Correlation between preimplantation genetic diagnosis for chromosomal aneuploidies and the efficiency of establishing human ES cell lines

There are several sources from which human embryonic stem cell (hESC) lines can be generated: surplus embryos after in vitro fertilization procedures, one- and three-pronuclear zygotes, early arrested or highly fragmented embryos that have reached the blastocyst stage, or otherwise chromosomally or genetically abnormal embryos after preimplantation genetic diagnosis (PGD). We report on the efficiency of establishing hESC lines from blastocysts with proven meiotic or mitotic errors after sequential testing of both polar bodies and blastomere analysis on day 3. The success rate of establishing hESC lines originating from blastocysts carrying a meiotic error was as low as 2.4% and differed significantly from the success rate of establishing hESC lines originating from blastocysts with balanced meiotic errors (21.6%) or mitotic errors (after sequential testing (9.1%) and after blastomere testing alone (12.2%)). This suggests that it may be reasonable to apply sequential PGD prior to the initiation of hESC culture. Information about the karyotype may in the future help refine the methods and possibly improve the efficiency by which hESC lines are derived from embryos with prezygotic abnormalities. Additionally, it may in general prove very difficult to obtain abnormal hESC lines for scientific study from aneuploid PGD embryos, which will limit our ability to study the biological consequences of chromosomal abnormalities. Furthermore, the success rates for generating aneuploid cell lines originating from fertilized oocytes carrying a prezygotic nondisjunction error seem to mirror the miscarriage rates during pregnancy of embryos carrying such errors.     

Derivation of new human embryonic stem cell lines from preimplantation genetic screening and diagnosis-analyzed embryos

In this study, we focused on the derivation of human embryonic stem cell (hESC) from preimplantation genetic screening (PGS)-analyzed and preimplantation genetic diagnosis (PGD)-analyzed embryos. Out of 62 fresh PGD/PGS-analyzed embryos, 22 embryos reached the blastocyst stage. From 12 outgrowth blastocysts, we derived four hESC lines onto a feeder layer. Surprisingly, karyotype analysis showed that hESC lines derived from aneuploid embryos had diploid female karyotype. One hESC line was found to carry a balanced Robertsonian translocation. All the cell lines showed hESC markers and had the pluripotent ability to differentiate into derivatives of the three embryonic germ layers. The established lines had clonal propagation with 22–31% efficiency in the presence of ROCK inhibitor. These results further indicate that hESC lines can be derived from PGD/PGS-analyzed embryos that are destined to be discarded and can serve as an alternative source for normal euploid lines.     

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.     

从植入前遗传学诊断异常的胚胎中分离人胚胎干细胞系

在体外受精过程中,通过胚胎植入前遗传性诊断（PGD）对有遗传风险患者的胚胎进行植入前活检和遗传学分析,选择无遗传性疾病的胚胎植入子宫,而PGD诊断异常的胚胎则会被丢弃。本研究尝试将PGD异常胚胎用于分离人胚胎干细胞,以获得携带遗传缺陷的人胚胎干细胞系。利用荧光原位杂交技术对第3-5天胚胎进行PGD检测,结果异常的胚胎进一步用于分离获取胚胎干细胞系,然后对h ES细胞系进行核型及干细胞表面标记、多能性基因表达、端粒酶活性以及分化能力等特征性鉴定。总共从13个PGD异常胚胎中分离获得8个人胚胎干细胞系,建系效率为61.5%,其中1个核型正常,5个核型异常。说明利用PGD异常胚胎可以获得携带遗传缺陷的人胚胎干细胞系,不仅为评估PGD技术临床结论的准确性提供了一种新方法,更重要的是为研究各种遗传性疾病的发病机理提供了有效的细胞模型。     

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.     

