Thymopentin enhances the generation of T-cell lineage derived from human embryonic stem cells in vitro.

Thymopentin is a group of biologically active peptide secreted mainly by the epithelial cells of thymic cortex and medulla. Whether it promotes T cells production from human embryonic stem cells(hESCs) in vitro remains an elusive issue. In the present study, we develop a novel strategy that enhances T-cell lineage differentiation of hESCs in collagen matrix culture by sequential cytokine cocktails treatment combined with thymopentin stimulation. We observed that approximately 30.75% cells expressed CD34 on day 14 of the cultures and expressed the surface markers of erythroid, lymphoid and myeloid lineages. The results of colony assays and gene expressions by RT-PCR analysis also demonstrated that hematopoietic progenitor cells (HPCs) derived from hESCs were capable of multi-lineage differentiation. Further study revealed that culturing with thymopentin treatment, the CD34(+)CD45RA(+)CD7(+) cells sorted from HPCs expressed T-cell-related genes, IKAROS, DNTT, TCRγ and TCRß, and T-cell surface markers, CD3, cytoplasmic CD3, CD5, CD27, TCRγδ, CD4 and CD8. The differentiated cells produced the cytokines including IFN-γ, IL-2 and TNF-α in response to stimulation, providing the evidence for T-cell function of these cells. In conclusion, thymopentin enhances T-cell lineage differentiation from hESCs in vitro by mimicking thymus peptide environment in vivo.

Mesoderm is committed to hemato-endothelial and cardiac lineages in human embryoid bodies by sequential exposure to cytokines.

Human embryonic stem (hES) cells can differentiate into cells of the three germ layers in vitro and serve as a powerful resource to study mechanisms involved in cell fate decisions. However, it is difficult to promote the directed and efficient differentiation of hES cells toward a specific lineage. Here we establish a stepwise strategy for generating hemato-endothelial and cardiac precursors from hES cells in single culture conditions. The efficiency of committing hES cells to three cell lineages was significantly higher with our approach than with exposure to single or multiple cytokines. Efficiency was determined using quantitative analysis by gene expression, flow cytometry, and colony assays. Several cytokines were sufficient to drive the efficient differentiation of hES cells into specific lineages. Each of these factors appeared to regulate specific steps of differentiation: BMP4 promoted the efficient formation of mesoderm; bFGF induced the differentiation of these mesodermal precursors to the hemangioblast fate; VEGF and TPO were required for the production of committed hematopoietic progenitors. This stepwise control of differentiation in vitro leads to a high frequency of hemato-endothelial and cardiac precursors derived from hES cells and offers a unique model for studying the molecular and cellular events that regulate hematopoiesis and cardiogenesis.

[Comparison of GnRH antagonist fixed protocol and GnRH agonists long protocol in infertile patients with normal ovarian reserve function in their first in vitro fertilization-embryo transfer cycle].

