Symposium SM01—Materials Modulating Stem Cells and Immune Response

Intestinal organoids capture essential features of the intestinal epithelium such as folding of the crypt, spatial compartmentalization of different cell types, and cellular movements from crypt to villus-like domains. Each of these processes and their coordination in time and space requires patterned physical forces that are currently unknown. In this study, we map the three-dimensional cell-ECM and cell-cell forces in mouse intestinal organoids grown on soft hydrogels. We show that these organoids exhibit a non-monotonic stress distribution that defines mechanical and functional compartments. The stem cell compartment pushes the ECM and folds through apical constriction, whereas the transit amplifying zone pulls the ECM and elongates through basal constriction. Tension measurements establish that the transit amplifying zone isolates mechanically the stem cell compartment and the villus-like domain. A 3D vertex model shows that the shape and force distribution of the crypt can be largely explained by cell surface tensions following the measured apical and basal actomyosin density. Finally, we show that cells are pulled out of the crypt along a gradient of increasing tension, rather than pushed by a compressive stress downstream of mitotic pressure as previously assumed. Our study unveils how patterned forces enable folding and collective migration in the intestinal crypt.

How adhesive forces are transduced and integrated into biochemical signals at focal adhesions (FAs) is poorly understood. Using cells adhering to deformable micropillar arrays, we demonstrate that traction force and FAK localization and traction force and Y397-FAK phosphorylation are linearly coupled at individual FAs on stiff, but not soft, substrates. Similarly, FAK phosphorylation increases linearly with external forces applied to FAs using magnetic beads. This mechanosignaling coupling requires actomyosin contractility, talin-FAK binding, and full-length vinculin that binds talin and actin. Using an in vitro 3D biomimetic wound healing model, we show that force-FAK coupling coordinates cell migration and tissue-scale forces to promote microtissue repair. A simple kinetic binding model of talin-FAK binding interactions under force can recapitulate the experimental observations. This study provides insights on how talin and vinculin convert forces into FAK signaling events regulating cell migration and tissue repair.

Human induced pluripotent stem cells (hiPSCs) attract interest to develop patient-specific therapeutic tools for their clinical applications [1]. The in vitro culture of hiPSCs with high quality is dependent on the signals from their extracellular environments [2, 3]. In this study, we explore the effect of cyclic cold shock on hiPSCs behavior. A computer-controlled thermochamber was employed to exert cyclic cold stress on hiPSCs, in which temperature was alternately varied between 10 °C and 37 °C, whereby each temperature was kept for 30 min. The expression of pluripotent markers was evaluated 24 h after culture either under constant temperature at 37 °C or under cyclic temperature changes (ΔT). An atomic force microscope (AFM) was used to detect changes in cellular mechanics and the decellularized extracellular matrix (ECM). Immunofluorescence analysis showed that the expression of pluripotent markers, NANOG and SOX2, was maintained within 24 h of culture. A slight change in Young's modulus of hiPSCs cultured under different conditions was observed. This observation might be a result of an alteration in the arrangement of the actin cytoskeleton revealed by the immunofluorescence analysis. Notably, the topography of decellularized ECM showed an increase in surface roughness in the ΔT group. The average surface roughness (Ra) and roughness mean square (Rms) of decellularized surfaces under the ΔT condition were 31.56 ± 2.82 nm and 38.07 ± 3.58 nm, while under the constant temperature were 39.52 ± 2.83 nm and 49.14 ± 4.23 nm. This result suggested that the component and/or the arrangement of ECM protein was altered by the cold stress. This was supported by the enzyme-linked immunosorbent assay (ELISA), which showed the cyclic cold shock promoted the secretion of laminin. The presented findings provide an insight into the interactions between hiPSCs and factors of their microenvironment, which would contribute to a better understanding of the interactions between hiPSCs and their local microenvironment.

Reference
1. Rowe, R.G. and G.Q. Daley, Induced pluripotent stem cells in disease modelling and drug discovery. Nature Reviews Genetics, 2019. 20(7): p. 377-388.
2. Garreta, E., et al., Fine tuning the extracellular environment accelerates the derivation of kidney organoids from human pluripotent stem cells. Nature materials, 2019. 18(4): p. 397-405.
3. Han, U., et al., Construction of nano-scale cellular environments by coating a multilayer nanofilm on the surface of human induced pluripotent stem cells. Nanoscale, 2019. 11(28): p. 13541-13551.
1. Rowe, R.G. and G.Q. Daley, Induced pluripotent stem cells in disease modelling and drug discovery. Nature Reviews Genetics, 2019. 20(7): p. 377-388.

