RECENT PUBLICATIONS
Article List
- Human embryonic stem cell lines generated without embryo destruction. “Cell Stem Cells.” In press. Epub 2008 Jan 10.
- Disruption of apical-basal polarity of human embryonic stem cells enhances hematoendothelial differentiation. Regenerative Medicine, Jan 2006, Vol. 1, No. 1, Pages 95-101.
- Serum-free derivation of human embryonic stem cell lines on human placental fibroblast feeders. Fertil Steril. 2005 May;83(5):1517-29.
- Derivation of hESC from intact blastocysts. Current Protocols in Stem Cell Biology, John Wiley & Sons
- First derivation in Spain of human embryonic stem cell lines: use of long-term cryopreserved embryos and animal-free conditions. Fertil Steril. 2005 Jan;83(1):246-9.
- Trophoblast L-selectin-mediated adhesion at the maternal-fetal interface. Science. 2003 Jan 17;299(5605):405-8.
- Derivation and characterization of three new Spanish human embryonic stem cell lines (VAL -3 -4 -5) on human feeder and in serum-free conditions. Reprod Biomed Online. 2006 Dec;13(6):875-86.
- Culture of human embryonic stem cells and the extracellular matrix microenvironment. Regenerative Medicine, Jan 2006, Vol. 1, No. 1, Pages 95-101.
Article
Human embryonic stem cell lines generated without embryo destruction
Source: “Cell Stem Cells.” In press. Epub 2008 Jan 10.
Contributors: Chung Y, Klimanskaya I, Becker S, Li T, Maserati M, Lu S-J, Zdravkovic T, Ilic D, Genbacev O, Fisher S, Krtolica A, and Lanza R.
Affiliations: Advanced Cell Technology, Worcester, MA 01605, USA; Department of Cell and Tissue Biology, University of California, San Francisco, CA 94143, USA; StemLifeLine, San Carlos, CA 94070, USA.
Abstract
To date, the derivation of all human embryonic stem cell (hESC) lines has involved the destruction of embryos. We previously demonstrated that hESCs can be generated from single blastomeres. In that study, multiple cells were removed from each embryo and none of the embryos was allowed to continue development. Here we report the derivation of the first five hESC lines without destruction of embryos. Single blastomeres were removed from the embryos using a technique similar to PGD. The biopsied embryos were grown to the blastocyst stage and frozen. The blastomeres were cultured using a new approach aimed at recreating the ICM niche. Trophectodermal-vesicle development was eliminated, exponentially improving the efficiency of the hESC derivation to rates reported for whole embryo derivations. The lines derived from the extracted blastomeres maintained normal karyotype and markers of pluripotency for up to more than 50 passages, and differentiated into all three germ layers, including insulin-producing cells, neurons, cartilage, bone, beating heart cells, and other cells of therapeutic importance.
Article
Disruption of apical-basal polarity of human embryonic stem cells enhances hematoendothelial differentiation.
Source: “Stem Cells; 2007 Sep;25(9):2215-23.
Pub Med ID: 17569786
Contributors:
Krtolica A, Genbacev O, Escobedo C, Zdravkovic T, Nordstrom A, Vabuena D, Nath A, Simon C, Mostov K, Fisher SJ.
Affiliations: StemLifeLine Inc., San Carlos, CA 94070 & Department of Cell and Tissue Biology University of California, San Francisco, CA 94143, USA.
Abstract
During murine development, the formation of tight junctions and acquisition of polarity are associated with allocation of the blastomeres on the outer surface of the embryo to the trophoblast lineage, whereas the absence of polarization directs cells to the inner cell mass. Here, we report the results of ultrastructural analyses that suggest a similar link between polarization and cell fate in human embryos. In contrast, the five human embryonic stem cell (hESC) lines displayed apical-basal, epithelial-type polarity with electron-dense tight junctions, apical microvilli, and asymmetric distribution of organelles. Consistent with these findings, molecules that are components of tight junctions or play regulatory roles in polarization localized to the apical regions of the hESCs at sites of cell-cell contact. The tight junctions were functional, as shown by the ability of hESC colonies to exclude the pericellular passage of a biotin compound. Depolarization of hESCs produced multilayered aggregates of rapidly proliferating cells that continued to express transcription factors that are required for pluripotency at the same level as control cells. However, during embryoid body formation, depolarized cells differentiated predominantly along mesenchymal lineage and
Article
Culture of human embryonic stem cells and the extracellular matrix microenvironment
Source: Regenerative Medicine, Jan 2006, Vol. 1, No. 1, Pages 95-101.
Contributors: Dusko Ilic
Affiliations: StemLifeLine, Inc.
