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An Integrated Miniature Bioprocessing for Personalized Human Induced Pluripotent Stem Cell Expansion and Differentiation into Neural Stem Cells.

ABSTRACT: Human induced pluripotent stem cells (iPSCs) are ideal cell sources for personalized cell therapies since they can be expanded to generate large numbers of cells and differentiated into presumably all the cell types of the human body in vitro. In addition, patient specific iPSC-derived cells induce minimal or no immune response in vivo. However, with current cell culture technologies and bioprocessing, the cost for biomanufacturing clinical-grade patient specific iPSCs and their derivatives are very high and not affordable for majority of patients. In this paper, we explored the use of closed and miniature cell culture device for biomanufacturing patient specific neural stem cells (NSCs) from iPSCs. We demonstrated that, with the assist of a thermoreversible hydrogel scaffold, the bioprocessing including iPSC expansion, iPSC differentiation into NSCs, the subsequent depletion of undifferentiated iPSCs from the NSCs, and concentrating and transporting the purified NSCs to the surgery room, could be integrated and completed within two closed 15?ml conical tubes.

SUBMITTER: Lin H

PROVIDER: S-EPMC5216399 | biostudies-literature | 2017 Jan

REPOSITORIES: biostudies-literature

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An Integrated Miniature Bioprocessing for Personalized Human Induced Pluripotent Stem Cell Expansion and Differentiation into Neural Stem Cells.

Lin Haishuang H Li Qiang Q Lei Yuguo Y

Scientific reports 20170106

Human induced pluripotent stem cells (iPSCs) are ideal cell sources for personalized cell therapies since they can be expanded to generate large numbers of cells and differentiated into presumably all the cell types of the human body in vitro. In addition, patient specific iPSC-derived cells induce minimal or no immune response in vivo. However, with current cell culture technologies and bioprocessing, the cost for biomanufacturing clinical-grade patient specific iPSCs and their derivatives are ...[more]

PMID: 28057917

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Project description:Efficient and effective methods for converting human induced pluripotent stem cells into differentiated derivatives are critical for performing robust, large-scale studies of development and disease modelling, and for providing a source of cells for regenerative medicine. Here, we describe a 14-day neural differentiation protocol which allows for the scalable, simultaneous differentiation of multiple iPSC lines into cortical neural stem cells We currently employ this protocol to differentiate and compare sets of engineered iPSC lines carrying loss of function alleles in developmental disorder associated genes, alongside isogenic wildtype controls. Using RNA sequencing (RNA-Seq), we can examine the changes in gene expression brought about by each disease gene knockout, to determine its impact on neural development and explore mechanisms of disease. The 10-day Neural Induction period uses the well established dual-SMAD inhibition approach combined with Wnt/β-Catenin inhibition to selectively induce formation of cortical NSCs. This is followed by a 4-day Neural Maintenance period facilitating NSC expansion and rosette formation, and NSC cryopreservation. We also describe methods for thawing and passaging the cryopreserved NSCs, which are useful in confirming their viability for further culture. Routine implementation of immunocytochemistry Quality Control confirms the presence of PAX6-positive and/or FOXG1-positive NSCs and the absence of OCT4-positive iPSCs after differentiation. RNA-Seq, flow cytometry, immunocytochemistry (ICC) and RT-qPCR provide additional confirmation of robust presence of NSC markers in the differentiated cells. The broader utility and application of our protocol is demonstrated by the successful differentiation of wildtype iPSC lines from five additional independent donors. This paper thereby describes an efficient method for the production of large numbers of high purity cortical NSCs, which are widely applicable for downstream research into developmental mechanisms, further differentiation into postmitotic cortical neurons, or other applications such as large-scale drug screening experiments.

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.

Dataset Information

An Integrated Miniature Bioprocessing for Personalized Human Induced Pluripotent Stem Cell Expansion and Differentiation into Neural Stem Cells.

Publications

An Integrated Miniature Bioprocessing for Personalized Human Induced Pluripotent Stem Cell Expansion and Differentiation into Neural Stem Cells.

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