Colonies of induced pluripotent stem cells (iPSCs) reveal aspects of self-organization even under culture conditions that maintain pluripotency. To investigate the dynamics of this process under spatial confinement, we used either polydimethylsiloxane (PDMS) pillars or micro-contact printing of vitronectin. There was a progressive upregulation of OCT4, E-cadherin, and NANOG within 70 µm from the outer rim of iPSC colonies. Single-cell RNA-sequencing demonstrated that OCT4^high subsets have pronounced up-regulation of the TGF-β pathway, particularly of NODAL and its inhibitor LEFTY, at the rim of the colonies. Furthermore, calcium-dependent cell-cell interactions were found to be relevant for the self-organization. Interestingly, after 5 to 7 days, the iPSC colonies detached spontaneously from micro-contact printed substrates to form 3D aggregates. This new method allowed generation of embryoid bodies (EBs) of controlled size, without any enzymatic or mechanical treatment. Within the early 3D aggregates, the radial organization and differential gene expression continued in analogy to the changes observed during self-organization of iPSC colonies. Our results provide further insight into the gradual self-organization within iPSC colonies and at their transition into EBs.

Panel discussion

Hydrogels are the ubiquitous three-dimensional networks that can store a great amount of water in their porous scaffolds, holding great potential for bio-related applications.[1] In particular, their biophysical similarity to living tissues and extracellular matrix has made them valuable for tissue engineering, drug/gene delivery, biocatalysis, and regenerative medicine. However, the preparation of robust hydrogels in a mild fashion remains a technical challenge. Typically, hydrogels are prepared by physical interactions or chemical reactions. However, physically crosslinked hydrogels are generally limited by the instability due to their weak interactions, while chemical approaches are often associated with the problems of involving toxic reagents and harsh reaction conditions. Therefore, there has been a quest to establish a reliable and efficient methodology to construct stable hydrogels under physiological conditions.

Here, we demonstrate an enzymatic approach to creating hydrogels to encapsulate living cells and biocatalytically active enzymes for regenerative therapy, catalysis, and antibacterial study, respectively.[2-4] In the first study, the hydrogels were prepared using horseradish peroxidase (HRP) to enzymatically crosslink phenolic derivatized dendritic polyglycerol polymer into hydrogels in a buffer.[2] Our results showed that the resultant gels were not only biocompatible to fibroblast murine cells but also allowed for their encapsulation with high viability over 48 hours. This successful demonstration suggests the potential of our hydrogels for future use in regenerative medicine. Furthermore, we used the same enzymatic crosslinking method and materials to prepare nanogels for the encapsulation of HRP and Candida antarctica lipase B, respectively.[3] The advantage of this method is the mild condition that maintains the good enzyme activity, and more importantly, the process doesn’t require any quench steps, thus substantially simplifying purifications. Therefore, this study proves a new concept of “enzymatic nanogelations” for biocatalysis. Lastly, the enzymatically crosslinked hydrogels were explored for antibacterial applications.[4] Similar to the previous, hydrogels were crosslinked on glass slides by HRP, but differently, a second enzyme, glucose oxidase (GOD), was co-encapsulated. The existence of GOD could generate toxic H2O2 when supplying glucose, and kill Pseudomonas putida (Gram-negative) by 100% and Staphylococcus aureus (Gram-positive) by ≥40%, respectively.

Overall, we show a facile method to prepare hydrogels using enzymatic crosslinking for diverse applications. The success of this method suggests its great potential in future biomedicine and biotechnology.

Reference.
1. Seliktar, D., Designing cell-compatible hydrogels for biomedical applications. Science 2012, 336 (6085), 1124-1128.
2. Wu, C.; Strehmel, C.; Achazi, K.; Chiappisi, L.; Dernedde, J.; Lensen, M. C.; Gradzielski, M.; Ansorge-Schumacher, M. B.; Haag, R., Enzymatically crosslinked hyperbranched polyglycerol hydrogels as scaffolds for living cells. Biomacromolecules 2014, 15 (11), 3881-3890.
3. Wu, C.; Böttcher, C.; Haag, R., Enzymatically crosslinked dendritic polyglycerol nanogels for encapsulation of catalytically active proteins. Soft Matter 2015, 11 (5), 972-980.
4. Wu, C.; Schwibbert, K.; Achazi, K.; Landsberger, P.; Gorbushina, A.; Haag, R., Active antibacterial and antifouling surface coating via a facile one-step enzymatic cross-linking. Biomacromolecules 2017, 18 (1), 210-216.

