Biology subjects

Stavish, D.

Publications and source records attributed to Stavish, D..

10 recordsLinked to original sources

bioRxiv2024

Chromosome X Dosage Modulates Development of Aneuploidy in Genetically Diverse Mouse Embryonic Stem Cells

The genetic integrity of pluripotent stem cells (PSC) is critical to their applications in research and therapy, but it is compromised by frequent development of structural chromosome variants associated with malignancy. Many cell lines exhibit remarkable genetic stability, but little is known about the basis of the known variation in genomic integrity amongst different PSC isolates. Here we identify aneuploidies using RNA-seq and proteomics data from a panel of mouse embryonic stem cell (mESC) lines derived from 170 Diversity Outbred mice. We found 62 lines with detectable aneuploid subpopulations and a subset of originally XX lines that lost one Chromosome X (XO). Strikingly, a much lower proportion of XX lines were aneuploid, compared to XY or XO lines. Two single-cell RNA-seq data sets demonstrated that aneuploid XY DO mESC also show lower Chromosome X gene expression, and a prospective study confirmed that XY mESC accumulate higher aneuploid proportions in culture than isogenic XX lines. We identify potential mechanisms for this protective effect of X chromosome dosage, including our findings that the lines with two active X Chromosomes have a higher proportion of 2-cell-like cells, a state associated with maintenance of genetic integrity of mESC, and that they show differential expression of X-linked tumor suppressor genes associated with the DNA damage response. HighlightsO_LIFirst genetic analysis of predisposition to aneuploidy in pluripotent stem cell cultures C_LIO_LIChromosomal regions duplicated in aneuploid mouse embryonic stem cells are syntenic with regions overrepresented in human pluripotent stem cell lines bearing recurrent genetic abnormalities C_LIO_LIX-Chromosome dosage strongly influences susceptibility to aneuploidy in mouse embryonic stem cells and to a lesser degree in human pluripotent stem cells C_LIO_LIXX mouse embryonic stem cell lines show a higher proportion of cells in 2 cell-like state and higher expression of tumor suppressor genes associated with DNA damage response C_LI

genetics↗

bioRxiv2023

Aberrant Wnt activation in recurrent genetically variant human pluripotent stem cells impairs cardiomyocyte differentiation and phenotype

Human pluripotent stem cell (hPSC)-derived cardiomyocytes have emerged as powerful tools for disease modelling and cell therapy. The production of cardiomyocytes from hPSCs typically requires expanding large numbers of hPSCs and maintaining them in culture for extended periods of time. This in turn predisposes hPSCs to the acquisition of non-random genetic changes, including recurrent gains of chromosome 1q. Here, we show that gain of chromosome 1q in hPSCs affects both the efficiency of differentiation to cardiomyocytes and phenotype of the differentiated cells. Mechanistically, we show that aberrant activation of the Wnt signalling pathway underpins the skewed differentiation of variant 1q hPSCs. Collectively, our data demonstrates that the presence of genetically variant cells in cultures is a significant concern for production of hPSC-derived cardiomyocytes for research or clinical applications. Further, our results suggest new approaches for removing genetically variant cells for future clinical applications.

cell biology↗

bioRxiv2023

Cytogenetic resource enables mechanistic resolution of changing trends in human pluripotent stem cell aberrations linked to feeder-free culture

Since the first derivation of human pluripotent stem cells (hPSCs), the number of culture conditions has steadily increased, making hPSC culture more facile. Nonetheless, there remains the persistent issue of culture-acquired genetic changes, hampering the reproducibility of hPSC research and jeopardising their clinical use. Here, we utilised comprehensive karyotyping datasets from over 20,000 hPSC cultures sampled under different conditions to ascertain association of genetic changes with specific culture regimens. We found condition-dependent patterns of aberrations, with higher prevalence of chromosome 1q gains in recent years, associated with increased use of contemporary, feeder-free cultures. Mechanistically, we show the context-dependent selection of 1q variants is mainly driven by MDM4, a gene amplified in many cancers, located on chromosome 1q. To facilitate reproducibility of hPSC research and their safe clinical utility, we provide a unique hPSC karyotype resource for informing the risk assessment of genetic aberrations and developing strategies to suppress their occurrence.

