- Email: [email protected]
ASCR-014 New methods for culturing high proliferative potential-endothelial colony-forming cells and human umbilical vein endothelial cells
Abstracts - Aspire: Optimizing infertility management in Asia
IVF were plated on mitomycin-treated human fetal fibroblast feeder cell dishes after zona removal by brief 0.4% pronase incubation. Derivation medium was KO-DMEM, 2 mmol/l glutamine, 50 IU/ml penicillin and 50 mg/ml streptomycin, 1s MEM-amino acids, 20% Knock-Out Serum Replacement (KSR) and 4 ng/ml basic fibroblast growth factor (bFGF). The culture of outgrowths and subsequent putative ESC lines was likewise performed in MINC incubators in 5% O2. The outgrowths were passaged mechanically until ES-like cells were observed and subsequently characterized to verify that they were true ES cells. Characterization included staining for enzyme alkaline phosphatase and immunocytochemical analysis for undifferentiated ESC markers SSEA-4, Oct-4 and Tra-1–60. Karyotyping of the cell lines was performed first between passages 4–6, and after that approximately every 15–20 passages. Results: Six PGD embryos were plated, resulting in six outgrowths. From these, three ESC lines were established. The initial analyses of the embryos producing the ESC lines comprised one triploidy affecting all trophectoderm cells sampled (the derived cell line was 47,XX,+16) and two cases of ‘chaotic’ karyotypes, i.e. different trophectoderm cells displayed different aneuploidies (from which in each case the derived cell lines were normal female, i.e. 46,XX). Identical karyotyping results were achieved in at least three subsequent karyotypings of the cell lines. Conclusions: PGD-analysed IVF embryos can yield ESC lines with high efficiency. As just one or a few pluripotent blastomeres can be enough to produce an ESC line, and because of the high prevalence of mosaicism in IVF embryos, the resulting cell lines often have a karyotype that is normal or consistent with early embryonic or fetal development. This outcome almost certainly reflects the positive selection pressures favouring normal cells during stem cell derivation culture. With appropriate culture conditions, abnormal embryos that are clinically unusable can nevertheless be a useful source of normal ESC lines. ASCR-012 Molecular cloning and characterization analysis of mPESCRG1, a novel gene expressed specifically in mouse embryonic stem cells Du J1, Nie ZY, Lin G, Lu GX Institute of Human Reproduction and Stem Cell Engineering, Central South University 1Correspondence: [email protected] Introduction: In a previous study, a novel gene, Homo sapiens pluripotent embryonic stem cell-related gene (hPESCRG1), was cloned which was expressed specifically in human embryonic stem cells (ESC), based on an EST CF948547 up-regulating in ESC from subtractive library between ESC and its spontaneously differentiated cells. In this study, it was intended to clone the mouse homologue gene of hPESCRG1 and analyse its characteristics. Materials/Methods: Using the sequence of hPESCRG1 as a seed sequence, a novel gene expressed specifically in mouse embryonic stem cells (mESC) was cloned by bioinformatics method and molecular biological technique. Its 5a terminal sequence was cloned rapidly by SMART (switching mechanism at 5a end of the RNA transcript) technique combined with the mouse genome draft. Its expression profile was analysed by reverse transcription polymerase chain reaction (RT-PCR), northern blot and in-situ hybridization, and subcellular location was determined by enhanced green fluorescent protein (EGFP) eukaryotic expression system. Results: The full-length cDNA sequence of a novel gene mPESCRG1 (mouse sapiens pluripotent embryonic stem cell-related gene) was cloned successfully. Its GenBank accession number was AY561246; cDNA length, 1941 bp; chromosomal mapping was located in 16B5; and the putative protein contained 301 amino acids with a molecular weight of 34,116 Da. The protein primary structure of this gene contained a SAP DNA-binding motif and it was subcellularly located in nuclei. Expression analysis showed that the gene was expressed specifically in mESC, with a lack of detectable transcript in various adult mouse tissues, such as ovary, testis, brain, heart, liver, kidney and spleen and mouse embryonic fibrocytes (mEFC) by northern blot. RT-PCR showed that the gene was expressed in mouse blastocyst cells. The in-situ hybridization results showed strong signals in mESC and no signals in mEFC using an oligo-nucleotide probe of the mPESCRG1 gene. The transfection