Adherent cell production is hindered by the use of solid substrates or hydrogels for culture and expansion. Such materials and platforms are difficult to scale up and parallelise. In contrast, liquid-liquid technologies and microdroplet platforms have been applied very successfully in the field of Chemical Engineering for the scale up of synthesis and purification of fine chemicals, therapeutics, polymers and nanomaterials. Yet, their use in the field of biotechnologies is restricted to the high throughput screening of single planktonic cells. However, we recently reported that the culture of adherent cells at the surface of liquids [1, 2], previously observed with fibroblasts, was mediated by the self-assembly of protein nanosheets forming mechanically strong interfaces [3, 4]. This enabled the adhesion of stem cells such as keratinocytes and mesenchymal stem cells and the regulation of their spreading via the classic integrin and acto-myosin machinery, allowing the retention of stem cell phenotypes despite the extreme compliance of liquid substrates [5]. We now examine the chemical design of protein nanosheets systematically and identify groups of pro-surfactant molecules effectively supporting stem cell expansion at liquid interfaces. Surprisingly, the shear moduli of associated interfaces does not correlate with cell expansion. Instead, we observe that the viscoelastic behaviour of protein nanosheets correlates with stem cell proliferation. We identify that particularly stiff nanosheets that display poor elasticity do not support stem cell expansion and that this phenomenon is associated with the formation of brittle domains that can relax and dissipate energy in response to cell mediated forces. Hence the multi-scale viscoelasticity of liquid-liquid interfaces, rather than shear moduli, is the primary physical determinant of stem cell proliferation. Based on neutron reflectometry, TEM and polarised optical microscopy, we report that this behaviour is controlled by the nanoscale organisation of prosurfactant-mediated rigid domains. To demonstrate the capacity to expand stem cells at liquid interfaces whilst retaining their phenotype, and the relevance of these systems for stem cell manufacturing, we characterised the expansion of mesenchymal stem cells over multiple passages, their retention of stem cell markers and ability to differentiate into multiple lineages. Cells cultured on nanosheet-stabilised emulsions displayed comparable phenotype in long-term expansion compared to stem cells cultured on 2D plastic and solid microcarriers. Overall, our results pave the way to the use of microdroplet technologies and liquid-liquid interfaces in the field of tissue engineering and for stem cell technologies.

1. Keese et al. Proc. Natl. Acad. Sci., 1983. 80, 5622. 2. Keese et al. Science, 1983. 219, 1448. 3. Kong, D., et al. Nano Lett., 2018. 18, 1946. 4. Kong, D., et al. Faraday Discuss., 2017. 204, 367. 5. Kong, D., et al. ACS Nano, 2018. 12, 9206.

Investigating the mechanical crosstalk between cells and their surrounding environment is fundamental to understand the influence of forces on cell functions and responses. In attempt to better elucidate the material – cell interaction in-vitro during adhesion process, here we report a new concept for a reliable and dynamic skin-over-liquid system for studying mechanobiology. It is made of a periodic array of highly compliant microbumps actuated through electrode-free electrohydrodynamic (EHD) pressure. These structures are highly repeatable and capable of swelling and deflating easily under a simple thermal stimulation driven by the pyroelectric effect, thus providing an innovative and easy tool that can be actively controlled at the microscale. To confirm the formation of the cytoskeleton structures after the stimulation, a fluorescence imaging system was used as a control to visualize actin filaments. The strategy here proposed portends broad applicability to investigate the correlation between the mechanical stress applied to cells by swelling these microbumps and their cytoskeleton assembly process. Moreover, the preliminary results are promising and permit us to consider skin-over-liquid platform as an easy assay for cell nanomechanic investigations in the future.

References
[1] Gennari, O., Rega, R., Mugnano, M., Oleandro, E., Mecozzi, L., Pagliarulo, V., Mazzon, E., Bramanti, A., Vettoliere, A., Granata, C. and Ferraro, P., 2019. A skin-over-liquid platform with compliant microbumps actuated by pyro-EHD pressure. NPG Asia Materials, 11(1), pp.1-8.
[2] Fusco, S., Memmolo, P., Miccio, L., Merola, F., Mugnano, M., Paciello, A., Ferraro, P. and Netti, P.A., 2016. Nanomechanics of a fibroblast suspended using point-like anchors reveal cytoskeleton formation. RSC advances, 6(29), pp.24245-24249.
[3] Marchesano, V., Gennari, O., Mecozzi, L., Grilli, S., & Ferraro, P. (2015). Effects of lithium niobate polarization on cell adhesion and morphology. ACS applied materials & interfaces, 7(32), 18113-18119.
[4] Rega, R., Gennari, O., Mecozzi, L., Grilli, S., Pagliarulo, V., & Ferraro, P. (2016). Bipolar patterning of polymer membranes by pyroelectrification. Advanced Materials, 28(3), 454-459.

