Claudia Spits Team

Genome integrity in human pluripotent cells

The unique characteristics of human pluripotent stem cells (hPSC) such as human embryonic stem cells (hESC) and more recently, induced pluripotent stem cells (hiPSC), have made them not only attractive as a potential source of cells in regenerative medicine, but also as a research tool for studying early human developmental processes and human disorders.

hESC and hiPSC are kept in culture for long periods of time without this apparently affecting their self-renewal and pluripotent capacities. Nevertheless, we and others have found that these cells accumulate numerous genetic and epigenetic abnormalities, some of which are highly recurrent. Despite that chromosome abnormalities may influence the functional characteristics of hPSC, both in the pluripotent state and upon differentiation, only a handful of studies have investigated this topic. Also, there is very limited information on the possible mechanisms behind the origin of these mutations, and the key driver genes of the selective advantage they appear to confer to the cells.

Taking into account the potential applications of hPSC, their significant genome instability raises concerns for their safety in therapy or as suitable research models. In this context, it is necessary to gain a better insight on the biological meaning of the different types of abnormalities and to evaluate their impact on the differentiation capacity and malignant potential of the cells. Furthermore, understanding the causes and mechanisms of selective advantage of this mutations in culture will allow us to develop more optimal hPSC culture conditions that help preventing genetic drift in the cells.

Conversely, this genome instability it also opens new research venues. For instance, hPSC carrying genetic abnormalities can be used to identify novel gene functions. It is likely that chromosome regions recurrently involved in abnormalities harbor genes of high significance for pluripotent cells. In this case, hPSC provide a good model to study early developmental processes, or even tumorigenesis.

Our research focuses on three points:

  • We study the causes and extend of genome instability in hPSC
  • We investigate the mechanisms and genes behind the selective advantage of these mutant cells
  • We study the impact of these abnormalities on the differentiation capacity of the cells

Genome integrity in hPSC

Over the years, the body of evidence that hPSC display significant genome instability in culture has grown exponentially. In the first work we published on this topic (Spits et al., 2008, Nature Biotechnology), we studied the VUB hESC lines by array-based comparative genomic hybridization (array-CGH). We showed that hESC frequently undergo chromosomal changes, most of them below the resolution of G-banding. We were the first reporting the duplication hotspot in 20q11.21, a finding nummerous laboratories worlwide confirmed, including the study by the International Stem Cell Initiative (ISCI), in which we also participated. In this study, 125 hESC lines worldwide were screened, and this aberration was found in over 20% of the lines (Amps et al., 2011, Nature Biotechnology). A second novel recurrent abnormality we found was the formation of a derivative chromosome 18 with a relatively small common region of deletion.

Image from Spits et al., 2008 (Nat Biotech). a: aCGH results for chromosome 20 for VUB01 passage 276 showing a duplication of 20q11.21. b: FISH on hESC interphase nuclei showing two cells containing the duplication 20q11.21, and two normal cells. The FISH was performed using one of the clones located in the duplication, labeled in spectrum orange. c: real-time PCR results for VUB01, comparing the expression of DNMT3B before and after the duplication of 20q11.21. d: Derivative chromosomes 18 of VUB04_CF, VUB13_FXS and VUB26 by G-banding.

These abnormalities are likely to appear as single events in the culture, and will take over the culture if they provide a selective advantage to the cells. In the lab, we set to investigate the mutation rate of hESC by using single-cell array-CGH, and compared them to somatic cell populations (Jacobs et al., 2014, Nature Communications). We studied the genetic content of 92 individual human cells, including fibroblasts, amniocytes and hESCs. We found that human somatic and embryonic stem cell cultures show significant fractions of cells carrying unique megabase-scale chromosomal abnormalities, forming genetic mosaics that could not have been detected by conventional cytogenetic methods. These findings are confirmed by studying seven clonal hESC sub-lines by aCGH. Furthermore, fluorescent in situ hybridisation reveals an increased instability of the subtelomeric regions in hESC as compared to somatic cells.

