Catalase-Laden Microdevices for Cell-Mediated Enzyme Delivery.

Enzymes have been used to treat various human diseases and traumas. However, the therapeutic utility of free enzymes is impeded by their short circulation time, lack of targeting ability, immunogenicity, and inability to cross biological barriers. Cell-mediated drug delivery approach offers the unique capability to overcome these limitations, but the traditional cell-mediated enzyme delivery techniques suffer from drawbacks such as risk of intracellular degradation of and low loading capacity for the payload enzyme. This article presents the development of a novel cell-mediated enzyme delivery technique featuring the use of micrometer-sized disk-shaped particles termed microdevices. The microdevices are fabricated by layer-by-layer assembly and soft lithography with catalase being used as a model therapeutic enzyme. The amount of catalase in the microdevices can be controlled with the number of catalase layers. Catalase in the microdevices is catalytically active, and active catalase is slowly released from the microdevices. Moreover, cell-microdevice complexes are produced by attaching the catalase-laden microdevices to the external surface of both K562 cells and mouse embryonic stem cells. This technique is potentially applicable to other enzymes and cells and promises to be clinically useful.

Intracellular labeling of mouse embryonic stem cell-derived neural progenitor aggregates with micron-sized particles of iron oxide.

BACKGROUND AIMS: Pluripotent stem cell (PSC)-derived neural progenitor cells (NPCs) represent an unlimited source for the treatment of various neurological disorders. NPCs are usually derived from PSCs through the formation of embryoid body (EB), an aggregate structure mimicking embryonic development. This study investigated the effect of labeling multicellular EB-NPC aggregates with micron-sized particles of iron oxide (MPIO) for cell tracking using magnetic resonance imaging (MRI). METHODS: Intact and dissociated EB-NPC aggregates were labeled with various concentrations of MPIOs (0, 2.5, 5 and 10 µg Fe/mL). The labeled cells were analyzed by fluorescent imaging, flow cytometry and in vitro MRI for labeling efficiency and detectability. Moreover, the biological effects of intracellular MPIO on cell viability, cytotoxicity, proliferation and neural differentiation were evaluated. RESULTS: Intact EB-NPC aggregates showed higher cell proliferation and viability compared with the dissociated cells. Despite diffusion limitation at low MPIO concentration, higher concentration of MPIO (i.e., 10 µg Fe/mL) was able to label EB-NPC aggregates at similar efficiency to the single cells. In vitro MRI showed concentration-dependent MPIO detection in EB-NPCs over 2.0-2.6 population doublings. More important, MPIO incorporation did not affect the proliferation and neural differentiation of EB-NPCs. CONCLUSIONS: Multicellular EB-NPC aggregates can be efficiently labeled and tracked with MPIO while maintaining cell proliferation, phenotype and neural differentiation potential. This study demonstrated the feasibility of labeling EB-NPC aggregates with MPIO for cellular monitoring of in vitro cultures and in vivo transplantation.

Catalytic polymer self-cleavage for CO2 generation before combustion empowers materials with fire safety.

Polymeric materials, rich in carbon, hydrogen, and oxygen elements, present substantial fire hazards to both human life and property due to their intrinsic flammability. Overcoming this challenge in the absence of any flame-retardant elements is a daunting task. Herein, we introduce an innovative strategy employing catalytic polymer auto-pyrolysis before combustion to proactively release CO2, akin to possessing responsive CO2 fire extinguishing mechanisms. We demonstrate that potassium salts with strong nucleophilicity (such as potassium formate/malate) can transform conventional polyurethane foam into materials with fire safety through rearrangement. This transformation results in the rapid generation of a substantial volume of CO2, occurring before the onset of intense decomposition, effectively extinguishing fires. The inclusion of just 1.05 wt% potassium formate can significantly raise the limiting oxygen index of polyurethane foam to 26.5%, increase the time to ignition by 927%, and tremendously reduce smoke toxicity by 95%. The successful application of various potassium salts, combined with a comprehensive examination of the underlying mechanisms, underscores the viability of this strategy. This pioneering catalytic approach paves the way for the efficient and eco-friendly development of polymeric materials with fire safety.

3D bioprinting of human neural tissues with functional connectivity.

