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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.
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Células Madre Embrionarias/citología , Linfopoyesis/efectos de los fármacos , Linfocitos T/citología , Timopentina/farmacología , Antígenos CD34/biosíntesis , Antígenos CD34/metabolismo , Antígenos CD7/metabolismo , Antígenos de Superficie/biosíntesis , Linaje de la Célula , Células Cultivadas , Células Madre Hematopoyéticas/citología , Humanos , Interferón gamma/biosíntesis , Interleucina-2/biosíntesis , Antígenos Comunes de Leucocito/metabolismo , Células Madre Pluripotentes/citología , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
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.
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Linaje de la Célula/fisiología , Citocinas/fisiología , Cuerpos Embrioides/citología , Células Madre Embrionarias/citología , Células Endoteliales/citología , Células Endoteliales/fisiología , Corazón/embriología , Células Madre Hematopoyéticas/citología , Diferenciación Celular/fisiología , Células Cultivadas , Cuerpos Embrioides/fisiología , Células Madre Embrionarias/fisiología , Células Madre Hematopoyéticas/fisiología , Humanos , Mesodermo/citología , Mesodermo/embriología , Miocardio/citologíaRESUMEN
With WO3/BiVO4/MXene ternary composite layers as a working electrode, a smart volumetric photoelectrochemical system using electrostatic bias voltage inducted by atmospheric electric field was developed. Under single sun illumination and 0.8 V hardwired bias, the current response of the ternary electrode is 1.15 mA cm-2, which is 1.31 times higher than that of the WO3/BiVO4 electrode, mainly due to the higher charge transfer rate between the MXene layer and the BiVO4 structure. Further, the response of the ternary electrode increases to 1.39 mA cm-2 at an extra atmospheric electric field of 1100 V m-1. It can be demonstrated that the effect of the atmospheric electric field can be regarded as an extra hardwired bias of 0.101 V in the system. The experimental results reveal that the native carriers, including inducted electron/holes in MXene and BiVO4, and carriers in the electrolyte, are all effectively excited by the electrostatic induction of atmospheric electric field.
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BACKGROUND: Maternal diabetes compromises the quality and developmental potential of oocytes. Therefore, it is important to study how to ameliorate the adverse effects of diabetes on oocyte quality. Epigallocatechin gallate (EGCG) has a variety of physiological activities, including anti-inflammatory, antioxidant, and anti-diabetes. In the present study, we evaluated the effect of EGCG on the maturation of diabetic oocytes in vitro. OBJECTIVE: Investigating the role of EGCG in restoring the adverse effects of diabetes on oocyte quality. METHODS: Diabetes mouse model was established by a single injection of streptozotocin (STZ). Oocytes were collected and matured in vitro with/without EGCG in M16 medium. RESULTS: Compared with control, diabetic oocytes have a higher frequency of spindle defects and chromosome misalignment, but EGCG effectively reduces the incidence of oocytes with abnormal spindle assembly and chromosome mismatches. Moreover, the abnormal mitochondrial membrane potential (MMP) of diabetic oocytes is significantly alleviated by EGCG, and the reduced expression of genes regulating mitochondrial fusion (Mfn1 and Mfn2) and fission (Drp1) in diabetic oocytes is significantly increased while EGCG is added. EGCG also decreases the higher level of reactive oxygen species (ROS) in diabetic oocytes that may be regulated by the increased expression of superoxide dismutase 1 (Sod1) and superoxide dismutase 2 (Sod2). EGCG can also reduce the DNA damage of diabetic oocytes. CONCLUSIONS: Our results suggest that EGCG, at least partially, improve the quality of diabetic oocytes.
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Catequina , Diabetes Gestacional , Ratones , Femenino , Humanos , Embarazo , Animales , Oocitos , Antioxidantes/farmacología , Catequina/farmacologíaRESUMEN
SCOPE: Tea is a popular beverage worldwide and has many health functions. Protocatechuic acid (PCA) is an important bioactive component of tea and has benefit to health. In some cases, oocytes after ovulation may miss the optimal fertilization time and enter a postovulatory ageing process. Therefore, to investigate the role of PCA in delaying oocyte ageing is aimed. METHODS AND RESULTS: Metaphase II (MII) oocytes aged in vitro are randomly divided into three groups: control, aged, and aged + PCA. PCA treatment (30 µM) reduces the fragmentation rate and the incidence of abnormal spindle morphology and chromosome misalignment of oocytes aged 24 h in vitro. The mitochondrial dysfunction of aged oocytes, such as decreased mitochondrial membrane potential and excessive accumulation of reactive oxygen (ROS), is also alleviated by PCA. PCA also delays apoptosis of aged oocytes, and improves the sperm binding capacity. Otherwise, aged oocytes treated with PCA have a higher fertilization rate and blastocyst rate compared with untreated aged oocytes in vitro. CONCLUSION: PCA is an important bioactive ingredient of tea that improves aged oocyte quality, suggesting that PCA is available to improve the quality of aged oocytes in vitro.