Abstract
Among the major obstacles impeding successful derivation and continuous culture of human embryonic stem cells (hESC) fortherapeutic purposes, are the presence of feeder cells andfeeder-conditioned media of animal origin. The risk of contaminationwith xenopathogens makes hESC cultured in this way unsafe for futureuse in regenerative medicine. A holy grail for investigators in thefield will be to establish and maintain new hESC lines in completelyfeeder-free and serum-free defined conditions. Recently, propagationof hESC has become possible, using mammalian- or human-derivedextracellular matrix (ECM) and conditioned medium from feeder cells.In addition, providing a three-dimensional ECM environment can evensupport the derivation of new hESC. In this review, we examine recentadvances in the use and development of substrates suitable for thederivation and maintenance of hESC, and our current understanding ofthe effects of a three-dimensional ECM milieu on cellular behavior.
Article
Serum-free derivation of human embryonic stem cell lines on human placental fibroblast feeders.
Source: Fertil Steril. 2005 May;83(5):1517-29.
Pub Med ID: 15866593
Contributors: Genbacev O, Krtolica A, Zdravkovic T, Brunette E, Powell S, Nath A, Caceres E, McMaster M, McDonagh S, Li Y, Mandalam R, Lebkowski J, Fisher SJ.
Affiliations: Department of Cell and Tissue Biology, Program in Human Stem Cell Biology, and Developmental and Stem Cell Biology Program, University of California San Francisco, San Francisco, California 94143-0512, USA.
Abstract
OBJECTIVE: To derive new human embryonic stem cell (hESC) lines on pathogen-free human placental fibroblast feeders under serum-free conditions. Because the embryo develops in close contact with extraembryonic membranes, we hypothesized that placental mesenchyme might replicate the stem cell niche in situ.
DESIGN: We isolated and characterized human placental fibroblast lines from individual donors and tested their ability to support growth of federally registered hESC lines. Moreover, we performed extensive pathogen testing to ensure their suitability as feeders for the derivation of therapy-grade hESCs.
RESULT: Human placental fibroblasts were comparable or superior to mouse embryo fibroblasts as hESC feeders. We used these qualified placental fibroblasts to derive two new hESC lines in knockout Dulbecco’s modified Eagle’s medium with serum-free 20% knockout serum replacement. The cells, which had a normal karyotype, were grown for more than 25 passages, expressed markers of stemness including Oct-3/4, Tra 1-60, Tra 1-80, and SSEA-4, exhibited high telomerase activity, and differentiated in vitro and in vivo into cells derived from all three germ layers, confirming their pluripotency. Additionally, newly derived hESCs were adapted to growth on a human placental laminin substrate in a defined medium.
CONCLUSION: To our knowledge, this is the first report of hESC derivation in the absence of serum on qualified pathogen-free human feeders.
Article
Derivation of hESC from intact blastocysts.
Source: Current Protocols in Stem Cell Biology, John Wiley & Sons
Contributors: Dusko Ilic, Olga Genbacev, and Ana Krtolica
Affiliations: StemLifeLine Inc., San Carlos, CA 94070 &University of California, San Francisco, CA 94143
Abstract
We describe protocols for human embryo culturing and derivation of human embryonic stem cells from the intact blastocyst. We begin with the description of the culturing methods to obtain human blastocysts using pronuclear or cleavage stage embryos left over after in vitro fertilization. We then describe methods that can be used to derive human embryonic stem cell lines from the blastocyst without trophectoderm removal. We also include discussion of the critical steps and parameters such as zona pellucida removal, embryo quality assessment, feeder selection, when and how to transfer early embryonic outgrowths. In addition, we provide protocols for embryo thawing, seeding of feeder cells, gelatin coating of plates, cleavage and blastocyst stage embryo grading, preparation and storage of reagents and solutions. We finish with discussion of the alternative derivation approaches and the timeline for the procedures.
Article
First derivation in Spain of human embryonic stem cell lines: use of long-term cryopreserved embryos and animal-free conditions.
Source: Fertil Steril. 2005 Jan;83(1):246-9.
Pub Med ID: 15652923
Contributors: Simon C, Escobedo C, Valbuena D, Genbacev O, Galan A, Krtolica A, Asensi A, Sanchez E, Esplugues J, Fisher S, Pellicer A.
Affiliations: Valencia Stem Cell Bank, Centro Superior de Alta Tecnologia, Valencia, Spain. csimon@ivi.es
Abstract
The first two human embryonic stem cell lines (VAL-1 and VAL-2) have been derived in Spain with long-term cryopreserved embryos under animal-free conditions. In the first series, 40 human embryos that had been cryopreserved at day 2 of development were thawed after >5 years. A derivation efficiency of 5% per frozen embryo or 12.5% per blastocyst was obtained.