OBJECTIVE: To compare the clinical outcomes of gonadotropin-releasing hormone (GnRH) antagonist (GnRH-ant) fixed protocol with GnRH agonist (GnRH-a) long protocol in infertile patients with normal ovarian reserve function in their first in vitro fertilization-embryo transfer (IVF-ET) cycle, and to explore the feasibility and advantage of GnRH antagonist protocol performed in normal responders. METHODS: From January 2011 to June 2011, 771 infertile women with normal ovarian reserve function underwent their first IVF or intracytoplasmic sperm injection (ICSI) cycles in Peking University Third Hospital, which were divided into 245 cycles in GnRH-ant fixed protocol group (GnRH-ant group) and 526 cycles in GnRH-a long protocol group (GnRH-a group). The data of general demographic, treatment and clinical outcome were compared between two groups. RESULTS: Age, infertile duration, body mass index (BMI), baseline serum follicle-stimulating hormone (FSH) and estradiol levels between two groups did not reached statistical difference (P > 0.05). The level of estradiol was (12 289 ± 6856) pmol/L in GnRH-ant group and (14 934 ± 8007) pmol/L in GnRH-a group at day of hCG injection. The mean length of stimulation was (10.3 ± 1.2) days in GnRH-ant group and (12.8 ± 1.6) days in GnRH-a group. The dose of gonadotropin was (2013 ± 607) U in GnRH-ant group and (2646 ± 913) U in GnRH-a group. The number of ovum was 15 ± 7 in GnRH-ant group and 17 ± 8 in GnRh-a group. Those clinical parameter all reached statistical difference (P < 0.05). The number of embryo was 7 ± 4 in GnRH-ant group and 8 ± 5 in GnRH-a group, the rate of clinical pregnancy was 40.9% (94/230) in GnRH-ant group and 45.6% (216/474) in GnRH-a group, the rate of implantation was 26.1% (128/490) in GnRH-ant group and 30.9% (307/994) in GnRH-a group, the rate of continuing pregnancy was 38.7% (89/230) in GnRH-ant group and 42.6% (202/474)in GnRH-a group, those parameter did not reach statistical difference (P > 0.05). The rate of moderate or severe ovarian hyperstimulation syndrome was 2.4% (6/245) in GnRH-ant group and 4.2% (22/526) in GnRH-a group, which did not show significant difference (P > 0.05). CONCLUSION: In the first IVF or ICSI cycle of the patients with normal ovarian reserve function, the fixed GnRH-ant protocol could get the same satisfied clinical outcome, and it is more economic, convenient and safer compared with low dose depot GnRH-a long protocol.

Early embryonic sensitivity to cyclophosphamide in cardiac differentiation from human embryonic stem cells.

hESCs (human embryonic stem cells) can differentiate into tissue derivatives of all three germ layers in vitro and mimic the development of the embryo in vivo. In this study, we have investigated the potential of an hESC-based assay for the detection of toxicity to cardiac differentiation in embryonic development. First of all, we developed the protocol of cardiac induction from hESCs according to our previous work and distinguished cardiac precursor cells and late mature cardiomyocytes from differentiated cells, demonstrated by the Q-PCR (quantitative real-time PCR), immunocytochemistry and flow cytometry analysis. In order to test whether CPA (cyclophosphamide) induces developmental and cellular toxicity in the human embryo, we exposed the differentiating cells from hESCs to CPA (a well-known proteratogen) at different stages. We have found that a high concentration of CPA could inhibit cardiac differentiation of hESCs. Two separate exposure intervals were used to determine the effects of CPA on cardiac precursor cells and late mature cardiomyocytes respectively. The cardiac precursor cells were sensitive to CPA in non-cytotoxic concentrations for the expression of the cardiac-specific mRNA markers Nkx2.5 (NK2 transcription factor related, locus 5), GATA-4 (GATA binding protein 4 transcription factor) and TNNT2 (troponin T type 2). Non-cytotoxic CPA concentrations did not affect the mRNA markers' expression in late mature cardiomyocytes, indicating that cardiac precursors were more sensitive to CPA than late cardiomyocytes in cardiogenesis. We set up the in vitro developmental toxicity test model so as to reduce the number of test animals and expenses without compromising the safety of consumers and patients. Furthermore, such in vitro methods may be possibly suited to test a large number of chemicals than the classical employed in vivo tests.

Ghrelin promotes differentiation of human embryonic stem cells into cardiomyocytes.

