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Human mesenchymal stromal cells (hMSCs) are mechanically sensitive undergoing phenotypic alterations when subjected to shear stress, cell aggregation, and substrate changes encountered in 3D dynamic bioreactor cultures. However, little is known about how bioreactor microenvironment affects the secretion and cargo profiles of hMSC-derived extracellular vesicles (EVs) including the subset, “exosomes”, which contain therapeutic proteins, nucleic acids, and lipids from the parent cells. In this study, bone marrow–derived hMSCs were expanded on 3D Synthemax II microcarriers in the PBS mini 0.1L Vertical-Wheel bioreactor system under variable shear stress levels at 25, 40, and 64 RPM (0.1–0.3 dyn/cm(2)). The bioreactor system promotes EV secretion from hMSCs by 2.5-fold and upregulates the expression of EV biogenesis markers and glycolysis genes compared to the static 2D culture. The microRNA cargo was also altered in the EVs from bioreactor culture including the upregulation of miR-10, 19a, 19b, 21, 132, and 377. EV protein cargo was characterized by proteomics analysis, showing upregulation of metabolic, autophagy and ROS-related proteins comparing with 2D cultured EVs. In addition, the scalability of the Vertical-Wheel bioreactor system was demonstrated in a 0.5L bioreactor, showing similar or better hMSC-EV secretion and cargo content compared to the 0.1L bioreactor. This study advances our understanding of bio-manufacturing of stem cell-derived EVs for applications in cell-free therapy towards treating neurological disorders such as ischemic stroke, Alzheimer's disease, and multiple sclerosis.

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.

Human mesenchymal stem or stromal cells (hMSCs) are known for their potential in regenerative medicine due to their differentiation abilities, secretion of trophic factors, and regulation of immune responses in damaged tissues. Due to the limited quantity of hMSCs typically isolated from bone marrow, other tissue sources, such as adipose tissue-derived mesenchymal stem cells (hASCs), are considered a promising alternative. However, differences have been observed for hASCs in the context of metabolic characteristics and response to in vitro culture stress compared to bone marrow derived hMSCs (BM-hMSCs). In particular, the relationship between metabolic homeostasis and stem cell functions, especially the immune phenotype and immunomodulation of hASCs, remains unknown. This study thoroughly assessed the changes in metabolism, redox cycles, and immune phenotype of hASCs during in vitro expansion. In contrast to BM-hMSCs, hASCs did not respond to culture stress significantly during expansion as limited cellular senescence was observed. Notably, hASCs exhibited the increased secretion of pro-inflammatory cytokines and the decreased secretion of anti-inflammatory cytokines after extended culture expansion. The NAD+/NADH redox cycle and other metabolic characteristics associated with aging were relatively stable, indicating that hASC functional decline may be regulated through an alternative mechanism rather than NAD+/Sirtuin aging pathways as observed in BM-hMSCs. Furthermore, transcriptome analysis by mRNA-sequencing revealed the upregulation of genes for pro-inflammatory cytokines/chemokines and the downregulation of genes for anti-inflammatory cytokines for hASCs at high passage. Proteomics analysis indicated key pathways (e.g., tRNA charging, EIF2 signaling, protein ubiquitination pathway) that may be associated with the immune phenotype shift of hASCs. Together, this study advances our understanding of the metabolism and senescence of hASCs and may offer vital insights for the biomanufacturing of hASCs for clinical use.

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.

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.

A presente dissertação é um diálogo com os estudos e as pesquisas que se ocupam com a construção da representação simbólica da família Wolff/Wolf em três obras distintas, de autores da literatura brasileira: Um Rio Imita o Reno (1938) de Vianna Moog, A Asa Esquerda do Anjo (1981) de Lya Luft e Valsa para Bruno Stein (1986) de Charles Kiefer. Desta maneira, nosso objetivo geral é analisar como se dá o relacionamento intertextual entre as famílias presentes nos textos, as quais possuem o mesmo sobrenome de origem alemã Wolff/Wolf. Sendo assim, analisaremos não apenas o sobrenome dessas famílias, mas também as questões inerentes aos textos literários, como símbolos e estrutura familiar, pois com as personagens dessas obras podemos analisar e indagar o contato dessas famílias de origem alemã com a cultura brasileira, resultando, desse modo, em uma hibridização de sua cultura, com a perda de elementos culturais alemães, assimilando, portanto, novos aspectos oriundos da cultura brasileira. Das Ziel dieser Arbeit ist, drei verschiedene Werke von drei unterschiedlichen Autoren der brasilianischen Literatur zu analysieren: Um Rio Imita o Reno (1938) von Vianna Moog, A Asa Esquerda do Anjo (1981) von Lya Luft und Valsa para Bruno Stein (1986) von Charles Kiefer. Die intertextuelle Beziehung existiert wegen der Familien, die einen gleichen deutschen Familiennamen haben, „Wolf“. Es werden nicht nur die Familiennamen analysiert, als auch inhärente Themen der literarischen Texte, wie: Symbole, Familienstruktur, und ihren Kontakt mit der brasilianischen Kultur, wodurch sich eine Hybridisierung ihrer Kultur kristalisiert, die wegen des Verlustes ihrer deutschen kulturellen Elemente stattfindet. Dadurch entstehen neue Aspkete in dieser hybriden Kultur.