Optogenetic control of Wnt signaling models cell-intrinsic embryogenic patterning using 2D human pluripotent stem cell culture.

In embryonic stem cell (ESC) models for early development, spatially and temporally varying patterns of signaling and cell types emerge spontaneously. However, mechanistic insight into this dynamic self-organization is limited by a lack of methods for spatiotemporal control of signaling, and the relevance of signal dynamics and cell-to-cell variability to pattern emergence remains unknown. Here, we combine optogenetic stimulation, imaging and transcriptomic approaches to study self-organization of human ESCs (hESC) in two-dimensional (2D) culture. Morphogen dynamics were controlled via optogenetic activation of canonical Wnt/ß-catenin signaling (optoWnt), which drove broad transcriptional changes and mesendoderm differentiation at high efficiency (>99% cells). When activated within cell subpopulations, optoWnt induced cell self-organization into distinct epithelial and mesenchymal domains, mediated by changes in cell migration, an epithelial to mesenchymal-like transition and TGFß signaling. Furthermore, we demonstrate that such optogenetic control of cell subpopulations can be used to uncover signaling feedback mechanisms between neighboring cell types. These findings reveal that cell-to-cell variability in Wnt signaling is sufficient to generate tissue-scale patterning and establish a hESC model system for investigating feedback mechanisms relevant to early human embryogenesis.

Gene Editing to Generate Versatile Human Pluripotent Stem Cell Reporter Lines for Analysis of Differentiation and Lineage Tracing.

Transcription factors (TFs) are potent proteins that control gene expression and can thereby drive cell fate decisions. Fluorescent reporters have been broadly knocked into endogenous TF loci to investigate the biological roles of these factors; however, the sensitivity of such analyses in human pluripotent stem cells (hPSCs) is often compromised by low TF expression levels and/or reporter silencing. Complementarily, we report an inducible and quantitative reporter platform based on the Cre-LoxP recombination system that enables robust, quantifiable, and continuous monitoring of live hPSCs and their progeny to investigate the roles of TFs during human development and disease. Stem Cells 2019;37:1556-1566.

Engineering biomaterials to control the neural differentiation of stem cells.

Stem cells with the potential for neural differentiation are a promising therapeutic avenue both for treating neurological disease and as a system to advance our fundamental understanding of disease biology in vitro. Precisely controlled extracellular environments that recapitulate critical aspects of embryonic development or the adult stem cell niche are necessary to ensure effective differentiation into the desired cell type. Biomaterials in particular have enabled new avenues for directing stem cell differentiation through the precise presentation of biochemical and biophysical cues. Furthermore, as translation of stem cell technologies necessitates the need for scalable cultures, biomaterials will continue to be valuable tools for guiding stem cell behavior in scalable, complex, three-dimensional cultures. In this review, we highlight the critical signals that guide neurogenesis and how biomaterials can be used to control and direct the neural differentiation of pluripotent and adult stem cells. In addition, we discuss recent new technologies that are further advancing material-based regulation of stem cells. Finally, we highlight the current state of the field and how next-generation biomaterials can enable scalable stem cell culture for cell replacement therapies as well as emerging advanced tissue models for studying tissue morphogenesis and disease pathology.

Enhanced Immunosuppression of T Cells by Sustained Presentation of Bioactive Interferon-γ Within Three-Dimensional Mesenchymal Stem Cell Constructs.

The immunomodulatory activity of mesenchymal stem/stromal cells (MSCs) to suppress innate and adaptive immune responses offers a potent cell therapy for modulating inflammation and promoting tissue regeneration. However, the inflammatory cytokine milieu plays a critical role in stimulating MSC immunomodulatory activity. In particular, interferon-γ (IFN-γ)-induced expression of indoleamine 2,3-dioxygenase (IDO) is primarily responsible for MSC suppression of T-cell proliferation and activation. Although pretreatment with IFN-γ is commonly used to prime MSCs for immunomodulatory activity prior to transplantation, the transient effects of pretreatment may limit the potential of MSCs to potently modulate immune responses. Therefore, the objective of this study was to investigate whether microparticle-mediated presentation of bioactive IFN-γ within three-dimensional spheroidal MSC aggregates could precisely regulate and induce sustained immunomodulatory activity. Delivery of IFN-γ via heparin-microparticles within MSC aggregates induced sustained IDO expression during 1 week of culture, whereas IDO expression by IFN-γ-pretreated MSC spheroids rapidly decreased during 2 days. Furthermore, sustained IDO expression induced by IFN-γ-loaded microparticles resulted in an increased and sustained suppression of T-cell activation and proliferation in MSC cocultures with CD3/CD28-activated peripheral blood mononuclear cells. The increased suppression of T cells by MSC spheroids containing IFN-γ-loaded microparticles was dependent on induction of IDO and supported by affecting monocyte secretion from pro- to anti-inflammatory cytokines. Altogether, microparticle delivery of IFN-γ within MSC spheroids provides a potent means of enhancing and sustaining immunomodulatory activity to control MSC immunomodulation after transplantation and thereby improve the efficacy of MSC-based therapies aimed at treating inflammatory and immune diseases. Stem Cells Translational Medicine 2017;6:223-237.

