3D Stem Cell Research Articles

Designing highly customizable human based platforms for cell culture using proteins from the amniotic membrane

In the past few years researchers have witnessed a paradigm shift in the development of biomaterials for drug discovery, tissue engineering, and regenerative medicine. After the great advances resulting from the transition of the 2D to the 3D, the new focus has been to increase the clinical relevance of such systems, as well as avoid the use of animals, by developing platforms that better replicate the human physiology in vitro. In this sense, we envisage the use of human matrices extracted from ethically sourced and readily available tissues as an optimal and promising alternative to currently used approaches. Hereupon, we report for the first time the chemical modification of human ECM proteins from the amniotic membrane (AM) with photoresponsive groups to produce bioinks and hydrogel precursors to engineer customizable platforms that are representative of native tissues and capable of supporting long-term cell culture. Our results demonstrated an efficient decellularization, liquefaction and functionalization of AM-derived ECM with methacryloyl domains (AMMA), with production of stable and versatile hydrogels. Mechanical characterization evidenced an increased compression strength as a function of methacrylation degree and decellularized ECM concentration. Three-dimensional (3D) stem cell culture in the AMMA hydrogels resulted in viable and proliferative cells up to 7 days; moreover, the mouldable character of the hydrogel precursors permits the processing of patterned hydrogel constructs allowing the control over cellular alignment and elongation, or microgels with highly tunable shape.

Controlling Morphology and Functions of Cardiac Organoids by Two-Dimensional Geometrical Templates

Traditionally, tissue-specific organoids are generated as 3D aggregates of stem cells embedded in Matrigel or hydrogels, and the aggregates eventually end up a spherical shape and suspended in the matrix. Lack of geometrical control of organoid formation makes these spherical organoids limited for modeling the tissues with complex shapes. To address this challenge, we developed a new method to generate 3D spatial-organized cardiac organoids from 2D micropatterned human induced pluripotent stem cell (hiPSC) colonies, instead of directly from 3D stem cell aggregates. This new approach opens the possibility to create cardiac organoids that are templated by 2D non-spherical geometries, which potentially provides us a deeper understanding of biophysical controls on developmental organogenesis. Here, we designed 2D geometrical templates with quadrilateral shapes and pentagram shapes that had same total area but different geometrical shapes. Using this templated substrate, we grew cardiac organoids from hiPSCs and collected a series of parameters to characterize morphological and functional properties of the cardiac organoids. In quadrilateral templates, we found that increasing the aspect ratio impaired cardiac tissue 3D self-assembly, but the elongated geometry improved the cardiac contractile functions. However, in pentagram templates, cardiac organoid structure and function were optimized with a specific geometry of an ideal star shape. This study will shed a light on “organogenesis-by-design” by increasing the intricacy of starting templates from external geometrical cues to improve the organoid morphogenesis and functionality.

Mesenchymal stem cell-encapsulated cellulose nanofiber microbeads and enhanced biological activities by hyaluronic acid incorporation

Cell microencapsulation is a process to entrap viable and functional cells within a biocompatible and semi-permeable matrix to provide a favorable microenvironment to the cells. Cellulose nanofiber (CNF), a low-cost and sustainable cellulose-derived natural polymer, has been studied as a matrix for 3D stem cell culture in the form of a bulk hydrogel. Here, the preparation of CNF microbeads for the long-term 3D culture of human adipose-derived stem cells (hADSCs) was demonstrated. Furthermore, hyaluronic acid (HA) was physically incorporated into the stem cell encapsulated CNF microbeads with various molecular weights and concentrations to investigate its potential in enhancing the cellular bioactivities. The beneficial effects of HA incorporation on encapsulated cells were significant compared to CNF microbeads, especially with 700 kDa molecular weight and 0.2% in concentration in terms of cell proliferation (~2 times) and VEGF secretion (~2 times) while maintaining their stemness. All the results demonstrated that the HA-incorporated CNF microbeads could serve as a promising microencapsulation matrix for hADSCs.

