Biomedical subjects

Outi Hovatta

Publications and source records attributed to Outi Hovatta.

At least 19 recordsLinked to original sources

PubMed2007

A meta-analysis of human embryonic stem cells transcriptome integrated into a web-based expression atlas.

Microarray technology provides a unique opportunity to examine gene expression patterns in human embryonic stem cells (hESCs). We performed a meta-analysis of 38 original studies reporting on the transcriptome of hESCs. We determined that 1,076 genes were found to be overexpressed in hESCs by at least three studies when compared to differentiated cell types, thus composing a "consensus hESC gene list." Only one gene was reported by all studies: the homeodomain transcription factor POU5F1/OCT3/4. The list comprised other genes critical for pluripotency such as the transcription factors NANOG and SOX2, and the growth factors TDGF1/CRIPTO and Galanin. We show that CD24 and SEMA6A, two cell surface protein-coding genes from the top of the consensus hESC gene list, display a strong and specific membrane protein expression on hESCs. Moreover, CD24 labeling permits the purification by flow cytometry of hESCs cocultured on human fibroblasts. The consensus hESC gene list also included the FZD7 WNT receptor, the G protein-coupled receptor GPR19, and the HELLS helicase, which could play an important role in hESCs biology. Conversely, we identified 783 genes downregulated in hESCs and reported in at least three studies. This "consensus differentiation gene list" included the IL6ST/GP130 LIF receptor. We created an online hESC expression atlas, http://amazonia.montp.inserm.fr, to provide an easy access to this public transcriptome dataset. Expression histograms comparing hESCs to a broad collection of fetal and adult tissues can be retrieved with this web tool for more than 15,000 genes.

Cell Differentiation↗

PubMed2007

Neurogenic neuroepithelial and radial glial cells generated from six human embryonic stem cell lines in serum-free suspension and adherent cultures.

The great potential of human embryonic stem (hES) cells offers the opportunity both for studying basic developmental processes in vitro as well as for drug screening, modeling diseases, or future cell therapy. Defining protocols for the generation of human neural progenies represents a most important prerequisite. Here, we have used six hES cell lines to evaluate defined conditions for neural differentiation in suspension and adherent culture systems. Our protocol does not require fetal serum, feeder cells, or retinoic acid at any step, to induce neural fate decisions in hES cells. We monitored neurogenesis in differentiating cultures using morphological (including on-line follow up), immunocytochemical, and RT-PCR assays. For each hES cell line, in suspension or adherent culture, the same longitudinal progression of neural differentiation occurs. We showed the dynamic transitions from hES cells to neuroepithelial (NE) cells, to radial glial (RG) cells, and to neurons. Thus, 7 days after neural induction the majority of cells were NE, expressing nestin, Sox1, and Pax6. During neural proliferation and differentiation, NE cells transformed in RG cells, which acquired vimentin, BLBP, GLAST, and GFAP, proliferated and formed radial scaffolds. gamma-Aminobutyric acid (GABA)-positive and glutamate positive neurons, few oligodendrocyte progenitors and astrocytes were formed in our conditions and timing. Our system successfully generates human RG cells and could be an effective source for neuronal replacement, since RG cells predominantly generate neurons and provide them with support and guidance.

Cell Adhesion↗

PubMed2006

Ovarian wedge resection restores fertility in estrogen receptor beta knockout (ERbeta-/-) mice.

