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Differentiating cerebellar organoids can be challenging due to complex cell organization and structure in the cerebellum. Different approaches were investigated to recapitulate differentiation process of the cerebellum from human-induced pluripotent stem cells (hiPSCs) without high efficiency. This study was carried out to test the hypothesis that the combination of different signaling factors including retinoic acid (RA), Wnt activator, and sonic hedgehog (SHH) activator promotes the cerebellar differentiation of hiPSCs. Wnt, RA, and SHH pathways were activated by CHIR99021 (CHIR), RA, and purmorphamine (PMR), respectively. Different combinations of the morphogens (RA/CHIR, RA/PMR, CHIR/PMR, and RA/CHIR/PMR) were utilized, and the spheroids (day 35) were characterized for the markers of three cerebellum layers (the molecular layer, the Purkinje cell layer, and the granule cell layer). Of all the combinations tested, RA/CHIR/PMR promoted both the Purkinje cell layer and the granule cell layer differentiation. The cells also exhibited electrophysiological characteristics using whole-cell patch clamp recording, especially demonstrating Purkinje cell electrophysiology. This study should advance the understanding of different signaling pathways during cerebellar development to engineer cerebellum organoids for drug screening and disease modeling. IMPACT STATEMENT: This study investigated the synergistic effects of retinoic acid, Wnt activator, and sonic hedgehog activator on cerebellar patterning of human-induced pluripotent stem cell (hiPSC) spheroids and organoids. The results indicate that the combination promotes the differentiation of the Purkinje cell layer and the granule cell layer. The cells also exhibit electrophysiological characteristics using whole-cell patch clamp recording, especially demonstrating Purkinje cell electrophysiology. The findings are significant for understanding the biochemical signaling of three-dimensional microenvironment on neural patterning of hiPSCs for applications in organoid engineering, disease modeling, and drug screening.

Pericytes (PCs) are a type of perivascular cells that surround endothelial cells of small blood vessels. In the brain, PCs show heterogeneity depending on their position within the vasculature. As a result, PC interactions with surrounding endothelial cells, astrocytes, and neuron cells play a key role in a wide array of neurovascular functions such as regulating blood–brain barrier (BBB) permeability, cerebral blood flow, and helping to facilitate the clearance of toxic cellular molecules. Therefore, a reliable method of engineering brain-specific PCs from human induced pluripotent stem cells (hiPSCs) is critical in neurodegenerative disease modeling. This review summarizes brain-specific PC differentiation of hiPSCs through mesoderm and neural crest induction. Key signaling pathways (platelet-derived growth factor-B [PDGF-B], transforming growth factor [TGF]-β, and Notch signaling) regulating PC function, PC interactions with adjacent cells, and PC differentiation from hiPSCs are also discussed. Specifically, PDGF-BB-platelet-derived growth factor receptor β signaling promotes PC cell survival, TGF-β signal transduction facilitates PC attachment to endothelial cells, and Notch signaling is critical in vascular development and arterial-venous specification. Furthermore, current challenges facing the use of hiPSC-derived PCs are discussed, and their ongoing uses in neurodegenerative disease modeling are identified. Further investigations into PCs and surrounding cell interactions are needed to characterize the roles of brain PCs in various neurodegenerative disorders. IMPACT STATEMENT: This article summarizes the work related to brain-specific pericytes (PCs) derived from human pluripotent stem cells (hPSCs). In particular, key signaling pathways regulating PC function, PC interactions with adjacent cells, and PC differentiation from hPSCs were discussed. Furthermore, current challenges facing the use of hPSC-derived PCs were identified, and their ongoing uses in neurodegenerative disease modeling were discussed. The review highlights the important role of cell–cell interactions in blood–brain barrier (BBB) models and neurodegeneration. The summarized findings are significant for establishing pluripotent stem cell-based BBB models toward the applications in drug screening and disease modeling.

Extracellular vesicles (EVs), including exosomes and microvesicles, are found to play an important role in various biological processes and maintaining tissue homeostasis. Because of the protective effects, stem cell-derived EVs can be used to reduce oxidative stress and apoptosis in the recipient cells. In addition, EVs/exosomes have been used as directional communication tools between stem cells and parenchymal cells, giving them the ability to serve as biomarkers. Likewise, altered EVs/exosomes can be utilized for drug delivery by loading with proteins, small interfering RNAs, and viral vectors, in particular, because EVs/exosomes are able to cross the blood–brain barrier. In this review article, the properties of human induced pluripotent stem cell (iPSC)-derived EVs are discussed. The biogenesis, that is, how EVs originate in the endosomal compartment or from the cell layer of microvesicles, EV composition, the available methods of purification, and characterizations of EVs/exosomes are summarized. In particular, EVs/exosomes derived from iPSCs of different lineage specifications and the applications of these stem cell-derived exosomes in neurological diseases are discussed. IMPACT STATEMENT: In this review, we summarized the work related to extracellular vesicles (EVs) derived from human pluripotent stem cells (hPSCs). In particular, EVs/exosomes derived from hPSCs of different lineage specifications and the applications of these stem cell-derived exosomes in neurological diseases are discussed. The results highlight the important role of cell-cell interactions in neural cellular phenotype and neurodegeneration. The findings reported in this article are significant for pluripotent stem cell-derived cell-free products toward applications in stem cell-based therapies.

