An alternative splicing switch regulates embryonic stem cell pluripotency and reprogramming.

Alternative splicing (AS) is a key process underlying the expansion of proteomic diversity and the regulation of gene expression. Here, we identify an evolutionarily conserved embryonic stem cell (ESC)-specific AS event that changes the DNA-binding preference of the forkhead family transcription factor FOXP1. We show that the ESC-specific isoform of FOXP1 stimulates the expression of transcription factor genes required for pluripotency, including OCT4, NANOG, NR5A2, and GDF3, while concomitantly repressing genes required for ESC differentiation. This isoform also promotes the maintenance of ESC pluripotency and contributes to efficient reprogramming of somatic cells into induced pluripotent stem cells. These results reveal a pivotal role for an AS event in the regulation of pluripotency through the control of critical ESC-specific transcriptional programs.

Highly efficient reprogrammable mouse lines with integrated reporters to track the route to pluripotency.

Revealing the molecular events associated with reprogramming different somatic cell types to pluripotency is critical for understanding the characteristics of induced pluripotent stem cell (iPSC) therapeutic derivatives. Inducible reprogramming factor transgenic cells or animals-designated as secondary (2°) reprogramming systems-not only provide excellent experimental tools for such studies but also offer a strategy to study the variances in cellular reprogramming outcomes due to different in vitro and in vivo environments. To make such studies less cumbersome, it is desirable to have a variety of efficient reprogrammable mouse systems to induce successful mass reprogramming in somatic cell types. Here, we report the development of two transgenic mouse lines from which 2° cells reprogram with unprecedented efficiency. These systems were derived by exposing primary reprogramming cells containing doxycycline-inducible Yamanaka factor expression to a transient interruption in transgene expression, resulting in selection for a subset of clones with robust transgene response. These systems also include reporter genes enabling easy readout of endogenous Oct4 activation (GFP), indicative of pluripotency, and reprogramming transgene expression (mCherry). Notably, somatic cells derived from various fetal and adult tissues from these 2° mouse lines gave rise to highly efficient and rapid reprogramming, with transgene-independent iPSC colonies emerging as early as 1 wk after induction. These mouse lines serve as a powerful tool to explore sources of variability in reprogramming and the mechanistic underpinnings of efficient reprogramming systems.

Stem cell bioengineering: building from stem cell biology.

New fundamental discoveries in stem cell biology have yielded potentially transformative regenerative therapeutics. However, widespread implementation of stem-cell-derived therapeutics remains sporadic. Barriers that impede the development of these therapeutics can be linked to our incomplete understanding of how the regulatory networks that encode stem cell fate govern the development of the complex tissues and organs that are ultimately required for restorative function. Bioengineering tools, strategies and design principles represent core components of the stem cell bioengineering toolbox. Applied to the different layers of complexity present in stem-cell-derived systems - from gene regulatory networks in single stem cells to the systemic interactions of stem-cell-derived organs and tissues - stem cell bioengineering can address existing challenges and advance regenerative medicine and cellular therapies.

A Myc enhancer cluster regulates normal and leukaemic haematopoietic stem cell hierarchies.

The transcription factor Myc is essential for the regulation of haematopoietic stem cells and progenitors and has a critical function in haematopoietic malignancies. Here we show that an evolutionarily conserved region located 1.7 megabases downstream of the Myc gene that has previously been labelled as a 'super-enhancer' is essential for the regulation of Myc expression levels in both normal haematopoietic and leukaemic stem cell hierarchies in mice and humans. Deletion of this region in mice leads to a complete loss of Myc expression in haematopoietic stem cells and progenitors. This caused an accumulation of differentiation-arrested multipotent progenitors and loss of myeloid and B cells, mimicking the phenotype caused by Mx1-Cre-mediated conditional deletion of the Myc gene in haematopoietic stem cells. This super-enhancer comprises multiple enhancer modules with selective activity that recruits a compendium of transcription factors, including GFI1b, RUNX1 and MYB. Analysis of mice carrying deletions of individual enhancer modules suggests that specific Myc expression levels throughout most of the haematopoietic hierarchy are controlled by the combinatorial and additive activity of individual enhancer modules, which collectively function as a 'blood enhancer cluster' (BENC). We show that BENC is also essential for the maintenance of MLL-AF9-driven leukaemia in mice. Furthermore, a BENC module, which controls Myc expression in mouse haematopoietic stem cells and progenitors, shows increased chromatin accessibility in human acute myeloid leukaemia stem cells compared to blasts. This difference correlates with MYC expression and patient outcome. We propose that clusters of enhancers, such as BENC, form highly combinatorial systems that allow precise control of gene expression across normal cellular hierarchies and which also can be hijacked in malignancies.

Author Correction: A Myc enhancer cluster regulates normal and leukaemic haematopoietic stem cell hierarchies.

In the originally published version of this Letter, ref. 43 was erroneously provided twice. In the 'Estimation of relative cell-type-specific composition of AML samples' section in the Methods, the citation to ref. 43 after the GEO dataset GSE24759 is correct. However, in the 'Mice' section of the Methods, the citation to ref. 43 after 'TAMERE' should have been associated with a new reference1. The original Letter has been corrected online (with the new reference included as ref. 49).