Probing how human neural networks operate is hindered by the lack of reliable human neural tissues amenable to the dynamic functional assessment of neural circuits. We developed a 3D bioprinting platform to assemble tissues with defined human neural cell types in a desired dimension using a commercial bioprinter. The printed neuronal progenitors differentiate into neurons and form functional neural circuits within and between tissue layers with specificity within weeks, evidenced by the cortical-to-striatal projection, spontaneous synaptic currents, and synaptic response to neuronal excitation. Printed astrocyte progenitors develop into mature astrocytes with elaborated processes and form functional neuron-astrocyte networks, indicated by calcium flux and glutamate uptake in response to neuronal excitation under physiological and pathological conditions. These designed human neural tissues will likely be useful for understanding the wiring of human neural networks, modeling pathological processes, and serving as platforms for drug testing.

3D Bioprinting of Human Neural Tissues with Functional Connectivity.

Probing how the human neural networks operate is hindered by the lack of reliable human neural tissues amenable for dynamic functional assessment of neural circuits. We developed a 3D bioprinting platform to assemble tissues with defined human neural cell types in a desired dimension using a commercial bioprinter. The printed neuronal progenitors differentiate to neurons and form functional neural circuits in and between tissue layers with specificity within weeks, evidenced by the cortical-to-striatal projection, spontaneous synaptic currents and synaptic response to neuronal excitation. Printed astrocyte progenitors develop into mature astrocytes with elaborated processes and form functional neuron-astrocyte networks, indicated by calcium flux and glutamate uptake in response to neuronal excitation under physiological and pathological conditions. These designed human neural tissues will likely be useful for understanding the wiring of human neural networks, modeling pathological processes, and serving as platforms for drug testing.

Generation of Cerebral Cortical Neurons from Human Pluripotent Stem Cells in 3D Culture.

Human forebrain cortical neurons are essential for fundamental functions like memory and consciousness. Generation of cortical neurons from human pluripotent stem cells provides a great source for creating models specific to cortical neuron diseases and for developing therapeutics. This chapter describes a detailed and robust method for generating human mature cortical neurons from stem cells in 3D suspension culture.

Neural lineage differentiation of human pluripotent stem cells: Advances in disease modeling.

Brain diseases affect 1 in 6 people worldwide. These diseases range from acute neurological conditions such as stroke to chronic neurodegenerative disorders such as Alzheimer's disease. Recent advancements in tissue-engineered brain disease models have overcome many of the different shortcomings associated with the various animal models, tissue culture models, and epidemiologic patient data that are commonly used to study brain disease. One innovative method by which to model human neurological disease is via the directed differentiation of human pluripotent stem cells (hPSCs) to neural lineages including neurons, astrocytes, and oligodendrocytes. Three-dimensional models such as brain organoids have also been derived from hPSCs, offering more physiological relevance due to their incorporation of various cell types. As such, brain organoids can better model the pathophysiology of neural diseases observed in patients. In this review, we will emphasize recent developments in hPSC-based tissue culture models of neurological disorders and how they are being used to create neural disease models.

Generation of locus coeruleus norepinephrine neurons from human pluripotent stem cells.

Central norepinephrine (NE) neurons, located mainly in the locus coeruleus (LC), are implicated in diverse psychiatric and neurodegenerative diseases and are an emerging target for drug discovery. To facilitate their study, we developed a method to generate 40-60% human LC-NE neurons from human pluripotent stem cells. The approach depends on our identification of ACTIVIN A in regulating LC-NE transcription factors in dorsal rhombomere 1 (r1) progenitors. In vitro generated human LC-NE neurons display extensive axonal arborization; release and uptake NE; and exhibit pacemaker activity, calcium oscillation and chemoreceptor activity in response to CO2. Single-nucleus RNA sequencing (snRNA-seq) analysis at multiple timepoints confirmed NE cell identity and revealed the differentiation trajectory from hindbrain progenitors to NE neurons via an ASCL1-expressing precursor stage. LC-NE neurons engineered with an NE sensor reliably reported extracellular levels of NE. The availability of functional human LC-NE neurons enables investigation of their roles in psychiatric and neurodegenerative diseases and provides a tool for therapeutics development.

The Use of Pluripotent Stem Cell-Derived Organoids to Study Extracellular Matrix Development during Neural Degeneration.