Summary Several kidney diseases including congenital nephrotic syndrome, Alport syndrome, and diabetic nephropathy are linked to podocyte dysfunction. Human podocytopathies may be modeled in either primary or immortalized podocyte cell lines. Human induced pluripotent stem cell (hiPSC)-derived podocytes are a source of human podocytes, but the existing protocols have variable efficiency and expensive media components. We developed an accelerated, feeder-free protocol for deriving functional, mature podocytes from hiPSCs in only 12 days, saving time and money compared with other approaches., Graphical abstract, Highlights • Specific steps with media formulations to differentiate podocytes from human iPSCs • The accelerated process mimics the phases of kidney development • Instructions for marker analysis of the defined cell types present at each stage • Validation of podocyte function includes marker analysis and FITC-albumin uptake, Several kidney diseases including congenital nephrotic syndrome, Alport syndrome, and diabetic nephropathy are linked to podocyte dysfunction. Human podocytopathies may be modeled in either primary or immortalized podocyte cell lines. Human induced pluripotent stem cell (hiPSC)-derived podocytes are a source of human podocytes, but the existing protocols have variable efficiency and expensive media components. We developed an accelerated, feeder-free protocol for deriving functional, mature podocytes from hiPSCs in only 12 days, saving time and money compared with other approaches.

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 Aβ42), and heparinase (digests HSPGs) treatments were investigated. The results indicate that the cortical organoids derived from AD-associated hiPSCs exhibit a high level of Aβ42 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.

Astrocytes are vital components in neuronal circuitry and there is increasing evidence linking the dysfunction of these cells to a number of central nervous system diseases. Studying the role of these cells in human brain function in the past has been difficult due to limited access to the human brain. In this study, human induced pluripotent stem cells were differentiated into astrospheres using a hybrid plating method, with or without dual SMAD inhibition. The derived cells were assessed for astrocytic markers, brain regional identity, phagocytosis, calcium-transient signaling, reactive oxygen species production, and immune response. Neural degeneration was modeled by stimulation with amyloid-β (Aβ) 42 oligomers. Finally, co-culture was performed for the derived astrospheres with isogenic neurospheres. Results indicate that the derived astroglial cells express astrocyte markers with forebrain dorsal cortical identity, secrete extracellular matrix, and are capable of phagocytosing iron oxide particles and responding to Aβ42 stimulation (higher oxidative stress, higher TNF-α, and IL-6 expression). RNA-sequencing results reveal the distinct transcriptome of the derived cells responding to Aβ42 stimulation for astrocyte markers, chemokines, and brain regional identity. Co-culture experiments show the synaptic activities of neurons and the enhanced neural protection ability of the astroglial cells. This study provides knowledge about the roles of brain astroglial cells, heterotypic cell-cell interactions, and the formation of engineered neuronal synapses in vitro. The implications lie in neurological disease modeling, drug screening, and studying progression of neural degeneration and the role of stem cell microenvironment. Impact Statement Human pluripotent stem cell-derived astrocytes are a powerful tool for disease modeling and drug screening. However, the properties regarding brain regional identity and the immune response to neural degeneration stimulus have not been well characterized. Results of this study indicate that the derived astroglial cells express astrocyte markers with forebrain dorsal cortical identity, secrete extracellular matrix (ECM), and are capable of phagocytosing iron oxide particles and responding to amyloid-β oligomers, showing the distinct transcriptome in astrocyte markers, chemokines, and brain regional identity. This study provides knowledge about the roles of brain astroglial cells, heterotypic cell-cell interactions, and engineering neural tissues in vitro.

