Distinct transcriptional signatures of aneuploidy in murine pluripotent cell populations

Grant no: BB/D526261/1Genomic integrity in mouse embryonic and induced pluripotent stem cells can be compromised by factors such as extended time in culture and cellular reprogramming. Surprising, only a few studies have thus far examined the accumulation of chromosomal imbalances in mouse pluripotent populations upon prolonged propagation in vitro. It is presumed that specific recurring genetic changes can confer selective growth advantage and resistance to apoptosis and/or differentiation to the affected cells, although the genes that drive these processes remain elusive. The presence of these changes in published studies can confound the analysis of the data and hinder the reproducibility of the results. At the transcriptional level, aneuploidy manifests as large chromosomal regions of aberrant gene expression. This thesis presents a method to identify these regions in large-scale datasets and interrogate for recurrent patterns. The present analysis shows that over half of the 315 mouse pluripotent samples examined carry whole or partial-chromosome spanning clusters of aberrant transcription. Furthermore, there are common gene expression changes across samples with any type of predicted aneuploidy and samples with chromosome-specific aberrations. These transcriptional signatures have been used to train classification models which can predict aneuploid samples with over 90% accuracy. This is an important step towards the development of a low-cost and reliable transcriptional validation assay for the presence of aneuploidy

Reprogramming roadblocks are system-dependent

Since the first generation of induced pluripotent stem cells (iPSCs), several reprogramming systems have been used to study its molecular mechanisms. However, the system of choice largely affects the reprogramming efficiency, influencing our view on the mechanisms. Here, we demonstrate that reprogramming triggered by less efficient polycistronic reprogramming cassettes not only highlights mesenchymal-to-epithelial transition (MET) as a roadblock but also faces more severe difficulties to attain a pluripotent state even post-MET. In contrast, more efficient cassettes can reprogram both wild-type and Nanog−/− fibroblasts with comparable efficiencies, routes, and kinetics, unlike the less efficient reprogramming systems. Moreover, we attribute a previously reported variation in the N terminus of KLF4 as a dominant factor underlying these critical differences. Our data establish that some reprogramming roadblocks are system dependent, highlighting the need to pursue mechanistic studies with close attention to the systems to better understand reprogramming

Intrinsic factors and the embryonic environment influence the formation of extragonadal teratomas during gestation

Background: Pluripotent cells are present in early embryos until the levels of the pluripotency regulator Oct4 drop at the beginning of somitogenesis. Elevating Oct4 levels in explanted post-pluripotent cells in vitro restores their pluripotency. Cultured pluripotent cells can participate in normal development when introduced into host embryos up to the end of gastrulation. In contrast, pluripotent cells efficiently seed malignant teratocarcinomas in adult animals. In humans, extragonadal teratomas and teratocarcinomas are most frequently found in the sacrococcygeal region of neonates, suggesting that these tumours originate from cells in the posterior of the embryo that either reactivate or fail to switch off their pluripotent status. However, experimental models for the persistence or reactivation of pluripotency during embryonic development are lacking. Methods: We manually injected embryonic stem cells into conceptuses at E9.5 to test whether the presence of pluripotent cells at this stage correlates with teratocarcinoma formation. We then examined the effects of reactivating embryonic Oct4 expression ubiquitously or in combination with Nanog within the primitive streak (PS)/tail bud (TB) using a transgenic mouse line and embryo chimeras carrying a PS/TB-specific heterologous gene expression cassette respectively. Results: Here, we show that pluripotent cells seed teratomas in post-gastrulation embryos. However, at these stages, induced ubiquitous expression of Oct4 does not lead to restoration of pluripotency (indicated by Nanog expression) and tumour formation in utero, but instead causes a severe phenotype in the extending anteroposterior axis. Use of a more restricted T(Bra) promoter transgenic system enabling inducible ectopic expression of Oct4 and Nanog specifically in the posteriorly-located primitive streak (PS) and tail bud (TB) led to similar axial malformations to those induced by Oct4 alone. These cells underwent induction of pluripotency marker expression in Epiblast Stem Cell (EpiSC) explants derived from somitogenesis-stage embryos, but no teratocarcinoma formation was observed in vivo. Conclusions: Our findings show that although pluripotent cells with teratocarcinogenic potential can be produced in vitro by the overexpression of pluripotency regulators in explanted somitogenesis-stage somatic cells, the in vivo induction of these genes does not yield tumours. This suggests a restrictive regulatory role of the embryonic microenvironment in the induction of pluripotency