Influenza remains one of the most severe threats to public health. Influenza mucosal immunity could confer broad cross-protection against influenza viruses. Intranasal (i.n.) vaccination with recombinant protein/peptide vaccines is a safe and promising strategy in the prevention of influenza virus infection due to the capability of generating both systemic immunity and local mucosal immunity. However, the intranasally administrated protein/peptide vaccines are poorly immunogenic due to the harsh and tolerogenic mucosal environment. Adjuvants could program the dimension and magnitude of antigen-specific immune responses and are helpful boosters to promote rapid and strong immune responses with a dose sparing effect. Nevertheless, few mucosal adjuvants are currently licensed for human use.
Nanoparticles are a special and promising class of vaccine adjuvants as it could not only serve as effective delivery carriers for antigen and/or molecular adjuvants but also synergistically work with molecular adjuvants like TLR agonists in a whole system. Polyethyleneimine (PEI) is a kind of organic polycations that could form nanoscale complexes with viral glycoprotein antigens. In our study, we prepared PEI-hemagglutinin (HA) nanocomplexes with or without CpG ODN molecules, and comprehensively studied the humoral and cellular immune responses after i.n. vaccination with the nanocomplexes. Our results showed that the PEI-HA nanocomplexes induced significantly enhanced mucosal and systematic antibody responses than soluble HA antigens. The combination of PEI and CpG in PEI-HA/CpG further boosted the responses. Furthermore, we would investigate the induced cellular responses and study the cross-protection efficacy of these nanocomplexes against different influenza strains in mice. The role of PEI, CpG and the nanoparticulate feature in regulating the immune responses would be further discussed. Our study will provide perspectives to guide the development of effective mucosal adjuvants for influenza vaccines.

We will discuss a straightforward method for the synthesis of a water-soluble C₆₀ fullerene derivative (F1) with five attached residues of phosphonic acid. Self-assembly of the synthesized compound in an aqueous solution leads to the formation of various nanostructures, as observed by AFM and DLS methods.
Antiviral screening of the synthesized compound in vitro demonstrated its activity against ten different viruses such as Feline coronavirus, Influenza A virus (H3N2 and H1N1), Influenza B virus, Varicella zoster virus, Cytomegalovirus, Herpes simplex virus (HSV-1, HSV-2), Cowpox virus, Feline herpes virus, and Human immunodeficiency virus (HIV-1, HIV-2).
The most unexpected effect of the water-soluble fullerene derivative F1 was its ability to promote myogenic differentiation of human mesenchymal stem cells (MSCs). In the absence of the fullerene derivative, stem cells from adipose tissue partially differentiate into adipocytes in the non-differentiating culture medium. During prolonged cultivation of MSCs in the presence of F1, the morphology of the cells changes spectacularly: fragments of stem cells with an ordered structure, which express markers of myogenic differentiation, appear. This kind of activity is unknown for all other reported water-soluble fullerene derivatives.
The obtained results open new horizons for the development of fullerene-based drugs for the regeneration of damaged muscle tissue and the treatment of severe muscle atrophy.
This work was supported by the Russian Science Foundation (project 19-13-00411).

The establishment of near-physiological in vitro thrombogenicity test systems for the evaluation of blood-contacting biomaterials requires co-cultivation of endothelialized materials and platelets (PLT) under blood flow-like conditions. Commercial perfusion systems for this purpose are equipped with gas plasma-treated cyclic olefin copolymer (COC)-based standard slides for endothelialization. Here, COC was characterized for physicochemical properties prior to co-culture studies. Moreover, endothelial culture conditions were systematically analyzed regarding PLT responses considering their sensitivity to external influences, both of their handling and their environment, and their central role in hemostasis and innate immunity.
A basic chemical characterization of COC and of its physical surface properties was performed. Human platelet rich plasma (PRP) was mixed with EGM-2 basal medium (BM) or with BM separately containing each of nine supplements or with BM containing all supplements together. PLT adherence analysis was carried out on COC and poly(tetrafluoro ethylene) (PTFE) as a control. PLT activation and aggregation were analyzed using light transmission aggregometry and flow cytometry (CD62P). COC endothelialized with human umbilical vein endothelial cells was co-cultivated with 150.000 PLT/µL for 1 h under static conditions and laminar flow (10 dyn/cm²).
The physical and chemical characteristics as determined were in line with COC’s cell adhesive properties. Medium supplements had no effect on PLT activation and aggregation. Regarding PLT adherence, supplements differentially affected PLT number, size and PLT covered area on COC and PTFE. EC count was significantly higher under laminar flow than under static conditions. PLT count and PLT covered area increased under static culture conditions compared to flow both on endothelialized and non-endothelialized surfaces.
Gas plasma-treated COC is suitable for the establishment of an in vitro thrombogenicity test system. Systematical medium analysis showed that according to the readouts employed here, the use of standard endothelial growth medium maintains functionality of PLT under EC compatible conditions without masking the differences of PLT adherence on different polymeric substrates. Moreover, improved EC attachment and reduced PLT adhesion under flow underlines the importance of blood flow-like conditions. These findings are important prerequisites for the assessment of polymer-based cardiovascular implant materials in contact with EC, PLT and, in the future, possibly also with further immune cells to grasp the complex role of blood homeostasis in contributing to thrombogenic events.

