Gains of 20q11.21 in human pluripotent stem cells: Insights from cancer research.

The genetic abnormalities observed in hPSC cultures worldwide have been suggested to pose an important hurdle in their safe use in regenerative medicine due to the possibility of oncogenic transformation by mutant cells in the patient posttransplantation. One of the best-characterized genetic lesions in hPSCs is the gain of 20q11.21, found in 20% of hPSC lines worldwide, and strikingly, also amplified in 20% of human cancers. In this review, we have curated the existing knowledge on the incidence of this mutation in hPSCs and cancer, explored the significance of chromosome 20q11.21 amplification in cancer progression, and reviewed the oncogenic role of the genes in the smallest common region of gain, to shed light on the significance of this mutation in hPSC-based cell therapy. Lastly, we discuss the state-of-the-art strategies devised to detect aneuploidies in hPSC cultures, avoid genetic changes in vitro cultures of hPSCs, and strategies to eliminate genetically abnormal cells from culture.

The consequences of recurrent genetic and epigenetic variants in human pluripotent stem cells.

It is well established that human pluripotent stem cells (hPSCs) can acquire genetic and epigenetic changes during culture in vitro. Given the increasing use of hPSCs in research and therapy and the vast expansion in the number of hPSC lines available for researchers, the International Society for Stem Cell Research has recognized the need to reassess quality control standards for ensuring the genetic integrity of hPSCs. Here, we summarize current knowledge of the nature of recurrent genetic and epigenetic variants in hPSC culture, the methods for their detection, and what is known concerning their effects on cell behavior in vitro or in vivo. We argue that the potential consequences of low-level contamination of cell therapy products with cells bearing oncogenic variants are essentially unknown at present. We highlight the key challenges facing the field with particular reference to safety assessment of hPSC-derived cellular therapeutics.

ESHRE survey results and good practice recommendations on managing chromosomal mosaicism.

STUDY QUESTION: How should ART/preimplantation genetic testing (PGT) centres manage the detection of chromosomal mosaicism following PGT? SUMMARY ANSWER: Thirty good practice recommendations were formulated that can be used by ART/PGT centres as a basis for their own policy with regards to the management of 'mosaic' embryos. WHAT IS KNOWN ALREADY: The use of comprehensive chromosome screening technologies has provided a variety of data on the incidence of chromosomal mosaicism at the preimplantation stage of development and evidence is accumulating that clarifies the clinical outcomes after transfer of embryos with putative mosaic results, with regards to implantation, miscarriage and live birth rates, and neonatal outcomes. STUDY DESIGN SIZE DURATION: This document was developed according to a predefined methodology for ESHRE good practice recommendations. Recommendations are supported by data from the literature, a large survey evaluating current practice and published guidance documents. The literature search was performed using PubMed and focused on studies published between 2010 and 2022. The survey was performed through a web-based questionnaire distributed to members of the ESHRE special interest groups (SIG) Reproductive Genetics and Embryology, and the ESHRE PGT Consortium members. It included questions on ART and PGT, reporting, embryo transfer policy and follow-up of transfers. The final dataset represents 239 centres. PARTICIPANTS/MATERIALS SETTING METHODS: The working group (WG) included 16 members with expertise on the ART/PGT process and chromosomal mosaicism. The recommendations for clinical practice were formulated based on the expert opinion of the WG, while taking into consideration the published data and results of the survey. MAIN RESULTS AND THE ROLE OF CHANCE: Eighty percent of centres that biopsy three or more cells report mosaicism, even though only 66.9% of all centres have validated their technology and only 61.8% of these have validated specifically for the calling of chromosomal mosaicism. The criteria for designating mosaicism, reporting and transfer policies vary significantly across the centres replying to the survey. The WG formulated recommendations on how to manage the detection of chromosomal mosaicism in clinical practice, considering validation, risk assessment, designating and reporting mosaicism, embryo transfer policies, prenatal testing and follow-up. Guidance is also provided on the essential elements that should constitute the consent forms and the genetic report, and that should be covered in genetic counselling. As there are several unknowns in chromosomal mosaicism, it is recommended that PGT centres monitor emerging data on the topic and adapt or refine their policy whenever new insights are available from evidence. LIMITATIONS REASONS FOR CAUTION: Rather than providing instant standardized advice, the recommendations should help ART/PGT centres in developing their own policy towards the management of putative mosaic embryos in clinical practice. WIDER IMPLICATIONS OF THE FINDINGS: This document will help facilitate a more knowledge-based approach for dealing with chromosomal mosaicism in different centres. In addition to recommendations for clinical practice, recommendations for future research were formulated. Following up on these will direct research towards existing research gaps with direct translation to clinical practice. Emerging data will help in improving guidance, and a more evidence-based approach of managing chromosomal mosaicism. STUDY FUNDING/COMPETING INTERESTS: The WG received technical support from ESHRE. M.D.R. participated in the EQA special advisory group, outside the submitted work, and is the chair of the PGT WG of the Belgian society for human genetics. D.W. declared receiving salary from Juno Genetics, UK. A.C. is an employee of Igenomix, Italy and C.R. is an employee of Igenomix, Spain. C.S. received a research grant from FWO, Belgium, not related to the submitted work. I.S. declared being a Co-founder of IVFvision Ltd, UK. J.R.V. declared patents related to 'Methods for haplotyping single-cells' and 'Haplotyping and copy number typing using polymorphic variant allelic frequencies', and being a board member of Preimplantation Genetic Diagnosis International Society (PGDIS) and International Society for Prenatal Diagnosis (ISPD). K.S. reported being Chair-elect of ESHRE. The other authors had nothing to disclose. DISCLAIMER: This Good Practice Recommendations (GPR) document represents the views of ESHRE, which are the result of consensus between the relevant ESHRE stakeholders and are based on the scientific evidence available at the time of preparation.  ESHRE GPRs should be used for information and educational purposes. They should not be interpreted as setting a standard of care or be deemed inclusive of all proper methods of care, or be exclusive of other methods of care reasonably directed to obtaining the same results. They do not replace the need for application of clinical judgement to each individual presentation, or variations based on locality and facility type.  Furthermore, ESHRE GPRs do not constitute or imply the endorsement, or favouring, of any of the included technologies by ESHRE.

