b (Kdm2b), both a frame-shifted Kdm2b null mutant and a Kdm2b enzyme activity disrupted mouse strain were obtained simultaneously. Moreover, sgRNA targeting flavin containing monooxygenases3 (Fmo3) gene and the corresponding single strand oligonucleotides (ssODN) donor template with point mutation were co-injected into the male pronucleus of one-cell mouse embryos stimulated HR-mediated repair mechanism. Genomic sequence analysis of F0 mice showed that frame-shifted Fmo3 knockout mouse and site-specific Fmo3 knock-in mouse with single base substitution were successfully generated, and these mutations could be stably transmitted to the next generation. Therefore, we successfully generated mouse strains containing site-specific mutations through HR-dependent and -independent DSB repair using the CRISPR/Cas9 system.

Comparison of the outcome of conventional in vitro fertilization and intracytoplasmic sperm injection in moderate male infertility from ejaculate.

OBJECTIVE: To evaluate whether couples with moderate male infertility should be treated with conventional in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). PATIENTS AND METHODS: A total of 249 couples with moderate male infertility undergoing their first IVF/ICSI cycle were enrolled in the study. The couples were divided into two groups according to the results of semen analysis: moderate oligozoospermia (O group) and moderate oligoasthenozoospermia (OA group). Sibling oocytes were randomized into groups to be inseminated either by conventional IVF or ICSI. Fertilization rate, embryo quality, implantation rate, and clinical pregnancy rate were examined. RESULTS: There was no difference in the fertilization, implantation, and pregnancy rates between conventional IVF and ICSI in either the O group or OA group (p > 0.05). Additionally, in the OA group, the good quality embryo rate was similar after IVF or ICSI (p > 0.05). However, in the O group, the good quality embryo rate was significantly higher after ICSI than after IVF (p < 0.05). CONCLUSIONS: Couples with moderate oligozoospermia or moderate oligoasthenozoospermia did not influence the major indices of IVF. Because of the uncertainties concerning the safety of ICSI, couples with moderate oligozoospermia or moderate oligoasthenozoospermia need not be subjected to this procedure.

Expression level of pluripotent genes in incomplete reprogramming.

OBJECTIVE: To compare the expression levels of pluripotent genes among incomplete reprogrammed colonies and induced pluripotent stem cells (iPSCs), to explore the relationship between the expression of pluripotent genes and incomplete reprogramming. METHODS: Four genes (Oct4, Sox2, Klf4, C-Myc) were introduced into human foreskin fibroblasts (HFFs) by retroviruses. The HFFs were induced to reprogramming. Different forms of colonies were picked up, analyzed, and compared with iPSCs from different aspects, including the morphology of clones, alkaline phosphatase (AP) staining, immuno-fluorescence, and Q-PCR. RESULTS: In the reprogramming process, different colonies were emerged, some of them exhibited typical human embryonic stem cell morphology (eg., compact colonies, high nucleus-to-cytoplasm ratios, and prominent nucleoli). However, these colonies couldn't maintain these characters after passage. There was an intermediate state, named partially reprogramming. Through analysis and identification, AP staining results were weakly positive, compared with iPSC colonies. The immuno-fluorescence staining demonstrated these colonies just expressed pluripotent protein Oct4. Q-PCR indicated that the expression of exogenous transcription factors was inappropriate, either at a high level or at a low level. Most of the endogenous pluripotency genes were expressed at a low level. CONCLUSIONS: It may be one of the causes of incomplete reprogramming that the exogenous pluripotent gene is low-expressed or over-expressed, and successful reprogramming may depend on a specific stoichiometric balance of Oct4, Sox2, Klf4 and c-Myc.

Cyclosporine A improves adhesion and invasion of mouse preimplantation embryos via upregulating integrin ß3 and matrix metalloproteinase-9.

