Guiding development using self-assembling cells

Abstract

Provided herein is a composition of cells that can self-assemble into a pre-determined architecture. In some embodiments, the composition may comprise: a population of progenitor cells and one or more populations of organizer cells that are engineered to express one or more cell adhesion molecules that mediate cell-cell interactions with the progenitor cells. In these embodiments, the progenitor cells and organizer cells self-assemble into an aggregate that has a pre-defined architecture. In some cases, at least one of the one or more populations of organizer cells may produce a morphogen, e.g., a diffusible molecule that secreted or released by one or more of the populations of organizer cells, that causes the progenitor cells to differentiate.

Description

[0001] Atty. Dkt: UCSF-753WO Client Ref.: SF-2024-020-2-PCT GUIDINGDEVELOPMENTUSINGSELF-ASSEMBLINGCELLSCROSS-REFERENCING This application claims the benefit of U.S. provisional application serial no. 63 / 537,453 filed on September 8, 2023, which application is incorporated by reference herein. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH This invention was made with government support under grant nos. RO1 DK130969 and R35 DE026602 awarded by The National Institutes of Health, and DBI-1548297 awarded by the National Science Foundation. The government has certain rights in the invention. INTRODUCTION Embryonic development is a complex yet highly stereotyped process. The reconstitution of development in vitro, however, still lacks much of this precision and reproducibility. A key feature missing in many in vitro developmental systems are spatially asymmetric instructive microenvironmental signals, such as those normally provided by natural developmental niches. In vivo niches usually contain developmental organizers (also referred to as signaling centers) – groups of cells present at a specific location in the developing embryo which produce highly directional instructive morphogen gradients that initiate and guide the development of neighboring embryonic cells. In contrast, common differentiation protocols that rely on morphogens provided isotropically in the media, are missing such spatial information. Microdevices, beads, and optogentic or chemogenetic induction systems provide ways in spatially control signals, but often come at a cost of decreased ease, throughput and reproducibility. This disclosure provides a solution to this problem. SUMMARY Recapitulation of tissue development in vitro relies primarily on treatment of progenitor cells with media-borne morphogens, and lacks the complex spatial instructions normally presented by native developmental niches. Here it is demonstrated that spatially asymmetric developmental niches can be created by engineering synthetic organizer cells programmed to Atty. Dkt: UCSF-753WO Client Ref.: SF-2024-020-2-PCT self-assemble around progenitor cells and provide spatially-defined morphogenic instructions. A toolkit of well-defined natural and engineered adhesion molecules was used to create diverse organizer cell geometries around the progenitor cells, allowing systematic generation of morphogen gradients with tunable position, amplitude, and signal types. As a test case, synthetic niches presenting the antagonistic morphogens Wnt3a and DKK1 were used to guide early embryonic development of pluripotent stem cells. Depending on the designed niche, various distinct morphological outcomes were observed, including formation of embryoids with a beating heart-like chamber and a diverse complement of cell lineages derived from mesoderm, endoderm and ectoderm. The resulting morphologies were reproducible in high-throughput 96 well format. Spatially programmable synthetic organizer cells thus provide a systematic approach to recapitulate, modify or study development in a controlled fashion. The concept that underlies certain aspects of this disclosure is that organizer cell adhesion properties can be engineered by expression of ‘new’ cell adhesion molecules (i.e., cell adhesion molecules that are not usually expressed in those cells, or synthetic cell adhesion molecules), which, in turn, determine the organizer cells self-assemble relative to the progenitor cells, and the other organizer cells. A composition of cells that can self-assemble into a pre-determined architecture is therefore provided. In some embodiments, the composition may comprise: a) a population of progenitor cells; and (b) a population of first organizer cells that are engineered to express one or more cell adhesion molecules that mediate cell-cell interactions with the progenitor cells. In these embodiments, the progenitor cells and organizer cells self-assemble into an aggregate that has a pre-defined architecture. In some embodiments, the first organizer cells may be additionally engineered to express one or more cell adhesion molecules that mediate cell-cell interactions between themselves (i.e., between the first organizer cells). In any embodiment, the composition may further comprise: (c) a population of second organizer cells, wherein the second population of organizer cells are engineered to express one or more cell adhesion molecules that recognizes a target on the surface of the progenitor cells and are additionally engineered to express one or more cell adhesion molecules that mediate interactions between the first organizer cells. By way of example, the aggregate may comprise (i) a cluster of progenitor cells, and (ii) one or more structures formed by the organizer cells, where the one or more structures Atty. Dkt: UCSF-753WO Client Ref.: SF-2024-020-2-PCT comprise: a shell that at least partially surrounds the cluster of progenitor cells, and / or one or more nodes that are attached to the progenitor cells. In some embodiments, the first organizer cells may produce a morphogen that causes the progenitor cells to differentiate. In some embodiments, the morphogen may be a diffusible molecule that is secreted or released by one or more of the populations of organizer cells, such as a member of the Wnt / beta-catenin, a member of the TGF-β (Transforming growth factor β) / BMP (Bone morphogenetic protein) family, a member of the Shh (sonic hedgehog) family, or retinoic acid or an analog thereof. In other embodiments, the morphogen may a juxtracrine signaling molecule, e.g., Notch, ephrin, semaphorin, a T-Cell Receptor (TCR) or a ligand thereof (e.g., Delta, Jagged, or Serrate, etc.). In some embodiments, the morphogen will form a concentration gradient across the cluster of progenitor cells. Also provided is a method for producing a cell aggregate. In some embodiments, the method may comprise incubating the composition of cells summarized in a medium under conditions under which the cells self-assemble into the aggregate. As noted above, in some embodiments the organizer cells expresses a morphogen. In these embodiments, the method may further comprise incubating the assembly in a medium, thereby causing at least one of the progenitor cells to differentiate. A cell aggregate is also provided. In some embodiments, this aggregate may comprise: (a) a cluster of progenitor cells and (b) one or more structures composed of organizer cells that are engineered to express one or more cell adhesion molecules that mediate cell-cell interactions with the progenitor cells. The aggregate is held together by cell-cell interactions. In some embodiments, the one or more structures may comprise: (i) a shell that at least partially surrounds the cluster of progenitor cells, and / or (ii) one or more nodes that are attached to the cluster of progenitor cells. In some cases, at least one of the one or more populations of organizer cells may express a morphogen. In these embodiments, the morphogen may cause differentiation of one or more of the progenitor cells in the cluster. A toolkit of synthetic cell adhesion molecules with tunable specificities and strengths has been developed. In this disclosure, the cell adhesion toolkit has been used engineer “organizer” cells that can self-assemble around progenitor cells in a wide variety of pre- determined, user-customizable architectures. These architectures can be harnessed to express morphogens in specific patterns (e.g., from one direction only, or in two opposing directions). Atty. Dkt: UCSF-753WO Client Ref.: SF-2024-020-2-PCT These cells provide a systematic, high-throughput, and highly reproducible approach to reconstitute, study and precisely modulate in vitro development. BRIEFDESCRIPTIONOFTHEDRAWINGSThe skilled artisan will understand that the drawings, described below, are for illustration purposes only. The drawings are not intended to limit the scope of the present teachings in any way. Fig. 1 shows the basic concepts of how organizer cells can guide cell development (last panel) relative to in vivo (first panel) and prior in vitro methods (middle panel). Figs. 2A-2E show how engineered cell adhesion interactions can be used to construct synthetic self-assembling organizers with diverse anisotropic geometries. Figs. 3A-3C show the spatial, amplitude and temporal control of a Wnt3a morphogen secreted from single node synthetic organizer. Figs. 4A-4E show a single Wnt3a node that consistently yields development of beating heart-like structure. Figs.5A-5D show a single Wnt3a node-generated structure that has myocardial, vascular, and hematopoetic lineages. Figs. 6A-6D show that multiple opposing synthetic organizers (Wnt3a vs DKK1) can reshape morphogen gradients and resulting cell lineages. Figs. 7A-7C show that multiple opposing synthetic organizers (Wnt3a vs DKK1) induce development with increased cell lineage diversity, yielding structures containing both cardiac and neuroectoderm cells. DEFINITIONSAs used herein, the terms "treatment," "treating," “treat” and the like, refer to obtaining a desired pharmacologic and / or physiologic effect and / or a response related to the treatment. The effect can be prophylactic in terms of completely or partially preventing a disease or symptom Atty. Dkt: UCSF-753WO Client Ref.: SF-2024-020-2-PCT thereof and / or can be therapeutic in terms of a partial or complete cure for a disease and / or adverse effect attributable to the disease. "Treatment," as used herein, covers any treatment of a disease in a mammal, particularly in a human, and includes: (a) preventing the disease from occurring in a subject which can be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing regression of the disease. A “therapeutically effective amount” or “efficacious amount” refers to the amount of an agent (including biologic agents, such as cells), or combined amounts of two agents, that, when administered to a mammal or other subject for treating a disease, is sufficient to effect such treatment for the disease. The “therapeutically effective amount” will vary depending on the agent(s), the disease and its severity and the age, weight, etc., of the subject to be treated. The terms “individual,” “subject,” “host,” and “patient,” used interchangeably herein, refer to a mammal, including, but not limited to, murines (e.g., rats, mice), non-human primates, humans, canines, felines, ungulates (e.g., equines, bovines, ovines, porcines, caprines), lagomorphs, etc. In some cases, the individual is a human. In some cases, the individual is a non-human primate. In some cases, the individual is a rodent, e.g., a rat or a mouse. In some cases, the individual is a lagomorph, e.g., a rabbit. The terms “synthetic”, “chimeric” and “engineered” as used herein generally refer to artificially derived polypeptides or polypeptide encoding nucleic acids that are not naturally occurring. Synthetic polypeptides and / or nucleic acids may be assembled de novo from basic subunits including, e.g., single amino acids, single nucleotides, etc., or may be derived from pre- existing polypeptides or polynucleotides, whether naturally or artificially derived, e.g., as through recombinant methods. Chimeric and engineered polypeptides or polypeptide encoding nucleic acids will generally be constructed by the combination, joining or fusing of two or more different polypeptides or polypeptide encoding nucleic acids or polypeptide domains or polypeptide domain encoding nucleic acids. Chimeric and engineered polypeptides or polypeptide encoding nucleic acids include where two or more polypeptide or nucleic acid “parts” that are joined are derived from different proteins (or nucleic acids that encode different proteins) as well as where the joined parts include different regions of the same protein (or nucleic acid encoding a protein) but the parts are joined in a way that does not occur naturally. Atty. Dkt: UCSF-753WO Client Ref.: SF-2024-020-2-PCT The term "recombinant", as used herein describes a nucleic acid molecule, e.g., a polynucleotide of genomic, cDNA, viral, semisynthetic, and / or synthetic origin, which, by virtue of its origin or manipulation, is not associated with all or a portion of the polynucleotide sequences with which it is associated in nature. The term recombinant as used with respect to a protein or polypeptide means a polypeptide produced by expression from a recombinant polynucleotide. The term recombinant as used with respect to a host cell or a virus means a host cell or virus into which a recombinant polynucleotide has been introduced. Recombinant is also used herein to refer to, with reference to material (e.g., a cell, a nucleic acid, a protein, or a vector) that the material has been modified by the introduction of a heterologous material (e.g., a cell, a nucleic acid, a protein, or a vector). The term “operably linked” refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. For instance, a promoter