On the origins and fate of chromosomal abnormalities in human preimplantation embryos: an unsolved riddle.

About 8 out of 10 human embryos obtained in vitro harbour chromosomal abnormalities of either meiotic or mitotic origin. Abnormalities of mitotic origin lead to chromosomal mosaicism, a phenomenon that has sparked much debate lately as it confounds results obtained through preimplantation genetic testing for aneuploidy (PGT-A). PGT-A in itself is still highly debated, not only on the modalities of its execution but also on whether it should be offered to patients at all. We will focus on post-zygotic chromosomal abnormalities leading to mosaicism. First, we will summarize what is known about the rates of chromosomal abnormalities at different developmental stages. Next, based on the current understanding of the origin and cellular consequences of chromosomal abnormalities, which is largely based on studies on cancer cells and model organisms, we will offer a number of hypotheses on which mechanisms may be at work in early human development. Finally, and very briefly, we will touch upon the impact our current knowledge has on the practice of PGT-A. What is the level of abnormal cells that an embryo can tolerate before it loses its potential for full development? And is blastocyst biopsy as harmless as it seems?

The Impact of Acquired Genetic Abnormalities on the Clinical Translation of Human Pluripotent Stem Cells.

Human pluripotent stem cells (hPSC) are known to acquire chromosomal abnormalities, which range from point mutations to large copy number changes, including full chromosome aneuploidy. These aberrations have a wide-ranging influence on the state of cells, in both the undifferentiated and differentiated state. Currently, very little is known on how these abnormalities will impact the clinical translation of hPSC, and particularly their potential to prime cells for oncogenic transformation. A further complication is that many of these abnormalities exist in a mosaic state in culture, which complicates their detection with conventional karyotyping methods. In this review we discuss current knowledge on how these aberrations influence the cell state and how this may impact the future of research and the cells' clinical potential.

MSH2 knock-down shows CTG repeat stability and concomitant upstream demethylation at the DMPK locus in myotonic dystrophy type 1 human embryonic stem cells.

