Establishment of human embryonic stem cell lines from frozen-thawed blastocysts using STO cell feeder layers

BACKGROUND: Recently, human embryonic stem (hES) cells have become very important resources for basic research on cell replacement therapy and other medical applications. The purpose of this study was to test whether pluripotent hES cell lines could be successfully derived from frozen-thawed embryos that were destined to be discarded after 5 years in a routine human IVF-embryo transfer programme and whether an STO cell feeder layer can be used for the culture of hES cells. METHODS: Donated frozen embryos (blastocysts or pronuclear) were thawed, and recovered or in vitro developed blastocysts were immunosurgically treated. All inner cell masses were cultured continuously on an STO cell feeder layer and then presumed hES cell colonies were characterized. RESULTS: Seven and two cell lines were established from frozen-thawed blastocysts (7/20, 35.0%) and pronuclear stage embryos (2/20, 10.0%), respectively. The doubling time of hES cells on the immortal STO cell feeder layer was approximately 36 h, similar to that of cells grown using fresh mouse embryonic fibroblast (MEF) feeder conditions. Subcultured hES cell colonies showed strong positive immunostaining for alkaline phosphatase, stage-specific embryonic antigen-4 (SSEA-4) and tumour rejection antigen 1-60 (TRA1-60) cell surface markers. Also, the hES colonies retained normal karyotypes and Oct-4 expression in prolonged subculture. When in vitro differentiation of hES cells was induced by retinoic acid, three embryonic germ layer cells were identified by RT-PCR or indirect immunocytochemistry. CONCLUSIONS: This study indicates that establishment of hES cells from frozen-thawed blastocysts minimizes the ethical problem associated with the use of human embryos in research and that the STO cell feeder layer can be used for the culture of hES cells.     

The derivation of two additional human embryonic stem cell lines from day 3 embryos with low morphological scores

BACKGROUND: Immune rejection can lead to the failure of human embryonic stem cell (hES cell) transplantation. One approach to address the problem is to establish hES cell line banks. Due to the limited source of human embryos and to ethical reasons, the hES cell lines are not readily available. This study was undertaken to determine whether discarded day 3 embryos with low morphological scores could develop into blastocysts and produce hES cell lines. METHODS: A total of 130 day 3 embryos with low morphological scores were cultured to blastocyst stage, and inner cell masses (ICM) were isolated by immunosurgery. Colonies derived from the ICM were passaged every 4-7 days and evaluated for cell surface markers, differentiation potentials and karyotypes. RESULTS: A total of 19 blastocysts were obtained from 130 embryos (quality score <16), which resulted in the formation of 10 ICM, and two cell lines. Both cell lines satisfied the criteria that characterize pluripotent hES cells. CONCLUSION: Our results suggest that a subset with poor quality day 3 embryos judged on the basis of morphological assessment can form blastocysts and give rise to hES cell lines.     

人胚胎干细胞饲养层培养体系的研究进展

人胚胎干细胞(hES细胞)来源于着床前人囊胚内细胞团(ICM),由于具有体外无限增殖和分化成3个胚层来源的各种细胞的潜能,使其成为当今生命科学的研究热点.建立一个理想的hES细胞培养体系是利用它的前提.目前,最常用的hES细胞的体外培养方式是将其培养在饲养层细胞上.迄今为止,已经有多种细胞用于hES细胞的体外培养.饲养...     

Derivation, characterization, and differentiation of human embryonic stem cells

The derivation of human embryonic stem (hES) cells establishes a new avenue to approach many issues in human biology and medicine for the first time. To meet the increased demand for characterized hES cell lines, we present the derivation and characterization of six hES cell lines. In addition to the previously described immunosurgery procedure, we were able to propagate the inner cell mass and establish hES cell lines from pronase-treated and hatched blastocysts. The cell lines were extensively characterized by expression analysis of markers characteristic for undifferentiated and differentiated hES cells, karyotyping, telomerase activity measurement, and pluripotency assays in vitro and in vivo. Whereas three of the cell lines expressed all the characteristics of undifferentiated pluripotent hES cells, one cell line carried a chromosome 13 trisomy while maintaining an undifferentiated pluripotent state, and two cell lines, one of which carried a triploid karyotype, exhibited limited pluripotency in vivo. Furthermore, we clonally derived one cell line, which could be propagated in an undifferentiated pluripotent state.     