is operably linked to a coding sequence if the promoter affects its transcription or expression. Operably linked nucleic acid sequences may but need not necessarily be adjacent. For example, in some instances a coding sequence operably linked to a promoter may be adjacent to the promoter. In some instances, a coding sequence operably linked to a promoter may be separated by one or more intervening sequences, including coding and non-coding sequences. Also, in some instances, more than two sequences may be operably linked including but not limited to e.g., where two or more coding sequences are operably linked to a single promoter. The terms “polynucleotide” and “nucleic acid,” used interchangeably herein, refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, this term includes, but is not limited to, single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. The terms “polypeptide,” “peptide,” and “protein”, used interchangeably herein, refer to a polymeric form of amino acids of any length, which can include genetically coded and non- genetically coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. The term includes fusion proteins, including, but not limited to, fusion proteins with a heterologous amino acid sequence, fusions Atty. Dkt: UCSF-753WO Client Ref.: SF-2024-020-2-PCT with heterologous and homologous leader sequences, with or without N-terminal methionine residues; immunologically tagged proteins; and the like. A "vector" or "expression vector" is a replicon, such as plasmid, phage, virus, or cosmid, to which another DNA segment, i.e. an "insert", may be attached so as to bring about the replication of the attached segment in a cell. The term “heterologous”, as used herein, means a nucleotide or polypeptide sequence that is not found in the native (e.g., naturally-occurring) nucleic acid or protein, respectively. Heterologous nucleic acids or polypeptide may be derived from a different species as the organism or cell within which the nucleic acid or polypeptide is present or is expressed. Accordingly, a heterologous nucleic acids or polypeptide is generally of unlike evolutionary origin as compared to the cell or organism in which it resides. The term “cell adhesion molecules that mediate cell-cell interactions” is intended to refer to cell adhesion molecules recognize (i.e., bind to) a target on the surface of another cell and, upon binding, can engage with and apply local control over the cytoskeleton, e.g., to reinforce the linkage and / or locally control cytoskeletal filament polymerization. These molecules will be described in greater detail below. The term “cells that are engineered to express a cell adhesion molecule” is meant cells that express a cell adhesion molecule that is ‘new’ to that cell (i.e., not expressed in non- recombinant versions of the same cell). The cell adhesion molecule may itself by wild-type but it may be under the control of different promoter compared to the endogenous gene that encodes the protein. In these embodiments, the cell adhesion molecule may be ‘ectopically’ expressed by the cells (where the cells would not usually express the cell adhesion molecule without the recombinant nucleic acid). In other embodiments, the cell adhesion molecule may be engineered, i.e., non-natural, synthetic or recombinant, which molecules are described in greater detail elsewhere in this disclosure. As would be understood, cells that are engineered to express a cell adhesion molecule contain a recombinant nucleic acid, which nucleic acid contains a coding sequence for the cell adhesion molecule operably linked to a promoter, where the promoter and coding sequence are not linked to one another in nature, either because the promoter is not natural, the coding sequence is not natural, both the promoter and coding sequence are not natural, or the promoter is not operably linked to the coding sequence in a wild type cell. Atty. Dkt: UCSF-753WO Client Ref.: SF-2024-020-2-PCT Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims. Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. It must be noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a cell” includes a plurality of such cells and reference to “the cell” includes reference to one or more cells and equivalents thereof known to those skilled in the art, and so forth. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation. It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in Atty. Dkt: UCSF-753WO Client Ref.: SF-2024-020-2-PCT the context of a single embodiment, may also be provided separately or in any suitable sub- combination. All combinations of the embodiments pertaining to the invention are specifically embraced by the present invention and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations of the various embodiments and elements thereof are also specifically embraced by the present invention and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed. DETAILEDDESCRIPTIONAs summarized above, this disclosure provides a composition of cells. In some embodiments, the composition may comprise: a) a population of progenitor cells; and (b) a population of first organizer cells that are engineered to express one or more cell adhesion molecules that mediate cell-cell interactions with the progenitor cells, i.e., one or more cell adhesion molecules that recognizes a target on the surface of the progenitor cells and engage with the cytoskeleton. The cell-cell interactions of these cells can be programmed such that the progenitor cells and organizer cells self-assemble into an aggregate that has a pre-defined architecture, where the term ‘aggregate’ is intended to mean that the cells adhere to one another via a combination of homotypic and heterotypic cell-cell interactions (which interactions may be natural, synthetic or any combination thereof) that program the architecture of the aggregate. In some cases, at least one of the populations of organizer cells may produce a morphogen, e.g., a diffusible molecule that secreted or released by one or more of the populations of organizer cells, that causes the progenitor cells to differentiate. In some embodiments, the aggregate may comprise a cluster of progenitor cells and one or more structures formed by the organizer cells, where the term ‘cluster’ is intended to mean that the progenitor cells are clumped together via homotypic cell-cell interactions. The cell-cell interactions that cause the progenitor cells to clumped may be mediated by endogenous (i.e., Atty. Dkt: UCSF-753WO Client Ref.: SF-2024-020-2-PCT wild type) cell adhesion molecules (because progenitor cells can clump together naturally) or, in some cases, engineered cell adhesion molecules. Therefore, in some embodiments, the progenitor cells may be non-recombinant (i.e., wild type), where the cell adhesion molecules that mediate homotypic interactions between the cells are naturally expressed by the cells. In other embodiments, the progenitor cells may be recombinant and may express a cell adhesion molecule (a wild type or engineered cell adhesion molecule) to facilitate homotypic interactions between the cells. In some embodiments, the progenitor cells may also express a target protein to which the organizer cells can bind. The structures formed by the organizer cells on the cluster of progenitor cells can be customized by customizing the cell-cell interactions of organizer cells using engineered cell adhesion molecules. For example, the organizer cells may also express cell adhesion molecules that mediate their self-clustering and / or interactions with another organizer cell population. In principle, the organizer cells can be engineered to have homotypic interactions, heterotypic interactions; or more than one type of cell adhesion interaction, In addition, organizer cells could also comprise multiple organizer cell types that either segregate from each other (i.e., do not recognize each other), homotypically adhere to each other (which allow organizer cell types to cluster), or heterotypically adhere to each other. The strength of these interactions can also be tailored to a desired end, where strong homotypic interactions are likely to produce clusters, weak interactions heterotypic interactions are more likely to produce shells, and a mixtures of cells that have heterotypic interactions of the same strength may result in a mosaic. These interactions determine how the cells self-assemble which, in turn, determines which structures and architecture will be produced. Such structures include, without limitation, a shell that at least partially surrounds the cluster of progenitor cells, a node (i.e., a three-dimensional mass of organizer cells) that are attached to the progenitor cells, a sandwich (multiple layers of organizer cells) that surround the progenitor cells, multiple nodes (e.g., two or three nodes) that are each attached to the progenitor cells or end-to end (i.e., in a string), etc. In some embodiments, the one or more structures formed by the organizer cells may comprise: a shell that at least partially surrounds the cluster of progenitor cells, and / or one or more nodes that are attached to the progenitor cells. As would be apparent, the architectures programmed by these interactions are numerous and Atty. Dkt: UCSF-753WO Client Ref.: SF-2024-020-2-PCT include other architecture in addition to the simple architectures that have been reduced to practice below. As demonstrated in the examples section of this disclosure, in some embodiments, the progenitor and organizer cells in the composition may be in the form of single cells in a cell suspension. In these embodiments, the cells self-assemble into an aggregate that has the desired architecture. In other embodiments, the cluster of progenitor cells may be pre-made before it is combined with the organizer cells. In these embodiments, the composition may comprise a cluster of progenitor cells and a suspension of organizer cells or a pre-made node of organizer cells, as desired. In these embodiments, the suspension of organizer cells may form a shell on the cluster of progenitor cells or the pre-made node of organizer cells may adhere to the cluster of progenitor cells. As such, in some embodiments, the composition may comprise a pre-made cluster of the progenitor cells and / or one or more pre-made nodes made up of the organizer cells. As noted above, at least one of the one or more populations of organizer cells may produce a morphogen, i.e., a cell fate regulator. In these embodiments, the morphogen causes one or more of the progenitor cells to differentiate (i.e., change identity) after the cells have self- assembled into the aggregate. The morphogen may be a diffusible molecule that is released by the organizer cells or a juxtracrine signaling molecule that is expressed on the surface of the organizer cells. If the morphogen is diffusible, then it may emanate from a restricted region of the aggregate and spread away from their source to form a concentration gradient. As the fate of each cell in the field depends on the concentration of the morphogen signal, the gradient may configure a pattern of development. In these embodiments, the morphogen may be a diffusible molecule that secreted or released by one or more of the populations of organizer cells. The morphogen may form a gradient. In some embodiments, the morphogen may be a member of the Wnt / beta-catenin, a member of the TGF-β (Transforming growth factor β) / BMP (Bone morphogenetic protein) family, a member of the Shh (Sonic hedgehog) family, or retinoic acid (or an analog thereof). Examples of morphogens are listed in Table 1 below: Wnt family (Wnt, Wnt2a, Wnt2b, Wnt3a, Wnt5a, Wnt10 etc.) Atty. Dkt: UCSF-753WO Client Ref.: SF-2024-020-2-PCT BMP antagonists: Noggin Activin A B C er of progenitor cells and at least two nodes of organizer cells that are on opposite sides of the cluster. In these embodiments, one node may produce a morphogen while the other node may produce an antagonist of the morphogen, thereby providing an effective gradient of the morphogen. In other embodiments, the morphogen may be a juxtracrine signaling molecule such as Notch, ephrin, semaphorin, a T-Cell Receptor (TCR) or a ligand thereof (e.g., Delta, Jagged, or Serrate, etc.). In any embodiment, the morphogen may be constitutively expressed or inducible (e.g., by an exogenous chemical). Inducible embodiments allow one to control expression of the morphogen temporally. In some embodiments, the morphogen may only be expressed when the organizer cells make physical contact with another cell, e.g., a progenitor cell. This can be accomplished using an engineered protease-mediated receptor (see, e.g., Morsut et al Cell 2016 164: 780–791 and Zhu et al Cell 2022185: 1431–1443, among others), for example. In some embodiments, the cell composition may comprise: (a) a population of progenitor cells that express a cell adhesion molecule that recognizes a target on the progenitor cells and causes the progenitor cells to form a cluster via homotypic cell-cell interactions; and (b) a Atty. Dkt: UCSF-753WO Client Ref.: SF-2024-020-2-PCT population of organizer cells that: (i) express a cell adhesion molecule that recognizes a target on the surface of the progenitor cells and causes the organizer cells to adhere to the cluster of progenitor cells via heterotypic cell-cell interactions; and either: (ii) additionally express a cell adhesion molecule that recognizes a target on the surface of the organizer cells and causes the organizer cells to form a node via homotypic cell-cell interactions; or (iii) do not additionally express a cell adhesion molecule that recognizes a target on the surface of the organizer cells, thereby causing the first cells to form a shell around the progenitor