BackgroundAutocrine & paracrine signaling are widespread both in vivo and in vitro, and are particularly important in embryonic stem cell (ESC) pluripotency and lineage commitment. Although autocrine signaling via fibroblast growth factor-4 (FGF4) is known to be required in mouse ESC (mESC) neuroectodermal specification, the question of whether FGF4 autocrine signaling is sufficient, or whether other soluble ligands are also involved in fate specification, is unknown. The spatially confined and closed-loop nature of diffusible signaling makes its experimental control challenging; current experimental approaches typically require prior knowledge of the factor/receptor in order to modulate the loop. A new approach explored in this work is to leverage transport phenomena at cellular resolution to downregulate overall diffusible signaling through the physical removal of cell-secreted ligands.Methodology/Principal FindingsWe develop a multiplex microfluidic platform to continuously remove cell-secreted (autocrine\paracrine) factors to downregulate diffusible signaling. By comparing cell growth and differentiation in side-by-side chambers with or without added cell-secreted factors, we isolate the effects of diffusible signaling from artifacts such as shear, nutrient depletion, and microsystem effects, and find that cell-secreted growth factor(s) are required during neuroectodermal specification. Then we induce FGF4 signaling in minimal chemically defined medium (N2B27) and inhibit FGF signaling in fully supplemented differentiation medium with cell-secreted factors to determine that the non-FGF cell-secreted factors are required to promote growth of differentiating mESCs.Conclusions/SignificanceOur results demonstrate for the first time that flow can downregulate autocrine\paracrine signaling and examine sufficiency of extracellular factors. We show that autocrine\paracrine signaling drives neuroectodermal commitment of mESCs through both FGF4-dependent and -independent pathways. Overall, by uncovering autocrine\paracrine processes previously hidden in conventional culture systems, our results establish microfluidic perfusion as a technique to study and manipulate diffusible signaling in cell systems.

Stem cells provide unique opportunities for understanding basic biology, for developing tissue models for drug testing, and for clinical applications in regenerative medicine. Despite the promise, the field faces significant challenges in identifying stem cell populations, controlling their fate, and characterizing their phenotype. These challenges arise because stem cells are ultimately functionally defined, and thus can often be identified only retrospectively. New technologies are needed that can provide surrogate markers of stem cell identity, can maintain stem cell state in vitro, and can better direct differentiation. In this review, we discuss the opportunities that microtechnologies, in particular, can provide to the unique qualities of stem cell biology. Microtechnology, by allowing organization and manipulation of cells and molecules at biologically relevant length scales, enables control of the cellular environment and assessment of cell functions and phenotypes with cellular resolution. This provides opportunities to, for instance, create more realistic stem cell niches, perform multi-parameter profiling of single cells, and direct the extracellular signals that control cell fate. All these features take place in an environment whose small size naturally conserves reagent and allows for multiplexing of experiments. By appropriately applying micro-scale engineering principles to stem cell research, we believe that significant breakthroughs can be made in stem cell research.

Understanding developmental biology requires knowledge of both the environmental factors regulating stem cell differentiation, which are increasingly being defined, and their spatial organization within a structurally heterogeneous niche, which is still largely unknown. Here we introduce spatially organized stem cell developmental models to interrogate the role of space in fate specification. Specifically, we developed Differential Environmental Spatial Patterning (δESP) to organize different microenvironments around single embryonic stem cell (ESC) colonies via sequential micropatterning. We first used δESP to decouple and understand the roles of cell organization and niche organization on ESCs deciding between self-renewal and differentiation fate choices. We then approximated in vitro an embryonic developmental step, specifically proximal-distal (PD) patterning of the mouse epiblast at pre-gastrulation, by spatially organizing two extraembryonic environments around ESCs, demonstrating that spatial organization of these three cell types is sufficient for PD patterns to form in vitro.

Juxtacrine cell-cell signaling mediated by the direct interaction of adjoining mammalian cells is arguably the mode of cell communication that is most recalcitrant to engineering. Overcoming this challenge is crucial for progress in biomedical applications, such as tissue engineering, regenerative medicine, immune system engineering and therapeutic design. Here, we describe the significant advances that have been made in developing synthetic platforms (materials and devices) and synthetic cells (cell surface engineering and synthetic gene circuits) to modulate juxtacrine cell-cell signaling. In addition, significant progress has been made in elucidating design rules and strategies to modulate juxtacrine signaling based on quantitative, engineering analysis of the mechanical and regulatory role of juxtacrine signals in the context of other cues and physical constraints in the microenvironment. These advances in engineering juxtacrine signaling lay a strong foundation for an integrative approach to utilizing synthetic cells, advanced ‘chassis’ and predictive modeling to engineer the form and function of living tissues.

CD8 + T cell responses are the foundation of the recent clinical success of immunotherapy in oncologic indications. Although checkpoint inhibitors have enhanced the activity of existing CD8 + T cell responses, therapeutic approaches to generate Ag-specific CD8 + T cell responses have had limited success. Here, we demonstrate that cytosolic delivery of Ag through microfluidic squeezing enables MHC class I presentation to CD8 + T cells by diverse cell types. In murine dendritic cells (DCs), squeezed DCs were ~1000-fold more potent at eliciting CD8 + T cell responses than DCs cross-presenting the same amount of protein Ag. The approach also enabled engineering of less conventional APCs, such as T cells, for effective priming of CD8 + T cells in vitro and in vivo. Mixtures of immune cells, such as murine splenocytes, also elicited CD8 + T cell responses in vivo when squeezed with Ag. We demonstrate that squeezing enables effective MHC class I presentation by human DCs, T cells, B cells, and PBMCs and that, in clinical scale formats, the system can squeeze up to 2 billion cells per minute. Using the human papillomavirus 16 (HPV16) murine model, TC-1, we demonstrate that squeezed B cells, T cells, and unfractionated splenocytes elicit antitumor immunity and correlate with an influx of HPV-specific CD8 + T cells such that >80% of CD8s in the tumor were HPV specific. Together, these findings demonstrate the potential of cytosolic Ag delivery to drive robust CD8 + T cell responses and illustrate the potential for an autologous cell-based vaccine with minimal turnaround time for patients.