Harnessing mesenchymal aggregation for engineered organ‐level regeneration: Recent progress and perspective

Stem cells, especially mesenchymal progenitors or mesenchymal stem cells (MSCs), possess an intrinsic property to form compact spheroid‐like assemblies, a phenomenon known as cell aggregation. In recent years, a growing body of researches have uncovered that this is a cross‐species conserved developmental event essential for initiating organogenesis in a variety of organs. Moreover, the self‐assembly property also contributes to the regenerative capacities of MSC aggregates in vivo with broad range of applications in tissue engineering. In this review, the principles of self‐assembled mesenchymal aggregation and its involvement in physiological organogenesis, as well as the construction approaches of engineering MSC aggregates and its application for organ regeneration are discussed. The authors aim to provide a speculative overview of the current understanding and the recent findings of cell aggregation, from both the developmental and the engineering perspectives, and thus offer insights into the understanding of stem cell biology and the establishment of novel organ regeneration strategies.


INTRODUCTION
Due to the increasing prevalence of trauma, congenital defects, and diseases, organ regeneration which ultimately involves the reinstitution of cellular and matrix integrity with restoration of physiological pattern and function of organs is in high demand.Over the last two decades, the rapid growth of stem cell research has been establishing an integrated understanding of cell fate specification and plasticity, leading to various regenerative approaches based on cellular reprogramming and stem cell transplantation. [1,2]eanwhile, advances in design, fabrication and manipulation of biomaterials, along with the emergence of 3D cell culture and bioprinting biotechnologies have substantially boosted stem cell-mediated tissue engineering practices with the promise to supply de novo regenerated organs in part or in toto. [3,4]Nevertheless, strategic and mechanistic challenges remain to be addressed before translational pro-gresses can be achieved for robust and effective clinical organ regeneration.
0][11] Notably, our understanding of the emergent principles by which the organs form through complex collective multicellular behaviours is greatly deepened by deciphering the early embryogenesis. [12,13]In general, the organs develop from densely-packed, integrated entity of progenitor clusters, termed the "organ buds", which are transformed from an assembly of loosely compacted immature cells, a process called self-assembled aggregation or condensation. [7,14,15] I G U R E 1 Mesenchymal aggregations in development (D) and regenerative or therapeutic applications (A).At the early embryonic stage, mesenchymal progenitors, or mesenchymal stem cells (MSCs) aggregate to initiate organogenesis.The mesenchymal aggregates may differentiate, undergo epithelial transition, or serve as the signalling niche for inducing epithelial patterning and driving co-aggregation of multiple lineage progenitors for organ bud formation.Accordingly, engineered MSC-based aggregates are established for extensive applications in tissue/organ regeneration and therapeutics.
[31][32][33] Intriguingly, after in vivo implantation, the MSC-based engineered aggregates are able to undergo subsequent differentiation, and induce vascularization and innervation putatively through paracrine mechanisms, implying recapitulation F I G U R E 2 Summary of the assembly mechanism and organogenetic function of mesenchymal aggregation.The mesenchymal progenitors or mesenchymal stem cells (MSCs) forming the mesenchymal aggregation are lineage-restricted while being heterogeneous, and their assembly depends on biochemical and biophysical regulation.Aggregated MSCs demonstrate collective behaviours and have been revealed as a representative self-organizing organogenetic centre, which not only dictates subsequent mesenchymal tissue formation and patterning, but also acts importantly as the signalling niche to orchestrate interlineage communication.
35][36] In this review, we discuss in detail the principles of selfassembled mesenchymal aggregation and how it dictates organogenesis in a variety of organs.We further highlight the engineering efforts aiming at harnessing this developmental program to regenerate functional and macroscale organ with recipient compatibility and sustainability, focusing on the construction approaches and the applicative effects.An overview of the current state-of-the-art update in development-inspired regeneration will help to establish translational promising strategies for clinical organ regeneration.

Specification and heterogeneity of mesenchymal progenitors
Most vertebrate organs are composed of the epithelium supported by mesenchymal stromal tissues that developmentally form by undifferentiated progenitor cells, namely the MSCs which continue to exist throughout the postnatal organism. [37,38]These cells, derived mostly from the mesoderm but also from the ectodermal neural crest in craniofacial regions, aggregate in the early embryonic stage to give rise to organs of the musculoskeletal system, such as the bone and the cartilage. [14]Aggregated MSCs further provide diverse niche signals to induce and pattern the adjoining epithelium in the developing tooth, foregut (which forms the lung, the intestine, et al.), urogenital ridge (which forms the kidney, the gonad, et al.), and integumentary system (which includes the skin, the hair, and the exocrine glands). [15,20,24,39,40]Typically recognized as highly proliferative, clonogenic, migratory, and multipotent primitive cells, MSCs are emergingly revealed to be lineage-restricted while being heterogeneous during organ development (Figure 2). [38,41,42]Understanding the specification and heterogeneity of these progenitors is thus a precedent for deciphering the principles of mesenchymal aggregation that underlie subsequent organogenesis.
Growing integrated efforts of single-cell analysis and lineage tracing studies have been providing a comprehensive picture of the MSC identities, their niche function and progenitor cell behaviours in developmental processes.Evidence from single-cell RNA sequencing reveals a surprising diversity of mesenchymal progenitor populations in organogenesis with stereotypic (e.g. in the skin and the intestine) or dynamic (e.g. in the lung) locations, patterns of movement and lineage boundaries, representing a mosaic of molecularly distinct cell compartments rather than a homogeneous pool of cells with broad potential (Figure 2). [38,39,41]Fate mapping of marked MSC subsets (e.g.][44][45] Intriguingly, as particularly shown in the hair follicle development, dermal MSCs are specified and aggregated prior to the niche fate acquisition for morphogenesis, which requires signals from pre-existing epithelial placodes, indicating the importance of interlineage regulation beyond a cell-autonomous process for mesenchymal aggregation. [10,39]Below we will further discuss the signals and the morphogenic principles driving mesenchymal aggregation.

