Generation of new human embryonic stem cell lines with diploid and triploid karyotypes

Human pluripotent embryonic stem cells (hESC) have great promise for research into human developmental biology and the development of cell therapies for the treatment of diseases. To meet the increased demand for characterized hESC lines, we present the derivation and characterization of five hESC lines on mouse embryonic fibroblast cells. Our stem cell lines are characterized by morphology, long-term expansion, and expression profiles of a number of specific markers, including TRA-1-60, TRA-1-81, alkaline phosphatase, connexin 43, OCT-4, NANOG, CXCR4, NODAL, LEFTY2, THY-1, TDGF1, PAX6, FOXD3, SOX2, EPHA2, FGF4, TAL1, AC133 and REX-1. The pluripotency of the cell line was confirmed by spontaneous differentiation under in vitro conditions. Whereas all of the cell lines expressed all the characteristics of undifferentiated pluripotent hESC, two of the cell lines carried a triploid karyotype.     

Cell engineering and genetic approaches to development of human embryonic stem cell models for genetic disorders

We introduced a novel approach for the establishment of genetically modified hESC lines, and have shown that mutant hESC may be derived from affected embryos after preimplantation genetic diagnosis (PGD) screening for a particular single gene disorder. Here we describe the procedure of embryo and cell manipulation, their diagnostic layout, and the analysis of the efficiency of embryo development and hESC establishment, as well as the developments for hESC derivation in animal-product-free conditions. Our study shows that a high efficiency of hESC derivation (50%) is especially crucial when working with rare and unique resources such as genetically screened embryos necessary for the derivation of hESC lines that represent specific genetic diseases.     

No relationship between embryo morphology and successful derivation of human embryonic stem cell lines

Background

The large number (30) of permanent human embryonic stem cell (hESC) lines and additional 29 which did not continue growing, in our laboratory at Karolinska Institutet have given us a possibility to analyse the relationship between embryo morphology and the success of derivation of hESC lines. The derivation method has been improved during the period 2002–2009, towards fewer xeno-components. Embryo quality is important as regards the likelihood of pregnancy, but there is little information regarding likelihood of stem cell derivation.

Methods

We evaluated the relationship of pronuclear zygote stage, the score based on embryo morphology and developmental rate at cleavage state, and the morphology of the blastocyst at the time of donation to stem cell research, to see how they correlated to successful establishment of new hESC lines.

Results

Derivation of hESC lines succeeded from poor quality and good quality embryos in the same extent. In several blastocysts, no real inner cell mass (ICM) was seen, but permanent well growing hESC lines could be established. One tripronuclear (3PN) zygote, which developed to blastocyst stage, gave origin to a karyotypically normal hESC line.

Conclusion

Even very poor quality embryos with few cells in the ICM can give origin to hESC lines.     

Laminin-511 expression is associated with the functionality of feeder cells in human embryonic stem cell culture

Fibroblast feeder cells play an important role in supporting the derivation and long term culture of undifferentiated, pluripotent human embryonic stem cells (hESCs). The feeder cells secrete various growth factors and extracellular matrix (ECM) proteins into extracellular milieu. However, the roles of the feeder cell-secreted factors are largely unclear. Animal feeder cells and use of animal serum also make current feeder cell culture conditions unsuitable for derivation of clinical grade hESCs. We established xeno-free feeder cell lines using human serum (HS) and studied their function in hESC culture. While human foreskin fibroblast (hFF) feeder cells were clearly hESC supportive, none of the established xeno-free human dermal fibroblast (hDF) feeder cells were able to maintain undifferentiated hESC growth. The two fibroblast types were compared for their ECM protein synthesis, integrin receptor expression profiles and key growth factor secretion. We show that hESC supportive feeder cells produce laminin-511 and express laminin-binding integrins α3ß1, α6ß1 and α7ß1. These results indicate specific laminin isoforms and integrins in maintenance of hESC pluripotency in feeder-dependent cultures. In addition, several genes with a known or possible role for hESC pluripotency were differentially expressed in distinct feeder cells.     

Gene expression profiles of human inner cell mass cells and embryonic stem cells

Human embryonic stem cell (hESC) lines are derived from the inner cell mass (ICM) of preimplantation human blastocysts obtained on days 5–6 following fertilization. Based on their derivation, they were once thought to be the equivalent of the ICM. Recently, however, studies in mice reported the derivation of mouse embryonic stem cell lines from the epiblast; these epiblast lines bear significant resemblance to human embryonic stem cell lines in terms of culture, differentiation potential and gene expression. In this study, we compared gene expression in human ICM cells isolated from the blastocyst and embryonic stem cells. We demonstrate that expression profiles of ICM clusters from single embryos and hESC populations were highly reproducible. Moreover, comparison of global gene expression between individual ICM clusters and human embryonic stem cells indicated that these two cell types are significantly different in regards to gene expression, with fewer than one half of all genes expressed in both cell types. Genes of the isolated human inner cell mass that are upregulated and downregulated are involved in numerous cellular pathways and processes; a subset of these genes may impart unique characteristics to hESCs such as proliferative and self-renewal properties.     

