The commercialization of allogeneic cell therapies, such as engineered T cells targeting malignant B cells, hinges on cost-effective manufacturing processes. Current therapies are often autologous, resulting in high variability and production costs. Pluripotent stem cells (PSCs) offer a potential solution due to their differentiation capacity and cell engineering amenability. Hematopoietic progenitor cells (HPCs), emerging from hemogenic endothelium (HE) through Notch signaling, are essential for T cell production. However, current methods for HPC generation are limited by the surface-bound Notch ligand DLL4. To address this, we developed a scalable process for generating T lymphoid-competent HPCs from PSCs. A serum- and feeder-free differentiation system in a dynamic suspension culture was employed. PSCs were aggregated and the immunophenotype of differentiating aggregates was tracked. Using a 2x2-factorial design, the protocol was optimized by evaluating the effects of split ratio and feeding frequency on HPC generation. In dynamic suspension culture, the aggregates contained up to 78.4% CD34+CD43− HE cells, with 93.5% expressing DLL4. These aggregates yielded CD34+CD43+CD45+ HPCs with a purity of 96.4%. These HPCs demonstrated the ability to develop into CD8+CD4−CD3+TCRαβ+ mature T cells. The modified protocol in a large-scale system generated 170±30 million HPCs, representing a 6-fold increase over historical results and a 14-fold reduction in reagent costs. This novel bioprocess offers a high-purity HPC source with T lymphoid competence. Our work demonstrates the technical feasibility of a cost-effective and renewable cell source for off-the-shelf immunotherapies. Future research will focus on modulating differentiation signals for further process understanding and intensification.

T cells develop from hematopoietic progenitors in the thymus and protect against pathogens and cancer. However, the emergence of human T cell-competent blood progenitors and their subsequent specification to the T lineage have been challenging to capture in real time. Here, we leveraged a pluripotent stem cell differentiation system to understand the transcriptional dynamics and cell fate restriction events that underlie this critical developmental process. Time-resolved single-cell RNA sequencing revealed that downregulation of the multipotent hematopoietic program, upregulation of >90 lineage-associated transcription factors, and cell-cycle exit all occur within a highly coordinated developmental window. Gene-regulatory network inference uncovered a role for YBX1 in T lineage specification. We mapped the differentiation cell fate hierarchy using transcribed lineage barcoding and discovered that mast and myeloid potential bifurcate from each other early in hematopoiesis, upstream of T lineage restriction. Our systems-level analyses provide a quantitative, time-resolved model of human T cell fate specification. A record of this paper’s transparent peer review process is included in the supplemental information.

Established cell-based therapies using haematopoietic stem and progenitor cells (HSPCs) and T cells have shown promising efficacy for treating blood disorders and cancer. However, access to these therapies is limited by the shortage of compatible donors and the complexity of patient-specific manufacturing processes. Human pluripotent stem cells (hPSCs) offer a renewable solution for generating HSPCs and T cells. While there have been attempts to differentiate hPSCs into various haematopoietic cell types, the intrinsic and extrinsic regulatory mechanisms that control the generation of definitive HSPCs and T cells are not fully known. Our lab has recently developed a robust, chemically defined protocol to generate T cell competent HSPCs from hPSCs. Using single-cell RNA-sequencing, we have identified differentially expressed genes across hemogenic endothelial cell, HSPC, mast cell, myeloid cell, erythroid cell, and progenitor T cell populations. To elucidate the roles of individual transcription factors (TFs) in HSPC emergence and T cell specification, we performed a CRISPR-Cas9 knockout screen targeting 70 TFs. Preliminary analysis confirmed the importance of known T-cell specific TFs GATA3 and TCF7, and we have identified additional top candidate genes including YBX1 to be essential for T cell development. Building on this foundation, we plan to perform multiplexed CRISPR screening read out by scRNA-seq to further examine the transcriptional networks that govern T cell specification. We have also utilized the computational methods such as NicheNet and IQCELL to infer ligand-receptor-target links and identify signalling pathways upstream of the effector TFs. This study contributes to our understanding of gene interactions orchestrating HSPC and T cell emergence, paving the way for scalable production of therapeutically relevant cell types in future stem cell therapy.

