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 human embryonic stem cells from day-8 blastocysts recovered after three-step in vitro culture

Human embryonic stem cells (hESCs) have been derived from the inner cell mass (ICM) of day 5-7 blastocysts and hold great promise for research into human developmental biology and the development of cell therapies for the treatment of human diseases. We report here that our novel three-step culture conditions successfully support the development of day-8 human blastocysts, which possess significantly (p <.01) more ICM cells than day-6 blastocysts. Plating of ICMs isolated from day-8 blastocysts resulted in the formation of a colony with hESC morphology from which a new hESC line (hES-NCL1) was derived. Our stem cell line is characterized by the expression of specific cell surface and gene markers: GTCM-2, TG343, TRA1-60, SSEA-4, alkaline phosphatase, OCT-4, NANOG, and REX-1. Cytogenetic analysis of the hESCs revealed that hES-NCL1 line has a normal female (46, XX) karyotype. The pluripotency of the cell line was confirmed by the formation of teratomas after injection into severely combined immunodeficient mice and spontaneous differentiation under in vitro conditions.     

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.     

Challenges and approaches to the culture of pluripotent human embryonic stem cells

Since the establishment of the first human embryonic stem cell (hESC) lines, several groups have described the derivation and culture of hESC lines in various culture conditions. In this review, we describe how hESC lines have been derived from the inner cell mass of blastocysts or morula-stage embryos and the culture conditions used. In order to be used for therapeutic purposes, the pluripotent hESC lines must be established and propagated according to good manufacturing practice quality requirements. In addition, any use of animal-derived components should be avoided to gain safer hESC lines for clinical purposes. Here, we will describe the development in derivation and chemically defined culturing conditions of hESC towards good manufacturing practice and discuss the future challenges for hESCs in clinical use. Similarly, we discuss the challenges and future directions in optimization of standard culture conditions of hESCs for research purposes.     

人胚胎干细胞原代克隆生长及其传代的研究

目的 评价人胚胎干细胞建系与囊胚质量、原代克隆生长的关系。方法 D3废弃胚胎成组共培养获得不同质量的囊胚，免疫刀去除囊胚滋养外胚层细胞后，将内细胞团(ICM)接种到饲养细胞层上生长、传代。结果 从质量好的囊胚得到的人胚胎干细胞传代的代数更多；原代克隆生长快的人胚胎干细胞传代效率更高。结论 人胚胎干细胞建系与囊胚质量、原代克隆生长情况密切相关。     

Mechanical isolation of the inner cell mass is effective in derivation of new human embryonic stem cell lines

BACKGROUND: For clinical grade human embryonic stem cell (hESC) lines, a robust derivation system without any substances having animal origin would be required. We have gradually improved our hESC derivations. Human skin fibroblasts were used as feeder cells in derivation of all our 25 permanent fully characterized hESC lines. In the first four derivations, fetal calf serum was used as a supplement in the medium, thereafter, serum replacement medium was used. Immunosurgery generally used for isolation of the inner cell mass (ICM) still involves animal serum and complement. METHODS: We developed a practical mechanical isolation method for the ICM. Two flexible metal needles with sharpened tips, 0.125 mm in diameter, were used to open the zona pellucida and extract the ICM under a stereomicroscope. Immunohistochemical and karyotype characterization of the new hESC lines was carried out, and pluripotency was tested in vitro (immunocytochemistry and RT-PCR) and in vivo (teratoma growth). RESULTS :Five hESC lines were obtained from 19 supernumerary blastocysts collected in 2005-2006 (26%), whereas in similar conditions, we obtained 16 lines from 100 blastocysts (16%) using immunosurgery in 2003-2005. The new lines had a normal karyotype and tissues originating from the three embryonic germ cell layers were present. CONCLUSIONS: Mechanical isolation of the ICM proved to be an effective way to derive new hESC lines. The technique is fast, does not require any extra investment and the xeno-components of immunosurgery could be avoided.     

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.     

