- Email: [email protected]
PS-1.4 Tracking stem cells in vivo
Abstracts - Aspire: ART Paving the way for new frontiers
factors including retinoic acid (RA), PGC enter meiosis as identified by chromosomal alignment and expression of meiotic genes. Although female PGC are more advanced in their invitro differentiation, meiosis is not successfully completed in both male and female PGC. It appears that full developmental potential of the female gamete from in-vivo- and in-vitroderived GS cells requires stimulation by factors controlling meiosis and follicular growth. The development of earlier stage in-vivo-derived PGC (E12.5) into primary and secondary oocytes requires the support of the complete cell population of the genital ridge followed by addition of growth factors (Obata et al., 2002). The presentation will include an overview on oocyte differentiation from stem cells and similarities and differences between in-vivo and in-vitro oocyte development, which may be the key to successful oogenesis in the culture dish PS-1.2 Sperm differentiation from stem cells Nayernia K NESCI and Institute of Human Genetics, University of Newcastle-upon-Tyne Stem cells offer substantial opportunities for providing welldefined differentiated cells for drug discovery, toxicology, and regenerative medicine, but the development of efficient techniques for controlling and directing their differentiation, present a substantial challenge. We developed a new promoter based genetic selection of germline stem cells (GSC). Germline stem cells, which can self-renew and generate gametes, are unique stem cells in that they are solely dedicated to transmit genetic information from generation to generation. Extensive studies on these two stem cell types in different organisms over the past few years have revealed some commonalities in the mechanisms controlling their self-renewal and differentiation. Furthermore, germline or somatic cells in various organisms and sexes also exhibit their own unique ways of regulating stem cell function. Mouse embryonic stem (ES) cells derive from the inner cell mass of the blastocyst and give rise to the three primitive embryonic layers, which later will form all the different tissue types of an adult. Embryonic stem cells are thus defined as totipotent cells. In vitro, these cells can give rise to all the somatic cells. We developed a strategy for the establishment of germline stem cell lines from embryonic stem cells. These cells are able to undergo meiosis, generate haploid male gametes in vitro and are functional, as shown by fertilization after intracytoplasmic injection into mouse oocytes. Molecular and cellular mechanisms underlying differentiation of ES to functional gametes should be elucidated in future research. In other approach, we show that bone marrow stem (BMS) cells are able to trans-differentiate into male germ cells. BMS cellderived germ cells expressed the known molecular markers of primordial germ cells. The ability to derive male germ cells from ES and BMS cells reveals novel aspects of germ cell development and opens the possibilities for use of these cells in reproductive medicine.
S-4 Reproductive BioMedicine Online, Vol. 16, Suppl. 2, April 2008
PS-1.3 Guided differentiation of human embryonic stem cells Chung H-M CHA Stem Cell Institute, Pochon CHA University College of Medicine Human embryonic stem cells (hESC) are established from the inner cell mass of preimplantation blastocysts. These cells exhibit unlimited proliferation and pluripotency, the ability to differentiate into all types of tissues, and are promising resources for regenerative medicine, which requires large numbers of a particular cell type. hESC are therefore unlimited sources for cell-based therapies if they can be successfully guided toward specific lineages with high populations. During hESC differentiation, embryoid bodies (EB), tissue-like spheroids of aggregated cells, are spontaneously formed from hESC in invitro suspension culture. Therefore, many studies have used EB to differentiate effectively hESC toward target lineages with high populations by altering their biological environments. Though several techniques have been developed, it remains challenging to efficiently differentiate hESC only toward specific lineages and to expand them to numbers required for cell therapy. Thus, differentiation and expansion have to be considered as primary goals prior to clinical applications of hESC. We focused on the development of functional endothelial or endothelial progenitor cells and their differentiation from human ES cells for therapeutic application. During the early stages of EB development, vasculogenesis takes place and blood vessels form through a multi-step process in which endothelial cell precursors differentiate, expand, and coalesce to form a network of primitive tubules. Endothelial cells play various important roles in blood vessels, including control of blood pressure, blood clotting, angiogenesis, and inflammation. Recently, many studies have reported that endothelial cell therapy is effective in regenerating damaged vessels and in curing vascular disease. Developing techniques of endothelial cell isolation and growth with high yield and purity will therefore facilitate therapies to cure various vascular diseases. In this presentation, the author will describe the