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One of the characteristic features of human embryonic stem cells (hESCs) is the competence for self-renewal and pluripotency. To date, little is known about cell cycle regulation in these cells and how the cell cycle machinery influences hESCs properties. A common feature of human, murine and primate ESCs is the presence of a short G1 phase, which has been viewed as a time window during which stem cells are exposed to differentiation signals. We used the hESCs differentiation model and comparisons to human embryonic carcinoma (EC) cells to study the key regulators of G1 to S transition in hESCs. Our studies show that hESCs express all G1-specific CYCLINs (D1, D2, D3 and E) and cyclin-dependent kinases (CDK) (CDK2, CDK4 and CDK6) at variable levels. In contrast to murine ESCs, most of the cell cycle regulators in hESCs show cell cycle-dependent expression, thus revealing important differences in the expression of cell cycle regulatory components between these two embryonic cell types. Knockdown of CDK2 using RNA interference resulted in hESCs arrest at G1 phase of the cell cycle and differentiation to extraembryonic lineages. This suggests an important role for CDK2 in cell cycle regulation in hESCs that are likely to bear significant impacts on the maintenance of their pluripotent phenotype.

Human pluripotent stem cells (hPSCs) have the potential to differentiate into all cell types, a property known as pluripotency. A deeper understanding of how pluripotency is regulated is required to assist in controlling pluripotency and differentiation trajectories experimentally. Mathematical modelling provides a non-invasive tool through which to explore, characterise and replicate the regulation of pluripotency and the consequences on cell fate. Here we use experimental data of the expression of the pluripotency transcription factor OCT4 in a growing hPSC colony to develop and evaluate mathematical models for temporal pluripotency regulation. We consider fractional Brownian motion and the stochastic logistic equation and explore the effects of both additive and multiplicative noise. We illustrate the use of time-dependent carrying capacities and the introduction of Allee effects to the stochastic logistic equation to describe cell differentiation. We conclude both methods adequately capture the decline in OCT4 upon differentiation, but the Allee effect model has the advantage of allowing differentiation to occur stochastically in a sub-set of cells. This mathematical framework for describing intra-cellular OCT4 regulation can be extended to other transcription factors and developed into predictive models.

p53 is an important regulator of normal cell response to stress and frequently mutated in human tumours. Here, we studied the effects of activation of p53 and its target gene p21 in human embryonic stem cells. We show that activation of p53 with small-molecule activator nutlin leads to rapid differentiation of stem cells evidenced by changes in cell morphology and adhesion, expression of cell-specific markers for primitive endoderm and trophectoderm lineages and loss of pluripotency markers. p21 is quickly and dose-dependently activated by nutlin. It can also be activated independently from p53 by sodium butyrate, which leads to the differentiation events very similar to the ones induced by p53. During differentiation, the activating phosphorylation site of CDK2 Thr-160 becomes dephosphorylated and cyclins A and E become degraded. The target for CDK2 kinase in p53 molecule, Ser-315, also becomes dephosphorylated. We conclude that the main mechanism responsible for differentiation of human stem cells by p53 is abolition of S-phase entry and subsequent stop of cell cycle in G0/G1 phase accompanied by p21 activation.

Human embryonic stem cells (hESC) and induced pluripotent stem cells (hiPSC) are characterised by an unusual and tightly regulated cell cycle that has been shown to be important for the maintenance of a pluripotent phenotype. Cyclin-dependant kinase 1 (CDK1) is a key player in cell cycle regulation and particularly mitosis; however, its role has not been studied previously in hESC and hiPSC. To investigate the impacts of CDK1 downregulation, we performed RNA interference studies which in addition to expected mitotic deficiencies revealed a large range of additional phenotypes related to maintenance of pluripotency, ability to repair double strand breaks (DSBs) and commitment to apoptosis. Downregulation of CDK1 led to the loss of typical pluripotent stem cell morphology, downregulation of pluripotency markers and upregulation of a large number of differentiation markers. In addition, human pluripotent stem cells with reduced CDK1 expression accumulated a higher number of DSBs were unable to activate CHK2 expression and could not maintain G2/M arrest upon exposure to ionising radiation. CDK1 downregulation led to the accumulation of cells with abnormal numbers of mitotic organelles, multiple chromosomal abnormalities and polyploidy. Furthermore, such cells demonstrated an inability to execute apoptosis under normal culture conditions, despite a significant increase in the expression of active PARP1, resulting in tolerance and very likely further propagation of genomic instabilities and ensuing of differentiation process. On the contrary, apoptosis but not differentiation, was the preferred route for such cells when they were subjected to ionising radiation. Together these data suggest that CDK1 regulates multiple events in human pluripotent stem cells ranging from regulation of mitosis, G2/M checkpoint maintenance, execution of apoptosis, maintenance of pluripotency and genomic stability.

Numerous biological approaches are available to characterise the mechanisms which govern the formation of human embryonic stem cell (hESC) colonies. To understand how the kinematics of single and pairs of hESCs impact colony formation, we study their mobility characteristics using time-lapse imaging. We perform a detailed statistical analysis of their speed, survival, directionality, distance travelled and diffusivity. We confirm that single and pairs of cells migrate as a diffusive random walk for at least 7 hours of evolution. We show that the presence of Cell Tracer significantly reduces hESC mobility. Our results open the path to employ the theoretical framework of the diffusive random walk for the prognostic modelling and optimisation of the growth of hESC colonies. Indeed, we employ this random walk model to estimate the seeding density required to minimise the occurrence of hESC colonies arising from more than one founder cell and the minimal cell number needed for successful colony formation. Our prognostic model can be extended to investigate the kinematic behaviour of somatic cells emerging from hESC differentiation and to enable its wide application in phenotyping of pluripotent stem cells for large scale stem cell culture expansion and differentiation platforms.

