Derivation and Differentiation of Human Pluripotent Stem Cells in Microfluidic Devices.

An integrative approach based on microfluidic design and stem cell biology enables capture of the spatial-temporal environmental evolution underpinning epigenetic remodeling and the morphogenetic process. We examine the body of literature that encompasses microfluidic applications where human induced pluripotent stem cells are derived starting from human somatic cells and where human pluripotent stem cells are differentiated into different cell types. We focus on recent studies where the intrinsic features of microfluidics have been exploited to control the reprogramming and differentiation trajectory at the microscale, including the capability of manipulating the fluid velocity field, mass transport regime, and controllable composition within micro- to nanoliter volumes in space and time. We also discuss studies of emerging microfluidic technologies and applications. Finally, we critically discuss perspectives and challenges in the field and how these could be instrumental for bringing about significant biological advances in the field of stem cell engineering.

High-efficiency cellular reprogramming with microfluidics.

We report that the efficiency of reprogramming human somatic cells to induced pluripotent stem cells (hiPSCs) can be dramatically improved in a microfluidic environment. Microliter-volume confinement resulted in a 50-fold increase in efficiency over traditional reprogramming by delivery of synthetic mRNAs encoding transcription factors. In these small volumes, extracellular components of the TGF-ß and other signaling pathways exhibited temporal regulation that appears critical to acquisition of pluripotency. The high quality and purity of the resulting hiPSCs (µ-hiPSCs) allowed direct differentiation into functional hepatocyte- and cardiomyocyte-like cells in the same platform without additional expansion.

Microfluidics for secretome analysis under enhanced endogenous signaling.

Cell secretome, the complex set of proteins that are secreted by the cells, is a fundamental mechanism of cell-cell communication both in vitro and in vivo. In vivo, the analysis of proteins secreted into body fluids can bring to the identification of biomarkers for important physiopathological conditions. However, due to the complexity of the protein content of body fluids, a better understanding of the secreted proteins by different cell types is highly desirable and can be performed in vitro for dissection. To this aim, microfluidic culture systems could be particularly relevant because of the accumulation of extrinsic endogenous signals at microliter scale, which better preserves the self-regulation occurring in the small interstitial spaces in vivo. In this work, we perform a quantitative study to compare the secretome in microfluidics and in a standard well plate. Human foreskin fibroblasts are used as a case study. This work also represents an important technological advance in terms of feasibility of high-throughput quantitative protein analyses in microfluidics.

Functional differentiation of human pluripotent stem cells on a chip.

Microengineering human "organs-on-chips" remains an open challenge. Here, we describe a robust microfluidics-based approach for the differentiation of human pluripotent stem cells directly on a chip. Extrinsic signal modulation, achieved through optimal frequency of medium delivery, can be used as a parameter for improved germ layer specification and cell differentiation. Human cardiomyocytes and hepatocytes derived on chips showed functional phenotypes and responses to temporally defined drug treatments.

Microfluidic technology enhances the potential of human pluripotent stem cells.

Since the discovery of human somatic cell reprogramming, human induced pluripotent stem cells (hiPSC) have been increasingly recognized as the landmark for development of organs-on-chip. hiPSCs show a remarkable plasticity that is related to their ability to promptly respond to the surrounding environment. In vitro, the soluble culture microenvironment, with its critical balance between exogenous and cell-secreted factors, plays a great role in inducing hiPSC response, for both preserving pluripotency and controlling differentiation stages. Exploring the complexity of hiPSC microenvironment requires new experimental tools, as a tight control is limited within conventional culture dishes. Microfluidic technology is particularly attractive in hiPSC research because of its ability to mimic specific environmental cues by accurate control of soluble factors with high spatiotemporal resolution and in a high-throughput fashion. In this review, we highlight recent progress in hiPSC research enabled by microfluidic technology as well as new emerging scenarios.

Single-cell PCR analysis of murine embryonic stem cells cultured on different substrates highlights heterogeneous expression of stem cell markers.

