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Human mesenchymal stem cell (hMSC) derived extracellular vesicles (EVs) have shown therapeutic potential in recent studies. However, the corresponding therapeutic components are largely unknown, and scale-up production of hMSC EVs is a major challenge for translational applications. In the current study, hMSCs were grown as 3D aggregates under wave motion to promote EV secretion. Results demonstrate that 3D hMSC aggregates promote activation of the endosomal sorting complexes required for transport (ESCRT)-dependent and -independent pathways. mRNA sequencing revealed global transcriptome alterations for 3D hMSC aggregates. Compared to 2D-hMSC-EVs, the quantity of 3D-hMSC-EVs was enhanced significantly (by 2-fold), with smaller sizes, higher miR-21 and miR-22 expression, and an altered protein cargo (e.g., upregulation of cytokines and anti-inflammatory factors) uncovered by proteomics analysis, possibly due to altered EV biogenesis. Functionally, 3D-hMSC-EVs rejuvenated senescent stem cells and exhibited enhanced immunomodulatory potentials. In summary, this study provides a promising strategy for scalable production of high-quality EVs from hMSCs with enhanced therapeutic potential.

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.

Stem cells possess unique properties of self-renewal and multilineage differentiation potentials. In addition, stem cells also exhibit important trophic function, by secreting large numbers of regulatory molecules such as cytokines and extracellular vesicles (EVs). Among them, exosomal miRNAs (exo-miRNAs), a type of small noncoding RNAs sorted in EVs, play an important role in the regulation of gene expression in various biological processes through cell–cell communications. As incorporated into EVs, exo-miRNAs can be delivered to recipient cells as cargos to regulate cellular behaviors. This chapter reviews the function of exo-miRNAs in EVs derived from stem cells, including mesenchymal stem cells and pluripotent stem cells, and summarizes current knowledge on the characteristics of miRNA expression at undifferentiated and lineage-primed stages of stem cells. Furthermore, the role of stem-cell-derived exo-miRNAs in regulating the phenotype of somatic and progenitor cells is also analyzed in order to elucidate their participations and the therapeutic function in specific diseases. Lastly, this work summarizes current approaches and progresses in engineering exo-miRNAs and EVs derived by stem cells toward potential clinical applications.

Human mesenchymal stem cells (hMSCs), including human adipose tissue-derived stem cells (hASCs), as well as the secreted extracellular vesicles (EVs), are promising therapeutics in treating inflammatory and neural degenerative diseases. However, prolonged expansion can lead to cellular senescence characterized by a gradual loss of self-renewal ability while altering secretome composition and EV generation. Additionally, hMSCs are highly sensitive to biophysical microenvironment in bioreactor systems utilized in scaling production. In this study, hASCs grown on Plastic Plus or Synthemax II microcarriers in a spinner flask bioreactor (SFB) system were compared to traditional 2D culture. The SFB microenvironment was found to increase the expression of genes associated with hASC stemness, nicotinamide adenine dinucleotide (NAD+) metabolism, glycolysis, and the pentose phosphate pathway as well as alter cytokine secretion (e.g., PGE2 and CXCL10). Elevated reactive oxidative species levels in hASCs of SFB culture were observed without increasing rates of cellular senescence. Expression levels of Sirtuins responsible for preventing cellular senescence through anti-oxidant and DNA repair mechanisms were also elevated in SFB cultures. In particular, the EV biogenesis genes were significantly upregulated (3-10 fold) and the EV production increased 40% per cell in SFB cultures of hASCs. This study provides advanced understanding of hASC sensitivity to the bioreactor microenvironment for EV production and bio-manufacturing towards the applications in treating inflammatory and neural degenerative diseases.