development of an efficient method to obtain highly purified endothelial cells (EC) and vascular angiogenic progenitor cells (VAPC) from HESC and discuss whether HESC-EC or VAPC has therapeutic potential for the treatment of ischemia in hindlimb ischemic animal models. This work was supported by a grant (SC2190) from the Stem Cell Research Centre of the 21C Frontier R and D Program funded by the Ministry of Science and Technology, Republic of Korea. PS-1.4 Tracking stem cells in vivo Radda G Singapore Bioimaging Consortium, Head of the Department of Physiology, Anatomy and Genetics, University of Oxford The Singapore Bioimaging Consortium (SBIC) was set up in the first instance to develop facilities and research programmes for in-vivo imaging of model organisms for human disease and for coordinating, at the national level, bioimaging research in Singapore. Its main biomedical targets are cancer, metabolic medicine and regenerative medicine. Research into stem cell biology is well advanced
Abstracts - Aspire: ART Paving the way for new frontiers
in Singapore, hence the third target for SBIC. Magnetic resonance imaging and spectroscopy (MRI/MRS), optical imaging, SPECT and PET are all under development in Singapore and in principle each of these techniques can be used to monitor the fate of cells and in particular stem cells when injected into animal models. Among the different approaches, monitoring GFP labelled cells (often requiring invasive observations) and MRI have been most widely used. Some specific examples that will be discussed, including following the migration of stem cells to the infracted region in an animal model of stroke and a detailed study from Oxford on the use of iron particles for the non-invasive monitoring by MRI of bone marrow stromal cell engraftment into infarcted rodent hearts. Cardiac function can be simultaneously examined in a quantitative manner. PS-2.1 Predictive factors of ovarian reserve Howles CM Merck Serono International SA, Geneva In ART, the ideal ‘yield’ of oocytes varies according to chronological and ovarian age; in women less than 40 years, 8–10 oocytes (range: 5–14) per stimulation cycle have been considered to be adequate (Popovic-Todorovic, 2003a; Yih, 2005; van der Gaast, 2006). An excessive number of retrieved oocytes combined with an ongoing pregnancy can put the patient at risk of ovarian hyperstimulation syndrome (OHSS) (Delvigne and Rozenberg, 2002). At the other extreme, being able to predict a poor ovarian response would enable clinicians to manage both the treatment cycle and the patients’ expectations proactively (Hendriks, 2007). Selecting the correct dose of recombinant human (r-h) FSH to retrieve an ‘acceptable’ number of oocytes for ART and to achieve development of a single dominant follicle for OI is, however, complex. To predict each patient’s ovarian response to pharmaceutical agents and to individualize the starting dose of exogenous FSH, various clinical, endocrine, ovarian ultrasonographic and genetic characteristics have been explored. Several putative characteristics have been assessed together in prediction models with varied success (PopovicTodorovic, 2003a). This model was tested prospectively with a comparative group receiving 150 IU FSH/day in a twosite clinical study giving very encouraging results (PopovicTodorovic, 2003b). However, the patient population included women up to 39 years of age, which could have complicated data interpretation as patients over 35 are normally given a higher starting dose than 150 IU. With the caveats above in mind, patient-specific variables available readily that are most predictive of successful treatment have been assessed recently (Howles, 2006), these were (in order): baseline serum FSH in the early follicular phase, body mass index (BMI), age, and antral follicle count (AFC) assessed at baseline. There have also been attempts to determine predictors of response to FSH in CC resistant PCOS women. Imani et al. (2002), found that BMI, cycle history, ovarian response to clomiphene citrate as well as basal serum FSH values were all significantly correlated and a model was developed to predict the individual FSH response dose. A recent prospective study using this model found the predictive performance was poor, the FSH predictive dose being higher than the actual dose
required (Van Wely, 2006). In order to further investigate this issue, an analysis of a large database was undertaken to determine which patient factors (clinical and laboratory parameters) are related to the likelihood of successful ovulation. These data additionally confirmed the importance of BMI as a major determinant of ovulation but also revealed for the first time that higher basal FSH and basal AFC are also related to the response to a standard starting dose of 75 IU r-HFSH The results of this analysis will be presented in detail and implications for clinical management discussed (Howles, 2007). PS-2.2 Use of a simple algorithm to predict the FSH starting dose in ART patients Trew G Department of Reproductive Medicine, Hammersmith Hospital, London At present there is still considerable variation on how the prescribing physician determines the optimal gonadotrophin dose for ART stimulation, in spite of increasing pressure from medical societies and agencies for evidence-based treatment guidelines. Recently, work