BACKGROUND INFORMATION: In the last few years, recent evidence has revealed that inside an apparently homogeneous cell population there indeed appears to be heterogeneity. This is particularly true for embryonic stem (ES) cells where markers of pluripotency are dynamically expressed within the single cells. In this work, we have designed and tested a new set of primers for multiplex PCR detection of pluripotency markers expression, and have applied it to perform a single-cell analysis in murine ES cells cultured on three different substrates that could play an important role in controlling cell behaviour and fate: (i) mouse embryonic fibroblast (MEF) feeder layer, as the standard method for ES cells culture; (ii) Matrigel coating; (iii) micropatterned hydrogel. RESULTS: Compared with population analysis, using a single-cell approach, we were able to evaluate not only the number of cells that maintained the expression of a specific gene but, most importantly, how many cells co-expressed different markers. We found that micropatterned hydrogel seems to represent a good alternative to MEF, as the expression of stemness markers is better preserved than in Matrigel culture. CONCLUSIONS: This single-cell assay allows for the assessment of the stemness maintenance at a single-cell level in terms of gene expression profile, and can be applied in stem cell research to characterise freshly isolated and cultured cells, or to standardise, for instance, the method of culture closely linked to the transcriptional activity and the differentiation potential.

Stochastic model-assisted development of efficient low-dose viral transduction in microfluidics.

Adenoviruses are commonly used in vitro as gene transfer vectors in multiple applications. Nevertheless, issues such as low infection efficiency and toxicity effects on host cells have not been resolved yet. This work aims at developing a new versatile tool to enhance the expression of transduced genes while working at low viral doses in a sequential manner. We developed a microfluidic platform with automatically controlled sequential perfusion stages, which includes 10 independent channels. In addition, we built a stochastic mathematical model, accounting for the discrete nature of cells and viruses, to predict not only the percentage of infected cells, but also the associated infecting-virus distribution in the cell population. Microfluidic system and mathematical model were coupled to define an efficient experimental strategy. We used human foreskin fibroblasts, infected by replication-incompetent adenoviruses carrying EGFP gene, as the testing system. Cell characterization was performed through fluorescence microscopy, followed by image analysis. We explored the effect of different aspects: perfusion, multiplicity of infection, and temporal patterns of infection. We demonstrated feasibility of performing efficient viral transduction at low doses, by repeated pulses of cell-virus contact. This procedure also enhanced the exogenous gene expression in the sequential microfluidic infection system compared to a single infection at a higher, nontoxic, viral dose.

Cellular population dynamics shape the route to human pluripotency.

Human cellular reprogramming to induced pluripotency is still an inefficient process, which has hindered studying the role of critical intermediate stages. Here we take advantage of high efficiency reprogramming in microfluidics and temporal multi-omics to identify and resolve distinct sub-populations and their interactions. We perform secretome analysis and single-cell transcriptomics to show functional extrinsic pathways of protein communication between reprogramming sub-populations and the re-shaping of a permissive extracellular environment. We pinpoint the HGF/MET/STAT3 axis as a potent enhancer of reprogramming, which acts via HGF accumulation within the confined system of microfluidics, and in conventional dishes needs to be supplied exogenously to enhance efficiency. Our data suggest that human cellular reprogramming is a transcription factor-driven process that it is deeply dependent on extracellular context and cell population determinants.

Micropatterning topology on soft substrates affects myoblast proliferation and differentiation.

Micropatterning techniques and substrate engineering are becoming useful tools to investigate several aspects of cell-cell interaction biology. In this work, we rationally study how different micropatterning geometries can affect myoblast behavior in the early stage of in vitro myogenesis. Soft hydrogels with physiological elastic modulus (E = 15 kPa) were micropatterned in parallel lanes (100, 300, and 500 µm width) resulting in different local and global myoblast densities. Proliferation and differentiation into multinucleated myotubes were evaluated for murine and human myoblasts. Wider lanes showed a decrease in murine myoblast proliferation: (69 ± 8)% in 100 µm wide lanes compared to (39 ± 7)% in 500 µm lanes. Conversely, fusion index increased in wider lanes: from (46 ± 7)% to (66 ± 7)% for murine myoblasts, and from (15 ± 3)% to (36 ± 2)% for human primary myoblasts, using a patterning width of 100 and 500 µm, respectively. These results are consistent with both computational modeling data and conditioned medium experiments, which demonstrated that wider lanes favor the accumulation of endogenous secreted factors. Interestingly, human primary myoblast proliferation is not affected by patterning width, which may be because the high serum content of their culture medium overrides the effect of secreted factors. These data highlight the role of micropatterning in shaping the cellular niche through secreted factor accumulation, and are of paramount importance in rationally understanding myogenesis in vitro for the correct design of in vitro skeletal muscle models.

Extracellular phosphoprotein regulation is affected by culture system scale-down.