Growth rate of human preimplantation embryos is sex dependent after ICSI but not after IVF

BACKGROUND: There is concern that IVF and/or ICSI might have an adverse effect on embryonic development via epigenetic alterations. Such alterations might also be involved in the sex-related growth differences in preimplantation embryos found in some animal species. In the present study we analysed cell numbers of human male and female surplus embryos that developed to the blastocyst stage after either IVF or ICSI in order to investigate possible sex-dependent differential growth rates. METHODS: Blastocysts resulting from surplus embryos obtained after either IVF or ICSI during a 5 year study period were analysed using fluorescence in situ hybridization (FISH). RESULTS: The number of cells and sex could be determined in 330 blastocysts collected from 92 IVF cycles and in 322 blastocysts collected from 121 ICSI cycles. Whereas female and male embryos originating from IVF showed comparable mean log cell numbers per embryo +/- SEM (3.76+/-0.05 in 147 female and 3.72+/-0.04 in 183 male embryos), significant differences were observed in embryos originating from ICSI (3.57+/-0.05 in 162 female and 3.90+/-0.03 in 160 male embryos). The sex-related growth difference was significantly greater in ICSI than in IVF embryos. In a subset of 84 embryos, inner cell mass (ICM) and trophectoderm (TE) were analysed separately. A significantly higher mean log cell number of TE cells in ICSI male embryos was found as compared to their female counterparts (3.44+/-0.12 in 16 female and 3.90+/-0.11 in 29 male embryos), whereas this difference was not found in IVF embryos. CONCLUSION: A clear sex-related growth difference was found in human blastocysts originating from ICSI, but not in blastocysts originating from IVF. It is as yet unknown which mechanism is responsible for our findings. We hypothesize that the ICSI procedure might interfere with the process of imprinted X-inactivation.     

A culture system using human foreskin fibroblasts as feeder cells allows production of human embryonic stem cells

BACKGROUND: Human embryonic stem (hES) cell lines were first cultured using fetal mouse fibroblasts as feeder cells. To avoid feeders and to reduce the amount of xeno-components, Matrigel- and laminin-coated dishes, and conditioned mouse feeder cell medium have been used, and hES cells have also been cultured on human fetal muscle and skin, and adult Fallopian tube epithelial cells. METHODS: We used post-natal, commercially available human foreskin fibroblasts as feeder cells. Inner cell masses (ICM) were isolated from five supernumerary blastocysts, obtained as donations from couples undergoing IVF treatment. RESULTS: Two ICM showed continuous growth. One line, HS181, has been in culture for 41 weeks with a doubling time of 24-36 h. It continues to express stem cell markers alkaline phosphatase, Oct-4, stage-specific embryonic antigen (SSEA)-4 and tumour-related antigen (TRA)-1-60. The karyotype is 46,XX. Pluripotency was demonstrated by teratoma formation in immunodeficient mice. In high-density cultures, spontaneous differentiation to beating cells and neuron-like cells was seen. The second line, HS207, was cultured for 9 weeks and cryopreserved, as were samples of line HS181. Both lines began to grow after thawing. CONCLUSIONS: We used successfully human foreskin fibroblasts as feeder cells for derivation and continued undifferentiated growth of hES cells. These feeder cells are convenient for IVF units, because no fetal human tissues or tissue from operations are needed.     