The mechanism that causes the Alzheimer's disease (AD) pathologies, including amyloid plaque, neurofibrillary tangles, and neuron death, is not well understood due to the lack of robust study models for human brain. Three-dimensional organoid systems based on human pluripotent stem cells (hPSCs) have shown a promising potential to model neurodegenerative diseases, including AD. These systems, in combination with engineering tools, allow in vitro generation of brain-like tissues that recapitulate complex cell-cell and cell-extracellular matrix (ECM) interactions. Brain ECMs play important roles in neural differentiation, proliferation, neuronal network, and AD progression. In this contribution related to brain ECMs, recent advances in modeling AD pathology and progression based on hPSC-derived neural cells, tissues, and brain organoids were reviewed and summarized. In addition, the roles of ECMs in neural differentiation of hPSCs and the influences of heparan sulfate proteoglycans, chondroitin sulfate proteoglycans, and hyaluronic acid on the progression of neurodegeneration were discussed. The advantages that use stem cell-based organoids to study neural degeneration and to investigate the effects of ECM development on the disease progression were highlighted. The contents of this article are significant for understanding cell-matrix interactions in stem cell microenvironment for treating neural degeneration.

Studying Heterotypic Cell⁻Cell Interactions in the Human Brain Using Pluripotent Stem Cell Models for Neurodegeneration.

Human cerebral organoids derived from induced pluripotent stem cells (iPSCs) provide novel tools for recapitulating the cytoarchitecture of the human brain and for studying biological mechanisms of neurological disorders. However, the heterotypic interactions of neurovascular units, composed of neurons, pericytes (i.e., the tissue resident mesenchymal stromal cells), astrocytes, and brain microvascular endothelial cells, in brain-like tissues are less investigated. In addition, most cortical organoids lack a microglia component, the resident immune cells in the brain. Impairment of the blood-brain barrier caused by improper crosstalk between neural cells and vascular cells is associated with many neurodegenerative disorders. Mesenchymal stem cells (MSCs), with a phenotype overlapping with pericytes, have promotion effects on neurogenesis and angiogenesis, which are mainly attributed to secreted growth factors and extracellular matrices. As the innate macrophages of the central nervous system, microglia regulate neuronal activities and promote neuronal differentiation by secreting neurotrophic factors and pro-/anti-inflammatory molecules. Neuronal-microglia interactions mediated by chemokines signaling can be modulated in vitro for recapitulating microglial activities during neurodegenerative disease progression. In this review, we discussed the cellular interactions and the physiological roles of neural cells with other cell types including endothelial cells and microglia based on iPSC models. The therapeutic roles of MSCs in treating neural degeneration and pathological roles of microglia in neurodegenerative disease progression were also discussed.

Cell population balance of cardiovascular spheroids derived from human induced pluripotent stem cells.

Stem cell-derived cardiomyocytes and vascular cells can be used for a variety of applications such as studying human heart development and modelling human disease in culture. In particular, protocols based on modulation of Wnt signaling were able to produce high quality of cardiomyocytes or vascular cells from human pluripotent stem cells (hPSCs). However, the mechanism behind the development of 3D cardiovascular spheroids into either vascular or cardiac cells has not been well explored. Hippo/Yes-associated protein (YAP) signaling plays important roles in the regulation of organogenesis, but its impact on cardiovascular differentiation has been less evaluated. In this study, the effects of seeding density and a change in YAP signaling on 3D cardiovascular spheroids patterning from hPSCs were evaluated. Compared to 2D culture, 3D cardiovascular spheroids exhibited higher levels of sarcomeric striations and higher length-to-width ratios of α-actinin+ cells. The spheroids with high seeding density exhibited more α-actinin+ cells and less nuclear YAP expression. The 3D cardiovascular spheroids were also treated with different small molecules, including Rho kinase inhibitor (Y27632), Cytochalasin D, Dasatinib, and Lysophosphatidic acid to modulate YAP localization. Nuclear YAP inhibition resulted in lower expression of active ß-catenin, vascular marker, and MRTF, the transcription factor mediated by RhoGTPases. Y27632 also promoted the gene expression of MMP-2/-3 (matrix remodeling) and Notch-1 (Notch signaling). These results should help our understanding of the underlying effects for the efficient patterning of cardiovascular spheroids after mesoderm formation from hPSCs.

Derivation of Cortical Spheroids from Human Induced Pluripotent Stem Cells in a Suspension Bioreactor.