Our previous study has demonstrated cyclosporin A (CsA) promotes the migration and invasiveness of human first-trimester trophoblast cells in vitro. Here, we further investigated the effect of CsA on the early implantation in vitro of mouse embryo. Female C57 mice were superovulated and mated, and then two-cell embryos were harvested from the oviducts and sequentially cultured in vitro in G1 and G2 media with 0, 0.1, 1.0 or 10 µM of CsA. Blastocyte formation, blastocyte cell number and apoptosis, embryo hatching were assessed in 4-6 dpc. The adhesion and stretching growth of hatched embryos in laminin coated dishes were evaluated from 5 dpc to 8 dpc, and the expressions of implantation serine proteinase 1 (ISP1), integrin (itg) ß3 and matrix metalloproteinase (MMP)-9 were determined by real time PCR and immunofluorescence, respectively. We showed there was no significant difference in blastocyst formation rates, hatching rates, number of whole embryonic cells, apoptotic cells, and distribution of inner cell masses (ICMs) and trophoblasts (TB) between the CsA- and control-treated groups. Expression of ISP1 mRNA was unaffected on 5 dpc. After hatching, adhesion rate of 7 dpc significantly increased in 0.1 and 1.0 µM of CsA treatment, and embryo area of 8 dpc stretch growing on laminin were increased in 1.0 µM of CsA. The mRNA and protein expression of itgß3 and MMP-9 on 7 dpc blastocyst were up-regulated. In conclusion, CsA in low dosage up-regulates itgß3 and MMP-9 expression, and enhances embryonic adhesion and invasion, which is beneficial to the embryo implantation.

Outcome of conventional IVF and ICSI on sibling oocytes in moderate ligoasthenozoospermia.

OBJECTIVE: To evaluate the outcomes of patients with moderate oligoasthenozoospermia treated with conventional in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). METHODS: A total of 99 couples with moderate oligoasthenozoospermia undergoing their first IVF/ICSI cycle were included in the study. Sibling oocytes were randomized to be inseminated either by conventional IVF or ICSI. Fertilization rate, cleavage rate, embryo quality, implantation rate, and clinical pregnancy rate were examined. RESULTS: There was no difference in the fertilization rate, cleavage rate, implantation rate, and pregnancy rate between conventional IVF and ICSI (P>0.05). The good quality embryo rate was significant difference between after IVF and after ICSI (P<0.05). CONCLUSIONS: Couples with moderate oligoasthenozoospermia did not influence the major indices of IVF and the uncertainties concerning the safety of ICSI, couples with moderate oligoasthenozoospermia need not be subjected to ICSI.

Trophoblasts-derived chemokine CCL24 promotes the proliferation, growth and apoptosis of decidual stromal cells in human early pregnancy.

Chemokine CCL24 is the second member of eotaxins, a group of eosinophils' selectively chemoattractants. Via binding to its only receptor CCR3, CCL24 mainly mediates atopic disorders, parasitic infections and systemic diseases. It is well-known that CCR3 is expressed at the maternal-fetal interface; nevertheless whether CCL24 is located there and which role CCL24/CCR3 axis played is unclear. In this article, we assessed the expression of CCL24 and CCR3 in decidual stromal cells (DSCs) and trophoblasts, investigated the effects of DSCs-trophoblasts contact and pregnancy-associated hormones on the expression of CCR3 by DSCs, and last examined the role of trophoblasts-derived CCL24 on the proliferation, cell numbers and apoptosis of DSCs in vitro. We found that trophoblasts secrete chemokine CCL24, whereas DSCs express receptor CCR3. DSCs and trophoblasts co-culture had an raised level of CCL24 in culture supernatants, and the expression of CCR3 on DSCs was also obviously improved. Estrogen, progesterone and hCG up-regulated the expression of CCR3 on DSCs at appropriate concentration. CCL24 increased the proliferation and apoptosis of DSCs, whereas on the whole it promoted the number of DSCs. Thus, we conclude that by secreting CCL24 trophoblasts could promote the growth of DSCs; pregnancy associated environments such as DSCs-trophoblasts contact and hormones increased local CCL24/CCR3, which means a beneficial factor for the process of decidualization in human early pregnancy.