Ovulation rarely occurs in mice in which the estrogen receptor beta (ERbeta) gene has been inactivated (ERbeta-/- mice). Here, we investigated whether this subfertility is due to a defect in the ovary itself or to more general endocrine changes in ERbeta-/- mice. We transplanted ERbeta-/- ovaries into WT mice and WT ovaries into ERbeta-/- mice. Upon mating with ERbeta-/- males, fertility increased from 20% in control intact ERbeta-/- group to 40% in the WT recipients with ERbeta-/- ovaries. The transplantation procedure was not efficient, and when WT ovaries were transplanted into WT mice, fertility was only 36%. Surgical ovarian wedge resection, a procedure which induces ovulation in anovulatory women with polycystic ovarian syndrome, resulted in 100% fertility of ERbeta-/- mice. In ERbeta-/- mice, as the follicles enlarged, the thecal layer remained very compact (revealed by H&E and collagen staining), and there was no increase in vascularization (measured as smooth muscle actin). In addition, there was an increase in PDGF receptor alpha (PDGFRalpha) and a decrease in PDGFbeta expression in the granulosa cells, similar to what has been found in follitropin receptor knockout mice. After wedge resection, expression of both smooth muscle actin and PDGFRs was normalized. During normal follicular development, increased vascularization of the thecal layer is a prerequisite for further follicular growth. We suggest that the defect in ERbeta-/- mouse ovaries is a failure of communication between the granulosa and thecal layers. The follicles do not mature because of insufficient blood supply. This problem is overcome by stimulating neovascularization by simple wedge resection of the ovaries.

Animals↗

PubMed2006

Distinct sets of developmentally regulated genes that are expressed by human oocytes and human embryonic stem cells.

OBJECTIVE: To identify genes that are expressed differently during final oocyte maturation and early embryonic development in humans. DESIGN: Comparison of gene expression profiles of human germinal vesicle oocytes (hGVO), human embryonic stem cells (hESC) and human foreskin fibroblasts. SETTING: Research centers and a fertility unit in a university hospital. PATIENT(S): Fifty-five healthy women donated 76 hGVO. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Gene expression profiles were analyzed and compared with the use of microarray and reverse-transcription polymerase chain reaction. RESULT(S): Altogether, 10,183 genes were expressed in hGVO, and 45% of these genes were unclassified by biologic function. Four oocyte-specific genes (MATER, ZAR1, NPM2 and FIGLA) were detected in hGVO for the first time. Known components of 4 signaling pathways (MOS-MPF, transforming growth factor-beta, WNT, and NOTCH) were also found expressed in hGVO, with some components detected in hGVO for the first time. Distinct sets of genes that were revealed by comparison of expression profiles between hGVO, hESC, and human foreskin fibroblasts appear to be involved in oocyte maturation and early embryonic development. CONCLUSION(S): We obtained, for the first time, a large amount of information on gene expression of hGVO as compared with hESC. These data, from a unique research material-human oocytes, can now be used to understand the molecular mechanisms of early human development.

Adult↗

PubMed2006

Exclusion of coding-region mutations in luteinizing hormone and follicle-stimulating hormone receptor genes as the cause of ovarian hyperstimulation syndrome.

OBJECTIVE: To sequence the coding regions of the luteinizing hormone receptor (LHR) and follicle-stimulating hormone receptor (FSHR) genes to find out if polymorphisms in them are responsible for the severe form of ovarian hyperstimulation syndrome (OHSS) in Swedish patients. DESIGN: A mutation analysis of gonadotropin receptor genes from women undergoing gonadotropin treatment. SETTING: The Fertility Unit of Karolinska University Hospital Huddinge, Stockholm, Sweden. PATIENT(S): A set of 10 well-characterized patients with severe OHSS, and 10 control women who did not develop OHSS after FSH stimulation. An additional 11 patients and 41 control women were screened for a two-amino-acid insertion in the first exon of the LHR gene. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Changes in the sequence of the receptor genes between patients and controls. RESULT(S): No association was found between polymorphisms of the coding region of LHR or FSHR genes and the development of OHSS. Incidence of the two-amino-acid insertion in the first exon of the LHR gene was slightly higher in patients than in controls, but no statistically significant difference was seen. CONCLUSION: LHR and FSHR coding polymorphisms are not a major cause of severe OHSS in Swedish patients.

Adult↗

PubMed2006

Expression of leukemia inhibitory factor and its receptors is increased during differentiation of human embryonic stem cells.