BACKGROUND: Phosphorylated proteins are known to be present in multiple body fluids in normal conditions, and abnormally accumulated under some pathological conditions. The biological significance of their role in the extracellular space has started being elucidated only recently, for example in bone mineralization, neural development, and coagulation. Here, we address some criticalities of conventional culture systems for the study of the extracellular regulation of phosphorylation. METHODS: We make use of microfluidics to scale-down the culture volume to a size comparable to the interstitial spaces occurring in vivo. The phosphoprotein content of conditioned media was analyzed by a colorimetric assay that detects global phosphorylation. RESULTS: We found that miniaturization of the culture system increases phosphoprotein accumulation. Moreover, we demonstrated that in conventional culture systems dilution affects the extent of the phosphorylation reactions occurring within the extracellular space. On the other hand, in microfluidics the phosphorylation status was not affected by addition of adenosine triphosphate (ATP) and FAM20C Golgi Associated Secretory Pathway Kinase (FAM20C) ectokinase, as if their concentration was already not limiting for the phosphorylation reaction to occur. CONCLUSIONS: The volume of the extracellular environment plays a role in the process of extracellular phosphorylation due to its effect on the concentration of substrates, enzymes and co-factors. GENERAL SIGNIFICANCE: Thus, the biological role of extracellular phosphoregulation may be better appreciated within a microfluidic culture system.

Micropillar-based phenotypic screening platform uncovers involvement of HDAC2 in nuclear deformability.

Nuclear deformation is an essential phenomenon allowing cell migration and can be observed in association with pathological conditions such as laminopathies, neurodegenerative disorders and diabetes. Abnormal nuclear morphologies are a hallmark of cancer progression and nuclear deformability is a necessary feature for metastatic progression. Nevertheless, the cellular processes and the key molecular components controlling nuclear shape are poorly understood, in part due to a limited availability of assays that allow high-throughput screening of nuclear morphology-phenotypes. In this study, we explore the application of micropillared substrates as the basis for a phenotypic screening platform aimed at identifying novel determinants of nuclear morphology. We designed PDMS substrates to maximize simplicity in image acquisition and analyses, and in a small-scale screening of inhibitors targeting chromatin-modifying enzymes, we identify histone deacetylation as cellular process involved in nuclear deformation. With increasingly specific targeting approaches, we identify HDAC2 as a novel player in controlling nuclear morphology through gene transcription repression. This study shows the effectiveness of micropillar-based substrates to act as phenotypic drug screening platforms and opens a new avenue in the identification of genes involved in determining the nuclear shape.

3D ECM-rich environment sustains the identity of naive human iPSCs.

The establishment of in vitro naive human pluripotent stem cell cultures opened new perspectives for the study of early events in human development. The role of several transcription factors and signaling pathways have been characterized during maintenance of human naive pluripotency. However, little is known about the role exerted by the extracellular matrix (ECM) and its three-dimensional (3D) organization. Here, using an unbiased and integrated approach combining microfluidic cultures with transcriptional, proteomic, and secretome analyses, we found that naive, but not primed, hiPSC colonies are characterized by a self-organized ECM-rich microenvironment. Based on this, we developed a 3D culture system that supports robust long-term feeder-free self-renewal of naive hiPSCs and also allows direct and timely developmental morphogenesis simply by modulating the signaling environment. Our study opens new perspectives for future applications of naive hiPSCs to study critical stages of human development in 3D starting from a single cell.

Synchronization between peripheral circadian clock and feeding-fasting cycles in microfluidic device sustains oscillatory pattern of transcriptome.

The circadian system cyclically regulates many physiological and behavioral processes within the day. Desynchronization between physiological and behavioral rhythms increases the risk of developing some, including metabolic, disorders. Here we investigate how the oscillatory nature of metabolic signals, resembling feeding-fasting cycles, sustains the cell-autonomous clock in peripheral tissues. By controlling the timing, period and frequency of glucose and insulin signals via microfluidics, we find a strong effect on Per2::Luc fibroblasts entrainment. We show that the circadian Per2 expression is better sustained via a 24 h period and 12 h:12 h frequency-encoded metabolic stimulation applied for 3 daily cycles, aligned to the cell-autonomous clock, entraining the expression of hundreds of genes mostly belonging to circadian rhythms and cell cycle pathways. On the contrary misaligned feeding-fasting cycles synchronize and amplify the expression of extracellular matrix-associated genes, aligned during the light phase. This study underlines the role of the synchronicity between life-style-associated metabolic signals and peripheral clocks on the circadian entrainment.

SARS-CoV-2 infection and replication in human gastric organoids.