Intrinsic properties and external factors determine the differentiation bias of human embryonic stem cell lines

A major goal of human embryonic stem cell (hESC) research is to regulate differentiation through external means to generate specific cell types with high purity for regenerative medicine applications. Although all hESC lines express pluripotency‐associated genes, their differentiation ability to various lineages differs considerably. We have compared spontaneous differentiation propensity of the two hESC lines, RelicellhES1 and BG01. Spontaneous differentiation of hESC lines grown in different media conditions was followed by differentiation using two methods. Kinetic data generated by real‐time gene expression studies for differentiated cell types were analyzed, and confirmed at protein levels. Both cell lines showed upregulation of genes associated with the 3 germ layers, although stark contrast was evident in the magnitude of upregulation of lineage specific genes. A distinct difference was also found in the rate at which the pluripoteny factors, Oct‐4 and Nanog, were downregulated during differentiation. Once differentiation was initiated, both Oct‐4 and Nanog gene expression was drastically reduced in RelicellhES1, whereas a gradual decrease was observed in BG01. A clear trend is seen in RelicellhES1 to differentiate into neuroectodermal and mesenchymal lineages, whereas BG01 cells are more prone to mesoderm and endoderm development. In addition, suspension versus plated methods of cell culture significantly influenced the outcome of differentiation of certain types of cells. Results obtained by spontaneous differentiation of hESCs were also amplified by induced differentiation. Thus, differential rates of downregulation of pluripotency markers along with culture conditions seem to play an important role in determining the developmental bias of human ES cell lines.     

In vitro culture period but not the passage number influences the capacity of chimera production of inner cell mass and its deriving cells from porcine embryos

Mammalian embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass (ICM) of the blastocyst. These cells are able to proliferate continuously without differentiation in vitro under suitable conditions. Their capacity of pluripotency in differentiation will be resumed when they are reintroduced into host embryos, when they will contribute to the embryonic development to form chimeric individuals. Manipulation of ES cells has been mainly established from studies in the mouse, and is powerful in the production of transgenic animals. Porcine ICM-derived cell lines possess the same cellular morphology and in vitro behavior as those of murine ES cells, but have lower efficiency in chimera formation when reintroduced into host embryos. This study was to determine the influences of passage number and the duration of in vitro culture on the capacity of porcine ICM-derived cells in the generation of chimeric embryos. The results showed that when passage number of porcine ICM-derived cells was less than 15, there were no detrimental effects on its integration ability. Extending the culture time up to 6 days in each passage of porcine ICM-derived cells impaired its integration capacity into the host blastocyst. Porcine ICM-derived cells cultured for more than 4 days in each passage should not be used for blastocyst injection if high efficiency of chimera production is to be achieved.     

Derivation, culture, and characterization of VUB hESC lines

In this report, we present the derivation and characterization of 15 hESC lines established at the Vrije Universiteit Brussel, Belgium in collaboration with the Universitair Ziekenhuis Brussel, Belgium, using surplus in vitro fertilization embryos and embryos carrying monogenic disorders donated for research. Four lines were derived from blastocyst-stage embryos presumed to be genetically normal, and 11 hESC lines were obtained from embryos shown to carry genetic mutations by preimplantation genetic diagnosis. All the lines express markers of pluripotency as determined by immunocytochemistry and RT-PCR, and formed teratomas when injected into SCID mice. All VUB hESC lines, except for VUB17, are reported in the European hESC registry and are available upon request after signing a Material Transfer Agreement from the VUB (contact person: Prof. Dr. Karen Sermon; Karen.Sermon@uzbrussel.be).     

High-efficiency derivation of human embryonic stem cell lines following pre-implantation genetic diagnosis

Pre-implantation genetic diagnosis allows the characterisation of embryos that carry a gene responsible for a severe monogenic disease and to transfer to the mother’s uterus only the unaffected one(s). The genetically affected embryos can be used to establish human embryonic stem cell (hESC) lines. We are currently establishing a cell bank of ESC lines carrying specific disease-causing mutant genes. These cell lines are available to the scientific community. For this purpose, we have designed a technique that requires only minimal manipulation of the embryos. At the blastocyst stage, we just removed the zona pellucida before seeding the embryo as a whole on a layer of feeder cells. This approach gave a good success rate (>20%), whatever the quality of the embryos, and allowed us to derive 11 new hESC lines, representing seven different pathologies. Full phenotypic validation of the cell lines according to ISCI guidelines confirmed their pluripotent nature, as they were positive for hESC markers and able to differentiate in vitro in all three germ layers derivatives. Nine out of 11 stem cell lines had normal karyotypes. Our results indicate that inner cell mass isolation is not mandatory for hESC derivation and that minimal manipulation of embryos can lead to high success rate.     