Expression of fibronectin and a fibronectin-binding molecule during preimplantation development in the mouse

Using an indirect immunofluorescent technique, expression ofcell surface fibronectin and a cell surface fibronectin-bindingmolecule was studied during mouse embryo preimplantation development.We also studied the expression of fibronectin on immunosurgicallyisolated inner cell masses (ICMs) and regenerated mouse blastocysts.Fibronectin and the fibronectin-binding molecule were not detectedat the morula stage. From the early to late blastocyst stage,fibronectin expression increased on the trophectoderm. Expressionof the fibronectin-binding molecule was found only in the polartrophectoderm region of the early blastocyst, then in the polarand mural trophectoderm regions of the middle blastocyst. Inthe late blastocyst stage, this fibronectin-binding moleculewas only present in the mural trophectoderm. Fibronectin expressionby ICMs of early blastocysts was more intense than that of lateblastocysts. After 24 h of culture, 10% of ICMs isolated fromearly blastocysts regenerated a trophectoderm which stainedintensively for fibronectin in the mural and polar trophectodermregions. After 48 h of culture, regenerated blastocyst-likestructures closely resembled the normally obtained late blastocystsand stained for fibronectin in the mural and polar trophectodermregions. The significance of the results is discussed in relationto mouse embryo development, trophectoderm formation and blastocystimplantation.     

Derivation of a xeno-free human embryonic stem cell line

Elimination of all animal material during both the derivation and long-term culture of human embryonic stem cells (hESCs) is necessary prior to future application of hESCs in clinical cell therapy. The potential consequences of transplanting xeno-contaminated hESCs into patients, such as an increased risk of graft rejection [Stem Cells 2006; 24:221-229] and the potential transfer of nonhuman pathogens, make existing hESC lines unsuitable for clinical applications. To avoid xeno-contamination during derivation and culture of hESCs, we first developed a xeno-free medium supplemented with human serum, which supports long-term (>50 passages) culture of hESCs in an undifferentiated state. To enable derivation of new xeno-free hESCs, we also established xeno-free human foreskin fibroblast feeders and replaced immunosurgery, which involves the use of guinea pig complement, with a modified animal-product-free derivation procedure. Here, we report the establishment and characterization (>20 passages) of a xeno-free pluripotent diploid normal hESC line, SA611.     

Blastocyst development after assisted reproduction using spermatozoa obtained after testicular stem cell transplantation in mice

BACKGROUND: Since its introduction in 1994, testicular stemcell transplantation (TSCT) has been widely used for research.This technique may also become important for preserving fertilityin pre-pubertal cancer patients. Therefore, it is necessaryto investigate the safety aspects of reproduction using spermatozoaobtained after TSCT. In this study, preimplantation developmentof mouse embryos, using spermatozoa obtained after TSCT, wasexamined. METHODS: TSCT-derived spermatozoa were used for IVFand ICSI. Embryos were cultured for five days until they reachedblastocyst stage and were evaluated by differential staining.RESULTS: IVF revealed significantly lower fertilization anddevelopment rates after TSCT-IVF compared to control-IVF. Blastocystsderived from TSCT-IVF had significantly lower inner cell massnumbers (ICMs) and lower ICM/trophectoderm (TE) ratios comparedto control-IVF blastocysts. No differences in fertilizationand development rates were observed between TSCT-ICSI and control-ICSI,and blastocyst quality in the transplanted group was similarto that of the control blastocysts. CONCLUSION: Our study showedthat after TSCT-IVF, fertilization and preimplantation developmentwere disturbed and blastocysts showed reduced ICM and ICM/TEratio. However, after TSCT-ICSI, both fertilization and preimplantationdevelopment were normal and blastocyst formation was comparableto control-ICSI.     

Derivation of new human embryonic stem cell lines reveals rapid epigenetic progression in vitro that can be prevented by chemical modification of chromatin