Human Embryonic Stem Cell Lines with Lesions in FOXP3 and NF1

Human embryonic stem cells (hESCs) are derived from the inner cell mass (ICM) of blastocyst staged embryos. Spare blastocyst staged embryos were obtained by in vitro fertilization (IVF) and donated for research purposes. hESCs carrying specific mutations can be used as a powerful cell system in modeling human genetic disorders. We obtained preimplantation genetic diagnosed (PGD) blastocyst staged embryos with genetic mutations that cause human disorders and derived hESCs from these embryos. We applied laser assisted micromanipulation to isolate the inner cell mass from the blastocysts and plated the ICM onto the mouse embryonic fibroblast cells. Two hESC lines with lesions in FOXP3 and NF1 were established. Both lines maintain a typical undifferentiated hESCs phenotype and present a normal karyotype. The two lines express a panel of pluripotency markers and have the potential to differentiate to the three germ layers in vitro and in vivo. The hESC lines with lesions in FOXP3 and NF1 are available for the scientific community and may serve as an important resource for research into these disease states.     

Efficient derivation of Chinese human embryonic stem cell lines from frozen embryos

Human embryonic stem (hES) cells are pluripotent cells derived from the inner cell mass of blastocysts. Their unique properties of self-renewal and pluripotency make them an attractive tool for basic research as well as a potential cell resource for therapy. However, each hES cell line demonstrates different identity. It is desirable to obtain more fully characterized hES cell lines with newly developed technologies associated with hES cell culture. Here, we report our experience of efficient derivation of three new Chinese hES cell lines (SHhES2, SHhES3, and SHhES4) from in vitro fertilization discarded embryos donated by women with polycystic ovary syndrome. These cell lines were derived under conditions minimizing exposure to animal components and maintained at an undifferentiated state for long-term culture. They retained a normal karyotype and expressed ALP, OCT4, SOX2, SSEA-4, TRA-1-60 and TRA-1-81. RT-PCR analysis also revealed high expression levels of pluripotency markers such as OCT4, LEFTY A, SOX2, TDGF-1, THY1, FGF4, NANOG, and REX1. When suspended in low-attachment culture dishes, embryoid bodies formed and were comprised of various differentiated cell types from all three embryonic germ layers. However, well-shaped teratomas were only harvested from line SHhES2, not from SHhES3 and SHhES4, indicating that the differentiation ability in vivo differs among the three cell lines. Collectively, the three new hES cell lines were established and fully characterized. The effort paves the way toward generating hES cell lines without contamination by animal components. All of these cell lines are available by contact Ying Jin at yjin@sibs.ac.cn.     

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).     

Degeneration pattern in somatic embryos of <Emphasis Type="Italic">Pinus sylvestris</Emphasis> L.

Somatic embryos can be used for propagating forest trees vegetatively, which is of great importance for capturing the genetic gain in breeding programs. However, many economically important Pinus species are difficult or impossible to propagate via somatic embryogenesis. In order to get a better understanding of the difficulties to propagate Pinus species via somatic embryogenesis, we are studying the developmental pathway of somatic embryos in different cell lines. In a previous study, we showed that the morphology of early somatic embryos in Scots pine (Pinus sylvestris) differs between cell lines giving rise to normal or abnormal cotyledonary embryos. In this study, we have compared the proliferation and degeneration pattern of early and late embryos in a normal and abnormal cell line. In both cell lines, a high frequency of the embryos degenerated. Among the degenerating embryos, two main degeneration patterns could be distinguished. In the normal cell line, the embryos degenerated similar to how the subordinate embryos are degraded in the seed. In the abnormal cell line, the degeneration of the embryos resulted in a continuous loop of embryo degeneration and differentiation of new embryos. We observed a similar degeneration pattern when embryogenic tissue was initiated from megagametophytes containing zygotic embryos at the stage of cleavage polyembryony. Based on our results, we suggest that the degeneration pattern in abnormal cell lines starts during initiation of embryogenic cultures.     

Karyotypically normal and abnormal human embryonic stem cell lines derived from PGD-analyzed embryos

