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Published in final edited form as: Curr Opin Biomed Eng. 2024 Aug 24;32:100554. doi: 10.1016/j.cobme.2024.100554

Synthetically programming natural cell-cell communication pathways for tissue engineering

Leah A Wallach 1,2, Connor D Thomas 1,2, Pulin Li 1,2
PMCID: PMC12246774  NIHMSID: NIHMS2095445  PMID: 40655075

Abstract

Tissue patterning, the process of localizing different cell types to the right place, is critical for tissue function and thus a central goal for tissue engineering. Developing embryos employ diverse cell interaction-based mechanisms to robustly pattern tissues, such as specifying different regions of the central nervous system and aligning all the hair cells in the inner ear. These events range in lengthscale and must all be specified with cell-level precision, imposing challenges for recreating such patterns in vitro using conventional engineering approaches. Synthetic developmental biology as an emerging field provides a complementary approach for patterning tissues, by harnessing the molecular mechanisms used by natural tissues to program self-organizing behavior of the cells. Here we review advances in adapting these modules to program cells in culture. These modules could potentially be used for biomedical tissue engineering, as a complement to existing methods for generating morphologically complex multi-cell-type tissues in vitro.

Spatially arranging different cell types into patterns to recapitulate natural tissues is an important goal in tissue engineering. Many current methods rely on either a “top-down” (directly-imposed cell organization) or “bottom-up” (minimally-guided tissue growth) approach to generate spatially complex multicellular tissues. “Top-down” approaches manually determine the exact spatial pattern of a tissue. Examples include 3-D bioprinting, patterned substrates, and microfluidic cell placement (Reddy et al. 2023; Jammalamadaka and Tappa 2018; Warmflash et al. 2014; Yue et al. 2021). These approaches offer the greatest control over precise tissue size, shape, and cell count, and are particularly useful for arranging cell types that lack the inherent capability of self-organizing into spatial patterns. However, these methods are still limited by spatial precision of the initial patterning mechanism, throughput, and issues of cost, technical complexity, and cell viability (Reddy et al. 2023; Lee, Ng, and Yeong 2019). Meanwhile, other tissues, particularly organoid models, use a “bottom-up” approach. Many features of spatially complex multilayered structures, like the intestinal villus, can be grown in vitro from stem/progenitor cells, sometimes even from a single cell (Taelman, Diaz, and Guiu 2022). These approaches, relying on the self-organizing capability of the stem/progenitor cells and their differentiated progenies, yield realistic aspects of tissue morphologies that would be difficult to engineer manually. However, the lack of direct control over tissue geometry and the stochasticity of the self-organizing process limit the reproducibility, although some of the issues can be greatly ameliorated by imposing microfluidic or mechanical signals (Sun et al 2023, Gjorevski et al 2022; Hofer and Lutolf 2021). Additionally, these “bottom-up” strategies are difficult to deploy when the source cells cannot spontaneously self-organize, as in joint tissue models or others that combine several tissue types, and thus are limited to a subset of tractable organs (Y. Hu et al. 2023; Hofer and Lutolf 2021).

Herein, we discuss recent advances from the emerging field of synthetic developmental biology, which uses molecular reconstitution and protein engineering techniques to program self-organizing capabilities of cells. (Schlissel and Li 2020; McNamara, Ramm, and Toettcher 2023; Trentesaux et al. 2023). Many of these studies are carried out through building controllable, engineered versions of the developmental signaling machinery that patterns tissues in vivo. In this review, we will discuss the engineering of newly understood developmental interactions, focusing in particular on the areas of cell-cell signaling, cell adhesion, and cell polarity (see Figure). Each of these mechanisms impart cellular precision to the developing tissue organization. We focus on modular patterning circuits which can be adapted from the original biological contexts to generate patterns in a dish with the precision and reproducibility found in embryos. These methods share traits of both “top-down” and “bottom-up” approaches. We propose that these modules bridge the gap between “top-down” and “bottom-up” tissue engineering for biomedical applications, serving as a new set of methods that can solve problems in bioengineering and help uncover fundamental principles of embryo development.