experiment mediated by lipofection showed that mPESCRG1 protein presented mainly in nuclei, which was consistent with the bioinformatics prediction. Conclusions: mPESCRG1 is a novel gene expressed specifically in mESC and mouse blastocyst cells, which might be related to self-renewal or proliferation of mESC and the early development of mouse embryo. ASCR-013 Molecular cloning and characterization of HESPRG3 gene promoter Miao CX1, Lin G, Nie CY, Dai C, Lu GX2 Institute of Human Reproduction and Stem Cell Engineering, Central South University Correspondence: [email protected]; 2 [email protected]. cn Introduction: HESPRG3 is a new gene related to human embryonic stem cells (ESC) and is thought to play an important role in the development of ES cells and in cell proliferation. HESPRG3 (GenBank accession number AY508815) whose cDNA length is 2307 bp contains 11 exons and 10 introns, with a chromosome locus at 1p34.2. The putative protein contains 280 amino acids. HESPRG3 has been demonstrated to express highly in human undifferentiated ES cells compared with the differentiated ES cells, and lacks detectable transcription in human multiple tissues of both the adult and fetus aborted at pregnancy over 5 months. To elucidate molecular mechanisms underlying the transcriptional regulation of HESPRG3 gene, approximately 2.0 kb of the 5a-flanking sequence of HESPRG3 was cloned and characterized. Materials/Methods: 2.0 kb of the 5a-flanking region of HESPRG3 was isolated from the human genomic total DNA by nested polymerase chain reaction (PCR). Using promoter deletion analysis, based on the luciferase reporter gene, a set of 5a deletion fragments from 2.0 kb upstream region of HESPRG3 were constructed with pGL3-Basic. Via the transient transfection/dual reporter assay, the promoter activities of the above constructs were vigorously tested. Applying the classical 5a-RACE technique, identification of the transcriptional start sites of the HESPRG3 gene was performed. A number of important transcription factor binding sites were analysed by the bioinformation software. The cis-elements within the promoter region were determined by electrophoretic mobility shift assay (EMSA) and mutation. Results: The basal promoter of HESPRG3 is located at the 269/143-bp region, which drives the transcription of reporter gene in both human embryo stem and multiple tumour cells; the highest activity region is located in the 1071-bp fragment. The transcription start site of HESPRG3 is located at 200 bp, 194 bp and 175 bp upstream of the translation start site. EMSA and mutants show the promoter region have five potent transcription factors, which could function as key regulators for activation of the HESPRG3 gene. Conclusions: The HESPRG3 promoter and cis-acting elements of HESPRG3 have been identified. The results establish the basis for further studies on the mechanism regulating the expression of the HESPRG3 gene. ASCR-014 New methods for culturing high proliferative potential-endothelial colony-forming cells and human umbilical vein endothelial cells Sun X1, Lu GX Institute of Human Reproduction and Stem Cell Engineering, Central South University 1Correspondence: [email protected] Introduction: Endothelial cells, especially human umbilical vein endothelial cells (HUVEC) and endothelial progenitor cells (EPC), are frequently used in many laboratories, but are usually expensive and inconvenient to culture. HUVEC proliferate within 10 passages, and high proliferative potential-endothelial colony-forming cells (HPP-ECFC) from umbilical cord blood are commonly cultured in endothelial growth medium (EGM-2), which is restricted for import in some countries. This study tested two kinds of suitable media for culturing HUVEC and HPP-ECFC, which are economical and can effectively support cell proliferation.
