Abstract
Neural stem/progenitor cell (NSPC) transplantation is a promising therapy for spinal cord injury (SCI). However, little is known about NSPC from the adult human spinal cord as a donor source. We demonstrate for the first time that multipotent and self-renewing NSPC can be cultured, passaged and transplanted from the adult human spinal cord of organ transplant donors. Adult human spinal cord NSPC require an adherent substrate for selection and expansion in EGF (epidermal growth factor) and FGF2 (fibroblast growth factor) enriched medium. NSPC as an adherent monolayer can be passaged for at least 9 months and form neurospheres when plated in suspension culture. In EGF/FGF2 culture, NSPC proliferate and primarily express nestin and Sox2, and low levels of markers for differentiating cells. Leukemia inhibitory factor (LIF) promotes NSPC proliferation and significantly enhances GFAP expression in hypoxia. In differentiating conditions in the presence of serum, these NSPC show multipotentiality, expressing markers of neurons, astrocytes, and oligodendrocytes. Dibutyryl cyclic AMP (dbcAMP) significantly enhances neuronal differentiation. We transplanted the multipotent NSPC into SCI rats and show that the xenografts survive, are post-mitotic, and retain the capacity to differentiate into neurons and glia.Together, these findings reveal that multipotent self-renewing NSPC cultured and passaged from adult human spinal cords of organ transplant donors, respond to exogenous factors that promote selective differentiation, and survive and differentiate after transplantation into the injured spinal cord.
Highlights
Despite advances in medical and surgical care, current clinical therapies for spinal cord injury (SCI) are limited [1,2]
We examined the effects of 10% BIT 9500 serum (StemCell Technologies, Vancouver BC) and 10 ng/ml leukemia inhibitory factor (LIF) (Millipore, Temecula, CA) in growth factor enriched serum-free medium (SFM) to promote the expansion of Neural stem/ progenitor cells (NSPC) cultured as suspension cultures in uncoated tissue culture flasks
From the first 3 donors, we cultured the cells in suspension culture in growth factor enriched SFM, using methods similar to our previous work with adult rat spinal cord NSPC [11,13]
Summary
Despite advances in medical and surgical care, current clinical therapies for spinal cord injury (SCI) are limited [1,2]. Human embryonic stem cellderived oligodendrocyte progenitor cells and fetal NSPC are already in Phase 1 clinical trials. Spinal cord derived NSPC are region-specific, and may respond more appropriately to the intrinsic micro-environment of the injured spinal cord. Lower vertebrates such as urodele amphibians can regenerate their spinal cord even after full transection. This is in part due to the ependymal cells lining the central canal which proliferate, migrate, and differentiate into neurons and glia and regenerate the spinal cord [9,10]. We and other groups have shown that transplantation of adult rat spinal cord NPSC into SCI rats promoted functional recovery [15,16,17]
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