Human umbilical cord Wharton's jelly stem cells and its conditioned medium support hematopoietic stem cell expansion ex vivo.

Bone marrow mesenchymal stromal cells (BMMSCs) have been used as feeder support for the ex vivo expansion of hematopoietic stem cells (HSCs) but have the limitations of painful harvest, morbidity, and risk of infection to the patient. This prompted us to explore the use of human umbilical cord Wharton's jelly MSCs (hWJSCs) and its conditioned medium (hWJSC-CM) for ex vivo expansion of HSCs in allogeneic and autologous settings because hWJSCs can be harvested in abundance painlessly, are proliferative, hypoimmunogenic, and secrete a variety of unique proteins. In the presence of hWJSCs and hWJSC-CM, HSCs put out pseudopodia-like outgrowths and became highly motile. Time lapse imaging showed that the outgrowths helped them to migrate towards and attach to the upper surfaces of hWJSCs and undergo proliferation. After 9 days of culture in the presence of hWJSCs and hWJSC-CM, MTT, and Trypan blue assays showed significant increases in HSC numbers, and FACS analysis generated significantly greater numbers of CD34(+) cells compared to controls. hWJSC-CM produced the highest number of colonies (CFU assay) and all six classifications of colony morphology typical of hematopoiesis were observed. Proteomic analysis of hWJSC-CM showed significantly greater levels of interleukins (IL-1a, IL-6, IL-7, and IL-8), SCF, HGF, and ICAM-1 compared to controls suggesting that they may be involved in the HSC multiplication. We propose that cord blood banks freeze autologous hWJSCs and umbilical cord blood (UCB) from the same umbilical cord at the same time for the patient for future ex vivo HSC expansion and cell-based therapies.

Statins inhibit the growth of variant human embryonic stem cells and cancer cells in vitro but not normal human embryonic stem cells.

BACKGROUND AND PURPOSE: Statins inhibit proliferation of various human cancer cell lines in vitro. As human embryonic stem cells (hESCs) possess neoplastic-like properties we have evaluated the role of various statins on karyotypically normal hESCs (HES3 and BG01), abnormal hESCs (BG01V) and breast adenocarcinoma cells (MCF-7) to evaluate whether the mode of action of the statins was via a stemness pathway. EXPERIMENTAL APPROACH: All cell lines were treated with simvastatin, pravastatin, lovastatin and mevastatin (1 micromol x L(-1) to 20 micromol x L(-1)) up to 7 days and their effects on cell proliferation, cell cycle, apoptosis and pluripotency studied. KEY RESULTS: All four statins did not inhibit HES3 and BG01 proliferation, but BG01V and MCF-7 were inhibited by simvastatin, lovastatin and mevastatin. These inhibitory effects were reversed by the endogenous isoprenoids, farnesylpyrophosphate and geranylgeranylpyrophosphate. Terminal deoxynucleotidyl transferase biotin-dUTP nick end labelling and cell cycle assay confirmed apoptosis in BG01V and MCF-7. Stem cell surface markers [stage-specific embryonic antigen-4, tumour rejection antigen-1-81, octamer-4 (OCT-4)] were expressed in HES3 and BG01, but not in BG01V cells, even after prolonged treatment with simvastatin. In BG01V and MCF-7, the pro-apoptotic Bcl-2-associated X protein genes were up-regulated, while the antiapoptotic BCL2 and SURVIVIN genes were down-regulated. Expression of the stemness-related genes namely, the growth differentiation factor-3, NANOG and OCT-4 was decreased in BG01V compared with BG01 and HES3. CONCLUSIONS AND IMPLICATIONS: Normal hESCs were resistant to prolonged exposure to statins over a range of doses, compared with BG01V and MCF-7, probably because of genetic and behavioural differences. The statins not only have anti-cancer properties but can suppress abnormal hESCs thus promoting growth of normal hESCs in vitro.

Comparative growth behaviour and characterization of stem cells from human Wharton's jelly.

