title: Rapid generation of sub-type, region-specific neurons and neural networks from human pluripotent stem cell-derived neurospheres.
abstract: Stem cell-based neuronal differentiation has provided a unique opportunity for disease modeling and regenerative medicine. Neurospheres are the most commonly used neuroprogenitors for neuronal differentiation, but they often clump in culture, which has always represented a challenge for neurodifferentiation. In this study, we report a novel method and defined culture conditions for generating sub-type or region-specific neurons from human embryonic and induced pluripotent stem cells derived neurosphere without any genetic manipulation. Round and bright-edged neurospheres were generated in a supplemented knockout serum replacement medium (SKSRM) with 10% CO2, which doubled the expression of the NESTIN, PAX6 and FOXG1 genes compared with those cultured with 5% CO2. Furthermore, an additional step (AdSTEP) was introduced to fragment the neurospheres and facilitate the formation of a neuroepithelial-type monolayer that we termed the "neurosphederm". The large neural tube-type rosette (NTTR) structure formed from the neurosphederm, and the NTTR expressed higher levels of the PAX6, SOX2 and NESTIN genes compared with the neuroectoderm-derived neuroprogenitors. Different layers of cortical, pyramidal, GABAergic, glutamatergic, cholinergic neurons appeared within 27 days using the neurosphederm, which is a shorter period than in traditional neurodifferentiation-protocols (42-60 days). With additional supplements and timeline dopaminergic and Purkinje neurons were also generated in culture too. Furthermore, our in vivo results indicated that the fragmented neurospheres facilitated significantly better neurogenesis in severe combined immunodeficiency (SCID) mouse brains compared with the non-fragmented neurospheres. Therefore, this neurosphere-based neurodifferentiation protocol is a valuable tool for studies of neurodifferentiation, neuronal transplantation and high throughput screening assays.
authors.affiliation: College of Veterinary Medicine, Western University of Health Sciences, Pomona, CA 91766-1854, USA.
authors.forename: Aynun N
authors.initials: AN
authors.lastname: Begum
authors.affiliation: Graduate College of Biomedical Sciences, Western University of Health Sciences, Pomona, CA 91766, USA.
authors.forename: Caleigh
authors.initials: C
authors.lastname: Guoynes
authors.affiliation: College of Veterinary Medicine, Western University of Health Sciences, Pomona, CA 91766-1854, USA.
authors.forename: Jane
authors.initials: J
authors.lastname: Cho
authors.affiliation: College of Veterinary Medicine, Western University of Health Sciences, Pomona, CA 91766-1854, USA; Graduate College of Biomedical Sciences, Western University of Health Sciences, Pomona, CA 91766, USA.
authors.forename: Jijun
authors.initials: J
authors.lastname: Hao
authors.affiliation: College of Pharmacy, Western University of Health Sciences, Pomona, CA 91766, USA.
authors.forename: Kabirullah
authors.initials: K
authors.lastname: Lutfy
authors.affiliation: College of Veterinary Medicine, Western University of Health Sciences, Pomona, CA 91766-1854, USA; Graduate College of Biomedical Sciences, Western University of Health Sciences, Pomona, CA 91766, USA. Electronic address: yhong@westernu.edu.
authors.forename: Yiling
authors.initials: Y
authors.lastname: Hong
mesh_terms: D002454:Cell Differentiation; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism*; D055495:Neurogenesis / Q000276:immunology*; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D044968:Regenerative Medicine
keywords: Human embryonic stem cells; Induced pluripotent stem cells; Neuroectoderm; Neurogenesis; Neurons; Neurosphere