Myotonic dystrophy type 1 (DM1) is caused by expansion of a CTG repeat in the DMPK gene, where expansion size and somatic mosaicism correlates with disease severity and age of onset. While it is known that the mismatch repair protein MSH2 contributes to the unstable nature of the repeat, its role on other disease-related features, such as CpG methylation upstream of the repeat, is unknown. In this study, we investigated the effect of an MSH2 knock-down (MSH2KD) on both CTG repeat dynamics and CpG methylation pattern in human embryonic stem cells (hESC) carrying the DM1 mutation. Repeat size in MSH2 wild-type (MSH2WT) and MSH2KD DM1 hESC was determined by PacBio sequencing and CpG methylation by bisulfite massive parallel sequencing. We found stabilization of the CTG repeat concurrent with a gradual loss of methylation upstream of the repeat in MSH2KD cells, while the repeat continued to expand and upstream methylation remained unchanged in MSH2WT control lines. Repeat instability was re-established and biased towards expansions upon MSH2 transgenic re-expression in MSH2KD lines while upstream methylation was not consistently re-established. We hypothesize that the hypermethylation at the mutant DM1 locus is promoted by the MMR machinery and sustained by a constant DNA repair response, establishing a potential mechanistic link between CTG repeat instability and upstream CpG methylation. Our work represents a first step towards understanding how epigenetic alterations and repair pathways connect and contribute to the DM1 pathology.

Human pluripotent stem cells in regenerative medicine: where do we stand?

Human pluripotent stem cells have the capacity to self-renew indefinitely and the ability to differentiate into all cell types of a human body. These characteristics instill them with an enormous promise in regenerative medicine, where they could be used in cell, tissue and even organ-based replacement therapy. In this review, we discuss their potential clinical applications and the advantages and pitfalls for the different types of human pluripotent stem cells to transition from the bench to the bedside. We provide an overview of the current clinical trials, and the specific challenges we are still facing, including immune compatibility, suboptimal differentiation, risk of tumor formation and genome instability.

Random Mutagenesis, Clonal Events, and Embryonic or Somatic Origin Determine the mtDNA Variant Type and Load in Human Pluripotent Stem Cells.

In this study, we deep-sequenced the mtDNA of human embryonic and induced pluripotent stem cells (hESCs and hiPSCs) and their source cells and found that the majority of variants pre-existed in the cells used to establish the lines. Early-passage hESCs carried few and low-load heteroplasmic variants, similar to those identified in oocytes and inner cell masses. The number and heteroplasmic loads of these variants increased with prolonged cell culture. The study of 120 individual cells of early- and late-passage hESCs revealed a significant diversity in mtDNA heteroplasmic variants at the single-cell level and that the variants that increase during time in culture are always passenger to the appearance of chromosomal abnormalities. We found that early-passage hiPSCs carry much higher loads of mtDNA variants than hESCs, which single-fibroblast sequencing proved pre-existed in the source cells. Finally, we show that these variants are stably transmitted during short-term differentiation.

Genetic and epigenetic factors which modulate differentiation propensity in human pluripotent stem cells.

BACKGROUND: Human pluripotent stem cell (hPSC) lines are known to have a bias in their differentiation. This gives individual cell lines a propensity to preferentially differentiate towards one germ layer or cell type over others. Chromosomal aberrations, mitochondrial mutations, genetic diversity and epigenetic variance are the main drivers of this phenomenon, and can lead to a wide range of phenotypes. OBJECTIVE AND RATIONALE: Our aim is to provide a comprehensive overview of the different factors which influence differentiation propensity. Specifically, we sought to highlight known genetic variances and their mechanisms, in addition to more general observations from larger abnormalities. Furthermore, we wanted to provide an up-to-date list of a growing number of predictive indicators which are able to identify differentiation propensity before the initiation of differentiation. As differentiation propensity can lead to difficulties in both research as well as clinical translation, our thorough overview could be a useful tool. SEARCH METHODS: Combinations of the following key words were applied as search criteria in the PubMed database: embryonic stem cells, induced pluripotent stem cells, differentiation propensity (also: potential, efficiency, capacity, bias, variability), epigenetics, chromosomal abnormalities, genetic aberrations, X chromosome inactivation, mitochondrial function, mitochondrial metabolism, genetic diversity, reprogramming, predictive marker, residual stem cell, clinic. Only studies in English were included, ranging from 2000 to 2017, with a majority ranging from 2010 to 1017. Further manuscripts were added from cross-references. OUTCOMES: Differentiation propensity is affected by a wide variety of (epi)genetic factors. These factors clearly lead to a loss of differentiation capacity, preference towards certain cell types and oftentimes, phenotypes which begin to resemble cancer. Broad changes in (epi)genetics, such as aneuploidies or wide-ranging modifications to the epigenetic landscape tend to lead to extensive, less definite changes in differentiation capacity, whereas more specific abnormalities often have precise ramifications in which certain cell types become more preferential. Furthermore, there appears to be a greater, though often less considered, contribution to differentiation propensity by factors such as mitochondria and inherent genetic diversity. Varied differentiation capacity can also lead to potential consequences in the clinical translation of hPSC, including the occurrence of residual undifferentiated stem cells, and the transplantation of potentially transformed cells. WIDER IMPLICATIONS: As hPSC continue to advance towards the clinic, our understanding of them progresses as well. As a result, the challenges faced become more numerous, but also more clear. If the transition to the clinic is to be achieved with a minimum number of potential setbacks, thorough evaluation of the cells will be an absolute necessity. Altered differentiation propensity represents at least one such hurdle, for which researchers and eventually clinicians will need to find solutions. Already, steps are being taken to tackle the issue, though further research will be required to evaluate any long-term risks it poses.