Mesenchymal stem cell (MSC)-based therapy has shown great promises in various animal disease models. However, this therapeutic potency has not been well claimed when applied to human clinical trials. This is due to both the availability of MSCs at the time of administration and lack of viable expansion strategies. MSCs are very susceptible to in vitro culture environment and tend to adapt the microenvironment which could lead to cellular senescence and aging. Therefore, extended in vitro expansion induces loss of MSC functionality and its clinical relevance. To combat this effect, this work assessed a novel cyclical aggregation as a means of expanding MSCs to maintain stem cell functionality. The cyclical aggregation consists of an aggregation phase and an expansion phase by replating the dissociated MSC aggregates onto planar tissue culture surfaces. The results indicate that cyclical aggregation maintains proliferative capability, stem cell proteins, and clonogenicity, and prevents the acquisition of senescence. To determine why aggregation was responsible for this phenomenon, the integrated stress response pathway was probed with salubrial and GSK-2606414. Treatment with salubrial had no significant effect, while GSK-2606414 mitigated the effects of aggregation leading to in vitro aging. This method holds the potential to increase the clinical relevance of MSC therapeutic effects from small model systems (such as rats and mice) to humans, and may open the potential of patient-derived MSCs for treatment thereby removing the need for immunosuppression.

Human mesenchymal stem cells (hMSCs) are a promising candidate in cell therapy as they exhibit multilineage differentiation, homing to the site of injury, and secretion of trophic factors that facilitate tissue healing and/or modulate immune response. As a result, hMSC-derived products have attracted growing interests in preclinical and clinical studies. The development of hMSC culture platforms for large-scale biomanufacturing is necessary to meet the requirements for late-phase clinical trials and future commercialization. Microcarriers in stirred-tank bioreactors have been widely utilized in large-scale expansion of hMSCs for translational applications because of a high surface-to-volume ratio compared to conventional 2D planar culture. However, recent studies have demonstrated that microcarrier-expanded hMSCs differ from dish- or flask-expanded cells in size, morphology, proliferation, viability, surface markers, gene expression, differentiation potential, and secretome profile which may lead to altered therapeutic potency. Therefore, understanding the bioprocessing parameters that influence hMSC therapeutic efficacy is essential for the optimization of microcarrier-based bioreactor system to maximize hMSC quantity without sacrificing quality. In this review, biomanufacturing parameters encountered in planar culture and microcarrier-based bioreactor culture of hMSCs are compared and discussed with specific focus on cell-adhesion surface (e.g., discontinuous surface, underlying curvature, microcarrier stiffness, porosity, surface roughness, coating, and charge) and the dynamic microenvironment in bioreactor culture (e.g., oxygen and nutrients, shear stress, particle collision, and aggregation). The influence of dynamic culture in bioreactors on hMSC properties is also reviewed in order to establish connection between bioprocessing and stem cell function. This review addresses fundamental principles and concepts for future design of biomanufacturing systems for hMSC-based therapy.

Stem cells, including mesenchymal stem cells and pluripotent stem cells, have attracted considerable attention in tissue engineering and regenerative medicine primarily because of their unique ability in self-renewal and multilineage differentiation. However, stem cells also have important secretory functions that form a specialized in vivo microenvironment and direct tissue development and regeneration. Extracellular matrices (ECMs) derived from stem cells retain the functional properties of their native environment and exhibit unique signaling that mediates stem cell self-renewal and lineage commitment. Stem cell-derived ECMs (scECMs) also have tunable properties corresponding to their developmental stages, suggesting that their lineage- and developmental specificity can be engineered for a wide range of applications. Hence, there is a growing interest in reconstructing stem cell microenvironment through decellularization and obtaining decellularized matrices that exhibit unique biological properties. This article summarizes recent advances in the use and understanding of scECMs. Moreover, future directions to extend the spectrum of applications of stem-derived ECMs in tissue engineering by comprehensively elucidating and engineering their regulatory function is highlighted. Impact statement Stem cells bear unique potency for multilineage differentiation as well as the capacity to secrete a vast amount of regulatory molecules. At different developmental stages, the extracellular matrices (ECMs) secreted by stem cells regulate their microenvironment and direct tissue development. The decellularization of stem cells effectively preserves ECM functional properties and can provide suitable templates to regulate stem cell fate decision, which can hardly be reproduced using single ECM proteins or synthetic scaffolds. This review highlights the unique regulatory functions of stem cell-derived ECMs, which can serve as novel sources of highly bioactive materials for tissue engineering and cell therapy.