Human embryonic stem cells (hESC) face ethical sensitivities and the problem of teratoma formation. Although Wharton's jelly stem cells (WJSC), also of embryonic origin, may not face such ethical concerns, it is not definitely known whether under hESC culture conditions they would be as pluripotent as hESC. WJSC grown on plastic showed two types of morphology (epithelioid and short fibroblastic) in primary culture depending on the culture medium used, and only fibroblastic morphology when passaged. When grown in the presence of hESC medium on mouse feeder cells, they produced atypical colonies containing hESC-like cells with high-nuclear cytoplasmic ratios and prominent nucleoli. They were positive for the hESC markers Tra-1-60, Tra-1-81, SSEA-1, SSEA-4, Oct-4 and alkaline phosphatase, negative for SSEA-3, showed normal karyotypes, developed embryoid body (EB)-like structures, did not produce teratomas in SCID mice and differentiated into neuronal derivatives. They were also positive for the mesenchymal CD markers (CD105, CD90, CD44), negative for CD34 and HLA, and although nine out of 10 embryonic stem cell genomic markers were detectable, these were expressed at low levels. WJSC are thus not as pluripotent as hESC but widely multipotent, and have the advantages of being able to be scaled up easily and not inducing teratomas.

Nine-day-old human embryo cultured in vitro: a clue to the origins of embryonic stem cells.

The aims of this study were to investigate whether the human embryo could sustain development beyond the blastocyst stage in vitro and to identify the precise origins of embryonic stem cells (ES cells) from the embryoblast. A frozen-thawed 4-cell embryo was cultured to the post-blastocyst stage. This 9-day-old embryo presented a solid mass of inner cells (resembling a tumour) surrounded by surface trophoblast cells. Clumps of multinucleated syncytiotrophoblast cells were evident at one pole. Most cells resembled those of blastocysts. However, there were groups of comparatively undifferentiated cells within the inner cell mass somewhat resembling ES cells documented previously, that might give a clue as to their origins. The embryo attempted to form an amnion with a cavity, but did not present a bilaminar, discoidal structure as expected in week 2 of development, and hence was abnormal.

Does sperm morphology play a significant role in increased sex chromosomal disomy? A comparison between patients with teratozoospermia and OAT by FISH.

Eight semen samples from men with teratozoospermia (T), along with samples from 3 men with normal fertility and 5 samples from men with oligoasthenoteratozoospermia (OAT) were analyzed for X and Y chromosomal anomalies with the use of fluorescently labeled centromeric probes. This study was undertaken to determine whether patients with abnormal sperm morphology (teratozoospermia) have increased or decreased incidence of a sex chromosomal anomaly. The sex chromosome disomy for the T samples was 0.36% and for the OAT group it was 0.61%, compared with baseline value for the normozoospermia group (0.09%).

Comparative evaluation of fresh and washed human sperm cryopreserved in vapor and liquid phases of liquid nitrogen.

Infectious organisms such as hepatitis B were recently shown to survive in liquid nitrogen. To prevent cross-contamination of semen samples via liquid nitrogen, studies were undertaken to evaluate human sperm survival in the vapor phase of liquid nitrogen at -189 degrees C. The study was conducted in 2 separate experiments. In the first experiment, a total of 30 unwashed, fresh semen samples (15 normozoospermic and 15 oligozoospermic) were evaluated for motility, vitality, and morphology after freeze-thaw survival in vaporous (-189 degrees C) and liquid nitrogen (-196 degrees C; control) phases. Similar evaluations were carried out in a second experiment on 27 samples (15 normozoospermic and 12 oligozoospermic) that were previously washed by the swim-up method. Motile sperm recovery rates were significantly different between liquid and vapor phases (unwashed, normozoospermic: 42.76% +/- 3.23% vs 45.52% +/- 4.44%, P < .05; washed, normozoospermic: 34.44% +/- 4.41% vs 37.58% +/- 3.90%, P < .05; unwashed, oligozoospermic: 16.53% +/- 3.34% vs 18.25% +/- 4.36%, P < .05; washed, oligozoospermic: 10.32% +/- 2.54% vs 12.25% +/- 2.81%, P < .05). Recovery rates for motility were much higher for unwashed samples compared with washed semen samples. In all experiments the recovery of normal and live forms showed no significant differences between the vapor and liquid nitrogen storage phases (P > .05). The results demonstrate that both washed and unwashed human sperm survive satisfactorily with good recovery in the vapor of liquid nitrogen and can be recommended for future storage in medically assisted conception programs.

