Morphological and molecular expression patterns of neural precursor cells derived from human fetal spinal cord in two-, three-dimensional, and organoid culture environments

Recently, interest in three-dimensional (3D) cell or tissue organoids that may, in vitro, overcome not only the practical problems associated with fetal tissue transplantation, but also provide a potential source for the regeneration of injured spinal cords, has been increasing steadily. In this study, we showed that human neural precursor cells (hNPCs) derived from the fetal spinal cord could be incubated in serum free medium at two dimensional (2D), three dimensional (3D) and tissue organoid-systems. Additionally, we investigated morphological changes over time along with the expression of proteoglycans, collagen, or myelin in 2D, 3D and tissue-like organoids. 2D cells exhibited a spindle-shaped morphology with classic hill and valley growth patterns, while 3D cells grew as clusters of undifferentiated cells and cell sheets (tissue organoids) that gradually rolled up like a carpet without forming a circular cell mass. Immunostaining was performed to demonstrate the expression of TUJ-1, MAP-2, GAD 65/67 and ChAT in 2D cells or tissue-like organoids, which stained positively for them. In addition, we observed the immunoreactivity of HNu, NG2, TUJ-1, and GFAP in tissue-like organoids. The organoid culture system studied in our work may be used as therapeutic agents for spinal cord injury (SCI), and as raw materials needed for development of new medicines to improve human responses and cure diseases.