Figure. Developmental patterning mechanisms in a dish.

Figure.

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(A) Differential cadherin surface adhesion leads to demixing of cells into distinct, sharply separated populations. In vitro, this can give rise to new signaling outcomes at the interface of cells. Image adapted with permission from Glykofrydis et al. 2021. Copyright 2021. American Chemical Society. (B) A tissue with a single region of morphogen-secreting cells adopts very different patterns depending on the combinations of shuttling proteins. Different modules lengthen, shorten, or shift the gradient by tunable amounts. Images adapted from Zhu et al. 2023 and Schlissel et al. 2024. (C) Single cell polarity: A polarity-protein-oligomerizing cap inducibly creates a subcellular signal that directs the orientation of cell divisions. Multicellular polarity: Asymmetric distribution of PCP proteins provide a cue for larger scale organization.

Engineering cell adhesion for sorting cells into spatially distinct domains

Many tissue functions depend on maintaining distinct populations of cell types that remain in close proximity without mixing. However, cell growth, movement and stochastic fate decisions can lead to intermixing of adjacent cell types even after each cell has been initially placed in its correct position in the tissue, whether by a cell fate decision or a human-imposed pattern. For example, the neural tube, which gives rise to the central nervous system, must be patterned in defined, distinct stripes from ventral to dorsal domains to ensure that the resulting neurons develop in the correct locations (Sagner and Briscoe 2019). A similar example in engineered tissue would be an engineered muscle and tendon, in which the two tissues must have mechanical contact, but the growth of tendon within the muscle, or vice versa, would impair normal mechanical function. In both cases, tissues benefit from the ability to “sort” misplaced cells into their correct locations

One of the most successful engineering approaches for creating sharp spatial domains is adhesion-based cell sorting, in which cells with differently adhesive surfaces will rearrange to maximize contact with equally adhesive neighbors. In recent years, this has been shown to have an essential role in the patterning of both embryos and engineered systems, such as the gastruloids that resemble gastrulating embryos (Tsai et al. 2020; McNamara et al. 2023). For example, neuronal specification is initially noisy, with tiny variations in Hedgehog signal dose leading to mixed populations of different fates, but cells subsequently self-sort into distinct territories for each neuron type (Sagner and Briscoe 2019; Xiong et al. 2013). Recent work has shown that this sorting is due to differential cadherin expression in response to Hedgehog, which allows all cells adopting the same fate to self-organize into a single shared compartment of the tissue (Tsai et al. 2020).

Control of cadherin-based cell-cell adhesion is one of the most well-studied and well-developed cases of adapting an endogenous patterning circuit for engineered effects (Leckband and de Rooij 2014). Initially, engineered cadherin expression was used to demonstrate that differential adhesion was sufficient to cause cell sorting (Foty and Steinberg 2005). Since then, multiple labs have shown that, by engineering relative adhesive strengths and ratios of different cell populations, it is possible to create repeatable, spatially complex structures from “blank-slate” cells, particularly in conjunction with other spatial patterning modules such as signaling ligands (Cachat et al. 2016; Toda et al. 2018). Recently, this cell sorting mechanism has been used to create spatially localized signaling sources for controlled patterning of three-dimensional tissues. In natural embryos, a single localized source of Wnt ligands induces the formation of a single primitive streak, a group of cells that migrate underneath the surface layer of cells to form the internal structure of the organism. When engineering embryo-like structures (“embryoids”) from embryonic stem cells (ESCs) in a dish, HEK cells engineered to secrete Wnt ligands can be mixed with ESCs to act as the ligand source and break the symmetry (Sagy et al. 2019). However, without controlling the localization of the HEK cells, embryoids have multiple sources of Wnt and induce primitive streak in multiple locations. Engineering the sender HEK cells to express P-cadherin results in the self-sorting of all HEK cells to a single location when mixed with E-cadherin-expressing ESCs, creating more consistent patterning and morphology of these embryoids (Glykofrydis et al. 2021).