Biochemical cues and the organizational mechanisms
Most of our understanding of the sequential and reciprocal interactions between the epithelium and the mesenchyme in directing organogenesis is originated from classical mesenchymal/epithelial tissue recombination assays in model organs, such as the hair follicle and the tooth. [46,47]Accumulated evidence reveals that while the "first signal" of morphogenesis is emanated from the mesenchyme to act on the unspecified epithelium, the subsequent formation of recognizable epithelial signaling centers, for example, the hair placodes and the tooth enamel knots, play essential roles to initiate and maintain mesenchymal aggregation for organ patterning and morphogenesis. [10,48]Early identification of diffusible morphogens and functional analysis of ligands and receptors, combined with experiments using gene knockout and spontaneous mutant mice, suggest that Wnt ligands (Wnts), Hedgehogs (HHs), fibroblast growth factors (FGFs), and bone morphogenetic proteins (BMPs) are key epithelial signals for synergistic induction of mesenchymal aggregation in the hair follicle, the tooth and the intestine (Figure 2). [10,24,39,49,50][56] Particularly in the limb skeleton and hair follicle morphogenesis, multiscale contributions, and interplay between two patterning mechanisms for mesenchymal aggregation are revealed.][56] Consequently, the MSCs exhibit haptotaxis to migrate till close surface contact and undergo cell shape changes to form a dense cell cluster, in which they exit the cell cycle and concomitantly acquire the progenitor and niche fates. [10,24,49,55,57]Meanwhile, mechanical compression occurs with deposition of the ECM, which is involved in the self-organization process of mesenchymal aggregation and will be discussed in the following section.

Mechanical compaction and ECM deposition
Mesenchymal progenitors/MSCs possess an internal potential to aggregate and during this process they are able to draw in adjacent interlineage cells to achieve mechanosensation and morphogenesis concomitantly (Figure 2). [7,15,22][60] In addition, cellular contractility generated by the actomyosin plays an important role in the spontaneous aggregation of mesenchymal progenitors, which accounts for the initiation of the primordium as follicles emerge. [22]Myosin-driven mechanical compaction of the mesenchyme is also critical for the folding of the epithelial-mesenchymal interface in the embryonic gut villus formation. [59]Furthermore, it has been revealed in the developing skeleton and tooth that compression within mesenchymal aggregation leads to RhoA activity and Rho kinase (ROCK)-mediated actin reorganization which participates in cell fate specification by modulating downstream molecular signalling. [15,61]Therefore, while it is still not possible to separate each effect induced by changes of cell size, shape, and density in mesenchymal aggregation, mechanotransduction through the cytoskeleton is one representative mechanism.
Biophysical stimuli that regulate cell behaviours are also defined by factors including tissue stiffness, geometry, and topology, some of which especially in the mesenchymal aggregates are provided by the ECM. [58]As the crucial noncellular component of tissues, the ECM stands as a scaffold for cellular adhesion and is involved in mechanotransduction and mechanoregulation, which plays indispensable roles in organ morphogenesis during embryonic development. [62]][65][66] The ECM formation is enhanced in mechanical compaction of MSCs, and the cell-ECM interactions are necessary for the boundary setting and the growth of the aggregation, and the fate acquisition of MSCs in the skeleton, the skin and the tooth. [15,21,59,63,64,66]Shyer and colleagues further reveal that an appropriate rigidity of ECM promotes the formation of mesenchymal aggregates, which competes with cellular contractility to mechanically resemble a Turing model of patterning in the morphogenesis of hair follicles, as confirmed by using MSCs in culture. [7,22]Collectively, principles of mesenchymal aggregation are uncovered at the cellular and molecular levels with regulation by both biochemical and biophysical cues to constitute the organizational patterns as a perquisite of organogenesis (Figure 2).

