Immune-infiltrated kidney organoid-on-chip model for assessing T cell bispecific antibodies.

T cell bispecific antibodies (TCBs) are the focus of intense development for cancer immunotherapy. Recently, peptide-MHC (major histocompatibility complex)-targeted TCBs have emerged as a new class of biotherapeutics with improved specificity. These TCBs simultaneously bind to target peptides presented by the polymorphic, species-specific MHC encoded by the human leukocyte antigen (HLA) allele present on target cells and to the CD3 coreceptor expressed by human T lymphocytes. Unfortunately, traditional models for assessing their effects on human tissues often lack predictive capability, particularly for "on-target, off-tumor" interactions. Here, we report an immune-infiltrated, kidney organoid-on-chip model in which peripheral blood mononuclear cells (PBMCs) along with nontargeting (control) or targeting TCB-based tool compounds are circulated under flow. The target consists of the RMF peptide derived from the intracellular tumor antigen Wilms' tumor 1 (WT1) presented on HLA-A2 via a bivalent T cell receptor-like binding domain. Using our model, we measured TCB-mediated CD8+ T cell activation and killing of RMF-HLA-A2-presenting cells in the presence of PBMCs and multiple tool compounds. DP47, a non-pMHC-targeting TCB that only binds to CD3 (negative control), does not promote T cell activation and killing. Conversely, the nonspecific ESK1-like TCB (positive control) promotes CD8+ T cell expansion accompanied by dose-dependent T cell-mediated killing of multiple cell types, while WT1-TCB* recognizing the RMF-HLA-A2 complex with high specificity, leads solely to selective killing of WT1-expressing cells within kidney organoids under flow. Our 3D kidney organoid model offers a platform for preclinical testing of cancer immunotherapies and investigating tissue-immune system interactions.

Functional maturation of kidney organoid tubules: PIEZO1-mediated Ca2+ signaling.

Kidney organoids cultured on adherent matrices in the presence of superfusate flow generate vascular networks and exhibit more mature podocyte and tubular compartments compared with static controls (Homan KA, Gupta N, Kroll KT, Kolesky DB, Skylar-Scott M, Miyoshi T, Mau D, Valerius MT, Ferrante T, Bonventre JV, Lewis JA, Morizane R. Nat Methods 16: 255-262, 2019; Takasato M, Er PX, Chiu HS, Maier B, Baillie GJ, Ferguson C, Parton RG, Wolvetang EJ, Roost MS, Chuva de Sousa Lopes SM, Little MH. Nature 526: 564-568, 2015.). However, their physiological function has yet to be systematically investigated. Here, we measured mechano-induced changes in intracellular Ca2+ concentration ([Ca2+]i) in tubules isolated from organoids cultured for 21-64 days, microperfused in vitro or affixed to the base of a specimen chamber, and loaded with fura-2 to measure [Ca2+]i. A rapid >2.5-fold increase in [Ca2+]i from a baseline of 195.0 ± 22.1 nM (n = 9; P ≤ 0.001) was observed when microperfused tubules from organoids >40 days in culture were subjected to luminal flow. In contrast, no response was detected in tubules isolated from organoids <30 days in culture. Nonperfused tubules (41 days) subjected to a 10-fold increase in bath flow rate also exhibited a threefold increase in [Ca2+]i from baseline (P < 0.001). Mechanosensitive PIEZO1 channels contribute to the flow-induced [Ca2+]i response in mouse distal tubule (Carrisoza-Gaytan R, Dalghi MG, Apodaca GL, Kleyman TR, Satlin LM. The FASEB J 33: 824.25, 2019.). Immunodetectable apical and basolateral PIEZO1 was identified in tubular structures by 21 days in culture. Basolateral PIEZO1 appeared to be functional as basolateral exposure of nonperfused tubules to the PIEZO1 activator Yoda 1 increased [Ca2+]i (P ≤ 0.001) in segments from organoids cultured for >30 days, with peak [Ca2+]i increasing with advancing days in culture. These results are consistent with a maturational increase in number and/or activity of flow/stretch-sensitive Ca2+ channels, including PIEZO1, in tubules of static organoids in culture.

Organoid-on-a-chip model of human ARPKD reveals mechanosensing pathomechanisms for drug discovery.

Organoids serve as a novel tool for disease modeling in three-dimensional multicellular contexts. Static organoids, however, lack the requisite biophysical microenvironment such as fluid flow, limiting their ability to faithfully recapitulate disease pathology. Here, we unite organoids with organ-on-a-chip technology to unravel disease pathology and develop therapies for autosomal recessive polycystic kidney disease. PKHD1-mutant organoids-on-a-chip are subjected to flow that induces clinically relevant phenotypes of distal nephron dilatation. Transcriptomics discover 229 signal pathways that are not identified by static models. Mechanosensing molecules, RAC1 and FOS, are identified as potential therapeutic targets and validated by patient kidney samples. On the basis of this insight, we tested two U.S. Food and Drug Administration-approved and one investigational new drugs that target RAC1 and FOS in our organoid-on-a-chip model, which suppressed cyst formation. Our observations highlight the vast potential of organoid-on-a-chip models to elucidate complex disease mechanisms for therapeutic testing and discovery.

