Hydrogel-Based Bioprocess for Scalable Manufacturing of Human Pluripotent Stem Cell-Derived Neural Stem Cells

Neural stem cells derived from human pluripotent stem cells (hPSC-NSCs) are of great value for modeling diseases, developing drugs, and treating neurological disorders. However, manufacturing high-quantity and -quality hPSC-NSCs, especially for clinical applications, remains a challenge. Here, we report a chemically defined, high-yield, and scalable bioprocess for manufacturing hPSC-NSCs. hPSCs are expanded and differentiated into NSCs in microscale tubes made with alginate hydrogels. The tubes are used to isolate cells from the hydrodynamic stresses in the culture vessel and limit the radial diameter of the cell mass to less than 400 μm to ensure efficient mass transport during the culture. The hydrogel tubes provide uniform, reproducible, and cell-friendly microspaces and microenvironments for cells. With this new technology, we showed that hPSC-NSCs could be produced in 12 days with high viability (∼95%), high purity (>90%), and high yield (∼5 × 108 cells/mL of microspace). The volumetric yield is about 250 times more than the current state-of-the-art. Whole transcriptome analysis and quantitative real-time polymerase chain reaction showed that hPSC-NSCs made by this process had a similar gene expression to hPSC-NSCs made by the conventional culture technology. The produced hPSC-NSCs could mature into both neurons and glial cells in vitro and in vivo. The process developed in this paper can be used to produce large numbers of hPSC-NSCs for various biomedical applications in the future

Hydrogel-Based Bioprocess for Scalable Manufacturing of Human Pluripotent Stem Cell-Derived Neural Stem Cells

Neural stem cells derived from human pluripotent stem cells (hPSC-NSCs) are of great value for modeling diseases, developing drugs, and treating neurological disorders. However, manufacturing high-quantity and -quality hPSC-NSCs, especially for clinical applications, remains a challenge. Here, we report a chemically defined, high-yield, and scalable bioprocess for manufacturing hPSC-NSCs. hPSCs are expanded and differentiated into NSCs in microscale tubes made with alginate hydrogels. The tubes are used to isolate cells from the hydrodynamic stresses in the culture vessel and limit the radial diameter of the cell mass to less than 400 μm to ensure efficient mass transport during the culture. The hydrogel tubes provide uniform, reproducible, and cell-friendly microspaces and microenvironments for cells. With this new technology, we showed that hPSC-NSCs could be produced in 12 days with high viability (∼95%), high purity (>90%), and high yield (∼5 × 108 cells/mL of microspace). The volumetric yield is about 250 times more than the current state-of-the-art. Whole transcriptome analysis and quantitative real-time polymerase chain reaction showed that hPSC-NSCs made by this process had a similar gene expression to hPSC-NSCs made by the conventional culture technology. The produced hPSC-NSCs could mature into both neurons and glial cells in vitro and in vivo. The process developed in this paper can be used to produce large numbers of hPSC-NSCs for various biomedical applications in the future

Establishment of a Human iPSC- and Nanofiber-Based Microphysiological Blood–Brain Barrier System

