Differentiation, polarization, and migration of human induced pluripotent stem cell-derived neural progenitor cells co-cultured with a human glial cell line with radial glial-like characteristics
Introduction
During embryonic corticogenesis, neural progenitor cells (NPCs) and neuronal cells have complicated interactions with glial cells. In particular, radial glia (RG) cells construct the framework and control the alignment of neuronal cells during embryonic CNS development [1]. Glial cells are also important for the differentiation of NPCs derived from pluripotent stem cells (PSCs) and induced pluripotent stem cells (iPSCs) [2]. It is now possible to investigate developmental processes of the human central nervous system (CNS) and the pathogenesis of neural disorders using human PSCs [3], [4], [5], [6]. iPSC-derived NPCs (iPSC-NPCs) can produce both neuronal and glial cells; however, they tend to remain neurogenic for a prolonged period [7], [8]. Extensive research has led to methods for promoting iPSC differentiation toward specific lineages. However, the differentiation propensities of the iPSCs and iPSC-NPCs are not completely controlled. Furthermore, iPSC-NPCs produce neuronal cells with disordered and randomly arranged neurites, which complicates the examination of neurite morphology and dynamics. The disorderly arrangement of neurites in in vitro cultures may be due to the absence of the radial glial scaffold that is abundant during in vivo corticogenesis.
This shortage of glial scaffolds in vitro is addressed by co-culturing iPSC-NPCs with glial cells. Human iPSC-NPCs can be induced to develop into mature neurons by culturing them with mouse primary astrocytes [2]. However, the isolation of primary glial cells for each experiment is time-consuming and laborious. Thus, the use of a human glial cell line as a scaffold for iPSC-NPC differentiation may be a more efficient approach.
Here, we established a co-culture system using iPSC-NPCs and a unique glial cell line, GDC90, which promoted iPSC-NPC differentiation, polarization, and migration. Use of this unique human glial cell line may shed light on the detailed morphological behaviors of iPSC-NPCs and contribute to their application in regenerative medicine.
Section snippets
Human tissues and cells
This study was carried out in accordance with the principles of the Helsinki Declaration, and approval for the use of human tissues and cells was obtained from the ethical committees of Osaka National Hospital and Keio University School of Medicine (Nos. 94, 110, 146, IRB0713). Surgically removed primary brain tumor tissues were collected at the Osaka National Hospital with written informed consent.
Human iPSC clone 201B7 [9], obtained from the RIKEN cell bank (Tsukuba, Japan), was propagated on
Isolation and establishment of fluorescently labeled human GDCs
We isolated a cell line, GDC90, from the tumor specimen of a 74-year-old female glioblastoma multiforme (GBM) patient. To distinguish GDC90 cells from host or co-cultured cells, we used the PB transposon vector which contains a tdTomato-expressing cassette driven by the CAG promoter, a neomycin-resistance gene cassette, and the PB terminal repeats sequence (Fig. 1A). Following nucleofection of this vector and neomycin selection, GDC90 cells were isolated that stably expressed tdTomato and
Discussion
Here, we established a unique human glial cell line, GDC90, which exhibited a radial glial-like molecular signature, a polarized shape, and radially spreading processes. Compared with the other glial cell lines tested, GDC90 cells showed a higher affinity for iPSC-NPCs, consistent with a previous report indicating that various glioma cell lines have different affinities for NSCs [16]. Consistent with this higher affinity, GDC90 cells expressed more N-cadherin than the other glioma-derived cell
Conclusion
We established a unique human glial cell line, GDC90, and examined its effects on iPSC-NPC differentiation, polarization, and migration. This simple technique has the potential to accelerate the research and development of iPSC-based disease modeling and regenerative medicine.
Acknowledgments
We thank all the members of Dr. Kanemura’s and Prof. Okano’s laboratories. This study was supported by the Research Center Network for Realization of Regenerative Medicine, Japan Science and Technology Agency (JST), the JSPS KAKENHI Grant Number 24592181, Japan, the Research on Applying Health Technology, Health and Labour Sciences Research Grants, the Ministry of Health, Labour and Welfare of Japan, and the Advanced Research for Medical Products Mining Programme of the National Institute of
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