Human embryonic stem cells (hESCs) are pluripotent cell types derived from the inner cell mass of human blastocysts. Recent data indicate that the majority of established female XX hESC lines have undergone X chromosome inactivation (XCI) prior to differentiation, and XCI of hESCs can be either XIST-dependent (class II) or XIST-independent (class III). XCI of female hESCs precludes the use of XX hESCs as a cell-based model for examining mechanisms of XCI, and will be a challenge for studying X-linked diseases unless strategies are developed to reactivate the inactive X. In order to recover nuclei with two active X chromosomes (class I), we developed a reprogramming strategy by supplementing hESC media with the small molecules sodium butyrate and 3-deazaneplanocin A (DZNep). Our data demonstrate that successful reprogramming can occur from the XIST-dependent class II nuclear state but not class III nuclear state. To determine whether these small molecules prevent XCI, we derived six new hESC lines under normoxic conditions (UCLA1-UCLA6). We show that class I nuclei are present within the first 20 passages of hESC derivation prior to cryopreservation, and that supplementation with either sodium butyrate or DZNep preserve class I nuclei in the self-renewing state. Together, our data demonstrate that self-renewal and survival of class I nuclei are compatible with normoxic hESC derivation, and that chemical supplementation after derivation provides a strategy to prevent epigenetic progression and retain nuclei with two active X chromosomes in the self-renewing state.     

Ultrastructure of the embryonic stem cells of the 8-day pig blastocyst before and after in vitro manipulation: development of junctional apparatus and the lethal effects of PBS mediated cell-cell dissociation

Ultrastructural examination of 8-day hatched pig blastocysts (large and small), their cultured inner cell mass (ICM), and cultured epiblast tissue (embryonic stem cells) was undertaken to assess the development of epiblast cell junctions and cytoskeletal elements. In small blastocysts, epiblast cells had no desmosomes or tight junction (TJ) connections and few organized microfilament bundles, whereas in large blastocysts the epiblast cells were connected by TJ and desmosomes with associated microfilaments. ICM isolation by immunodissection damaged the endoderm cells beneath the trophectoderm cells but did not appear to damage the epiblast cells or their associated endoderm cells. Epiblast cells in cultured ICMs were similar in character to those in the intact large blastocyst except that perinuclear microfilaments were observed. Isolated pig epiblasts, cultured for approximately 36 hr on STO feeder layers, formed a monolayer whose cells were connected by TJ, adherens junctions and desmosomes with prominent microfilament bundles running parallel to the apical cytoplasmic membranes. Perinuclear microfilaments were a consistent feature in the approximately 36 hr cultured epiblast cells. A feature characteristic of differentiation into notochordal cells, i.e., a solitary cilium, was also observed in the cultured epiblast. Exposure of the cultured epiblast cells to Ca(++)-Mg(++)-free phosphate buffered saline (PBS) for 5-10 min resulted in extensive cell blebbing and lysis. The results may indicate that pig epiblast cells could be more easily dissociated from early blastocysts ( approximately 400 microm in diameter) if immunodissection damage to the ICM can be avoided. It may be difficult, however, to establish them as embryonic stem cell lines because the cultured pig epiblast cells were easily lysed by standard cell-cell dissociation methods.     

Optimized Protocol for Derivation of Human Embryonic Stem Cell Lines

For the past 12 years, the biology and applications of human embryonic stem cells (hESCs) have received great attention from the scientific community. Derivatives of the first hESC line obtained by J. Thomson's group (Science 282(5391):1145-1147, 1998) have been used in clinical trials in patients with spinal cord injury, and other hESC lines have now been used to generate cells for use in treating blindness (Lancet 379(9817):713-720, 2012). In addition to the classical protocol based on mouse or human feeder layers using open culture methods (In Vitro Cellular & Developmental Biology - Animal 46(3-4):386-394, 2010; Stem Cells 23(9):1221-1227, 2005; Nature Biotechnology 24(2):185-187, 2006; Human Reproduction 21(2):503-511, 2006; Human Reproduction 20(8):2201-2206, 2005; Fertility and Sterility 83(5):1517-1529, 2005), novel hESC lines have been derived xeno-free (without using animal derived reagents) (PLoS One 5 (4):1024-1026, 2010), feeder-free (without supporting cell monolayers) (Lancet 365(9471):1601-1603, 2005), in microdrops under oil (In Vitro Cellular & Developmental Biology - Animal 46(3-4):236-41, 2010) and in suspension with ROCK inhibitor (Nature Biotechnology 28(4):361-4, 2010). Regardless of the culture system, successful hESC derivation usually requires optimization of embryo culture, the careful and timely isolation of its inner cell mass (ICM), and precise culture conditions up to the establishment of pluripotent cell growth during hESC line derivation. Herein we address the crucial steps of the hESC line derivation protocol, and provide tips to apply quality control to each step of the procedure.     