Modification of the cell surface with nanoparticles (NP) can be of interest for a wide variety of biomedical applications. An attractive therapeutic strategy involving conjugation of NP to living cells is to exploit the tissue homing properties of specific cell types for targeted delivery of drugs loaded into NP (e.g. ex vivo engineering of tumor infiltrating T lymphocytes and CAR T cells). Cell surface conjugation requires either a covalent or non-covalent reaction. Non-covalent conjugation with ligand decorated NP is challenging and involve a dynamic equilibrium between the bound and unbound state. By contrast, covalent NP conjugation where reactive groups on the NP surface react with a complementary reaction partners in the cellular glycocalyx, results in a permanently bound state of NP. Current routes for cell surface conjugation predominantly make use of natural occurring lysines and cysteines on the cell surface and respectively activated esters and maleimides on the surface of NP. However also these routes tend to be challenging due to slow reaction kinetics, random conjugation to proteins in the glycocalyx etc. Therefore, there is strong need for new strategies that allow for efficient covalent cell surface conjugation. In particular, bio-orthogonal click reactions are an attractive alternative and in this context, introducing azides on the cells surface through metabolic labelling with azido sugars is attractive to introduce unnatural functional groups on the cell surface that can be addressed further on using biorthogonal strain promoted azide-alkyne cycloaddition (SPAAC) click chemistry. To explore this strategy, we used dibenzyl cyclooctyne (DBCO) functionalized lipid nanoparticles (LNP) formed by electrostatic complexation of an ionizable lipid and a double stranded RNA synthetic analogue, polyinosinic: polycytidylic acid (polyIC).Interestingly, we found that, whereas the reaction between small molecule dye-labeled DBCO and azido-labeled cells is extremely fast and efficient, reaction between DBCO-LNP and azide-labelled cells is rather inefficient, leading to a low degree of functionalization and high unspecific background adsorption of LNP to cells when attempting at optimizing conjugation conditions. We attribute this to a combination of sterical hindrance and the low concentration and slow Brownian motion of the reaction partners. This issue could be circumvented by designing LNP that contain a diisopropylamino-functionalized lipid that has a pKa below physiological pH. A short co-incubation time at pH 7.4 shows almost no association between DBCO-LNP and azido-labelled cells, but at pH 5, where the diisopropylamino moieties bear a cationic charge, strong LNP adsorption takes place due to electrostatic interaction. Due to the close contact between DBCO-LNP and azide-labelled cells, SPAAC takes place and LNP remain permanently bound to the cell surface. However, this route leads to an extremely high non-specific background adsorption of control non-SPAAC-reactive LNP and on control cells, despite multiple washing steps with neutral buffer. To tackle the issue of low reactivity and high non-specific binding, we explored the ultra-fast bio-orthogonal inverse electron demand Diels–Alder (IEDDA) reaction between tetrazines (Tz) and trans-cyclooctene (TCO). We developed a 2-step conjugation sequence in which metabolically azide labelled cells first react with sulfo-6-methyl-tetrazine-DBCO (Tz-DBCO), followed by co-incubation with TCO-LNP. Eventually, this strategy gives a highly specific cell surface conjugation of LNP, with very low non-specific background fluorescence for controls cells and for control LNP lacking TCO, as evidenced by flow cytometry and confocal microscopy. Hence, we believe that this last strategy holds a general applicability for cell surface modification in view of adoptive cell transfer therapy with NP-backpacked cells.

Stem cells are highly relevant in tissue regeneration due to their self-renewal capacity and differentiation potential. Given the capacity of stem cells to sense the environmental cues and to convert the perceived information to intracellular signaling cascades, the use of external stimulation through biomaterials to regulate stem cells has become a safe and effective approach to guide them towards a desired function and lineage commitment. We designed and fabricated polymer based cell culture substrates with well-defined geometric micro-wells (size, shape) to exhibit static mechanical cues or thermally controlled artificial muscles as dynamic substrates. We investigated how the stem cells respond to these mechanical stimuli provided by the biomaterial and elucidated the underlying mechanism.
The micro-topography of the differently structured substrates exerted highly distinct effects on the major cellular process of mesenchymal stem cells (MSCs), including cell morphology, cytoskeleton organization, focal adhesion distribution, migration, gene expression, proliferation, and differentiation. Taking advantage of an AB copolymer network with a thermally controlled shape-memory actuation capability, we studied MSC response to the thermal (environmental temperature change) and mechanical (substrate shape change) stimuli. The combinational and synergetic effect of the dual stimuli as well as the activated signaling cascades were explored. The Ca²⁺ dynamics was shown to regulate the intercellular connection of mechano- and thermosensing components, promoting the osteogenic differentiation of MSCs.
These results provide a fundamental principle for design and application of advanced multifunctional biomaterials in regenerative medicine.

