Cellular composition of long‐term human spinal cord‐ and forebrain‐derived neurosphere cultures

Abstract

In vitro expanded neural precursor cells (NPCs) may provide a stable source for cell therapy. In search of the optimal cell source for spinal cord repair, we investigated influences of gestational age, regional heterogeneity, and long-term in vitro propagation. The cellular content of neurosphere cultures prior to and after in vitro differentiation was studied by immunocytochemistry and flow cytometry. Human forebrain and spinal cord NPCs deriving from first-trimester tissue were cultured as neurospheres in the presence of epidermal growth factor, basic fibroblast growth factor, and ciliary neurotrophic factor. Proteins characteristic for embryonic stem cells, i.e., Tra-1-60, Tra-1-81, and SSEA-4, were present in approximately 0.5% of the cells in donor tissues and neurospheres. The proportions of nestin- and proliferating cell nuclear antigen-immunoreactive (IR) cells were also maintained, whereas the CD133-IR population increased in vitro. Glial fibrillary acidic protein-IR cells increased in number, and in contrast the fraction of beta-tubulin III-IR cells decreased, at and beyond passage 5 in spinal cord but not forebrain cultures. However, dissociated and in vitro-differentiated forebrain- and spinal cord-derived neurospheres generated similar proportions of neurons, astrocytes, and oligodendrocytes. Gestational age of the donor tissue, which ranged from 4.5 to 12 weeks for forebrain and from 4.5 to 9.5 weeks for spinal cord, did not affect the proportion of cells with different phenotypes in culture. Thus, cellular composition of human neurosphere cultures differs as a result of long-term in vitro propagation and regional heterogeneity of source tissue, despite expansion under equal culture conditions. This could in turn imply that human spinal cord and forebrain NPCs present different repair potentials in in vivo settings.

Multiplex mapping of chromatin accessibility and DNA methylation within targeted single molecules identifies epigenetic heterogeneity in neural stem cells and glioblastoma

“…2A) (Fig. 2C), which encodes the cell surface antigen CD133, expressed in up to 40% of cultured NSC (Piao et al 2006;Sun et al 2009). All analyzed PROM1 sequences had low or no methylation (Fig.…”

Phenotypic characterization of neural stem cells from human fetal spinal cord: Synergistic effect of LIF and BMP4 to generate astrocytes

“…These findings are consistent with earlier investigations which have shown that NSCs derived from human spinal cord tissue demonstrate a reduced capacity to generate the oligodendrocytic phenotype and as a consequence an increase in the number of GFAP expressing cells with passage (Chandran et al, 2004;Quinn et al, 1999;Svendsen et al, 1998). Comparing long term cultures of human NSCs derived from brain to those from the corresponding spinal cord, Piao et al found that the brain derived neurospheres generated consistent numbers of neural phenotypes while the spinal cord derived neurospheres showed a significant decrease in bIII-tubulin 1 and increase in GFAP 1 cells by passage 5 and at passage 10 the O4 1 cells only equated to 0.5% of the total cell numbers (Piao et al, 2006). Other studies of neurospheres derived from fetal and embryonic human spinal cord have found no evidence for the oligodendrocytic phenotype after differentiation in vitro (Barami et al, 2001a,b;Iwanami et al, 2005;Walder and Ferretti, 2004).…”

Evaluation of human fetal neural stem/progenitor cells as a source for cell replacement therapy for neurological disorders: Properties and tumorigenicity after long‐term in vitro maintenance

“…However, previous in vitro studies showed that subjecting hNS/PCs to multiple passages for up to 300 days in vitro (DIV) reduces their growth rate and alters their neurogenic potential (Caldwell et al, 2001;Kanemura et al, 2002;Piao et al, 2006;Wright et al, 2006;Anderson et al, 2007). Neither the proliferative and differentiation properties nor the in vivo dynamics have been reported for hNS/ PCs cultured for longer than 300 DIV.…”