cells. In some embodiments, the cell composition may comprise: (a) a population of progenitor cells that express a cell adhesion molecule that recognizes a target on the progenitor cells and causes the progenitor cells to form a cluster via homotypic cell-cell interactions; and (b) a population of first organizer cells that: (i) express a cell adhesion molecule that recognizes a target on the surface of the progenitor cells and causes the first organizer cells to adhere to the cluster of progenitor cells via heterotypic cell-cell interactions; and (ii) additionally express a cell adhesion molecule that recognizes a target on the surface of the first organizer cells and causes the first organizer cells to form a node via homotypic cell-cell interactions; or (c) a population of second organizer cells that: (i) express a cell adhesion molecule that recognizes a target on the surface of the progenitor cells and causes the second organizer cells to adhere to the cluster of progenitor cells via heterotypic cell-cell interactions; and (ii) do not express a cell adhesion molecule that recognizes a target on the surface of the second organizer cells, thereby allowing the first cells to form a shell around the progenitor cells. As noted above, at least one of the cell adhesion molecules of the progenitor cells, first cells and / or the second cells is an engineered cell adhesion molecule. engineered cell adhesion molecules are described in WO2022094416 and Stevens et al (Nature 2023614: 144–152), which are incorporated by reference herein for disclosure of such molecules. Engineered cell adhesion molecules generally comprises three domains: an extracellular binding domain, one or more transmembrane domains and an intracellular domain, where the wherein the extracellular binding domain and the intracellular binding domain of the fusion protein are “heterologous” i.e., not from the same native cell adhesion molecule. For example, the extracellular binding domain may comprise the antigen-binding domain of an antibody (e.g., a scFv or nanobody) or a dimerization domain, for example. In some cases, the target for the engineered cell adhesion Atty. Dkt: UCSF-753WO Client Ref.: SF-2024-020-2-PCT molecule on the surface another cell may be endogenous to the cell or added to the other cell by recombinant means. The intracellular domain of the engineered cell adhesion molecule, in the other hand, may be the intracellular domain of a naturally-occurring cell adhesion molecule or a variant thereof that retains its ability to engage with and apply local control over the cytoskeleton, e.g., to reinforce the linkage and / or locally control cytoskeletal filament polymerization. Thus, the intracellular domain of the cell adhesion molecule is capable of signaling to and reorganizing the cytoskeleton of the cell upon specific binding of the first binding moiety of the cell adhesion molecule to a second binding moiety. Because signaling of many cell adhesion molecules is triggered by physical force (e.g., a force that “tugs” at the molecule), the second binding moiety that activates signaling should be at least partially immobilized or tethered, i.e., not in solution. For example, the second binding moiety that triggers signaling may be on the surface of another cell or tissue scaffold, or tethered to another cell or tissue scaffold, for example. The engineered cell adhesion molecule also contains one or more transmembrane domains (which should be in between the extracellular and intracellular domains). In some embodiments, e.g., the engineered cell adhesion molecule may have a single transmembrane domain. In other embodiments, the engineered cell adhesion molecule may have multiple transmembrane domains. The transmembrane domain of an engineered cell adhesion molecule can be the transmembrane domain of a naturally-occurring cell adhesion molecule (e.g., the same naturally-occurring cell adhesion molecule as the intracellular domain). However, this is not necessary because the transmembrane domain can be readily designed using hydrophobic amino acids or a transmembrane domain from another transmembrane protein can be used. As would be apparent, the fusion protein may have other sequences, e.g., linkers, effector domains, signaling domains, etc., in addition to the domains that are specifically described herein. In addition to embodiments in which the cells adhere to each other directly, the cells may also adhere to each other directly via a soluble bridging molecule, e.g., a soluble protein, to which two engineered cell adhesion molecules bind. In these embodiments, the engineered cell adhesion molecules on the different cells may bind to different sites (e.g., epitopes) on the soluble bridging molecule. How cell adhesion proteins engage with and apply local control over the cytoskeleton, e.g., to reinforce the linkage and / or and recruit additional cytoskeleton to the area has been well Atty. Dkt: UCSF-753WO Client Ref.: SF-2024-020-2-PCT studied. For example, Harburger et al (Journal of Cell Science 2009122: 159-163) provides a detailed review of the mechanism by which integrins are able to signal from extracellular binding to cytoskeletal reorganization; Liu et al (Journal of Cell Science 2000113: 3563-3571) describes the structure / function relationship of integrins, particularly details the known binding partners of the intracellular sequence of integrins; Hoffman et al (Quant. Cell Biology 201525: 803-814) describes the mechanism of cadherin intracellular signaling and how it is mechanically regulated; Beutel et al (Cell 2019179: 923-936) describes the phase-separation-based mechanism by which JAMs are able to recruit the scaffold protein ZO-1 and signal; and Lawson et al (Pharmacol Rep. 200961: 22-32) describes the intracellular signaling pathways that are activated by ICAM signaling. It is noted that some cadherin / integrin and protocadherin / e- cadherin chimeras retain their ability to signal (Geiger et al J. Cell Science 1992103, 943-951 and Obato et al J. Cell Science 1995108: 3765-3773). In any embodiment, the intracellular domain may be selected from Table 1 of WO2022094416, where the amino acid sequence of the intracellular domain may be at least 80%, at least 90%, at least 95% or identical to a human sequence found in that table. In these embodiments, the intracellular domain may be an intracellular domain of a cell adhesion molecule selected from Table 1 of WO2022094416, or a variant thereof that retains the ability to engage with the cytoskeleton The sequences of hundreds of wild-type human cell adhesion molecules have been deposited into the Genbank database. The intracellular and intracellular domains of these proteins should be readily identified because the transmembrane domain is straightforward to identify in these molecules. Many mammalian orthologs of these proteins exist. Exemplary families of intracellular domains from wild-type cell adhesion molecules are set forth below: 1. Cadherins (e.g. CDH1, CDH2….CDH29) (i.e. CDH 1 through 29) 2. Cadherin related family-CDHR (CDHR1-16) 3. Desmoglein (DSG1, DSG2, DSG3, DSG4) 4. Desmocollin (DSC1, DSC2, DSC3) 5. Integrin beta chain (e.g. ITGB1, ITGB2…, ITGB8) 6. Integrin alpha chain (e.g. ITGA1, ITGA2…ITGA11, ITGAE) 7. Junction Adhesion molecules (e.g. JAMA, JAMB, JAMC, JAML Atty. Dkt: UCSF-753WO Client Ref.: SF-2024-020-2-PCT 8. CXADR 9. Protocadherins (e.g. pcdha1, pcdhb1, pcdhg1) 10. Immunoglobulin adhesion molecules (e.g. ICAM1…ICAM5, VCAM1, MAdCAM-1, NCAM1..NCAM-2, CD2, CD58, CD48, CD150, ALCAM, CD96, CD226, CD229, NRCAM, PVR, CD200, BCAM, PECAM1, MPZL2, MAG) 11. IGSFs (IGSF1-IGSF6, IGSF8, IGSF9, IGSF11-IGSF13, IGSF16, IGSF23) 12. CD44 13. Claudins (e.g. CLDN1, CLDN2..CLDN25) 14. Syndecan (SDC1, SDC2, SDC3, SDC4) 15. Occludin 16. Ephrins (e.g. EFNA, EFNB) 17. Eph receptors ( EPHA, EPHB) 18. Nephrin 19. Neurexin (NRXN1, NRXN2, NRXN3) 20 . Neuroligin (NLGN1, NLGN2, NLGN3, NLGN4X) 21. Contactin associated protein (CNTNAP1, CNTNAP2, CNTNAP3, CNTNAP4, CNTNAP5, CNTNAP3B) 22. Notch ligands (e.g. DLL1, JAG1) 23. Selectins (e.g. E-selectin, P-selectin, L-selectin, SELPLG) 24. SELPLG (PSGL-1) 25. Nck 26. DSCAM, DSCAML1 27. Nectin 28. CD4, CD34 29. EPCAM, Trop2, ESAM 30. AMIGO1, AMIGO2, AMIGO3 31. SMAGP 32. CEACAM1-CEACAM21, CERCAM 33. Lectins (CD209, SIGLEC1-SIGLEC15, CLECL1, MRC1, MRC2 34. SLAMF1-9 35. MCAM Atty. Dkt: UCSF-753WO Client Ref.: SF-2024-020-2-PCT 36. TENM1-4 37. HEPACAM 38. CD99, CD99L2 39. FLRT, FLRT2, FLRT3 40. L1 family (NFASC, CHL1, L1CAM, BOC) 41. BVES 42. NPTN 43. Netrin receptor (DCC, UNC5A, UNC5B, UNC5C, UNC5D) 44. PTPRM 45. ASTN1 46. CD47, SIRPA 47. CD93 48. CD99 49. Plexin family (plxna1-4, plxnb1-3, plxnd1) 50. SCARF1, SCARF2 51. CD22 52. Leukocyte immunoglobulin like receptor (LILRA1, LILRA2, LILRA3, LILRA4, LILRA5, LILRA6, LILRB1, LILRB2, LILRB3, LILRB4, LILRB5) 53. CD302 54. FGFRL1 55. Leucine rich repeat neuronal (LRRN1, LRRN2, LRRN3, LRRN4, LRRN4CL) 56. Sperm acrosome associated (SPACA1-9) 57. Calsyntenin (CLSTN1, CLSTN2, CLSTN3) 58. Ninjurin (NINJ1, NINJ2) 59. DGCR2 60. Endoglin 61. FGFRL1 62. Mucins 63. NckAP1, NckAP1L 64. ZAN Atty. Dkt: UCSF-753WO Client Ref.: SF-2024-020-2-PCT An engineered cell adhesion molecule is recombinant in the sense that within a fusion protein the extracellular and intracellular domains are not from the same native (i.e., wild-type) cell adhesion molecule. In some embodiments, the engineered cell adhesion molecule may be a chimeric cell adhesion molecule, where the extracellular and intracellular domains are from different cell adhesion molecules. In any embodiment, the engineered cell adhesion molecule may be a chimeric cell adhesion molecule, where the extracellular and intracellular domains are from the same class of cell adhesion molecules, where the classes are numbered above. In any embodiment, the engineered cell adhesion molecule may be a chimeric cell adhesion molecule, where the extracellular and intracellular domains are from different classes of cell adhesion molecules, where the classes are numbered above. In any embodiment, the fusion may or may not be a fusion between a cadherin family member and a cadherin family member, for example. In any embodiment, the fusion may or may not be a fusion between any of the classes of cell adhesion molecule listed above. In any embodiment, the extracellular binding domain of the fusion protein may not be at least 90% or 95% identical to an extracellular binding domain of any of the adhesion molecules listed above, e.g., not an extracellular binding domain of a cadherin selected from CDH1, CDH2….CDH29, etc. or a protocadherin or integrin, etc. (i.e., the extracellular binding domain of naturally occurring adhesion molecule of Table 1 or a variant thereof that has at least 90% or 95% sequence identity to the extracellular domain of a naturally-occurring naturally occurring adhesion molecule). As noted above, in some embodiments, the extracellular binding domain may comprise the antigen-binding domain of an antibody (e.g., a scFv or nanobody) or a dimerization domain, for example, which binds to a cognate antigen / dimerization domain on another cell. s As noted above, the engineered cell adhesion molecules may be tuned, e.g., to provide a suitable level of binding strength, etc. In any embodiment, the cells may be in vitro. However, in vivo embodiments are also envisioned. For example, in conjunction with progenitor cells, organizer cells with engineered adhesion and morphogen properties can be used to drive development and regeneration in vivo, e.g., by implanting the cells or an aggregate of the same (or organoids made from the same) into a subject. The progenitor cells may pluripotent cells such as embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs), or the progenitor cells may be adult stem cells (ASCs). Atty. Dkt: UCSF-753WO Client Ref.: SF-2024-020-2-PCT Embryonic Stem Cells (ESC) may be derived from inner cell mass (ICM) of pre-implantation blastocyst-stage embryos. These cells can be potentially be used in allogeneic / ‘off-the-shelf’ (donor-derived) cell therapies. In some cases, such cells may be modified for immune compatibility (primarily deletion / disruption of HLA-I and HLA-II classes) (see Kim Stem Cell Reviews and Reports 17 (2021): 1053-1067. Induced Pluripotent Stem Cells (iPSCs) may be derived from at least skin, lung, heart, stomach, brain, liver, blood, kidney or muscle cells, where stemness and pluripotency can be induced by expression of Yamanaka factors (Oct 3 / 4 , Sox2, Klf4, and c-Myc) among others. These cells can generally be used for autologous (self- derived) or allogeneic (donor-derived) cell therapies. See WO 2010 / 017562A2 and Liu, Gele, et al. "Advances in pluripotent stem cells: history, mechanisms, technologies, and applications." Stem cell reviews and reports 16 (2020): 3-32. Adult stem cells include bronchioalveolar stem cells (BASCs), bulge epithelial stem cells (bESCs), corneal epithelial stem cells (CESCs), cardiac stem cells (CSCs), epidermal neural crest stem cells (eNCSCs), embryonic stem cells (ESCs), endothelial progenitor cells (EPCs), intestinal stem cells, hepatic oval cells (HOCs), hematopoetic stem cells (HSCs), keratinocyte stem cells (KSCs), mesenchymal stem cells (MSCs), neuronal stem cells (NSCs), pancreatic stem cells (PSCs), retinal stem cells (RSCs), endothelial stem cells, neural stem cells, olfactory adult stem cells, neural crest stem cells, testicular cells and skin-derived precursors (SKPs). Progenitor cells include endothelial progenitor cells, muscle stem cells, pancreatic progenitor cells, cardiac progenitor cells, ectodermal cells, mesodermal cells, and endodermal cells. A list of exemplary progenitor cells can be found Table 2 below: embryonic stem cells