Myocyte transplantation for myocardial repair: a few good cells can mend a broken heart.

Cell transplantation is a potential therapeutic approach for patients with chronic myocardial failure. Experimental transplantation of neonatal and fetal cardiac myocytes showed that the grafted cells can functionally integrate with and augment the function of the recipient heart. Clinical application of this approach will be limited by shortage of donors, chronic rejection, and because it is ethically contentious. By contrast skeletal myoblasts (satellite cells) are abundant and can be grafted successfully into the animal's own heart even after genetic manipulation in vitro. Functional integration of myoblasts, however, is hampered by the lack of intercellular gap junction communication and the difference in excitation-contraction coupling between skeletal and cardiac myocytes. In experimental studies several other cell types have been used to augment cardiac function. In this review we discuss the published results of myocyte transplantation with emphasis on potential sources of cells, the ethics of using donor embryonic and fetal cardiomyocytes, genetic transformation of skeletal myoblasts for myocardial repair, and the functional benefits of cell transplantation to the failing heart.

Ultrastructural observations of enzymatically treated human blastocysts: zona-free blastocyst transfer and rescue of blastocysts with hatching difficulties.

Enzymatic treatment of the zona pellucida to either soften or remove totally the zona before blastocyst transfer has resulted in high implantation rates. The zona is usually completely dissolved after 1.5 min exposure with 10 IU pronase at 37 degrees C. Since there may be concerns that pronase treatment for periods of 1.5 min or longer may cause adverse effects on the trophectoderm (TE) and inner cell mass (ICM), the changes to human blastocysts exposed to different time intervals of pronase were investigated. Of 18 blastocysts exposed to pronase for 1.5 min, the zona was completely dissolved and no changes were observed by light microscopy (LM) or transmission electron microscopy (TEM), compared with 11 naturally hatched untreated blastocysts (controls). In another five blastocysts exposed to pronase for 2 min, no LM changes were observed but subtle TEM changes such as fewer bundles of tonofibrils attached to desmosomes were observed. When three other blastocysts were exposed to pronase for 5 min, the blastocoele collapsed, and the TE cells started to show blebbing under LM. Under TEM, the cytoplasm of TE cells was extensively vacuolated and many TE cells showed cytoplasmic blebbing towards the blastocoele. However, the epithelium was uninterrupted with intact tight junctions and desmosomes. Of a separate group of 44 blastocysts cultured in vitro, 54.5% had hatching difficulties when monitored from day 5 to day 8 and 80% of these could be rescued by removal of the zona with pronase for 1.5 min prior to extensive degeneration taking place. The results confirm that the optimal time for softening or complete removal of the zona before transfer was around 1.5 min and that enzymatic treatment was a safe, non-invasive procedure to remove the zona of blastocysts. The human embryonic TE is a very hardy, robust epithelium that withstands pronase treatment.

Discrepancy between cytogenetic and FISH results on an amniotic fluid sample of 45,X/46,X,idic(Y)(p11).

The presence of abnormal ultrasound markers showing a thick nuchal fold with short middle phalanx of the fifth finger in an otherwise normal-appearing female fetus led to the sampling of amniotic fluid at 16 weeks gestation. Cytogenetic analysis with routine G-banding showed a 45,X karyotype in all 20 cells analysed from two flasks. However, fluorescent in situ hybridization on uncultured cells showed presence of a Y signal in 9 cells, 11 cells showing a single signal for the X. A cytogenetic analysis of the fetal blood at 23 weeks confirmed the presence of two cell lines, 45,X and 46,X, idic(Y)(p11). The couple opted to have the pregnancy terminated. However, the fetus was not available to carry out confirmatory tests.

Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro.

We describe the derivation of pluripotent embryonic stem (ES) cells from human blastocysts. Two diploid ES cell lines have been cultivated in vitro for extended periods while maintaining expression of markers characteristic of pluripotent primate cells. Human ES cells express the transcription factor Oct-4, essential for development of pluripotential cells in the mouse. When grafted into SCID mice, both lines give rise to teratomas containing derivatives of all three embryonic germ layers. Both cell lines differentiate in vitro into extraembryonic and somatic cell lineages. Neural progenitor cells may be isolated from differentiating ES cell cultures and induced to form mature neurons. Embryonic stem cells provide a model to study early human embryology, an investigational tool for discovery of novel growth factors and medicines, and a potential source of cells for use in transplantation therapy.

