The molecular and cellular signals that guide T-cell development from hematopoietic stem and progenitor cells (HSPCs) remain poorly understood. The thymic microenvironment integrates multiple niche molecules to potentiate T-cell development in vivo. Recapitulating these signals in vitro in a stromal cell-free system has been challenging and limits T-cell generation technologies. Here, we describe a fully defined engineered in vitro niche capable of guiding T-lineage development from HSPCs. Synergistic interactions between Notch ligand Delta-like 4 and vascular cell adhesion molecule 1 (VCAM-1) were leveraged to enhance Notch signaling and progenitor T-cell differentiation rates. The engineered thymus-like niche enables in vitro production of mouse Sca-1cKit and human CD34 HSPC-derived CD7 progenitor T-cells capable of in vivo thymus colonization and maturation into cytokine-producing CD3 T-cells. This engineered thymic-like niche provides a platform for in vitro analysis of human T-cell development as well as clinical-scale cell production for future development of immunotherapeutic applications.

Populations of cells create local environments that lead to emergent heterogeneity. This is particularly evident in human pluripotent stem cells (hPSCs) where microenvironmental heterogeneity limits cell fate control. We have developed a high-throughput platform to screen hPSCs in configurable micro-environments, enabling the optimization of colony size, cell density, and additional parameters for rapid and robust cell fate responses. Single-cell protein expression profiling revealed that Oct4 and Sox2 co-staining discriminate pluripotent, neuroectoderm, primitive streak, and extraembryonic cell fates, allowing dose responses of 27 developmental factors to simultaneously delineate lineage-specific concentration optima. This platform also enabled quantification of endogenous signaling pathway activation and differentiation bias (fingerprinting). Short-term (48 h) fingerprinting is predictive of definitive endoderm induction efficiency across 12 cell lines and was used a priori to rescue long-term (>18 day) differentiation of a cell line reticent to cardiac induction. These findings facilitate high-throughput hPSC-based screening and quantification of lineage induction bias.

We demonstrate derivation of induced pluripotent stem cells (iPSCs) from terminally differentiated mouse cells in serum- and feeder-free stirred suspension cultures. Temporal analysis of global gene expression revealed high correlations between cells reprogrammed in suspension and cells reprogrammed in adhesion-dependent conditions. Suspension (S) reprogrammed iPSCs (SiPSCs) could be differentiated into all three germ layers in vitro and contributed to chimeric embryos in vivo. SiPSC generation allowed for efficient selection of reprogramming factor expressing cells based on their differential survival and proliferation in suspension. Seamless integration of SiPSC reprogramming and directed differentiation enabled the scalable production of functionally and phenotypically defined cardiac cells in a continuous single cell- and small aggregate-based process. This method is an important step towards the development of a robust PSC generation, expansion and differentiation technology.

(TCM) S U M M A R Y Embryonic stem cells (ESCs) provide a convenient model to probe the molecular and cellular dynamics of developmental cell morphogenesis. ESC differentiation in vitro via embryoid bodies (EBs) recapitulates many aspects of early stages of development, including the epithelial-mesenchymal transition (EMT) of pluripotent cells into more differentiated progeny. Hyaluronan and versican are important extracellular mediators of EMT processes, yet the temporal expression and spatial distribution of these extracellular matrix (ECM) molecules during EB differentiation remains undefined. Thus, the objective of this study was to evaluate the synthesis and organization of hyaluronan and versican by using murine ESCs during EB differentiation. Hyaluronan and versican (V0 and V1 isoforms), visualized by immunohistochemistry and evaluated biochemically, accumulated within EBs during the course of differentiation. Interestingly, increasing amounts of a 70-kDa proteolytic fragment of versican were also detected over time, along with ADAMTS-1 and -5 protein expression. ESCs expressed each of the hyaluronan synthases (HAS) -1, -2, and -3 and versican splice variants (V0, V1, V2, and V3) throughout EB differentiation, but HAS-2, V0, and V1 were expressed at significantly increased levels at each time point examined. Hyaluronan and versican exhibited overlapping expression patterns within EBs in regions of low cell density, and versican expression was excluded from clusters of epithelial (cytokeratin-positive) cells but was enriched within the vicinity of mesenchymal (N-cadherin-positive) cells. These results indicate that hyaluronan and versican synthesized by ESCs within EB microenvironments are associated with EMT processes and furthermore suggest that endogenously produced ECM molecules play a role in ESC differentiation. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials. (J Histochem Cytochem 58:345-358, 2010)