OBJECTIVE: To investigate gene expression profiles during the early spontaneous differentiation of human embryonic stem cells (hESCs), with particular emphasis on leukemia inhibitory factor (LIF)-induced pathways and the ultrastructural surface morphology of the undifferentiated and spontaneously differentiated hESCs. DESIGN: Prospective experimental study. SETTING: University laboratory. PATIENT(S): Four hESC cell lines. INTERVENTION(S): The effect of LIF on receptor expression level was studied in cultures. MAIN OUTCOME MEASURE(S): Gene expression in the hESC line HS237 was analyzed using microarrays. Real-time reverse-transcription polymerase chain reaction was used to validate the microarray results in four hESC lines (HS181, HS235, HS237, HS293). Immunohistochemistry was used to assay LIF, LIF receptor, and gp130 protein expression. Cell surface morphology was studied using scanning electron microscopy. RESULT(S): The expression of LIF, LIF receptor, and gp130 messenger RNA and protein was increased in spontaneously differentiated HS237 cells compared with undifferentiated cells, with high expression of an inhibitor of LIF-mediated signaling, suppressor of cytokine signaling-1, in undifferentiated hESCs. Genes, those expressed specifically and those shared in undifferentiated hESCs, differentiated cells, and in fibroblasts, were identified. Supplementation with LIF did not affect the LIF receptor expression. CONCLUSION(S): The expression of LIF and its receptors is low in undifferentiated hESCs but increases during differentiation. Added LIF does not prevent spontaneous differentiation. Suppressor of cytokine signaling-1 may prevent LIF signaling in hESCs.

Cell Differentiation↗

PubMed2006

The derivation of clinical-grade human embryonic stem cell lines.

The pluripotent nature of human embryonic stem cells (hESC) has attracted great interest in using them as a source of cells or tissue in cell therapy. However, in order to be used in regenerative medicine, the pluripotent hESC lines should be established and propagated according to good manufacturing practice quality requirements. The cultures should be animal substance free in order to exclude the risk of infections and immunogenity. They should also be genetically and epigenetically normal. The detailed molecular mechanisms of their pluripotency are still not defined. Using human feeder cells, a medium containing only human proteins, the mechanical isolation of the inner cell mass and mechanical passaging of hESC, is a safe option until a functional defined medium containing physiological concentrations of regulatory factors is available.

Cell Line↗

PubMed2006

Coxsackievirus and adenovirus receptor (CAR) is expressed in male germ cells and forms a complex with the differentiation factor JAM-C in mouse testis.

The coxsackievirus and adenovirus receptor (CAR) is a transmembrane protein important for viral binding to target cells. Using RT-PCR, Western analysis, GST pull-down assay and indirect immunofluorescence, it was shown that CAR is expressed in male germ cells from mice, rats, and humans. CAR was detected in round spermatids in the testis as well as in purified, mature spermatozoa. The two membrane-bound isoforms of CAR occupied different subcellular sites in the acrosomal region of the spermatozoa. CAR was exposed on the surface of acrosome-reacted, but not acrosome-intact cells. Two CAR-binding proteins belonging to the ligand-of-numb protein-X (LNX) family also occupied distinct regions in spermatozoa. Finally, co-immunoprecipitation experiments demonstrated an interaction between CAR and JAM-C, a protein required for spermatid differentiation. Together, these findings imply a function for CAR in male fertility. The results also suggest that CAR in spermatozoa is inaccessible to adenovirus-based gene therapy vectors, and that the risk of germ line infection therefore is low.

Acrosome↗

PubMed2006

Glycodelin is present in pinopodes of receptive-phase human endometrium and is associated with down-regulation of progesterone receptor B.

