Harnessing synthetic biology to engineer organoids and tissues

Abstract

The development of an organism depends on intrinsic genetic programs of progenitor cells and their spatiotemporally complex extrinsic environment. Ex vivo generation of organoids from progenitor cells provides a platform for recapitulating and exploring development. Current approaches rely largely on soluble morphogens or engineered biomaterials to manipulate the physical environment, but the emerging field of synthetic biology provides a powerful toolbox to genetically manipulate cell communication, adhesion, and even cell fate. Applying these modular tools to organoids should lead to a deeper understanding of developmental principles, improved organoid models, and an enhanced capability to design tissues for regenerative purposes.

Figure 1.. Reconstituting spatiotemporal complexity of development in vitro

(A) Schematic of in vivo development of a gastrulating embryo with the epiblast (progenitor cells, in pink) receiving chemical and mechanical cues from the extraembryonic tissues surrounding it. (B) In contrast, classic in vitro models of development consist of isolated progenitor cells and lack the environmental cues that drive native development. (C) These cues are supplemented in vitro by adding soluble morphogens to the organoid media to favor specific cell fates. Organoids can also be embedded hydrogels or materials that can provide desired mechanical cues.

Figure 2.. Emerging toolbox to create regulatory cell-cell interactions

(A) Schematic illustration of optogenetic chimeric receptors. When cells express these receptors, they respond to light and activate the downstream signaling without a morphogen. Optogenetic chimeric receptors are typically designed as the cytoplasmic regions of morphogen receptors fused with a light-responsible element, such as a light-oxygen-voltage domain. Light triggers hetero- or homodimerization of the receptors, which activates the downstream signaling. (B) Diagram of synNotch receptor system. synNotch is composed of an extracellular antigen recognition domain, such as scFv, a central regulatory domain in transmembrane domain, and an orthogonal transcription factor (TF). When synNotch detects the antigen on the sender cells (blue), the TF s released by cleavage. (C) Illustration of synthetic diffusive morphogen system using a synthetic receptor, such as MESA. In this system, synthetic receptor dimerization occurs upon synthetic ligand binding, and such dimerization causes cleavage of the intracellular domain of the receptor, releasing the TF to activate its target gene. In all figures, activated cells are colored in red. (D) Design of synthetic cell-cell adhesion molecules. Extracellular domains of native adhesion proteins are replaced by specific protein-protein interactions, such as GFP and anti-GFP nanobody. (E) Multi Fate system is a synthetic circuit that controls multi-fates of mammalian cells for a long term. In MultiFate, TFs (“A” and “B”) homodimerize to self-activate and mutually inhibit one another by heterodimerization.

Figure 3.. Constructing simple synthetic developmental networks with modular cell-cell interaction

(A) Schematic illustration of engineering self-organizing multi-layered spheroids. Sender cells expressing a ligand (blue) induce a synNotch-expressing receiver cell(green) to cell(green) to express homotypic adhesion molecule (yellow) and heterotypic adhesion molecule (red). When we mix these cell types together, the cell population starts to organize to form a core aggregate of receiver cells surrounded by sender cells (blue). (B) Examples of user-defined organizers. (Left) Shh-producing hPSC aggregate is used as a local source of Shh, acting as one pole of the developing forebrain organoid. (Right) Strategy to generate a Wnt and Nodal gradient in an embryoid model. mESCs treated with BMP4 are used as an engineered morphogen signaling center. mESCs close to the signaling center differentiate into mesoderm, resembling the posterior region of a mouse embryo. (C) The synthetic diffusible communication system generates an artificial morphogen gradient. By modulating expression levels of morphogens, we can tune the gradient patters (right).

Figure 4.. Tissue or organ design could follow the path of the more mature field of molecular design

(A) As the field of molecular engineering matures, we have improved our understanding of complex molecular structures composed of synthetic proteins or nucleic acids. Understanding the interactions and driving forces of molecular structure allows for the development of improved native-based structures and the design of custom protein or DNA structures that do not already exist in nature. (B) Likewise, as the field of tissue development matures, so should our ability to predict cell fates and behaviors and to generate more native-like and functional organoids with higher complexity and reproducibility. These structures can be used to acquire a better understanding of development and design improved organoids and organs with custom modular properties. The applications of such designer tissues range from drug testing to transplantation and therapy.