AIM: Ghrelin is involved in regulating the differentiation of mesoderm-derived precursor cells. The aim of this study was to investigate whether ghrelin modulated the differentiation of human embryonic stem (hES) cells into cardiomyocytes and, if so, whether the effect was mediated by growth hormone secretagogue receptor 1α (GHS-R1α). METHODS: Cardiomyocyte differentiation from hES cells was performed according to an embryoid body (EB)-based protocol. The cumulative percentage of beating EBs was calculated. The expression of cardiac-specific markers including cardiac troponin I (cTnI) and α-myosin heavy chain (α-MHC) was detected using RT-PCR, real-time PCR and Western blot. The dispersed beating EBs were examined using immunofluorescent staining. RESULTS: The percentage of beating EBs and the expression of cTnI were significantly increased after ghrelin (0.1 and 1 nmol/L) added into the differentiation medium. From 6 to 18 d of differentiation, the increased expression of cTnI and α-MHC by ghrelin (1 nmol/L) was time-dependent, and in line with the alteration of the percentages of beating EBs. Furthermore, the dispersed beating EBs were double-positively immunostained with antibodies against cTnI and α-actinin. However, blockage of GHS-R1α with its specific antagonist D-[lys(3)]-GHRP-6 (1 µmol/L) did not alter the effects of ghrelin on cardiomyocyte differentiation. CONCLUSION: Our data show that ghrelin enhances the generation of cardiomyocytes from hES cells, which is not mediated via GHS-R1α.

Normal birth from cryopreserved embryos after intracytoplasmic sperm injection of frozen semen into vitrified human oocytes.

OBJECTIVE: To describe the birth achieved from frozen embryos after intracytoplasmic sperm injection (ICSI) of donor sperm into vitrified oocytes. PATIENT: A 25-year-old woman whose husband was azoospermic undergoing IVF therapy. METHODS: Oocytes collected after ovarian stimulation were vitrified, thawed, and fertilized by frozen donor sperm. RESULTS: Nineteen oocytes were vitrified and all survived after thawing. Thirteen of the 19 oocytes that underwent ICSI with donors sperm were successfully fertilized. Twelve embryos were cryopreserved again by conventional slow-freezing protocol because of uterine bleeding on the day of transfer. Three thawed embryos were transferred, and a normal male with an infant karyotype of 46,XY was delivered. CONCLUSION: This case report demonstrates effective oocyte cryopreservation by vitrification.

[Follicular stimulating hormone receptor gene C566T mutation in premature ovarian failure].

OBJECTIVE: To investigate the incidence of follicular stimulating hormone receptor (FSHR) gene C566T mutation in Chinese women with premature ovarian failure (POF) and to explore the etiologies of POF. METHODS: This case-control study was carried out between 73 Chinese women with idiopathic POF (POF group) and 35 controls (control group), including 25 normal females with a regular menstrual history and 10 normal post-menopause women. DNA was extracted from the peripheral blood of patients and controls. The exon 7 of FSHR gene was amplified by PCR. PCR products were subsequently digested by the enzyme BsmI and then subjected to electrophoresis on agarose gels and stained with ethidium bromide to determine the C566T mutation. DNA samples of random sampling were further analysed by sequencing the PCR products to confirm the mutation. RESULTS: BsmI digestion resulting in two fragments of 51 and 27 base pairs was noted for all 73 POF patients and 35 controls. PCR sequencing confirmed that the 566 allele of FSHR gene is C, demonstrating normal FSHR allele. CONCLUSIONS: No FSHR gene C566T mutation is present in POF patients and controls. FSHR C566T mutation may be rare in Chinese women with POF.

Neural precursors derived from human embryonic stem cells.

Human embryonic stem (hES) cells provide a promising supply of specific cell types for transplantation therapy. We presented here the method to induce differentiation of purified neural precursors from hES cells. hES cells (Line PKU-1 and Line PKU-2) were cultured in suspension in bacteriological Petri dishes, which differentiated into cystic embryoid bodies (EBs). The EBs were then cultured in N2 medium containing bFGF in poly-L-lysine-coated tissue culture dishes for two weeks. The central, small cells with 2-3 short processes of the spreading outgrowth were isolated mechanically and replated. The resulting neurospheres were cultured in suspension for 10 days, then dissociated into single cell suspension with a Pasteur pipette and plated. Cells grew vigorously in an attached way and were passed every 4-5 days. Almost all the cells were proved nestin positive by immunostaining. Following withdrawal of bFGF, they differentiated into neurons expressing beta-tubulin isotype(III), GABA, serotonin and synaptophysin. Through induction of PDGF-AA, they differentiated into astrocytes expressing GFAP and oligodendrocytes expressing O4. The results showed that hES cells can differentiate into typical neural precursors expressing the specific marker nestin and capable of generating all three cell types of the central nervous system (CNS) in vitro.