OBJECTIVE: To test the hypothesis that glycodelin is localized on pinopodes and correlates with temporal immunostaining of leukemia inhibitory factor (LIF), LIF receptor (LIFR), and progesterone receptor B (PRB). DESIGN: Prospective clinical study. SETTING: Hospital-based reproductive health unit and research laboratories. PATIENT(S): Twenty-five healthy fertile women with normal menstrual cycles. INTERVENTION(S): Endometrial biopsy specimens were obtained from healthy fertile women in the luteal phase of the menstrual cycle. MAIN OUTCOME MEASURE(S): Immunohistochemical staining of glycodelin, ultrastructural immunostaining of glycodelin, and double staining of glycodelin and PRB. RESULT(S): Glycodelin is present in the glands when pinopodes appear. Glycodelin is localized on pinopodes but is also secreted from luminal epithelial cells regardless of pinopode formation. There was a negative correlation between glycodelin secretion from the glands and PRB staining. A weak correlation between the presence of LIFR (but not LIF) and glycodelin was found. CONCLUSION(S): Pinopode appearance, intense staining of LIFR in pinopodes and glycodelin staining in the glands are synchronized events. Down-regulation of PRB in the endometrium is concomitant with the presence of glycodelin in the endometrium, suggesting interaction.

Adult↗

PubMed2006

Human embryonic stem cells are immunogenic in allogeneic and xenogeneic settings.

Recent studies have suggested that human embryonic stem cells (HESC) are immune-privileged and may thereby circumvent rejection. The expression of immunologically active molecules was studied by DNA microarray analysis and by flow cytometry. HESC were transplanted into immunologically competent mice and traced by fluorescence in-situ hybridization (FISH) and immunohistochemistry. The ability of HESC to directly and indirectly induce immune responses in CD4+ T-cells from naive and transplanted mice was studied. Their ability to induce immune responses of human CD4+ T-cells, when cultured in the presence of dendritic cells (DC) syngeneic to responder T-cells, was also analysed. HESC demonstrated expression of HLA class I and HLA class II genes, but the cell surface expression of HLA class II molecules was low even after incubation with IFNgamma. In wild-type mice, HESC could be demonstrated by FISH until 3 days after transplantation and were surrounded by heavy infiltrates of T-cells and macrophages. HESC induced a similar immune response as human fibroblast cells (HFib) on naive and immunized T-cells, both directly and in the presence of syngeneic DC. A similar response was observed in the allogeneic setting. It is concluded that HESC are immunologically inert and do not inhibit immune responses during direct or indirect antigen presentation, and they were acutely rejected in a xenogeneic setting.

Animals↗

PubMed2006

Derivation of human embryonic stem cell lines, towards clinical quality.

Human embryonic stem (hES) cells offer an excellent source of cells for transplantation in the treatment of severe diseases. To be clinically safe, the lines have to be derived using strict quality criteria and good manufacturing practice. Animal proteins are immunogenic and may contain microbes, and they should not be used in establishing or propagating hES cells. Derivation systems have been improved towards clinical quality by establishing all 25 hES cell lines using human skin fibroblasts as feeder cells instead of mouse fibroblasts. A further 21 cell lines have been established using synthetic serum instead of fetal calf serum in the medium. In the five latest derivations, the inner cell mass was isolated mechanically instead of by immunosurgery (animal antibodies). Feeder-free derivation would be optimal, but it is not yet considered safe. Clinical-quality lines can be derived by establishing the skin fibroblast feeders in the good manufacturing practice laboratory with human serum in the medium, and by establishing the hES cells on such feeders. In this process, a serum replacement that contains only human protein can be used, the inner cell mass has to be isolated mechanically, and the colonies have to be split mechanically for passaging. Somatic cell nuclear transfer would help to overcome rejection of transplanted cells.

Cell Culture Techniques↗

PubMed2006

Prostatic volume in young adults after treatment of cryptorchidism in childhood.

OBJECTIVE: To examine factors affecting prostatic growth in young men treated for cryptorchidism in childhood. MATERIAL AND METHODS: Prostatic volume was measured in 76 formerly cryptorchid 16-28-year-old men treated by means of orchidopexy and/or hormonally for cryptorchidism in childhood. Correlations between prostatic volume and different hormonal parameters, sperm quality and body dimensions were investigated. RESULTS: There were no significant correlations between adult prostatic volume and age, body dimensions or testicular size. There was, however, a significant positive correlation between prostatic volume and semen volume (R = 0.341; p = 0.0143), as well as a negative correlation between prostatic volume and serum testosterone concentration (R = 0.459; p = 0.0008). There was a trend towards smaller prostatic volumes in men who had received human chorionic gonadotropin as hormonal treatment than in those who had been treated with surgery only (median 9.5 vs 12 ml; p = 0.1595). CONCLUSIONS: Measured prostatic volumes were small in previously cryptorchid men. However, small prostatic volumes did not result from low testosterone levels.