Hydrolytic degradation of commercially available 3D printing filament, i.e. poly (lactic acid) with broad molecular weight distribution was induced by incubating 3D-printed parts in deionized water at 3 temperatures. Small changes in orthogonal dimensions occurred due to relaxation of printing stresses, but no mass or volume loss were detected over the time-frame of the experiments. Molecular weight decreased while polydispersity remained constant. The most sensitive measure of degradation was found to be nondestructive, small-amplitude oscillatory tensile measurements. A rapid decay of tensile storage modulus was found with an exponential decay time constant of about an hour. This work demonstrates that practical monitoring of commercially available PLA degradation can be achieve with linear viscoelastic measurements of modulus.

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.

The human brain formation involves complicated processing, which is regulated by a gene regulatory network influenced by different signaling pathways. The cross-regulatory interactions between elements of different pathways affect the process of cell fate assignment during neural and astroglial tissue patterning. In this study, the interactions between Wnt and Notch pathways, the two major pathways that influence neural and astroglial differentiation of human induced pluripotent stem cells (hiPSCs) individually, were investigated. In particular, the synergistic effects of Wnt-Notch pathway on the neural patterning processes along the anterior–posterior or dorsal–ventral axis of hiPSC-derived cortical spheroids were explored. The human cortical spheroids derived from hiPSCs were treated with Wnt activator CHIR99021 (CHIR), Wnt inhibitor IWP4, and Notch inhibitor (N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester [DAPT]) individually, or in combinations (CHIR + DAPT, IWP4 + DAPT). The results suggest that CHIR + DAPT can promote Notch signaling, similar or higher than CHIR alone, whereas IWP4 + DAPT reduces Notch activity compared to IWP4 alone. Also, CHIR + DAPT promoted hindbrain marker HOXB4 expression more consistently than CHIR alone, while IWP4 + DAPT promoted Olig2 expression, indicating the synergistic effects distinctly different from that of the individual small molecule. In addition, IWP4 simultaneously promoted dorsal and ventral identity. The patterned neural spheroids can be switched for astroglial differentiation using bone morphogenetic protein 4. This study should advance the derivations of neurons, astroglial cells, and brain region-specific organoids from hiPSCs for disease modeling, drug screening, as well as for hiPSC-based therapies. IMPACT STATEMENT: Wnt signaling plays a central role in neural patterning of human pluripotent stem cells. It can interact with Notch signaling in defining dorsal–ventral and rostral–caudal (or anterior–posterior) axis of brain organoids. This study investigates novel Wnt and Notch interactions (i.e., Wntch) in neural patterning of dorsal forebrain spheroids or organoids derived from human induced pluripotent stem cells. The synergistic effects of Wnt activator or inhibitor with Notch inhibitor were observed. This study should advance the derivations of neurons, astroglial cells, and brain region-specific organoids from human stem cells for disease modeling and drug screening, as well as for stem cell-based therapies. The results can be used to establish better in vitro culture methods for efficiently mimicking in vivo structure of central nervous system.

Human mesenchymal stem cells (hMSCs) are well known in cell therapy due to their secretion of trophic factors, multipotent differentiation potential, and ability for self-renewal. As a result, the number of clinical trials has been steadily increasing over the last decade highlighting the need for in vitro systems capable of producing large quantities of cells to meet growing demands. However, hMSCs are highly sensitive to microenvironment conditions, including shear stress caused by dynamic bioreactor systems, and can lead to alteration of cellular homeostasis. In this study, hMSCs were expanded on microcarriers within a 125 mL spinner flask bioreactor system. Our results demonstrate a three-fold expansion over seven days. Furthermore, our results show that culturing hMSCs in the microcarrier-based suspension bioreactor (compared to static planar culture) results in smaller cell size and higher levels of reactive oxidative species (ROS) and ROS regulator Sirtuin-3, which have implications on the nicotinamide adenine dinucleotide metabolic pathway and metabolic homeostasis. In addition, hMSCs in the bioreactor showed the increased Prostaglandin E(2) secretion as well as reduced the Indoleamine-pyrrole 2,3-dioxygenase secretion upon stimulus with interferon gamma. The results of this study provide understanding of potential hMSC physiology alterations impacted by bioreactor microenvironment during scalable production of hMSCs for biomanufacturing and clinical trials.