Establishment and characterization of human embryonic stem cell lines, Turkey perspectives

Human embryonic stem cells (hESC), which are derived from the inner cell mass (ICM) of blastocyst stage embryos, are of great importance because of their unpredictable two unique features: their differentiation ability into all types of cells derived from three germ layers and their potentially unlimited capacity of self renewing with stable karyotype. These distinguished properties make hESC very promising cell source for regenerative medicine, tissue replacement therapies, and drug screening studies as well as genomics. However, due to the several technical problems, such as risk of teratoma formation, immune response, and unknown genetic pathways for lineage specific differentiation, and ethical drawbacks of their using in clinical treatments, hESC researches are still waiting to advance beyond to animal trials and drug studies. During the last decade, more than 300 new hESC lines have been derived and published by researchers worldwide. However, despite their similar well-known unique properties, recent studies reported that hESC lines have very individual properties and are differed from each other with regards to their differentiation ability and gene expression profiles. Therefore, all hESC lines should be characterized in detail and then registered in a stem cell bank for generating global database. In this report, the characteristic of hESC lines, which were established in Istanbul Memorial Hospital between 2003 and 2005, and derivation methods were described in detail to inform researchers and to facilitate new prospective cooperative studies.     

Derivation of human embryonic stem cell lines

Human embryonic stem cells (hESC) are undifferentiated cells derived from an early embryo that can grow in vitro indefinitely, while retaining their capability to differentiate into specialized somatic cell types. Over the last decade there has been great interest in derivation and culture of these cells, as they can potentially provide a supply of readily available differentiated cells and tissues of all types to be used for therapeutic purposes in cell transplantation in humans, as well as for other medical uses such as drug discovery. The source of hESC lines is usually excess human embryos from in vitro fertilization treatments, although novel ways of producing hESCs have been suggested recently. The actual methods of hESC derivation have not changed greatly since the first report by Thomson et al. in 1998 . However, the main emphasis over the last several years has been in finding defined conditions for derivation and culture of hESCs, because to enable the clinical use of hESC for cell transplantation, the use of animal derived biological components is no longer acceptable. For basic research, the aim is to replace even human derived materials with completely defined systems. In this paper we describe methods utilized in our laboratory for hESC derivation and describe two studies conducted in an attempt to improve derivation efficiency and to enable research outcomes to be achieved using fewer embryos.     

Derivation of a new human embryonic stem cell line, Endeavour-2, and its characterization

Here, we describe the derivation of a novel human embryonic stem cell (hESC) line, Endeavour-2 (E-2), propagated on human fetal fibroblasts (HFF) in a serum-replacement media. The inner cell mass (ICM) was manually dissected from the blastocyst without using immunodissection and, therefore, antibodies from animal sources. A total of 20 embryos were thawed and cultured, eight embryos were hatched, and five ICMs were obtained. They were transferred onto HFF used as feeder layer, and one colony representing the initial cell proliferation of a new hESC line, E-2, was obtained. The newly emerged hESC colony was passaged first by physical dissection and subsequently by enzymatic dissociation. E-2 has been in culture for over 6 months and has been shown to possess typical features of a pluripotent hESC line including expression of stem cell surface markers (SSEA4, TRA-160, and integrin alpha-6), intracellular alkaline phosphatase, and pluripotency gene markers, OCT4 and NANOG. This hESC line shows lineage-specific differentiation into various representative cell types expressing markers characteristic of the three somatic germ layers under both in vitro and in vivo conditions. E-2 line shows a normal karyotype (46 XX) and has been successfully cryopreserved and thawed several times using slow-freezing procedures. E-2 adds to the repertoire of existing hESC lines for research and development purposes in the field of regenerative medicine.     

Derivation and characterization of three new human embryonic stem cell lines in Finland

Human embryonic stem cell (hESC) lines can be established from the preimplantation embryos. Due to their ability to differentiate into all three embryonic layers, hESC are of significant interest as a renewable source of cell material for different applications, especially for cell replacement therapy. Since the establishment of the first hESC lines in 1998, several studies have described the derivation and culture of new hESC lines using various derivation methods and culture conditions. Our group has currently established eight new hESC lines of which three of the latest ones are described in a more detailed way in this report. The described lines have been established using mechanical derivation methods for surplus bad quality embryos and culture conditions containing human foreskin fibroblast feeder cells and serum-free culture medium. All the new lines have a normal karyotype and typical hESC characteristics analyzed in vitro. The described hESC lines are available for research purposes upon request (www.regea.fi).     

