In vitro human cell-based models: What can they do and what are their limitations?

It is said that all models are wrong, but some are useful. In vitro human cell-based models are a prime example of this maxim. We asked researchers: when is your model system useful? How can it be made more useful? What are its limitations?

PI3Kγ in B cells promotes antibody responses and generation of antibody-secreting cells.

The differentiation of naive and memory B cells into antibody-secreting cells (ASCs) is a key feature of adaptive immunity. The requirement for phosphoinositide 3-kinase-delta (PI3Kδ) to support B cell biology has been investigated intensively; however, specific functions of the related phosphoinositide 3-kinase-gamma (PI3Kγ) complex in B lineage cells have not. In the present study, we report that PI3Kγ promotes robust antibody responses induced by T cell-dependent antigens. The inborn error of immunity caused by human deficiency in PI3Kγ results in broad humoral defects, prompting our investigation of roles for this kinase in antibody responses. Using mouse immunization models, we found that PI3Kγ functions cell intrinsically within activated B cells in a kinase activity-dependent manner to transduce signals required for the transcriptional program supporting differentiation of ASCs. Furthermore, ASC fate choice coincides with upregulation of PIK3CG expression and is impaired in the context of PI3Kγ disruption in naive B cells on in vitro CD40-/cytokine-driven activation, in memory B cells on toll-like receptor activation, or in human tonsillar organoids. Taken together, our study uncovers a fundamental role for PI3Kγ in supporting humoral immunity by integrating signals instructing commitment to the ASC fate.

Profiling Ssb-Nascent Chain Interactions Reveals Principles of Hsp70-Assisted Folding.

The yeast Hsp70 chaperone Ssb interacts with ribosomes and nascent polypeptides to assist protein folding. To reveal its working principle, we determined the nascent chain-binding pattern of Ssb at near-residue resolution by in vivo selective ribosome profiling. Ssb associates broadly with cytosolic, nuclear, and hitherto unknown substrate classes of mitochondrial and endoplasmic reticulum (ER) nascent proteins, supporting its general chaperone function. Ssb engages most substrates by multiple binding-release cycles to a degenerate sequence enriched in positively charged and aromatic amino acids. Timely association with this motif upon emergence at the ribosomal tunnel exit requires ribosome-associated complex (RAC) but not nascent polypeptide-associated complex (NAC). Ribosome footprint densities along orfs reveal faster translation at times of Ssb binding, mainly imposed by biases in mRNA secondary structure, codon usage, and Ssb action. Ssb thus employs substrate-tailored dynamic nascent chain associations to coordinate co-translational protein folding, facilitate accelerated translation, and support membrane targeting of organellar proteins.

The Hsp70 chaperone network.

The 70-kDa heat shock proteins (Hsp70s) are ubiquitous molecular chaperones that act in a large variety of cellular protein folding and remodelling processes. They function virtually at all stages of the life of proteins from synthesis to degradation and are thus crucial for maintaining protein homeostasis, with direct implications for human health. A large set of co-chaperones comprising J-domain proteins and nucleotide exchange factors regulate the ATPase cycle of Hsp70s, which is allosterically coupled to substrate binding and release. Moreover, Hsp70s cooperate with other cellular chaperone systems including Hsp90, Hsp60 chaperonins, small heat shock proteins and Hsp100 AAA+ disaggregases, together constituting a dynamic and functionally versatile network for protein folding, unfolding, regulation, targeting, aggregation and disaggregation, as well as degradation. In this Review we describe recent advances that have increased our understanding of the molecular mechanisms and working principles of the Hsp70 network. This knowledge showcases how the Hsp70 chaperone system controls diverse cellular functions, and offers new opportunities for the development of chemical compounds that modulate disease-related Hsp70 activities.

Spatiotemporally resolved colorectal oncogenesis in mini-colons ex vivo.