Extracellular vesicles (EVs) contribute to a variety of signaling processes and the overall physiological and pathological states of stem cells and tissues. Human induced pluripotent stem cells (hiPSCs) have unique characteristics that can mimic embryonic tissue development. There is growing interest in the use of EVs derived from hiPSCs as therapeutics, biomarkers, and drug delivery vehicles. However, little is known about the characteristics of EVs secreted by hiPSCs and paracrine signaling during tissue morphogenesis and lineage specification. Methods: In this study, the physical and biological properties of EVs isolated from hiPSC-derived neural progenitors (ectoderm), hiPSC-derived cardiac cells (mesoderm), and the undifferentiated hiPSCs (healthy iPSK3 and Alzheimer&rsquo, s-associated SY-UBH lines) were analyzed. Results: Nanoparticle tracking analysis and electron microscopy results indicate that hiPSC-derived EVs have an average size of 100&ndash, 250 nm. Immunoblot analyses confirmed the enrichment of exosomal markers Alix, CD63, TSG101, and Hsc70 in the purified EV preparations. MicroRNAs including miR-133, miR-155, miR-221, and miR-34a were differently expressed in the EVs isolated from distinct hiPSC lineages. Treatment of cortical spheroids with hiPSC-EVs in vitro resulted in enhanced cell proliferation (indicated by BrdU+ cells) and axonal growth (indicated by &beta, tubulin III staining). Furthermore, hiPSC-derived EVs exhibited neural protective abilities in A&beta, 42 oligomer-treated cultures, enhancing cell viability and reducing oxidative stress. Our results demonstrate that the paracrine signaling provided by tissue context-dependent EVs derived from hiPSCs elicit distinct responses to impact the physiological state of cortical spheroids. Overall, this study advances our understanding of cell‒cell communication in the stem cell microenvironment and provides possible therapeutic options for treating 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.

Brain spheroids or organoids derived from human pluripotent stem cells (hiPSCs) are still not capable of completely recapitulating in vivo human brain tissue, and one of the limitations is lack of microglia. To add built-in immune function, coculture of the dorsal forebrain spheroids with isogenic microglia-like cells (D-MG) was performed in our study. The three-dimensional D-MG spheroids were analyzed for their transcriptome and compared with isogenic microglia-like cells (MG). Cortical spheroids containing microglia-like cells displayed different metabolic programming, which may affect the associated phenotype. The expression of genes related to glycolysis and hypoxia signaling was increased in cocultured D-MG spheroids, indicating the metabolic shift to aerobic glycolysis, which is in favor of M1 polarization of microglia-like cells. In addition, the metabolic pathways and the signaling pathways involved in cell proliferation, cell death, PIK3/AKT/mTOR signaling, eukaryotic initiation factor 2 pathway, and Wnt and Notch pathways were analyzed. The results demonstrate the activation of mTOR and p53 signaling, increased expression of Notch ligands, and the repression of NF-κB and canonical Wnt pathways, as well as the lower expression of cell cycle genes in the cocultured D-MG spheroids. This analysis indicates that physiological 3-D microenvironment may reshape the immunity of in vitro cortical spheroids and better recapitulate in vivo brain tissue function for disease modeling and drug screening.

Human induced pluripotent stem cells (hiPSCs) have special ability to self-assemble into neural spheroids or mini-brain-like structures. During the self-assembly process, Wnt signaling plays an important role in regional patterning and establishing positional identity of hiPSC-derived neural progenitors. Recently, the role of Wnt signaling in regulating Yes-associated protein (YAP) expression (nuclear or cytoplasmic), the pivotal regulator during organ growth and tissue generation, has attracted increasing interests. However, the interactions between Wnt and YAP expression for neural lineage commitment of hiPSCs remain poorly explored. The objective of this study is to investigate the effects of Wnt signaling and YAP expression on the cellular population in three-dimensional (3D) neural spheroids derived from hiPSCs. In this study, Wnt signaling was activated using CHIR99021 for 3D neural spheroids derived from human iPSK3 cells through embryoid body formation. Our results indicate that Wnt activation induces nuclear localization of YAP and upregulates the expression of HOXB4, the marker for hindbrain/spinal cord. By contrast, the cells exhibit more rostral forebrain neural identity (expression of TBR1) without Wnt activation. Cytochalasin D was then used to induce cytoplasmic YAP and the results showed the decreased HOXB4 expression. In addition, the incorporation of microparticles in the neural spheroids was investigated for the perturbation of neural patterning. This study may indicate the bidirectional interactions of Wnt signaling and YAP expression during neural tissue patterning, which have the significance in neurological disease modeling, drug screening, and neural tissue regeneration.

Human pluripotent stem cells (hPSCs) have shown the ability to self-organize into different types of neural organoids (e.g., whole brain organoids, cortical spheroids, midbrain organoids etc.) recently. The extrinsic and intrinsic signaling elicited by Wnt pathway, Hippo/Yes-associated protein (YAP) pathway, and extracellular microenvironment plays a critical role in brain tissue morphogenesis. This article highlights recent advances in neural tissue patterning from hPSCs, in particular the role of Wnt pathway and YAP activity in this process. Understanding the Wnt-YAP interactions should provide us the guidance to predict and modulate brain-like tissue structure through the regulation of extracellular microenvironment of hPSCs.

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 β (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.