Abstract
Homogeneous culture of neural precursor cells (NPCs) derived from human pluripotent stem cells (hPSCs) would provide a powerful tool for biomedical applications. However, previous efforts to expand mechanically dissected neural rosettes for cultivation of NPCs remain concerns regarding non-neural cell contamination. In addition, several attempts to purify NPCs using cell surface markers have not demonstrated the expansion capability of the sorted cells. In the present study, we show that polysialic acid-neural cell adhesion molecule (PSA-NCAM) is detected in neural rosette cells derived from hPSCs, and employ PSA-NCAM as a marker for purifying expandable primitive NPCs from the neural rosettes. PSA-NCAM-positive NPCs (termed hNPCPSA-NCAM+) were isolated from the heterogeneous cell population of mechanically harvested neural rosettes using magnetic-based cell sorting. The hNPCPSA-NCAM+ extensively expressed neural markers such as Sox1, Sox2, Nestin, and Musashi-1 (80∼98% of the total cells) and were propagated for multiple passages while retaining their primitive characteristics in our culture condition. Interestingly, PSA-NCAM-negative cells largely exhibited characteristics of neural crest cells. The hNPCPSA-NCAM+ showed multipotency and responsiveness to instructive cues towards region-specific neuronal subtypes in vitro. When transplanted into the rat striatum, hNPCPSA-NCAM+ differentiated into neurons, astrocytes, and oligodendrocytes without particular signs of tumorigenesis. Furthermore, Ki67-positive proliferating cells and non-neural lineage cells were rarely detected in the grafts of hNPCPSA-NCAM+ compared to those of neural rosette cells. Our results suggest that PSA-NCAM-mediated cell isolation provides a highly expandable population of pure primitive NPCs from hPSCs that will lend themselves as a promising strategy for drug screening and cell therapy for neurodegenerative disorders.
Highlights
Proper methods for differentiation and long term culture of neural precursors cells (NPCs) from human pluripotent stem cells such as human embryonic stem cells and human induced pluripotent stem cells are prerequisites for new drug screening and cell-based replacement therapies of the nervous system
Neural rosettes that appeared in the center of attached embryoid bodies (EBs) colonies were carefully isolated using pulled glass pipettes from the surrounding flat cells, and small rosette clumps were seeded on Matrigelcoated dishes after gentle trituration, and cultured in DMEM/ F12 supplemented with 16 N2, 16 B27 (Invitrogen) plus 20 ng/ml basic fibroblast growth factor (FGF)
We previously reported that the simultaneous inhibition of bone morphogenetic protein (BMP) and activin/nodal signals by the small molecules, dorsomorphin (DM) and SB431542 (SB) during embryoid body (EB) culture facilitated neural differentiation in the various human pluripotent stem cells (hPSCs) lines [21]
Summary
Proper methods for differentiation and long term culture of neural precursors cells (NPCs) from human pluripotent stem cells (hPSCs) such as human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) are prerequisites for new drug screening and cell-based replacement therapies of the nervous system. The neural rosette is the distinctive cellular structure that is morphologically similar to the developing neural tube, arising during the initial neuralization process of ESCs [1,10] These cells could be continuously expanded in the presence of basic fibroblast growth factor (FGF) and epidermal growth factor (EGF), and differentiated into neurons and astrocytes in vitro as well as in vivo. Koch et al succeeded in partially answering this question by deriving long-term self-renewing neural stem cells with the characteristics of neural rosette cells from hESCs [5] These cells responded to regional cues inducing region-specific neuronal subtypes, and showed successful functionality of in vivo integration without tumor formation
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