During tissue development, progenitor stem cells form functional tissue with high cellular diversity and intricate micro and macro architecture. Current approaches have attempted to replicate this process with materials cues or through spontaneous cell self-organization. However, it has become increasingly clear that cell-directed and material-directed organization are both required to achieve native structure and function. Here, I will present a biomanufacturing method to deposit stem cells in freeform within a granular matrix while spatially guiding their differentiation. The directed combination of varied microgels establish a 3D bioprinting support medium with gradients of mechanical stiffness and growth factor release. The packed suspension of gelatin methacrylate microgels crosslink together under near UV light, locking the cells and gradients in place. And controlled deposition of mesenchymal stem cells across these gradients within the gel enables guided differentiation to osteogenic, adipogenic, or chondrogenic phenotypes. As a demonstration of this method, we combine a gradient of stiffness and growth factor release with a gradient of hyaluronic acid to create a model meniscus. We anticipate this material platform will open new avenues for regenerative medicine and fundamental studies on the role matrix cues play in stem cell differentiation.

Citrate-based biodegradable polymer has recently joined in a handful of biodegradable synthetic polymers used in Food and Drug Administration (FDA) approved implantable medical devices such as orthopedic fixation devices. Citrate, historically known as an intermediate in the Krebs cycle, is a multifunctional, nontoxic, readily available, and inexpensive cornerstone monomer used in the design of citrate-based biomaterials. We have recently identified citrate as an osteo-promotive factor and revealed a key link between citrate metabolism and its downstream effects on osteogenic differentiation of human mesenchymal stem cells (hMSCs), named metabonegenic regulation. Our studies show that extracellular citrate uptake through solute carrier family 13, member 5 (SLC13a5) supported osteogenic differentiation via regulation of energy-producing metabolic pathways, which led to elevated cell energy status to fuel osteo-differentiation of hMSCs with high metabolic demands. We have also identified other factors such as phosphoserine (PSer) that can synergize with citrate to exhibit concerted action not only in metabonegenic potential for orthopedic regeneration, but also in functionalizing materials with imaging capability for materials tracking and monitoring. This unique citrate metabonegenic regulation has allowed us to design new citrate-based platform biomaterials to meet the dynamic biological, biochemical, and biophysical needs in tissue regeneration. The recent regulatory success of citate-based biomaterials makes them promising candidate materials for technology translation and commercialization. In this presentation, a methodology for the design of citrate-based biomaterials in various regenerative engineering applications with a focus on orthopedic applications will be discussed.

Developing interfaces between active materials and biological structures is becoming increasingly important for manipulating and interrogating living systems for use in advanced biological engineering. We have developed techniques for transferring graphene-based thin film materials from one substrate to another without needing to use harsh chemicals to detach the films from their initial substrates. This allows us to interface a number of materials directly with biological substrates including live stem cells without sacrificing viability. We found that partially reduced graphene oxide can be used as a transferrable surface for printing a number of materials, including metallic structures, fluorescent cell dyes, and phase-separated block copolymers. The mild transfer techniques we have developed allow us to easily probe biological systems at the scale of individual cells, and we can also use our transferrable block copolymer technology to lithographically pattern novel hierarchical structures on arbitrary substrates. These capabilities offer a cheap and easy way to exert spatiotemporal control over single cells and cell-cell interactions.

Collagen is one of the most abundant extracellular matrix of animal tissues and also serves as the main structural material by providing mechanical environments for cells. Especially, collagen hydrogels are widely used as scaffold materials in vitro since the chemo-mechanical environments they give the cells are almost identical to those in vivo. Thus, providing physical reliability of the collagen hydrogel presents significant challenges for tissue engineering applications, and it is essential to understand its mechanical behavior and deformation mechanisms. In this study, we aim to investigate the mechanical behavior of collagen hydrogels under quasi-static uniaxial tensile strain to present the complex tempo-mechanical response of biomaterials apart from the rheometric response. A combination of tensile testing in a hydrogel-friendly hydrated environment and microstructure analysis including structural parameters and strain evolution demonstrates the mechanism of tensile properties, complementarily. When the tensile strain is applied, the stress-strain relationship of collagen hydrogels shows a non-linear response with strain-stiffening. This non-linearity of the physical properties of collagen hydrogel comes from the shift of deformation mechanism and the unfolding dominated deformation of the collagen fiber network is completed as the very first stage, the mechanical response gradually turns to the stretching dominated deformation of the collagen fibers by aligning their microstructure. These results illustrate that collagen hydrogels respond to tensile strain with two main different deformation mechanisms, unfolding and stretching of collagen fibers, which shows the non-linear stress-strain relationship, and the topology of collagen hydrogels can control the mechanical response. Furthermore, it is a new approach to physical behavior for soft and hydrated materials that can be applied throughout the bioengineering field.

Olfactory ecto-mesenchymal stem cells (OE-MSCs) exert protective effects in some autoimmune diseases, partly through the regulation of activation, proliferation and differentiation of lymphocytes. Extracellular vesicles (EVs), including exosomes, released by MSCs contain bioactive factors, such as microRNAs, proteins, lipids, and some studies have confirmed that the immunoregulatory function of MSCs was mediated by exosomes. However, it is unclear whether OE-MSC-derived exosomes (OE-MSC-Exos) influence regulatory B cells in collagen-induced arthritis (CIA). In this study, we found that OE-MSC-Exos could promote the expansion of interleukin-10-producing B cells (B10 cells) with regulatory function in an Epstein-Barr virus-induced gene 3 (EBI3)-dependent pathway. In vivo, intravenous injection of OE-MSC-Exos into CIA mice significantly inhibited the disease progression and reduced the severity of both clinical score and joint damage. Taken together, our findings identified that OE-MSC-Exos might become a new cell-free therapy for the treatment of RA and other inflammatory diseases.