Article
Trophoblast L-selectin-mediated adhesion at the maternal-fetal interface.
Source: Science. 2003 Jan 17;299(5605):405-8.
Pub Med ID: 12532021
Contributors: Genbacev OD, Prakobphol A, Foulk RA, Krtolica AR, Ilic D, Singer MS, Yang ZQ, Kiessling LL, Rosen SD, Fisher SJ.
Affiliations: Departments of Stomatology, Anatomy, and Pharmaceutical Chemistry, University of California, San Francisco, CA 94143, USA.
Abstract
Trophoblast adhesion to the uterine wall is the requisite first step of implantation and, subsequently, placentation. At the maternal-fetal interface, we investigated the expression of selectin adhesion systems that enable leukocyte capture from the bloodstream. On the maternal side, human uterine epithelial cells up-regulated selectin oligosaccharide-based ligands during the window of receptivity. On the fetal side, human trophoblasts expressed L-selectin. This ligand-receptor system was functional, because beads coated with the selectin ligand 6-sulfo sLe(x) bound to trophoblasts, and trophoblasts bound to ligand-expressing uterine luminal epithelium in tissue sections. These results suggest that trophoblast L-selectin mediates interactions with the uterus and that this adhesion mechanism may be critical to establishing human pregnancy.
Article
Derivation and characterization of three new Spanish human embryonic stem cell lines (VAL -3 -4 -5) on human feeder and in serum-free conditions.
Source: Reprod Biomed Online. 2006 Dec;13(6):875-86.
Pub Med ID: 17169214
Contributors: Valbuena D, Galan A, Sanchez E, Poo ME, Gomez E, Sanchez-Luengo S, Melguizo D, Garcia A, Ruiz V, Moreno R, Pellicer A, Simon C.
Affiliations: Banco Nacional de Lineas Celulares, Nodo de Valencia, Centro de Investigacion Principe Felipe (CIPF), Valencia, Spain.
Abstract
A total of 184 human embryos, frozen for >5 years, were donated; informed consent was obtained according to Spanish law 45/2003. Survival rate was 40% and three out of 24 blastocysts (12.5%) developed into putative hESC lines, named VAL-3, VAL-4, and VAL-5. The derivation process was performed on microbiologically tested and irradiated human foreskin fibroblasts and designed to minimize contact with xeno-components in knockout DMEM supplemented with knockout serum replacement, and basic fibroblast growth factor. Fingerprinting and HLA typing of the cell lines allowed their identification and traceability. Karyotype was normal for VAL-3 (46XY), VAL-4 (46XX) and VAL-5 (46XX). All three hESC lines expressed specific markers for non-differentiation (Nanog, stage-specific embryonic antigen-4 [SSEA-4], tumour-related antigen [TRA]-1-60, and TRA-1-81) and were negative for SSEA-1. RT-PCR further demonstrated the expression of Oct-4, Sox2, Rex-1, Nanog, Cripto, Thy-1, and Lefty-A. Furthermore, they were found to be negative for classical differentiation markers such as neurofilament heavy chain (ectoderm), renin (mesoderm), and amylase (endoderm). All three cell lines displayed high levels of telomerase activity, and were shown to successfully overcome cryopreservation and thawing. Finally, these three new hESC lines have demonstrated the potential to differentiate in vitro and in vivo (teratoma formation) into cell types originating from all three germ layers.
Article
Culture of human embryonic stem cells and the extracellular matrix microenvironment
Source: Regenerative Medicine, Jan 2006, Vol. 1, No. 1, Pages 95-101.
Contributors: Dusko Ilic
Affiliations: StemLifeLine, Inc.
Abstract
Among the major obstacles impeding successful derivation and continuous culture of human embryonic stem cells (hESC) fortherapeutic purposes, are the presence of feeder cells andfeeder-conditioned media of animal origin. The risk of contaminationwith xenopathogens makes hESC cultured in this way unsafe for futureuse in regenerative medicine. A holy grail for investigators in thefield will be to establish and maintain new hESC lines in completelyfeeder-free and serum-free defined conditions. Recently, propagationof hESC has become possible, using mammalian- or human-derivedextracellular matrix (ECM) and conditioned medium from feeder cells.In addition, providing a three-dimensional ECM environment can evensupport the derivation of new hESC. In this review, we examine recentadvances in the use and development of substrates suitable for thederivation and maintenance of hESC, and our current understanding ofthe effects of a three-dimensional ECM milieu on cellular behavior.
