Elucidating the Reprogramming Mechanism: Selection Pressures Direct a Poised Subpopulation of Somatic Cells to an Induced Pluripotent Stem Cell State

Through the overexpression of key transcription factors – Oct4, Klf4, c-Myc, and Sox2 (OKMS) – mouse somatic cells can be reprogrammed to an induced pluripotent stem cell (iPSC) state, enabling them to give rise to all cell types of the body. However, the process of reprogramming has remained elusive and it is unclear whether all somatic cells traverse the same trajectory following OKMS transgene overexpression. The reprogramming process has been mapped by conducting ‘omics analyses of bulk reprogramming culture over time. These population-averaged datasets describe reprogramming as a stepwise process where somatic cells transition through a (transgene-dependent) pre-iPSC state to finally stabilise in a (transgene-independent) iPSCs state. On the other hand, single mouse somatic cells reprogrammed in isolation exhibit stochastic reprogramming latencies. As a result, cells experiencing asynchronous reprogramming dynamics are expected to give rise to heterogeneity in bulk reprogramming culture. Here, we aim to consolidate the apparent dichotomy between stepwise cell state changes observed in population reprogramming with the stochasticity of isolated clonal reprogramming by investigating heterogeneity in mouse reprogramming culture. We first develop a surface marker strategy to track transgene-dependent and -independent iPSCs, enabling us to assess heterogeneity with ease. This marker strategy also shows utility in separating naïve and primed pluripotent states in mouse and human culture. We next combine this surface marker-based approach with clonal tracking and mathematical modeling to investigate the reprogramming trajectory of individual reprogramming clones in bulk culture. In doing so, we uncover clonal competition as a novel driver of population reprogramming dynamics. In fact, we show that a subset of poised somatic cells gives rise to clones that have an elite propensity to dominate bulk reprogramming culture and contribute to the iPSC fraction. We also show, for the first time, that a few clones overtake population reprogramming culture, thus making population-averaged data clonally biased. This work provides novel insights into the role of cell competition in driving heterogeneity in reprogramming culture, providing a new lens with which to interpret population-averaged ‘omics datasets. Taken together, we show that not all reprogramming clones exhibit equivalent behaviour in multicellular contexts, underscoring the need to consider the competition potential of iPSCs.Ph.D.2018-12-19 00:00:0

Context-aware synthetic biology by controller design: Engineering the mammalian cell

The rise of systems biology has ushered a new paradigm: the view of the cell as a system that processes environmental inputs to drive phenotypic outputs. Synthetic biology provides a complementary approach, allowing us to program cell behavior through the addition of synthetic genetic devices into the cellular processor. These devices, and the complex genetic circuits they compose, are engineered using a design-prototype-test cycle, allowing for predictable device performance to be achieved in a context-dependent manner. Within mammalian cells, context effects impact device performance at multiple scales, including the genetic, cellular, and extracellular levels. In order for synthetic genetic devices to achieve predictable behaviors, approaches to overcome context dependence are necessary. Here, we describe control systems approaches for achieving context-aware devices that are robust to context effects. We then consider cell fate programing as a case study to explore the potential impact of context-aware devices for regenerative medicine applications

Systematic characterization of methods for inducing human naïve pluripotency [Registered Report Stage 1 Protocol]

ABSTRACT Since their derivation, human pluripotent stem cells (hPSCs) have been maintained in a “primed” pluripotent state in vitro, corresponding to embryonic cells of the post-implantation blastocyst. To mimic cells of the pre-implantation blastocyst, the field has devised protocols to revert primed hPSCs to an earlier embryonic, “naïve”, state that can be used to derive in vitro blastocyst-like structures, “blastoids”, providing an interesting model of human pre-implantation embryos. Despite their utility in understanding fundamental cell biology and early embryology, in vitro naïve hPSCs are prone to karyotypic abnormalities and the efficacy of their derivation varies across protocols and cell lines. To promote their reproducibility and reliability in the field, we here apply two widely adopted methods for naïve induction on several hPSC lines varying initial culture conditions while systematically assessing the quality of resulted cells by transcriptional profiling, karyotyping, and blastoid formation ability.</p

A stepwise model of reaction-diffusion and positional information governs self-organized human peri-gastrulation-like patterning