Collective behaviours of aggregated MSCs
Since the first report describing mesenchymal aggregation as a crucial feature of cartilage formation in 1925 by Fell, an increasing number of studies have focused on its characteristics, mechanisms and implications in the early F I G U R E 3 Self-assembled mesenchymal aggregates in dictating organogenesis.Critically attributed to the extracellular matrix (ECM) cue stimuli, metabolic switch and cell-cell interaction, mesenchymal aggregates show collective behaviours including increased multi-lineage plasticity, retained cellular stemness, promoted paracrine secretion, and spatiotemporal regulation of gene expressions.These behaviours underlie the key roles of mesenchymal aggregates as either the tissue progenitors or the organogenetic niche.For the tissue progenitor function, specifically in the skeleton development, mesenchymal aggregates undergo osteogenesis for mediating intramembranous ossification (IMO) or chondrogenesis for initiating endochondral ossification (ECO).In driving the nephrogenesis, the metanephric mesenchymal progenitors aggregate at the cap of the epithelial ureteric bud (UB) and transform into the pretubular aggregates for mesenchymal-epithelial transition (MET) to generate renal vesicles.For the organogenetic niche function, based on cytokine-or extracellular vesiclemediated inter-lineage communication, mesenchymal aggregates regulate specification and patterning of the epithelium in skin, hair, tooth, and exocrine gland morphogenesis.Mesenchymal aggregates also induce angiogenesis for organ bud formation.organogenesis of vertebrates. [14,19,67]It has been revealed that the proliferation activity of MSCs is density-dependent.During the aggregation process in vitro, the mitosis of MSCs is gradually suppressed with the convergence and cellcell contact of MSCs. [24,57,67]Researchers have constructed engineered MSC aggregates which partially mimic or reconstitute the key features of mesenchymal aggregation in vitro.Notably, despite the cellular heterogeneity, engineered MSC aggregates constitute of cohesive multicellular units that behave as a group (Figure 2). [7,15,68]70][71] With the recent discovery of the in situ signature of aggregated MSCs (e.g.FOXD1 and Tbx18 for dermal condensates, Pax9 for dental mesenchymal condensates) and the application of the fluorescence-activated cell-sorting (FACS) strategy, future work will be feasible to directly analyze collective behaviours of developing mesenchymal aggregates. [10,15,43,45]ide from the gene expression changes, studies have further suggested potential regulatory factors for the acquisition of homeostasis and the configuration of plasticity by mesenchymal aggregates. [14,19]It has been reported that the metabolic state of MSCs is dynamic and varies with cellular density (Figure 3).During aggregation process, the mitochondrial morphology of MSCs has been revealed to be changed, resulting in reduced mitochondrial membrane potential and controlled autophagic flux.Consequently, aggregated MSCs undergo a metabolic switch from oxidative phosphorylation to anaerobic glycolysis, which plays a crucial role in the enhancement of their in vivo properties and functions. [70,72,73]For mechanisms underlying the collective behaviours, researchers have focused on microenvironmental communication in engineered MSC aggregates, which is essential for the organization and function of multicellular systems and is orchestrated by multiple modes of signals (Figure 3).These communicational cues of aggregated MSCs mainly include substrate-mediated stimuli from the ECM and cell-cell interactions mediated by cytoskeletal actin microfilaments that can transfer cytoplasm and organelles, such as gap junctions (e.g.][76][77] Notably, although that paracrine cytokines are also secreted by MSCs and are involved in regulating collective decisions in the organ level, their roles in mesenchymal aggregation remain elusive. [78,79]he evidence indicates that mesenchymal aggregates preferentially use direct contact-based communication modes for concerted actions, which are readily accessible and highly efficient in this particular context.

Lineage commitment as the tissue progenitors
In this section, we will briefly summarize current knowledge of the roles and mechanisms of mesenchymal aggregates as the tissue progenitors in the development of representative organs: the bone, where they proceed to overt differentiation for osteogenesis and chondrogenesis; and the kidney, where they undergo a necessary mesenchymal-epithelial transition (MET) for nephrogenesis (see reviews for general information on the development of these organs [80,81] ) (Figure 2 and Figure 3).Especially, the embryonic bone formation occurs in two distinct processes, respectively intramembranous ossification (IMO) of flat bones (e.g. the skull), in which aggregated MSCs directly differentiate into osteoblasts started from the centre, and endochondral ossification (ECO) of long bones (e.g. the limb), in which aggregated MSCs firstly differentiate into chondrocytes to form a cartilage intermediate (Figure 3). [14,81]Lineage commitment of MSCs toward osteoblasts and chondrocytes is sequentially controlled by a series of master transcription factors, such as Runx2 and Osterix/Sp7 for osteogenesis and Sox9 and Col2a1 for chondrogenesis, and is orchestrated by multiple signalling including HH, Notch, Wnt, BMP, and FGF pathways. [81,82]][84][85] However, while the mechanisms underlying lineage commitment of MSCs have been revealed by many studies, how MSCs exit the aggregation state remains rather elusive in this field.
The mature kidney of mammals, that is, the metanephros, develop by the epithelial ureteric bud (UB) from the Wolffian duct, which forms the collecting system, and mesenchymal progenitors from the intermediate mesoderm (IM), which are programmed to undergo MET in response to inductive signals of the UB and form the nephrons (Figure 3). [80]In particular, the metanephric mesenchymal progenitors aggregate at the cap around the tips of epithelial branches of the UB and transform into the pretubular aggregates for MET to generate renal vesicles (Figure 3). [17,86,87]The MET process is notable for cell morphological changes with activation of epitheliumspecific genes, such as those encoding for cytokeratins and tight junctions, and inactivation of mesenchyme-featured genes, such as those encoding for collagens. [80,88]][91] Wnt9b, which is expressed in the UB, functions upstream of Wnt4, and effects of Wnts on aggregation and nephrogenesis of the metanephric mesenchymal progenitors are mediated by both the canonical and non-canonical pathways. [88,90]While other regulatory factors are involved in the MET, these studies establish a mechanistic framework that interlineage communication underlies the fate commitment of mesenchymal aggregates as the tissue progenitors in organogenesis. [80]