Laser-assisted derivation of human embryonic stem cell lines from IVF embryos after preimplantation genetic diagnosis

BACKGROUND: Human embryonic stem cells (hESCs) suitable for future transplantation therapy should preferably be developed in an animal-free system. Our objective was to develop a laser-based system for the isolation of the inner cell mass (ICM) that can develop into hESC lines, thereby circumventing immunosurgery that utilizes animal products. METHODS: Hatching was assisted by micromanipulation techniques through a laser-drilled orifice in the zona pellucida of 13 abnormal preimplantation genetic diagnosed blastocysts. ICMs were dissected from the trophectoderm by a laser beam and plated on feeders to derive hESC lines. RESULTS: eight ICMs were isolated from nine hatched blastocysts and gave rise to three hESC lines affected by myotonic dystrophy type 1, hemophilia A and a carrier of cystic fibrosis 405 + 1G > A mutation. Five blastocysts that collapsed during assisted hatching or ICM dissection were plated whole, giving rise to an additional line affected by fragile X. All cell lines expressed markers of pluripotent stem cells and differentiated in vitro and in vivo into the three germ layers. CONCLUSIONS: These hESC lines can serve as an important model of the genetic disorders that they carry. Laser-assisted isolation of the ICMs may be applied for the derivation of new hESC lines in a xeno-free system for future clinical applications.     

Derivation of a diploid human embryonic stem cell line from a mononuclear zygote

BACKGROUND: IVF occasionally produces aneuploid zygotes with one or three pronuclei (PN). Routinely, these zygotes are discarded. The aim of this work was to establish human embryonic stem cell (hESC) lines from blastocysts resulting from abnormal fertilization. METHODS: Abnormally fertilized zygotes were cultured to the blastocyst stage and, following zona pellucida digestion, zona-free blastocysts were placed on a mouse feeder layer. Culture of hESCs was carried out as described earlier. RESULTS: Six out of the nine developing blastocysts attached to the feeder layer. One hESC line, originating from a mononuclear zygote following ICSI, was successfully derived. This line displayed typical phenotype and embryonic surface markers, and exhibited the potential to develop into all three embryonic germ layers both in vitro (by embryoid body formation) and in vivo (teratoma generation). Genetic examination revealed normal diploid karyotype and heterozygotic appearance for metachromatic leukodystrophy (MLD). CONCLUSION: This method, which requires neither immuno nor mechanical removal of the trophectoderm, may facilitate the derivation of hESC lines in general, and those from abnormal embryos in particular. Furthermore, it is shown that aneuploid zygotes can be used as a source for normal hESC derivation and hold the potential to generate aneuploid hESC lines for research purposes.     

Differentiation prevents assessment of neural stem cell pluripotency after blastocyst injection

Earlier studies reported that neural stem (NS) cells injected into blastocysts appeared to be pluripotent, differentiating into cells of all three germ layers. In this study, we followed in vitro green fluorescent protein (GFP)-labeled NS and embryonic stem (ES) cells injected into blastocysts. Forty-eight hours after injection, significantly fewer blastocysts contained GFP-NS cells than GFP-ES cells. By 96 hours, very few GFP-NS cells remained in blastocysts compared with ES cells. Moreover, 48 hours after injection, GFP-NS cells in blastocysts extended long cellular processes, ceased expressing the NS cell marker nestin, and instead expressed the astrocytic marker glial fibrillary acidic protein. GFP-ES cells in blastocysts remained morphologically undifferentiated, continuing to express the pluripotent marker stage-specific embryonic antigen-1. Selecting cells from the NS cell population that preferentially formed neurospheres for injection into blastocysts resulted in identical results. Consistent with this in vitro behavior, none of almost 80 mice resulting from NS cell-injected blastocysts replaced into recipient mothers were chimeric. These results strongly support the idea that NS cells cannot participate in chimera formation because of their rapid differentiation into glia-like cells. Thus, these results raise doubts concerning the pluripotency properties of NS cells.     