Efficient derivation of embryonic stem cells by inhibition of glycogen synthase kinase-3

Embryonic stem (ES) cells are derived from the inner cell mass (ICM) of blastocysts. The use of ES cells as a source of differentiated cells holds great promise for cell transplantation therapy. The efficiency of ES cell derivation is affected by genetic variation in mice; that is, some mouse strains, such as C57BL/6, are amenable to ES cell derivation, whereas others, such as BALB/c, are refractory. Developing an efficient method to establish ES cells from strains of various genetic backgrounds should be valuable for derivation of ES cells in various mammalian species, including human. Although it is well-established that various signaling pathways, including phosphoinositide 3-kinase (PI3K)/Akt and Wnt/beta-catenin, regulate the maintenance of ES cell pluripotency, little is known about the signaling pathways involved in the derivation of ES cells from ICMs. In this study, we demonstrated that inhibition of glycogen synthase kinase-3 (GSK-3), one of the crucial molecules in the regulation of the Wnt/beta-catenin, Hedgehog, and Notch signaling pathways, dramatically augmented ES cell derivation from both C57BL/6 and BALB/c mouse strains. In contrast, Akt signaling activation enhanced the growth of ICM but did not increase the efficiency of ES cell derivation. Our study establishes an efficient means for ES cell derivation by pharmacological inhibition of GSK-3.     

Fertilization and early embryolgoy: Lineage tracing demonstrates that blastomeres of early cleavage-stage human pre-embryos contribute to both trophectoderm and inner cell mass

We injected a fluorescent lineage tracer (Texas Red-lysinedextran)into individual blastomeres of donated human diploid 2- to 8-cellpre-embryos and cultured them to blastocysts. Once pre-embryosreached the expanded blastocyst stage, they were fixed and examinedin a scanning confocal microscope to identify the location offluorescent tracer. In successfully injected pre-embryos thatdeveloped to expanded blastocysts, we found that randomly injectedblastomeres formed both trophectoderm (TE) and inner cell mass(ICM). More labelled progeny were found in TE than in ICM. Ourresults show that individual early blastomeres are not yet committedto form either TE or ICM but instead can form both rudiments.     

Stereological study of mouse uterine and in vitro grown blastocysts: Cell numbers and volumes

Mouse blastocysts were studied to determine if there were differences in cell number and volumes between those that were (1) derived from the uterus prior to implantation on the afternoon of day 4 of pregnancy and (2) those that were cultured for 72 hr from two-cell-stage embryos. Blastocysts were fixed, embedded in resin, and serially sectioned at 1.5 or 2 μm. Photographic prints of alternate sections were used to count the numbers of inner cell mass (ICM) and trophectoderm cells. Cavalieri's direct estimator was applied to the same prints to estimate the volume of the whole blastocyst. Point counting was used to determine the volumes of the ICM, trophectoderm, and zona pellucida. The number of cells and size of the ICM were similar between the two groups of blastocysts, although it was found that the ICM of uterine embryos that did not have a zona pellucida were smaller than the ICM of those that did. There were twice as many trophectoderm cells in the blastocysts that were cultured from two-cell embryos, and these cells were also found to be larger. Furthermore, the volume of the zona pellucida was less in the uterine blastocysts. This study indicates that, while trophectoderm proliferation is enhanced in vitro, the ICM is more constant and thus may be self-regulating and independent of the growth conditions of the blastocyst as a whole. This study also suggests partial zona lysis occurs in utero and occurs either at a reduced rate or not at all in vitro. © 1993 Wiley-Liss, Inc.     

Ultrastructure of the embryonic stem cells of the 8‐day pig blastocyst before and after in vitro manipulation: Development of junctional apparatus and the lethal effects of PBS mediated cell–cell dissociation