Adolescent↗

PubMed2006

Kit ligand and c-Kit are expressed during early human ovarian follicular development and their interaction is required for the survival of follicles in long-term culture.

The receptor tyrosine c-Kit and its cognate ligand, c-Kit ligand (KL, stem cell factor, SCF), are involved in ovarian follicular development in several animal species. We studied the expression of KL and c-Kit using in situ hybridization and immunohistochemistry in donated human ovarian cortical tissue. The KL transcripts were expressed in granulosa cells of primary follicles, whereas the expression of c-Kit was confined to the oocyte and granulosa cells in primary and secondary follicles. We employed an ovarian organ culture using firstly serum-containing and then serum-free medium to study the effects of KL and an anti-c-Kit antibody, ACK2, on the development and survival of ovarian follicles in vitro. Culture of ovarian cortical slices for 7 days resulted in a 37% increase in the number of primary follicles and a 6% increase in secondary follicles. The proportion of viable follicles decreased in all cultures. The addition of KL (1, 10 and 100 ng/ml) into the culture media did not affect the developmental stages of the follicles or the proportion of atretic follicles. Inclusion of ACK2 (800 ng/ml) in the culture medium significantly increased the proportion of atretic follicles on days 7 (49 vs 28% in control cultures) and 14 (62 vs 38%) of culture. In conclusion, c-Kit and KL are expressed in human ovaries during follicular development. Blocking the c-Kit receptor induces follicular atresia. The KL/c-Kit signaling system is likely to control the survival of human ovarian follicles during early follicular development.

Antibodies, Monoclonal↗

PubMed2006

Culture conditions for human embryonic stem cells.

Human embryonic stem cell (hESC) lines have been derived and cultured in variable conditions. The idea behind derivation of hESC lines is to use them in human cell transplantation after differentiation, but already now these cells are widely used for research purposes. Despite similarities among the established lines, important differences have been reported between them, and it has been difficult to compare the results obtained using different lines. Recent optimization of hESC culture conditions has moved from cultures on mouse embryonic fibroblasts (MEFs) in fetal bovine serum-containing medium towards feeder-free culture methods using more defined animal substance-free cultures. The aim has been to establish robust and cost-effective systems for culturing these cells and eliminate the risk of infection transmitted by animal pathogens and immunoreactions caused by animal substances in cell cultures before clinical treatment. It is important to take these modifications into account when carrying out research using these cells. It is known that culture conditions influence gene expression and, hence, probably many properties of the cells. Optimization and standardization of culture methods is needed for research as well as for clinical purposes.

Cell Culture Techniques↗

PubMed2005

Unique gene expression signature by human embryonic stem cells cultured under serum-free conditions correlates with their enhanced and prolonged growth in an undifferentiated stage.

Understanding the interaction between human embryonic stem cells (hESCs) and their microenvironment is crucial for the propagation and the differentiation of hESCs for therapeutic applications. hESCs maintain their characteristics both in serum-containing and serum-replacement (SR) media. In this study, the effects of the serum-containing and SR culture media on the gene expression profiles of hESCs were examined. Although the expression of many known embryonic stem cell markers was similar in cells cultured in either media, surprisingly, 1,417 genes were found to be differentially expressed when hESCs cultured in serum-containing medium were compared with those cultured in SR medium. Several genes upregulated in cells cultured in SR medium suggested increased metabolism and proliferation rates in this medium, providing a possible explanation for the increased growth rate of nondifferentiated cells observed in SR culture conditions compared with that in serum medium. Several genes characteristic for cells with differentiated phenotype were expressed in cells cultured in serum-containing medium. Our data clearly indicate that the manipulation of hESC culture conditions causes phenotypic changes of the cells that were reflected also at the level of gene expression. Such changes may have fundamental importance for hESCs, and gene expression changes should be monitored as a part of cell culture optimization aiming at a clinical use of hESCs for cell transplantation.