3D hESC exosomes enriched with miR-6766-3p ameliorates liver fibrosis by attenuating activated stellate cells through targeting the TGF\u03b2RII-SMADS pathway

BackgroundExosomes secreted from stem cells exerted salutary effects on the fibrotic liver. Herein, the roles of exosomes derived from human embryonic stem cell (hESC) in anti-fibrosis were extensively investigated. Compared with two-dimensional (2D) culture, the clinical and biological relevance of three-dimensional (3D) cell spheroids were greater because of their higher regeneration potential since they behave more like cells in vivo. In our study, exosomes derived from 3D human embryonic stem cells (hESC) spheroids and the monolayer (2D) hESCs were collected and compared the therapeutic potential for fibrotic liver in vitro and in vivo.ResultsIn vitro, PKH26 labeled-hESC-Exosomes were shown to be internalized and integrated into TGFβ-activated-LX2 cells, and reduced the expression of profibrogenic markers, thereby regulating cellular phenotypes. TPEF imaging indicated that PKH26-labeled-3D-hESC-Exsomes possessed an enhanced capacity to accumulate in the livers and exhibited more dramatic therapeutic potential in the injured livers of fibrosis mouse model. 3D-hESC-Exosomes decreased profibrogenic markers and liver injury markers, and improved the level of liver functioning proteins, eventually restoring liver function of fibrosis mice. miRNA array revealed a significant enrichment of miR-6766-3p in 3D-hESC-Exosomes, moreover, bioinformatics and dual luciferase reporter assay identified and confirmed the TGFβRII gene as the target of miR-6766-3p. Furthermore, the delivery of miR-6766-3p into activated-LX2 cells decreased cell proliferation, chemotaxis and profibrotic effects, and further investigation demonstrated that the expression of target gene TGFβRII and its downstream SMADs proteins, especially phosphorylated protein p-SMAD2/3 was also notably down-regulated by miR-6766-3p. These findings unveiled that miR-6766-3p in 3D-hESC-Exosomes inactivated SMADs signaling by inhibiting TGFβRII expression, consequently attenuating stellate cell activation and suppressing liver fibrosis.ConclusionsOur results showed that miR-6766-3p in the 3D-hESC-Exosomes inactivates smads signaling by restraining TGFβRII expression, attenuated LX2 cell activation and suppressed liver fibrosis, suggesting that 3D-hESC-Exosome enriched-miR-6766-3p is a novel anti-fibrotic therapeutics for treating chronic liver disease. These results also proposed a significant strategy that 3D-Exo could be used as natural nanoparticles to rescue liver injury via delivering antifibrotic miR-6766-3p.Graphical

TO901317 activation of LXR-dependent pathways mitigate amyloid-beta peptide-induced neurotoxicity in 3D human neural stem cell culture scaffolds and AD mice

Alzheimer's disease (AD) is the major cause of neurodegeneration worldwide and is characterized by the accumulation of amyloid beta (Aβ) in the brain, which is associated with neuronal loss and cognitive impairment. Liver X receptor (LXR), a critical nuclear receptor, and major regulator in lipid metabolism and inflammation, is suggested to play a protective role against the mitochondrial dysfunction noted in AD. In our study, our established 3D gelatin scaffold model and a well characterized in vivo (APP/PS1) murine model of AD were used to directly investigate the molecular, biochemical and behavioral effects of neuronal stem cell exposure to Aβ to improve understanding of the in vivo etiology of AD. Herein, human neural stem cells (hNSCs) in our 3D model were exposed to Aβ, and had significantly decreased cell viability, which correlated with decreased mRNA and protein expression of LXR, Bcl-2, CREB, PGC1α, NRF-1, and Tfam, and increased caspase 3 and 9 activities. Cotreatment with a synthetic agonist of LXR (TO901317) significantly abrogated these Aβ-mediated effects in hNSCs. Moreover, TO901317 cotreatment both significantly rescues hNSCs from Aβ-mediated decreases in ATP levels and mitochondrial mass, and significantly restores Aβ-induced fragmented mitochondria to almost normal morphology. TO901317 cotreatment also decreases tau aggregates in Aβ-treated hNSCs. Importantly, TO901317 treatment significantly alleviates the impairment of memory, decreases Aβ aggregates and increases proteasome activity in APP/PS1 mice; whereas, these effects were blocked by cotreatment with an LXR antagonist (GSK2033). Together, these novel results improve our mechanistic understanding of the central role of LXR in Aβ-mediated hNSC dysfunction. We also provide preclinical data unveiling the protective effects of using an LXR-dependent agonist, TO901317, to block the toxicity observed in Aβ-exposed hNSCs, which may guide future treatment strategies to slow or prevent neurodegeneration in some AD patients.

