Downloaded from http://cshperspectives.cshlp.org/ on October 1, 2021 - Published by Cold Spring Harbor Laboratory Press In Vitro Models for Neurogenesis Hassan Azari1,2 and Brent A. Reynolds1 1Department of Neurosurgery, McKnight Brain Institute, University of Florida, Gainesville, Florida 32611 2Neural Stem Cell and Regenerative Neuroscience Laboratory, Department of Anatomical Sciences & Shiraz Stem Cell Institute, Shiraz University of Medical Sciences, Shiraz, Iran Correspondence: [email protected] The process of generating new neurons of different phenotype and function from undiffer- entiated stem and progenitor cells starts at very early stages of development and continues in discrete regions of the mammalian nervous system throughout life. Understanding mecha- nisms underlying neuronal cell development, biology, function, and interaction with other cells, especially in the neurogenic niche of fully developed adults, is important in defining and developing new therapeutic regimes in regenerative neuroscience. Studying these complex and dynamic processes in vivo is challenging because of the complexity of the nervous system and the presence of many known and unknown confounding variables. However, the challenges could be overcome with simple and robust in vitro models that more or less recapitulate the in vivo events. In this work, we will present an overview of present available in vitro cell-based models of neurogenesis. he central nervous system (CNS) is one of ent in the CNS, play a significant role in assist- Tthe most complex and intriguing organs ing neuronal cells to fulfill their proper function in mammalians, and its development, function, in a homeostatic and balanced microenviron- and pathology has attracted the attention of ment (Kettenmann et al. 1996; Araque and Na- many scientists throughout centuries. One of varrete 2010; Perry and Teeling 2013; Zabel and the amazing phenomena that occur in the CNS Kirsch 2013). Hence, as neurons are the primary is the process of new nerve cell generation or functional units, many of the diseases and dis- neurogenesis (Morrens et al. 2012; Jessberger orders of the CNS are associated with neuronal and Gage 2014). Neuronal cells are the building cell loss and dysfunction (Amor et al. 2010). blocks of the nervous system, enabling it to es- Understanding the root causes and, therefore, tablish a highly complex wiring system with finding meaningful therapies for many CNS the ability to receive, integrate, and respond to diseases is dependent on our understanding of a variety of stimuli in a timely and highly orga- the generation of the neuronal cells in associa- nized fashion. Other neural cell types, such as tion with other cells, mechanisms of their func- astrocytes and oligodendrocytes, and also the tion, maintenance, turnover, and replacement nonneural cells, such as microglia, endothelial, in normal and diseased conditions. Studying fibroblasts, and blood cells, which are also pres- all these processes in vivo is a daunting task, Editors: Fred H. Gage, Gerd Kempermann, and Hongjun Song Additional Perspectives on Neurogenesis available at www.cshperspectives.org Copyright # 2016 Cold Spring Harbor Laboratory Press; all rights reserved; doi: 10.1101/cshperspect.a021279 Cite this article as Cold Spring Harb Perspect Biol 2016;8:a021279 1 Downloaded from http://cshperspectives.cshlp.org/ on October 1, 2021 - Published by Cold Spring Harbor Laboratory Press H. Azari and B.A. Reynolds considering the complexity and dynamic nature cells a unique cell source to model early stages of the nervous system. To facilitate understand- of nervous system development and studying ing the complex process of neurogenesis, in vi- production of different neuronal subtypes and tro assays and methodologies have been de- also finding optimal conditions to generate veloped to recapitulate in vivo processes, while these cells at a large scale with high purity for at the same time decreasing some of the as- cell therapy approaches. Three main culture sociated complexities (reductionist approach). systems are used to generate neural cells from In this article, we present an overview of cur- the pluripotent stem cells, which include em- rently available in vitro cell-based neurogenesis bryoid body (EB) formation (Schulz et al. models. 