Pax2/5 and Pax6 subdivide the early neural tube into three domains. Using the stromal feeder-based differentiation approach [stromal derived induction activity (SDIA)-mediated protocols) the differentiation of neuroepithelial cells from hESCs has been achieved by their cocultivation on MS5 or PA6 stromal cells in serum-free medium. The radially organized cells of these structures can create a lumen, similar to the VZ of a developing brain (Elkabetz et al., 2008; Ziv et al., 2015). doi: 10.1002/dvdy.24035, Takahashi, K., Tanabe, K., Ohnuki, M., Narita, M., Ichisaka, T., Tomoda, K., et al. Avoid repeated freeze/thaw cycles. BMP-signaling is involved in the regulation of neurodevelopmental processes (progenitor proliferation, differentiation, and apoptosis) (Hegarty et al., 2013). Coat the surface of the culture vessel with the working solution of CELLStart CTS (14 mL for T-75, 7 mL for T-25, 3.5 mL for 60-mm dish, 2 mL for 35-mm dish). (2018) proposed a method of intracerebral implantation of brain organoids grown in vitro into the brains of mice. In addition, the distribution and gradients of morphogens and signaling molecules that the cells exchange during differentiation are dispersed and are very different from those in vivo. Biol. Proc. Developmental Biology, 9th Edn. (2006). (2002). In mice, FGF-2 induces the proliferation of neuroepithelial cells and neural precursors that can be isolated from the embryonic spinal cord, telencephalon and mesencephalon (Murphy et al., 1990; Raballo et al., 2000). * You can prepare a 30 g/mL T3 stock solution (1,000X) in distilled water. Several studies on isolated embryonic and adult mouse NSCs have revealed that FGF-2 and epidermal growth factor (EGF) are involved in their proliferation (Reynolds et al., 1992; Vescovi et al., 1993; Gritti et al., 1996; Reynolds and Weiss, 1996). 11995), Dulbeccos Phosphate-Buffered Saline (D-PBS) (Cat. A., Tetzlaff, W., and Weiss, S. (1992). doi: 10.1093/cercor/bhn260, Kunath, T., Saba-El-Leil, M. K., Almousailleakh, M., Wray, J., Meloche, S., and Smith, A. doi: 10.1038/35067582, Amoureux, M. C., Cunningham, B. doi: 10.1242/dev.081133, Vescovi, A. L., Reynolds, B. Res. Oligodendrocyte differentiation medium requires supplementation of Neurobasal medium with B-27, GlutaMAX-I, and T3. (2014) applied the Dual SMAD inhibition protocol to EBs formed from patient-derived iPSCs, and the differentiation of neurons was initiated by the action of the factors BDNF, GDNF, and cAMP. These neural rosettes, as in the other protocols, expressed ZO-1, PLZF, and Dach1 (Fedorova et al., 2019). Hum. Sci. The first mouse iPSCs were obtained from fibroblasts in the Yamanaka laboratory, using retroviral transfection of the pluripotency genes (Oct3/4, Sox2, c-Myc, and Klf4) (Takahashi and Yamanaka, 2006). Conversion of embryonic stem cells into neuroectodermal precursors in adherent monoculture. Biochem. The third type of NSC is the basal (intermediate) progenitor cell (IPC). J. Neurosci. However, these organoids have several limitations. Cell Death Dis. 31, 36833695. Later, the suggestion regarding the inhibition of TGF--signaling was supported by experiments to obtain NSCs from hESCs cultured on serum-free medium in the presence of Noggin (Pera et al., 2004; Itsykson et al., 2005). (2001). Mix gently. After 2 days, change the medium to neural differentiation medium. (2005). 31, 215225. Methods such as fluorescence-activated cell sorting (FACS) and MACS may be suitable for this purpose. The TGF-/Activin/Nodal pathway is required for the self-renewal of ESCs and maintaining their pluripotency (James et al., 2005). 