Tailoring the proliferation of fibroblast cells by multiresponsive and thermosensitive stem cells composite F127 hydrogel containing folic acid.MgO:ZnO/chitosan hybrid microparticles for skin regeneration

In this study, biodegradable and thermosensitive F127 hydrogel containing folic acid.MgO:ZnO/chitosan hybrid particles (FMZC) was fabricated as a 3D mesenchymal stem cells (MSCs) delivery vehicle for regenerative medicine and wound healing purposes, in such a way to be responsive to lysozyme and UVA irradiation. The results showed that F127 hydrogel containing FMZC is a suitable and nontoxic construct for encapsulation of MSCs in the presence of lysozyme and UVA irradiation, bearing high stem cell viability and proliferation. The final hydrogel, MSC&FMZC, in response to lysozyme induced a higher proliferation rate and migration in human foreskin fibroblast cells (HFF). These phenomena were attributed to the released F.MgO:ZnO nanocomposites from chitosan microparticles and paracrine factors from MSCs within the hydrogel, resulting in synergistic biological effects. Moreover, lysozyme-treated MSC&FMZC hydrogel showed higher antibacterial and anti-biofilm activity against both Gram-positive and Gram-negative bacteria than bare hydrogel. However, a significant increase in the antibacterial activity of MSC&FMZC was observed as the treated bacteria were subjected to UVA irradiation owing to the photocatalytic activity of F.MgO:ZnO nanocomposites. Regarding the antibacterial activity and stimulating skin cell behavior of MSC&FMZC hydrogel that can promote the regenerative activities of skin, it could be considered as a promising scaffold for bacteria-accompanied wound healing.

Investigating developmental effects of PFAS using a 3D human induced pluripotent stem cell differentiation model

Investigating developmental effects of PFAS using a 3D human induced pluripotent stem cell differentiation model

Increased connectivity of hiPSC-derived neural networks in multiphase granular hydrogel scaffolds

To reflect human development, it is critical to create a substrate that can support long-term cell survival, differentiation, and maturation. Hydrogels are promising materials for 3D cultures. However, a bulk structure consisting of dense polymer networks often leads to suboptimal microenvironments that impedes nutrient exchange and cell-to-cell interaction. Herein, granular hydrogel-based scaffolds were used to support 3D human induced pluripotent stem cell (hiPSC)-derived neural networks. A custom designed 3D printed toolset was developed to extrude hyaluronic acid hydrogel through a porous nylon fabric to generate hydrogel granules. Cells and hydrogel granules were combined using a weaker secondary gelation step, forming self-supporting cell laden scaffolds. At three and seven days, granular scaffolds supported higher cell viability compared to bulk hydrogels, whereas granular scaffolds supported more neurite bearing cells and longer neurite extensions (65.52 ± 11.59 μm) after seven days compared to bulk hydrogels (22.90 ± 4.70 μm). Long-term (three-month) cultures of clinically relevant hiPSC-derived neural cells in granular hydrogels supported well established neuronal and astrocytic colonies and a high level of neurite extension both inside and beyond the scaffold. This approach is significant as it provides a simple, rapid and efficient way to achieve a tissue-relevant granular structure within hydrogel cultures.