35
Abstracts - Aspire: Optimizing infertility management in Asia
Materials/Methods: Newborn human umbilical cord vein was digested with collagenase IV for 30 min to get HUVEC. The culture medium was composed of MCDB131, 10% FBS, rhEGF, dexamethasone, penicillin and streptomycin. Cells were allowed to adhere to the plate for 6 h and then the non-adherent cells were aspirated. Mononuclear cells (MNC) were separated from human umbilical cord blood by density gradient centrifugation. MNC were then resuspended in MCDB131 including 20% FBS, L-glutamine, heparin sodium, hydrocortisone, ascorbic acid and endothelial cell growth supplement (ECGS), and seeded onto cell culture plates pre-coated with fibronectin. Non-adherent cells were removed after 5 days of culture. HPP-ECFC forming colonies were achieved 10–14 days later. The endothelial cell population was identified by testing their karyotype, immunophenotype, Matrigel assays and uptake of DiI-acetylated-low density lipoprotein (DiI-Ac-LDL). Results: A single HUVEC has been estimated to undergo up to a 3 s 1019fold expansion during 107 days of culture using the present approach, and seems to still have high proliferative potential. Hitherto, a single HPPECFC can generate as many as 108 progeny in vitro in 35 days. Both kinds of cells were of normal karyotype after long-term culture. They express PECAM1, VE-cadherin and vWF, and can form capillary-like formations on Matrigel. They also have the ability to incorporate DiIAc-LDL. Conclusions: Emerging evidence to support the use of these endothelial cells for angiogenic therapies, tissue engineering, pharmacological assays or as vehicle cells for gene therapies is compelling. The described approaches to culture HUVEC and HPP-ECFC from umbilical cord blood make experiments as convenient and economical as using endothelial cell lines, with more convincing results. ASCR-015 Suspending-culture supports transdifferentiation from human amniotic epithelial cells to insulin-secreting cells Wang J1, Hu LS, Sun X, Lu GX Institute of Human Reproduction and Stem Cell Engineering, Central South University 1Correspondence: [email protected] Introduction: Human amniotic epithelial cells (HAEC) constitute the inner layer of the amnion, generated from amnioblasts on day 8 after fertilization. It has long been proposed that amniotic epithelial cells may potentially differentiate into various organs, including the heart, liver and brain. As HAEC do not express the HLA-A, -B, -C and -DR antigens, they might be an excellent source of seed cells for cell-replacement therapy in the clinical practice, once differentiation is directed towards a specially functional cell type. An inductive method was introduced for transdifferentiation from the HAEC to insulin-secreting cells. Materials/Methods: Human amniotic membrane was mechanically peeled from the chorion of a placenta obtained from an uncomplicated elective Caesarean section with the informed consent of each donor patient. The HAEC were trypsinized into single cells after a 30-min digestion by the trypsin–EDTA twice, and then cultured under an AE culture medium including epidermal growth factor (EGF) and DMEM–F12 for 3–4 days. The first stage of induction was performed by suspending culture cells for 7 days by transferring cells into a new plate with 0.8% agar under an inductive system consisting of N2, B27, 10 Mg/ml nicotinamide and DMEM–F12, during which the single cell formed into spheric cell clusters. After which, cells were processed into the second inductive stage for another 7 days, with a new inductive system consisting of 20 Mg/ml EGF and 10 Mg/ml nicotinamide. The cell markers of the islet cells such as nestin and insulin were examined by the immunocytochemistry and then the insulin-stimulating experiment was performed by culturing the cells with phosphate-buffer solution (PBS) containing 5% glucose for 2 h. Results: The immunocytochemistry results showed that the spheric cell clusters were nestin and insulin positive. When the cells were cultured with PBS containing 5% glucose, the spheric cell clusters could secrete high concentrations of insulin, which was more than 300 IU per 100 cell clusters per ml. The results were significantly better than that of the control group. Conclusions: HAECs could be extended easily in vitro; the method of inductive differentiation form the HAECs to insulin-secreting cells could