Interlineage communication as the organogenetic niche
][94] These organs share common molecular and cellular mechanisms of morphogenesis, as previously reviewed. [95,96]In the Avian skin, Shyer et al. reveals an intriguing mechanism whereby activation of the canonical Wnt signalling as indicated by nuclear translocation and Srcdependent phosphorylation of epidermal β-catenin, acts as a sensor of mechanical compression triggered by aggregation of dermal progenitors and leads to upregulation of the downstream follicle gene expression. [22]Ho et al. further unravel that an integrated BMP and mechanical signalling produced by aggregated mesenchymal progenitors regulates epithelial FGF20 to break the symmetry for periodic patterning of feathers. [49]In the mouse skin and hair follicle development, epithelial Wnt signalling is also regulated by Wnt ligands and modulators derived from the aggregated mesenchymal progenitors. [23,94]xcept morphogens and mechanical stimuli, extracellular vesicles (EVs), which are cell-released membrane-bounded nanoparticles with various cargos, also plays a pivotal role in mediating the interlineage crosstalk during organogenesis (Figure 3). [97,98]In specific, exosomes, the most widely studied population of EVs, have been reported to transport mesenchyme-derived microRNAs to the epithelium in morphogenesis of the tooth and the salivary gland. [92,93]In the developing tooth, miR-135a-carrying exosomes are diffused through the basement membrane and are endocytosed by cells of the epithelium or the mesenchyme with reciprocal preference instead of self-uptake for regulation of cell differentiation and ECM synthesis, which is disrupted in vivo by genetic or pharmacological attenuation of exosomal secretion. [93]In the developing submandibular gland, exosomal miR-133b-3p is delivered from mesenchymal aggregates to the epithelium, in which they regulate DNA methylation to potentiate epithelial progenitor cell proliferation. [92,97]Mesenchymal aggregation-based interlineage crosstalk in organogenesis additionally involves angiogenic regulation in organ bud formation (Figure 3).While the forming of mesenchymal aggregation requires vascular regression, the avascular aggregated MSCs are capable of regulating vascular patterning via a direct Sox9-governed, vascular endothelial growth factor (VEGF)-based long-range mechanism, which coordinates vascular and skeletal development in the developing limb buds. [16,99]Vascularization and innervation also spatiotemporally accompany with mesenchymal aggregation in the tooth germ, suggesting a common mechanism for organ bud formation. [100]03]

ENGINEERING MESENCHYMAL STEM CELL AGGREGATION FOR ORGAN REGENERATION: APPROACHES AND EFFECTS
The developmental processes of organs have established promising framework for the design and engineering of a myriad of regenerative organ constructs, which holds promise of recapitulating the desirable organogenetic traits including the physiological anatomy, regulatory mechanisms and functional robustness. [4,26]Notably, proof-of-concept studies have demonstrated that the key features of mesenchymal aggregation, an early critical event in organogenesis, have been to some extent reconstituted in vitro and the resulting engineering constructs exert potent regenerative potential in multiple major organs in animals, with positive clinical outcomes being achieved firstly in the oral system (Table 1). [7,15,29,33,59]We have recently proposed the word "cytocondensoids" to conceptually define the engineered aggregate-mimetic cell assembly, [18] which are an in vitro constructed entity in 3D compacted forms composed of single (homotypic) or multiple (heterotypic) cellular and noncellular elements (e.g. the ECM).In specific, engineered aggregates are semi-autonomous and primitive since they are constructed through stem cell self-assembly facilitated by mechanochemical cues without induction of morphogenesis in vitro and usually not involving genetic, epigenetic or pharmacological modification of MSCs, thus possessing the potential to initiate regenerative cascades imitating organogenesis in vivo (Figure 4).Given the findings that implanted engineered aggregates are able to integrate with the host niche and coordinate host interaction which concertedly restore the components, architecture, and function of defected organs, they are highly anticipated to become a novel paradigm for organ replacement therapy, tissue defect repair, and disease treatments.

Homotypic engineered aggregates generated by self-assembly of MSCs
After embryogenesis, MSCs continue to reside in a wide spectrum of postnatal organs as archetypal multipotent progenitors for tissue repair and homeostatic maintenance. [37,42]espite rare in situ, these cells are easily accessible, readily proliferative and characterized by stem cell properties in vitro, and are capable of multi-differentiation and environmental regulation with limited immunogenicity or tumorigenicity when transplanted in vivo, thus having become the ideal subject of extensive investigation in tissue engineering and regenerative medicine. [1,104]Notably, cultured MSCs at high cell density demonstrate potent and unique capacity of self-organizing aggregation with increasing cell-cell and cell-ECM interactions, which upon appropriate biochemical and biophysical cues will develop into the homotypic engineered mesenchymal aggregates. [6,29,105][111][112] These characteristics make the diverse forms of engineered mesenchymal aggregates particularly advantageous for translational applications (Figure 4).
Although a number of methodologies have been reported for 3D culture and engineering of stem cells (reviewed in reference [113]), as a perspective and function-oriented strategy connecting organ development and regeneration, engineered mesenchymal aggregates summarized in this study will be based on integrated techniques with clear reference to cell aggregation in organogenesis and identifiable outcomes in regenerative applications (Table 1).In this regard, the combinatorial use of morphogenetic and mechanical cues along with support of suitable synthetic ECM scaffolds has been particularly tested.The group led by Boerckel and Alsberg have mimicked the cellular organization and lineage progression of the early limb bud using self-assembled MSC aggregates, which are engineered in vitro in response to local TGF-β1 and/or BMP-2 incorporated into gelatin microspheres or mineral-coated microparticles and are primed to ECO by dynamic mechanical loading in vivo. [6,29,114]Even without morphogen applications, spheroid MSC aggregation in AggreWells with bioactive hydrogel and hydroxyapatites has been reported to recapitulate IMO, and engineered mesenchymal aggregates assembled in development-emulated ECM demonstrate strengthened chondrogenesis and osteogenesis in vivo. [110,115,116]Mechanochemical induction of mesenchymal aggregation in skeletogenesis has also been revealed to be leveraged by fabricated scaffolds. [117,118]With differential up-bottom or bottom-up design approaches being developed, engineered mesenchymal aggregate-biomaterial composites hold the potential to build up millimetre-sized geometrically shaped viable organ constructs in future studies. [119,120]omotypic engineered mesenchymal aggregates can form by forced aggregating methods in vitro without xenogeneic materials, thus being safe and biocompatible for the translational use (Figure 4).][123][124] In specific, MSCs derived from different species and sources have been used, which are enriched for specific subsets and delivered via multiple Engineering mesenchymal aggregation for organ regeneration.The engineered mesenchymal aggregates are an integrated, 3D structured, semi-autonomous (forming by mechanochemical cue-facilitated stem cell self-assembly), and primitive cell collections, which possess the potential of initiating regenerative cascades akin to organogenesis in vivo.Accordingly, the application of engineered mesenchymal aggregates for regenerating functional and endurable macroscale organs requires both the reestablishment of physiological tissue pattern and the ultimate integration of graft into the recipient organism.Engineered mesenchymal aggregates are assembled by single (homotypic MSCs) or multiple (heterotypic) cellular types through various different approaches.
routes, and morphogens and the bioprinting technique can further be applied for construction of scaffold-free mesenchymal aggregate graft (Table 1). [125,126]Of note, the above methods use engineered extrinsic stimuli to promote MSC assembly, for which the resulting mesenchymal aggregates are lack of abundant ECM due to excessive spatial confinement or short cultural duration. [107,127]ntriguingly, during the self-assembly process on culture dish surfaces, MSCs possess innate ability to deposit considerable amount of ECM, thus producing mesenchymal aggregates with more physiologically relevant features and are favourable for regeneration application (Figure 4).][130] Briefly, MSCs are culture-expanded, and the second or third passage cells are digested and seeded into the six-well plate.When the cells achieved approximately 80% confluence, the culture medium was changed to inducing medium containing 100 μg/mL vitamin C, which was refreshed every 2 days.After 7-10 days of incubation, the engineered mesenchymal aggregates that are dense and integrated assemblies are observed.Additionally, a series of small molecular compounds, such as resveratrol and melatonin, can be used to optimize the stemness and function of aggregated MSCs.][133][134][135][136] It should be noted that  [132] Copyright 2021, Elsevier Ltd.
several important factors should be taken into account when choosing the source cells.First, whether the cells express classic MSC markers, and are highly proliferative and able to self-aggregate.Second, whether the cells possess the potential to differentiate into target cell types.Third, whether the cells are safe, abundant, and non-invasively/easily harvested.Last but not least, whether the cells are taken from autologous, allogenic, or xenogeneic sources.In this aspect, the condition of the donors is also an important factor.Molecular profiling of the various homotypic engineered MSC aggregates and detailed mechanistic investigations on their assembly and fates in comparison with the developing mesenchymal aggregates will further enlighten plausible engineering construction strategies and set up feasible organspecific engineered mesenchymal aggregates for macroscale regeneration.