Enhanced mechanosensing of cells in synthetic 3D matrix with controlled biophysical dynamics

3D culture of cells in designer biomaterial matrices provides a biomimetic cellular microenvironment and can yield critical insights into cellular behaviours not available from conventional 2D cultures. Hydrogels with dynamic properties, achieved by incorporating either degradable structural components or reversible dynamic crosslinks, enable efficient cell adaptation of the matrix and support associated cellular functions. Herein we demonstrate that given similar equilibrium binding constants, hydrogels containing dynamic crosslinks with a large dissociation rate constant enable cell force-induced network reorganization, which results in rapid stellate spreading, assembly, mechanosensing, and differentiation of encapsulated stem cells when compared to similar hydrogels containing dynamic crosslinks with a low dissociation rate constant. Furthermore, the static and precise conjugation of cell adhesive ligands to the hydrogel subnetwork connected by such fast-dissociating crosslinks is also required for ultra-rapid stellate spreading (within 18 h post-encapsulation) and enhanced mechanosensing of stem cells in 3D. This work reveals the correlation between microscopic cell behaviours and the molecular level binding kinetics in hydrogel networks. Our findings provide valuable guidance to the design and evaluation of supramolecular biomaterials with cell-adaptable properties for studying cells in 3D cultures.

3D bioprinted human iPSC-derived somatosensory constructs with functional and highly purified sensory neuron networks

Engineering three-dimensional (3D) sensible tissue constructs, along with the complex microarchitecture wiring of the sensory nervous system, has been an ongoing challenge in the tissue engineering field. By combining 3D bioprinting and human pluripotent stem cell (hPSC) technologies, sensible tissue constructs could be engineered in a rapid, precise, and controllable manner to replicate 3D microarchitectures and mechanosensory functionalities of the native sensory tissue (e.g. response to external stimuli). Here, we introduce a biofabrication approach to create complex 3D microarchitecture wirings. We develop an hPSC-sensory neuron (SN) laden bioink using highly purified and functional SN populations to 3D bioprint microarchitecture wirings that demonstrate responsiveness to warm/cold sense-inducing chemicals and mechanical stress. Specifically, we tailor a conventional differentiation strategy to our purification method by utilizing p75 cell surface marker and DAPT treatment along with neuronal growth factors in order to selectively differentiate neural crest cells into SNs. To create spatial resolution in 3D architectures and grow SNs in custom patterns and directions, an induced pluripotent stem cell (iPSC)-SN-laden gelatin bioink was printed on laminin-coated substrates using extrusion-based bioprinting technique. Then the printed constructs were covered with a collagen matrix that guided SNs growing in the printed micropattern. Using a sacrificial bioprinting technique, the iPSC-SNs were seeded into the hollow microchannels created by sacrificial gelatin ink printed in the gelatin methacryloyl supporting bath, thereby demonstrating controllability over axon guidance in curved lines up to several tens of centimeters in length on 2D substrates and in straight microchannels in 3D matrices. Therefore, this biofabrication approach could be amenable to incorporate sensible SN networks into the engineered skin equivalents, regenerative skin implants, and augmented somatosensory neuro-prosthetics that have the potential to regenerate sensible functions by connecting host neuron systems in injured areas.

Kinship of conditionally immortalized cells derived from fetal bone to human bone-derived mesenchymal stroma cells

The human fetal osteoblast cell line (hFOB 1.19) has been proposed as an accessible experimental model for study of osteoblast biology relating to drug development and biomaterial engineering. For their multilineage differentiation potential, hFOB has been compared to human mesenchymal progenitor cells and used to investigate bone-metabolism in vitro. Hereby, we studied whether and to what extent the conditionally immortalized cell line hFOB 1.19 can serve as a surrogate model for bone-marrow derived mesenchymal stromal cells (bmMSC). hFOB indeed exhibit specific characteristics reminiscent of bmMSC, as colony formation, migration capacity and the propensity to grow as multicellular aggregates. After prolonged culture, in contrast to the expected effect of immortalization, hFOB acquired a delayed growth rate. In close resemblance to bmMSC at increasing passages, also hFOB showed morphological abnormalities, enlargement and finally reduced proliferation rates together with enhanced expression of the cell cycle inhibitors p21 and p16. hFOB not only have the ability to undergo multilineage differentiation but portray several important aspects of human bone marrow mesenchymal stromal cells. Superior to primary MSC and osteoblasts, hFOB enabled the generation of continuous cell lines. These provide an advanced basis for investigating age-related dysfunctions of MSCs in an in vitro 3D-stem cell microenvironment.