During the current SARS-CoV-2 pandemic, intravenous infusion of mesenchymal stromal cells (MSCs) is emerging as one of the promising therapeutic tools for treating the severe respiratory syndrome in COVID-19, which mainly relies on the regenerative capacity and superior immunomodulatory function of MSCs [1, 2]. The transplanted MSCs were found to be recruited at the interface of alveoli and surrounding capillaries, where respiratory airway structures are exposed to periodic heat exchange and mechanical stresses with respiratory cycles [2, 3]. Toll-like receptor (TLR) triggers innate immune response against virus including SARS-CoV-2 [4, 5]. TLR also plays a key role in regulation of differentiation and immune polarization of MSCs, which highly associated with the MSC therapeutic efficacy. However, TLR expression profiles and distributions are varying in MSCs not only depending on the cell sources but also the culture conditions [6]. To date, little is known about the influence of the thermomechanical cues on regulating the TLRs in MSCs. Here, thermally-controlled polymer sheets with programmable actuation capacity (cPCLBA) were created to autonomously synchronize and transfer the thermomechanical signal to MSCs. With the periodic temperature change between 10 °C and 37 °C, 14 ± 1% elongation of programmed sheets can be achieved at both macro- and micro-scales. As a result, in response to thermomechanical stimuli, the proportion of cells expressing SARS-CoV-2 responsive TLRs (TLR4+TLR7+) were significantly increased from 69 ± 5% to 81 ± 3%, while the TLR3+TLR9+ cells that sensing double-stranded RNA and unmethylated DNA virus was decreased from 41 ± 5% to 26 ± 4%. Moreover, the internalization of cell surface expressed TLR4 was promoted and the intracellular TLR4 was co-localized with TLR7, and retained closely to the endoplasmic reticula where viral entry, replication and assembly takes place [7]. In summary, the programmed cPCLBA sheet provided a simple, cost effective and efficient respiratory cycle model, which synchronized and transduced the thermomechanical stimuli into MSCs. Such stimuli were able to accumulate TLR4 and TLR7 at endoplasmic reticula and selectively amplified the TLR4+TLR7+ MSCs, which can specifically react to single-stranded RNA virus including SARS-CoV-2. These results highlight the potential application of boosting the anti-viral immunity via preconditioning of MSCs with thermomechanical cues prior to cell infusion.

References:

1. Chowdhury, M.A., et al., Immune response in COVID-19: A review. J Infect Public Health, 2020.
2. Leng, Z., et al., Transplantation of ACE2(-) Mesenchymal Stem Cells Improves the Outcome of Patients with COVID-19 Pneumonia. Aging Dis, 2020. 11(2): p. 216-228.
3. Garcia, C.S., et al., Understanding the mechanisms of lung mechanical stress. Braz J Med Biol Res, 2006. 39(6): p. 697-706.
4. Patra, R., N. Chandra Das, and S. Mukherjee, Targeting human TLRs to combat COVID-19: A solution? J Med Virol, 2020.
5. Heil, F., et al., Species-specific recognition of single-stranded RNA via toll-like receptor 7 and 8. Science, 2004. 303(5663): p. 1526-9.
6. Seo, Y., T.H. Shin, and H.S. Kim, Current Strategies to Enhance Adipose Stem Cell Function: An Update. Int J Mol Sci, 2019. 20(15).
7. Inoue, T. and B. Tsai, How viruses use the endoplasmic reticulum for entry, replication, and assembly. Cold Spring Harb Perspect Biol, 2013. 5(1): p. a013250. <div id="gtx-trans" style="position: absolute; left: 625px; top: 233px;"> <div class="gtx-trans-icon"> </div> </div>

The task of mechanosensing of cells involves dynamic assembly of various tension-transmission supramolecular linkages, which enable the cells to properly sense and respond to the level of tension in the linkages. A tension-transmission linkage typically consists of a few non-covalently linked proteins, in which the domains and protein-protein interfaces are subject to dynamic fluctuation of intracellular tensions. This leads to highly complex tension-dependent connectivity of the supramolecular linkages, conformational changes of the domains and the resulting tension-dependent interactions of binding sites in the linkage with signalling proteins. Increasing evidence has suggested that protein-protein interfaces and protein domains in various mechanosensing tension-transmission supramolecular linkages have been evolved with special structural and topological features that impart them with special mechanical properties. In this talk, I will introduce our recent single-molecule and theoretical investigations of the effects of the structural-topological features shared by many tension bearing protein-protein interfaces and protein domains on the mechanical stability and mechanosensing function of tension-transmission supramolecular linkages.