Three-dimensional organoid culture technologies have revolutionized cancer research by allowing for more realistic and scalable reproductions of both tumour and microenvironmental structures1-3. This has enabled better modelling of low-complexity cancer cell behaviours that occur over relatively short periods of time4. However, available organoid systems do not capture the intricate evolutionary process of cancer development in terms of tissue architecture, cell diversity, homeostasis and lifespan. As a consequence, oncogenesis and tumour formation studies are not possible in vitro and instead require the extensive use of animal models, which provide limited spatiotemporal resolution of cellular dynamics and come at a considerable cost in terms of resources and animal lives. Here we developed topobiologically complex mini-colons that are able to undergo tumorigenesis ex vivo by integrating microfabrication, optogenetic and tissue engineering approaches. With this system, tumorigenic transformation can be spatiotemporally controlled by directing oncogenic activation through blue-light exposure, and emergent colon tumours can be tracked in real-time at the single-cell resolution for several weeks without breaking the culture. These induced mini-colons display rich intratumoural and intertumoural diversity and recapitulate key pathophysiological hallmarks displayed by colorectal tumours in vivo. By fine-tuning cell-intrinsic and cell-extrinsic parameters, mini-colons can be used to identify tumorigenic determinants and pharmacological opportunities. As a whole, our study paves the way for cancer initiation research outside living organisms.

Embryo-uterine interaction coordinates mouse embryogenesis during implantation.

Embryo implantation into the uterus marks a key transition in mammalian development. In mice, implantation is mediated by the trophoblast and is accompanied by a morphological transition from the blastocyst to the egg cylinder. However, the roles of trophoblast-uterine interactions in embryo morphogenesis during implantation are poorly understood due to inaccessibility in utero and the remaining challenges to recapitulate it ex vivo from the blastocyst. Here, we engineer a uterus-like microenvironment to recapitulate peri-implantation development of the whole mouse embryo ex vivo and reveal essential roles of the physical embryo-uterine interaction. We demonstrate that adhesion between the trophoblast and the uterine matrix is required for in utero-like transition of the blastocyst to the egg cylinder. Modeling the implanting embryo as a wetting droplet links embryo shape dynamics to the underlying changes in trophoblast adhesion and suggests that the adhesion-mediated tension release facilitates egg cylinder formation. Light-sheet live imaging and the experimental control of the engineered uterine geometry and trophoblast velocity uncovers the coordination between trophoblast motility and embryo growth, where the trophoblast delineates space for embryo morphogenesis.

Thymic epithelial organoids mediate T-cell development.

Although the advent of organoids has opened unprecedented perspectives for basic and translational research, immune system-related organoids remain largely underdeveloped. Here, we established organoids from the thymus, the lymphoid organ responsible for T-cell development. We identified conditions enabling mouse thymic epithelial progenitor cell proliferation and development into organoids with diverse cell populations and transcriptional profiles resembling in vivo thymic epithelial cells (TECs) more closely than traditional TEC cultures. In contrast to these two-dimensional cultures, thymic epithelial organoids maintained thymus functionality in vitro and mediated physiological T-cell development upon reaggregation with T-cell progenitors. The reaggregates showed in vivo-like epithelial diversity and the ability to attract T-cell progenitors. Thymic epithelial organoids are the first organoids originating from the stromal compartment of a lymphoid organ. They provide new opportunities to study TEC biology and T-cell development in vitro, paving the way for future thymic regeneration strategies in ageing or acute injuries.

The unfolding story of a redox chaperone.

Oxidative stress, especially in combination with heat stress, poses a life-threatening challenge to many organisms by causing protein misfolding and aggregation. In this issue, Reichmann et al. demonstrate how a destabilized linker region of the bacterial chaperone Hsp33 prevents aggregation of a denatured protein by stabilizing structural elements.

Hsp70- and Hsp90-Mediated Regulation of the Conformation of p53 DNA Binding Domain and p53 Cancer Variants.

The activity of the tumor suppressor p53 has to be timed and balanced closely to prevent untimely induction of cell death. The stability of p53 depends on the ubiquitin ligase Mdm2 but also on Hsp70 and Hsp90 chaperones that interact with its DNA binding domain (DBD). Using hydrogen exchange mass spectrometry and biochemical methods, we analyzed conformational states of wild-type p53-DBD at physiological temperatures and conformational perturbations in three frequent p53 cancer mutants. We demonstrate that the Hsp70/Hdj1 system shifts the conformational equilibrium of p53 toward a flexible, more mutant-like, DNA binding inactive state by binding to the DNA binding loop. The analyzed cancer mutants are likewise destabilized by interaction with the Hsp70/Hdj1 system. In contrast, Hsp90 protects the DBD of p53 wild-type and mutant proteins from unfolding. We propose that the Hsp70 and Hsp90 chaperone systems assume complementary functions to optimally balance conformational plasticity with conformational stability.

Molecular mechanisms of heat shock factor 1 regulation.