The blood–brain barrier (BBB) is an active and complex diffusion barrier that separates the circulating blood from the brain and extracellular fluid, regulates nutrient transportation, and provides protection against various toxic compounds and pathogens. Creating an in vitro microphysiological BBB system, particularly with relevant human cell types, will significantly facilitate the research of neuropharmaceutical drug delivery, screening, and transport, as well as improve our understanding of pathologies that are due to BBB damage. Currently, most of the in vitro BBB models are generated by culturing rodent astrocytes and endothelial cells, using commercially available transwell membranes. Those membranes are made of plastic biopolymers that are nonbiodegradable, porous, and stiff. In addition, distinct from rodent astrocytes, human astrocytes possess unique cell complexity and physiology, which are among the few characteristics that differentiate human brains from rodent brains. In this study, we established a novel human BBB microphysiologocal system, consisting of a three-dimensionally printed holder with a electrospun poly­(lactic-<i>co</i>-glycolic) acid (PLGA) nanofibrous mesh, a bilayer coculture of human astrocytes, and endothelial cells, derived from human induced pluripotent stem cells (hiPSCs), on the electrospun PLGA mesh. This human BBB model achieved significant barrier integrity with tight junction protein expression, an effective permeability to sodium fluorescein, and higher transendothelial electrical resistance (TEER) comparing to electrospun mesh-based counterparts. Moreover, the coculture of hiPSC-derived astrocytes and endothielial cells promoted the tight junction protein expression and the TEER value. We further verified the barrier functions of our BBB model with antibrain tumor drugs (paclitaxel and bortezomib) and a neurotoxic peptide (amyloid β 1-42). The human microphysiological system generated in this study will potentially provide a new, powerful tool for research on human BBB physiology and pathology

Establishment of a Human iPSC- and Nanofiber-Based Microphysiological Blood–Brain Barrier System

The blood–brain barrier (BBB) is an active and complex diffusion barrier that separates the circulating blood from the brain and extracellular fluid, regulates nutrient transportation, and provides protection against various toxic compounds and pathogens. Creating an in vitro microphysiological BBB system, particularly with relevant human cell types, will significantly facilitate the research of neuropharmaceutical drug delivery, screening, and transport, as well as improve our understanding of pathologies that are due to BBB damage. Currently, most of the in vitro BBB models are generated by culturing rodent astrocytes and endothelial cells, using commercially available transwell membranes. Those membranes are made of plastic biopolymers that are nonbiodegradable, porous, and stiff. In addition, distinct from rodent astrocytes, human astrocytes possess unique cell complexity and physiology, which are among the few characteristics that differentiate human brains from rodent brains. In this study, we established a novel human BBB microphysiologocal system, consisting of a three-dimensionally printed holder with a electrospun poly­(lactic-co-glycolic) acid (PLGA) nanofibrous mesh, a bilayer coculture of human astrocytes, and endothelial cells, derived from human induced pluripotent stem cells (hiPSCs), on the electrospun PLGA mesh. This human BBB model achieved significant barrier integrity with tight junction protein expression, an effective permeability to sodium fluorescein, and higher transendothelial electrical resistance (TEER) comparing to electrospun mesh-based counterparts. Moreover, the coculture of hiPSC-derived astrocytes and endothielial cells promoted the tight junction protein expression and the TEER value. We further verified the barrier functions of our BBB model with antibrain tumor drugs (paclitaxel and bortezomib) and a neurotoxic peptide (amyloid β 1-42). The human microphysiological system generated in this study will potentially provide a new, powerful tool for research on human BBB physiology and pathology

Single-cell trajectory analysis shows epidermal stem cells differentiate into keratinocytes in both fresh and cryopreserved samples.

Single-cell trajectory analysis shows epidermal stem cells differentiate into keratinocytes in both fresh and cryopreserved samples.</p

Quality control parameters of fresh and cryopreserved (frozen) pig skin cells, including the number of genes, UMIs, and % of mitochondrial gene in each cell.

(a) pre-cut-off and (b) post-cut-off data are shown. 4500 genes/cell, 30,000 molecules/cell, and 10% mitochondrial genes are set as the maximum threshold to exclude the doublets, multiples, and low-quality single cells. Each dot represents one cell. (TIF)</p

Fresh and cryopreserved cells have similar gene expression profiles.

(a) The pseudo-bulk expression profiles of fresh and cryopreserved cells are compared using correlation scatter plots. The profiles correlate well (R = 0.981). (b) Analysis of the highly variable genes shows fresh and cryopreserved samples share the highly variable features with similar variance and average expression. (c) UAMP shows fresh and cryopreserved samples have similar structures.</p

Markers used for annotating cell clusters.

(TIF)</p

Top 10 marker genes for each cell type.

(TIF)</p