[0002] Atty. Dkt: UCSF-753WO Client Ref.: SF-2024-020-2-PCT endodermal cells ectodermal cells nt and may naturally express a cell adhesion molecule that mediates cell-cell interactions between themselves, thereby causing them to cluster. In some embodiments, the progenitor cells may be engineered to express one or more cell adhesion molecules (which may be wild-type or engineered) that facilitate homotypic interactions or a target protein to which the organizer cells can bind, for example. The organizer cells can be any cell. In any embodiment, the organizer cells could be progenitor cells. In alternative embodiments, the organizer cells may be a somatic cell, e.g. a fibroblast, a hematopoietic cell, a neuron, a pancreatic cell, a muscle cell, a bone cell, a hepatocyte, a pancreatic cell, an epithelial cell, an endothelial cell, epithelial cell, an adipocyte, a cardiomyocyte, a T cell, a B cell, an osteocyte, or the like. Organizer cells can be grown as a suspension, although this is not necessary. While the number of progenitor and organizer cells in the composition may vary, in some embodiments, a composition may contain at least 10 cells of each type. For example, a composition may contain 10 to 100,000 progenitor cells (e.g., 10-1000, or 50-500 progenitor Atty. Dkt: UCSF-753WO Client Ref.: SF-2024-020-2-PCT cells) and, independently, 10 to 100,000 organizer cells (e.g., 100-10,000, 100-50,000 progenitor cells). As would be apparent, different numbers of cells (e.g., more or less than the numbers defined in these ranges) may be necessary depending on the cell types, the morphogens, and the particular developmental context. Also provided is a method for assembling cells. This method may comprise incubating the composition of cells described above in a medium under conditions in which the cells self- assemble into an aggregate. As noted above, the self-assembly process may be mediated by homotypic interactions between the progenitor cells, heterotypic interactions between the progenitor cells and the organizer cells and, in some embodiments, homotypic interactions between the progenitor cells and the organizer cells. As noted above, the aggregate may be made from single cells in solution or, in certain cases, the progenitor cells may be pre-assembled into a cluster prior to contact with the organizer cells. In embodiments that make use of a node of organizer cells, the node may be pre-assembled into the node prior to contact with the progenitor cells. In some embodiments, the method may comprise: (a) producing a population of cells by admixing: (i) a population of self-adhering progenitor cells (which can be in the form of a cell suspension or, alternatively, pre-assembled into a cluster); and (ii) one or more populations of organizer cells that produce an engineered cell adhesion molecule (which can be in the form of a cell suspension or, alternatively, pre-assembled into a node) and (b) incubating the composition under conditions by which the cells self-assemble to produce an aggregate that has a pre-defined architecture. In these embodiments, the cell-cell interactions of the progenitor cells and organizer cells are configured such that the progenitor cells and organizer cells self-assemble into an aggregate that has a pre-defined architecture. In embodiments in which one or more of the one or more populations of organizer cells expresses a morphogen the method may further comprise incubating the aggregate in a medium, thereby causing at least one of the progenitor cells to differentiate into another cell type, e.g., a cell of an internal organ (such as liver, kidney, stomach, or lung), a skeletal cell, a muscle cell, a blood cell, a cell of a particular tissue (e.g., skin, etc.), a neuronal cell or a fat cell. As illustrated in the examples section of this disclosure, the method may be used to make organoids (which are mini, simplified three-dimensional versions of an organ that mimic the key functional, structural and biological complexity of that organ). For example, the method may be used to Atty. Dkt: UCSF-753WO Client Ref.: SF-2024-020-2-PCT make a brain, kidney, lung, intestine, stomach, liver, or heart organoid if programed appropriately. A cell aggregate is also provided. In these embodiments, the aggregate may comprise (a) a cluster of progenitor cells; and (b) one or more structures composed of organizer cells that comprise an engineered cell adhesion molecule that recognizes a target on the surface of the progenitor cells. The cells in the aggregate are held together by cell-cell interactions, some of which are synthetic. As noted above, in some embodiments, the one or more structures may comprise: (i) a shell that at least partially surrounds the cluster of progenitor cells, and / or (ii) one or more nodes that are attached to the cluster of progenitor cells. As noted above, in some embodiments, at least one of the one or more populations of organizer cells expresses a morphogen, and wherein the morphogen causes differentiation of one or more of the progenitor cells in the cluster of progenitor cells. As such, in some embodiments, the aggregate may comprise (a) a cluster of progenitor cells; (b) one or more structures composed of organizer cells that comprise an engineered cell adhesion molecule that recognizes a target on the surface of the progenitor cells and that produce a morphogen and (c) differentiated cells (where those cells have become differentiated by the morphogen). EXAMPLES The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. Standard abbreviations may be used, e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s or sec, second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb, kilobase(s); bp, base pair(s); nt, nucleotide(s); i.m., intramuscular(ly); i.p., intraperitoneal(ly); s.c., subcutaneous(ly); and the like. Atty. Dkt: UCSF-753WO Client Ref.: SF-2024-020-2-PCT MATERIALS AND METHODS Cell lines and culture conditions The following ESC lines were used: - pTCF-mCherry reporter mESCs with membrane nfGFP and H2B-iRFP expression, generated by piggyBac transposon. - Brachyruy-mCherry::Sox1-EGFP reporter mESCs with membrane nfGFP and H2B-iRFP expression, generated by piggyBac transposon. - Kdr-EGFP reporter mESCs with membrane nfGFP and H2B-iRFP expression, generated by piggyBac transposon. Also, the following synthetic organizer cells were generated: - Wnt3a node: L929 cells expressing P-cad and synCAM: Lag16-ICAM1 with overexpressing Wnt3a-IRES-tagBFP2 upon DOX induction, generated by Lenti-virus transduction. - Wnt3a shell: L929 cells expressing synCAM: Lag16-ITGB2 with overexpressing Wnt3a-IRES- tagBFP2 upon DOX induction, generated by Lenti-virus transduction. - Dkk1 node: L929 cells expressing P-cad and synCAM:Lag16-ICAM1, and iRFP with overexpressing Dkk1-IRES-tagBFP2 upon GRZ induction, generated by Lenti-virus transduction. - Dkk1 shell: L929 cells expressing synCAM: Lag16-ITGB2 and iRFP with overexpressing Dkk1- IRES-tagBFP2 upon GRZ induction, generated by Lenti-virus transduction. - L929 cells expressing P-cad and tagBFP2, generated by Lenti-virus transduction. - L929 cells expressing K-cad and tagBFP2, generated by Lenti-virus transduction. - L929 cells expressing N-cad and tagBFP2, generated by Lenti-virus transduction. - L929 cells expressing E-cad and tagBFP2, generated by Lenti-virus transduction. - L929 cells expressing synCAM: Lag16-tether and tagBFP2, generated by Lenti-virus transduction. - L929 cells expressing synCAM: Lag16-DLL and tagBFP2, generated by Lenti-virus transduction. - L929 cells expressing synCAM: Lag16-ICAM1 and tagBFP2, generated by Lenti-virus transduction. - L929 cells expressing synCAM: Lag16-ECAD and tagBFP2, generated by Lenti-virus transduction. - L929 cells expressing synCAM: Lag16-ITGB1 and tagBFP2, generated by Lenti-virus transduction. - L929 cells expressing synCAM: Lag16-ITGB2 and tagBFP2, generated by Lenti-virus transduction. mESCs were cultured at 37°C and 5% CO2 in 2iLIF medium (DMEM + Glutamax (10564-011; Gibco) supplemented by 10% embryonic stem cell qualified FBS (Gibco), 0.1% 2- Atty. Dkt: UCSF-753WO Client Ref.: SF-2024-020-2-PCT Mercaptoethanol (21985-023; Gibco), 1% MEM Non-Essential Amino Acid (NEAA) (11140- 050; Gibco), and 1% Sodium Pyruvate (11360-070; Gibco), 3 μM CHIR99021 (Chir) (Stem Cell Technologies), 1 μM PD0305901 (Stem Cell Technologies), 0.01 μg / ml LIF (Stem Cell Technologies), and 1% penicillin-streptomycin (151140-122; Gibco)). Prior to plating mESCs, add 1 mL of 0.1% gelatin (07903; Stem Cell Technologies) to a 12-well plate and let sit at least for an 1 hr. Cells were passaged every other day with TrypLE Express (Gibco). If the cells were not being passaged, the media was replaced with fresh 2iLIF medium. L929 cells were cultured in D10 medium (DMEM(1x) + Glutamax (10564-011; Gibco) supplemented by 10% FBS (89510; University of California, San Francisco [UCSF] Cell Culture Facility), and 1% penicillin-streptomycin (151140-122; Gibco)). Cells were cultured in a 37°C incubator at 5% CO2atmosphere. Gene construction and cloning All the plasmids were constructed by using the Mammalian Cloning Toolkit (MTK). In brief, each element (promoter, coding region, 3”UTR) was introduced into the MTK system either by PCR or gene block (IDT). A library of circuit components was assembled into transcriptional units with a BsaI Golden Gate reaction. After bacterial transformation, plasmid DNAs were extracted (QIAprep Spin Miniprep Kit; 27106) and their sequence were verified. Establishing synthetic organizer cell line - transduction plasmids using Lentivirus To generate synthetic organizer cells, constructs were introduced into L929 cells using Lentivirus transduction. Lentivirus was produced using HEK293T lentiviral packaging cells (Takara bio, Cat# 632180) that were seeded in 6-well plates at 7x 105cells / well and 24 hours later transfected with a pHR lentiviral backbone and viral packaging plasmids pCMVdr8.91 and pMD2.g using TransIT-VirusGEN (22063557; Mirus) following the manufacturer’s protocol. 48 hours after transfection viral supernatant was collected and passed through a 0.45 mm filter prior to use with L929 cells and used immediately for transduction. L929 cells were plated in a 12 well plate in 500 µL of 1.0x105cells / mL and 500 µL of Lentiviral supernatant. 