Cellular composition of long-term human spinal cord- and forebrain-derived neurosphere cultures.
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.
Piao
Jing-Hua
JH
Neurotec Department, Division of Neurodegeneration and Neuroinflammation, Karolinska Institutet, Stockholm, Sweden. jing-hua.piao@ki.se
Odeberg
Jenny
J
Samuelsson
Eva-Britt
EB
Kjaeldgaard
Anders
A
Falci
Scott
S
Seiger
Ake
A
Sundström
Erik
E
Akesson
Elisabet
E
D000367:Age Factors; D001253:Astrocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D015415:Biomarkers / Q000378:metabolism; D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D006801:Humans; D009414:Nerve Growth Factors / Q000494:pharmacology; D009416:Nerve Regeneration / Q000502:physiology; D009419:Nerve Tissue Proteins / Q000378:metabolism; D009457:Neuroglia / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D009836:Oligodendroglia / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D039904:Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism*; D016548:Prosencephalon / Q000166:cytology / Q000196:embryology* / Q000378:metabolism; D018874:Spheroids, Cellular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D013116:Spinal Cord / Q000166:cytology / Q000196:embryology* / Q000378:metabolism; D033581:Stem Cell Transplantation / Q000379:methods
D015415:Biomarkers; D009414:Nerve Growth Factors; D009419:Nerve Tissue Proteins