[Establishment and characteristics of clonal human embryonic stem cell lines].

OBJECTIVE: To establish clonal human embryonic stem cell lines and investigate their biological characteristics. METHODS: Cells were derived from one inner cell mass of human blastocyst, multiplied for 20 passages, and then dissociated into single cell suspension by digestion with 0.5% trypsin. Single cell was picked up and plated into individual well of a 96-well plate containing feeder-layers directly under a dissection microscope. The outgrowth clones were passed by treatment with collagenase. Surface markers were detected by cytochemistry and histoimmunochemistry. Karyotypes were tested using standard G-banding techniques. The pluripotency was analyzed by inoculating cells into severe combined immunodeficient (SCID) mice. RESULTS: Two clonal human embryonic stem cell lines were established. Cells of these two lines possess the characteristics and differentiating potencies: normal 46 XX karyotypes; expressing a series of surface markers such as: alkaline phosphotase, stage-specific embryonic antigen (SSEA)-4, tumor recognition antigen (TRA)-1-60, TRA-1-81 etc; and forming teratomas comprising derivatives of three embryonic germ layers such as neural tissue, cartilage, squamous epithelium and columnar epithelium when injected into SCID mice. CONCLUSIONS: The two single cell-cloned human embryonic stem (hES) cell lines were derived in our laboratory. The cells possess stable biological characteristics of undifferentiated hES cells.

[Human embryonic stem cells forming embryoid bodies comprising multiple types of cells].

OBJECTIVE: To investigate the pluripotency of human embryonic stem cells in vitro. METHODS: Human embryonic stem cells were cultured in suspension without feeder layers and bFGF in medium. mRNAs of different types of cells were analyzed by RT-PCR. RESULTS: When Human embryonic stem cells were cultured in suspension without feeder layers and bFGF in medium, they formed floating aggregates termed embryoid bodies (EBs), in which many precursors and functional cells were detected by RT-PCR, such as neural and islet precursors, neurons, insulin-secreting cells,glucagon-secreting cells and liver cells et al. CONCLUSION: Human embryonic stem cells are able to form embryoid bodies naturally in vitro which express specific markers of many types of precursors and mature functional cells.

[Establishment of a single cell-cloned mouse embryonic stem cell line].

OBJECTIVE: To establish embryonic stem cells culture system and methods. METHODS: Inner cell masses were isolated from blastocysts of 3.5 day-old 129SvJ mice by immunosurgery, seeded onto gamma-irradiated mouse fibroblasts feeder layer. The outgrowths were passed by digestion with trypsin. Surface markers were identified by cytochemistry and immunohistochemistry. Karyotype was tested with standard G-banding technique. Differential potency was tested both in vivo and in vitro. RESULTS: One single cell cloned mouse embryonic stem cell line G(11) was established, which had normal 40XY karyotype and proliferated rigorously for a long time, expressed surface markers such as AP and SSEA-1, and was able to differentiate into many cell types both in vivo and in vitro. CONCLUSION: Mouse embryonic stem cell culture system has been established in our laboratory.

[Influence of cryoprotectant and cooling rate in vitrification method on the spindles of rabbit oocytes].

OBJECTIVE: To investigate the influence of cryoprotectants and cooling rates in vitrification method on the spindles of rabbit M II oocytes. METHODS: Rabbit oocytes were verified by using cryoloop with ethylene glycol (EG) singly or EG combined with dimethyl sulphoxide (DMSO) as cryoprotectants, and cooled by taking oocytes directly into liquid nitrogen or by vitrification machine. After frozen rabbit oocytes thawed, the microtubulin and chromosome of the spindles were fixation and stained by immunofluorescent method. Confocal microscope was used to reveal spindle configuration. RESULTS: In the two protocols of single EG used and EG combined with DMSO, the spindles were severely injured. But in protocol of EG combined with DMSO and at ultra-rapid cooling rate, the normal configuration of spindle rate of thawed rabbit oocytes was similar to that of the control group. CONCLUSION: The protocol of EG combined with DMSO as cryoprotectants and with extremely high cooling rate by vitrification machine can produce the best effect on conservation of spindle configuration in vitrification of rabbit oocytes.