Regulation of human and mouse oocyte maturation in vitro with 6-dimethylaminopurine.

It has been postulated that premature shortening of the oocyte growth phase due to the recovery of oocytes from small diameter follicles may be responsible for the developmental anomalies associated with in-vitro maturation. 6-Dimethylaminopurine (DMAP) was used to artificially lengthen the pre-maturation period of oocyte growth, in vitro, by inhibiting germinal vesicle breakdown in mouse and human oocytes. DMAP inhibited the meiotic maturation of mouse and human oocytes and the inhibition was fully reversible. The timing of polar body extrusion was accelerated in mouse oocytes following the withdrawal of DMAP; however, the kinetics of nuclear maturation in human oocytes was unaffected by exposure to DMAP. All mouse and human DMAP-treated oocytes that matured to metaphase II expressed histone H1 kinase activity. Fertilization rates in both DMAP-treated and control mouse and human oocytes were comparable, and human embryonic development was similar in control and DMAP-treated oocytes. However, blastocyst development was significantly reduced in DMAP-treated mouse oocytes (P < 0.05). It is concluded that lengthening the prematuration growth phase, by temporarily inhibiting kinase activity with DMAP, does not directly improve oocyte developmental competence but provides a useful tool for further investigating meiotic and developmentally related events in vitro by manipulating meiotic resumption.

Effects of peritoneal macrophages from women with endometriosis on endometrial cellular proliferation in an in vitro coculture model.

OBJECTIVE: To study the effects of peritoneal macrophages on endometrial cellular proliferation in an in vitro coculture model and to compare the magnitude of these effects between macrophages from women with endometriosis and normal women. DESIGN: Controlled study of peritoneal macrophage function. SETTING: University hospital. PATIENT(S): Patients with a normal peritoneal cavity (n = 15) and with pelvic endometriosis (n = 20) undergoing laparoscopy. INTERVENTION(S): Peritoneal macrophages were cocultured with endometrial epithelial and stromal cells; endometrial cell cultures without macrophage coculture acted as controls. MAIN OUTCOME MEASURE(S): Endometrial cellular proliferation measured by 3H-thymidine incorporation. RESULT(S): Endometrial epithelial cells cocultured with peritoneal macrophages from women with endometriosis showed significantly increased proliferation compared with cocultures using macrophages from normal women when assessed at 24 hours (1.56 versus 1.03 times, respectively, over control) and at 72 hours (1.55 versus 1.10 times over control). Endometrial stromal cells cocultured with peritoneal macrophages from women with endometriosis similarly exhibited increased proliferation compared with cocultures using macrophages from normal women when assessed at 24 hours (1.65 versus 1.17 times over control) and at 72 hours (1.65 versus 1.21 times over control). CONCLUSION(S): Peritoneal macrophages of patients with endometriosis stimulate cellular proliferation of endometrial epithelial and stromal cells in vitro.

Comparative evaluation of two density gradient preparations for sperm separation for medically assisted conception.

To evaluate and optimize the sperm separation efficiency of a novel silane-coated silica bead (Puresperm), serial studies were carried out to compare the various sperm parameters between: (i) three-layer (90%-70%-40%) Puresperm and three-layer (90%-70%-40%) conventional polyvinylpyrrolidone (PVP)-coated silica bead (Percoll) gradients; (ii) three-layer (90%-70%-40%) and two-layer (90%-45%) Puresperm gradients and separately the same for Percoll; and (iii) large (3.0 ml) and small (0.75 ml) semen loading volumes on three-layer Puresperm gradients. Normozoospermic semen samples were treated and analysed in 12 replicates for each experiment. Manual evaluation of concentration, percentage motility, percentage vitality, percentage normal morphology; computer-assisted semen analysis evaluation of concentration, percentage motility, grade of motility, motion characteristics (curvilinear velocity, linearity, amplitude of lateral head velocity, beat cross frequency, percentage hyperactivation); and yields from the initial semen samples were compared. Percoll was found to be superior to Puresperm in concentration, percentage motility, percentage vitality and yields after three-layer density gradient centrifugation. There were no significant differences in sperm parameters between two- and three-layer Percoll gradients, but three-layer Puresperm gradients behaved significantly better than two-layer gradients. Large semen volume loads on three-layer Puresperm gradients resulted in greater sperm concentrations, percentage motility, percentage vitality and percentage normal morphology, but small semen volume loads produced greater yields of good-quality spermatozoa. In the light of Percoll being withdrawn from the shelf for the use of assisted reproduction because of the presence of PVP, three-layer Puresperm gradients with large semen loading volumes appear to be an attractive alternative for sperm separation in medically assisted conception.