36
be a very useful tool for clinical cell replacement to heal type 1 diabetes. ASCR-016 VLA-4 and LFA-1 involved in homing endothelial progenitor cells into ischaemic tissue Duan HX1, Cheng LM, Wang J, Lu GX Institute of Human Reproduction and Stem Cell Engineering, Central South University 1Correspondence: [email protected] Introduction: Endothelial progenitor cell (EPC) transplantation has opened a new field for ischaemic disease therapy because they can promote the neovascularization of ischaemic tissue. However, the mechanisms are not yet understood regarding EPC adhering to and migrating across the endothelial cells, which is important for EPC in vessels to participate in angiogenesis. The objective of this study was to investigate the molecular mechanism of ‘homing’ to ischaemial tissue of EPCs, specifically EPC adhesion to and migration across the endothelial cells of capillaries of ischaemic tissue. Materials/Methods: The mononuclear cells were isolated form human umbilical cord blood and cultured in EGM-2 (endothelial cell complete culture system). The markers of EPC were analysed by fluorescenceactivated cell sorting (FACS) and the potential of angiogenesis was examined in vitro on Matrigel. Adhesion molecules, such as VLA-4 and LFA-1 in EPCs, VCAM-1, ICAM-1 and ICAM-2 in mice bone marrow endothelial cell line, and endothelial cells in ischaemic tissue, were detected by FACS and by immunocytochemistry (ICC) respectively. The monoclonal antibodies were used to block the adhesion molecules in adhesion and migration experiments in vitro. Results: EPC expressed markers of endothelial cells, such as CD31, KDR and CD144, and showed strong proliferation and colonogenic potential. Further, EPC expressed VLA-4, LFA-1 and CXCR4, and the endothelial cells of ischaemic tissue expressed the receptor of those adhesion molecules, VCAM-1, ICAM-1 and ICAM-2. Antibodies of VLA-4 and LFA-1 decreased the number of EPC adhering to and migrating across endothelial cells in vitro. Conclusion: VLA-4 and LFA-1 are involved in the ‘homing’ of EPC into the ischaemic tissue, partly through affecting EPC adhesion to and migration across endothelial cells. EMB-001 Aneuploidy rate in patients of different age groups undergoing IVF Khoo G1, Tan R, Tee ST, Chong WA Damansara Fertility Centre 1Correspondence: [email protected] Introduction: Preimplantation genetic diagnosis (PGD) is used to reduce aneuploidy rate, miscarriage rate, allow sex selection and increase implantation rate by selecting chromosomally normal embryos for transfer. The objective of this study was to evaluate the percentage of abnormal embryos determined by PGD at Damansara Fertility Centre according to different age groups. Materials/Methods: The data were collected from January 1 to October 31, 2005. All cases were divided into four age groups: <35, 35–37, 38– 40, and >40 years old. PGD on day 3, day 5 or day 6 were performed. For day 3 PGD, one blastomere was biopsied, whereas for day 5 or day 6 PGD, 3–5 trophectoderm cells were biopsied. All specimens were fixed and hybridized for aneuploidy screening for chromosomes 13, 18, 21, X and Y. Results: Forty-eight patients had PGD. A total of 166 embryos were biopsied, of which 138 (83.1%) yielded results, whereas 28 (16.9%) were indeterminate. Of those that yielded results, 53 (38.4%) were chromosomally abnormal embryos. Of the 48 cycles, 14 were from patients <35 years old, 16 from 35–37 years old, 13 from age group 38–40 years old, and five from the age group >40 years old. The percentages of abnormal embryos in these age groups were 30.6, 36.2, 43.8 and 70% respectively. Patients <38 years old had clinical pregnancy rate per cases reached embryo transfer of 47.6% (10/21) and delivery rate/ongoing pregnancy rate per cases reached embryo transfer of 42.9% (9/21). However, patients ≥38 years old had clinical pregnancy rate per cases reached embryo transfer of 36.4% (4/11) and delivery rate/