cell biology↗

bioRxiv2023

Notch signalling influences cell fate decisions and HOX gene induction in axial progenitors

The generation of the post-cranial embryonic body relies on the coordinated production of spinal cord neurectoderm and presomitic mesoderm cells from neuromesodermal progenitors (NMPs). This process is orchestrated by pro-neural and pro-mesodermal transcription factors that are co-expressed in NMPs together with Hox genes, which are critical for axial allocation of NMP derivatives. NMPs reside in a posterior growth region, which is marked by the expression of Wnt, FGF and Notch signalling components. While the importance of Wnt and FGF in influencing the induction and differentiation of NMPs is well established, the precise role of Notch remains unclear. Here, we show that the Wnt/FGF-driven induction of NMPs from human embryonic stem cells (hESCs) relies on Notch signalling. Using hESC-derived NMPs and chick embryo grafting, we demonstrate that Notch directs a pro-mesodermal character at the expense of neural fate. We show that Notch also contributes to activation of HOX gene expression in human NMPs, partly in a non cell-autonomous manner. Finally, we provide evidence that Notch exerts its effects via the establishment of a negative feedback loop with FGF signalling.

developmental biology↗

bioRxiv2022

Neuroblastoma-associated chromosomal aberrations drive cell identity loss in human neural crest via disruption of developmental regulators

Early childhood tumours arise from transformed embryonic cells, which often carry large copy number alterations (CNA). However, it remains unclear how CNAs contribute to embryonic tumourigenesis due to a lack of suitable models. Here we employ female human embryonic stem cell (hESC) differentiation and single-cell transcriptome and epigenome analysis to assess the effects of chromosome 17q/1q gains, which are prevalent in the embryonal tumour neuroblastoma (NB). We show that CNAs impair the specification of trunk neural crest (NC) cells and their sympathoadrenal derivatives, the putative cells-of-origin of NB. This effect is exacerbated upon overexpression of MYCN, whose amplification co-occurs with CNAs in NB. Moreover, CNAs potentiate the pro-tumourigenic effects of MYCN and mutant NC cells resemble NB cells in tumours. These changes correlate with a stepwise aberration of developmental transcription factor networks. Together, our results sketch a mechanistic framework for the CNA-driven initiation of embryonal tumours.

cancer biology↗

bioRxiv2022

HDAC6 inhibition partially alleviates mitochondrial trafficking defects and restores motor function in human motor neuron and zebrafish models of Charcot-Marie-Tooth Disease Type 2A

Charcot Marie Tooth Disease (CMT) is a group of inherited progressive conditions affecting distal motor and sensory neurons, leading to muscle weakness, pain and loss of sensation in limbs. There are currently no treatments for this debilitating disease. To investigate disease mechanisms and facilitate treatment discovery, here we developed an in vitro model for CMT type 2A by introducing the patient-specific MFN2R94Q mutation into human embryonic stem cells (hESCs). Isogenic mutant and wild-type hESCs differentiated to spinal motor neurons with similar efficiency and gave rise to functional motor neurons in vitro. However, MFN2R94Q/+ spinal motor neurons displayed impaired mitochondrial trafficking, resulting in reduced numbers of mitochondria in distal parts of axons. Importantly, we showed that mitochondrial trafficking defects can be alleviated by treatment with an HDAC6 inhibitor. Chemical and genetic inhibition of HDAC6 also significantly rescued the motor phenotype in a zebrafish CMT2A model. Taken together, our study reveals a mutation-specific insight into CMT2A disease mechanism and confirms HDAC6 as a promising target for further therapeutic development.

cell biology↗

bioRxiv2019

Genetically variant human pluripotent stem cells selectively eliminate wild-type counterparts through YAP-mediated cell competition