Because of the parallels between the chromosomal abnormalities in hPSC and in cancerous cells, 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.(Nguyen et al., Molecular Human Reproduction, 2014)

Next to abnormalties of the nuclear DNA, in recent work we studied the mitochondrial genome of human embryonic stem cells by long-range PCR and sanger sequencing. We found that all tested human embryonic stem cell lines were heteroplasmic for different large mtDNA deletions. We found these mutations back as early as three passages after derivation, and that time in culture did not appear to affect the load. This observation suggested that the mutations were not de novo, but inherited from the embryos from which the lines were derived. Finally, we found that these mutations did not impair cell differentiation, and they were still present in the differentiatied progeny (Van Haute et al., Nature Biotechnology, 2014)

Image from Van Haute et al., 2014 (Nat Biotech). Results for seven control samples and three passages of the hESC line VUB01. The analysis was done by a long-range PCR that generates a fragment of 8.7 kb for the wild-type allele. mwm, molecular weight marker X in base pairs (Roche). Control samples: lane 1, mouse DNA to test the human-specific nature of the primer set; lane 2, DNA from cultured fibroblasts; lane 3, cultured amniocytes. Lanes 4 to 6, DNA from peripheral blood of three healthy individuals; lane 7, peripheral blood DNA from a patient that carries a 2,690-bp deletion with a mutation load of >95%. The different lanes for the same hESC passage are the results of three PCR replicates of the same DNA sample. Through a stochastic effect and owing to the low frequency of each individual deletion, each PCR amplifies different deletions; the most frequent are visible in all replicates.

Ongoing research

Currently, we are investigating the mechanisms of origin of small chromosomal abnormalities in hPSC. We are evaluating the impact of the culture conditions on the integrity of the nuclear DNA of the cells, and studying the possibility of developing more optimal culture systems that prevent genome instability.

Selective advantage and differentiation potential of cells with recurrent chromosome abnormalities

The recurrent nature of many of these abnormalities opens the question on which selective advantage this abnormalities confer the cells. Interestingly, many of these abnormalities are also recurrently found in cancer cells, highligthing the parallels between cancer and stem cell biology.

Gain of 20q11.21 is one of the chromosomal abnormality most recurrently found in human pluripotent stem cells and cancers, strongly suggesting that this mutation confers a proliferative or survival advantage to these cells. In recent work (Nguyen et al., 2014, Molecular Human Reproduction) we studied three of our hESC lines that acquired a gain of 20q11.21 during in vitro culture. The study of the mRNA gene expression levels of the loci located in the common region of duplication showed that HM13, ID1, BCL2L1, KIF3B and the immature form of the micro-RNA miR-1825 were up-regulated in mutant cells. ID1 and BCL2L1 were further studied as potential drivers of the phenotype of hESC with a 20q11.21 gain. We found no increase in the protein levels of ID1, nor the downstream effects expected from over-expression of this gene. On the other hand, hESC with a gain of 20q11.21 had on average a 3-fold increase of Bcl-xL (the anti-apoptotic isoform of BCL2L1) protein levels. The mutant hESC underwent 2- to 3-fold less apoptosis upon loss of cell-to-cell contact and were 2-fold more efficient in forming colonies from a single cell. The key role of BCL2L1 in this mutation was further confirmed by transgenic over-expression of BCL2L1 in the wild-type cells, leading to apoptosis-resistant cells, and BCL2L1-knock-down in the mutant hESC, resulting in a restoration of the wild-type phenotype. This resistance to apoptosis supposes a significant advantage for the mutant cells, explaining the high frequency of gains of 20q11.21 in human pluripotent stem cells.

Ongoing research

After completing our work on identifying the key driver gene of the gains of 20q11.21, our attention has moved to another recurrent chromosome abnormality, namely the loss of 18q. Remarkably, and in analogy to the gains of 20q11.21, this abnormality is also recurrent in human cancers. Up to now, in hPSC, this abnormality has not been fully characterized, and there is no knowledge on the phenotype of the cells and its selective advantage, nor on its key driver gene(s).

Regarding the impact of these chromosomal changes, we are currently investigating if the gain of 20q11.21 and the loss of 18q change the differentiation capacity of hESC.

Publications

Human embryonic stem cells show low-grade microsatellite instability. Nguyen HT*, Markouli C*, Geens M, Barbe L, Sermon K, Spits C. Mol Hum Reprod. 2014 Oct;20(10):981-9. * Joint first authorship.