The mechanism by which morphogenetic signals engage the regulatory networks responsible for early embryonic tissue patterning is incompletely understood. Here, we developed a minimal gene regulatory network (GRN) model of human pluripotent stem cell (hPSC) lineage commitment and embedded it into "cellular" agents that respond to a dynamic morphogenetic signaling microenvironment. Simulations demonstrated that GRN wiring had significant non-intuitive effects on tissue pattern order, composition, and dynamics. Experimental perturbation of GRN connectivities supported model predictions and demonstrated the role of OCT4 as a master regulator of peri-gastrulation fates. Our so-called GARMEN strategy provides a multiscale computational platform to understand how single-cell-based regulatory interactions scale to tissue domains. This foundation provides new opportunities to simulate the impact of network motifs on normal and aberrant tissue development.

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.

The emergence of the anterior-posterior body axis during early gastrulation constitutes a symmetry-breaking event, which is key to the development of bilateral organisms, and its mechanism remains poorly understood. Two-dimensional gastruloids constitute a simple and robust framework to study early developmental events in vitro. Although spontaneous symmetry breaking has been observed in three dimensional (3D) gastruloids, the mechanisms behind this phenomenon are poorly understood. We thus set out to explore whether a controllable 2D system could be used to reveal the mechanisms behind the emergence of asymmetry in patterned cellular structures. We first computationally simulated the emergence of organization in micro-patterned mouse pluripotent stem cell (mPSC) colonies using a Turing-like activator-repressor model with activator-concentration-dependent flux boundary condition at the colony edge. This approach allows the self-organization of the boundary conditions, which results in a larger variety of patterns than previously observed. We found that this model recapitulated previous results of centro-symmetric patterns in large colonies, and also that in simulated small colony sizes, patterns with spontaneous asymmetries emerged. Model analysis revealed reciprocal effects between diffusion and size of the colony, with model-predicted asymmetries in small pattern sizes being dominated by diffusion, and centro-symmetric patterns being size-dominated. To test these predictions, we performed experiments on micro-patterned mPSC colonies of different sizes stimulated with Bone Morphogenetic Protein 4 (BMP4), and used Brachyury (BRA)-GFP expressing cells as pattern readout. We found that while large colonies showed centro-symmetric BRA patterns, the probability of colony polarization increased with decreasing sizes, with a maximum polarization frequency of 35% at ∼200μm. These results indicate that a simple molecular activator-repressor system can provide cells with collective features capable of initiating a body-axes plan, and constitute a theoretical foundation for the engineering of asymmetry in developmental systems.

During development, state transitions are coordinated through changes in the identity of molecular regulators in a cell state- and dose specific manner. The ability to rationally engineer such functions 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 discrete cell state, and upon state detection, produce fine-tuned effector proteins in a programmable manner. Effectively, these gene circuits convert a discrete (digital-like) cell state into an analog signal by merging AND-like logic integration of endogenous miRNAs (classifiers) with a miRNA-mediated output fine-tuning technology (miSFITs). Using an automated miRNA identification and model-guided circuit optimization approach, we were able to produce robust cell state specific and graded output production in undifferentiated hPSC. We further finely controlled the levels of endogenous BMP4 secretion, which allowed us to document the effect of endogenous factor secretion in comparison to exogenous factor addition on early tissue development using the hPSC-derived gastruloid system. Our work provides the first demonstration of a discrete-to-analog signal conversion circuit operating in living hPSC, and a platform for customized cell state-specific control of desired physiological factors, laying the foundation for programming cell compositions in hPSC-derived tissues and beyond. Graphical Abstract