Human induced pluripotent stem cells (hiPSCs) emerge as a promising source to construct human brain-like tissues, spheroids, or organoids in vitro for disease modeling and drug screening. A suspension bioreactor can be used to generate large size of brain organoids from hiPSCs through enhanced diffusion, but the influence of a dynamic bioreactor culture environment on neural tissue patterning from hiPSCs has not been well understood. The objective of this study is to assess the influence of a suspension bioreactor culture on cortical spheroid (i.e., forebrain-like aggregates) formation from hiPSCs. Single undifferentiated hiPSK3 cells or preformed embryoid bodies were inoculated into the bioreactor. Aggregate size distribution, neural marker expression (e.g., Nestin, PAX6, ß-tubulin III, and MAP-2), and cortical tissue patterning markers (e.g., TBR1, BRN2, SATB2, and vGlut1) were evaluated with static control. Bioreactor culture was found to promote the expression of TBR1, a deep cortical layer VI marker, and temporally affect SATB2, a superficial cortical layer II-IV marker that appears later according to inside-out cortical tissue development. Prolonged culture after 70 days showed layer-specific cortical structure in the spheroids. Differential expression of matrix metalloproteinase-2 and -3 was also observed for bioreactor and static culture. The altered expression of cortical markers by a suspension bioreactor indicates the importance of culture environment on cortical tissue development from hiPSCs.

Modeling Neurodegenerative Microenvironment Using Cortical Organoids Derived from Human Stem Cells.

Alzheimer's disease (AD) is one of the most common neurodegenerative disorders and causes cognitive impairment and memory deficits of the patients. The mechanism of AD is not well known, due to lack of human brain models. Recently, mini-brain tissues called organoids have been derived from human induced pluripotent stem cells (hiPSCs) for modeling human brain development and neurological diseases. Thus, the objective of this research is to model and characterize neural degeneration microenvironment using three-dimensional (3D) forebrain cortical organoids derived from hiPSCs and study the response to the drug treatment. It is hypothesized that the 3D forebrain organoids derived from hiPSCs with AD-associated genetic background may partially recapitulate the extracellular microenvironment in neural degeneration. To test this hypothesis, AD-patient derived hiPSCs with presenilin-1 mutation were used for cortical organoid generation. AD-related inflammatory responses, matrix remodeling and the responses to DAPT, heparin (completes with heparan sulfate proteoglycans [HSPGs] to bind Aß42), and heparinase (digests HSPGs) treatments were investigated. The results indicate that the cortical organoids derived from AD-associated hiPSCs exhibit a high level of Aß42 comparing with healthy control. In addition, the AD-derived organoids result in an elevated gene expression of proinflammatory cytokines interleukin-6 and tumor necrosis factor-α, upregulate syndecan-3, and alter matrix remodeling protein expression. Our study demonstrates the capacity of hiPSC-derived organoids for modeling the changes of extracellular microenvironment and provides a potential approach for AD-related drug screening.

Fast Generation of Functional Subtype Astrocytes from Human Pluripotent Stem Cells.

Differentiation of astrocytes from human pluripotent stem cells (hPSCs) is a tedious and variable process. This hampers the study of hPSC-generated astrocytes in disease processes and drug development. By using CRISPR/Cas9-mediated inducible expression of NFIA or NFIA plus SOX9 in hPSCs, we developed a method to efficiently generate astrocytes in 4-7 weeks. The astrocytic identity of the induced cells was verified by their characteristic molecular and functional properties as well as after transplantation. Furthermore, we developed a strategy to generate region-specific astrocyte subtypes by combining differentiation of regional progenitors and transgenic induction of astrocytes. This simple and efficient method offers a new opportunity to study the fundamental biology of human astrocytes and their roles in disease processes.

Pluripotent stem cell expansion and neural differentiation in 3-D scaffolds of tunable Poisson's ratio.

Biophysical properties of the scaffolds such as the elastic modulus, have been recently shown to impact stem cell lineage commitment. On the other hand, the contribution of the Poisson's ratio, another important biophysical property, to the stem cell fate decision, has not been studied. Scaffolds with tunable Poisson's ratio (ν) (termed as auxetic scaffolds when Poisson's ratio is zero or negative) are anticipated to provide a spectrum of unique biophysical 3-D microenvironments to influence stem cell fate. To test this hypothesis, in the present work we fabricated auxetic polyurethane scaffolds (ν=0 to -0.45) and evaluated their effects on neural differentiation of mouse embryonic stem cells (ESCs) and human induced pluripotent stem cells (hiPSCs). Compared to the regular scaffolds (ν=+0.30) before auxetic conversion, the auxetic scaffolds supported smaller aggregate formation and higher expression of ß-tubulin III upon neural differentiation. The influences of pore structure, Poisson's ratio, and elastic modulus on neural lineage commitment were further evaluated using a series of auxetic scaffolds. The results indicate that Poisson's ratio may confound the effects of elastic modulus, and auxetic scaffolds with proper pore structure and Poisson's ratio enhance neural differentiation. This study demonstrates that tuning the Poisson's ratio of the scaffolds together with elastic modulus and microstructure would enhance the capability to generate broader, more diversified ranges of biophysical 3-D microenvironments for the modulation of cellular differentiation. STATEMENT OF SIGNIFICANCE: Biophysical signaling from the substrates and scaffolds plays a critical role in neural lineage commitment of pluripotent stem cells. While the contribution of elastic modulus has been well studied, the influence of Poisson's ratio along with microstructure of the scaffolds remains unknown largely due to the lack of technology to produce materials with tailorable Poisson's ratio. This study fabricated auxetic polyurethane scaffolds with different elastic modulus, Poisson's ratio and microstructure and evaluated neural differentiation of pluripotent stem cells. The findings add a novel angle to understand the impact of biophysical microenvironment on stem cell fate decisions.