Comparison of human blastulation rates and total cell number in sequential culture media with and without co-culture.

Recent interest in delayed embryo transfers necessitated the evaluation of two improved in-vitro systems that could generate viable blastocysts. A total of 178 two-pronucleated embryos (entire cohorts) from 19 patients was cultured in IVF50 medium (100 microl) under oil for 24 h until day 2. Each patient's day 2 embryos were then equally allotted to two in-vitro systems. Embryos in system A were grown until the morning of day 3 on Vero cells covered with IVF50 medium (100 microl) under oil. The medium was then replaced on day 3 with a 1:1 mixture (100 microl) of IVF50:S2 medium and on day 4 with S2 medium only. The same culture protocol was used for system B without Vero cells. Throughout the 5 days all dishes were housed in sealed humidified modular chambers containing a triple gas atmosphere. Separately, 175 spare embryos from 80 patients were grown in system A and B up to days 6 and 7 for total cell number (TCN) analysis. Blastulation rates were not significantly different between system A and B (67.4 versus 68.5%; P > 0.01) although co-cultured embryos cleaved slightly faster by day 4. The overall pregnancy and implantation rates were 52.0% and 32.1% for the 19 patients each of whom received a mixed cohort of three day 5 embryos from both systems. TCN values for the day 6 and 7 blastocysts from both systems were high and increased steadily from days 6-7 and from expanded to hatching stages. There were no significant differences in TCN for day 6 expanded blastocysts between the two systems although day 6 hatching and hatched co-cultured blastocysts had greater values than non-co-cultured blastocysts (246.0 +/- 18.5 and 236.7 +/- 17.8 versus 173.0 +/- 13.5 and 166.5 +/- 16.0; P < 0.01). The results demonstrated that the culture protocol using the sequential IVF50-S2 media combination was a good substitute for Vero cell co-culture for the transfer of viable day 3-6 embryos.

Blastocyst transfer after enzymatic treatment of the zona pellucida: improving in-vitro fertilization and understanding implantation.

It has been shown recently that delayed transfers improve implantation rates in assisted reproductive technology programmes. In a prospective study, the pregnancy rates and safety of outcome were evaluated in a group of patients after the transfer of day 5 blastocysts with enzymatic treatment of the zona pellucida. Nineteen women with a mean age of 32.6+/-5.2 years and mean 2.1+/-2.2 repeated attempts had blastocyst transfers with a mean number of 2.5+/-0.7 embryos replaced per patient. The clinical pregnancy rates per cycle/transfer and implantation rate were 53% and 33%, respectively. The multiple pregnancy rate was 40% (two pregnancies were triplets). The pregnancy and implantation rates were very much higher than observed for most assisted reproduction technology centres. The 'in-vitro implantation' rates of zona-free blastocysts on a variety of feeder monolayers was 92%, offering some thoughts as to the role of the zona and interaction of the inner cell mass and trophoectoderm with the endometrium in implantation. Based on the in-vitro studies and the high multiple pregnancy rates, it appears that zona-manipulated blastocysts implant relatively well and there would be a need to reduce the number of transferred embryos to one or two, thus reducing multiple pregnancies and having spare blastocysts available for cryopreservation. The results also suggest that using the embryo culture protocol and method of transfer in the present study offers encouraging improvements to assisted reproduction technology, and enzymatic treatment of the zona may allow better anchorage and dialogue of the embryo with the endometrium, helping us to improve and understand implantation.

