A comparison between the Felix™ electrophoretic system of sperm isolation and conventional density gradient centrifugation: a multicentre analysis.

PURPOSE: Developing optimized techniques for the isolation of human spermatozoa possessing low levels of DNA damage is an important objective for the ART industry. The purpose of this study was to compare a novel electrophoretic system (Felix™) of sperm isolation with a conventional method involving density gradient centrifugation (DGC). METHODS: Five international ART Centres in Australia, India, Sweden, the USA, and China have collaborated in order to compare the quality of the sperm populations isolated by Felix™ and DGC in terms of processing time, sperm concentration, motility, vitality, and DNA integrity as assessed by 3 methods: SCSA, Halo, and TUNEL. RESULTS: Across all centers, 112 comparisons were performed. Although significant differences were noted between centers in terms of the quality of the semen samples subjected for analysis, overall, both methods were equally capable of isolating populations of spermatozoa exhibiting high levels of vitality and progressive motility. The absolute numbers of spermatozoa recovered were significantly (p < 0.001) lower with the Felix™ device although sperm quality was higher with 4/5 centers reporting a significant improvement in DNA integrity relative to DGC (p < 0.01-p < 0.001). In practical terms, the Felix™ device featured a standardized 6 min preparation time whereas clinical DGC protocols varied from center to center but generally took around 40 min to complete. CONCLUSIONS: The Felix™ device is a positive technical development capable of isolating suspensions of highly motile spermatozoa exhibiting low levels of DNA damage in a fraction of the time taken by conventional procedures such as DGC.

Clonal derivation and characterization of human embryonic stem cell lines.

Human embryonic stem cells (hESC) are isolated as clusters of cells from the inner cell mass of blastocysts and thus should formally be considered as heterogeneous cell populations. Homogenous hESC cultures can be obtained through subcloning. Here, we report the clonal derivation and characterization of two new hESC lines from the parental cell line SA002 and the previously clonally derived cell line AS034.1, respectively. The hESC line SA002 was recently reported to have an abnormal karyotype (trisomy 13), but within this population of cells we observed rare individual cells with an apparent normal karyotype. At a cloning efficiency of 5%, we established 33 subclones from SA002, out of which one had a diploid karyotype and this subline was designated SA002.5. From AS034.1 we established one reclone designated AS034.1.1 at a cloning efficiency of 0.1%. These two novel sublines express cell surface markers indicative of undifferentiated hESC (SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81), Oct-4, alkaline phosphatase, and they display high telomerase activity. In addition, the cells are pluripotent and form derivatives of all three embryonic germ layers in vitro as well as in vivo. These results, together with the clonal character of SA002.5 and AS034.1.1 make these homogenous cell populations very useful for hESC based applications in drug development and toxicity testing. In addition, the combination of the parental trisomic hESC line SA002 and the diploid subclone SA002.5 provides a unique experimental system to study the molecular mechanisms underlying the pathologies associated with trisomy 13.

Human embryonic stem cells and chromosome stability.

The use of human embryonic stem cells (HESC) in research is increasing exponentially and HESC will certainly be of importance in biological, clinical and toxicological research for many years to come. Once established, HESC lines are expected to be chromosomally stable. However, our own experience of culturing HESC and some published reports indicate that HESC may show chromosomal instability while being cultured continuously in vitro. We conclude that the effects of different culture techniques and long-term culture on the chromosome stability of HESC still remain to be elucidated and we recommend regular analysis of the chromosome constitution in cell lines using traditional karyotyping, CGH, FISH and PCR. We also recommend freezing of HESC at low passage number and in larger batches after thawing and expansion in order to secure material in case mutations occur in the cell line at a later stage of culture.

Transplantation of human embryonic stem cells onto a partially wounded human cornea in vitro.