Engineered adhesion not only allows the control of initial cell arrangement, but also rearrangements in response to orthogonal stimuli, such as synthetic Notch signal (Toda et al. 2018). In this circuit, different synthetic ligand-receptor pairs induce distinct levels and combinations of cadherin expression. After the first round of stimulation and self-organization, the cells’ new direct contacts activate a different cadherin expression state. This process creates equilibrium states with reproducible single-cell resolution patterning of cell fates. By coupling signaling and changes in adhesiveness, it is possible to generate spatial complexity that might be difficult to achieve by directly imposing spatial patterning from the outside. However, it is worth noting that adhesion-based sorting relies on cell movement, and therefore, the efficiency of sorting depends on the number of cells and the distance between them, which may be why these mechanisms seem typically used for sharpening an already-defined boundary between adjacent cell populations in a relatively small region of tissue (tens of microns long) (Tordoff et al 2021, Tsai et al. 2020).

Engineering morphogen-mediated communication to control pattern size and shape

Another molecular module that can create spatial patterns is morphogens, a group of secreted signaling ligands that can travel up to hundreds of microns to mediate long-distance cell-cell communication. Within the patterning field, relatively homogenous populations of cells make different fate decisions based on the concentration and/or duration of the morphogen signal they receive, allowing two adjacent cells to become different cell types (Briscoe and Small 2015). Despite the spatially inhomogeneous nature of morphogens, most tissue engineering applications administer morphogen ligands as recombinant proteins in a spatially homogeneous manner, providing limited control and consistency of the resulting spatial organization (Quadrato and Arlotta 2017). Recent developments have demonstrated the feasibility of creating and controlling the spatial gradients of natural morphogens in engineered systems, and ongoing work in engineering features like diffusivity and feedback shows future potential to create complex, multi-ligand systems (Li et al. 2018; Sekine, Shibata, and Ebisuya 2018; Toda et al. 2020).

One important engineering consideration is how to control the lengthscale and shape of morphogen gradients in a quantitative and predictable way. Indeed, an eventual goal of multi-ligand patterning, such as the complexities of Turing patterning, would require highly precise control of not only individual species behavior but specific tuning of their relative behavior. During development, a single ligand can form gradients of many sizes ranging from tens to hundreds of microns in length (Farin et al. 2016; Pani and Goldstein 2018). These gradients are formed with immense precision, able to define cell fate at a single-cell resolution based on gradient length and localization (Merle et al 2024, Gregor et al 2007). At the same time, the shape and size of these morphogen gradients have proved to be highly tunable within and across organisms. These mechanisms, many of which arise from precise combinations of secreted proteins or extracellular matrix components, are proving tractable to human engineering.

One mechanism involves secreted proteins that directly bind to the morphogens and move with them in the extracellular space, altering their diffusion properties. As a result, they can tune the lengthscale and steepness of the morphogen gradients. Some of the secreted modulators are highly specific to each pathway. For example, BMP is a family of morphogens that is widely and ubiquitously expressed in the dorsal region of Drosophila embryos, and yet BMP response is sharply confined to a much narrower region (O’Connor et al. 2006). This observation suggests that BMP ligands need to be “shuttled” and enriched in a location away from the source (Ben-Zvi and Barkai 2010). The secreted protein, Chordin, performs this “shuttling” function by binding to the BMP ligand, enhancing its diffusion by protecting it from receptor binding, and then releasing the ligand upon its cleavage. This diffusion-enhancing function allows for localized BMP release without signaling response in the intervening space (Shilo et al. 2013). Importantly, these protein-protein interactions have been fully reconstituted in cell culture, allowing direct control over the length, shape, and peak amplitude location of spatially graded BMP signals (Zhu et al. 2023). This work demonstrates that shuttler expression is a highly engineerable system for directly tuning BMP-induced tissue patterning. In the case of Hedgehog family morphogens, another extracellular diffusible protein, Scube, promotes both the secretion and diffusion of Hedgehog (Collins et al. 2023; Schlissel et al. 2024). Scube performs this function by acting as a catalyst to accelerate the transition of Hedgehog molecules between membrane-confined and freely diffusing populations (Schlissel et al. 2024). The modular nature of this mode of regulation, and its recurrence in many contexts, suggests that morphogen pathways may be generally tunable using shuttling proteins. It may even be possible to develop artificial shuttling protein circuits with novel desired properties to modulate the spatiotemporal dynamics of natural or synthetic signaling ligands.