The appearance of genetic changes in human pluripotent stem cells (hPSCs) presents a concern for their use in research and regenerative medicine. Variant hPSCs harbouring recurrent culture-acquired aneuploidies display growth advantages over wild-type diploid cells, but the mechanisms yielding a drift from predominantly wild-type to variant cell populations remain poorly understood. Here we show that the dominance of variant clones in mosaic cultures is enhanced through competitive interactions resulting in elimination of wild-type cells. This elimination occurs through corralling and mechanical compression by faster growing variants, causing a redistribution of F-actin and sequestration of YAP in the cytoplasm that induces apoptosis in wild-type cells. Importantly, YAP overexpression in wild-type cells is sufficient to alleviate their loser phenotype. Our results demonstrate that hPSC fate is coupled to mechanical cues imposed by neighbouring cells and reveal that hijacking this mechanism allows variants to achieve clonal dominance in cultures.

cell biology↗

bioRxiv2019

Nucleosides rescue replication-mediated genome instability of human pluripotent stem cells

Human pluripotent stem cells (PSC) often acquire genetic changes on prolonged culture, which pose concerns for their use in research and regenerative medicine (Amps et al., 2011, Seth et al., 2011). The acquisition of these changes during culture necessarily first requires mutation and then selection of those mutations that provide a growth advantage. Whilst selection accounts for the recurrent nature of the variants commonly reported (Draper et al., 2004, Olariu et al., 2010), the mechanisms of mutation in PSC remain largely elusive. Here we show that, in contrast to somatic cells, human PSC have an increased susceptibility to DNA damage and mitotic errors, both of which are caused by heightened replication stress in PSC and this can be alleviated by culture with exogenous nucleosides. These results reflect the requirement for rapid replication of human PSC enabled by a truncated G1 (Becker et al., 2006, Becker et al., 2010) that impairs the preparation of these cells for the ensuing DNA replication. A similar relationship has been shown in relation to chromosomal instability in cancer cells (Burrell et al., 2013, Wilhelm et al., 2019) but PSC differ by replication stress triggering apoptosis (Desmarais et al., 2012, Desmarais et al., 2016). Nevertheless, evasion of this response still leads to the appearance of genetic variants that are of concern for regenerative medicine. The inclusion of nucleosides into culture media greatly improves the efficiency of human PSC culture and minimises the acquisition of genomic damage.

cell biology↗

bioRxiv2019

Generation and Trapping of a Mesoderm Biased State of Human Pluripotency

We postulate that exit from pluripotency involves intermediates that retain pluripotency while simultaneously exhibiting lineage-bias. Using a MIXL1 reporter, we explored mesoderm lineage-bias within the human pluripotent stem cell compartment. We identified a substate, which at the single cell level coexpresses pluripotent and mesodermal gene expression programs. Functionally these cells could initiate stem cell cultures and exhibited mesodermal bias in differentiation assays. By promoting mesodermal identity through manipulation of WNT signalling while preventing exit from pluripotency using lysophosphatidic acid, we could trap and maintain cells in a lineage-biased stem cell state through multiple passages. These cells correspond to a normal state on the differentiation trajectory, the plasticity of which is evidenced by their reacquisition of an unbiased state upon removal of differentiation cues. The use of cross-antagonistic signalling to trap pluripotent stem cell intermediates with different lineage-bias may have general applicability in the efficient production of cells for regenerative medicine.

cell biology↗

bioRxiv2018

Human axial progenitors generate trunk neural crest cells

The neural crest (NC) is a multipotent embryonic cell population generating distinct cell types in an axial position-dependent manner. The production of NC cells from human pluripotent stem cells (hPSCs) is a valuable approach to study human NC biology. However, the origin of human trunk NC remains undefined and therefore current in vitro differentiation strategies induce only a modest yield of trunk NC cells. Here we show that hPSC-derived axial progenitors, the posteriorly-located drivers of embryonic axis elongation, give rise to trunk NC cells and their derivatives. Moreover, we define the molecular signatures associated with the emergence of human NC cells of distinct axial identities in vitro. Collectively, our findings indicate that there are two routes toward a human post-cranial NC state: the birth of cardiac and vagal NC is facilitated by retinoic acid-induced posteriorisation of an anterior precursor whereas trunk NC arises within a pool of posterior axial progenitors.

developmental biology↗