48 hours after infection, the virus media were removed, and cells were replaced with 1 mL of D10. Cells were split if they reached approximately 80% confluence. After Lentiviral transduction, positive cells were selected using Puromycin antibiotics (A11138-03; Gibco) or Hygromycin (10687010; Invitrogen). Positive cells were examined under the Opera Phenix with 5x air-immersion objective lens using brightfield and BFP Atty. Dkt: UCSF-753WO Client Ref.: SF-2024-020-2-PCT channels. After the selection, single clonal cell line was selected by plating a single cell per well in 96-well plate. Phenotypes of each clone, such as morphologies and BFP expression level, were examined under the Opera Phenix with 5x air-immersion objective lens using brightfield and BFP channels. Establishing mESC line - transduction plasmids using Nucleofection Nucleofection is used to transduce plasmids into mESCs, employing the piggyBac transposon system. Plasmids were prepared by mixing 1 µg of piggyBac plasmid, 1 ug of transposase plasmid, 82 µL of nucleofector solution and 18 µL of supplement. Following the preparation of the master mix, mESCs are collected from their 12-well plate and detached using 300 µL of TrypLE (12604-013; Gibco). After waiting for 3-5 minutes, 1 mL of media was added, and the cells were transferred to a 15 ml conical tube. The cells were spun down at 400g for 4 minutes. The supernatant was removed, and cells were resuspended in 1 mL of media. Cells were diluted to 1 x 105cells / mL. After spinning down the cells, the cells were resuspended with 110 µL of the prepared plasmid mix and transfer to the P3 Primary cell 4D nucleofector X kits cuvette. Nucleofection was carried out using the Lonza 4D Nucleofector Core Unit with CG104 program. Subsequently, the cells were diluted with 1 mL of 2iLIF mESC media and plate into 2 wells of a 12 well plate (500 µL each), followed by the addition of 500 µL of 2iLIF mESC media. Synthetic niche construction around a mESC core and morphogen induction Making aggregates of mESCs and synthetic organizers were based on the original gastruloid protocol. In short, mESCs and L929 cells were detached from tissue culture plates with TypLE and collected with 2iLIF medium for mESC or D10 medium for L929. After Centrifugation at 500g for 3 minutes, the cells were washed once with N2B27 medium. N2B27 media is based on DMEM / F12 (1x) (11330-032; Gibco) and Nuerobasal Medium (21103-049; Gibco), supplemented by, 1% B27 supplement (50X)(12587-010; Gibco), 0.5% N2 supplement (100x) (17502-048; Gibco), 1% MEM Non-Essential Amino Acid (NEAA) (11140-050; Gibco), 1% Sodium Pyruvate (11260-070; Gibco), , 0.5% Glutamax (35050-061; Gibco), 0.2% 2- Mercaptoethanol (21985-023; Gibco), and 1% penicillin-streptomycin (151140-122; Gibco). The cells were then resuspended in N2B27 medium, and the cell concentrations were determined. The cells were diluted to experimental amounts: 300-600 cells / well for mESC, 50- 100 cells / well for Nodes, and 10-50 cells / well for shells, and 40 µL / well of mESCs and Atty. Dkt: UCSF-753WO Client Ref.: SF-2024-020-2-PCT synthetic organizer shells were plated into an ultra-low attachment round bottom 96 well plate (7007; Costar), or 40 µL / well of synthetic organizer nodes were plated into an ultra-low attachment round bottom 384 well plate. The Plates were spun down at 500g for 1 minute and were kept in a 37°C incubator at 5% CO2 atmosphere. The following day, nodes were transferred to the 96-well plate with the mESCs. There should be 80 µL (single node or single shell) or 120 µL (2 nodes or node & shell) of medium after transferring nodes. To induce Wnt3a in L929 cells, 40 µL of N2B27 medium containing doxycycline (D3447, Sigma) at final concentration of 200 ng / mL was added to wells. To induce Dkk1 in L929 cells, 40 µL of N2B27 medium with grazoprevir (MK-5172, MedChemExpress) at final concentration of 0.8 uM was added to wells. Live imaging Plates were imaged on the Perkin Elmer Opera Phenix High Content Screening System using 10x air objective (NA = 0.3) lens with 2-pixel binning. Images were captured sequentially in a multi-stacks, five-channel mode. The detailed imaging conditions were as follows: tagBFP for 500 millisecond (ms) exposure time, EGFP for 400 ms, mCherry for 1200 ms, iRFP670 for 400 ms, bright-field for 100 ms. Laser powers were 50% for all wavelengths. Z-stacks were set up to start at -10.0 µm with each plane 7.4 µm apart, capturing 16 planes in total. Calcium imaging Calcium transients were imaged in beating phenotypes on day 8, stained with Fluo-4 AM (F14217, Thermo Fisher Scientific) using a confocal microscope (Nikon, T2). One µM of Fluo-4 AM was added to the samples and incubated for at least 30 minutes. Then, the samples were washed twice with N2B27 medium and transferred to a µ-slide 8 well (ibidi). Using a 20x Air objective lens, several 10 seconds recordings were acquired for each sample at a frame rate of 10 frames per second. Fluorescence intensities were quantified with Fiji software to measure the signal intensity (F) and background (F0). Immunostaining Whole mount immunostaining was performed based on previous papers. Briefly, samples were transferred to F-bottom 96 well plate using a p200 pipette with the tip cut-off at the 50 µL position. The collected samples were washed once in PBS and then fixed in 4% PFA for 30 minutes at room temperature. After fixation, the sample were washed twice with PBS and stored in PBS until staining was performed. the samples were permeabilized by three 20 minutes Atty. Dkt: UCSF-753WO Client Ref.: SF-2024-020-2-PCT incubation in 1.0% Triton-X / PBS and blocked in Universal Blocking Solution (UBS: 1.0 g / dL BSA, 0.2 g / dL evaporated nonfat milk, 0.5% Triton X-100, 1% DMSO in PBS) for 1 hour at room temperature. Primary antibodies were incubated in UBS for 48-72 hours at 4˚C. Subsequently, the samples were washed three times with UBS for 10 minutes at room temperature. Afterward, secondary antibodies diluted in UBS were incubated for 48 hours at 4˚C. Twenty-four hours after secondary antibodies addition, DRAQ5 (at a 500-fold dilution) was added to stain the nuclei. After overnight incubation at 4˚C, samples were washed three times with PBS and transferred to µ-slide 8 well ibiTreat slide for imaging. Imaging fixed samples Immunostained samples were imaged at the UCSF Nikon Imaging Center using a Nikon Ti2-E microscope equipped with Crest L-FOV spinning disk and C2 confocal with DUVb detector (Crest / C2). Images that capture an entire sample were taken with Crest spinning disk confocal using 10x Air (NA= 0.45) objective lens. For the detailed image around a chamber, C2 point-scanning confocal with 10xAir (NA= 0.45) objective lens was used. MULTI-seq sample preparation Individual embryoids were labeled with unique lipid-modified oligonucleotide (LMOs) to multiplex multiple samples for MULTI-seq. In brief, around 10 embryoids in each condition were transferred to a 96-well U-bottom plate and were digested using TrypLE. The cells were then washed with BGJb medium and labeled with 10 uL of 2.5 uM anchor LMO-barcode for 5 min on ice. Then, 10 uL of 2.5 uM co-anchor LMO were added to each cell pool, and samples were labeled for another 5 min on ice. Following LMO labeling, cells were washed with 1% BSA in PBS to quench LMOs. After washing, the cells were resuspended in FACS buffer with DAPI and live embryoid cells were sorted using BD FACS Aria. L929 node cells were sorted separately and mixed with embryoid cells at a 2% rati. Transcripts of each cell were barcoded with 10× Genomics Chromium system at the UCSF 10x Genomic core. Sequencing was carried out at the UCSF sequencing core with NovaSeq X. Analyzing Positions and Morphologies Imaging data acquired through Opera Phenix microscope were exported as TIFF files and reconstituted as a 4D TIFF file (xyzt) for each color channel, using a custom Python script. Each 3D stack of images was transformed into a 2D maximum projection along the z-axis using the numpy library in Python. To discern cell morphologies, a thresholding technique was Atty. Dkt: UCSF-753WO Client Ref.: SF-2024-020-2-PCT applied using the skimage.filter.threshold function. Optimal thresholding methods were evaluated using skimage.filteres.try_all_threshold prior to final selection. Binary images were generated based on the chosen threshold, from which features characterizing cell morphologies were extracted. Measuring relative positions of two nodes on embryoid The centroids of both embryoid and two nodes were determined utilizing skimage.measure.centroid function. Subsequently, angles between the two nodes were computed based on their positional data in relation to the embryoid. Quantification of major and minor axis of embryoids Morphological attributes of embryoids were quantified using the skimage.measre.regionprops functions, and their elongations were assessed by calculating the ratio of the major axis length to the minor axis length. Measuring TCF signals To quantify TCF signals, z-slices encompassing the central region of the embryoid were extracted from the 3D TIFF files. Subsequently, a 2D maximum projection along the z axis was generated based on the extracted 3D files. Fluorescence intensity profiles of pTCF-mCherry were assessed in Fiji using a rectangular region of interest (ROI) that encompassed the embryoid. RESULTS Fig. 1 shows the basic concepts of how organizer cells can guide cell development (last panel) relative to in vivo and prior in vitro methods. Using engineered cell adhesion code to specify self-organizing architectures around stem cell core. The differential adhesion hypothesis postulates that multicellular systems self- organize into a minimal energy structure that is determined by the relative strengths and of homotypic and heterotypic cell adhesion interactions between the cell species. A toolkit of both natural and synthetic adhesion molecules that can be used to tune both homotypic and heterotropic adhesion specificity and strength was developed, allowing one to program self- organization of engineered multi-cellular systems. This adhesion toolkit was used to engineer a simple L929 fibroblast cell line to self-organize around mouse embryonic stems cells in a user defined manner (Fig. 2A). To simplify engineering of interactions between L929 cells and mESCs, a mESC line expressing non-fluorescent GFP molecule on its surface was created. The Atty. Dkt: UCSF-753WO Client Ref.: SF-2024-020-2-PCT surface GFP could then be used as an interaction handle to interact