IVF were plated on mitomycin-treated human fetal fibroblast feeder cell dishes after zona removal by brief 0.4% pronase incubation. Derivation medium was KO-DMEM, 2 mmol/l glutamine, 50 IU/ml penicillin and 50 mg/ml streptomycin, 1s MEM-amino acids, 20% Knock-Out Serum Replacement (KSR) and 4 ng/ml basic fibroblast growth factor (bFGF). The culture of outgrowths and subsequent putative ESC lines was likewise performed in MINC incubators in 5% O2. The outgrowths were passaged mechanically until ES-like cells were observed and subsequently characterized to verify that they were true ES cells. Characterization included staining for enzyme alkaline phosphatase and immunocytochemical analysis for undifferentiated ESC markers SSEA-4, Oct-4 and Tra-1–60. Karyotyping of the cell lines was performed first between passages 4–6, and after that approximately every 15–20 passages. Results: Six PGD embryos were plated, resulting in six outgrowths. From these, three ESC lines were established. The initial analyses of the embryos producing the ESC lines comprised one triploidy affecting all trophectoderm cells sampled (the derived cell line was 47,XX,+16) and two cases of ‘chaotic’ karyotypes, i.e. different trophectoderm cells displayed different aneuploidies (from which in each case the derived cell lines were normal female, i.e. 46,XX). Identical karyotyping results were achieved in at least three subsequent karyotypings of the cell lines. Conclusions: PGD-analysed IVF embryos can yield ESC lines with high efficiency. As just one or a few pluripotent blastomeres can be enough to produce an ESC line, and because of the high prevalence of mosaicism in IVF embryos, the resulting cell lines often have a karyotype that is normal or consistent with early embryonic or fetal development. This outcome almost certainly reflects the positive selection pressures favouring normal cells during stem cell derivation culture. With appropriate culture conditions, abnormal embryos that are clinically unusable can nevertheless be a useful source of normal ESC lines. ASCR-012 Molecular cloning and characterization analysis of mPESCRG1, a novel gene expressed specifically in mouse embryonic stem cells Du J1, Nie ZY, Lin G, Lu GX Institute of Human Reproduction and Stem Cell Engineering, Central South University 1Correspondence: [email protected] Introduction: In a previous study, a novel gene, Homo sapiens pluripotent embryonic stem cell-related gene (hPESCRG1), was cloned which was expressed specifically in human embryonic stem cells (ESC), based on an EST CF948547 up-regulating in ESC from subtractive library between ESC and its spontaneously differentiated cells. In this study, it was intended to clone the mouse homologue gene of hPESCRG1 and analyse its characteristics. Materials/Methods: Using the sequence of hPESCRG1 as a seed sequence, a novel gene expressed specifically in mouse embryonic stem cells (mESC) was cloned by bioinformatics method and molecular biological technique. Its 5a terminal sequence was cloned rapidly by SMART (switching mechanism at 5a end of the RNA transcript) technique combined with the mouse genome draft. Its expression profile was analysed by reverse transcription polymerase chain reaction (RT-PCR), northern blot and in-situ hybridization, and subcellular location was determined by enhanced green fluorescent protein (EGFP) eukaryotic expression system. Results: The full-length cDNA sequence of a novel gene mPESCRG1 (mouse sapiens pluripotent embryonic stem cell-related gene) was cloned successfully. Its GenBank accession number was AY561246; cDNA length, 1941 bp; chromosomal mapping was located in 16B5; and the putative protein contained 301 amino acids with a molecular weight of 34,116 Da. The protein primary structure of this gene contained a SAP DNA-binding motif and it was subcellularly located in nuclei. Expression analysis showed that the gene was expressed specifically in mESC, with a lack of detectable transcript in various adult mouse tissues, such as ovary, testis, brain, heart, liver, kidney and spleen and mouse embryonic fibrocytes (mEFC) by northern blot. RT-PCR showed that the gene was expressed in mouse blastocyst cells. The in-situ hybridization results showed strong signals in mESC and no signals in mEFC using an oligo-nucleotide probe of the mPESCRG1 gene. The transfection