Generation of Neural Progenitor Spheres from Human Pluripotent Stem Cells in a Suspension Bioreactor.

Conventional two-dimensional (2-D) culture systems cannot provide large numbers of human pluripotent stem cells (hPSCs) and their derivatives that are demanded for commercial and clinical applications in in vitro drug screening, disease modeling, and potentially cell therapy. The technologies that support three-dimensional (3-D) suspension culture, such as a stirred bioreactor, are generally considered as promising approaches to produce the required cells. Recently, suspension bioreactors have also been used to generate mini-brain-like structure from hPSCs for disease modeling, showing the important role of bioreactor in stem cell culture. This chapter describes a detailed culture protocol for neural commitment of hPSCs into neural progenitor cell (NPC) spheres using a spinner bioreactor. The basic steps to prepare hPSCs for bioreactor inoculation are illustrated from cell thawing to cell propagation. The method for generating NPCs from hPSCs in the spinner bioreactor along with the static control is then described. The protocol in this study can be applied to the generation of NPCs from hPSCs for further neural subtype specification, 3-D neural tissue development, or potential preclinical studies or clinical applications in neurological diseases.

Neural patterning of human induced pluripotent stem cells in 3-D cultures for studying biomolecule-directed differential cellular responses.

INTRODUCTION: Appropriate neural patterning of human induced pluripotent stem cells (hiPSCs) is critical to generate specific neural cells/tissues and even mini-brains that are physiologically relevant to model neurological diseases. However, the capacity of signaling factors that regulate 3-D neural tissue patterning in vitro and differential responses of the resulting neural populations to various biomolecules have not yet been fully understood. METHODS: By tuning neural patterning of hiPSCs with small molecules targeting sonic hedgehog (SHH) signaling, this study generated different 3-D neuronal cultures that were mainly comprised of either cortical glutamatergic neurons or motor neurons. RESULTS: Abundant glutamatergic neurons were observed following the treatment with an antagonist of SHH signaling, cyclopamine, while Islet-1 and HB9-expressing motor neurons were enriched by an SHH agonist, purmorphamine. In neurons derived with different neural patterning factors, whole-cell patch clamp recordings showed similar voltage-gated Na(+)/K(+) currents, depolarization-evoked action potentials and spontaneous excitatory post-synaptic currents. Moreover, these different neuronal populations exhibited differential responses to three classes of biomolecules, including (1) matrix metalloproteinase inhibitors that affect extracellular matrix remodeling; (2) N-methyl-d-aspartate that induces general neurotoxicity; and (3) amyloid ß (1-42) oligomers that cause neuronal subtype-specific neurotoxicity. CONCLUSIONS: This study should advance our understanding of hiPSC self-organization and neural tissue development and provide a transformative approach to establish 3-D models for neurological disease modeling and drug discovery. STATEMENT OF SIGNIFICANCE: Appropriate neural patterning of human induced pluripotent stem cells (hiPSCs) is critical to generate specific neural cells, tissues and even mini-brains that are physiologically relevant to model neurological diseases. However, the capability of sonic hedgehog-related small molecules to tune different neuronal subtypes in 3-D differentiation from hiPSCs and the differential cellular responses of region-specific neuronal subtypes to various biomolecules have not been fully investigated. By tuning neural patterning of hiPSCs with small molecules targeting sonic hedgehog signaling, this study provides knowledge on the differential susceptibility of region-specific neuronal subtypes derived from hiPSCs to different biomolecules in extracellular matrix remodeling and neurotoxicity. The findings are significant for understanding 3-D neural patterning of hiPSCs for the applications in brain organoid formation, neurological disease modeling, and drug discovery.

Crosslinking of extracellular matrix scaffolds derived from pluripotent stem cell aggregates modulates neural differentiation.