PURPOSE: The aim of this study was to investigate whether cells originating from human embryonic stem cells (hESCs) could be successfully transplanted onto a partially wounded human cornea. A second aim was to study the ability of the transplanted cells to differentiate into corneal epithelial-like cells. METHODS: Spontaneously, differentiated hESCs were transplanted onto a human corneal button (without limbus) with the epithelial layer partially removed. The cells were cultured on Bowman's membrane for up to 9 days, and the culture dynamics documented in a time-lapse system. As the transplanted cells originated from a genetically engineered hESC line, they all expressed green fluorescent protein, which facilitated their identification during the culture experiments, tissue preparation and analysis. To detect any differentiation into human corneal epithelial-like cells, we analysed the transplanted cells by immunohistochemistry using antibodies specific for CK3, CK15 and PAX6. RESULTS: The transplanted cells established and expanded on Bowman's membrane, forming a 1-4 cell layer surrounded by host corneal epithelial cells. Expression of the corneal marker PAX6 appeared 3 days after transplantation, and after 6 days, the cells were expressing both PAX6 and CK3. CONCLUSION: This shows that it is possible to transplant cells originating from hESCs onto Bowman's membrane with the epithelial layer partially removed and to get these cells to establish, grow and differentiate into corneal epithelial-like cells in vitro.

Screening ethnically diverse human embryonic stem cells identifies a chromosome 20 minimal amplicon conferring growth advantage.

The International Stem Cell Initiative analyzed 125 human embryonic stem (ES) cell lines and 11 induced pluripotent stem (iPS) cell lines, from 38 laboratories worldwide, for genetic changes occurring during culture. Most lines were analyzed at an early and late passage. Single-nucleotide polymorphism (SNP) analysis revealed that they included representatives of most major ethnic groups. Most lines remained karyotypically normal, but there was a progressive tendency to acquire changes on prolonged culture, commonly affecting chromosomes 1, 12, 17 and 20. DNA methylation patterns changed haphazardly with no link to time in culture. Structural variants, determined from the SNP arrays, also appeared sporadically. No common variants related to culture were observed on chromosomes 1, 12 and 17, but a minimal amplicon in chromosome 20q11.21, including three genes expressed in human ES cells, ID1, BCL2L1 and HM13, occurred in >20% of the lines. Of these genes, BCL2L1 is a strong candidate for driving culture adaptation of ES cells.

The establishment of 20 different human embryonic stem cell lines and subclones; a report on derivation, culture, characterisation and banking.

This report summarises our efforts in deriving, characterising and banking of 20 different human embryonic stem cell lines. We have derived a large number of human embryonic stem cell lines between 2001 and 2005. One of these cell lines was established under totally xeno-free culture conditions. In addition, several subclones have been established, including a karyoptypical normal clone from a trisomic mother line. A master cell banking system has been utilised in concert with an extensive characterisation programme, ensuring a supply of high quality pluripotent stem cells for further research and development. In this report we also present the first data on a proprietary novel antibody, hES-Cellect, that exhibits high specificity for undifferentiated hES cells. In addition to the traditional manual dissection approach of propagating hES cells, we here also report on the successful approaches of feeder-free cultures as well as single cell cultures based on enzymatic digestion. All culture systems used as reported here have maintained the hES cells in a karyotypical normal and pluripotent state. These systems also have the advantage of being the principal springboards for further scale up of cultures for industrial or clinical applications that would require vastly more cells that can be produced by mechanical means.

Chromosomal integrity maintained in five human embryonic stem cell lines after prolonged in vitro culture.

There have been recent reports of human embryonic stem cell (hESC) lines developing chromosomal aberrations after long-term culture, indicating an unstable genomic status due to the in vitro milieu. This raises concern, since it would limit their use in therapeutics. In this study the chromosomal status of five well-characterized hESC lines, SA002, SA002.5, AS034.1.1, SA121 and SA461, was monitored during long-term in vitro culture. The criteria of defined hESCs were met by all of the five hESC lines (four diploid and one trisomic for chromosome 13). The genomes were screened for chromosomal aberrations and rearrangements using comparative genomic hybridization (CGH), interphase fluorescence in situ hybridization (FISH) and traditional karyotyping on several occasions while in culture. The genomic integrity was shown to be maintained after repeated freeze-thaw procedures and continuous culture in vitro for up to 22 months (148 passages). We discuss the most common de novo chromosomal aberrations reported in hESCs, as well as their possible origin.