Agonists of toll-like receptors (TLRs) form an important class of adjuvants employed in cancer immunotherapies and prophylactic vaccines. Among the various TLR agonists, polyriboinosinic acid:polyribocytidylic acid (poly(I:C)), a synthetic mimic of viral dsRNA, has been shown to activate and boost antigen cross-presentation by dendritic cell (DCs), promote NK cell cytotoxicity as well as effect direct tumor-killing to generate tumor-associated antigens for DC presentation. Poly(I:C) is recognized by the endosomal receptor TLR3 as well as cytosolic receptors such as RIG-I and MDA-5. Poly(I:C) binding to its receptor initiates a cascade of signaling pathways resulting in the activation of NF-κB and production of pro-inflammatory cytokines (TLR3), or IRF and production of type-I interferons (MDA-5). Despite its potent immunogenic properties, safety concerns including inadvertent immune over-stimulation, induction of autoimmune diseases, and short half-life in vivo have limited the scope of poly(I:C). Several strategies including liposomal and polymeric encapsulation, polycationic complexes or polyplexes, and surface-assembly on nanoparticles (NPs) or microparticles (MPs) have been explored to overcome these issues.

Here, we present a comparative study of poly(I:C) polyplexes and layer-by-layer (LbL)-assembled NPs/MPs carrying poly(I:C) to selectively activate the TLR3 and MDA-5 receptors. Poly(I:C) delivery platforms were fabricated using three classes of polycations: polypeptides, polysaccharides, and synthetic polymers. Dynamic light scattering, zeta potential measurements, gel electrophoresis, fluorophore exclusion titration assay, circular dichroism, and isothermal titration calorimetry were utilized to characterize the polyplexes and assess the structural integrity of poly(I:C) complexed with different polycations. LbL particles were built on a negatively-charged core using the polycations to electrostatically assemble poly(I:C) on the particle surface. HEK-Blue mTLR3 reporter cell line was used to assess the ability of polyplexes and LbL particles to activate the endosomal TLR3 receptors. For the polyplexes with varying poly(I:C):polycation ratios, TLR3 activation was found to be roughly inversely correlated with the poly(I:C) binding strength of the polycations. While poly(I:C)-loaded LbL particles were as effective as free poly(I:C) in activating TLR3 when in direct contact with the cells, significant loss of bioactivity was observed when the cells and the particles were separated using a trans-well. This was likely due to enhanced cellular uptake of the LbL particles or degradation of poly(I:C) released from the LbL particles in the latter case. The selectivity of polyplexes and LbL particles toward TLR3 and MDA-5 receptors was assessed in A549 dual NF-κB-SEAP and IRF-luciferase reporter cell line. Activation of the two pathways was further correlated with cellular uptake and trafficking of the polyplexes and LbL particles. We elucidate the role of the delivery mechanism and polycationic carriers in stabilizing poly(I:C) against serum nucleases, reducing cellular toxicity and selectivity toward endosomal or cytosolic receptors. The presented biophysical studies provide critical insight into the design of poly(I:C) delivery platforms toward more potent adjuvants.

Modulating effector immune cells via monoclonal antibodies (mAbs) and facilitating co-engagement of T cells and tumor cells via CAR T cells or bispecific T cell-engaging antibodies are two typical cancer immunotherapy approaches^1-3. We speculated that immobilizing two types of mAbs against effector cells and tumor cells onto single nanoparticle could integrate the functions of these two approaches, as the engineered formulation (immunomodulating nano-adaptor, imNA) could potentially associate with both cells and bridge them together like an ‘adaptor’ while maintaining the immunomodulatory properties of parental mAbs. However, existing mAbs immobilization strategies mainly rely on chemical reaction, a process that is rough and difficult to control. Here, we built up a versatile antibody immobilization platform by conjugating anti-IgG (Fc specific) antibody (αFc) onto nanoparticle surface (αFc-NP), and confirmed that αFc-NP could conveniently and efficiently immobilize two types of mAbs through Fc-specific noncovalent interactions to form imNAs. The superiority of imNAs over the mixture of parental mAbs were validated in T cell-, natural killer cell- and macrophage-mediated antitumor immune responses in multiple murine tumor models.

1. Velasquez, M.P., Bonifant, C.L. & Gottschalk, S. Redirecting T cells to hematological malignancies with bispecific antibodies. Blood 131, 30-38 (2018).
2. Slaney, C.Y., Wang, P., Darcy, P.K. & Kershaw, M.H. CARs versus BiTEs: a comparison between T cell-redirection strategies for cancer treatment. Cancer Discov. 8, 1-11 (2018).
3. Sadelain, M., Riviere, I. & Riddell, S. Therapeutic T cell engineering. Nature 545, 423-431 (2017).