How position-dependent cell fate acquisition occurs during embryogenesis is a central question in developmental biology. To study this process, we developed a defined, high-throughput assay to induce peri-gastrulation-associated patterning in geometrically confined human pluripotent stem cell (hPSC) colonies. We observed that, upon BMP4 treatment, phosphorylated SMAD1 (pSMAD1) activity in the colonies organized into a radial gradient. We developed a reaction-diffusion (RD)-based computational model and observed that the self-organization of pSMAD1 signaling was consistent with the RD principle. Consequent fate acquisition occurred as a function of both pSMAD1 signaling strength and duration of induction, consistent with the positional-information (PI) paradigm. We propose that the self-organized peri-gastrulation-like fate patterning in BMP4-treated geometrically confined hPSC colonies arises via a stepwise model of RD followed by PI. This two-step model predicted experimental responses to perturbations of key parameters such as colony size and BMP4 dose. Furthermore, it also predicted experimental conditions that resulted in RD-like periodic patterning in large hPSC colonies, and rescued peri-gastrulation-like patterning in colony sizes previously thought to be reticent to this behavior.This work was funded by the Canadian Institutes for Health Research and Medicine by Design, a Canada First Research Excellence Program at the University of Toronto.M.T.received funding from CREATE Materials, Mimetics, and Manufacturing from the Natural Sciences and Engineering Research Council of Canada. P.W.Z. is the Canada Research Chair in Stem Cell Engineering

Searching for Superspreaders: Identifying Epidemic Patterns Associated with Superspreading Events in Stochastic Models

The importance of host transmissibility in disease emergence has been demonstrated in historical and recent pandemics that involve infectious individuals, known as superspreaders, who are capable of transmitting the infection to a large number of susceptible individuals. To investigate the impact of superspreaders on epidemic dynamics, we formulate deterministic and stochastic models that incorporate differences in superspreaders versus nonsuperspreaders. In particular, continuous-time Markov chain models are used to investigate epidemic features associated with the presence of superspreaders in a population. We parameterize the models for two case studies, Middle East respiratory syndrome (MERS) and Ebola. Through mathematical analysis and numerical simulations, we find that the probability of outbreaks increases and time to outbreaks decreases as the prevalence of superspreaders increases in the population. In particular, as disease outbreaks occur more rapidly and more frequently when initiated by superspreaders, our results emphasize the need for expeditious public health interventions

Cell competition during reprogramming gives rise to dominant clones

International audienceThe ability to generate induced pluripotent stem cells from differentiated cell types has enabled researchers to engineer cell states. Although studies have identified molecular networks that reprogram cells to pluripotency, the cellular dynamics of these processes remain poorly understood. Here, by combining cellular barcoding, mathematical modeling, and lineage tracing approaches, we demonstrate that reprogramming dynamics in heterogeneous populations are driven by dominant “elite” clones. Clones arise a priori from a population of poised mouse embryonic fibroblasts derived from Wnt1-expressing cells that may represent a neural crest–derived population.This work highlights the importance of cellular dynamics in fate programming outcomes and uncovers cell competition as a mechanism by which cells with eliteness emerge to occupy and dominate the reprogramming niche

High-throughput micropatterning platform reveals Nodal-dependent bisection of peri-gastrulation-associated versus preneurulation-associated fate patterning.

In vitro models of postimplantation human development are valuable to the fields of regenerative medicine and developmental biology. Here, we report characterization of a robust in vitro platform that enabled high-content screening of multiple human pluripotent stem cell (hPSC) lines for their ability to undergo peri-gastrulation-like fate patterning upon bone morphogenetic protein 4 (BMP4) treatment of geometrically confined colonies and observed significant heterogeneity in their differentiation propensities along a gastrulation associable and neuralization associable axis. This cell line-associated heterogeneity was found to be attributable to endogenous Nodal expression, with up-regulation of Nodal correlated with expression of a gastrulation-associated gene profile, and Nodal down-regulation correlated with a preneurulation-associated gene profile expression. We harness this knowledge to establish a platform of preneurulation-like fate patterning in geometrically confined hPSC colonies in which fates arise because of a BMPs signalling gradient conveying positional information. Our work identifies a Nodal signalling-dependent switch in peri-gastrulation versus preneurulation-associated fate patterning in hPSC cells, provides a technology to robustly assay hPSC differentiation outcomes, and suggests conserved mechanisms of organized fate specification in differentiating epiblast and ectodermal tissues

CD24 tracks divergent pluripotent states in mouse and human cells

Reprogramming is a dynamic process that can result in multiple pluripotent cell types emerging from divergent paths. Cell surface protein expression is a particularly desirable tool to categorize reprogramming and pluripotency as it enables robust quantification and enrichment of live cells. Here we use cell surface proteomics to interrogate mouse cell reprogramming dynamics and discover CD24 as a marker that tracks the emergence of reprogramming-responsive cells, while enabling the analysis and enrichment of transgene-dependent (F-class) and -independent (traditional) induced pluripotent stem cells (iPSCs) at later stages. Furthermore, CD24 can be used to delineate epiblast stem cells (EpiSCs) from embryonic stem cells (ESCs) in mouse pluripotent culture. Importantly, regulated CD24 expression is conserved in human pluripotent stem cells (PSCs), tracking the conversion of human ESCs to more naive-like PSC states. Thus, CD24 is a conserved marker for tracking divergent states in both reprogramming and standard pluripotent culture