On the statistical analysis of single cell lineage trees

Stem cells play a central role in the regeneration and repair of multicellular organisms. However, it remains far from trivial to reliably identify them. Despite decades of work, current techniques to isolate hematopoietic stem cells (HSCs) based on cell-surface markers only result in 50% purity, i.e. half of the sorted cells are not stem cells when functionally tested. Modern microscopy techniques allow us to follow single cells and their progeny for up to weeks in vitro, while recording the cell fates and lifetime of each individual cell. This cell tracking generates so-called lineage trees. Here, we propose statistical techniques to determine if the initial cell in a lineage tree was a HSC. We apply these techniques to murine hematopoietic lineage trees, revealing that 18% of the trees in our HSC dataset display a unique signature, and this signature is compatible with these trees having started from a true stem cell. Assuming 50% purity of HSC empirical datasets, this corresponds to a 0.35 power of the test, and the type-1-error is estimated to be 0.047. In summary, this study shows that statistical analysis of lineage trees could improve the classification of cells, which is currently done based on bio-markers only. Our statistical techniques are not limited to mammalian stem cell biology. Any type of single cell lineage trees, be it from bacteria, single cell eukaryotes, or single cells in a multicellular organism can be investigated. We expect this to contribute to a better understanding of the molecules influencing cellular dynamics at the single cell level.ISSN:0022-5193ISSN:1095-854

CSF-1-induced Src signaling can instruct monocytic lineage choice.

Controlled regulation of lineage decisions is imperative for hematopoiesis. Yet, the molecular mechanisms underlying hematopoietic lineage choices are poorly defined. CSF-1, the cytokine acting as the principal regulator of monocyte/macrophage development, has been shown to be able to instruct the lineage choice of uncommitted granulocyte macrophage progenitors towards a monocyte/macrophage fate. However, the intracellular signaling pathways involved are unknown. CSF-1 activates a multitude of signaling pathways resulting in a pleiotropic cellular response. The precise role of individual pathways within this complex and redundant signaling network is dependent on cellular context and not well understood. Here, we addressed which CSF-1-activated pathways are involved in transmitting the lineage-instructive signal in primary bone marrow-derived granulocyte macrophage progenitors. While its loss is compensated for by alternative signaling activation mechanisms, Src family kinase signaling is sufficient to transmit the CSF-1 lineage instructive signal. Moreover, c-Src activity is sufficient to drive macrophage fate, even in non-myeloid cells

Simple derivation of transgene-free iPS cells by a dual recombinase approach.

Mammalian cells can be reprogrammed into induced pluripotent stem cells (iPSCs), a valuable tool for in vitro disease modeling and regenerative medicine. These applications demand for iPSCs devoid of reprogramming factor transgenes, but current procedures for the derivation of transgene-free iPSCs are inefficient and cumbersome. Here, we describe a new approach for the simple derivation of transgene-free iPSCs by the sequential use of two DNA recombinases, C31 Integrase and Cre, to control the genomic insertion and excision of a single, non-viral reprogramming vector. We show that such transgene-free iPSCs exhibit gene expression profiles and pluripotent developmental potential comparable to genuine, blastocyst-derived embryonic stem cells. As shown by a reporter iPSC line for the differentiation into midbrain dopaminergic neurons, the dual recombinase approach offers a simple and efficient way to derive transgene-free iPSCs for studying disease mechanisms and cell replacement therapies