has been carried out to determine predictive factors of ovarian response and to development predictive models; however these include variables that are not routinely measured in standard clinical practice (e.g. total Doppler score, anti-Müllerian hormone, serum testosterone). The CONSORT (CONsistency in r-hFSH Starting dOses for Individualized tReatmenT) programme was initiated to address these issues. Data were analysed from 1378 women under the age of 35 years, who received stimulation with recombinant follicle-stimulating hormone (r-hFSH) alone for ART. Of 15 variables identified, the most important predictive factors routinely measured were identified (basal FSH, body mass index, age and antral follicle count) and used to generate an FSH starting dose algorithm, the ‘CONSORT calculator’ (Howles et al., 2006). To validate this model a prospective, open-label, multicentre study of ART 18 centres worldwide was conducted in which 172 were enrolled. All cycles were stimulated using the GONAL-f® filled-by-mass (FbM) pre-filled pen, which provides precise and accurate dosing and enables FSH dosing in relatively small increments. Of these, after down-regulation with a GnRH agonist (long protocol), 165 were allocated and received the following seven different individualized starting doses of r-hFSH FbM according to the CONSORT calculator: 75 IU (n = 48); 112.5 IU (n = 45); 150 IU (n = 34); 187.5 IU (n = 24), 225 IU (n = 10), 262.5 IU (n = 2), 300 IU (n = 2). Based on statistical considerations, only dosing groups with at least five patients per group were analysed (ITT population, n = 161). The overall median number of embryos transferred was 2.0. Clinical pregnancy rates per cycle started were 31.3%, 31.1%, 35.3%, 50.0% and 20% per r-hFSH FbM starting dose of 75 IU, 112.5 IU, 150 IU, 187.5 IU and 225 IU, respectively (overall mean pregnancy rate was 34.2%, n = 161). One early and one late onset case of severe OHSS were reported, in patients allocated to the 75 IU and 187.5 IU group respectively. Further results of the study will be presented in detail during the presentation. Overall, by using the CONSORT calculator an adequate oocyte yield was achieved, accompanied by high pregnancy rates. Notably, over 90% of patients required
S-5 Reproductive BioMedicine Online, Vol. 16, Suppl. 2, April 2008
factors including retinoic acid (RA), PGC enter meiosis as identified by chromosomal alignment and expression of meiotic genes. Although female PGC are more advanced in their invitro differentiation, meiosis is not successfully completed in both male and female PGC. It appears that full developmental potential of the female gamete from in-vivo- and in-vitroderived GS cells requires stimulation by factors controlling meiosis and follicular growth. The development of earlier stage in-vivo-derived PGC (E12.5) into primary and secondary oocytes requires the support of the complete cell population of the genital ridge followed by addition of growth factors (Obata et al., 2002). The presentation will include an overview on oocyte differentiation from stem cells and similarities and differences between in-vivo and in-vitro oocyte development, which may be the key to successful oogenesis in the culture dish PS-1.2 Sperm differentiation from stem cells Nayernia K NESCI and Institute of Human Genetics, University of Newcastle-upon-Tyne Stem cells offer substantial opportunities for providing welldefined differentiated cells for drug discovery, toxicology, and regenerative medicine, but the development of efficient techniques for controlling and directing their differentiation, present a substantial challenge. We developed a new promoter based genetic selection of germline stem cells (GSC). Germline stem cells, which can self-renew and generate gametes, are unique stem cells in that they are solely dedicated to transmit genetic information from generation to generation. Extensive studies on these two stem cell types in different organisms over the past few years have revealed some commonalities in the mechanisms controlling their self-renewal and differentiation. Furthermore, germline or somatic cells in various organisms and sexes also exhibit their own unique ways of regulating stem cell function. Mouse embryonic stem (ES) cells derive from the inner cell mass of the blastocyst and give rise to the three primitive embryonic layers, which later will form all the different tissue types of an adult. Embryonic stem cells are thus defined as totipotent cells. In vitro, these cells can give rise to all the somatic cells. We developed a strategy for the establishment of germline stem cell lines from embryonic stem cells. These cells are able to undergo meiosis, generate haploid male gametes in vitro and are functional, as shown by fertilization after intracytoplasmic injection into mouse oocytes. Molecular and cellular mechanisms underlying differentiation of ES to functional gametes should be elucidated in future research. In other approach, we show that bone marrow stem (BMS) cells are able to trans-differentiate into male germ cells. BMS cellderived germ cells expressed the known molecular markers of primordial germ cells. The ability to derive male germ cells from ES and BMS cells reveals novel aspects of germ cell development and opens the possibilities for use of these cells in reproductive medicine.