Although a normal karyotype is generally a requirement for stem cell lines, new applications are likely to emerge for stem cells with defined chromosomal aneuploidies. We therefore investigated the use of embryos found to be aneuploid on biopsy followed by preimplantation genetic diagnosis (PGD) with fluorescent in situ hybridization (FISH), and developmentally arrested embryos for stem cell derivation. Eleven stem cell lines were obtained from 41 embryos in 36 cultures, with higher success rate achieved from PGD-analyzed, developmentally advanced embryos (45%) than from clinically unsuitable non-PGD embryos (13%). The resulting stem cell lines were karyotyped, and surprisingly, six of the nine lines from aneuploid embryos as well as both lines from non-PGD embryos were karyotypically normal. Three lines from PGD embryos were aneuploid exhibiting trisomy 5, trisomy 16, and an isochromosome 13, respectively. None of the aneuploid lines presented the same anomally as the original PGD analysis. Our study has three important implications. First, we confirm the ability to produce stem cell lines from PGD-tested embryos as well as developmentally abnormal embryos, offering specialty stem cell lines for research into the clinically important aneuploidies. Second, we observe that stem cell derivation from apparently aneuploid embryos is often thwarted by underlying mosaicism and emerging dominance of the stem cell line by karyotypically normal cells. The corollary, however, is that regular production of normal stem cell lines from developmentally abnormal embryos ordinarity discarded opens a new source of embryos for stem cells, whether for research or for eventual therapeutic use within the donating families.     

Comparative analysis of the developmental competence of three human embryonic stem cell lines in vitro

One of the goals of stem cell technology is to control the differentiation of human embryonic stem cells (hESCs), thereby generating large numbers of specific cell types for many applications including cell replacement therapy. Although individual hESC lines resemble each other in expressing pluripotency markers and telomerase activity, it is not clear whether they are equivalent in their developmental potential in vitro. We compared the developmental competence of three hESC lines (HSF6, Miz-hES4, and Miz-hES6). All three generated the three embryonic germ layers, extraembryonic tissues, and primordial germ cells during embryoid body (EB) formation. However, HSF6 and Miz-hES6 readily formed neuroectoderm, whereas Miz-hES4 differentiated preferentially into mesoderm and endoderm. Upon terminal differentiation, HSF6 and Miz-hES6 produced mainly neuronal cells whereas Miz-hES4 mainly formed mesendodermal derivatives, including endothelial cells, leukocyte progenitors, hepatocytes, and pancreatic cells. Our observations suggest that independently-derived hESCs may differ in their developmental potential.     

Mesenchymal differentiation propensity of a human embryonic stem cell line

Objectives: To characterize basal differentiation tendencies of a human embryonic stem (hES) cell line, KCL‐002. Materials and methods: In vitro specification and differentiation of hES cells were carried out using embryoid body (EB) cultures and tests of pluripotency and in vivo differentiation were performed by teratoma assays in SCID mice. Real‐time PCR, immunohistochemistry, flow cytometry and histological analyses were used to identify expression of genes and proteins associated with the ectodermal, endodermal and mesodermal germ layers. Results: Undifferentiated KCL‐002 cells expressed characteristic markers of pluripotent stem cells such as Nanog, Sox‐2, Oct‐4 and TRA 1‐60. When differentiated in vitro as EB cultures, expression of pluripotency, endodermal and ectodermal markers decreased rapidly. In contrast, mesodermal and mesenchymal markers such as VEGFR‐2, α‐actin and vimentin increased during EB differentiation as shown by qPCR, immunostaining and flow cytometric analyses. Teratoma formation in SCID mice demonstrated the potential to form all germ layers in vivo with a greater proportion of the tumours containing mesenchymal derivatives. Conclusions: The data presented suggest that the KCL‐002 hES cell line is pluripotent and harbours a bias in basal differentiation tendencies towards mesodermal and mesenchymal lineage cells. Characterizing innate differentiation propensities of hES cell lines is important for understanding heterogeneity between different cell lines and for further studies aimed at deriving specific lineages from hES cells.     

Hemato-endothelial differentiation from lentiviral-transduced human embryonic stem cells retains durable reporter gene expression under the control of ubiquitin promoter