[Expression of cystic fibrosis transmembrane conductance regulator in human endometrium].

OBJECTIVE: To study expression of cystic fibrosis transmembrane conductance regulator (CFTR) in human endometrium. METHODS: The expression of CFTR mRNA and protein from 50 samples of normal cyclic human endometrium was examined by in situ hybridization, immunohistochemistry and Western blotting respectively. RESULTS: CFTR mRNA and protein expressions were only detected in the endometrial glandular cells with cyclic changes. CFTR mRNA could be detected from the midproliferative phase with the highest level found in the late proliferative phase, significantly higher than those of late-secretory phase endometrium (P < 0.05). While a large quantity of CFTR protein were seen in late proliferative phase and still presented in the secretive and menstrual phases. Western blotting analysis demonstrated that human endometrium expressed the special CFTR band at 170 000. CONCLUSION: The expression of CFTR in human endometrium presented in glandular cells and the amounts were varied cyclically. The abundant CFTR mRNAs and proteins around the ovulatory period may drive Na(+) and fluid from plasma into the uterine lumen to make optimal electrolyte composition and sufficient fluid volume for sperm migration.

[Immunohistochemical expression of integrin alphav, beta5 and beta3 in ovarian epithelial carcinoma: correlation with drug resistance of chemotherapy].

OBJECTIVE: To investigate the possible correlation between the expression of integrin alphav, beta5 and beta3 in tumor tissues and the response to chemotherapy and survival of patients with ovarian epithelial carcinoma. METHODS: Seventy-seven patients with ovarian epithelial carcinoma from January 1996 to December 2002 were entered into the study. All subjects were matched up with the inclusion criteria and followed up till December 2003. Each patient received cytoreductive surgery and systematic chemotherapy. Patients were divided into drug resistant group (n = 26) and drug sensitive group (n = 51). The antigens of integrin alphav, beta5 and beta3 in ovarian tumour tissues were tested by immunohistochemistry. Logistic regression was employed to analyze the correlation between drug resistance and factors including patients' age, clinical stage, pathological degree and type of tumors, chemotherapy methods, operative results and the expression of integrin alphav, beta5 and beta3 in tumors. The disease prognosis was analyzed by multivariate COX regression. RESULTS: In the drug resistance group, the expressions of integrin alphav and beta5 were significantly higher than that in the drug sensitive group (P = 0.004 and 0.001). The expression of integrin beta3 was not statistically different in the two groups (P = 0.668). According to multivariate analysis, the expression of integrin alphav and beta5 and clinical stage were three independent factors correlated with drug resistance and disease prognosis for the patients with ovarian epithelial carcinoma (P = 0.014, 0.030, 0.042 for drug resistance; and P = 0.045, 0.031, 0.001 for prognosis respectively). CONCLUSION: The high expressions of integrin alphav and beta5 in ovarian epithelial carcinoma are risk factors for drug resistance and poor disease prognosis.

[Progress on Y chromosome microdeletions and male infertility].

About 10%-15% of azoospermic and 5%-10% of severely oligozoospermic men with idiopathic infertility have Yq microdeletions which could be transmitted to their male offspring by means of ICSI. We review present studies about Yq microdeletions including incidence, region, correlations between genotype and phenotype, the mechanism of Yq deletions and try to further understand the cause of male infertility as well as provide a new way for diagnosis and therapy.

Dynamic expression of microRNAs during the differentiation of human embryonic stem cells into insulin-producing cells.