用低形态学评分D3胚胎建立人胚胎干细胞系

目的 寻找人胚胎干细胞(hESC)建系材料来源.方法 选用IVF低形态学评分的D3胚胎行序贯囊胚培养,用免疫外科的方法去除滋养细胞,将得到的内细胞团(ICM)接种于丝裂霉素C灭活的小鼠胚胎成纤维细胞(MEFs)上培养5～8 d,每4～7 d传代1次,分别取不同代的hESC进行碱性磷酸酶(AKP)染色、转录因子OCT-4、阶段特异性胚胎抗原(SSEA)SSEA-4、SSEA-1、肿瘤排斥抗原(TRA)TRA-1-60、TAR-1-81、核型及体内外分化全能性鉴定.结果 130枚废弃的D3低形态学评分(评分＜16)的胚胎培养出囊胚19枚,获得原代克隆5个,成功培养出两株hESC系,它们具有hESC的共同的生物学特性.结论 部分低形态学评分的D3废弃胚胎可发育成囊胚.囊胚形成率与形态学评分相关,这些胚胎可作为建立hESC系的材料来源之一.     

Whole-blastocyst culture followed by laser drilling technology enhances the efficiency of inner cell mass isolation and embryonic stem cell derivation from good- and poor-quality mouse embryos: new insights for derivation of human embryonic stem cell lines

The optimization of human embryonic stem (hES) cell line derivation methods is challenging because many worldwide laboratories have neither access to spare human embryos nor ethical approval for using supernumerary human embryos for hES cell derivation purposes. Additionally, studies performed directly on human embryos imply a waste of precious human biological material. In this study, we developed a new strategy based on the combination of whole-blastocyst culture followed by laser drilling destruction of the trophoectoderm for improving the efficiency of inner cell mass (ICM) isolation and ES cell derivation using murine embryos. Embryos were divided into good- and poor-quality embryos. We demonstrate that the efficiency of both ICM isolation and ES cell derivation using this strategy is significantly superior to whole-blastocyst culture or laser drilling technology itself. Regardless of the ICM isolation method, the ES cell establishment depends on a feeder cell growth surface. Importantly, this combined methodology can be successfully applied to poor-quality blastocysts that otherwise would not be suitable for laser drilling itself nor immunosurgery in an attempt to derive ES cell lines due to the inability to distinguish the ICM. The ES cell lines derived by this combined method were characterized and shown to maintain a typical morphology, undifferentiated phenotype, and in vitro and in vivo three germ layer differentiation potential. Finally, all ES cell lines established using either technology acquired an aneuploid karyotype after extended culture periods, suggesting that the method used for ES cell derivation does not seem to influence the karyotype of the ES cells after extended culture. This methodology may open up new avenues for further improvements for the derivation of hES cells, the majority of which are derived from frozen, poor-quality human embryos.     

Human Embryonic St me Cell Lines fromthe Chinese Population and Differentiation to Liver and Muscle Cell Types

We have established 6 hES cell lines from IVF surplus blastocysts. Characterization of these lines have shown that 4 of the 6 lines meet all of the criterion (Science) for hES cell lines and 2 of them display most characteristics of hES cells but do not form teratoma. In order to produce hES cell lines without using mouse feeders, we have produced a hES cell line using feeders derived from hES cells themselves, and showed that hES-derived feeders are capable of supporting the derivation of new hES cell line...     

Cryopreservation of biopsied embryos at the blastocyst stage

BACKGROUND: The availability of an efficient cryopreservation program is especially important in the case of embryos that have undergone blastomere biopsy for PGD. Unfortunately, the freezing/thawing of biopsied embryos has given disappointing results when performed at the cleavage stage. In this study, embryos diagnosed as normal after PGD were grown to the blastocyst stage, frozen and thawed for successive frozen embryo transfer. METHODS: A total of 34 patients performed a thawing cycle in which 47 blastocysts were thawed. The cryopreservation solutions were based on HEPES-buffered medium supplemented with human serum albumin (HSA), sucrose and 1,2-propanediol. The same protocol was applied to embryos from 88 IVF/ICSI patients, which underwent 92 thawing cycles with 150 thawed blastocysts. RESULTS: The survival rate was similar in the two groups (53% after PGD and 58% in IVF/ICSI cycles), as well as the cumulative pregnancy rate per patient (59% after PGD versus 47% in IVF/ICSI cycles), despite a higher maternal age and a lower proportion of embryos available for transfer or cryopreservation in the PGD group. CONCLUSIONS: Neither the survival rate nor the subsequent development and chances of implantation, differed between embryos frozen at the blastocyst stage following biopsy and those frozen intact.     