with synthetic adhesion molecules bearing a set of well-characterized anti-GFP nanobodies (in principle, an endogenous mESC surface molecule could be used instead). L929 variants were engineered to form either “node” or “shell” structures (Figs. 2A and 2B). A node is a cluster of organizer cells that interacts with the mESC’s at a single point. A shell is a single layer of organizer cells surrounding the entire mESC spheroid. Such architectures was specified by expressing the proper combinations of homotypic and heterotypic adhesion molecules. Programming formation of node organizers To make a node structure, L929 cells that express N, K or P cadherins, homophilic cadherins that will yield node clustering while still maintaining sorting from the E cadherin expressing mESCs were used. It was found that P cad yielded the best segregation. Whether the node could be stably tethered to the mESC spheroid in the presence of a heterotypic synCAM in the L929 node cells (here the synCAM binds the surface GFP ligand engineered on the mESCs) was tested. Thus, anti-GFP synCAMs were expressed in the L929 node cells, in addition to P cad. Screening multiple synCAMs, it was found that the synCAM with the intracellular domain from ICAM-1 yielded the most well-formed mESC-tethered node (Figs. 2A and 2C). When mESCs and the node cells were mixed, they self-organized into a single node attached to the core spheroid (Fig. 2D). Nonetheless, it was found that node formation was much (5-10 times) faster and more consistent (fewer double node structures) if the node and mESC clusters were preformed first, and then mixed (Fig. 2D). It was found that by independently pre-forming two separate nodes and mixing them with an ESC cluster, two-node structures, where each node was composed of different cells (e.g., producing different morphogens), could be reliably generated. This architecture allows one to program self-assembly of structures with multiple distinct synthetic organizer nodes. Programming a shell organizer. Anti-GFP synCAMs that when expressed in L929 cells led to the formation of a shell around the GFP+ mESC spheroid were then screened. While many synCAMs led to a clumpy interaction with mESC’s, synCAMs with the integrin ß1 or integrin ß 2 intracellular domains consistently yielded single layer shells. When the shell L929 cells were mixed with GFP+ mESCs, they were able to self-organize into a core-shell Atty. Dkt: UCSF-753WO Client Ref.: SF-2024-020-2-PCT architecture (Figs. 2C and 2D). By using multiple L929 organizer cell populations, structures with one to two nodes and a shell could be programmed. Adhesion constrains spatial relationship of nodes and mESCs. When structures with two distinct nodes attached to the mESC cluster were built, it was found that the radial separation of the two nodes could vary between ~90-180 degrees. Notably, however, when such assemblies were tracked over several days, it was observed that nodes attached with the ICAM- 1 synCAM showed significantly less radial angle change over multiple days, compared to nodes attached with a tether molecule (anti-GFP nanobody and TM region, but without any intracellular signaling domain) or lacking any anti-GFP molecule (Fig. 2E). This result suggests that a key advantage of using strong adhesion interactions (one that strongly engage the cytoskeleton) to link organizer nodes to the mESC core, is that they lock the nodes into more fixed positions. Nodes with higher mobility along the mESC core surface might blur much of the positional information encoded in the nodes over the slow timescale of development. Wnt3a producing organizers generate distinct morphogen gradient patterns. A key morphogen, Wnt3a, was expressed from either a synthetic node or synthetic shell and used a proximal Wnt response reporter (pTCF-mCherry) in the mESC’s to determine what kind of morphogen gradient was produced (Fig. 3A). When the mESC core was stimulated with the small molecule Wnt agonist, CHIR99021, in the media, the Wnt reporter showed isotropic activation throughout. Stimulation with Wnt3a in the media led to symmetric reporter activation, but with higher activation near the surface. Using a Wnt3a producing shell organizer, a similar symmetric pattern of activity, higher at the surface, was also observed. In contrast, using a Wnt3a producing single node, a sharp, asymmetric graded response in the mESC core was observed (Fig. 3A). Tunability of organizer payloads: amplitude and dynamic control. Wnt expression by the synthetic organizers was controlled by a small molecule induced promoter (pTRE or pUAS, controlled by addition of doxycycline (DOX) or grazoprevir (GRZ) , respectively). With this kind of system, one can control the amplitude of morphogen expression by adding different amounts of induce molecule (Fig. 3B). One can also control the timing of morphogen expression by adding each inducer molecule after different delay times (Fig. 3C). Thus, this system provides the ability to tune morphogen amplitude and dynamic control (e.g. relative Atty. Dkt: UCSF-753WO Client Ref.: SF-2024-020-2-PCT timing of production of two different morphogens) in an easy and flexible manner, compatible with high-throughput studies. Directional elongation of embryoid structure with Wnt3a node. By tracking the development of these different organizer architectures (which provide Wnt3a in different spatial patterns), it was observed that over the course of 5 days, only the Wnt3a node induced structure showed a distinctive elongation of the resulting embryoid (Fig. 3C). This elongation occurred in the direction of the Wnt3a gradient produced by the single node. Embryoids lacking this Wnt3a gradient did not show elongation. Wnt3a node reproducibly yields beating heart-like chamber. To further track development of the single Wnt3a node embryoid, development over 8 days starting with mESC’s with a mesodermal reporter, Kdr-GFP (VEGFR2, Flk-1) was tested. In the single node structure, it was observed KDR+ cells forming in the center of the embryoid at days 5 and 6. On day 7, these KDR+ cells migrated towards the distal pole (opposite the Wnt3a node) and coalesced to form what appear to be tubular structures. These cells then organized into a chambered heart-like structure (day 8) which showed regular beating (Figs. 4A and 4B). In contrast, although KDR+ cells were also induced in embryoids treated with uniform Wnt3a in the media on days 4-6, these cells did not show any coherent organization into tubes or a chamber, but instead faded away in intensity by day 8 (Fig.4A). Thus, the anisotropic gradient of Wnt3a produced by the synthetic node appears to be critical for formation of the heart-like chamber. Observing 93 embryoids treated with the single Wnt3a synthetic node, it was found that formation of a beating chamber occurred with ~11% frequency (Fig. 4C), with ~60% of the embryoids showing beating (without a clear chamber) and ~27% showing no detectable beating. Staining with anti-actin antibodies and DAPI (nuclear stain) shows that the chamber is a fully enclosed chamber (Fig. 4D). To further analyze the beating, imaging using the fluorescent Ca++indicator, Fluo-4 AM, was performed, which showed oscillatory Ca++flux with a regular period of 1.4 sec (~42 beats per min.) (Fig. 4E). Beating clearly only occurs in a defined region of the embryoid. In summary, the Wnt3a gradient produced by the single node organizer could consistently lead to the formation of embryoids with a defined, closed heart-like structure with a regular Ca++associated beating rhythm. Atty. Dkt: UCSF-753WO Client Ref.: SF-2024-020-2-PCT Myocardial and hematopoetic cell types are found in the Single Wnt3a node induced embryoids. Single cell RNAseq (scRNA-seq) was performed on the embryoids on day 8, to determine what cell types were present. As shown in the UMAP plot (Fig. 5A), a wide variety of cell types are observed, including a variety of mesodermal lineages, notably fetal ventricular myocardium, vascular endothelium, and blood / hematopoetic cells. As shown in the feature plots (Fig. 5B), the fetal cardiac cells express the pan-cardiac markers Tnnt2 (Cardiac muscle troponin T) and Nkx2-5 (homeobox transcription factor regulating heart development), and the vertricular markers Myl2 (Myosin Light Chain 2) and Irx4 (Iroguois homeobox 4). The blood cells express the blood progenitor markers Itga2b (Integrin Subunit Alpha 2), Runx1 (runt-related transcription factor 1), and Gata1 (GATA Binding Protein 1), as well as he embryonic hemoglobin isoforms Hbb-y (hemoglobin Y, beta-like embryonic chain), Hbb-bh1 (hemoglobin Z, beta-like embryonic chain), and Hba-x (hemoglobin X, alpha-like embryonic chain). (REFS) To further localize where cardiac cells are found, immunohistochemical staining for the cardiac transcription factor Nkx2.5 and cardiac troponin (cTnT) was performed (Fig. 5C). These distinct markers only stain the region of the beating chambered heart-like structure. The embryoids were stained for the endothelial marker PECAM1 (Platelet And Endothelial Cell Adhesion Molecule 1). Costaining of the cardiac marker cTnT and the endothelial marker producing opposing factors Wnt3a vs DKK1. It was then explored whether more complex synthetic organizer structures could modulate embryoid development. This work involved combining one set of organizer cells that produce Wnt3a, and a second set of organizer cells that secrete the Wnt inhibitor, DKK1 (Dickkopf-related protein 1). One hypothesis was that the combination of Wnt and its inhibitor, produced in the right spatial patterns, could lead to even organizer structures: single Wnt3a node; dual Wnt3a vs DKK1 node; Wnt3a shell vs DKK1 Atty. Dkt: UCSF-753WO Client Ref.: SF-2024-020-2-PCT node. On day 4 after induction, output from the Wnt activity reporter (TCF promoter) imaged and found that the addition of the second organizer cell population could dramatically shift the Wnt activity gradient. In particular, the architecture with two opposing (Wnt vs DKK1) nodes, yield a sharper Wnt activity gradient that was more stably maintained (see Fig. 3d – sharp Wnt activity gradient is observed on day 3, but becomes more uniform on days 4 and 5). Thus, the interplay of two opposing synthetic nodes can yield a sharper gradient that is more stably maintained over time. We then followed the development of these two-organizer induced embryoids using mESC’s containing early cell fate