To thrive and to fulfill their functions, cells need to maintain proteome homeostasis even in the face of adverse environmental conditions or radical restructuring of the proteome during differentiation. At the center of the regulation of proteome homeostasis is an ancient transcriptional mechanism, the so-called heat shock response (HSR), orchestrated in all eukaryotic cells by heat shock transcription factor 1 (Hsf1). As Hsf1 is implicated in aging and several pathologies like cancer and neurodegenerative disorders, understanding the regulation of Hsf1 could open novel therapeutic opportunities. In this review, we discuss the regulation of Hsf1's transcriptional activity by multiple layers of control circuits involving Hsf1 synthesis and degradation, conformational rearrangements and post-translational modifications (PTMs), and molecular chaperones in negative feedback loops.

An engineered multicellular stem cell niche for the 3D derivation of human myogenic progenitors from iPSCs.

Fate decisions in the embryo are controlled by a plethora of microenvironmental interactions in a three-dimensional niche. To investigate whether aspects of this microenvironmental complexity can be engineered to direct myogenic human-induced pluripotent stem cell (hiPSC) differentiation, we here screened murine cell types present in the developmental or adult stem cell niche in heterotypic suspension embryoids. We identified embryonic endothelial cells and fibroblasts as highly permissive for myogenic specification of hiPSCs. After two weeks of sequential Wnt and FGF pathway induction, these three-component embryoids are enriched in Pax7-positive embryonic-like myogenic progenitors that can be isolated by flow cytometry. Myogenic differentiation of hiPSCs in heterotypic embryoids relies on a specialized structural microenvironment and depends on MAPK, PI3K/AKT, and Notch signaling. After transplantation in a mouse model of Duchenne muscular dystrophy, embryonic-like myogenic progenitors repopulate the stem cell niche, reactivate after repeated injury, and, compared to adult human myoblasts, display enhanced fusion and lead to increased muscle function. Altogether, we provide a two-week protocol for efficient and scalable suspension-based 3D derivation of Pax7-positive myogenic progenitors from hiPSCs.

Homeostatic mini-intestines through scaffold-guided organoid morphogenesis.

Epithelial organoids, such as those derived from stem cells of the intestine, have great potential for modelling tissue and disease biology1-4. However, the approaches that are used at present to derive these organoids in three-dimensional matrices5,6 result in stochastically developing tissues with a closed, cystic architecture that restricts lifespan and size, limits experimental manipulation and prohibits homeostasis. Here, by using tissue engineering and the intrinsic self-organization properties of cells, we induce intestinal stem cells to form tube-shaped epithelia with an accessible lumen and a similar spatial arrangement of crypt- and villus-like domains to that in vivo. When connected to an external pumping system, the mini-gut tubes are perfusable; this allows the continuous removal of dead cells to prolong tissue lifespan by several weeks, and also enables the tubes to be colonized with microorganisms for modelling host-microorganism interactions. The mini-intestines include rare, specialized cell types that are seldom found in conventional organoids. They retain key physiological hallmarks of the intestine and have a notable capacity to regenerate. Our concept for extrinsically guiding the self-organization of stem cells into functional organoids-on-a-chip is broadly applicable and will enable the attainment of more physiologically relevant organoid shapes, sizes and functions.

Molecular Mechanism of J-Domain-Triggered ATP Hydrolysis by Hsp70 Chaperones.

Efficient targeting of Hsp70 chaperones to substrate proteins depends on J-domain cochaperones, which in synergism with substrates trigger ATP hydrolysis in Hsp70s and concomitant substrate trapping. We present the crystal structure of the J-domain of Escherichia coli DnaJ in complex with the E. coli Hsp70 DnaK. The J-domain interacts not only with DnaK's nucleotide-binding domain (NBD) but also with its substrate-binding domain (SBD) and packs against the highly conserved interdomain linker. Mutational replacement of contacts between J-domain and SBD strongly reduces the ability of substrates to stimulate ATP hydrolysis in the presence of DnaJ and compromises viability at heat shock temperatures. Our data demonstrate that the J-domain and the substrate do not deliver completely independent signals for ATP hydrolysis, but the J-domain, in addition to its direct influence on Hsp70s catalytic center, makes Hsp70 more responsive for the hydrolysis-inducing signal of the substrate, resulting in efficient substrate trapping.

Hsp90 Breaks the Deadlock of the Hsp70 Chaperone System.