Establishment of pluripotent cell lines from vertebrate species--present status and future prospects

Pluripotent embryonic stem (ES) cells are undifferentiated cell lines derived from early embryos and are capable of unlimited undifferentiated proliferation in vitro. They retain the ability to differentiate into all cell types including germ cells in chimeric animals in vivo, and can be induced to form derivatives of all three germ layers in vitro. Mouse ES cells represent one of the most important tools in genetic research. Major applications include the targeted mutation of specific genes by homologous recombination and the discovery of new genes by gene trap strategies. These applications would be of high interest for other model organisms and also for livestock species. However, in spite of tremendous research activities, no proven ES cells colonizing the germ line have been established for vertebrate species other than mouse and chicken thus far. This review summarizes the current status of deriving pluripotent embryonic stem cell lines from vertebrates and recent developments in nuclear transfer technology, which may provide an alternative tool for genetic modification of livestock animals.     

Birth of a healthy infant following trophectoderm biopsy from blastocysts for PGD of beta-thalassaemia major

PGD is a well accepted reproductive choice for couples at genetic risk and involves the diagnosis and transfer of unaffected IVF embryos. PGD for monogenetic diseases is most commonly accomplished by the biopsy of one or two blastomeres from cleavage stage embryos, followed by PCR-based protocols. However, PCR-based DNA analysis of one or two cells is subject to several problems, including total PCR failure, or failure of one allele to amplify. Trophectoderm biopsy at the blastocyst stage enables the removal of more than two cells for diagnosis while being non-invasive to the inner cell mass which is destined for fetal development. The aim of this study was to develop a safe, reliable technique for the biopsy of trophectoderm cells from human blastocysts. This case report demonstrates that removal of trophectoderm cells prior to blastocyst transfer is compatible with implantation and development to term. Here we report successful PGD for beta-thalassaemia following trophectoderm cell biopsy from blastocysts and the birth of a healthy infant.     

Derivation and characterization of two sibling human embryonic stem cell lines from discarded grade III embryos

Human embryonic stem (hES) cells are a valuable tool for studying human development in addition to their potential applications in regenerative medicine and drug discovery. The role of genetic background and epigenetic influences in development as well as in response to external influences such as drugs and therapies is well recognized. The great ethnic diversity in the Indian subcontinent translates to interindividual variability in drug response and disease susceptibility. For these reasons, new hES cell lines representing Indian genetic diversity will be valuable in studies of tissue-differentiation, cellular-function and for aspects of characterization of responses to drugs. We have derived two new hES cell lines, BJNhem19 and BJNhem20 from the inner cell mass (ICM) of discarded grade III human embryos that were not suitable for in vitro fertility treatment. Human leukocyte antigen (HLA) isotype analysis shows that they are genetically distinct from existing hES cell lines. Short tandem repeat (STR) analysis shows that the two cell lines are derived from sibling embryos. These cell lines show an undifferentiated phenotype in culture for more than 65 passages, show normal karyotype and express pluripotency markers such as TRA-1-60, TRA-1-81, stage-specific embryonic antigen-4 (SSEA-4), alkaline phosphatase, DNMT3B, GABRB3, GDF3, OCT4, NANOG, SOX2, TERF1, TDGF, LEFTA, THY1, and REX1. While both cell lines can differentiate into derivatives of all three germ layers in vitro, only BJNhem20 can form teratomas when transplanted into mice. We observe an increased frequency of cardiomyocyte differentiation from BJNhem20 embryoid bodies in feeder-free cultures upon induction with DMSO. Cardiomyocytes purified from such cultures survive and show rhythmic contractions for several weeks in culture. These hES cell lines have been accepted for deposit in the U.K. Stem Cell Bank and will be a useful resource for the international stem cell community.