At various developmental stages, pluripotent stem cells (PSCs) and their progeny secrete a large amount of extracellular matrices (ECMs) which could interact with regulatory growth factors to modulate stem cell lineage commitment. ECMs derived from PSC can be used as unique scaffolds that provide broad signaling capacities to mediate cellular differentiation. However, the rapid degradation of ECMs can impact their applications as the scaffolds for in vitro cell expansion and in vivo transplantation. To address this issue, this study investigated the effects of crosslinking on the ECMs derived from embryonic stem cells (ESCs) and the regulatory capacity of the crosslinked ECMs on the proliferation and differentiation of reseeded ESC-derived neural progenitor cells (NPCs). To create different biological cues, undifferentiated aggregates, spontaneous embryoid bodies, and ESC-derived NPC aggregates were decellularized. The derived ECMs were crosslinked using genipin or glutaraldehyde to enhance the scaffold stability. ESC-derived NPC aggregates were reseeded on different ECM scaffolds and differential cellular compositions of neural progenitors, neurons, and glial cells were observed. The results indicate that ESC-derived ECM scaffolds affect neural differentiation through intrinsic biological cues and biophysical properties. These scaffolds have potential for in vitro cell culture and in vivo tissue regeneration study. STATEMENT OF SIGNIFICANCE: Dynamic interactions of acellular extracellular matrices and stem cells are critical for lineage-specific commitment and tissue regeneration. Understanding the synergistic effects of biochemical, biological, and biophysical properties of acellular matrices would facilitate scaffold design and the functional regulation of stem cells. The present study assessed the influence of crosslinked embryonic stem cell-derived extracellular matrix on neural differentiation and revealed the synergistic interactions of various matrix properties. While embryonic stem cell-derived matrices have been assessed as tissue engineering scaffolds, the impact of crosslinking on the embryonic stem cell-derived matrices to modulate neural differentiation has not been studied. The results from this study provide novel knowledge on the interface of embryonic stem cell-derived extracellular matrix and neural aggregates. The findings reported in this manuscript are significant for stem cell differentiation toward the applications in stem cell-based drug screening, disease modeling, and cell therapies.

The microenvironment of embryoid bodies modulated the commitment to neural lineage postcryopreservation.

Neural progenitor cells are usually derived from pluripotent stem cells (PSCs) through the formation of embryoid bodies (EBs), the three-dimensional (3D) aggregate-like structure mimicking embryonic development. Cryo-banking of EBs is a critical step for sample storage, process monitoring, and preservation of intermediate cell populations during the lengthy differentiation procedure of PSCs. However, the impact of microenvironment (including 3D cell organization and biochemical factors) of EBs on neural lineage commitment postcryopreservation has not been well understood. In this study, intact EBs (I-E) and dissociated EBs (D-E) were compared for the recovery and neural differentiation after cryopreservation. I-E group showed the enhanced viability and recovery upon thaw compared with D-E group due to the preservation of extracellular matrix, cell-cell contacts, and F-actin organization. Moreover, both I-E and D-E groups showed the increased neuronal differentiation and D-E group also showed the enhanced astrocyte differentiation after thaw, probably due to the modulation of cellular redox state indicated by the expression of reactive oxygen species. In addition, mesenchymal stem cell secretome, known to bear a broad spectrum of protective factors, enhanced EB recovery. Taken together, EB microenvironment plays a critical role in the recovery and neural differentiation postcryopreservation.

Labeling pluripotent stem cell-derived neural progenitors with iron oxide particles for magnetic resonance imaging.

Due to the unlimited proliferation capacity and the unique differentiation ability of pluripotent stem cells (PSCs), including both embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), large numbers of PSC-derived cell products are in demand for applications in drug screening, disease modeling, and especially cell therapy. In stem cell-based therapy, tracking transplanted cells with magnetic resonance imaging (MRI) has emerged as a powerful technique to reveal cell survival and distribution. This chapter illustrated the basic steps of labeling PSC-derived neural progenitors (NPs) with micron-sized particles of iron oxide (MPIO, 0.86 µm) for MRI analysis. The protocol described PSC expansion and differentiation into NPs, and the labeling of the derived cells either after replating on adherent surface or in suspension. The labeled cells can be analyzed using in vitro MRI analysis. The methods presented here can be easily adapted for cell labeling in cell processing facilities under current Good Manufacturing Practices (cGMP). The iron oxide-labeled NPs can be used for cellular monitoring of in vitro cultures and in vivo transplantation.