experiment mediated by lipofection showed that mPESCRG1 protein presented mainly in nuclei, which was consistent with the bioinformatics prediction. Conclusions: mPESCRG1 is a novel gene expressed specifically in mESC and mouse blastocyst cells, which might be related to self-renewal or proliferation of mESC and the early development of mouse embryo. ASCR-013 Molecular cloning and characterization of HESPRG3 gene promoter Miao CX1, Lin G, Nie CY, Dai C, Lu GX2 Institute of Human Reproduction and Stem Cell Engineering, Central South University Correspondence: [email protected]; 2 [email protected]. cn Introduction: HESPRG3 is a new gene related to human embryonic stem cells (ESC) and is thought to play an important role in the development of ES cells and in cell proliferation. HESPRG3 (GenBank accession number AY508815) whose cDNA length is 2307 bp contains 11 exons and 10 introns, with a chromosome locus at 1p34.2. The putative protein contains 280 amino acids. HESPRG3 has been demonstrated to express highly in human undifferentiated ES cells compared with the differentiated ES cells, and lacks detectable transcription in human multiple tissues of both the adult and fetus aborted at pregnancy over 5 months. To elucidate molecular mechanisms underlying the transcriptional regulation of HESPRG3 gene, approximately 2.0 kb of the 5a-flanking sequence of HESPRG3 was cloned and characterized. Materials/Methods: 2.0 kb of the 5a-flanking region of HESPRG3 was isolated from the human genomic total DNA by nested polymerase chain reaction (PCR). Using promoter deletion analysis, based on the luciferase reporter gene, a set of 5a deletion fragments from 2.0 kb upstream region of HESPRG3 were constructed with pGL3-Basic. Via the transient transfection/dual reporter assay, the promoter activities of the above constructs were vigorously tested. Applying the classical 5a-RACE technique, identification of the transcriptional start sites of the HESPRG3 gene was performed. A number of important transcription factor binding sites were analysed by the bioinformation software. The cis-elements within the promoter region were determined by electrophoretic mobility shift assay (EMSA) and mutation. Results: The basal promoter of HESPRG3 is located at the 269/143-bp region, which drives the transcription of reporter gene in both human embryo stem and multiple tumour cells; the highest activity region is located in the 1071-bp fragment. The transcription start site of HESPRG3 is located at 200 bp, 194 bp and 175 bp upstream of the translation start site. EMSA and mutants show the promoter region have five potent transcription factors, which could function as key regulators for activation of the HESPRG3 gene. Conclusions: The HESPRG3 promoter and cis-acting elements of HESPRG3 have been identified. The results establish the basis for further studies on the mechanism regulating the expression of the HESPRG3 gene. ASCR-014 New methods for culturing high proliferative potential-endothelial colony-forming cells and human umbilical vein endothelial cells Sun X1, Lu GX Institute of Human Reproduction and Stem Cell Engineering, Central South University 1Correspondence: [email protected] Introduction: Endothelial cells, especially human umbilical vein endothelial cells (HUVEC) and endothelial progenitor cells (EPC), are frequently used in many laboratories, but are usually expensive and inconvenient to culture. HUVEC proliferate within 10 passages, and high proliferative potential-endothelial colony-forming cells (HPP-ECFC) from umbilical cord blood are commonly cultured in endothelial growth medium (EGM-2), which is restricted for import in some countries. This study tested two kinds of suitable media for culturing HUVEC and HPP-ECFC, which are economical and can effectively support cell proliferation.