Programming Cellular Alignment in Engineered Cardiac Tissue via Bioprinting Anisotropic Organ Building Blocks.

The ability to replicate the 3D myocardial architecture found in human hearts is a grand challenge. Here, the fabrication of aligned cardiac tissues via bioprinting anisotropic organ building blocks (aOBBs) composed of human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) is reported. A bioink composed of contractile cardiac aOBBs is first generated and aligned cardiac tissue sheets with linear, spiral, and chevron features are printed. Next, aligned cardiac macrofilaments are printed, whose contractile force and conduction velocity increase over time and exceed the performance of spheroid-based cardiac tissues. Finally, the ability to spatially control the magnitude and direction of contractile force by printing cardiac sheets with different aOBB alignment is highlighted. This research opens new avenues to generating functional cardiac tissue with high cell density and complex cellular alignment.

The prognostic value of fat invasion and tumor expansion in the hilar veins in pT3a renal cell carcinoma.

PURPOSE: The 7th TNM classification summarizes renal cell carcinoma (RCC) with perirenal (PFI) and/or sinus fat invasion (SFI) as well as hilar vein involvement (RVI) as pT3a tumors. In this study, we aimed to determine the prognostic value of fat invasion (FI) in the different compartments and RVI for medium-term cancer-specific-survival (CSS) in pT3a RCC. MATERIALS AND METHODS: Patients with pT3a RCC were identified using an institutional database. All original pathological reports were reclassified according to the 7th TNM edition. The prognostic value of FI as well as divided into PFI, SFI, combined PFI + SFI, and RVI for CSS was assessed using univariate and multivariate Cox-regression analysis. Survival was estimated using the Kaplan-Meier method. RESULTS: Median follow-up in 184 pT3a tumors was 38 months. FI was detectable in 153 patients (32.7% PFI, 45.1% SFI, 22.2% PFI + SFI), 31 patients showed RVI alone. Combined PFI + SFI increased the risk of cancer-related death compared to PFI (HR 3.11, p < 0.01), SFI (HR 1.84, p = 0.023) or sole RVI (HR 2.12, p = 0.025). In multivariate analysis, a combined PFI + SFI vs. PFI or SFI as the only compartment involved was confirmed as independent prognostic factor (HR 1.83, p = 0.029). Patients with FI and simultaneous RVI had significantly shorter CSS (HR 2.63, p < 0.01). In an unweighted model, the difference between patients with combined PFI + SFI and RVI and those with PFI alone was highest (HR 4.01, p = 0.029). CONCLUSIONS: These results underline the subdivision of pT3a RCC depending on the location of FI and RVI for patient stratification.

Flow-enhanced vascularization and maturation of kidney organoids in vitro.

Kidney organoids derived from human pluripotent stem cells have glomerular- and tubular-like compartments that are largely avascular and immature in static culture. Here we report an in vitro method for culturing kidney organoids under flow on millifluidic chips, which expands their endogenous pool of endothelial progenitor cells and generates vascular networks with perfusable lumens surrounded by mural cells. We found that vascularized kidney organoids cultured under flow had more mature podocyte and tubular compartments with enhanced cellular polarity and adult gene expression compared with that in static controls. Glomerular vascular development progressed through intermediate stages akin to those involved in the embryonic mammalian kidney's formation of capillary loops abutting foot processes. The association of vessels with these compartments was reduced after disruption of the endogenous VEGF gradient. The ability to induce substantial vascularization and morphological maturation of kidney organoids in vitro under flow opens new avenues for studies of kidney development, disease, and regeneration.

Vocabulary as a Central Link between Phonological Working Memory and Narrative Competence: Evidence from Monolingual and Bilingual Four-Year-Olds from Different Socioeconomic Backgrounds.

Phonological working memory capacity, vocabulary size, and narrative competence are important skills in children's L1 and L2 acquisition, which may vary as a function of their language background and socioeconomic status (SES). We investigated test data of 56 typically developing 4-year-old kindergarten children from two SES and two language backgrounds: 29 children (15 higher SES, 14 lower SES) were monolingual German-speaking, and 27 children (14 higher SES, 13 lower SES) were successive Turkish-German bilinguals. The tests comprised a non-word repetition task testing phonological working memory, receptive vocabulary tests (in L1 and L2), and a narrative task. We investigated the effects of SES and language background on children's test performance. Results indicate that SES was a highly significant factor for phonological working memory and vocabulary in the monolingual children, but not in the bilingual children. Although the items of the non-word repetition task followed German phonotactic structure, lower SES (LSES) L2 children did not differ significantly from their monolingual LSES peers, demonstrating that there was no bilingual working memory disadvantage in the LSES group. A significant effect of language background was found for German vocabulary and for all categories of narrative competence, but only two slight SES effects on narrative competence. Significant correlations were found between phonological working memory and vocabulary as well as between vocabulary and narrative competence, but not between phonological working memory and narrative competence. Results suggest that phonological working memory and narrative competence are different domains of language awareness, and that vocabulary may act as the central variable mediating between them.