S-4 Reproductive BioMedicine Online, Vol. 16, Suppl. 2, April 2008
PS-1.3 Guided differentiation of human embryonic stem cells Chung H-M CHA Stem Cell Institute, Pochon CHA University College of Medicine Human embryonic stem cells (hESC) are established from the inner cell mass of preimplantation blastocysts. These cells exhibit unlimited proliferation and pluripotency, the ability to differentiate into all types of tissues, and are promising resources for regenerative medicine, which requires large numbers of a particular cell type. hESC are therefore unlimited sources for cell-based therapies if they can be successfully guided toward specific lineages with high populations. During hESC differentiation, embryoid bodies (EB), tissue-like spheroids of aggregated cells, are spontaneously formed from hESC in invitro suspension culture. Therefore, many studies have used EB to differentiate effectively hESC toward target lineages with high populations by altering their biological environments. Though several techniques have been developed, it remains challenging to efficiently differentiate hESC only toward specific lineages and to expand them to numbers required for cell therapy. Thus, differentiation and expansion have to be considered as primary goals prior to clinical applications of hESC. We focused on the development of functional endothelial or endothelial progenitor cells and their differentiation from human ES cells for therapeutic application. During the early stages of EB development, vasculogenesis takes place and blood vessels form through a multi-step process in which endothelial cell precursors differentiate, expand, and coalesce to form a network of primitive tubules. Endothelial cells play various important roles in blood vessels, including control of blood pressure, blood clotting, angiogenesis, and inflammation. Recently, many studies have reported that endothelial cell therapy is effective in regenerating damaged vessels and in curing vascular disease. Developing techniques of endothelial cell isolation and growth with high yield and purity will therefore facilitate therapies to cure various vascular diseases. In this presentation, the author will describe the development of an efficient method to obtain highly purified endothelial cells (EC) and vascular angiogenic progenitor cells (VAPC) from HESC and discuss whether HESC-EC or VAPC has therapeutic potential for the treatment of ischemia in hindlimb ischemic animal models. This work was supported by a grant (SC2190) from the Stem Cell Research Centre of the 21C Frontier R and D Program funded by the Ministry of Science and Technology, Republic of Korea. PS-1.4 Tracking stem cells in vivo Radda G Singapore Bioimaging Consortium, Head of the Department of Physiology, Anatomy and Genetics, University of Oxford The Singapore Bioimaging Consortium (SBIC) was set up in the first instance to develop facilities and research programmes for in-vivo imaging of model organisms for human disease and for coordinating, at the national level, bioimaging research in Singapore. Its main biomedical targets are cancer, metabolic medicine and regenerative medicine. Research into stem cell biology is well advanced
Abstracts - Aspire: ART Paving the way for new frontiers
in Singapore, hence the third target for SBIC. Magnetic resonance imaging and spectroscopy (MRI/MRS), optical imaging, SPECT and PET are all under development in Singapore and in principle each of these techniques can be used to monitor the fate of cells and in particular stem cells when injected into animal models. Among the different approaches, monitoring GFP labelled cells (often requiring invasive observations) and MRI have been most widely used. Some specific examples that will be discussed, including following the migration of stem cells to the infracted region in an animal model of stroke and a detailed study from Oxford on the use of iron particles for the non-invasive monitoring by MRI of bone marrow stromal cell engraftment into infarcted rodent hearts. Cardiac function can be simultaneously examined in a quantitative manner. PS-2.1 Predictive factors of ovarian reserve Howles CM Merck Serono International SA, Geneva In ART, the ideal ‘yield’ of oocytes varies according to chronological and ovarian age; in women less than 40 years, 8–10 oocytes (range: 5–14) per stimulation cycle have been considered to be adequate (Popovic-Todorovic, 2003a; Yih, 2005; van der Gaast, 2006). An excessive number of retrieved oocytes combined with an ongoing pregnancy can put the patient at risk of ovarian hyperstimulation syndrome (OHSS) (Delvigne and Rozenberg, 2002). At the other extreme, being able to predict a poor ovarian response would enable clinicians to manage both the treatment cycle and the patients’ expectations proactively (Hendriks, 2007). Selecting the correct dose of recombinant human (r-h) FSH to retrieve an ‘acceptable’ number of oocytes for ART and to