35
Abstracts - Aspire: Optimizing infertility management in Asia
Materials/Methods: Newborn human umbilical cord vein was digested with collagenase IV for 30 min to get HUVEC. The culture medium was composed of MCDB131, 10% FBS, rhEGF, dexamethasone, penicillin and streptomycin. Cells were allowed to adhere to the plate for 6 h and then the non-adherent cells were aspirated. Mononuclear cells (MNC) were separated from human umbilical cord blood by density gradient centrifugation. MNC were then resuspended in MCDB131 including 20% FBS, L-glutamine, heparin sodium, hydrocortisone, ascorbic acid and endothelial cell growth supplement (ECGS), and seeded onto cell culture plates pre-coated with fibronectin. Non-adherent cells were removed after 5 days of culture. HPP-ECFC forming colonies were achieved 10–14 days later. The endothelial cell population was identified by testing their karyotype, immunophenotype, Matrigel assays and uptake of DiI-acetylated-low density lipoprotein (DiI-Ac-LDL). Results: A single HUVEC has been estimated to undergo up to a 3 s 1019fold expansion during 107 days of culture using the present approach, and seems to still have high proliferative potential. Hitherto, a single HPPECFC can generate as many as 108 progeny in vitro in 35 days. Both kinds of cells were of normal karyotype after long-term culture. They express PECAM1, VE-cadherin and vWF, and can form capillary-like formations on Matrigel. They also have the ability to incorporate DiIAc-LDL. Conclusions: Emerging evidence to support the use of these endothelial cells for angiogenic therapies, tissue engineering, pharmacological assays or as vehicle cells for gene therapies is compelling. The described approaches to culture HUVEC and HPP-ECFC from umbilical cord blood make experiments as convenient and economical as using endothelial cell lines, with more convincing results. ASCR-015 Suspending-culture supports transdifferentiation from human amniotic epithelial cells to insulin-secreting cells Wang J1, Hu LS, Sun X, Lu GX Institute of Human Reproduction and Stem Cell Engineering, Central South University 1Correspondence: [email protected] Introduction: Human amniotic epithelial cells (HAEC) constitute the inner layer of the amnion, generated from amnioblasts on day 8 after fertilization. It has long been proposed that amniotic epithelial cells may potentially differentiate into various organs, including the heart, liver and brain. As HAEC do not express the HLA-A, -B, -C and -DR antigens, they might be an excellent source of seed cells for cell-replacement therapy in the clinical practice, once differentiation is directed towards a specially functional cell type. An inductive method was introduced for transdifferentiation from the HAEC to insulin-secreting cells. Materials/Methods: Human amniotic membrane was mechanically peeled from the chorion of a placenta obtained from an uncomplicated elective Caesarean section with the informed consent of each donor patient. The HAEC were trypsinized into single cells after a 30-min digestion by the trypsin–EDTA twice, and then cultured under an AE culture medium including epidermal growth factor (EGF) and DMEM–F12 for 3–4 days. The first stage of induction was performed by suspending culture cells for 7 days by transferring cells into a new plate with 0.8% agar under an inductive system consisting of N2, B27, 10 Mg/ml nicotinamide and DMEM–F12, during which the single cell formed into spheric cell clusters. After which, cells were processed into the second inductive stage for another 7 days, with a new inductive system consisting of 20 Mg/ml EGF and 10 Mg/ml nicotinamide. The cell markers of the islet cells such as nestin and insulin were examined by the immunocytochemistry and then the insulin-stimulating experiment was performed by culturing the cells with phosphate-buffer solution (PBS) containing 5% glucose for 2 h. Results: The immunocytochemistry results showed that the spheric cell clusters were nestin and insulin positive. When the cells were cultured with PBS containing 5% glucose, the spheric cell clusters could secrete high concentrations of insulin, which was more than 300 IU per 100 cell clusters per ml. The results were significantly better than that of the control group. Conclusions: HAECs could be extended easily in vitro; the method of inductive differentiation form the HAECs to insulin-secreting cells could