Branching development of early post-implantation human embryonic-like tissues in 3D stem cell culture

Human embryonic stem cells (hESCs) have the intrinsic capacity to self-organize and generate patterned tissues. In vitro models that coax hESCs to form embryonic-like structures by modulating physical environments and priming with chemical signals have become a powerful tool for dissecting the regulatory mechanisms underlying early human development. Here we present a 3D suspension culture system of hESCs that can generate post-implantation, pre-gastrulation embryonic-like tissues in an efficient and controllable manner. The efficiency of the development of asymmetric tissues, which mimic the post-implantation, pre-gastrulation amniotic sac, was about 50% in the 3D suspension culture. Quantitative imaging profiling and unsupervised trajectory analysis revealed that hESC aggregates first entered into a transitional stage expressing Brachyury (or T), before their development branched into different paths to develop into asymmetric embryonic-like tissues, amniotic-like tissues, and mesodermal-like tissues, respectively. Moreover, the branching developmental trajectory of embryonic-like structures was affected by the initial cell seeding density or cluster size of hESCs. A higher percentage of amniotic-like tissues was observed under a small initial cell seeding density of hESCs. Conversely, a large initial cell seeding density of hESCs promoted the development of mesodermal-like tissues. Intermediate cell seeding densities of hESCs in the 3D suspension culture promoted the development of asymmetric embryonic-like tissues. Our results suggest that hESCs have the intrinsic capability to sense the initial cell population size, which in turn regulates their differentiation and self-organization into different embryonic-like tissues. Our 3D suspension culture thus provides a promising experimental tool to study the interplay between tissue topology and self-organization and progressive embryonic development using in vitro hESC-based models.

Developmental effects of PFOS, PFOA and GenX in a 3D human induced pluripotent stem cell differentiation model

Polyfluoroalkyl substances (PFASs), including perfluorooctanesulfonic acid (PFOS) and perfluorooctanoic acid (PFOA), are persistent pollutants routinely found in human blood. PFASs have been associated with health issues such as decreased birth weight and impaired vaccination response in children. Substitutes to these PFASs, such as ammonium 2,3,3,3-tetrafluoro-2-(heptafluoropropoxy)propanoate (GenX) have been introduced, although hazard information is limited. Human induced pluripotent stem cell (hiPSC) based models are valuable for studying these compounds, as they mimic human embryonic development. We used our recently developed PluriBeat assay to investigate PFOS, PFOA and GenX for effects on early embryonic development in vitro. In our assay hiPSCs go through the early stages of embryonic development in 3D cultures of embryoid bodies (EBs) that mimic the human blastocyst until they finally form beating cardiomyocytes. Both PFOS and PFOA had a strong effect on cardiomyocyte differentiation at non-cytotoxic concentrations, with PFOS being more potent than PFOA. Moreover, both compounds decreased EB size at the highest test concentrations. GenX induced a weak concentration-dependent effect on differentiation of one hiPSC line, but not of another. Transcriptional analysis of mRNA from the cardiomyocytes showed that PFOS increased expression of the early cardiac marker ISL1, whereas PFOA decreased expression of the cardiomyocyte marker MYH7. This suggest that PFOS and PFOA perturb cardiomyocyte differentiation by disrupting molecular pathways similar to those taking place in the developing embryo. Based on these findings, we conclude that our PluriBeat assay has the potential to become a valuable, sensitive model system for elucidating embryotoxic effects of PFASs in future.

Bioactive Decellularized Extracellular Matrix Derived from 3D Stem Cell Spheroids under Macromolecular Crowding Serves as a Scaffold for Tissue Engineering.