Ultrastructural examination of 8‐day hatched pig blastocysts (large and small), their cultured inner cell mass (ICM), and cultured epiblast tissue (embryonic stem cells) was undertaken to assess the development of epiblast cell junctions and cytoskeletal elements. In small blastocysts, epiblast cells had no desmosomes or tight junction (TJ) connections and few organized microfilament bundles, whereas in large blastocysts the epiblast cells were connected by TJ and desmosomes with associated microfilaments. ICM isolation by immunodissection damaged the endoderm cells beneath the trophectoderm cells but did not appear to damage the epiblast cells or their associated endoderm cells. Epiblast cells in cultured ICMs were similar in character to those in the intact large blastocyst except that perinuclear microfilaments were observed. Isolated pig epiblasts, cultured for ～36 hr on STO feeder layers, formed a monolayer whose cells were connected by TJ, adherens junctions and desmosomes with prominent microfilament bundles running parallel to the apical cytoplasmic membranes. Perinuclear microfilaments were a consistent feature in the ～36 hr cultured epiblast cells. A feature characteristic of differentiation into notochordal cells, i.e., a solitary cilium, was also observed in the cultured epiblast. Exposure of the cultured epiblast cells to Ca⁺⁺‐Mg⁺⁺‐free phosphate buffered saline (PBS) for 5–10 min resulted in extensive cell blebbing and lysis. The results may indicate that pig epiblast cells could be more easily dissociated from early blastocysts (～400 μm in diameter) if immunodissection damage to the ICM can be avoided. It may be difficult, however, to establish them as embryonic stem cell lines because the cultured pig epiblast cells were easily lysed by standard cell–cell dissociation methods. Anat Rec 264:101–113, 2001. © 2001 Wiley‐Liss, Inc.     

Enhancement of the propagation of human embryonic stem cells by modifications in the gel architecture of PMEDSAH polymer coatings

Well-defined culture conditions are essential for realizing the full potential of human embryonic stem cells (hESCs) in regenerative medicine where large numbers of cells are required. Synthetic polymers such as poly[2-(methacryloyloxy) ethyl dimethyl-(3-sulfopropyl) ammonium hydroxide] (PMEDSAH), offer multiple advantages over mouse embryonic fibroblasts (MEFs) and Matrigel™ for hESC culture and expansion. However, there is limited understanding of the mechanisms by which hESCs are propagated on synthetic polymers coatings. Here, the effects of PMEDSAH gel architecture on hESC self-renewal were determined. By increasing the atom transfer radical polymerization (ATRP) reaction time, the thickness of PMEDSAH was increased and its internal hydrogel architecture was modified, while maintaining its overall chemical structure. A 105 nm thick ATRP PMEDSAH coating showed a significant increase in the expansion rate of hESCs. Theoretical calculations suggested that 20,000 hESCs cultured on this substrate could be expanded up to 4.7 × 10⁹ undifferentiated cells in five weeks. In addition, hESCs grown on ATRP PMEDSAH coatings retained pluripotency and displayed a normal karyotype after long-term culture. These data demonstrate the importance of polymer physical properties in hESC expansion. This modification of PMEDSAH coatings may be used to obtain large populations of hESCs required for many applications in regenerative medicine.     

Restriction landmark genome scanning identifies culture-induced DNA methylation instability in the human embryonic stem cell epigenome

Widespread provision of human embryonic stem cells (hESCs) for therapeutic use, drug screening and disease modelling will require cell lines sustainable over long periods in culture. Since the short-term, in vitro culture of mammalian embryos can result in DNA methylation changes, the epigenetic stability of hESCs warrants investigation. Existing hESC lines have been derived and cultured under diverse conditions, providing the potential for programming differential changes into the epigenome that may result in inter-line variability over and above that inherited from the embryo. By examining the DNA methylation profiles of > 2000 genomic loci by Restriction Landmark Genome Scanning, we identified substantial inter-line epigenetic distance between six independently derived hESC lines. Lines were found to inherit further epigenetic changes over time in culture, with most changes arising in the earliest stages post-derivation. The loci affected varied between lines. The majority of culture-induced changes (82.3-87.5%) were stably inherited both within the undifferentiated cells and post-differentiation. Adapting a line to a serum-free culture system resulted in additional epigenetic instability. Overall 80.5% of the unstable loci uncovered in hESCs have been associated previously with an adult tumour phenotype. Our study shows that current methods of hESC propagation can rapidly programme stable and unpredictable epigenetic changes in the stem cell genome. This highlights the need for (i) novel screening strategies to determine the experimental utility and biosafety of hESCs and (ii) optimization and standardization of procedures for the derivation and culture of hESC lines that minimize culture-induced instability.