Biomarkers↗

PubMed2005

Gene expression signatures of seven individual human embryonic stem cell lines.

Identification of molecular components that define a pluripotent human embryonic stem cell (hESC) provides the basis for understanding the molecular mechanisms regulating the maintenance of pluripotency and induction of differentiation. We compared the gene expression profiles of seven genetically independent hESC lines with those of nonlineage-differentiated cells derived from each line. A total of 8,464 transcripts were expressed in all hESC lines. More than 45% of them have no yet-known biological function, which indicates that a high number of unknown factors contribute to hESC pluripotency. Among these 8,464 transcripts, 280 genes were specific for hESCs and 219 genes were more than twofold differentially expressed in all hESC lines compared with nonlineage-differentiated cells. They represent genes implicated in the maintenance of pluripotency and those involved in early differentiation. The chromosomal distribution of these hESC-enriched genes showed over-representation in chromosome 19 and under-representation in chromosome 18. Although the overall gene expression profiles of the seven hESC lines were markedly similar, each line also had a subset of differentially expressed genes reflecting their genetic variation and possibly preferential differentiation potential. Limited overlap between gene expression profiles illustrates the importance of cross-validation of results between different ESC lines.

Cell Differentiation↗

PubMed2005

Birth of a healthy infant after in vitro oocyte maturation and ICSI in a woman with diminished ovarian response: case report.

In vitro maturation of oocytes (IVM) has been developed as a treatment option for subjects with good prognosis in assisted reproduction. We present successful IVM treatment in connection with a woman from whom low numbers of embryos were obtained after repeated failed conventional IVF cycles. A 35 year old woman, after 5 years infertility and two intrauterine insemination and three conventional IVF cycles, underwent first an IVM cycle with low dose FSH stimulation, and after failure, another natural IVM cycle. Three oocytes were obtained. After 36 h of IVM the oocytes had reached metaphase II stage, and fertilization using ICSI resulted in one 4-cell stage embryo, which was transferred 2 days later. The result was an uneventful pregnancy and birth of a healthy female infant weighing 4150 g. IVM may be an option for women from whom only low numbers of oocytes are obtained after gonadotrophin stimulation.

Adult↗

PubMed2005

Optimizing cryopreservation of human testicular tissue: comparison of protocols with glycerol, propanediol and dimethylsulphoxide as cryoprotectants.

BACKGROUND: Cryopreservation of testicular tissue is an option in fertility preservation for pre-pubertal boys who will lose spermatogenic cells as a result of chemotherapy. We compared three different protocols and cryoprotectants in cryopreservation of testicular tissue. METHODS: Testicular tissue obtained from 16 infertile men was evaluated by light microscopy(LM), immunostaining against MAGE-A4, transmission electron microscopy (TEM) and organ culture. Seminiferous tubules (1312) from non-frozen (n = 16) and frozen-thawed samples (n = 34) were studied following cryopreservation using protocols with either 1,2-propanediol (PrOH), glycerol or dimethylsulphoxide (DMSO) as cryoprotectants. RESULTS: Normal structure was seen in 86 +/- 6% (mean +/- SD) of the fresh tissue. After freezing with DMSO, 70 +/- 6% and after PrOH, 37+/-3% of the tubules were judged to be good. When glycerol was used, the structure of the basal compartment of the tubules was severely damaged. The ultrastructure of the cryopreserved samples as revealed by TEM and MAGE-positive spermatogonia confirmed the findings. Cryopreserved Leydig cells maintained their morphology and ability to release testosterone in culture. CONCLUSION: DMSO as a cryoprotectant (at a 0.7 mol/l concentration) proved to maintain the structure of testicular tissue, especially spermatogonia, after cryopreservation better than PrOH or glycerol.

Adult↗