Human Stem Cells for Ophthalmology: Recent Advances in Diagnostic Image Analysis and Computational Modelling.

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.

Pathophysiology of aniridia-associated keratopathy: Developmental aspects and unanswered questions.

Aniridia, a rare congenital disease, is often characterized by a progressive, pronounced limbal insufficiency and ocular surface pathology termed aniridia-associated keratopathy (AAK). Due to the characteristics of AAK and its bilateral nature, clinical management is challenging and complicated by the multiple coexisting ocular and systemic morbidities in aniridia. Although it is primarily assumed that AAK originates from a congenital limbal stem cell deficiency, in recent years AAK and its pathogenesis has been questioned in the light of new evidence and a refined understanding of ocular development and the biology of limbal stem cells (LSCs) and their niche. Here, by consolidating and comparing the latest clinical and preclinical evidence, we discuss key unanswered questions regarding ocular developmental aspects crucial to AAK. We also highlight hypotheses on the potential role of LSCs and the ocular surface microenvironment in AAK. The insights thus gained lead to a greater appreciation for the role of developmental and cellular processes in the emergence of AAK. They also highlight areas for future research to enable a deeper understanding of aniridia, and thereby the potential to develop new treatments for this rare but blinding ocular surface disease.

A mathematical modelling framework for the regulation of intra-cellular OCT4 in human pluripotent stem cells.

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.

A systematic review of cellular therapies for the treatment of limbal stem cell deficiency affecting one or both eyes.

PURPOSE: This systematic review (SR) assessed the efficacy, safety and cost-effectiveness of cell-based therapy to manage limbal stem cell deficiency (LSCD), a sight-threatening orphan condition most frequently associated with severe chemical or thermal burns. LSCD has historically been treated by transplanting limbal tissue. In 1997, a new treatment, cultured limbal epithelial autografts, was described for unilateral LSCD. In cases of bilateral disease cultured autologous oral mucosa stem cells have been used. The relative efficacy of different cultured tissue procedures is unknown. METHODS: A protocol was registered with PROSPERO (CRD42017081117). Searches were conducted in 14 databases and 6 conference websites. Two reviewers independently selected studies, conducted data extraction and assessed risk of bias. One reviewer extracted individual patient data (IPD); a second checked extracted data. Data were assessed to determine the feasibility of statistical analysis, with Bayesian synthesis used to estimate improvement achieved by different treatments. RESULTS: Fifty-two studies were eligible for inclusion (1113 eyes); 41 studies (716 eyes) reported IPD. No evidence was identified on cost-effectiveness. This SR was unable to confirm that any of the types of ex vivo cultured stem cell transplants identified for LSCD treatment were statistically superior when assessed against the outcomes of interest. CONCLUSIONS: We believe this SR is the first to include IPD analysis of LSCD data. There is no evidence for the superiority of any method of limbal stem cell transplant. Confirmation of the safety and efficacy of this treatment modality is challenging due to heterogeneity within and between the studies identified. Therefore, recommendations for future research are proposed.

OCT4 expression in human embryonic stem cells: spatio-temporal dynamics and fate transitions.

The improved in vitro regulation of human embryonic stem cell (hESC) pluripotency and differentiation trajectories is required for their promising clinical applications. The temporal and spatial quantification of the molecular interactions controlling pluripotency is also necessary for the development of successful mathematical and computational models. Here we use time-lapse experimental data of OCT4-mCherry fluorescence intensity to quantify the temporal and spatial dynamics of the pluripotency transcription factor OCT4 in a growing hESC colony in the presence and absence of BMP4. We characterise the internal self-regulation of OCT4 using the Hurst exponent and autocorrelation analysis, quantify the intra-cellular fluctuations and consider the diffusive nature of OCT4 evolution for individual cells and pairs of their descendants. We find that OCT4 abundance in the daughter cells fluctuates sub-diffusively, showing anti-persistent self-regulation. We obtain the stationary probability distributions governing hESC transitions amongst the different cell states and establish the times at which pro-fate cells (which later give rise to pluripotent or differentiated cells) cluster in the colony. By quantifying the similarities between the OCT4 expression amongst neighbouring cells, we show that hESCs express similar OCT4 to cells within their local neighbourhood within the first two days of the experiment and before BMP4 treatment. Our framework allows us to quantify the relevant properties of proliferating hESC colonies and the procedure is widely applicable to other transcription factors and cell populations.

The recent advances in the mathematical modelling of human pluripotent stem cells.

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.

Seeding hESCs to achieve optimal colony clonality.

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.

Correlated random walks of human embryonic stem cells in vitro.

We perform a detailed analysis of the migratory motion of human embryonic stem cells in two-dimensions, both when isolated and in close proximity to another cell, recorded with time-lapse microscopic imaging. We show that isolated cells tend to perform an unusual locally anisotropic walk, moving backwards and forwards along a preferred local direction correlated over a timescale of around 50 min and aligned with the axis of the cell elongation. Increasing elongation of the cell shape is associated with increased instantaneous migration speed. We also show that two cells in close proximity tend to move in the same direction, with the average separation of [Formula: see text]m or less and the correlation length of around 25 µm, a typical cell diameter. These results can be used as a basis for the mathematical modelling of the formation of clonal hESC colonies.