[Effect of domestic highly purified urinary follicle stimulating hormone on outcomes of in vitro fertilization-embryo transfer in controlled ovarian stimulation].

OBJECTIVE: To investigate the effect of domestic urine-derived high-purity follicle- stimulating hormone (HP-FSH, Lishenbao) on the outcome of in vitro fertilization(IVF) embryo transfer (ET) in controlled ovarian stimulation (COS). METHODS: From 1 September 2010 to 31 March 2011, total of 3178 infertility patients from 14 Reproductive Center with IVF or intracytoplasmic sperm injection (ICSI) indications who accepted first IVF or ICSI cycle were studied retrospectively. Their causes of infertility include all infertility factors except ovulatory dysfunction infertility and uterine factor infertility. The only long luteal phase gonadotropin-releasing hormone agonist (GnRH-a) protocol was included. Patients were divided into 2 groups according to the type of follicle-stimulating hormone (FSH) agents used: 1932 cases in HP-FSH group and 1246 cases in recombinant FSH (rFSH)group. Patients in both groups were combined with human menopausal gonadotropin (hMG) at doses of 150 U when follicle with diameter reached to 14-16 mm. When 3 dominate follicle with diameter reached 18 mm, hCG at dose of 5000 to 10 000 U or recombinant hCG at dose of 250 µg was administered by intramuscular injection. After 34 to 36 hours, oocytes were obtained guided by ultrasound, then IVF-ET were underwent in their Reproductive Center. The primary endpoint was comparison of live birth rate between the two groups. The secondary endpoints were comparisons of clinical pregnancy rate, miscarriage rate, and implantation rate, as well as COS and IVF outcome between the two groups. RESULTS: (1) There were significantly differences in baseline characteristics of the patients between two groups. The mean age was elder(32 ± 4 versus 30 ± 4, P < 0.01) , the infertility duration was longer (5 ± 4 versus 5 ± 3, P < 0.01) , and antral follicle count (AFC) was less (11 ± 5 versus 13 ± 7, P < 0.01) in patients of HP-FSH group compared with those in patients of rFSH group. (2) As compared with rFSH, the total doses of gonadotropin needed was (2348 ± 1011) U in HP-FSH group versus (2022 ± 659) U in rFSH group, the number of oocytes 13 ± 6 in HP-FSH group and 14 ± 7 in rFSH group, the rate of embryo frozen cycle of 66.30% (1281/1932) in HP-FSH group and 74.88% (933/1246) in rFSH group, which all reached statistical difference (P < 0.01). However, there were no significant different implantation rate [30.49% (1111/3644) versus 32.45% (737/2271)] between two groups. The other clinical parameters did not show significant difference, including clinical pregnancy rate per started cycle [41.61% (804/1932) versus 41.97% (523/1246) ] , clinical pregnancy rate per ET cycle[46.58% (804/1726) versus 48.47% (523/1079)], live birth rate per started cycle[34.21% (661/1932) versus 34.19% (426/1246)], live birth rate per ET cycle [38.30% (661/1726) versus 39.48% (426/1079)], miscarriage rate[13.6% (109/804) versus 16.4% (86/523)], and moderate/severe ovarian hyperstimulation syndrome (OHSS) rate [5.80% (112/1932) versus 7.78% (97/1246)](P > 0.05).(3) Treatment cost: the cost of gonadotropins needed for the patients in HP-FSH group was lower than that in rFSH group (4005 ± 1650 versus 6482 ± 2095, P < 0.01). CONCLUSION: In IVF/ICSI treatment cycles, domestic HP-FSH has similar live birth rate and lower financial burden when compared with rFSH.

A whole-mechanical method to establish human embryonic stem cell line HN4 from discarded embryos.