High-resolution analysis of the subtelomeric regions of human embryonic stem cells.

The use of human embryonic stem cells (hESCs) in most applications is dependent on their undifferentiated proliferation in vitro. Recent studies have illustrated the possibility that chromosomal changes may occur in hESCs during in vitro propagation of these cells. However, no studies so far have screened for chromosomal abnormalities in hESCs using high-resolution techniques that can detect alterations on a few base-pair levels. We have used the recently developed multiplex ligation-dependent probe amplification procedure to analyze the possible occurrence of deletions or duplications in the subtelomeric regions of hESCs in early and late passages. In this study we show that no subtelomeric anomalies were detected in any of the nine hESC lines investigated, supporting the conclusion that hESCs, under appropriate conditions, maintain genomic stability during in vitro propagation.

A morphological and chromosomal study of blastocysts developing from morphologically suboptimal human pre-embryos compared with control blastocysts.

BACKGROUND: IVF laboratories performing embryo transfer at day 2 or 3 after fertilization are currently discarding pre-embryos considered suboptimal using morphological criteria. The objective of this study was to investigate whether blastocysts, cultured from such pre-embryos (surplus), were chromosomally and morphologically normal. As a control group we used morphologically good quality embryos (GQE), cultured to the blastocyst stage. METHODS: Human pre-embryos considered suboptimal were cultured to the blastocyst stage. As a control group, frozen-thawed pre-embryos of good quality were cultured under identical conditions. The chromosomal status of the blastocysts obtained was studied by multi-colour fluorescence in-situ hybridization for chromosomes 13, 16, 18, 21, 22, X and Y. RESULTS: There is, on average, a significantly higher degree of chromosomal aberrations in blastocysts derived from surplus pre-embryos compared to blastocysts derived from GQE, and the chromosomal aberrations are generally found in a higher number of blastomeres per blastocyst. In addition, blastocysts from surplus pre-embryos had significantly poorer morphology compared to GQE. Improvement in morphology and/or developmental rate in surplus pre-embryos between day 2 and day 3 did not predict a morphologically/chromosomally normal blastocyst. However, this study shows that close to half of the surplus pre-embryos that reach the blastocyst stage can be considered chromosomally normal when assessed for these seven chromosomes. Furthermore, we found that chromosomal aberrations were more concentrated in a particular cell population within blastocysts derived from GQE, compared with surplus blastocysts. CONCLUSIONS: The study suggests that even if the IVF laboratory is on average making the correct decision about the potential of a pre-embryo, surplus pre-embryos that might become chromosomally normal blastocysts are still being discarded.

Human blastocysts for the development of embryonic stem cells.

Establishment of human embryonic stem cells (hES) from surplus human IVF embryos has been successful when both fresh and frozen-thawed cleavage stage embryos have been cultured to the blastocyst stage. This study reports the characteristics of the starting material, the blastocysts, for hES cell lines that were first derived at the University of Gothenburg, Sahlgrenska University Hospital in 1999. Twenty-two hES cell lines were derived by Cellartis AB from 114 blastocysts, giving an overall success rate of 19.3%. The blastocysts from which the hES cell lines were established were of varying morphological quality, both fresh and frozen-thawed. Two techniques of hES establishment were applied, i.e. direct application of the blastocysts on feeder cells or the standard immunosurgery method. It was further found that the efficiency by which frozen-thawed embryos gave rise to new hES cell lines was 3.7 times better than with fresh surplus embryos. These findings suggest that frozen-thawed embryos are superior to fresh surplus human embryos in hES cell establishment, which also avoids specific ethical problems associated with embryo donation in a fresh IVF cycle.