Pre-conditioning mesenchymal stromal cell spheroids for immunomodulatory paracrine factor secretion.

BACKGROUND AIMS: Mesenchymal stromal cells (MSCs) exhibit the inherent potential to regulate multiple signaling pathways and cell types that contribute to the pathogenesis of inflammatory and immune diseases. However, more recent studies have suggested that the secretion of immunomodulatory factors by MSCs can be enhanced by three-dimensional aggregation or pro-inflammatory cytokine treatment. METHODS: Human MSC spheroids were formed by forced aggregation into agarose micro-wells and subsequently cultured in either minimal essential medium alpha supplemented with fetal bovine serum or serum-free, defined MesenCult-XF medium (STEMCELL Technologies, Vancouver, Canada). A subset of the spheroids were treated with pro-inflammatory cytokines interferon (IFN)-γ or tumor necrosis factor (TNF)-α or both for 4 days. Immunomodulatory factor (prostaglandin E2, indoleamine 2,3-dioxygenase, transforming growth factor-ß1 and interleukin-6) secretion was quantified after 4 days of culture, and the immunomodulatory activity of MSCs was assessed by quantifying activated macrophage expression of TNF-α after trans-well co-culture. RESULTS: Culturing human MSCs as three-dimensional aggregates increased secretion of immunomodulatory paracrine factors, which was enhanced further by treatment with IFN-γ and TNF-α, demonstrating that these parameters can synergistically enhance endogenous human MSC immunomodulatory properties. However, immunomodulatory factor secretion was found to be highly dependent on the composition of cell culture medium. Human MSCs cultured in MesenCult-XF medium displayed significantly less expression of prostaglandin E2, indoleamine 2,3-dioxygenase, transforming growth factor-ß1 and interleukin-6 compared with human MSCs cultured in medium supplemented with fetal bovine serum. Finally, pre-conditioning of human MSC spheroids with IFN-γ and TNF-α resulted in greater immunomodulatory activity in a macrophage co-culture assay. CONCLUSIONS: Altogether, engineering the environment of human MSCs to develop pre-conditioning strategies for enhancing human MSC immunomodulation may be a simple approach for improving MSC-based therapies for the treatment of inflammatory and immune diseases.

Structure-function relationships in the stem cell's mechanical world A: seeding protocols as a means to control shape and fate of live stem cells.

Shape and fate are intrinsic manifestations of form and function at the cell scale. Here we hypothesize that seeding density and protocol affect the form and function of live embryonic murine mesenchymal stem cells (MSCs) and their nuclei. First, the imperative for study of live cells was demonstrated in studies showing changes in cell nucleus shape that were attributable to fixation per se. Hence, we compared live cell and nuclear volume and shape between groups of a model MSC line (C3H10T1/2) seeded at, or proliferated from 5,000 cells/cm2 to one of three target densities to achieve targeted development contexts. Cell volume was shown to be dependent on initial seeding density whereas nucleus shape was shown to depend on developmental context but not seeding density. Both smaller cell volumes and flatter nuclei were found to correlate with increased expression of markers for mesenchymal condensation as well as chondrogenic and osteogenic differentiation but a decreased expression of pre-condensation and adipogenic markers. Considering the data presented here, both seeding density and protocol significantly alter the morphology of mesenchymal stem cells even at very early stages of cell culture. Thus, these design parameters may play a critical role in the success of tissue engineering strategies seeking to recreate condensation events. However, a better understanding of how these changes in cell volume and nucleus shape relate to the differentiation of MSCs is important for prescribing precise seeding conditions necessary for the development of the desired tissue type. In a companion study (Part B, following), we address the effect of concomitant volume and shape changing stresses on spatiotemporal distribution of the cytoskeletal proteins actin and tubulin. Taken together, these studies bring us one step closer to our ultimate goal of elucidating the dynamics of nucleus and cell shape change as tissue templates grow (cell proliferation) and specialize (cell differentiation).