COVID-19 typically manifests as a respiratory illness, but several clinical reports have described gastrointestinal symptoms. This is particularly true in children in whom gastrointestinal symptoms are frequent and viral shedding outlasts viral clearance from the respiratory system. These observations raise the question of whether the virus can replicate within the stomach. Here we generate gastric organoids from fetal, pediatric, and adult biopsies as in vitro models of SARS-CoV-2 infection. To facilitate infection, we induce reverse polarity in the gastric organoids. We find that the pediatric and late fetal gastric organoids are susceptible to infection with SARS-CoV-2, while viral replication is significantly lower in undifferentiated organoids of early fetal and adult origin. We demonstrate that adult gastric organoids are more susceptible to infection following differentiation. We perform transcriptomic analysis to reveal a moderate innate antiviral response and a lack of differentially expressed genes belonging to the interferon family. Collectively, we show that the virus can efficiently infect the gastric epithelium, suggesting that the stomach might have an active role in fecal-oral SARS-CoV-2 transmission.

Nuclear Morphological Remodeling in Human Granulocytes Is Linked to Prenylation Independently from Cytoskeleton.

Nuclear shape modulates cell behavior and function, while aberrant nuclear morphologies correlate with pathological phenotype severity. Nevertheless, functions of specific nuclear morphological features and underlying molecular mechanisms remain poorly understood. Here, we investigate a nucleus-intrinsic mechanism driving nuclear lobulation and segmentation concurrent with granulocyte specification, independently from extracellular forces and cytosolic cytoskeleton contributions. Transcriptomic regulation of cholesterol biosynthesis is equally concurrent with nuclear remodeling. Its putative role as a regulatory element is supported by morphological aberrations observed upon pharmacological impairment of several enzymatic steps of the pathway, most prominently the sterol ∆14-reductase activity of laminB-receptor and protein prenylation. Thus, we support the hypothesis of a nuclear-intrinsic mechanism for nuclear shape control with the putative involvement of the recently discovered GGTase III complex. Such process could be independent from or complementary to the better studied cytoskeleton-based nuclear remodeling essential for cell migration in both physiological and pathological contexts such as immune system function and cancer metastasis.

The Microfluidic Environment Reveals a Hidden Role of Self-Organizing Extracellular Matrix in Hepatic Commitment and Organoid Formation of hiPSCs.

The specification of the hepatic identity during human liver development is strictly controlled by extrinsic signals, yet it is still not clear how cells respond to these exogenous signals by activating secretory cascades, which are extremely relevant, especially in 3D self-organizing systems. Here, we investigate how the proteins secreted by human pluripotent stem cells (hPSCs) in response to developmental exogenous signals affect the progression from endoderm to the hepatic lineage, including their competence to generate nascent hepatic organoids. By using microfluidic confined environment and stable isotope labeling with amino acids in cell culture-coupled mass spectrometry (SILAC-MS) quantitative proteomic analysis, we find high abundancy of extracellular matrix (ECM)-associated proteins. Hepatic progenitor cells either derived in microfluidics or exposed to exogenous ECM stimuli show a significantly higher potential of forming hepatic organoids that can be rapidly expanded for several passages and further differentiated into functional hepatocytes. These results prove an additional control over the efficiency of hepatic organoid formation and differentiation for downstream applications.

Engineering a 3D in vitro model of human skeletal muscle at the single fiber scale.

The reproduction of reliable in vitro models of human skeletal muscle is made harder by the intrinsic 3D structural complexity of this tissue. Here we coupled engineered hydrogel with 3D structural cues and specific mechanical properties to derive human 3D muscle constructs ("myobundles") at the scale of single fibers, by using primary myoblasts or myoblasts derived from embryonic stem cells. To this aim, cell culture was performed in confined, laminin-coated micrometric channels obtained inside a 3D hydrogel characterized by the optimal stiffness for skeletal muscle myogenesis. Primary myoblasts cultured in our 3D culture system were able to undergo myotube differentiation and maturation, as demonstrated by the proper expression and localization of key components of the sarcomere and sarcolemma. Such approach allowed the generation of human myobundles of ~10 mm in length and ~120 µm in diameter, showing spontaneous contraction 7 days after cell seeding. Transcriptome analyses showed higher similarity between 3D myobundles and skeletal signature, compared to that found between 2D myotubes and skeletal muscle, mainly resulting from expression in 3D myobundles of categories of genes involved in skeletal muscle maturation, including extracellular matrix organization. Moreover, imaging analyses confirmed that structured 3D culture system was conducive to differentiation/maturation also when using myoblasts derived from embryonic stem cells. In conclusion, our structured 3D model is a promising tool for modelling human skeletal muscle in healthy and diseases conditions.

Decellularized skeletal muscles display neurotrophic effects in three-dimensional organotypic cultures.