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Envejecimiento , Semen , Femenino , Masculino , Animales , Ratones , Oocitos/metabolismo , Té/metabolismoRESUMEN
Repurposing existing drugs to inhibit SARS-CoV-2 infection in airway epithelial cells (AECs) is a quick way to find novel treatments for COVID-19. Computational screening has found dicoumarol (DCM), a natural anticoagulant, to be a potential SARS-CoV-2 inhibitor, but its inhibitory effects and possible working mechanisms remain unknown. Using air-liquid interface culture of primary human AECs, we demonstrated that DCM has potent antiviral activity against the infection of multiple Omicron variants (including BA.1, BQ.1 and XBB.1). Time-of-addition and drug withdrawal assays revealed that early treatment (continuously incubated after viral absorption) of DCM could markedly inhibit Omicron replication in AECs, but DCM did not affect the absorption, exocytosis and spread of viruses or directly eliminate viruses. Mechanistically, we performed single-cell sequencing analysis (a database of 77,969 cells from different airway locations from 10 healthy volunteers) and immunofluorescence staining, and showed that the expression of NAD(P)H quinone oxidoreductase 1 (NQO1), one of the known DCM targets, was predominantly localised in ciliated AECs. We further found that the NQO1 expression level was positively correlated with both the disease severity of COVID-19 patients and virus copy levels in cultured AECs. In addition, DCM treatment downregulated NQO1 expression and disrupted signalling pathways associated with SARS-CoV-2 disease outcomes (e.g., Endocytosis and COVID-19 signalling pathways) in cultured AECs. Collectively, we demonstrated that DCM is an effective post-exposure prophylactic for SARS-CoV-2 infection in the human AECs, and these findings could help physicians formulate novel treatment strategies for COVID-19.
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COVID-19 , Dicumarol , Humanos , SARS-CoV-2 , COVID-19/genética , EpitelioRESUMEN
Neutron scattering instruments play an important role in studying the inner structure of materials. A neutron beam monitor is a detector commonly used in a neutron scattering instrument. The detection efficiency for most neutron beam monitors is quite low (10-4-10-6). However, in some experiments with a low neutron flux, such as small angle neutron scattering (SANS) and inelastic neutron scattering experiments, a neutron beam monitor with a higher detection efficiency (â¼1% for thermal neutrons) is required to reduce the duration of the experiment. To meet this requirement, a ceramic gas electron multiplier-based neutron beam monitor equipped with a 1 µm 10B4C neutron converter was developed in this study. Its performance was determined both experimentally and in simulations. The detection efficiency in the wavelength range of 1.8-5.5 Å was measured experimentally and was confirmed by the simulation results. An algorithm based on event selection and position reconstruction was developed to improve the spatial resolution to about 1 mm full-width-half-maximum. The wavelength spectrum was measured in beamline 20 (BL20) and agreed well with the results obtained using a commercial monitor. The maximum counting rate was 1.3 MHz. The non-uniformity over the whole 100 × 100 mm2 active area was determined to be 1.4%. Due to the excellent performance of this monitor, it has been used in several neutron instruments, such as the SANS and the High-Energy Direct-Geometry Inelastic Spectrometer instruments in the China spallation neutron source.
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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.
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Fertilización In Vitro , Hormona Liberadora de Gonadotropina/agonistas , Hormona Liberadora de Gonadotropina/antagonistas & inhibidores , Infertilidad Femenina/terapia , Inducción de la Ovulación/métodos , Adulto , Gonadotropina Coriónica/administración & dosificación , Gonadotropina Coriónica/uso terapéutico , Protocolos Clínicos , Transferencia de Embrión , Estradiol/sangre , Femenino , Hormona Folículo Estimulante/sangre , Hormona Liberadora de Gonadotropina/administración & dosificación , Gonadotropinas/administración & dosificación , Gonadotropinas/uso terapéutico , Antagonistas de Hormonas/administración & dosificación , Antagonistas de Hormonas/uso terapéutico , Humanos , Síndrome de Hiperestimulación Ovárica/epidemiología , Síndrome de Hiperestimulación Ovárica/prevención & control , Embarazo , Índice de Embarazo , Resultado del TratamientoRESUMEN
BACKGROUND: Human sperm concentration and motility have dropped dramatically (50%) in the past few decades, and environmental factors are involved in this decline. Long non-coding RNAs (lncRNA) have been discovered to be involved in many cellular processes including spermatogenesis. OBJECTIVE: This investigation aimed to explore the role of lncRNA8276 in murine spermatogenesis. MATERIALS AND METHODS: The expression of lncRNA8276 was modified by knockdown or overexpression in mouse testes and spermatogonial stem cells (C18-4 cell line). Sperm quality was determined in the F0 and F1 generations of mice. Furthermore, the underlying mechanisms were studied through gene expression and/or protein expression of spermatogenesis-related genes and cell junction-related genes by different methods. RESULTS: In the current investigation, we discovered that sperm lncRNA8276 was decreased by NH3 /H2 S in three generations (F0, F1, and F2) of mouse sperm. In vivo testicular knockdown of lncRNA8276 led to a decline in sperm concentration and motility in both F0 (muF0) and F1 (muF1) generations Moreover, knockdown lncRNA8276 decreased the gene and protein levels of important genes related to cell-cell junctions and spermatogenesis. The data were further confirmed in mouse spermatogonia stem cell line C18-4 cells through knockdown of lncRNA8276. DISCUSSION AND CONCLUSION: Our study suggests that lncRNA8276 may be involved in cell-cell junction formation in the mouse testis to regulate spermatogenesis. It may be a target for the modification of spermatogenesis and male fertility, or male contraception. This investigation offers a potential therapeutic strategy for male infertility.