Synthetic gene circuits for cell state detection and protein tuning in human pluripotent stem cells.

During development, cell state transitions are coordinated through changes in the identity of molecular regulators in a cell type- and dose-specific manner. The ability to rationally engineer such transitions in human pluripotent stem cells (hPSC) will enable numerous applications in regenerative medicine. Herein, we report the generation of synthetic gene circuits that can detect a desired cell state using AND-like logic integration of endogenous miRNAs (classifiers) and, upon detection, produce fine-tuned levels of output proteins using an miRNA-mediated output fine-tuning technology (miSFITs). Specifically, we created an "hPSC ON" circuit using a model-guided miRNA selection and circuit optimization approach. The circuit demonstrates robust PSC-specific detection and graded output protein production. Next, we used an empirical approach to create an "hPSC-Off" circuit. This circuit was applied to regulate the secretion of endogenous BMP4 in a state-specific and fine-tuned manner to control the composition of differentiating hPSCs. Our work provides a platform for customized cell state-specific control of desired physiological factors in hPSC, laying the foundation for programming cell compositions in hPSC-derived tissues and beyond.

IQCELL: A platform for predicting the effect of gene perturbations on developmental trajectories using single-cell RNA-seq data.

The increasing availability of single-cell RNA-sequencing (scRNA-seq) data from various developmental systems provides the opportunity to infer gene regulatory networks (GRNs) directly from data. Herein we describe IQCELL, a platform to infer, simulate, and study executable logical GRNs directly from scRNA-seq data. Such executable GRNs allow simulation of fundamental hypotheses governing developmental programs and help accelerate the design of strategies to control stem cell fate. We first describe the architecture of IQCELL. Next, we apply IQCELL to scRNA-seq datasets from early mouse T-cell and red blood cell development, and show that the platform can infer overall over 74% of causal gene interactions previously reported from decades of research. We will also show that dynamic simulations of the generated GRN qualitatively recapitulate the effects of known gene perturbations. Finally, we implement an IQCELL gene selection pipeline that allows us to identify candidate genes, without prior knowledge. We demonstrate that GRN simulations based on the inferred set yield results similar to the original curated lists. In summary, the IQCELL platform offers a versatile tool to infer, simulate, and study executable GRNs in dynamic biological systems.

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.

A 17-gene stemness score for rapid determination of risk in acute leukaemia.

Refractoriness to induction chemotherapy and relapse after achievement of remission are the main obstacles to cure in acute myeloid leukaemia (AML). After standard induction chemotherapy, patients are assigned to different post-remission strategies on the basis of cytogenetic and molecular abnormalities that broadly define adverse, intermediate and favourable risk categories. However, some patients do not respond to induction therapy and another subset will eventually relapse despite the lack of adverse risk factors. There is an urgent need for better biomarkers to identify these high-risk patients before starting induction chemotherapy, to enable testing of alternative induction strategies in clinical trials. The high rate of relapse in AML has been attributed to the persistence of leukaemia stem cells (LSCs), which possess a number of stem cell properties, including quiescence, that are linked to therapy resistance. Here, to develop predictive and/or prognostic biomarkers related to stemness, we generated a list of genes that are differentially expressed between 138 LSC+ and 89 LSC- cell fractions from 78 AML patients validated by xenotransplantation. To extract the core transcriptional components of stemness relevant to clinical outcomes, we performed sparse regression analysis of LSC gene expression against survival in a large training cohort, generating a 17-gene LSC score (LSC17). The LSC17 score was highly prognostic in five independent cohorts comprising patients of diverse AML subtypes (n = 908) and contributed greatly to accurate prediction of initial therapy resistance. Patients with high LSC17 scores had poor outcomes with current treatments including allogeneic stem cell transplantation. The LSC17 score provides clinicians with a rapid and powerful tool to identify AML patients who do not benefit from standard therapy and who should be enrolled in trials evaluating novel upfront or post-remission strategies.

The optoelectronic microrobot: A versatile toolbox for micromanipulation.

Microrobotics extends the reach of human-controlled machines to submillimeter dimensions. We introduce a microrobot that relies on optoelectronic tweezers (OET) that is straightforward to manufacture, can take nearly any desirable shape or form, and can be programmed to carry out sophisticated, multiaxis operations. One particularly useful program is a serial combination of "load," "transport," and "deliver," which can be applied to manipulate a wide range of micrometer-dimension payloads. Importantly, microrobots programmed in this manner are much gentler on fragile mammalian cells than conventional OET techniques. The microrobotic system described here was demonstrated to be useful for single-cell isolation, clonal expansion, RNA sequencing, manipulation within enclosed systems, controlling cell-cell interactions, and isolating precious microtissues from heterogeneous mixtures. We propose that the optoelectronic microrobotic system, which can be implemented using a microscope and consumer-grade optical projector, will be useful for a wide range of applications in the life sciences and beyond.