Mieke Geens

Recurrent chromosomal abnormalities in human embryonic stem cells

Cultured human embryonic stem (hES) cells have a known predisposition to aneuploidy of chromosomes 12, 17 and X. We studied 17 hES cell lines by array-based comparative genomic hybridization (aCGH) and found that the cells accumulate other recurrent chromosomal abnormalities, including amplification at 20q11.21 and a derivative chromosome 18. These genomic changes have a variable impact at the transcriptional level.

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.

Autologous spermatogonial stem cell transplantation in man: current obstacles for a future clinical application

Fertility preservation is becoming an important issue in the management of the quality of life of prepubertal boys undergoing cancer treatment. At present, the only theoretical option for preservation of fertility in these boys is the preservation of the spermatogonial stem cells for autologous intratesticular stem cell transplantation. In animal models, this technique has shown promising results. However, before translation to the clinic, some major concerns should be evaluated. Improving the efficiency of the technique is one of the first goals for further research, besides evaluation of the safety of the clinical application. Also, the cryopreservation of the spermatogonial stem cells needs extra attention, since this first step will be crucial in the success of any clinical application. Another concern is the risk of malignant contamination of the testicular tissue in childhood cancer patients. Extensive research in this field and especially on the feasibility of decontaminating the testicular tissue will be inevitable. Another important, though overlooked, issue is the prevention of damage to the testicular niche cells. Finally, xenografting and in vitro proliferation/maturation of the spermatogonia should be studied as alternatives for the transplantation technique.

Spermatogonial survival in long-term human prepubertal xenografts.

Although childhood cancer treatments are yielding higher survival rates, sterility remains one of their major side effects. For prepubertal boys, there currently are no options to preserve fertility. Testicular tissue banking, together with subsequent grafting, may become a strategy in the future. In this study, prepubertal human testicular tissue was xenografted. Testicular tissue from two patients who had severe sickle-cell anemia and who needed to undergo chemotherapy and bone marrow transplantation was grafted onto the backs of six Swiss nude mice. Four months after grafting, spermatogonia could be observed by immunohistochemistry with MAGE-A4 antibodies, and Sertoli cells could be visualized by vimentin staining. Because both Sertoli cells and spermatogonia survived, tissue grafting may become a means for restoring future fertility in prepubertal male cancer patients.

Gain of 20q11.21 in human embryonic stem cells improves cell-survival by increased expression of Bcl-xL

Gain of 20q11.21 is a chromosomal abnormality that is recurrently found in human pluripotent stem cells and cancers, strongly suggesting that this mutation confers a proliferative or survival advantage to these cells. In this work we studied three human embryonic stem cell (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.

Totipotency and lineage segregation in the human embryo

During human preimplantation development the totipotent zygote divides and undergoes a number of changes that lead to the first lineage differentiation in the blastocyst displaying trophectoderm (TE) and inner cell mass (ICM) on Day 5. The TE is a differentiated epithelium needed for implantation and the ICM forms the embryo proper and serves as a source for pluripotent embryonic stem cells (ESCs). The blastocyst implants around Day 7. The second lineage differentiation occurs in the ICM after implantation resulting in specification of primitive endoderm and epiblast. Knowledge on human preimplantation development is limited due to ethical and legal restrictions on embryo research and scarcity of materials. Studies in the human are mainly descriptive and lack functional evidence. Most information on embryo development is obtained from animal models and ESC cultures and should be extrapolated with caution. This paper reviews totipotency and the molecular determinants and pathways involved in lineage segregation in the human embryo, as well as the role of embryonic genome activation, cell cycle features and epigenetic modifications.

Human embryonic stem cells commonly display large mitochondrial DNA deletions

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Sertoli cell-conditioned medium induces germ cell differentiation in human embryonic stem cells

PurposeTo investigate the spontaneous germ cell differentiation capacity of VUB hESC lines, develop a protocol for the induction of germ cell differentiation using conditioned medium from Sertoli cells (SCCM) and compare it to existing protocols.MethodshESC were allowed to differentiate spontaneously or after the addition of bone morphogenetic proteins (BMPs) and/or SCCM. VASA transcripts were measured by relative quantification real-time RT-PCR to determine the efficiency of germ cell differentiation.ResultsVUB hESC lines can differentiate spontaneously towards the germ cell lineage, however, more consistently in an embryoid body approach than in monolayer cultures. BMPs and SCCM significantly improve VASA expression, but do not have a synergistic effect. Direct contact of differentiating hESC with Sertoli cells does not improve VASA expression.ConclusionsSCCM contains inductive factors for germ cell differentiation and could represent an element for in-vitro differentiation to germ cells.

Higher-Density Culture in Human Embryonic Stem Cells Results in DNA Damage and Genome Instability

Summary Human embryonic stem cells (hESC) show great promise for clinical and research applications, but their well-known proneness to genomic instability hampers the development to their full potential. Here, we demonstrate that medium acidification linked to culture density is the main cause of DNA damage and genomic alterations in hESC grown on feeder layers, and this even in the short time span of a single passage. In line with this, we show that increasing the frequency of the medium refreshments minimizes the levels of DNA damage and genetic instability. Also, we show that cells cultured on laminin-521 do not present this increase in DNA damage when grown at high density, although the (long-term) impact on their genomic stability remains to be elucidated. Our results explain the high levels of genome instability observed over the years by many laboratories worldwide, and show that the development of optimal culture conditions is key to solving this problem.

Cell selection by selective matrix adhesion is not sufficiently efficient for complete malignant cell depletion from contaminated human testicular cell suspensions

OBJECTIVE To investigate whether a selective matrix adhesion-based protocol can enrich germ cells and deplete cancer cells from contaminated human testicular cell suspensions. DESIGN Artificially contaminated and control testicular cell suspensions were selectively cultured. After each experimental step, the presence of B cells was evaluated by fluorescence-activated cell sorting (FACS) and/or polymerase chain reaction (PCR). SETTING Academic medical center. PATIENT(S) Five vasectomy reversal patients willing to donate testicular tissue for research after informed consent. INTERVENTION(S) In vitro culture, magnetic-activated cell sorting (MACS), matrix adhesion, FACS, and PCR. MAIN OUTCOME MEASURE(S) FACS analysis for CD49f and human leukocyte antigen (HLA) class I expression; PCR analysis for a 120-bp fragment of the B-cell receptor. RESULT(S) With the use of a matrix adhesion-based protocol, CD49f(+) HLA class I(-) cells could be highly enriched, but HLA class I(+) CD49f(-) cells could still be detected. PCR proved that the cell suspensions contained malignant cells originating from the cell line used to contaminate them. CONCLUSION(S) Even though an efficient enrichment of germ cells was achieved, the resulting population was not pure. Malignant cells were detected in selected cell suspensions of all five patients, demonstrating that the efficacy of this protocol is insufficient for clinical application.