Human embryonic stem (hES) cells with the capacity of self-renewal and multilineage differentiation are promising sources for generation of pancreatic islet cells for cell replacement therapy in diabetes. Here we induced hES cells into insulin-producing cells (IPCs) in a stepwise process which recapitulated islet organogenesis by directing cells through the stages resembling definitive endoderm, gut-tube endoderm, pancreatic precursor and cells that expressed pancreatic endocrine hormones. The dynamic expression of microRNAs (miRNAs) during the differentiation was analyzed and was compared with that in the development of human pancreatic islets. We found that the dynamic expression patterns of miR-375 and miR-7 were similar to those seen in the development of human fetal pancreas, whereas the dynamic expression of miR-146a and miR-34a showed specific patterns during the differentiation. Furthermore, the expression of Hnf1ß and Pax6, the predicted target genes of miR-375 and miR-7, was reciprocal to that of miR-375 and miR-7. Over-expression of miR-375 down-regulated the expression of gut-endoderm/pancreatic progenitor specific markers Hnf1ß and Sox9. Therefore, the miRNAs may directly or indirectly regulate the expression of pancreatic islet organogenesis-specific transcription factors to control the differentiation and maturation of pancreatic islet cells.

Three step derivation of cartilage like tissue from human embryonic stem cells by 2D-3D sequential culture in vitro and further implantation in vivo on alginate/PLGA scaffolds.

In this study a three step culture system, 2D-3D sequential culture in vitro and further implantation in vivo was developed to induce human embryonic stem cells (hESCs) into cartilage like tissues. Five-day-old embryoid bodies were plated for chondrogenic induction for 27 days (step1), then the cells were suspended in alginate and seeded onto polylactic-co-glycolic acid (PLGA) scaffolds for 3D cultivation for 7 days (step 2) and the cells/alginate/PLGA complexes were further transplanted into nude mice for 8 weeks (step 3). At same time, some of complexes were cultured in vitro up to 8 weeks. At the end of step 1, cells exhibited fibroblast-like morphology and expressed chondrocyte-specific markers, Sox 9 and collagen II. During the following 8 weeks of 3D cultivation in vitro, cells displayed spherical morphology, decreased immunoreactivity to Sox-9 and increased one to collagen II, demonstrated further differentiation to mature chondrocyte. In implanted grafts, not only cells appeared typical chondrocytes shape and markers but also cartilage like tissues were formed. These results indicate that 2D-3D sequential culture in vitro is an efficient protocol to induce hESCs differentiates into chondrocytes, while the three step culture system may be an appropriate procedure to derive cartilage like tissues from hESCs.

The reversal of hyperglycaemia in diabetic mice using PLGA scaffolds seeded with islet-like cells derived from human embryonic stem cells.

Islet-like cells derived from embryonic stem (ES) cells may be a promising therapeutic option for future diabetes treatment. Here, we demonstrated a five-stage protocol with adding exendin-4 instead of nicotinamide finally could generate islet-like cells from human embryonic stem (ES) cells. Immunofluorescence analysis revealed a high percentage of c-peptide positive cells in the derivation. However, in addition to insulin/c-peptide, most cells also coexpressed PDX-1 (pancreas duodenum homeobox-1), glucagon, somatostatin or pancreatic polypeptide. Insulin and other pancreatic beta-cell-specific genes were all present in the differentiated cells. Insulin secretion could be detected and increased significantly by adding KCL in high glucose concentration in vitro. Furthermore, subcutaneous transplantation of scaffolds seeded with the islet-like cells or cell transplantation under kidney capsules for further differentiation in vivo could improve 6h fasted blood glucose levels and diabetic phenotypes in streptozotocin-induced diabetic SCID mice. More interestingly, blood vessels of host origin, characterized by mouse CD31 immunostaining, invaded the cell-scaffold complexes. This work reveals a five-stage protocol with adding exendin-4 may be an effective protocol on the differentiation of human ES cells into islet-like cells, and suggests scaffolds can serve as vehicles for islet-like cell transplantation.