4.1.2
Heterotypic engineered aggregates formed by MSC-supported co-aggregation Started from the 1960s, the organoid technology based on self-organization and induced lineage specification of pluripotent stem cell aggregates in vitro has provided an evolving paradigm toward regenerative therapy but has yet to be applied in humans, mainly because of the reproducibility and scalability challenges. [4,137]While organoids are miniatures recapitulating many aspects of mature organs or tissues, the engineered aggregates that we propose as "cytocondensoids" are mimics of developmental mesenchymal aggregates.Recently, emerging studies have demonstrated that large-scale self-organized primitive cell assemblies can be easily and robustly obtained through MSC-supported aggregation in optimal heterogeneous cell mixtures and be effectively used to potentially recapitulate organogenesis and achieve functional organ regeneration in vivo after transplantation (Table 1).In this regard, fundamental experiments have revealed that MSC-dependent cytoskeletal contraction and ECM deposition act as mechanical actuators of folding and tissue architecture reconstitution by complex cell compositions, in which incorporated cells serve as "passengers" of the mechanical stimuli for collective aggregation driven by postnatal MSCs. [7,59]Pioneer studies have also isolated specific mesenchymal and epithelial progenitors from the developing organs in situ and recombine them in culture to induce MSC-based spontaneous co-aggregation with patterning and morphogenic potential being preserved. [15,25]ccordingly, respective heterotypic engineered aggregates have been established via the "organ bud" and "organ germ" methodologies, as proposed by the Taniguchi and Takebe's group and the Tsuji's group (Figure 4). [7,25]s regional vascularization is of vital importance for the survival of regenerative implants and the functional integration with hosts, researchers have applied undifferentiated progenitor-derived heterotypic engineered aggregates formed by MSC and human umbilical vein endothelial cell (HUVEC) mixture spheroids for promoting angiogenesis and regeneration in vivo (Figure 6). [34,138]The mesenchymal progenitors used have been derived from specific tissues (e.g.cartilage progenitors), and the tissue specimens are adaptable for en bloc cryopreservation by a slow-freezing method to isolate cells after 30 days, which potentially facilitate the application and tissue-preference of this regenerative approach. [34]The MSC-HUVEC collective is able to further incorporate induced pluripotent stem cell (iPSC)committed progenitors, embryonic organ-specific progenitors, or dissociated functional adult cells, and spontaneously co-aggregate into macroscopically visible three-dimensional cell clusters, thus generating heterotypic engineered aggregates mimicking the organ buds for provoking concomitant differentiation and driving organogenesis with vascularization (Figure 7). [7,8,32,35]Ratios of the numbers of respective progenitor cells, culture time durations, and substrate stiffness should be context-dependently optimized. [7]Moreover, diverse tissue fragments from adult animals or human iPSCderived organoids can be applied for co-aggregation with MSCs-HUVECs for efficient regenerative applications. [31]SCs used for the organ bud aggregates engineering may also be specified from iPSCs. [5,32]Particularly of note, despite the involvement of iPSCs, in vitro morphogenic induction is dispensable for engineered aggregate establishment and regenerative application.These results indicate that the construction of heterotypic engineered aggregates share technical compatibility with the organoid technology, such as the cell aggregation methods, through which the primitive organ buds generated through the aggregation principle without advanced maturation might be a highly efficient approach toward the reconstitution of mature organ function after transplantation (Figure 4). [7]hile the homotypic engineered MSC aggregates are mainly available for regenerating mesodermal tissues/organs, the organ bud engineering is feasible for regenerating endodermal tissues/organs, including the liver, the lung, the intestine and the pancreas, and also conceptually proved for the kidney, the heart, and the brain regeneration (Table 1).The organ germ technology, on another front, is applicable for the regeneration of ectodermal appendage organs. [25]][141][142] Then, a 3D cell-manipulation method that involves sequential injection of MSCs and epithelial progenitors into collagen gel drops is used to reconstitute bioengineered germ-mimetic aggregates with the correct tissue compartmentalization, which are subjected to organ culture for several days in vitro before transplantation (Figure 8). [30,139,140]In specific, for hair follicle regeneration, the germ-like aggregates can be originated from cells of the neonatal or adult epidermis, dermis or follicles, but adult cells may show diminished efficacy of condensation and might follow distinct mechanisms of regeneration. [68,141,143]mportantly, the dermal condensation is further recapitulated in the heterotypic engineered aggregates, and the number of regenerated follicles is linearly correlated with that of the condensed MSC aggregates. [141]To this far, although that the multiple engineered aggregates established by the in vitro self-organization process is not identical to the biological aggregates in embryonic organ formation, they potentially harness the developmental aggregation principles and will be expected to reconstitute functional organs in vivo (Figure 4).

