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Organoids directly derived from L-MYC immortalized human neural stem cells (LMNSC01) model cerebral development in vitro

ABSTRACT: Developing platforms for ex vivo studies of human brain development and formulation of therapeutic strategies for a variety of conditions and diseases has been hampered by limited access to relevant human tissue and the necessity of using rodent models that imperfectly recapitulate human brain physiology. A promising advance is brain organoids that, to a greater extent than monolayer or spheroid cultures, recapitulate to varying extents the patterns of cell differentiation and morphological development characteristic of human brain. Here, we present an organoid-based research platform initiated using L-MYC immortalized human fetal neural stem cells (NSCs) (LMNSC01), and) and grown in a physiological 4% oxygen environment similar that in developing brain. Expanded LMNSC01 cells maintain genomic stability and multipotency, and are non-tumorigenic. Using authentic human NSCs as starting material eliminates the ex vivo programming and reprogramming required to achieve directed differentiation of human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs). LMNSC008 brain organoids were characterized using NanoString technology to profile gene expression for over 120 days in vitro and comparing these patterns to these same genes during normal brain development (BrainSpan database). We also visualized by immunofluorescence changes in expression and distribution of genes representative of these developmental processes along with morphological changes occurring over this same period. We observe parallel developmental patterns of gene expression between organoids and developing cortex for pathways involved in neuronal cytoskeleton, neuron-glia interaction, neural connectivity, neurotransmission, metabolism, axon and dendrite structure, tissue integrity, angiogenesis, and myelination. We suggest that LMNSC01 organoids offer an alternative to the challenges of iPSC programming and neural induction. Notable properties of this platform are its initiation with a line of authentic neural stem cells (LMNSC01), the consistency of the organoids produced, and favorable comparison of their gene expression patterns with those observed during normal fetal development.

ORGANISM(S): Homo sapiens

PROVIDER: GSE272682 | GEO | 2024/07/20

REPOSITORIES: GEO

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Project description:Neural stem cell (NSC) transplantation replaces damaged brain cells and provides disease-modifying effects in many neurological disorders. However, there has been no efficient way to obtain autologous NSCs in patients. Given that ectopic factors can reprogram somatic cells to be pluripotent, we attempted to generate human NSC-like cells by reprograming human fibroblasts. Fibroblasts were transfected with NSC line-derived cellular extracts and grown in neurosphere culture conditions. The cells were then analyzed for NSC characteristics, including neurosphere formation, gene expression patterns, and ability to differentiate. The obtained induced neurosphere-like cells (iNS), which formed daughter neurospheres after serial passaging, expressed neural stem cell markers, and had demethylated SOX2 regulatory regions, all characteristics of human NSCs. The iNS had gene expression patterns that were a combination of the patterns of NSCs and fibroblasts, but they could be differentiated to express neuroglial markers and neuronal sodium channels. These results show for the first time that iNS can be directly generated from human fibroblasts. Further studies on their application in neurological diseases are warranted. We generated induced neural stem cells (iNS) and compared the gene expressions with control cells, which included primary human dermal fibroblast cultured in DMEM media (FB), primary human dermal fibroblast cultured in neurosphere-forming condition (FB_con), and neural stem cell line (F3). The four cell lines (iNS, FB, FB_con, and F3) showed different gene expression patterns. In detail, RNA was isolated using Trizol (Invitrogen) according to the manufacturer's instructions, and was labeled and hybridized to an Affimetrix GeneChip human gene 1.0 ST array (Affimetrix, Santa Clara, CA, USA) as described according to the manufacturer’s instructions. Data were analyzed using Expression Console software version 1.1 (Affimetrix), in which gene expression ratios were normalized by Robust Multichip Analysis according to the manufacturer’s protocol.

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Organoids directly derived from L-MYC immortalized human neural stem cells (LMNSC01) model cerebral development in vitro

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