Acoustophoretic printing.

Droplet-based printing methods are widely used in applications ranging from biological microarrays to additive manufacturing. However, common approaches, such as inkjet or electrohydrodynamic printing, are well suited only for materials with low viscosity or specific electromagnetic properties, respectively. While in-air acoustophoretic forces are material-independent, they are typically weak and have yet to be harnessed for printing materials. We introduce an acoustophoretic printing method that enables drop-on-demand patterning of a broad range of soft materials, including Newtonian fluids, whose viscosities span more than four orders of magnitude (0.5 to 25,000 mPa·s) and yield stress fluids (τ0 > 50 Pa). By exploiting the acoustic properties of a subwavelength Fabry-Perot resonator, we have generated an accurate, highly localized acoustophoretic force that can exceed the gravitational force by two orders of magnitude to eject microliter-to-nanoliter volume droplets. The versatility of acoustophoretic printing is demonstrated by patterning food, optical resins, liquid metals, and cell-laden biological matrices in desired motifs.

Electro-mechanical conditioning of human iPSC-derived cardiomyocytes for translational research.

RATIONALE: Impaired maturation of human iPSC-derived cardiomyocytes (hiPSC-CMs) currently limits their use in experimental research and further optimization is required to unlock their full potential. OBJECTIVE: To push hiPSC-CMs towards maturation, we recapitulated the intrinsic cardiac properties by electro-mechanical stimulation and explored how these mimetic biophysical cues interplay and influence the cell behaviour. METHODS AND RESULTS: We introduced a novel device capable of applying synchronized electrical and mechanical stimuli to hiPSC-CM monolayers cultured on a PDMS membrane and evaluated effects of conditioning on cardiomyocyte structure and function. Human iPSC-CMs retained their cardiac phenotype and displayed adaptive structural responses to electrical (E), mechanical (M) and combined electro-mechanical (EM) stimuli, including enhanced membrane N-cadherin signal, stress-fiber formation and sarcomeric length shortening, most prominent under the EM stimulation. On the functional level, EM conditioning significantly reduced the transmembrane calcium current, resulting in a shift towards triangulation of intracellular calcium transients. In contrast, E and M stimulation applied independently increased the proportion of cells with L-Type calcium currents. In addition, calcium transients measured in the M-conditioned samples advanced to a more rectangular shape. CONCLUSION: The new methodology is a simple and elegant technique to systematically investigate and manipulate cardiomyocyte remodelling for translational applications. In the present study, we adjusted critical parameters to optimize a regimen for hiPSC-CM transformation. In the future, this technology will open up new avenues for electro-mechanical stimulation by allowing temporal and spatial control of stimuli which can be easily scaled up in complexity for cardiac development and disease modelling.

SuperCYP: a comprehensive database on Cytochrome P450 enzymes including a tool for analysis of CYP-drug interactions.

Much of the information on the Cytochrome P450 enzymes (CYPs) is spread across literature and the internet. Aggregating knowledge about CYPs into one database makes the search more efficient. Text mining on 57 CYPs and drugs led to a mass of papers, which were screened manually for facts about metabolism, SNPs and their effects on drug degradation. Information was put into a database, which enables the user not only to look up a particular CYP and all metabolized drugs, but also to check tolerability of drug-cocktails and to find alternative combinations, to use metabolic pathways more efficiently. The SuperCYP database contains 1170 drugs with more than 3800 interactions including references. Approximately 2000 SNPs and mutations are listed and ordered according to their effect on expression and/or activity. SuperCYP (http://bioinformatics.charite.de/supercyp) is a comprehensive resource focused on CYPs and drug metabolism. Homology-modeled structures of the CYPs can be downloaded in PDB format and related drugs are available as MOL-files. Within the resource, CYPs can be aligned with each other, drug-cocktails can be 'mixed', SNPs, protein point mutations, and their effects can be viewed and corresponding PubMed IDs are given. SuperCYP is meant to be a platform and a starting point for scientists and health professionals for furthering their research.

Early noun plurals in German: regularity, productivity or default?

The acquisition of German plurals has been the focus of controversy in the last decade. In this paper we claim that degree of productivity (i.e. the capacity of nouns to form potential plurals) plays a key role in determining pace of acquisition. A plural elicitation task was administered to 84 Viennese German-speaking children aged 2;6 to 6;0. Analyses of correct responses showed that the highest scores were obtained with -e plurals, followed by the plural markers -e + U, -er + U, -s and -(e)n. The lowest score was observed for pure Umlaut (U) plurals. Analyses suggested an impact of productivity on the number of correct scores: fully productive and productive plural patterns obtained higher correct scores than weakly productive and non-productive ones. The results of the study support our productivity scale and are compatible both with single-route models and with a race-model variant of the dual-route view.