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Adhesión Celular , ARN Largo no Codificante , Espermatogénesis , Animales , Adhesión Celular/genética , Humanos , Masculino , Ratones , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Semen , Espermatogénesis/genética , Espermatogonias , Testículo/metabolismoRESUMEN
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.
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Antineoplásicos Alquilantes/toxicidad , Diferenciación Celular/efectos de los fármacos , Ciclofosfamida/toxicidad , Células Madre Embrionarias/efectos de los fármacos , Miocitos Cardíacos/efectos de los fármacos , Biomarcadores/metabolismo , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Humanos , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , ARN Mensajero/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Pruebas de ToxicidadRESUMEN
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.
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Células Madre Embrionarias/citología , Ghrelina/metabolismo , Miocitos Cardíacos/citología , Receptores de Ghrelina/metabolismo , Diferenciación Celular , Línea Celular , Células Madre Embrionarias/metabolismo , Humanos , Miocitos Cardíacos/metabolismoRESUMEN
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.
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Criopreservación , Oocitos/fisiología , Resultado del Embarazo , Preservación de Semen , Inyecciones de Esperma Intracitoplasmáticas , Adulto , Azoospermia/terapia , Transferencia de Embrión , Femenino , Humanos , Masculino , Embarazo , Donantes de TejidosRESUMEN
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.
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Mutación Puntual , Insuficiencia Ovárica Primaria/genética , Receptores de HFE/genética , Adulto , Pueblo Asiatico/genética , Secuencia de Bases , Estudios de Casos y Controles , China , ADN/química , ADN/genética , Análisis Mutacional de ADN , Electroforesis en Gel de Poliacrilamida , Exones , Femenino , Frecuencia de los Genes , Humanos , Reacción en Cadena de la Polimerasa/métodos , Insuficiencia Ovárica Primaria/etnologíaRESUMEN
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.
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Embrión de Mamíferos/citología , Neuronas/citología , Células Madre/citología , Antígenos de Diferenciación/análisis , Astrocitos/química , Astrocitos/citología , Astrocitos/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Linaje de la Célula/efectos de los fármacos , Embrión de Mamíferos/efectos de los fármacos , Factor 2 de Crecimiento de Fibroblastos/farmacología , Proteína Ácida Fibrilar de la Glía/análisis , Humanos , Inmunohistoquímica , Neuronas/química , Neuronas/efectos de los fármacos , Oligodendroglía/química , Oligodendroglía/citología , Oligodendroglía/efectos de los fármacos , Factor de Crecimiento Derivado de Plaquetas/farmacología , Isoformas de Proteínas/análisis , Serotonina/análisis , Células Madre/efectos de los fármacos , Sinaptofisina/análisis , Tubulina (Proteína)/análisis , Ácido gamma-Aminobutírico/análisisRESUMEN
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.
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Diferenciación Celular/efectos de los fármacos , Embrión de Mamíferos/patología , Células Madre Embrionarias/patología , Cariotipificación/métodos , Células Madre/patología , Técnicas de Cultivo de Célula , Humanos , Antígenos Embrionarios Específico de EstadioRESUMEN
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.
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Desarrollo Embrionario , Células Madre Embrionarias/citología , Células Madre Pluripotentes/citología , Técnicas de Cultivo de Célula/métodos , Diferenciación Celular , Proliferación Celular , Células Cultivadas , HumanosRESUMEN
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.
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Blastocisto/citología , Células Madre Embrionarias/citología , Animales , Técnicas de Cultivo de Célula , Línea Celular , Separación Celular/métodos , Células Clonales/citología , Femenino , Ratones , Ratones Endogámicos ICR , EmbarazoRESUMEN
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.
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Criopreservación/métodos , Crioprotectores/farmacología , Oocitos/efectos de los fármacos , Huso Acromático/efectos de los fármacos , Animales , Dimetilsulfóxido/farmacología , Glicol de Etileno/farmacología , Femenino , Técnica del Anticuerpo Fluorescente , Microscopía Confocal , Oocitos/citología , Oocitos/metabolismo , Conejos , Huso Acromático/metabolismoRESUMEN
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.