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An integrated biomanufacturing platform for the large-scale expansion and neuronal differentiation of human pluripotent stem cell-derived neural progenitor cells.

ABSTRACT: Human pluripotent stem cell derived neural progenitor cells (hNPCs) have the unique properties of long-term in vitro expansion as well as differentiation into the various neurons and supporting cell types of the central nervous system (CNS). Because of these characteristics, hNPCs have tremendous potential in the modeling and treatment of various CNS diseases and disorders. However, expansion and neuronal differentiation of hNPCs in quantities necessary for these applications is not possible with current two dimensional (2-D) approaches. Here, we used a fully defined peptide substrate as the basis for a microcarrier (MC)-based suspension culture system. Several independently derived hNPC lines were cultured on MCs for multiple passages as well as efficiently differentiated to neurons. Finally, this MC-based system was used in conjunction with a low shear rotating wall vessel (RWV) bioreactor for the integrated, large-scale expansion and neuronal differentiation of hNPCs. Overall, this fully defined and scalable biomanufacturing system will facilitate the generation of hNPCs and their neuronal derivatives in quantities necessary for basic and translational applications. STATEMENT OF SIGNIFICANCE:In this work, we developed a microcarrier (MC)-based culture system that allows for the expansion and neuronal differentiation of human pluripotent stem cell-derived neural progenitor cells (hNPCs) under defined conditions. In turn, this MC approach was implemented in a rotating wall vessel (RWV) bioreactor for the large-scale expansion and neuronal differentiation of hNPCs. This work is of significance as it overcomes current limitations of conventional two dimensional (2-D) culture systems to enable the generation of hNPCs and their neuronal derivatives in quantities required for downstream applications in disease modeling, drug screening, and regenerative medicine.

SUBMITTER: Srinivasan G

PROVIDER: S-EPMC8171278 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundActivin A is a protein that participates principally in reproductive functions. In the adult brain, Activin is neuroprotective, but its role in brain development is still elusive.Methodology/principal findingsWe studied if Activin A influences proliferation, differentiation or survival in rat cerebrocortical neural progenitor cells (NPC). After stimulation of NPC with Activin A, phosphorylation and nuclear translocation of Smad 2/3 were induced. In proliferating NPC, Activin produced a significant decrease in cell area and also a discrete increase in the number of neurons in the presence of the mitogen Fibroblast Growth Factor 2. The percentages of cells incorporating BrdU, or positive for the undifferentiated NPC markers Nestin and Sox2, were unchanged after incubation with Activin. In differentiating conditions, continuous treatment with Activin A significantly increased the number of neurons without affecting astroglial differentiation or causing apoptotic death. In cells cultured by extended periods, Activin treatment produced further increases in the proportion of neurons, excluding premature cell cycle exit. In clonal assays, Activin significantly increased neuronal numbers per colony, supporting an instructive role. Activin-induced neurogenesis was dependent on activation of its receptors, since incubation with the type I receptor inhibitor SB431542 or the ligand-trap Follistatin prevented neuronal differentiation. Interestingly, SB431542 or Follistatin by themselves abolished neurogenesis and increased astrogliogenesis, to a similar extent to that induced by Bone Morphogenetic Protein (BMP)4. Co-incubation of these Activin inhibitors with the BMP antagonist Dorsomorphin restored neuronal and astrocytic differentiation to control levels.ConclusionsOur results show an instructive neuronal effect of Activin A in cortical NPC in vitro, pointing out to a relevant role of this cytokine in the specification of NPC towards a neuronal phenotype.

Project description:Mucopolysaccharidosis type IIIA (MPS IIIA) is characterised by a progressive neurological decline leading to early death. It is caused by bi-allelic loss-of-function mutations in SGSH encoding sulphamidase, a lysosomal enzyme required for heparan sulphate glycosaminoglycan (HS GAG) degradation, that results in the progressive build-up of HS GAGs in multiple tissues most notably the central nervous system (CNS). Skin fibroblasts from two MPS IIIA patients who presented with an intermediate and a severe clinical phenotype, respectively, were reprogrammed into induced pluripotent stem cells (iPSCs). The intermediate MPS IIIA iPSCs were then differentiated into neural progenitor cells (NPCs) and subsequently neurons. The patient derived fibroblasts, iPSCs, NPCs and neurons all displayed hallmark biochemical characteristics of MPS IIIA including reduced sulphamidase activity and increased accumulation of an MPS IIIA HS GAG biomarker. Proliferation of MPS IIIA iPSC-derived NPCs was reduced compared to control, but could be partially rescued by reintroducing functional sulphamidase enzyme, or by doubling the concentration of the mitogen fibroblast growth factor 2 (FGF2). Whilst both control heparin, and MPS IIIA HS GAGs had a similar binding affinity for FGF2, only the latter inhibited FGF signalling, suggesting accumulated MPS IIIA HS GAGs disrupt the FGF2:FGF2 receptor:HS signalling complex. Neuronal differentiation of MPS IIIA iPSC-derived NPCs was associated with a reduction in the expression of neuronal cell marker genes βIII-TUBULIN, NF-H and NSE, revealing reduced neurogenesis compared to control. A similar result was achieved by adding MPS IIIA HS GAGs to the culture medium during neuronal differentiation of control iPSC-derived NPCs. This study demonstrates the generation of MPS IIIA iPSCs, and NPCs, the latter of which display reduced proliferation and neurogenic capacity. Reduced NPC proliferation can be explained by a model in which soluble MPS IIIA HS GAGs compete with cell surface HS for FGF2 binding. The mechanism driving reduced neurogenesis remains to be determined but appears downstream of MPS IIIA HS GAG accumulation. Highlights • Induced pluripotent stem cells (iPSCs) were generated from MPS IIIA skin fibroblasts.• MPS IIIA iPSCs were successfully differentiated into neural progenitor cells (NPCs).• MPS IIIA NPC proliferation was reduced due to interference with FGF2 signalling.• Neurogenesis from MPS IIIA iPSC-derived NPCs is reduced via an unknown mechanism.

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An integrated biomanufacturing platform for the large-scale expansion and neuronal differentiation of human pluripotent stem cell-derived neural progenitor cells.

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