Gamma-irradiated human amniotic membrane decellularised with sodium dodecyl sulfate is a more efficient substrate for the ex vivo expansion of limbal stem cells.

The gold standard substrate for the ex vivo expansion of human limbal stem cells (LSCs) remains the human amniotic membrane (HAM) but this is not a defined substrate and is subject to biological variability and the potential to transmit disease. To better define HAM and mitigate the risk of disease transmission, we sought to determine if decellularisation and/or γ-irradiation have an adverse effect on culture growth and LSC phenotype. Ex vivo limbal explant cultures were set up on fresh HAM, HAM decellularised with 0.5M NaOH, and 0.5% (w/v) sodium dodecyl sulfate (SDS) with or without γ-irradiation. Explant growth rate was measured and LSC phenotype was characterised by histology, immunostaining and qRT-PCR (ABCG2, ΔNp63, Ki67, CK12, and CK13). Ƴ-irradiation marginally stiffened HAM, as measured by Brillouin spectromicroscopy. HAM stiffness and γ-irradiation did not significantly affect the LSC phenotype, however LSCs expanded significantly faster on Ƴ-irradiated SDS decellularised HAM (p<0.05) which was also corroborated by the highest expression of Ki67 and putative LSC marker, ABCG2. Colony forming efficiency assays showed a greater yield and proportion of holoclones in cells cultured on Ƴ-irradiated SDS decellularised HAM. Together our data indicate that SDS decellularised HAM may be a more efficacious substrate for the expansion of LSCs and the use of a γ-irradiated HAM allows the user to start the manufacturing process with a sterile substrate, potentially making it safer. STATEMENT OF SIGNIFICANCE: Despite its disadvantages, including its biological variability and its ability to transfer disease, human amniotic membrane (HAM) remains the gold standard substrate for limbal stem cell (LSC) culture. To address these disadvantages, we used a decellularised HAM sterilised by gamma-irradiation for LSC culture. We cultured LSCs on fresh HAM, HAM decellularised with NaOH, HAM decellularised with sodium dodecyl sulfate (SDS) and HAM decellularised with SDS and sterilised with gamma-irradiation. We demonstrated that although HAM decellularised with SDS and sterilised with gamma-irradiation is significantly stiffer this does not affect LSC culture growth rate or the phenotype of cultured LSCs. We therefore recommend the use of SDS decellularised gamma-irradiated HAM in future LSC clinical trials.

Dynamics of single human embryonic stem cells and their pairs: a quantitative analysis.

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.

CDK1 plays an important role in the maintenance of pluripotency and genomic stability in human pluripotent stem cells.

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.

A human iPSC model of Ligase IV deficiency reveals an important role for NHEJ-mediated-DSB repair in the survival and genomic stability of induced pluripotent stem cells and emerging haematopoietic progenitors.

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.

Expression and functional analysis of G1 to S regulatory components reveals an important role for CDK2 in cell cycle regulation in human embryonic stem cells.

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.

Activation of p53 by nutlin leads to rapid differentiation of human embryonic stem cells.

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.

Neural development by transplanted human embryonal carcinoma stem cells expressing green fluorescent protein.

For many years, researchers have investigated the fate and potential of neuroectodermal cells during the development of the central nervous system. Although several key factors that regulate neural differentiation have been identified, much remains unknown about the molecular mechanisms that control the fate and specification of neural subtypes, especially in humans. Human embryonal carcinoma (EC) stem cells are valuable research tools for the study of neural development; however, existing in vitro experiments are limited to inducing the differentiation of EC cells into only a handful of cell types. In this study, we developed and characterized a novel EC cell line (termed TERA2.cl.SP12-GFP) that carries the reporter molecule, green fluorescent protein (GFP). We demonstrate that TERA2.cl.SP12-GFP stem cells and their differentiated neural derivatives constitutively express GFP in cells grown both in vitro and in vivo. Cellular differentiation does not appear to be affected by insertion of the transgene. We propose that TERA2.cl.SP12-GFP cells provide a valuable research tool to track the fate of cells subsequent to transplantation into alternative environments and that this approach may be particularly useful to investigate the differentiation of human neural tissues in response to local environmental signals.

Overexpression of telomerase confers growth advantage, stress resistance, and enhanced differentiation of ESCs toward the hematopoietic lineage.

Embryonic stem cells (ESCs) are capable of extended self-renewal and maintenance of pluripotency even after many population doublings. This is supported by high levels of telomerase activity and enhanced antioxidant protection in ESCs, both of which are downregulated during differentiation. To examine the role of telomerase for ESC self-renewal and differentiation, we overexpressed the reverse transcriptase subunit (Tert) of murine telomerase in ESCs. Increased telomerase activity enhances the self-renewal ability of the Tert-overexpressing ESCs, improves their resistance to apoptosis, and increases their proliferation. The differentiated progeny of wild-type ESCs express little Tert and show shortening of telomeric overhangs. In contrast, the progeny of Tert-overexpressing ESCs maintain high telomerase activity, as well as the length of G-rich overhangs. In addition, these cells accumulate lower concentrations of peroxides than wild-type cells, implying greater resistance to oxidative stress. Finally, differentiation toward hematopoietic lineages is more efficient as a result of the continued expression of Tert. Microarray analysis revealed that overexpression of Tert altered expression of a variety of genes required for extended self-renewal and lifespan. Our results suggest that telomerase functions as a "survival enzyme" in ESCs and its differentiated progeny by protecting the telomere cap and by influencing the expression patterns of stress response and defense genes. This results in improved proliferation of ESCs and more efficient differentiation, and these results might have profound consequences for stem cell-replacement therapies.