Low-grade chromosomal mosaicism in human somatic and embryonic stem cell populations. Jacobs K, Mertzanidou A, Geens M, Thi Nguyen H, Staessen C, Spits C. Nat Commun. 2014 Jun 27;5:4227.

Gain of 20q11.21 in human embryonic stem cells improves cell survival by increased expression of Bcl-xL. Nguyen HT, Geens M, Mertzanidou A, Jacobs K, Heirman C, Breckpot K, Spits C. Mol Hum Reprod. 2014 Feb;20(2):168-77.

Human embryonic stem cells commonly display large mitochondrial DNA deletions. Van Haute L*, Spits C*, Geens M, Seneca S, Sermon K. Nat Biotechnol. 2013 Jan;31(1):20-3. * Joint first authorship.

Genetic and epigenetic instability in human pluripotent stem cells. Nguyen HT, Geens M, Spits C. Hum Reprod Update. 2013 Mar-Apr;19(2):187-205.

Screening ethnically diverse human embryonic stem cells identifies a chromosome 20 minimal amplicon conferring growth advantage.International Stem Cell Initiative, Amps K, Andrews PW, Anyfantis G, Armstrong L, Avery S, Baharvand H, Baker J, Baker D, Munoz MB, Beil S, Benvenisty N, Ben-Yosef D, Biancotti JC, Bosman A, Brena RM, Brison D, Caisander G, Camarasa MV, Chen J, Chiao E, Choi YM, Choo AB, Collins D, Colman A, Crook JM, Daley GQ, Dalton A, De Sousa PA, Denning C, Downie J, Dvorak P, Montgomery KD, Feki A, Ford A, Fox V, Fraga AM, Frumkin T, Ge L, Gokhale PJ, Golan-Lev T, Gourabi H, Gropp M, Lu G, Hampl A, Harron K, Healy L, Herath W, Holm F, Hovatta O, Hyllner J, Inamdar MS, Irwanto AK, Ishii T, Jaconi M, Jin Y, Kimber S, Kiselev S, Knowles BB, Kopper O, Kukharenko V, Kuliev A, Lagarkova MA, Laird PW, Lako M, Laslett AL, Lavon N, Lee DR, Lee JE, Li C, Lim LS, Ludwig TE, Ma Y, Maltby E, Mateizel I, Mayshar Y, Mileikovsky M, Minger SL, Miyazaki T, Moon SY, Moore H, Mummery C, Nagy A, Nakatsuji N, Narwani K, Oh SK, Oh SK, Olson C, Otonkoski T, Pan F, Park IH, Pells S, Pera MF, Pereira LV, Qi O, Raj GS, Reubinoff B, Robins A, Robson P, Rossant J, Salekdeh GH, Schulz TC, Sermon K, Sheik Mohamed J, Shen H, Sherrer E, Sidhu K, Sivarajah S, Skottman H, Spits C, Stacey GN, Strehl R, Strelchenko N, Suemori H, Sun B, Suuronen R, Takahashi K, Tuuri T, Venu P, Verlinsky Y, Ward-van Oostwaard D, Weisenberger DJ, Wu Y, Yamanaka S, Young L, Zhou Q. Nat Biotechnol. 2011 Nov 27;29(12):1132-44.

Derivation, culture, and characterization of VUB hESC lines. Mateizel I*, Spits C*, De Rycke M, Liebaers I, Sermon K. In Vitro Cellular and Developmental Biology-Animal. 2010, 46:300-8. * Joint first authorship.

Human embryonic stem cell lines derived from single blastomeres of two 4-cell stage embryos. Geens M, Mateizel I, Sermon K, De Rycke M, Spits C, Cauffman G, Devroey P, Tournaye H, Liebaers I, Van de Velde H. Human Reproduction. 2009, 11:2709-17.

Characterization of CD30 expression in hESC lines cultured in serum free media and mechanically passaged. Mateizel I, Spits C, Verloes A, Mertzanidou A, Liebaers I, Sermon K. Human Reproduction. 2009, 24:2477-89.

Recurrent chromosomal abnormalities in human embryonic stem cells. Spits C, Mateizel I, Geens M, Mertzanidou A, Staessen C, Vandeskelde Y, Van Der Elst J, Liebaers I, Sermon K. Nat Biotechnol. 2008,26:1361-1363.

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