DNA double strand breaks (DSBs) are the most common form of DNA damage and are repaired by non-homologous-end-joining (NHEJ) or homologous recombination (HR). Several protein components function in NHEJ, and of these, DNA Ligase IV is essential for performing the final ‘end-joining’ step. Mutations in DNA Ligase IV result in LIG4 syndrome, which is characterised by growth defects, microcephaly, reduced number of blood cells, increased predisposition to leukaemia and variable degrees of immunodeficiency. In this manuscript, we report the creation of a human induced pluripotent stem cell (iPSC) model of LIG4 deficiency, which accurately replicates the DSB repair phenotype of LIG4 patients. Our findings demonstrate that impairment of NHEJ-mediated-DSB repair in human iPSC results in accumulation of DSBs and enhanced apoptosis, thus providing new insights into likely mechanisms used by pluripotent stem cells to maintain their genomic integrity. Defects in NHEJ-mediated-DSB repair also led to a significant decrease in reprogramming efficiency of human cells and accumulation of chromosomal abnormalities, suggesting a key role for NHEJ in somatic cell reprogramming and providing insights for future cell based therapies for applications of LIG4-iPSCs. Although haematopoietic specification of LIG4-iPSC is not affected per se , the emerging haematopoietic progenitors show a high accumulation of DSBs and enhanced apoptosis, resulting in reduced numbers of mature haematopoietic cells. Together our findings provide new insights into the role of NHEJ-mediated-DSB repair in the survival and differentiation of progenitor cells, which likely underlies the developmental abnormalities observed in many DNA damage disorders. In addition, our findings are important for understanding how genomic instability arises in pluripotent stem cells and for defining appropriate culture conditions that restrict DNA damage and result in ex vivo expansion of stem cells with intact genomes.

Human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) have promising clinical applications which often rely on clonally-homogeneous cell populations. To achieve this, it is important to ensure that each colony originates from a single founding cell and to avoid subsequent merging of colonies during their growth. Clonal homogeneity can be obtained with low seeding densities; however, this leads to low yield and viability. It is therefore important to quantitatively assess how seeding density affects clonality loss so that experimental protocols can be optimised to meet the required standards. Here we develop a quantitative framework for modelling the growth of hESC colonies from a given seeding density based on stochastic exponential growth. This allows us to identify the timescales for colony merges and over which colony size no longer predicts the number of founding cells. We demonstrate the success of our model by applying it to our own experiments of hESC colony growth; while this is based on a particular experimental set-up, the model can be applied more generally to other cell lines and experimental conditions to predict these important timescales.

Purpose of Review To explore the advances and future research directions in image analysis and computational modelling of human stem cells (hSCs) for ophthalmological applications. Recent Findings hSCs hold great potential in ocular regenerative medicine due to their application in cell-based therapies and in disease modelling and drug discovery using state-of-the-art 2D and 3D organoid models. However, a deeper characterisation of their complex, multi-scale properties is required to optimise their translation to clinical practice. Image analysis combined with computational modelling is a powerful tool to explore mechanisms of hSC behaviour and aid clinical diagnosis and therapy. Summary Many computational models draw on a variety of techniques, often blending continuum and discrete approaches, and have been used to describe cell differentiation and self-organisation. Machine learning tools are having a significant impact in model development and improving image classification processes for clinical diagnosis and treatment and will be the focus of much future research.

Human pluripotent stem cells hold great promise for developments in regenerative medicine and drug design. The mathematical modelling of stem cells and their properties is necessary to understand and quantify key behaviours and develop non-invasive prognostic modelling tools to assist in the optimisation of laboratory experiments. Here, the recent advances in the mathematical modelling of hPSCs are discussed, including cell kinematics, cell proliferation and colony formation, and pluripotency and differentiation.

Background One of the main priorities of Dutch organisations providing shelter services is to develop evidence-based interventions in the care for abused women and homeless people. To date, most of these organisations have not used specific intervention models and the interventions which have been implemented rarely have an empirical and theoretical foundation. The present studies aim to examine the effectiveness of critical time intervention (CTI) for abused women and homeless people. Methods In two multi-centre randomised controlled trials we investigate whether CTI, a time-limited (nine month) outreach intervention, is more effective than care-as-usual for abused women and homeless people making the transition from shelter facilities to supported or independent housing. Participants were recruited in 19 women’s shelter facilities and 22 homeless shelter facilities across The Netherlands and randomly allocated to the intervention group (CTI) or the control group (care-as-usual). They were interviewed four times in nine months: once before leaving the shelter, and then at three, six and nine months after leaving the shelter. Quality of life (primary outcome for abused women) and recurrent loss of housing (primary outcome for homeless people) as well as secondary outcomes (e.g. care needs, self-esteem, loneliness, social support, substance use, psychological distress and service use) were assessed during the interviews. In addition, the model integrity of CTI was investigated during the data collection period. Discussion Based on international research CTI is expected to be an appropriate intervention for clients making the transition from institutional to community living. If CTI proves to be effective for abused women and homeless people, shelter services could include this case management model in their professional standards and improve the (quality of) services for clients. Trial registration NTR3463 and NTR3425