Scaffolds for tissue engineering aim to mimic the native extracellular matrix (ECM) that provides physical support and biochemical signals to modulate multiple cell behaviors. However, the majority of currently used biomaterials are oversimplified and therefore fail to provide a niche required for the stimulation of tissue regeneration. In the present study, 3D decellularized ECM (dECM) scaffolds derived from mesenchymal stem cell (MSC) spheroids and with intricate matrix composition are developed. Specifically, application of macromolecular crowding (MMC) to MSC spheroid cultures facilitate ECM assembly in a 3D configuration, resulting in the accumulation of ECM and associated bioactive components. Decellularized 3D dECM constructs produced under MMC are able to adequately preserve the microarchitecture of structural ECM components and are characterized by higher retention of growth factors. This results in a stronger proangiogenic bioactivity as compared to constructs produced under uncrowded conditions. These dECM scaffolds can be homogenously populated by endothelial cells, which direct the macroassembly of the structures into larger cell-carrying constructs. Application of empty scaffolds enhances intrinsic revascularization in vivo, indicating that the 3D dECM scaffolds represent optimal proangiogenic bioactive blocks for the construction of larger engineered tissue constructs.

Advanced Cardiac Models: Considerations for the Bioengineering of Advanced Cardiac In Vitro Models of Myocardial Infarction (Small 15/2021)

In article number 2003765, Carmine Gentile and co-workers describe the major pathophysiological features required for the bioengineering of a heart attack in a Petri dish. The article presents a thorough comparison of current in vitro, ex vivo, and in vivo models. It also explores the potential use of latest technologies for advanced cardiac models, such as 3D bioprinting, microfluidic devices and patient-derived stem cell, with the potential to develop personalized novel therapies for heart attack patients.

Three-dimensional model of glioblastoma by co-culturing tumor stem cells with human brain organoids.

ABSTRACTEmerging three-dimensional (3D) cultures of glioblastoma are becoming powerful models to study glioblastoma stem cell behavior and the impact of cell–cell and cell–microenvironment interactions on tumor growth and invasion. Here we describe a method for culturing human glioblastoma stem cells (GSCs) in 3D by co-culturing them with pluripotent stem cell-derived brain organoids. This requires multiple coordinated steps, including the generation of cerebral organoids, and the growth and fluorescence tagging of GSCs. We highlight how to recognize optimal organoid generation and how to efficiently mark GSCs, before describing optimized co-culture conditions. We show that GSCs can efficiently integrate into brain organoids and maintain a significant degree of cell fate heterogeneity, paving the way for the analysis of GSC fate behavior and lineage progression. These results establish the 3D culture system as a viable and versatile GBM model for investigating tumor cell biology and GSC heterogeneity.This article has an associated First Person interview with the first author of the paper.

Human liver model systems in a dish.

The human adult liver has a multi-cellular structure consisting of large lobes subdivided into lobules containing portal triads and hepatic cords lined by specialized blood vessels. Vital hepatic functions include filtering blood, metabolizing drugs, and production of bile and blood plasma proteins like albumin, among many other functions, which are generally dependent on the location or zone in which the hepatocyte resides in the liver. Due to the liver's intricate structure, there are many challenges to design differentiation protocols to generate more mature functional hepatocytes from human stem cells and maintain the long-term viability and functionality of primary hepatocytes. To this end, recent advancements in three-dimensional (3D) stem cell culture have accelerated the generation of a human miniature liver system, also known as liver organoids, with polarized epithelial cells, supportive cell types and extra-cellular matrix deposition by translating knowledge gained in studies of animal organogenesis and regeneration. To facilitate the efforts to study human development and disease using in vitro hepatic models, a thorough understanding of state-of-art protocols and underlying rationales is essential. Here, we review rapidly evolving 3D liver models, mainly focusing on organoid models differentiated from human cells.

Automated optimization of endoderm differentiation on chip.