The first 2 case reports of frozen embryo donation twin pregnancies in Singapore: hormonal profiles and obstetrical outcome.

A 35 year old women with premature ovarian failure and another 30 year old women with gonadal dysgenesis were the recipients of donated supernumerous frozen embryos after successfully prepared with cyclic steroid replacement therapy as described previously. One patient received 4 and the second 2 frozen embryos, transferred transcervically on the 3rd day of progesterone administration. Both patients had viable twin pregnancies. The plasma beta hCG levels for both patients at 2 weeks post replacement (4 weeks gestation) were lower than the median values in our normal, uncomplicated singleton pregnancy for the same gestation. The level after 4 weeks post-replacement (6 weeks gestation) became comparable. Plasma progesterone profiles suggested a level of above 70 ng/ml would be enough to support the twin pregnancies. The first patient developed antepartum haemorrhage of unknown origin at 34 weeks of gestation preceding preterm premature rupture of membranes and subsequently had preterm labour. The second patient developed proteinuric hypertension at 33 weeks of gestation. Both ended in a lower segment cesarean section. Both sets of twins and their mothers were discharged well.

Ongoing normal pregnancy after transfer of zona-free blastocysts: implications for embryo transfer in the human.

Recent reports suggest that transfer of day 5 blastocysts improves implantation rates in in-vitro fertilization programmes. This paper reports a successful ongoing pregnancy after the transfer of zona-free day 6 expanded and hatching blastocysts. The patient was 37 years old and had undergone six stimulated and two thaw cycles previously, all of which had failed. Stimulation was by down-regulation and oocytes recovered transvaginally by ultrasound guidance. Two pronuclear embryos were co-cultured on Vero cells to day 6. The zonae of two hatching and two fully expanded blastocysts were removed using 0.5% pronase, and the zona-free blastocysts were then transferred. Pregnancy was confirmed on day 18 with a positive human chorionic gonadotrophin (HCG) and ultrasound at 6 weeks showed a single healthy fetal heart inside a clear sac. At 14 weeks a triple test (oestriol, J-HCG and alpha-fetoprotein) was normal and at 22 weeks a detailed ultrasound scan showed no congenital anomalies. This is the first report in the human of a normal ongoing pregnancy after the transfer of zona-free day 6 embryos.

Analysis of cystic hygroma, ascitic, and pleural fluids by conventional lymphocyte culture and fluorescent in situ hybridization.

Serous fluids from cystic hygromas, pleural effusions, and ascites are an easily accessible and plentiful source of lymphocytes. The feasibility and reliability of using these as alternative sources to conventional amniotic fluid or fetal blood cultures have been studied here. In some cases of prenatal diagnosis, especially in pregnancies complicated by the presence of cystic hygromas and fetal hydrops, obtaining amniotic fluid or fetal blood can be difficult due to obstruction by the cyst or oligohydramnios. A total of 14 cases with fetal hydrops detected ultrasonigraphically between 15 and 33 weeks of pregnancy over a period of 1 year have been subjected to conventional amniotic fluid or fetal blood karyotyping, along with samples of fluids from cystic hygromas, ascites or pleural effusions as obtained. Pleural fluids (n = 4), cystic hygroma fluids (n = 5), and ascitic fluids (n = 6) were obtained. The culture failure rate was low, 2/14. Karyotypically, two of the fluids, both from cystic hygromas, were 45,X; the rest were normal. A rapid 1-day additional test of fluorescent in situ hybridization (FISH) was carried out on uncultured cells of the alternative fluids using probes for the most commonly occurring aneuploidies, 13, 18, 21, X and Y, with good results.

Fluorescent in-situ hybridization--some of its applications in clinical cytogenetics.

Fluorescent in-situ hybridization (FISH) is becoming more and more relevant as an important future tool in prenatal and pre-implantation genetic diagnosis and cancer cytogenetics. This review describes the FISH technique as applied to whole chromosome spreads and interphase cells and discusses its applications in clinical cytogenetics. Information is presented on the various types of probes and the subsequent hybridization and detection procedures. The potential use of this novel FISH technique in the diagnosis of numerical and structural chromosomal aberrations in routine karyotyping for prenatal diagnosis, tumour cytogenetics and pre-implantation genetic diagnosis is outlined.