reporters: Brachyury, a marker for mesendoderm (precursor to mesoderm and endoderm), and node and notochord at the later stage; and Sox1, a marker for ectoderm, particularly those lineages that give rise to neural fates (Fig. 6B). It was observed that the single Wnt3a producing node only induces Brachyury+ cells (orange), i.e. mesodermalfates. In contrast, the single DKK1 producing node only generates Sox1+ cells (green), i.e. ectodermal fates. However, in embryoids induced by both Wnt3a and DKK1 organizers (either Wnt3a shell / DKK1 node or Wnt3a node / DKK1 node) the simultaneous induction of Brachyury+ cells and Sox1+ cells within the same structure was observed. In both of these structures the formation of distinct Brachyury+ and Sox1+ segments was observed, with Brachyury signal closer to the Wnt3a organizer, and Sox1 signal closer to the DKK1 organizer. Timing of the two nodes can reshape development. The dual node (Wnt3a and DKK1) embryoids appeared to have the best balance between Brachyury+ and Sox1+ cell lineages, so how these embryoids would develop if the timing of morphogenic signaling by the two organizers was changed was then explored. Controlled timing of the two nodes could be precisely controlled because both are controlled by independent chemical inducers (DOX induction of Wnt; GRZ induction of DKK1). Development when DKK1 induction was delayed by 0,1, 2, and 3 days after Wnt3a induction was followed. It was found that variations in timing could alter the balance between Brachyury+ and Sox1+ cells. With no delay (0 days), embryoids in which Sox1+ cells were dominant were observed; by day 8 there were few Brachyury+ cells. In contrast, with a delay of DKK1 induction of 3 days, Brachyury+ cells were dominant; by day 8 there were very few Sox1+ cells. However, with delays of 1 to 2 days, significant numbers of both Brachyury+ cells and Sox1+ cells in the same embryoid were Atty. Dkt: UCSF-753WO Client Ref.: SF-2024-020-2-PCT observed (Fig. 6C). The resulting embryoids formed large elongated and complex structures, even in 96 well plates with limited space to grow. Interestingly, ~17% of these embryoids also showed beating heart-like structures, with a handful showing a chambered structure on day 8 (Fig. 6D). Dual node-induced embryoids show increased cell type diversity. To examine resulting cell type diversity in more detail, sc-RNAseq on dual node embryoids induced with a 1 day delay between Wnt3a and DKK1 was performed (Fig. 7A). Samples for analysis were collected on day 8 of development. To compare the cell types in the dual node embryoids with those found in single node embryoids, scRNA-seq was performed on embryoids induced by the single DKK1 node (single Wnt3a node data shown in Fig. 5A). Fig. 7B (and Fig. 5A) shows the UMAP analysis of these samples. The single Wnt3a node embryoids show largely mesodermal and endodermal cell fates, while the single DKK1 node embryoids show largely ectoderm fates (Fig. 7C). In contrast, in the dual (Wnt3a + DKK1) node embryoids a significantly more diverse cell population that has significant fractions of cells with both mesendoderm and ectoderm fate was observed. This expansion in cell types includes many neural lineages, such as neuroectoderm, early neurons, and neuroendocrine cells, compared to the single Wnt3a node embryoids. The sharper and more stable gradient that is generated by the dual antagonistic nodes – with a morphogen and its antagonist – leads to a wider range of cell types within the same developing structure. DISCUSSION Organizing the organizer using engineered cell adhesion. Here a powerful cell engineering approach to reconstitute development in vitro, based on engineering self-assembling developmental organizers is described. The concept of organizers - groups of cells that provide inductive signals that can mediate differentiation, patterning and organization of surrounding cells – has been a foundational principle of development since first proposed. Organizers are thought to create the coordinate systems that provide position information to organize development. Here, the principles of differential adhesion to engineer synthetic organizer cells are harnessed. Advances in precision cell engineering, particularly of synthetic cell adhesion molecules, now allow us to construct synthetic organizer cells that are programmed to spatially self-organize around progenitor cell spheroids. The ensemble of cells acts as a system that, Atty. Dkt: UCSF-753WO Client Ref.: SF-2024-020-2-PCT driven by systemwide energy minimization, self-organizes based on the energies of the homo- and heterotropic interactions between both organizer and ES cells. In short, this work suggests that the concepts of positional information and differential adhesion, although often thought of as separate, likely work together intimately to both create developmental organizers and to subsequently propagate their inductive effects through the tissue or embryo. More specifically, it as been shown that a library of natural and synthetic adhesion molecules can be used to engineer cell lines that self-organize around mouse embryonic stem cells. Diverse organizer architectures can be created. An anisotropic node, a cluster of cells that associates with the ES cells at a single point, can be created. Alternatively, an isotropic shell – an ensemble of cells that distribute in a thin layer around the ES cell spheroid – can be created. By using multiple organizer cell species together, multi-node or mixed node-shell architectures can be created. Although these architectures can be simple, they provide clear asymmetric organization, and they form through self-assembly. Unlike other techniques to stimulate cells in spatially defined ways, spatial localization of the organizer is an autonomous process requiring little to no user direction, making this approach both more robust and amenable to high- throughput studies. Given these architectures, any morphogen can be expressed from the engineered, self-assembling synthetic organizers. These organizers can create custom shaped morphogen gradients - the fields of positional information that guide development. Use of coordinated organizers that secrete antagonistic or complementary morphogens can even further increase positional information. Simple Wnt3a node organizer yields sophisticated cardiac development. The synthetic organizers create a far more anisotropic morphogen gradient compared to the more traditional in vitro approach of providing morphogen or chemical agonists in the media. When a shallow Wnt3a gradient is created using a single synthetic Wnt3a node, a highly complex developmental outcome in which a beating, chambered heart-like structure is formed in a specific region of the embryoid is observed. This heart-like chamber has fetal myocardial cells and blood cells, has an endothelial vascular network emerging from its base and extending throughout the embryoid. The tissue also beats in a regular fashion, showing Ca++ oscillations. It was hypothesized that it is the shallow and gradient produced by the organizer that allows this highly organized process of more native-like cardiac development to occur. Atty. Dkt: UCSF-753WO Client Ref.: SF-2024-020-2-PCT Antagonistic organizers lead to more complex developmental gradients and cell- type diversity. It was also found that combining multiple organizers that antagonize one another, can lead to even more refined or complex gradients, as in the case of combining an organizer producing Wnt3a with an organizer producing the Wnt inhibitor DKK1. In this case the generation of a stable Wnt activity gradient that spans from high activity at one pole, to no detectable activity at the opposite pole, is observed. This gradient leads to the generation of far more diverse cell lineages, including mesendoderm lineages near the high Wnt activity pole, and ectoderm lineages near the low Wnt activity pole. In contrast, the single Wnt3a or DKK1 nodes are dominated by only one of these lineages. Future implications. Although the synthetic organizers that have experimentally reduced to practice herein are relatively simple, they demonstrate the use of engineered cells to drive and guide development. As the tools of cell engineering continue to advance, it will be more refined and complex organizer architectures can be engineered. For example, it will be possible to engineer more dynamic synthetic organizers that function in a more complex stepwise manner. One feature of the organizer cells is that they act in a very autonomous manner, such that very little precision input is required by the experimentor. Thus, this approach is much more amenable to high-throughput formats. Here all studies were performed in 96 well formats. The resulting embryoids were not transferred and grown in larger wells or in roller bottles with advanced media, although such advanced approaches would likely lead to larger and more developed embryoids. These current approach is also highly reproducible. Although not perfect, a high percentage of assemblies (10-90%) form the target architecture, the target morphogen gradient, and the subsequent morphological output. Such advances will lead to far better and more reliable organoids for the study of development or model systems for studying disease or therapeutics. Ultimately it should be possible to apply some of these approaches to drive regenerative development in vivo, especially if organizer cells can be programmed to localize to sites of injury or degeneration and to establish engineered morphogenic gradients in situ. Another feature of the synthetic organizer cells is their tunability. By using different organizer architectures and morphogen combinations, one can systematically change the system and generate different morphogenic outcomes. Thus, these approaches offer new ways to study Atty. Dkt: UCSF-753WO Client Ref.: SF-2024-020-2-PCT fundamental developmental processes, not simply by tracking one natural trajectory, but also by making systematic changes to the system to see how this alters the trajectory. These approaches may allow one to far better map the landscapes that guide and keep development on track. Moreover, the tunability of these systems could allow the generation of tissues or organs with systematic alterations, some of which might be more useful for particular purposes. While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto.