Protein folding in the cell requires ATP-driven chaperone machines such as the conserved Hsp70 and Hsp90. It is enigmatic how these machines fold proteins. Here, we show that Hsp90 takes a key role in protein folding by breaking an Hsp70-inflicted folding block, empowering protein clients to fold on their own. At physiological concentrations, Hsp70 stalls productive folding by binding hydrophobic, core-forming segments. Hsp90 breaks this deadlock and restarts folding. Remarkably, neither Hsp70 nor Hsp90 alters the folding rate despite ensuring high folding yields. In fact, ATP-dependent chaperoning is restricted to the early folding phase. Thus, the Hsp70-Hsp90 cascade does not fold proteins, but instead prepares them for spontaneous, productive folding. This stop-start mechanism is conserved from bacteria to man, assigning also a general function to bacterial Hsp90, HtpG. We speculate that the decreasing hydrophobicity along the Hsp70-Hsp90 cascade may be crucial for enabling spontaneous folding.

Sensory tuning in neuronal movement commands.

Movement control is critical for successful interaction with our environment. However, movement does not occur in complete isolation of sensation, and this is particularly true of eye movements. Here, we show that the neuronal eye movement commands emitted by the superior colliculus (SC), a structure classically associated with oculomotor control, encompass a robust visual sensory representation of eye movement targets. Thus, similar saccades toward different images are associated with different saccade-related "motor" bursts. Such sensory tuning in SC saccade motor commands appeared for all image manipulations that we tested, from simple visual features to real-life object images, and it was also strongest in the most motor neurons in the deeper collicular layers. Visual-feature discrimination performance in the motor commands was also stronger than in visual responses. Comparing SC motor command feature discrimination performance to that in the primary visual cortex during steady-state gaze fixation revealed that collicular motor bursts possess a reliable perisaccadic sensory representation of the peripheral saccade target's visual appearance, exactly when retinal input is expected to be most uncertain. Our results demonstrate that SC neuronal movement commands likely serve a fundamentally sensory function.

Visual Feature Tuning Properties of Short-Latency Stimulus-Driven Ocular Position Drift Responses during Gaze Fixation.

Ocular position drifts during gaze fixation are significantly less well understood than microsaccades. We recently identified a short-latency ocular position drift response, of ∼1 min arc amplitude, that is triggered within <100 ms by visual onsets. This systematic eye movement response is feature-tuned and seems to be coordinated with a simultaneous resetting of the saccadic system by visual stimuli. However, much remains to be learned about the drift response, especially for designing better-informed neurophysiological experiments unraveling its mechanistic substrates. Here we systematically tested multiple new feature tuning properties of drift responses. Using highly precise eye tracking in three male rhesus macaque monkeys, we found that drift responses still occur for tiny foveal visual stimuli. Moreover, the responses exhibit size tuning, scaling their amplitude (both up and down) as a function of stimulus size, and they also possess a monotonically increasing contrast sensitivity curve. Importantly, short-latency drift responses still occur for small peripheral visual targets, which additionally introduce spatially directed modulations in drift trajectories toward the appearing peripheral stimuli. Drift responses also remain predominantly upward even for stimuli exclusively located in the lower visual field and even when starting gaze position is upward. When we checked the timing of drift responses, we found it was better synchronized to stimulus-induced saccadic inhibition than to stimulus onset. These results, along with a suppression of drift response amplitudes by peristimulus saccades, suggest that drift responses reflect the rapid impacts of short-latency and feature-tuned visual neural activity on final oculomotor control circuitry in the brain.

Self-organizing in vitro mouse neural tube organoids mimic embryonic development.

The embryonic neural tube is the origin of the entire adult nervous system, and disturbances in its development cause life-threatening birth defects. However, the study of mammalian neural tube development is limited by the lack of physiologically realistic three-dimensional (3D) in vitro models. Here, we report a self-organizing 3D neural tube organoid model derived from single mouse embryonic stem cells that exhibits an in vivo-like tissue architecture, cell type composition and anterior-posterior (AP) patterning. Moreover, maturation of the neural tube organoids showed the emergence of multipotent neural crest cells and mature neurons. Single-cell transcriptome analyses revealed the sequence of transcriptional events in the emergence of neural crest cells and neural differentiation. Thanks to the accessibility of this model, phagocytosis of migrating neural crest cells could be observed in real time for the first time in a mammalian model. We thus introduce a tractable in vitro model to study some of the key morphogenetic and cell type derivation events during early neural development.

Dissecting mechanisms of chamber-specific cardiac differentiation and its perturbation following retinoic acid exposure.