A High Proliferation Rate is Critical for Reproducible and Standardized Embryoid Body Formation from Laminin-521-Based Human Pluripotent Stem Cell Cultures.

When aiming for homogenous embryoid body (EB) differentiation, the use of equal-sized EBs is required to avoid a size-induced differentiation bias. In this study we developed an efficient and standardized EB formation protocol for human pluripotent stem cells (hPSC) cultured in a laminin-521-based xeno-free system. As the cell proliferation rate of the cells growing on laminin-521 strongly affected the efficiency of aggregate formation, we found that recently passaged cells, as well as the addition of ROCK inhibitor, were essential for reproducible EB formation from hPSC single-cell suspensions. EBs could be obtained in a variety of differentiation media, in 96-well round-bottom plates and in hanging drops. Gene expression studies on differentially sized EBs from three individual human embryonic stem cell lines demonstrated that the medium used for differentiation influenced the differentiation outcome to a much greater extent than the number of cells used for the initial EB formation. Our findings give a new insight into factors that influence the EB formation and differentiation process. This optimized method allows us to easily manipulate EB formation and provide an excellent starting point for downstream EB-based differentiation protocols.

Human embryonic stem cells show low-grade microsatellite instability.

It is well known that human embryonic stem cells (hESCs) frequently acquire recurrent chromosomal abnormalities, very reminiscent of those found in cancerous cells. Given the parallels between cancer and stem cell biology, we set out to investigate the occurrence of a common form of genome instability in tumors, namely microsatellite instability (MSI), in hESCs. MSI is caused by a deficiency in mismatch repair (MMR) genes, which leads to the accumulation of mutations during DNA replication. In this study, we analyzed up to 122 microsatellites in a total of 10 hESC lines, for 1-11 different passages, ranging from passage 7 to passage 334. In two lines, this revealed that two microsatellites had altered allelic patterns. Small-pool PCR for several microsatellites and testing of the Bethesda panel microsatellites (commonly used in cancer studies) revealed that, whilst MSI is common in all tested lines, it occurs at a very low and variable frequency, ranging from ∼1 to 20% of the total number of alleles. In cancerous cells, MSI leads to multiple large shifts in allele sizes within the majority of the cells, while hESCs show small changes in a minority of the cells. Since these genetic alterations do not consistently take over the culture, we assume that they are not concurrent with a selective advantage as it is in tumors. Finally, the MMR genes showed a very variable gene expression that could not be correlated with the variable (low) levels of MSI in the different hESC lines.

Huntington's and myotonic dystrophy hESCs: down-regulated trinucleotide repeat instability and mismatch repair machinery expression upon differentiation.

Huntington's disease (HD) and myotonic dystrophy (DM1) are caused by trinucleotide repeat expansions. The repeats show different instability patterns according to the disorder, cell type and developmental stage. Here we studied the behavior of these repeats in DM1- and HD-derived human embryonic stem cells (hESCs) before and after differentiation, and its relationship to the DNA mismatch repair (MMR). The relatively small (CAG)44 HD expansion was stable in undifferentiated and differentiated HD hESCs. In contrast, the DM1 repeat showed instability from the earliest passages onwards in DM1 hESCs with (CTG)250 or (CTG)1800. Upon differentiation the DM1 repeat was stabilized. MMR genes, including hMSH2, hMSH3 and hMSH6 were assessed at the transcript and protein levels in differentiated cells. The coincidence of differentiation-induced down-regulated MMR expression with reduced instability of the long expanded repeats in hESCs is consistent with a known requirement of MMR proteins for repeat instability in transgenic mice. This is the first demonstration of a correlation between altered repeat instability of an endogenous DM1 locus and natural MMR down-regulation, in contrast to the commonly used murine knock-down systems.