Organ regeneration mediated by engineered mesenchymal aggregates
In this section, we will focus on the regenerative outcomes that have been achieved by harnessing mesenchymal condensation (Table 1).According to the practice of regenerative medicine, we recommend that the achievement of functional and endurable macroscale organ regeneration requires both the re-establishment of physiological tissue pattern and the ultimate integration of graft into the recipient organism.In this regard, the engineered aggregates which remain primitive in culture and are able to initiate organogenesis as progenitors and coordinate recipient components as the signalling niche after transplantation, provide a promising paradigm for accomplishing the above goals (Figure 4).

TA B L E 1
A list of regenerative and therapeutic outcomes achieved by harnessing mesenchymal aggregation.

Homotypic engineered aggregates in tissue regeneration and niche regulation
Engineered MSC aggregates are primarily applied in mesodermal tissue/organ regeneration, such as the cartilage and the bone (Table 1).As shown, transplanted engineered mesenchymal aggregates undergo chondrogenesis in situ to repair full thickness critical-sized rabbit articular cartilage defects. [117]In critical-sized bone defects of rat femora, transplanted engineered mesenchymal aggregates further enhance endochondral bone regeneration while responding to mechanical stimuli, during which temporal effects of chondrogenic lineage commitment, bone matrix formation, and recipient vascular invasion are detected. [6,29] (A-E) Reproduced with permission. [7]Copyright 2015, Elsevier.
Engineered mesenchymal aggregates are also used for promoting critical-sized calvaria defect healing in rodents with regulation of osteogenesis, ECM deposition, and vascularization via the canonical Wnt signalling. [114,116,118,135,144][135] The communicational properties of transplanted engineered mesenchymal aggregates based on enhanced paracrine mechanisms are widely used in orchestrating recipient components in disease therapies (Table 1).The crosstalk between transplanted aggregates and recipient immune components further leads to amelioration of inflammatory tissue damages, including murine experimental colitis, peritonitis, and  (A-E) Reproduced under the terms of the Creative Commons Attribution 3.0 Unported Licence (http://creativecommons.org/licenses/by/3.0/). [30]Copyright 2013, The Author(s).
rhesus osteoarthritis, which might contribute to a beneficial microenvironment for tissue restoration. [106,108,123,135]][147][148][149] Therefore, introduced by the intrinsic MSC characteristics and strengthened during the aggregation process, the niche regulation efficacy of engineered mesenchymal aggregates has been potentially applied beyond regenerative organ defect healing to providing extensive translational therapeutics.
In the practice of dental regeneration, engineered mesenchymal aggregates have been revealed to re-exhibit tooth morphogenesis and undergo dentinogenesis after transplantation, which show efficacy in reconstructing the pulp/dentine complex and the periodontal ligament/bone composite. [129,136,150]Chen et al. further identify CD24a as a marker of a group of multipotent sphere-forming MSCs which reside in the mouse tooth germ and the human dental papilla, and are able to regenerate the pulp/dentine complex in ectopic root canals in vivo. [125]Moreover, our group have unravelled that the application of engineered mesenchymal aggregates based on autologous human deciduous pulp stem cells (hDPSCs) that are a population of highly proliferative, clonogenic cells with high differentiation potential reinstitutes physiological pulp pattern with odontoblasts lining the dentin wall in miniature pigs, thus verifying the efficacy for tooth pulp regeneration in the large animal. [33]We have further performed randomized clinical trial of school-aged patients with diagnosis of a traumatized permanent incisor tooth.Briefly, hDPSC aggregates are washed three times with phosphate buffered salin (PBS), resuspended in saline and transferred to the clinic on ice.Then, the periapical tissue is provoked to bleeding in the cervical portion of the root canal, and the hDPSC aggregates are collected and implanted into the root canal, with a plunger being used to pack the hDPSC aggregates into the apical end of the root.Results have shown that transplanted engineered mesenchymal aggregates successfully and safely regenerate physiologic-relevant whole pulp tissue, with ingrowth of blood vessels and innervations during the 12-month period. [33]The neovascularization might be attributed to EVs secreted by transplanted aggregated MSCs and encapsulating functional microRNAs and proteins. [36,151]Notably, the regenerated dental pulp exhibits normal function of maintaining continued root development as the length of the root is increased and the width of the apical foramina is reduced. [33]Furthermore, Guo et al. applied combination of engineered mesenchymal aggregates and decellularized tooth matrix (DTM) to regenerate 3D pulp and periodontal tissues equipped with vasculature and innervation of avulsed tooth after implantation into the alveolar bone in both preclinical pig model and clinical patients (Figure 5). [132]These results provide the pioneering clinical evidence that transplantation of homotypic engineered mesenchymal aggregates regenerates a de novo complete human organ with functional recovery (Table 1).