Human induced pluripotent stem cells (hiPSCs) can serve as an unlimited source to rebuild organotypic tissues in vitro. Successful engineering of functional cell types and complex organ structures outside the human body requires knowledge of the chemical, temporal, and spatial microenvironment of their in vivo counterparts. Despite an increased understanding of mouse and human embryonic development, screening approaches are still required for the optimization of stem cell differentiation protocols to gain more functional mature cell types. The liver, lung, pancreas, and digestive tract originate from the endoderm germ layer. Optimization and specification of the earliest differentiation step, which is the definitive endoderm (DE), is of central importance for generating cell types of these organs because off-target cell types will propagate during month-long cultivation steps and reduce yields. Here, we developed a microfluidic large-scale integration (mLSI) chip platform for combined automated three-dimensional (3D) cell culturing and high-throughput imaging to investigate anterior/posterior patterns occurring during hiPSC differentiation into DE cells. Integration of 3D cell cultures with a diameter of 150 μm was achieved using a U-shaped pneumatic membrane valve, which was geometrically optimized and fluidically characterized. Upon parallelization of 32 fluidically individually addressable cell culture unit cells with a total of 128 3D cell cultures, complex and long-term DE differentiation protocols could be automated. Real-time bright-field imaging was used to analyze cell growth during DE differentiation, and immunofluorescence imaging on optically cleared 3D cell cultures was used to determine the DE differentiation yield. By systematically alternating transforming growth factor β (TGF-β) and WNT signaling agonist concentrations and temporal stimulation, we showed that even under similar DE differentiation yields, there were patterning differences in the 3D cell cultures, indicating possible differentiation differences between established DE protocols. The automated mLSI chip platform with the general analytical workflow for 3D stem cell cultures offers the optimization of in vitro generation of various cell types for cell replacement therapies.

Thin fluorinated polymer film microcavity arrays for 3D cell culture and label-free automated feature extraction.

There is an increasing need for automated label-free morphometric analysis using brightfield microscopy images of 3D cell culture systems. This requires automated feature detection which can be achieved by improving the image contrast, e.g. by reducing the refractive index mismatch in the light path. Here, a novel microcavity platform fabricated using microthermoforming of thin fluorinated ethylene-propylene (FEP) films which match the refractive index of cell culture medium and provide a homogenous background signal intensity is described. FEP is chemically inert, mechanically stable and has been used as a substrate for light sheet microscopy. The microcavities promote formation of mouse embryonic stem cell (mESC) aggregates, which show axial elongation and germ layer specification similar to embryonic development. A label-free feature extraction pipeline based on a machine-learning plugin for FIJI is used to extract morphometric features from time-lapse imaging in a highly robust and reproducible manner. Lastly, the pipeline is utilized for testing the effect of the drug Latrunculin A on the mESC aggregates, highlighting the platform's potential for high-content screening (HCS) in drug discovery. This new microengineered tool is an important step towards label-free imaging of free-floating stem cell aggregates and paves the way for high-content drug testing and translational studies.

The miR-199a/214 Cluster Controls Nephrogenesis and Vascularization in a Human Embryonic Stem Cell Model.

SummaryMicroRNAs (miRNAs) are gene expression regulators and they have been implicated in acquired kidney diseases and in renal development, mostly through animal studies. We hypothesized that the miR-199a/214 cluster regulates human kidney development. We detected its expression in human embryonic kidneys by in situ hybridization. To mechanistically study the cluster, we used 2D and 3D human embryonic stem cell (hESC) models of kidney development. After confirming expression in each model, we inhibited the miRNAs using lentivirally transduced miRNA sponges. This reduced the WT1+ metanephric mesenchyme domain in 2D cultures. Sponges did not prevent the formation of 3D kidney-like organoids. These organoids, however, contained dysmorphic glomeruli, downregulated WT1, aberrant proximal tubules, and increased interstitial capillaries. Thus, the miR-199a/214 cluster fine-tunes differentiation of both metanephric mesenchymal-derived nephrons and kidney endothelia. While clinical implications require further study, it is noted that patients with heterozygous deletions encompassing this miRNA locus can have malformed kidneys.