Since the first human embryonic stem cell (hESC) line was generated by Thomson et al. (in Science 282:1145-1147, 1998), hundreds of hESC lines have been reported by different labs, providing resources for basic research and regenerative medicine as well. However it has been widely recognized that hESC lines varied on their properties, in terms of gene expression profile, epigenetic modify profile, and differentiation tendency. Generation of more hESC lines will largely enhance our knowledge of hESCs innate character. In this current work, we reported the generation of HN4, a hESC line derived from grade III IVF human embryo by using a mixture of human foreskin fibroblast (HFF) and mouse embryonic fibroblast (MEF) as feeder layers, and a whole-mechanical method in inner cell mass (ICM) isolation. HN4 satisfied the criteria of hESCs pluripotency, with high expression of hESC surface markers (SSEA-3, SSEA-4, TRA-1-60, TRA-1-81), transcription factors (OCT-4, NANOG, REX-1), and alkaline phosphatase. It is able to differentiate to three germ layer derivatives when cultured in vitro, or in teratoma formation. Moreover, it displayed promising potential in neural differentiation under a proper culture condition, suggesting the advantage of HN4 in further investigation. Additionally, the whole-mechanical protocol for ICM isolation facilitates hESC line generation for its ease to handle.

[Effects of Geranium sibirum extracts on liver metastases in nude mice with colonic cancer].

OBJECTIVE: To establish nude mice models with the liver metastases of colonic adenocarcinoma and study the effects of Geranium sibirum extracts on them. METHODS: Nude mice liver metastases model of colonic cancer was established with human colonic cancer cells line( Ls 174t) inoculated into mice spleen. 36 nude mice were randomly divided into 3 groups, containing control group, Geranium sibirum extracts group and hydroxycamptothecine group. The weight and size of the mice and growth of the carcinoma were recorded. All specimens were examined histologically. pS2 in blood in nude mice with liver metastasis of colonic carcinoma was detected with nested RT-PCR. RESULTS: The incidence of liver metastasis was 100% in this intrasplenic injection model. The pathological results showed that tumour cells of liver metastases were poorly differentiated human colonic adenocarcinoma. In Geranium sibirum extracts group, the tumor number and the weight of liver metastases were significantly lower than those in hydroxycamptothecine group (P < 0.05). Using semiquantitative examination,in Geranium sibirum extracts group,the relative value of pS2 expression in blood was significantly higher than that in hydroxycamptothecine group (P < 0.05). CONCLUSION: Geranium sibirum extracts can effectively inhibit the occurrence of liver metastases carcinoma and decrease the positive expression of pS2, it also has better effect than hydroxycamptothecine so that Geranium sibirum extracts may become the potential therapeutic strategy for liver metatstases of colonic cancer.

Effect of volatile oil of amomum on expressions of platelet activating factor and mastocarcinoma-related peptide in the gastric membrane of chronic gastritis patients with helicobacter-pylori infection.

OBJECTIVE: To observe the effect of volatile oil of amomum (VOA) on the expressions of mastocarcinoma-related peptide (PS2) and platelet activating factor (PAF) in helicobacter pyloriassociated gastritis (HPG) and to analyze its potential mechanism. METHODS: Eighty patients with HPG were randomly assigned to two groups, 42 patients in the treated group treated with 0.5 mL VOA, thrice per day; and the 38 patients in the control group receiving Western tertiary medicinal treatment. Gastroscopic picture and helicobacter pylori (HP) infection (by quick urease and Warthin-Starry stain) of the gastro-membrane, expressions of PS2 and PAF (by immunohistochemical assay and Western blotting) as well as the contents of aminohexose and phospholipid (by Neuhaus method) in the gastric membrane of all patients were detected before treatment and 4 weeks after treatment. The clinical efficacy in the two groups was compared. RESULTS: The total effective rate in the treated group was 88.1%, which was significantly higher than that in the control group (78.9%, P<0.05). After treatment, in the treated group, gastric membranous contents of aminohexose and phospholipid was increased, expression of PS2 elevated but that of PAF lowered, all showing significant difference as compared with those in the control group (P<0.01). In the control group, the expressions of PS2 and PAF changed insignificantly. The radical eliminating rate of HP in the treated group and the control group was insignificantly different between them (76.1% vs. 65.8%, P>0.05). CONCLUSION: The mechanism of VOA for anti-gastritis might be related with its action in increasing the expression of PS2 and decreasing the expression of PAF, and thus regulating the hydrophobicity of the gastric membrane.