Human embryonal carcinoma stem cells expressing green fluorescent protein form functioning neurons in vitro: a research tool for co-culture studies.

Neural differentiation is controlled by complex molecular mechanisms that determine cell fate and diversity within the nervous system. Interactions between developing tissues play an important role in regulating this process. In vitro co-culture experiments offer a method to study cell differentiation and function under controlled conditions, with the additional benefit of investigating how interactions between populations of cells influence cell growth and behavior. However, it can often be difficult to distinguish between populations of co-cultured cells. Here we report the development of a human embryonal carcinoma (EC) stem cell line (named TERA2.cl.SP12-GFP) that expresses the genetic marker, green fluorescent protein (GFP). Here, we demonstrate that TERA2.cl.SP12-GFP stem cells stably express GFP and that this remains detectable during retinoic acid-induced differentiation. Regulated expression of neural markers during cell development correlated with the formation of morphologically identifiable neurons. Populations of post-mitotic GFP-positive neurons were readily purified and electrophysiological characterization confirmed that such neurons were functionally active. Thus, cultured TERA2.cl.SP12-GFP cells can be readily distinguished from alternative cell types in vitro and provide an amenable system for live cell imaging to study the development and function of human neurons in isolation, and in co-culture with other tissue types.

Characterisation of Wnt gene expression during the differentiation of murine embryonic stem cells in vitro: role of Wnt3 in enhancing haematopoietic differentiation.

The first haematopoietic stem cells in mammalian and non-mammalian vertebrates are derived from mesoderm, therefore genes that are important in mesoderm patterning and formation might also play an essential role in haematopoietic stem cell commitment and differentiation. Several members of the Wnt gene family are expressed in very specific patterns in embryonic mesoderm and have previously been shown to act as haematopoietic growth factors. In order to investigate in detail the role that such secreted proteins play in the biology of early haematopoietic commitment we have used in vitro differentiation of murine embryonal stem (ES) as a model system. Using reverse-transcriptase polymerase chain reaction analysis we identified several candidate Wnt genes whose expression pattern was consistent with a role in generation, maintenance and/or differentiation of early haematopoietic progenitor cells including three genes previously shown to have a role in haematopoiesis (Wnt5a, Wnt2b and Wnt10b). The most interesting candidate was Wnt3, because of its strong and regulated expression during in vitro differentiation of murine ES cells as well as its early embryonic expression in mesoderm. Overexpression of Wnt3 was sufficient to cause a consistent increase in the number of embryoid bodies committing to haematopoiesis further strengthening the evidence that this protein can enhance haematopoietic commitment during in vitro differentiation of ES cells. In addition, overexpression of Wnt3 caused a marked upregulation of Brachyury expression, thus providing some evidence that Brachyury may be one of the target genes for the Wnt3 signalling pathway.

mTert expression correlates with telomerase activity during the differentiation of murine embryonic stem cells.

Telomerase, the enzyme which maintains the ends of linear chromosomes in eukaryotic cells, is found at low levels in somatic stem cells but while this is incapable of preventing the progressive erosion of telomeres occurring as a consequence of cell division, such cells show greater proliferative capacity than normal somatic cells hence examination of telomerase activity in such stem cells is of interest. Our aim in this work was to examine the relationship between expression of the reverse transcriptase component (mTert) of murine telomerase. We report here the insertion of a reporter cassette comprising a segment of the promoter sequence of murine Tert gene coupled to the coding sequence of green fluorescent protein (GFP) into murine embryonic stem (ES) cells and show that this is sufficient for mimicking the expression of mTert. We show that the expression of mTert is very closely linked to telomerase activity and that both are substantially reduced upon differentiation of ES cells into more committed lineages giving us a potential reporter system for the selection and isolation of ES cells possessing different levels of telomerase activity.

Searching the unknown with gene trapping.

dramatic increase in sequence information, both in the form of complementary DNA (cDNA) and genomic DNA, has created a huge gap between the discovery of genes and the process of identifying gene function. To fill this gap, the 'gene-trapping' approach has been developed; this combines into a single process the three stages of gene cloning, the study of the pattern of gene expression and the analysis of the respective mutant phenotype. Recent results indicate that gene trapping can be used successfully to clone specific genes that are involved in the development of the central nervous system, limbs and haematopoietic system. Continuous improvements in the design of trapping vectors, faster sequencing of cDNA clones and more-efficient in vitro pre-screening will certainly aid the large-scale trapping of mammalian genomes.