Conceptually similar, though biochemically different, are the abilities of the membrane-tethered or insoluble components of the extracellular matrix (ECM) to modulate morphogen movement (Hynes 2009). Unlike secreted modulators, these ECM components have been indicated to interact with multiple morphogens promiscuously, making them potentially “global modulators” of tissue patterning. For example, glypicans, membrane-anchored heparan-sulfate proteoglycans, are required for signaling in many developmental pathways. They have been implicated in regulating the transfer of ligands, such as Wnt and Hedgehog, between cells (B. Hu et al. 2021; McGough et al. 2020; Gude et al. 2023). Additionally, glypican has been recently demonstrated to serve as an essential coreceptor for Wnt signaling to directly facilitate intestinal organoid development (de Almeida Magalhaes et al. 2024). Given the importance of this cell-surface-anchored protein family, engineered control of ECM proteins may provide a significant improvement in signaling regulation compared to ligand transmission through either an uncharacterized mixture like Matrigel or a simplified hydrogel substrate. Better understanding of the biophysical movements that generate ECM-mediated morphogen spread will allow for precise tuning of the signal-conveying properties of an engineered tissue’s extracellular space.

The regulatory principles of natural morphogens also offer inspirations for ways to control bioorthogonal ligands. There is an emerging interest in the use of GFP as a morphogen, activating engineered GFP-binding receptors, and therefore generating a crosstalk-free ligand with the patterning abilities of any natural signaling pathway (Stapornwongkul et al. 2020; Toda et al. 2020). However, initial work has found GFP diffuses too quickly in the extracellular space to form gradients of biological lengthscales (Toda et al. 2020). Therefore, it is necessary to introduce additional constraints to regulate GFP diffusion, such as cell-surface binders or a hampering agarose layer (Stapornwongkul et al. 2020; Toda et al. 2020).

Adding new pathways to the toolkit: engineering cell polarity

Cell polarity at the single-cell level regulates cell shape, growth, and fate adoption, whereas coordinated polarity at the tissue level over mm scales is critical for tissue morphogenesis and function. For example, asymmetric cell divisions are essential for stem cell maintenance, ensuring that one daughter remains in contact with the stem cell niche while one leaves and differentiates (Yadlapalli and Yamashita 2012). Apical-basal (AB) polarity is necessary for epithelial layer integrity and signaling, while the planar cell polarity (PCP) pathway orients cells within the plane of a tissue to pattern functional structures like airway cilia and inner ear sensory hairs (Butler and Wallingford 2017; Bryant and Mostov 2008.) While polarity-capable cell lines can mimic in vivo AB polarization to varying degrees, complete polarization typically requires culture conditions that limit the downstream applications or coculture possibility (Bryant and Mostov 2008; Hagelaars et al. 2022). Epithelial organoids can fully apicobasally polarize, but often in stereotyped apical-in conformations, limiting access for measurements and manipulations (Taelman, Diaz, and Guiu 2022; Pianigiani and Roccio 2024). It is not currently clear to what extent apicobasal polarity in culture affects PCP, which is largely absent in cultured cells.