Skeletal muscle decellularization allows the generation of natural scaffolds that retain the extracellular matrix (ECM) mechanical integrity, biological activity, and three-dimensional (3D) architecture of the native tissue. Recent reports showed that in vivo implantation of decellularized muscles supports muscle regeneration in volumetric muscle loss models, including nervous system and neuromuscular junctional homing. Since the nervous system plays pivotal roles during skeletal muscle regeneration and in tissue homeostasis, support of reinnervation is a crucial aspect to be considered. However, the effect of decellularized muscles on reinnervation and on neuronal axon growth has been poorly investigated. Here, we characterized residual protein composition of decellularized muscles by mass spectrometry and we show that scaffolds preserve structural proteins of the ECM of both skeletal muscle and peripheral nervous system. To investigate whether decellularized scaffolds could per se attract neural axons, organotypic sections of spinal cord were cultured three dimensionally in vitro, in presence or in absence of decellularized muscles. We found that neural axons extended from the spinal cord are attracted by the decellularized muscles and penetrate inside the scaffolds upon 3D coculture. These results demonstrate that decellularized scaffolds possess intrinsic neurotrophic properties, supporting their potential use for the treatment of clinical cases where extensive functional regeneration of the muscle is required.

In vitro metabolic zonation through oxygen gradient on a chip.

Among the multiple metabolic signals involved in the establishment of the hepatic zonation, oxygen could play a key role. Indeed, depending on hepatocyte position in the hepatic lobule, gene expression and metabolism are differently affected by the oxygen gradient present across the lobule. The aim of this study is to understand whether an oxygen gradient, generated in vitro in our developed device, is sufficient to instruct a functional metabolic zonation during the differentiation of human embryonic stem cells (hESCs) from endoderm toward terminally differentiated hepatocytes, thus mimicking the in vivo situation. For this purpose, a microfluidic device was designed for the generation of a stable oxygen gradient. The oxygen gradient was applied to differentiating hESCs at the pre-hepatoblast stage. The definitive endoderm and hepatic endoderm cells were characterized by the expression of the transcription factor SOX-17 and alpha-fetoprotein (AFP). Immature and mature hepatocytes were characterized by hepatocyte nuclear factor 4-alpha (HNF-4α) and albumin (ALB) expression and also analyzed for cytochrome P450 (CYP3A4) zonation and glycogen accumulation through PAS staining. Metabolic zonated genes expression was assessed through quantitative real time PCR. Application of the oxygen gradient during differentiation induced zonated glycogen storage, which was higher in the hepatocytes grown in high pO2 compared to those grown in low pO2. The mRNA levels of glutamine synthetase (GLUL), beta-catenin (CTNNB) and its direct target cyclin D1 (CCND1) showed significantly higher expression in the cells grown in low pO2 compared to those grown in high pO2. On the contrary, carbamoyl-phosphate synthetase 1 (CPS1), ALB, the proliferative marker ki67 (MKI67) and cyclin A (CCNA) resulted to be significantly higher expressed in cells cultured in high pO2 compared to those cultured in low pO2. These results indicate that the oxygen gradient generated in our device can instruct the establishment of a functional metabolic zonation in differentiating hESCs. The possibility to obtain differentiated hepatocytes in vitro may allow in the future to deepen our knowledge about the physiology/pathology of hepatocytes in relation to the oxygen content.

Microfluidic reprogramming to pluripotency of human somatic cells.

Human induced pluripotent stem cells (hiPSCs) have a number of potential applications in stem cell biology and regenerative medicine, including precision medicine. However, their potential clinical application is hampered by the low efficiency, high costs, and heavy workload of the reprogramming process. Here we describe a protocol to reprogram human somatic cells to hiPSCs with high efficiency in 15 d using microfluidics. We successfully downscaled an 8-d protocol based on daily transfections of mRNA encoding for reprogramming factors and immune evasion proteins. Using this protocol, we obtain hiPSC colonies (up to 160 ± 20 mean ± s.d (n = 48)) in a single 27-mm2 microfluidic chamber) 15 d after seeding ~1,500 cells per independent chamber and under xeno-free defined conditions. Only ~20 µL of medium is required per day. The hiPSC colonies extracted from the microfluidic chamber do not require further stabilization because of the short lifetime of mRNA. The high success rate of reprogramming in microfluidics, under completely defined conditions, enables hundreds of cells to be simultaneously reprogrammed, with an ~100-fold reduction in costs of raw materials compared to those for standard multiwell culture conditions. This system also enables the generation of hiPSCs suitable for clinical translation or further research into the reprogramming process.