Lineage restriction of neuroepithelial precursor cells from fetal human spinal cord

Abstract

In the presence of epidermal growth factor (EGF) and/or fibroblast growth factor 2 (FGF2), neuroepithelial precursor cells from dissociated fetal human spinal cord are mitotically active and form free-floating spheres of undifferentiated cells. Proliferating cells were obtained in approximately 40% of preparations with each mitogen, were immunoreactive for the intermediate filament nestin, and did not express neuronal- or glial-specific markers. Early passage neuroepithelial precursor cells were pluripotent and differentiated into neurons expressing MAP2a,b, NF-M, and TuJ1, and GFAP-positive astrocytes; however, oligodendrocytes were never seen. As the cells were passaged from P0 to P4, the percentage of differentiating neurons significantly decreased and the prevalence of astrocytes significantly increased. While the majority of cell populations from individual preparations stopped proliferating between 3 and 6 passages, two expanding cell lines have been successfully expanded in EGF and FGF2 for over 25 passages and have been maintained in culture for over one year. These cells express nestin and not other cell-specific lineage markers. When differentiated, these neuroepithelial cell lines differentiate only into astrocytes, showing no expression of any neuronal marker. These data suggest that continued passage under these conditions preferentially selects for spinal cord neural precursors that are restricted to the astrocytic lineage. Despite the lineage restriction of later passage cell populations, these results provide a rationale for future investigation into the lineage potential of these cells in vivo following transplantation into the adult CNS, potentially as a therapeutic approach for traumatic injury and neurodegenerative disease.

Markers of Pluripotency and Differentiation in Human Neural Precursor Cells Derived from Embryonic Stem Cells and CNS Tissue

“…streptomycin (Cambrex Bio Science), and 8 ng/ml huHuman NPCs can also be isolated from the human man basic fibroblast growth factor (bFGF, R&D Sysembryonic or fetal CNS, or the adult CNS, and extems, Minneapolis, MN). Human ESC colonies were panded in vitro for prolonged periods (36,45). Although passaged by mechanical splitting at 5-7-day intervals these cells may have more limited differentiation potenand replated on fresh fibroblasts.…”

“…The neural differentiation was performed in two different media: neural differentiation medium tantly, whatever the cell type used for future clinical applications, large amounts of cells have to be produced (NDM) and neural stem cell medium (NSM). NDM contained DMEM/F-12 and neurobasal medium (1:1) supunder Good Manufacturing Practice (GMP) quality standards, which can be accomplished by modifying preplemented with 1 × B27, 1 × N2, 2 mM GlutaMax (Gibco Invitrogen), 25 U/ml penicillin/streptomycin viously published protocols (7,36,42). However, the main concern of hESC-derived cells in cell therapy is (Cambrex Bio Science), and 20 ng/ml bFGF.…”

Identification and characterization of neuronal precursors and their progeny from human fetal tissue

“…Multipotent human neural stem cells (NSCs) have been isolated from fetal human forebrain and spinal cord (Buc-Caron, 1995;Sabate et al, 1995;Svendsen et al, 1996Svendsen et al, , 1998 ChalmersRedman et al, 1997;Johansson et al, 1999; Vescovi et al, 1999a,b;Carpenter et al, 1999;Quinn et al, 1999;Piper et al, 2000) as well as the adult human subependymal zone and hippocampus (Kukekov et al, 1999; Roy et al, 2000a,b). Furthermore, phenotypically restricted lineages have also been reported for human brain tissue.…”

In Vitro Isolation and Expansion of Human Retinal Progenitor Cells

“…More importantly, replacement of damaged CNS or PNS neurons might be achieved by transplantation of these precursors. In human, neural stem and progenitor cells which can be maintained for extended passages in vitro have been identified from the brain (4,6,8,10,17,19,20,23,24,26) and spinal cord (25). These cells, similar to their in situ counterparts in the normal brain and spinal cord, retain the capabilities of multilineage differentiation after engraftment into the rodent brain and spinal cord (2,3,11,12,22,27,29,30,33).…”