achieve development of a single dominant follicle for OI is, however, complex. To predict each patient’s ovarian response to pharmaceutical agents and to individualize the starting dose of exogenous FSH, various clinical, endocrine, ovarian ultrasonographic and genetic characteristics have been explored. Several putative characteristics have been assessed together in prediction models with varied success (PopovicTodorovic, 2003a). This model was tested prospectively with a comparative group receiving 150 IU FSH/day in a twosite clinical study giving very encouraging results (PopovicTodorovic, 2003b). However, the patient population included women up to 39 years of age, which could have complicated data interpretation as patients over 35 are normally given a higher starting dose than 150 IU. With the caveats above in mind, patient-specific variables available readily that are most predictive of successful treatment have been assessed recently (Howles, 2006), these were (in order): baseline serum FSH in the early follicular phase, body mass index (BMI), age, and antral follicle count (AFC) assessed at baseline. There have also been attempts to determine predictors of response to FSH in CC resistant PCOS women. Imani et al. (2002), found that BMI, cycle history, ovarian response to clomiphene citrate as well as basal serum FSH values were all significantly correlated and a model was developed to predict the individual FSH response dose. A recent prospective study using this model found the predictive performance was poor, the FSH predictive dose being higher than the actual dose
required (Van Wely, 2006). In order to further investigate this issue, an analysis of a large database was undertaken to determine which patient factors (clinical and laboratory parameters) are related to the likelihood of successful ovulation. These data additionally confirmed the importance of BMI as a major determinant of ovulation but also revealed for the first time that higher basal FSH and basal AFC are also related to the response to a standard starting dose of 75 IU r-HFSH The results of this analysis will be presented in detail and implications for clinical management discussed (Howles, 2007). PS-2.2 Use of a simple algorithm to predict the FSH starting dose in ART patients Trew G Department of Reproductive Medicine, Hammersmith Hospital, London At present there is still considerable variation on how the prescribing physician determines the optimal gonadotrophin dose for ART stimulation, in spite of increasing pressure from medical societies and agencies for evidence-based treatment guidelines. Recently, work has been carried out to determine predictive factors of ovarian response and to development predictive models; however these include variables that are not routinely measured in standard clinical practice (e.g. total Doppler score, anti-Müllerian hormone, serum testosterone). The CONSORT (CONsistency in r-hFSH Starting dOses for Individualized tReatmenT) programme was initiated to address these issues. Data were analysed from 1378 women under the age of 35 years, who received stimulation with recombinant follicle-stimulating hormone (r-hFSH) alone for ART. Of 15 variables identified, the most important predictive factors routinely measured were identified (basal FSH, body mass index, age and antral follicle count) and used to generate an FSH starting dose algorithm, the ‘CONSORT calculator’ (Howles et al., 2006). To validate this model a prospective, open-label, multicentre study of ART 18 centres worldwide was conducted in which 172 were enrolled. All cycles were stimulated using the GONAL-f® filled-by-mass (FbM) pre-filled pen, which provides precise and accurate dosing and enables FSH dosing in relatively small increments. Of these, after down-regulation with a GnRH agonist (long protocol), 165 were allocated and received the following seven different individualized starting doses of r-hFSH FbM according to the CONSORT calculator: 75 IU (n = 48); 112.5 IU (n = 45); 150 IU (n = 34); 187.5 IU (n = 24), 225 IU (n = 10), 262.5 IU (n = 2), 300 IU (n = 2). Based on statistical considerations, only dosing groups with at least five patients per group were analysed (ITT population, n = 161). The overall median number of embryos transferred was 2.0. Clinical pregnancy rates per cycle started were 31.3%, 31.1%, 35.3%, 50.0% and 20% per r-hFSH FbM starting dose of 75 IU, 112.5 IU, 150 IU, 187.5 IU and 225 IU, respectively (overall mean pregnancy rate was 34.2%, n = 161). One early and one late onset case of severe OHSS were reported, in patients allocated to the 75 IU and 187.5 IU group respectively. Further results of the study will be presented in detail during the presentation. Overall, by using the CONSORT calculator an adequate oocyte yield was achieved, accompanied by high pregnancy rates. Notably, over 90% of patients required
S-5 Reproductive BioMedicine Online, Vol. 16, Suppl. 2, April 2008