2003; Elkabetz et al. 2008), coculture with cells, such as bone marrow stromal cells or their con- ditioned medium that potentiate neuralization IN VITRO NEUROGENESIS MODELS processes (Kawasaki et al. 2000; Vazin et al. Neurogenesis occurs throughout mammalian 2008), and monolayer culture systems (Ying life, mainly in embryonic, fetal, and neonatal et al. 2003; Gerrard et al. 2005). stages and to a lesser extent in the adult stage. In the embryonic development, the backbone Embryoid Body Formation of the nervous system is established through formation of neural plate, neural tube, and es- Differentiation through EB formation recapitu- tablishment of the rostrocaudal and anteropos- lates embryogenesis of different tissues originat- terior patterns (Stiles and Jernigan 2010). In fe- ing from all three germ layers including primi- tal and neonatal stages, the developing nervous tive neural tissue (Leahy et al. 1999). In the EB, system acquires its final shape and in the adult pluripotent stem cells spontaneously differen- stage, the nervous system is fully established and tiate into different cell lineages. Therefore, the the process of neurogenesis is limited to certain resulting neuroepithelial cells need further neu- discrete areas, such as the subventricular zone ral cell selection to enhance their purity. More- (SVZ) of the lateral ventricles toward the olfac- over, the process of neuralization with this tory bulb (Shen et al. 2008; Kriegstein and Alva- approach is lengthy with reduced control over rez-Buylla 2009) and subgranular zone (SGZ) the early phases of the process. Although factors of the dentate gyrus (DG) in the hippocampus such as retinoic acid (RA) have been used to (Kempermann et al. 2003; Seri et al. 2004). Each enhance neural differentiation in the EB (Okabe one of these stages could be modeled in vitro et al. 1996), this could affect neural patterning using pluripotent stem cells and adult neural and alter the identity of the resulting cells, such stem cells (NSCs). as suppression of forebrain neuronal identity (Kawasaki et al. 2000). Hence, the low efficien- cy of neural conversion, the need for subsequent USING PLURIPOTENT STEM CELLS AS lineage selection to achieve afinal homogeneous AN IN VITRO NEUROGENESIS MODEL neural cell population, and the complexity of In vitro models of embryonic neurogenesis and multicellular aggregates in the EB approach, formation of different neuronal phenotypes is makes this culture system difficult for studying mainly based on the usage of pluripotent stem cellular and molecular mechanisms underlying cells, such as embryonic stem cells (ESCs) neural cell development. (Zhang et al. 2001; Schulz et al. 2004; Zeng et al. 2004; Fathi et al. 2015) and induced plu- Coculture with Stromal Cells ripotent stem cells (iPSCs) (Lu et al. 2013; Compagnucci et al. 2014; Velasco et al. 2014). Induction of neural identity in mouse ESCs us- The ability to differentiate these cells into all ing stromal cells (such as PA6 and MS5) or their three germ layers, namely, the ectoderm, meso- conditioned medium has led to the establish- derm, and endoderm, makes pluripotent stem ment of a serum-free culture system that cir- 2 Cite this article as Cold Spring Harb Perspect Biol 2016;8:a021279 Downloaded from http://cshperspectives.cshlp.org/ on October 1, 2021 - Published by Cold Spring Harbor Laboratory Press In Vitro Neurogenesis cumvents the need for EB formation and short- rosettes (Ying et al. 2003; Gerrard et al. 2005; ens the length of the neuralization process dra- Koch et al. 2009). As this process occurs in a matically (Kawasaki et al. 2000; Vazin et al. chemically defined serum-free medium, it pro- 2008). The same methodology could also effi- vides a unique substrate to tease out signaling ciently generate neuroepithelial cells from pri- pathways and complex molecular mechanisms mate and human ESCs. Moreover, it has been underlying pluripotent stem-cell-derived neu- shown that stromal cells can induce the genera- rogenesis (Ying et al. 2003). For example, treat- tion of neuroepithelial cells through factors se- ment of murine ESCs with factors, such as Dkk1 creted bystromal cellsandcell-surface-anchored or lefty, to inhibit Wnt and nodal signaling en- molecules. Further studies have led to character- hances generation of neuroepithelial cells (Wa- ization of factors, such as Sonic hedgehog (Shh), tanabe et al. 2005). In addition, treatment of which are secreted from stromal cells, and play human ESCs with noggin dramatically reduces an essential role in acquiring a dopaminergic contamination of the final neural progenitors phenotype in human ESC (hESC)-derived neu- with derivatives of the extraembryonic endo- ral stem cells (Swistowska et al. 2010). Although derm (Gerrard et al. 2005). This monolayer cul- this methodology is simple, fast, and efficient in ture system facilitates visualization of neural generating neuroepithelial cells from murine conversion of the ES cells and provides research- and human ESCs, the approach is dependent ers with the opportunity to study factors and on feeder cells and their unidentified stromal- signaling pathways underlying human neural derived inducing activity (SDIA). tube formation, anteroposterior and