The specification of distinct cardiac lineages occurs before chamber formation and acquisition of bona fide atrial or ventricular identity. However, the mechanisms underlying these early specification events remain poorly understood. Here, we performed single cell analysis at the murine cardiac crescent, primitive heart tube and heart tube stages to uncover the transcriptional mechanisms underlying formation of atrial and ventricular cells. We find that progression towards differentiated cardiomyocytes occurs primarily based on heart field progenitor identity, and that progenitors contribute to ventricular or atrial identity through distinct differentiation mechanisms. We identify new candidate markers that define such differentiation processes and examine their expression dynamics using computational lineage trajectory methods. We further show that exposure to exogenous retinoic acid causes defects in ventricular chamber size, dysregulation in FGF signaling and a shunt in differentiation towards orthogonal lineages. Retinoic acid also causes defects in cell-cycle exit resulting in formation of hypomorphic ventricles. Collectively, our data identify, at a single cell level, distinct lineage trajectories during cardiac specification and differentiation, and the precise effects of manipulating cardiac progenitor patterning via retinoic acid signaling.

Deterministic scRNA-seq captures variation in intestinal crypt and organoid composition.

Single-cell RNA sequencing (scRNA-seq) approaches have transformed our ability to resolve cellular properties across systems, but are currently tailored toward large cell inputs (>1,000 cells). This renders them inefficient and costly when processing small, individual tissue samples, a problem that tends to be resolved by loading bulk samples, yielding confounded mosaic cell population read-outs. Here, we developed a deterministic, mRNA-capture bead and cell co-encapsulation dropleting system, DisCo, aimed at processing low-input samples (<500 cells). We demonstrate that DisCo enables precise particle and cell positioning and droplet sorting control through combined machine-vision and multilayer microfluidics, enabling continuous processing of low-input single-cell suspensions at high capture efficiency (>70%) and at speeds up to 350 cells per hour. To underscore DisCo's unique capabilities, we analyzed 31 individual intestinal organoids at varying developmental stages. This revealed extensive organoid heterogeneity, identifying distinct subtypes including a regenerative fetal-like Ly6a+ stem cell population that persists as symmetrical cysts, or spheroids, even under differentiation conditions, and an uncharacterized 'gobloid' subtype consisting predominantly of precursor and mature (Muc2+) goblet cells. To complement this dataset and to demonstrate DisCo's capacity to process low-input, in vivo-derived tissues, we also analyzed individual mouse intestinal crypts. This revealed the existence of crypts with a compositional similarity to spheroids, which consisted predominantly of regenerative stem cells, suggesting the existence of regenerating crypts in the homeostatic intestine. These findings demonstrate the unique power of DisCo in providing high-resolution snapshots of cellular heterogeneity in small, individual tissues.

Insulin Determines Transforming Growth Factor ß Effects on Hepatocyte Nuclear Factor 4α Transcription in Hepatocytes.

Loss of hepatocyte nuclear factor 4α (HNF4α) expression is frequently observed in end-stage liver disease and associated with loss of vital liver functions, thus increasing mortality. Loss of HNF4α expression is mediated by inflammatory cytokines, such as transforming growth factor (TGF)-ß. However, details of how HNF4α is suppressed are largely unknown to date. Herein, TGF-ß did not directly inhibit HNF4α but contributed to its transcriptional regulation by SMAD2/3 recruiting acetyltransferase CREB-binding protein/p300 to the HNF4α promoter. The recruitment of CREB-binding protein/p300 is indispensable for CCAAT/enhancer-binding protein α (C/EBPα) binding, another essential requirement for constitutive HNF4α expression in hepatocytes. Consistent with the in vitro observation, 67 of 98 patients with hepatic HNF4α expressed both phospho-SMAD2 and C/EBPα, whereas 22 patients without HNF4α expression lacked either phospho-SMAD2 or C/EBPα. In contrast to the observed induction of HNF4α, SMAD2/3 inhibited C/EBPα transcription. Long-term TGF-ß incubation resulted in C/EBPα depletion, which abrogated HNF4α expression. Intriguingly, SMAD2/3 inhibitory binding to the C/EBPα promoter was abolished by insulin. Two-thirds of patients without C/EBPα lacked membrane glucose transporter type 2 expression in hepatocytes, indicating insulin resistance. Taken together, these data indicate that hepatic insulin sensitivity is essential for hepatic HNF4α expression in the condition of inflammation.