Claims

Atty. Dkt: UCSF-753WO Client Ref.: SF-2024-020-2-PCT CLAIMS What is claimed is:

1. A composition of cells, comprising: (a) a population of progenitor cells; and (b) a population of first organizer cells that are engineered to express one or more cell adhesion molecules that mediate cell-cell interactions with the progenitor cells; wherein the progenitor cells and first organizer cells self-assemble into an aggregate that has a pre-defined architecture.

2. The composition of cells of claim 1, wherein the first organizer cells are additionally engineered to express one or more cell adhesion molecules that mediate cell-cell interactions between the first organizer cells.

3. The composition of claim 1 or 2, wherein the composition further comprises: (c) a population of second organizer cells, wherein the second population of organizer cells are engineered to express one or more cell adhesion molecules that recognizes a target on the surface of the progenitor cells and are either additionally engineered to express one or more cell adhesion molecules that mediate interactions between the first organizer cells or do not recognize the first organizer cells.

4. The composition of any prior claim, wherein the aggregate comprises: (i) a cluster of progenitor cells, and (ii) one or more structures formed by the organizer cells.

5. The composition of claim 4, wherein the one or more structures comprise: a shell that at least partially surrounds the cluster of progenitor cells, and / or one or more nodes that are attached to the progenitor cells.

6. The composition of claim 4 or 5, wherein the progenitor cells are held together in the cluster via homotypic interactions.Atty. Dkt: UCSF-753WO Client Ref.: SF-2024-020-2-PCT 7. The composition of any prior claim, wherein the composition comprises a pre-made cluster of progenitor cells made up of the cells of (a); and / or one or more pre-made nodes made up of the cells of (b).

8. The composition of any of claims 1-6, wherein the cells of (a) and (b) are single cells in a cell suspension.

9. The composition of any prior claim, wherein the first organizer cells produce a morphogen.

10. The composition of claim 9, where the morphogen is a diffusible molecule that secreted or released by one or more of the populations of organizer cells.

11. The composition of claim 10, wherein the morphogen is selected from a member of the Wnt / beta-catenin, a member of the TGF-β (Transforming growth factor β) / BMP (Bone morphogenetic protein) family, a member of the Shh (sonic hedgehog) family, and retinoic acid or an analog thereof, or a morphogen listed in Table 1.

12. The composition of claim 9, wherein the morphogen is a juxtracrine signaling molecule.

13. The composition of claim 10, wherein the juxtracrine signaling molecule is Notch, ephrin, semaphorin, a T-Cell Receptor (TCR).

14. The composition of any prior claim, wherein the progenitor cells are embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), or adult stem cells (ASCs), selected from Table 2.

15. The composition of any prior claim, wherein the composition comprises: (a) a population of progenitor cells that express a cell adhesion molecule that recognizes a target on the progenitor cells and causes the progenitor cells to form a cluster via homotypicAtty. Dkt: UCSF-753WO Client Ref.: SF-2024-020-2-PCT cell-cell interactions; and (b) a population of organizer cells that: (i) express a cell adhesion molecule that recognizes a target on the surface of the progenitor cells and causes the organizer cells to adhere to the cluster of progenitor cells via heterotypic cell-cell interactions; and either: (ii) additionally express a cell adhesion molecule that recognizes a target on the surface of the organizer cells and causes the organizer cells to form a node via homotypic cell-cell interactions; or (iii) do not additionally express a cell adhesion molecule that recognizes a target on the surface of the organizer cells, thereby causing the first cells to form a shell around the progenitor cells.

16. The composition of any prior claim, wherein the composition comprises: (a) a population of progenitor cells that express a cell adhesion molecule that recognizes a target on the progenitor cells and causes the progenitor cells to form a cluster via homotypic cell-cell interactions; and (b) a population of first organizer cells that: (i) express a cell adhesion molecule that recognizes a target on the surface of the progenitor cells and causes the first organizer cells to adhere to the cluster of progenitor cells via heterotypic cell-cell interactions; and (ii) additionally express a cell adhesion molecule that recognizes a target on the surface of the first organizer cells and causes the first organizer cells to form a node via homotypic cell-cell interactions; or (c) a population of second organizer cells that: (i) express a cell adhesion molecule that recognizes a target on the surface of the progenitor cells and causes the second organizer cells to adhere to the cluster of progenitor cells via heterotypic cell-cell interactions; and (ii) do not express a cell adhesion molecule that recognizes a target on the surface of the second organizer cells, thereby allowing the first cells to form a shell around the progenitor cells.Atty. Dkt: UCSF-753WO Client Ref.: SF-2024-020-2-PCT 17. A method for assembling cells, comprising: incubating the composition of cells of any prior claim in a medium under conditions by which the cells self-assemble into the aggregate.

18. The method of claim 17, wherein at least one of populations of organizer cells expresses a morphogen and the method further comprises incubating the aggregate in a medium, thereby causing at least one of the progenitor cells to differentiate.

19. The method of claim 17 or 18, wherein the progenitor cells develop into an organoid.

20. A cell aggregate, comprising: (a) a cluster of progenitor cells; and (b) one or more structures composed of organizer cells that are engineered to express one or more cell adhesion molecules that mediate cell-cell interactions with the progenitor cells; wherein the cell aggregate is held together by cell-cell interactions.

21. The cell assembly of claim 20, wherein the one or more structures comprises: (i) a shell that at least partially surrounds the cluster of progenitor cells, and / or (ii) one or more nodes that are attached to the cluster of progenitor cells 22. The cell assembly of claim 20 or 21, wherein at least one of the one or more populations of organizer cells expresses a morphogen, and wherein the morphogen causes differentiation of one or more of the progenitor cells in the cluster.

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