Embryonic stem cells (ESCs) can differentiate into all somatic cell types, thereby providing a robust cell source for regenerative medicine therapies. ESCs are commonly induced to differentiate via three-dimensional cell aggregates called embryoid bodies (EBs), which recapitulate cellular and molecular aspects of early tissue morphogenesis. Recent studies suggest that biomolecules synthesized by transplanted ESCs may provide instructive cues for tissue regeneration in vivo. Thus, the objective of this study was to acellularize EBs at different stages of differentiation in order to extract extracellular matrices containing ESC-derived biomolecules. Successive treatment with Triton X-100 and DNase significantly reduced the cellularity and completely inhibited the viability of EBs at various stages of differentiation. In addition, most DNA content (69-75%) was removed, while a portion of the original protein content (15-25%) was retained. Most importantly, extracellular matrix components produced by EBs were retained after acellularization. These results indicate that successful acellularization of EBs can be performed at various stages of differentiation to enable temporal modulation of acellular ECM composition. In addition, acellular matrices derived from EBs represent a novel route of obtaining molecular cues produced by ESCs actively undergoing morphogenesis, thus this technology may be relevant to the development of future regenerative medicine therapies.

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.

BackgroundCellular clearance of reactive oxygen species is dependent on a network of tightly coupled redox enzymes; this network rapidly adapts to oxidative conditions such as aging, viral entry, or inflammation. Current widespread use of shRNA as a means to perturb specific redox couples may be misinterpreted if the targeted effects are not monitored in the context of potential global remodeling of the redox enzyme network.ResultsStable cell lines containing shRNA targets for glutaredoxin 1, thioredoxin 1, or glucose-6-phosphate dehydrogenase were generated in order to examine the changes in expression associated with altering cytosolic redox couples. A qRT PCR array revealed systemic off-target effects of altered antioxidant capacity and reactive oxygen species formation. Empty lentiviral particles generated numerous enzyme expression changes in comparison to uninfected cells, indicating an alteration in antioxidant capacity irrespective of a shRNA target. Of the three redox couples perturbed, glutaredoxin 1, attenuation produced the most numerous off-target effects with 10/28 genes assayed showing statistically significant changes. A multivariate analysis extracted strong co-variance between glutaredoxin 1 and peroxiredoxin 2 which was subsequently experimentally verified. Computational modeling of the peroxide clearance dynamics associated with the remodeling of the redox network indicated that the compromised antioxidant capacity compared across the knockdown cell lines was unequally affected by the changes in expression of off-target proteins.ConclusionsOur results suggest that targeted reduction of redox enzyme expression leads to widespread changes in off-target protein expression, changes that are well-insulated between sub-cellular compartments, but compensatory in both the production of and protection against intracellular reactive oxygen species. Our observations suggest that the use of lentivirus can in itself have off-target effects on dynamic responses to oxidative stress due to the changes in species concentrations.

The thymus supports the development and differentiation of T cells, which are a central component of the mammalian adaptive immune system. From entry of hematopoietic progenitor cells into the thymus to their complex maturation sequence into naïve T cells, the role of the thymic microenvironment has been the subject of intense study. A pivotal aspect of this process is the activation of Notch receptors on progenitors by Delta-like (Dll) ligands present on thymic epithelial cells. Thus far, two approaches have been taken to create an artificial thymus, or mimic thymic function. One involves an in vitro cell-based system in which several key components are provided, including Dll and cytokines, to induce and support T cell lineage differentiation. The gold standard approach makes use of a bone marrow-derived cell line (OP9), ectopically expressing Dll (OP9-DL). A related method involves an in vitro cell-free system that provides a similar set of required signaling components. The second approach involves the generation of organized thymic tissue, which can be achieved by direct reprogramming of another cell type or via the differentiation of pluripotent stem cells (PSCs), either by the ectopic expression of the thymic master regulatory gene, FoxN1, in fibroblasts or by inducing the differentiation of PSCs using developmental cues, respectively. These approaches share a similar goal, to generate T cells from different sources of stem cells. However, the former takes advantage of cellular or molecular drivers of T-lineage differentiation, while the latter is focused on creating thymic tissues that would support T cell development.