Human embryonic stem (hES) cells are able to give rise to a variety of cell lineages under specific culture condition. An effective strategy for stable genetic modification in hES cells may provide a powerful tool for study of human embryogenesis and cell-based therapies. However, gene silences are documented in hES cells. In current study, we investigated whether genes controlled under ubiquitin promoter are expressed during hematopoietic-endothelial differentiation in hES cells. Undifferentiated hES cells (H1) were transduced by lentivirus encoding green fluorescent protein (GFP) gene under ubiquitin promoter. GFP-expressing hES cells (GFP-H1) were established after several rounds of mechanical selection under fluorescence microscope. GFP gene was stably expressed in hES cells throughout prolonged (> 50 passages) cultivation, and in differentiated embryo body (EB) and teratoma. Hematopoietic and endothelial markers, including KDR (VEGFR2), CD34, CD31, Tie-2, GATA-1 and GATA-2, were expressed at similar levels during hES cell differentiation in parent hES cells and GFP-H1 hES cells. CD34⁺ cells isolated from GFP-H1 hES cells were capable to generate hematopoietic colony-forming cells and tubular structure-forming cells. Differentiated GFP-EB formed vasculature structures in a semi-solid sprouting EB model. These results indicated that a transgene under ubiquitin promoter in lentiviral transduced hES cells retained its expression in undifferentiated hES cells and in hES-derived hematopoietic and endothelial cells. With the view of embryonic mesodermal developing events in humans, genetic modification of hES cells by lentiviral vectors provides a powerful tool for study of hematopoiesis and vasculogenesis.     

A Teratocarcinoma-Like Human Embryonic Stem Cell (hESC) Line and Four hESC Lines Reveal Potentially Oncogenic Genomic Changes

The first Swiss human embryonic stem cell (hESC) line, CH-ES1, has shown features of a malignant cell line. It originated from the only single blastomere that survived cryopreservation of an embryo, and it more closely resembles teratocarcinoma lines than other hESC lines with respect to its abnormal karyotype and its formation of invasive tumors when injected into SCID mice. The aim of this study was to characterize the molecular basis of the oncogenicity of CH-ES1 cells, we looked for abnormal chromosomal copy number (by array Comparative Genomic Hybridization, aCGH) and single nucleotide polymorphisms (SNPs). To see how unique these changes were, we compared these results to data collected from the 2102Ep teratocarcinoma line and four hESC lines (H1, HS293, HS401 and SIVF-02) which displayed normal G-banding result. We identified genomic gains and losses in CH-ES1, including gains in areas containing several oncogenes. These features are similar to those observed in teratocarcinomas, and this explains the high malignancy. The CH-ES1 line was trisomic for chromosomes 1, 9, 12, 17, 19, 20 and X. Also the karyotypically (based on G-banding) normal hESC lines were also found to have several genomic changes that involved genes with known roles in cancer. The largest changes were found in the H1 line at passage number 56, when large 5 Mb duplications in chromosomes 1q32.2 and 22q12.2 were detected, but the losses and gains were seen already at passage 22. These changes found in the other lines highlight the importance of assessing the acquisition of genetic changes by hESCs before their use in regenerative medicine applications. They also point to the possibility that the acquisition of genetic changes by ESCs in culture may be used to explore certain aspects of the mechanisms regulating oncogenesis.     

A Euploid Line of Human Embryonic Stem Cells Derived from a 43,XX,dup(9q),+12,-14,-15,-18,-21 Embryo

Aneuploid embryos diagnosed by FISH-based preimplantation genetic screening (PGS) have been shown to yield euploid lines of human embryonic stem cells (hESCs) with a relatively high frequency. Given that the diagnostic procedure is usually based on the analysis of 1–2 blastomeres of 5 to 10-cell cleavage-stage embryos, mosaicism has been a likely explanation for the phenomena. However, FISH-based PGS can have a significant rate of misdiagnosis, and therefore some of those lines may have been derived from euploid embryos misdiagnosed as aneuploid. More recently, coupling of trophectoderm (TE) biopsy at the blastocyst stage and array-CGH lead to a more informative form of PGS. Here we describe the establishment of a new line of hESCs from an embryo with a 43,XX,dup(9q),+12,-14,-15,-18,-21 chromosomal content based on array-CGH of TE biopsy. We show that, despite the complex chromosomal abnormality, the corresponding hESC line BR-6 is euploid (46,XX). Single nucleotide polymorphism analysis showed that the embryo´s missing chromosomes were not duplicated in BR-6, suggesting the existence of extensive mosaicism in the TE lineage.     

Distinct differentiation characteristics of individual human embryonic stem cell lines

Background  

Individual differences between human embryonic stem cell (hESC) lines are poorly understood. Here, we describe the derivation of five hESC lines (called FES 21, 22, 29, 30 and 61) from frozen-thawed human embryos and compare their individual differentiation characteristic.