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).

Human embryonic stem cell lines derived from single blastomeres of two 4-cell stage embryos.

BACKGROUND: Recently, we demonstrated that single blastomeres of a 4-cell stage human embryo are able to develop into blastocysts with inner cell mass and trophectoderm. To further investigate potency at the 4-cell stage, we aimed to derive pluripotent human embryonic stem cells (hESC) from single blastomeres. METHODS: Four 4-cell stage embryos were split on Day 2 of preimplantation development and the 16 blastomeres were individually cultured in sequential medium. On Day 3 or 4, the blastomere-derived embryos were plated on inactivated mouse embryonic fibroblasts (MEFs). RESULTS: Ten out of sixteen blastomere-derived morulae attached to the MEFs, and two produced an outgrowth. They were mechanically passaged onto fresh MEFs as described for blastocyst ICM-derived hESC, and shown to express the typical stemness markers by immunocytochemistry and/or RT-PCR. In vivo pluripotency was confirmed by the presence of all three germ layers in the teratoma obtained after injection in immunodeficient mice. The first hESC line displays a mosaic normal/abnormal 46, XX, dup(7)(q33qter), del(18)(q23qter) karyotype. The second hESC line displays a normal 46, XY karyotype. CONCLUSION: We report the successful derivation and characterization of two hESC lines from single blastomeres of four split 4-cell stage human embryos. These two hESC lines were derived from distinct embryos, proving that at least one of the 4-cell stage blastomeres is pluripotent.

Preimplantation genetic diagnosis for Marfan syndrome.

OBJECTIVE: To develop and apply efficient and reliable protocols for preimplantation genetic diagnosis (PGD) for Marfan syndrome. DESIGN: Two mutation-specific protocols were developed, and the markers D15S1028, D15S992, D15S196, D15S576, D15S123, and D15S143 were used to set up four multiplex polymerase chain reactions (PCRs). SETTING: Research Center Reproduction and Genetics. PATIENT(S): Ten couples carrying mutations in the FBN1 gene. INTERVENTION(S): Six PGD protocols were developed for 10 couples, and 7 of them underwent a total of 16 clinical cycles. MAIN OUTCOME MEASURE(S): Amplification, allele drop-out (ADO), and contamination rates during the preclinical assays. DNA analyses of blastomeres from embryos biopsied during PGD cycles. RESULT(S): Six different protocols were set up, with the main objective being to to use one protocol for several couples. A total of 16 PGD cycles were performed, which resulted in the delivery of an unaffected boy and three ongoing pregnancies. CONCLUSION(S): The development of single-cell multiplex PCRs for linked markers and its use in PGD reduce the workload of the genetic diagnostic laboratory as well as the average waiting time for patients. This approach also allows for the simultaneous and accurate detection of recombination, contamination, and ADO, thereby increasing the reliability of the diagnosis.

Single-cell chromosomal imbalances detection by array CGH.

Genomic imbalances are a major cause of constitutional and acquired disorders. Therefore, aneuploidy screening has become the cornerstone of preimplantation, prenatal and postnatal genetic diagnosis, as well as a routine aspect of the diagnostic workup of many acquired disorders. Recently, array comparative genomic hybridization (array CGH) has been introduced as a rapid and high-resolution method for the detection of both benign and disease-causing genomic copy-number variations. Until now, array CGH has been performed using a significant quantity of DNA derived from a pool of cells. Here, we present an array CGH method that accurately detects chromosomal imbalances from a single lymphoblast, fibroblast and blastomere within a single day. Trisomy 13, 18, 21 and monosomy X, as well as normal ploidy levels of all other chromosomes, were accurately determined from single fibroblasts. Moreover, we showed that a segmental deletion as small as 34 Mb could be detected. Finally, we demonstrated the possibility to detect aneuploidies in single blastomeres derived from preimplantation embryos. This technique offers new possibilities for genetic analysis of single cells in general and opens the route towards aneuploidy screening and detection of unbalanced translocations in preimplantation embryos in particular.