36
be a very useful tool for clinical cell replacement to heal type 1 diabetes. ASCR-016 VLA-4 and LFA-1 involved in homing endothelial progenitor cells into ischaemic tissue Duan HX1, Cheng LM, Wang J, Lu GX Institute of Human Reproduction and Stem Cell Engineering, Central South University 1Correspondence: [email protected] Introduction: Endothelial progenitor cell (EPC) transplantation has opened a new field for ischaemic disease therapy because they can promote the neovascularization of ischaemic tissue. However, the mechanisms are not yet understood regarding EPC adhering to and migrating across the endothelial cells, which is important for EPC in vessels to participate in angiogenesis. The objective of this study was to investigate the molecular mechanism of ‘homing’ to ischaemial tissue of EPCs, specifically EPC adhesion to and migration across the endothelial cells of capillaries of ischaemic tissue. Materials/Methods: The mononuclear cells were isolated form human umbilical cord blood and cultured in EGM-2 (endothelial cell complete culture system). The markers of EPC were analysed by fluorescenceactivated cell sorting (FACS) and the potential of angiogenesis was examined in vitro on Matrigel. Adhesion molecules, such as VLA-4 and LFA-1 in EPCs, VCAM-1, ICAM-1 and ICAM-2 in mice bone marrow endothelial cell line, and endothelial cells in ischaemic tissue, were detected by FACS and by immunocytochemistry (ICC) respectively. The monoclonal antibodies were used to block the adhesion molecules in adhesion and migration experiments in vitro. Results: EPC expressed markers of endothelial cells, such as CD31, KDR and CD144, and showed strong proliferation and colonogenic potential. Further, EPC expressed VLA-4, LFA-1 and CXCR4, and the endothelial cells of ischaemic tissue expressed the receptor of those adhesion molecules, VCAM-1, ICAM-1 and ICAM-2. Antibodies of VLA-4 and LFA-1 decreased the number of EPC adhering to and migrating across endothelial cells in vitro. Conclusion: VLA-4 and LFA-1 are involved in the ‘homing’ of EPC into the ischaemic tissue, partly through affecting EPC adhesion to and migration across endothelial cells. EMB-001 Aneuploidy rate in patients of different age groups undergoing IVF Khoo G1, Tan R, Tee ST, Chong WA Damansara Fertility Centre 1Correspondence: [email protected] Introduction: Preimplantation genetic diagnosis (PGD) is used to reduce aneuploidy rate, miscarriage rate, allow sex selection and increase implantation rate by selecting chromosomally normal embryos for transfer. The objective of this study was to evaluate the percentage of abnormal embryos determined by PGD at Damansara Fertility Centre according to different age groups. Materials/Methods: The data were collected from January 1 to October 31, 2005. All cases were divided into four age groups: <35, 35–37, 38– 40, and >40 years old. PGD on day 3, day 5 or day 6 were performed. For day 3 PGD, one blastomere was biopsied, whereas for day 5 or day 6 PGD, 3–5 trophectoderm cells were biopsied. All specimens were fixed and hybridized for aneuploidy screening for chromosomes 13, 18, 21, X and Y. Results: Forty-eight patients had PGD. A total of 166 embryos were biopsied, of which 138 (83.1%) yielded results, whereas 28 (16.9%) were indeterminate. Of those that yielded results, 53 (38.4%) were chromosomally abnormal embryos. Of the 48 cycles, 14 were from patients <35 years old, 16 from 35–37 years old, 13 from age group 38–40 years old, and five from the age group >40 years old. The percentages of abnormal embryos in these age groups were 30.6, 36.2, 43.8 and 70% respectively. Patients <38 years old had clinical pregnancy rate per cases reached embryo transfer of 47.6% (10/21) and delivery rate/ongoing pregnancy rate per cases reached embryo transfer of 42.9% (9/21). However, patients ≥38 years old had clinical pregnancy rate per cases reached embryo transfer of 36.4% (4/11) and delivery rate/