4.2.2
Heterotypic engineered aggregates in proof-of-concept organ regeneration The heterotypic MSC-driven organ bud aggregates are remarkably promising for life-maintaining solid organ regeneration (Table 1).Particularly, condensed liver buds composed of human MSCs, HUVECs, and iPSC-specified hepatic endoderm cells potentially recapitulate organogenetic interactions between endothelial and mesenchymal progenitors, and rapidly connect to the host vessels after implantation, which stimulates the maturation of engineered aggregates into tissues resembling the adult liver. [35]Importantly, the integrated liver performs specific functions including albumin production and drug metabolism, and it rescues the lethal liver failure when transplanted on the mesentery in mice. [35]ransplantation of organ bud aggregates also induces physiologically patterned and vascularized kidney formation with functional recovery of filtration and collection (Figure 7). [7]oreover, for pancreas islet regeneration, transplantation of aggregated MSCs and HUVECs with β cells or islet tissue fragments successfully regenerates islet-like tissue with restored tissue structure, insulin secretion, and functional vasculatures connected to the recipient circulatory system, which are thus effective under the mouse renal capsule in alleviating type 1 fulminant diabetes and promoting survival rates. [7,31]n addition, engineered aggregates established by HUVECs and adult cartilage progenitors, the specialized postnatal MSCs, reconstruct elastic cartilage with microvascular networks upon transplantation. [34]With both scalability and extensive availability, the organ bud aggregates have paved an avenue for future translational studies in large animals and in human.
Regeneration of the ectodermal appendage organs has been reported to be achieved by the organ germ aggregates for replacement therapies (Table 1).It has been reported that transplantation of germ aggregates into the alveolar bony holes in the lost tooth regions regenerates fully structured and functional bioengineered incisor and molar teeth. [25,140,142]otably, the regenerated teeth not only possess correct morphology and structure of hard and soft tissues with vasculatures and innervations, but also can erupt, establish occlusion, support mastication, respond to mechanical stimulation, and transduce neural perceptions. [25,140]The germ aggregate-bioengineered hair follicles also develop the correct structures and forms proper connections with surrounding host tissues, such as the epidermis, the muscle, and nerve fibres, which restore hair cycles and piloerection through the rearrangement of follicular stem cells and their niches. [25,141]Moreover, functional salivary and lacrimal glands are regenerated by engineered germ aggregates, which develop in vivo after transplantation and achieve sufficient functionality to fulfil physiological saliva and tear production (Figure 8). [30,139]Furthermore, transplantation of preassembled 3D spheroids of MSCs and HUVECs without tissue progenitors also exhibit notable proneurogenic, proangiogenic and neuroprotective effects in the brain of an ischemic stroke mouse model, resulting in favourable structural and motor function recovery (Figure 6). [138]Despite the acquired intriguing outcomes, it should be noticed that the translational application of this method is largely limited by the originality of mesenchymal and epithelial progenitors from the developing organ germs.Therefore, this methodology should be recognized as proof-of-concept and thus require substitute strategies for application, such as using the secretome or EVs for aggregate-extended cell-free regenerative therapeutics. [36,152]

CONCLUSIONS AND PERSPECTIVES
[155][156] Within recent years, advances in conceptual and mechanistic understanding as well as in research technology have dramatically promoted the crosstalk between developmental biology and regenerative medicine, leading to the emergency of the developmental tissue engineering that provides a promising paradigm for regenerating injured or diseased tissues and organs. [26,27,157]otably, self-assembled mesenchymal aggregation has drawn increasing attention, during which mesenchymal progenitors forms a compacted group of primitive cells with abundant ECM and contributing to both formation of mesenchymal tissue and orchestration of cross-lineage progenitor fate specification. [7,14,15]This has been gradually recognized as a cross-species conserved developmental event essential for initiating organogenesis in a variety of organs.Learning from the developmental aggregation program, accumulating engineering efforts have been devoted into the establishment of multiple types of engineered aggregates based on MSCs, which is broadly classified into homotypic aggregates generated by self-assembly of MSCs and heterotypic aggregates formed by MSC-supported co-aggregation with other lineage progenitors.While remaining immature in vitro, engineered mesenchymal aggregates exhibit high developmental plasticity and integrate well with the host components after transplantation. [6,30,31,33]Based on the current evidence, engineered mesenchymal aggregates hold high potential for robust macroscale organ regeneration with physiological hierarchical patterns and function.
It should be noted that many issues remain to be resolved before the optimized translational application of engineered mesenchymal aggregates.First of all, the physiological mechanistic basis of self-assembled aggregation formation and aggregation-mediated organogenesis is not fully understood.For instance, given the heterogeneity of MSCs, how distinct cells collectively coordinate to form aggregation, and the changes of heterogeneity during and after the aggregating process is unclear.Also, the biochemical and biophysical mechanism mediating the self-assembled aggregation of MSCs, and their crosstalk with the niche and other lineages remain elusive.In addition, despite the various methods to construct engineered mesenchymal aggregates, the cellular, and molecular states of in vitro constructed aggregates in comparison with the embryonic organ-specific aggregates need to be further revealed, which will facilitate the development of optimal engineering approach.Moreover, the in vivo regenerative integration behaviour of implanted engineered mesenchymal aggregates, especially how they integrate with recipient components and co-ordinately reinstitute the vasculo-immuno-neural niche, is largely unknown.Last but not least, clinical trials that are designed with well-structured protocol and standardized procedures for data collection and analysis, as well as maintain rigorous monitoring and quality control, and effective communication with participants, are in great need to promote the translational application of engineered mesenchymal aggregates.With the benefits from the progress of new technologies, such as single-cell and spatial multi-omics technologies, high-throughput livecell imaging, and stem cell gene editing, we are provided with tools to further elucidate the biological mechanism and optimize the engineering techniques, [41,158,159] which are expected to provide readily translational engineered mesenchymal aggregates for functional regeneration of robust clinical organs.