FGF2 secreting human fibroblast feeder cells: a novel culture system for human embryonic stem cells

Human embryonic stem cell (hESC) lines are traditionally derived and maintained on mouse embryonic fibroblasts (MEF) which are xenogeneic and enter senescence rapidly. In view of the clinical implications of hESCs, the use of human fibroblast as feeders has been suggested as a plausible alternative. However, use of fibroblast cells from varying sources leads to culture variations along with the need to add FGF2 in cultures to sustain ES cell pluripotency. In this study we report the derivation of FGF2 expressing germ layer derived fibroblast cells (GLDF) from hESC lines. These feeders could support the pluripotency, karyotypes and proliferation of hESCs with or without FGF2 in prolonged cultures as efficiently as that on MEF. GLDF cells were derived from embryoid bodies and characterized for expression of fibroblast markers by RT-PCR, Immunofluorescence and by flow cytometry for CD marker expression. The expression and secretion of FGF2 was confirmed by RT-PCR, Western blot, and ELISA. The hESC lines cultured on MEF and GLDF were analyzed for various stemness markers. These feeder cells with fibroblast cells like properties maintained the properties of hESCs in prolonged culture over 30 passages. Proliferation and pluripotency of hESCs on GLDF was comparable to that on mouse feeders. Further we discovered that these GLDF cells could secrete FGF2 and maintained pluripotency of hESC cultures even in the absence of supplemental FGF2. To our knowledge, this is the first study reporting a novel hESC culture system which does not warrant FGF2 supplementation, thereby reducing the cost of hESC cultures.     

Improved genetic manipulation of human embryonic stem cells

Low efficiency of transfection limits the ability to genetically manipulate human embryonic stem cells (hESCs), and differences in cell derivation and culture methods require optimization of transfection protocols. We transiently transferred multiple independent hESC lines with different growth requirements to standardized feeder-free culture, and optimized conditions for clonal growth and efficient gene transfer without loss of pluripotency. Stably transfected lines retained differentiation potential, and most lines displayed normal karyotypes.     

Chemical enhancement in embryo development and stem cell derivation from single blastomeres

Several chemicals targeting the mitogen-activated protein (MAP) kinase signaling pathway, which play an important role in regulating cell growth and differentiation, have shown enhancing effects on the development of the inner cell mass (ICM) and the derivation of ES cells. However, investigation of such chemicals on early embryonic development and the establishment of ES cell lines has not been elucidated. This study was aimed to determine if ACTH, MAP2K1 inhibitor [MAP2K1 (I)], and MAPK14 inhibitor [MAPK14 (I)] could enhance the development of the ICM in preimplantation mouse embryos and blastocyst outgrowths, and the establishment of ES cell lines from blastomeres of early embryos. We have demonstrated that both MAP2K1 (I) and MAPK14 (I) delay early embryo development and inhibit the development of embryos from early blastomeres. On the other hand, ACTH had a positive effect on embryos derived from early blastomeres. As a result, 17 ES cell lines were established. Among these ES cell lines, nine and five ES cell lines were established from single blastomeres of two-cell embryos with and without the supplement of ACTH, respectively. In addition to two-cell isolated blastomeres, three ES cell lines were established from blastomeres of four-cell embryos only with the supplement of ACTH. Our results suggest that ACTH can enhance the derivation of ES cells from single blastomere-derived embryos.     

Diverse Epigenetic Profile of Novel Human Embryonic Stem Cell Lines

Human embryonic stem cells (hESCs) are a promising model for studying mechanisms ofregulation of early development and differentiation. OCT4, NANOG, OCT4-related genes andsome others were recently described to be important in pluripotency maintenance. Lesser isknown about molecular mechanisms involved in their regulation. Apart from genetic regulationof gene expression epigenetic events, particularly methylation, play an important role in earlydevelopment. Using RT-PCR we studied the expression of pluripotency-related genes OCT4,NANOG, DPPA3, and DPPA5 during hESCs differentiation to embryoid bodies. Analysis ofmethylation profiles of promoter or putative regulatory regions of the indicated genesdemonstrated that expression of the pluripotency-maintaining genes correlated with theirmethylation status, whereas methylation of DPPA3 and DPPA5 varied between cell lines. Wepropose that DNA methylation underlies the developmental stage-specific mechanisms ofpluripotency-related genes expression and reactivation and may have an impact ondifferentiation potential of hESC lines.