Some advances have been made in engineering cell polarity. Landmark studies in budding yeast, which represent the early phase of the synthetic biology field, revealed fundamental principles of polarity emergence (Chau et al. 2012). Recent work on inducible polarity protein oligomerization shows a promising angle for engineered polarity in mammalian cells (Watson et al. 2023). To create single-cell polarity, cells were engineered to express a transmembrane protein that binds intracellularly to a cytosolic Par protein, and extracellularly to a linker protein that clusters the transmembrane proteins into networks (Watson et al. 2023). Par proteins self-organize into a single domain that then recruits dynein and reorients the mitotic spindle (Goldstein and Macara 2007). During mitosis, addition of this linker protein leads to the formation of an asymmetrically localized polarity domain that reorients cell division. This work demonstrates that the positive feedback loop created by Par protein clustering is sufficient to generate a single confined domain of asymmetry and recruit downstream cell division regulators. This module is cell-type-agnostic and reversible by controlled addition of the linker protein (Watson et al. 2023). This module could be combined with other methods of inducing oriented division, such as patterned surfaces or subcellular optogenetic stimulation (Okumura et al. 2018; Théry et al. 2007), or with spatially restricted expression of the linker, to predetermine the angle of the division axis. Oligomerization of multiple intracellular targets could enforce asymmetric inheritance of particular transcription factors. Finally, Par proteins also organize the AB polarity pathway, establishing a distinct “top” and “bottom” within epithelial and endothelial cell layers (Rodriguez-Boulan and Macara 2014). If used in epithelial cells, it is possible that this method could induce both apicobasal polarization and cell divisions, taking advantage of the multiple polarity pathways downstream of Par proteins.

Although polarity within a single cell has been successfully engineered, engineering coordinated polarity among many cells, such as PCP, has proven challenging. The signals that align intracellular planar asymmetry across all the cells in a tissue are still not fully understood, and appear to vary between tissues (Aw and Devenport 2017). Organoids modeling PCP-organized tissues, such as the sensory hair cells of the inner ear, fail to recreate cellular asymmetries (Pianigiani and Roccio 2024), and culture of primary cells that undergo PCP in vivo fail to show tissue-scale coordination (Heck 2018; Vladar et al. 2012). Reconstitutions in both the “core” planar polarity pathway and the Fat-Dachsous PCP pathway have helped to uncover the logic of polarity specification over 1–2 cell junctions, but have yet to identify a circuit capable of long-range patterning in vitro (Strutt et al. 2023;Loza et al. 2017). These limited successes suggest that generating planar polarized tissues might require additional mechanisms that can propagate the asymmetry over long distances.

Conclusion

In this review, we highlight recent progress in engineering native cell-cell interactions as part of synthetic reconstitution studies of developmental biology. The approaches we review here, rooted in engineered endogenous signal-regulatory components, are only a subset of the synthetic biology tools bridging developmental biology knowledge and biomedical tissue engineering. Fully synthetic ligand-receptor systems such as synNotch, optogenetic activation of patterning, and the use of mechanical and physical constraints have been previously reviewed for their own strengths and limitations (McNamara et al. 2023; Trentesaux et al. 2023). We focus here on engineering of natural patterning pathways due to the immense precision and amazing tunability which these pathways achieve in vivo, as well as the rich cellular phenotypes these pathways can directly regulate, relative to the simpler responses currently achieved by fully orthogonal signaling. The resulting patterns are simple enough to be reconstituted with only a small number of proteins, and to be described through mathematically tractable descriptions, yet are repurposed to achieve very different outcomes across tissues.

As we discuss, while some patterning modules are ready for use as engineering tools, there are still significant steps remaining in the development of these methods. Currently, many existing signaling modules fail to incorporate critical in vivo feedback loops, and more protein components may be needed to generate them. It will also be important to expand on the capability to interface multiple patterning modules together to achieve greater complexity and robustness. At the moment, this capacity is significantly limited by the efficiency and toxicity of engineering many genetic circuits into a single cell line, especially the primary cell types used in medical applications. Advances in genome engineering technology will be a vital part of making these modules practical tools for engineering. While further development is needed, the modularity and precision with which developmental circuits operate shows great promise far beyond their original tissue contexts, and we urge tissue engineers to take advantage of and help develop these toolkits for any application which would benefit from fine spatial control.

Acknowledgements:

This work was supported by National Institute of Health grants DP2HD108777 (PL), Allen Distinguished Investigator Award, a Paul G. Allen Frontiers Group advised grant of the Paul G. Allen Family Foundation (PL), and National Science Foundation GRFP (LAW).

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