C O N F L I C T O F I N T E R E S T S
The authors declare no conflicts of interests.

F I G U R E 5
Engineered aggregates by mesenchymal stem cell (MSC) spontaneous aggregation for functional tooth regeneration.(A) Schematic illustration of the construction strategy of the bioengineered tooth based on spontaneous aggregation of homotypic MSCs.(B, C) Representative hematoxylin and eosin (H&E) (upper) and Masson's (lower) staining images of the bioengineered incisor teeth at 6 months after implantation into alveolar bone of minipigs.Scale bars, 500 μm in low-magnification images as well as 30 μm (left) and 10 μm (right) in high-magnification images.(D, E) Representative cone-beam computed tomography (CBCT) (D) and 3D (E) images of the bioengineered incisor teeth at 12 months after implantation into root canal space of clinical patients.(A-E) Reproduced under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International license (https://creativecommons.org/licenses/by-nc-nd/4.0/).

F I G U R E 6
Engineered aggregates by mesenchymal stem cell (MSC) and human umbilical vein endothelial cell (HUVEC) co-aggregation for brain tissue regeneration.(A) Representative fluorescence images of engineered aggregates containing mixed red fluorescence protein (RFP)-positive MSCs (red) and CD31-positive HUVECs (green), with interconnected vessel-like structures formed within the engineered aggregates.Scale bars, 100 μm.(B) Representative fluorescence images showing the production of abundant extracellular matrix proteins, including collagen, fibronectin, and laminin, in the engineered aggregates.Scale bars, 100 μm.(C) Representative fluorescence images showing the production of abundant paracrine factors, including brain-derived neurotrophic factor (BDNF), vascular endothelial growth factor (VEGF), insulin-like growth factor-1 (IGF-1), hepatocyte growth factor (HGF), stromal cell-derived factor-1 (SDF-1), and tissue inhibitor of matrix metalloproteinase-3 (TIMP-3) in the engineered aggregates.Scale bars, 100 μm.(D) Representative magnetic resonance imaging (MRI) images of the brain tissue at 1 day and 14 days after administration of the engineered aggregates in a permanent distal middle cerebral artery occlusion mouse model as well as the corresponding quantitative analysis.(E) Representative crystal violet-stained serial coronal brain sections at 14 days after administration of the engineered aggregates as well as the corresponding quantitative analysis.Scale bar, 1 mm.(

F I G U R E 7
Construction of vascularized organ buds as heterotypic engineered aggregates.(A) Schematic illustration of the construction strategy.(B) Representative macroscopic images of the engineered multiple organ buds before (upper) and after (lower) aggregation.(C) Representative macroscopic images of the aggregated β cells, β cells and mesenchymal stem cells (MSCs), as well as β cells, MSCs, and human umbilical vein endothelial cells (HUVECs).Scale bars, 1 mm.(D) Representative fluorescence images of the engineered organ bud containing β cells (red), HUVECs (green), and unlabelled MSCs.Scale bars: 250 μm (left) and 100 μm (right).(E) Representative macroscopic and fluorescence images of the transplanted β cell-derived aggregates (upper two rows) and the HUVEC-absent aggregates (lower two rows).

F I G U R E 8
Engineered organ germ aggregates for functional salivary gland regeneration.(A) Representative phase-contrast images showing the construction strategy of a bioengineered salivary gland germ.Scale bars, 200 μm.(B) Representative phase-contrast images of the natural (upper) and the bioengineered (lower) submandibular glands on days 0, 1, 2, and 3 of organ culture.The submandibular glands were analyzed by hematoxylin and eosin (H&E) staining on day 3 of organ culture.Scale bars, 200 μm.(C) Schematic illustration of the transplantation procedure of the bioengineered salivary gland germ with the interepithelial tissue connecting plastic method.(D) Representative macroscopic images of the natural (left) and the transplanted bioengineered submandibular glands (middle and right) at days 0 and 30 post-transplantation, with FITC-conjugated gelatine injected from the host parotid duct (right).Scale bars, 1 mm.(E) Representative H&E staining (left three) and Periodic acid-Schiff (PAS) staining (right two) images of the natural (upper) and bioengineered (lower) submandibular glands.Higher magnification images in each box area are shown on the right side to the source images.Scale bar, 100 μm (left column from the left) and 25 μm (second and subsequent columns from the left).
Chen-Xi Zheng and Bing-Dong Sui contributed equally to this work.This work was supported by grants from the National Key Research and Development Program of China (2022YFA1104400), the National Natural Science Foundation of China (81930025, 82301028 and 82371020), the China Postdoctoral Science Foundation (BX20230485), and the Shaanxi Youth Science and Technology Rising Star Program (2023KJXX-027).