[Observation on the expression of PS2 and platelet activating factor affected by Jianwei Yuyang granules and study its potential mechanisim against peptic ulcer].

OBJECTIVE: To observe the influences of Jianwei Yuyang (JWYY) granules on expressions of PS2 and platelet activating factor (PAF), and analyze its potential mechanism. METHODS: 76 gastric ulcer patients were final diagnosed by gastroscope. They were randomly devided into 3 groups, including JWYY group (36 cases), Ranitidine group (36 cases) and Normal group (12 cases). Detection on Biospy Specimens of gastric mucosa to obseve the change of PAF, PS2 and the contents of aninohexose and phosphatide in ulcerated gastric mucosa for Immunohischemical and Western blotting. RESULTS: The contents of aninohexose, phosphatide of PU patients increased in JWYY group. There was significant difference between JWYY group and Ranitidine group (P < 0.01). The Immunohischemical and Western blotting methods showed that there was a linear correlation between the the contents of phosphatide, anino-hexose and the expression of PAF, PS2 in PU patients (P < 0.01). CONCLUSION: JWYY granule can prevent the occurrence and relaps of ulcer and affect the hydrophobicity of gastic mucosa and strengthen the stability of myxo-gellayer by reducing the expression of PAF, elevating the expression of PS2 and effecting the contents of phosphatide and aninohexose, that may be one of the mechanisms of JWYY to heal ulcer quickly.

[Committed hematopoeitic differentiation of embryonic stem cell].

OBJECTIVE: To explore a stable technology for inducing ES cell to committed hematopoietic differentiation. METHODS: The effects of various inductive factors on BLast Colony-Forming Cell (BL-CFC) were investigated. In vitro differentiative system for ES cells was employed in this study. RESULTS: A high linear correlation between the number of D3.5 EB-derived cells plated and the number of blast cell colonies was developed, r = 0.9931. With high frequency of blast colonies observed (1.08-1.2 colonies per 100 cells). 20%-30% D4T conditioned medium (D4T CM) showed the most significant growth potentials of blast colonies. D4T CM, EPO or KL alone had no blast colony growth promoting effect (P > 0.05). But VEGF alone had high significant blast colony growth promoting effect (P < 0.001). However, any two factors combination from above four factors exerted better growth promoting effect than VEGF alone (EPO + D4T CM, P < 0.05; KL + D4T CM, P < 0.01; VEGF + D4T CM, P < 0.001). There were no significant difference among VEGF + KL and EPO + D4T CM or KL + D4T CM, and KL + D4T CM (P > 0.05). While the combination of VEGF + D4T CM was better than KL + D4T CM, VEGF + KL or EPO + D4T CM (P < 0.001). Moreover, the combination of VEGF + KL + D4T CM + EPO, had the highest significant blast colony growth promoting effect (P < 0.001). And the highest frequency of blast colonies was observed (1.5-1.2 colonies per 100 cells). CONCLUSION: VEGF may be the main factor which stimulates the growth of significant numbers of blast cell colonies. D4T CM maybe contains strong cofactors. EPO and KL are the main factors for the induction of BL-CFC to committed hematopoietic differentiation. D3.5 EB-derived cells are more sensitive to various stimulators and have strong blast colony growth promoting effect than that of D3.25 EB-derived cells.