9:937. doi: 10.1038/s41419-018-0990-2, Sato, N., Meijer, L., Skaltsounis, L., Greengard, P., and Brivanlou, A. H. (2004). doi: 10.1016/s0896-6273(03)00116-8. Sox1 expression is also detected in adult neural progenitor cells and several types of neurons (Kan et al., 2007; Venere et al., 2012). However, do not split the cells once they reach day 910 of differentiation when they can get damaged during the passaging process. Mouse embryonic stem cells and reporter constructs to detect developmentally regulated genes. No. (2018). Exposure of the smNPCs to FGF-2 resulted in the formation of typical neural rosettes. doi: 10.1038/nprot.2017.152, Qian, X., Nguyen, H. N., Song, M. M., Hadiono, C., Ogden, S. C., Hammack, C., et al. 17, 50465061. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Dev. A role for Wnt signalling in self-renewal of haematopoietic stem cells. Note: You may observe a white precipitate when thawing StemPro Neural Supplement; this precipitate will disappear when the supplement is completely thawed or dissolved. This method allowed the generation of cerebral organoids with vascular structures, without inhibiting neuronal differentiation (Ham et al., 2020). doi: 10.1038/nature05934, Pasxca, A. M., Sloan, S. A., Clarke, L. E., Tian, Y., Makinson, C. D., Huber, N., et al. Biol. Cell 110, 385397. J. Comp. Maintenance of pluripotency in human and mouse embryonic stem cells through activation of Wnt signaling by a pharmacological GSK-3-specific inhibitor. doi: 10.1126/science.2497519, Gtz, M., Stoykova, A., and Gruss, P. (1998). Biol. (2020). doi: 10.1159/000017435, Kawasaki, H., Mizuseki, K., Nishikawa, S., Kaneko, S., Kuwana, Y., Nakanishi, S., et al. Coat the surface of the culture vessel (with or without cover slips) with the polyLornithine working solution (14 mL for T-75, 7 mL for T-25, 3.5 mL for 60-mm dish, 2 mL for 35-mm dish). Hedgehog signaling in the subventricular Zone is required for both the maintenance of stem cells and the migration of Newborn neurons. Adv. No. Nat. For example, the application of the Dual-SMAD inhibition protocol for forming EBs from iPSCs allows the generation of cortical spheroids, which can be maintained in neurobasal medium containing FGF-2, EGF, and B27 supplement (Pasxca et al., 2015). Nature 390, 465471. Activin/Nodal inhibition alone accelerates highly efficient neural conversion from human embryonic stem cells and imposes a caudal positional identity. (2001) achieved only 24% of Nestin-positive cells out of the total number of ESCs after 24 h (of which 70% did not survive). It should be particularly useful for researchers who are beginning investigations in this area of cell biology. Do not allow slides to dry. Pham et al. doi: 10.1242/dev.009324, Doetsch, F., Caill, I., Lim, D. A., Garca-Verdugo, J. M., and Alvarez-Buylla, A. If desired, add 1 mL of Antibiotic-Antimycotic solution per 100 mL of medium. doi: 10.1007/s12015-013-9482-z, Rodrigues, G. M., Rodrigues, C. A., Fernandes, T. G., Diogo, M. M., and Cabral, J. M. (2015b). Blood vessel formation in cerebral organoids formed from human embryonic stem cells. The method was named serum free culture of embryoid bodies (SFEB; Watanabe et al., 2005). The original SFEB protocol could provide 90% of cells with Sox1 expression by day 5 of mEB culture, but only 35% of the cells were Bf1-positive (Watanabe et al., 2005), while 6575% of the cells were Bf1-positive using the SFEBq modification (Eiraku et al., 2011). That is why it is necessary not only to develop one or several universal and reproducible protocols for obtaining NSCs in vitro from PSCs but also to select universal criteria for evaluating the NSC phenotypes and the methods for their selection. doi: 10.1038/nn.3041, Shi, Y., Sun, L., Wang, M., Liu, J., Zhong, S., Li, R., et al. Res. This cell type remains Nestin-positive and GFAP- and Vimentin-negative but differs from the precursor in its expression of polysialylated neural cell adhesion molecule (PSA-NCAM), doublecortin (DCX), and TuJ1 (-tubulin) (Doetsch et al., 1997; Francis et al., 1999). Dev. Cereb. ED read, edited, and approved the manuscript. doi: 10.1016/j.ydbio.2004.08.031, Venere, M., Han, Y. G., Bell, R., Song, J. S., Alvarez-Buylla, A., and Blelloch, R. (2012). It was found that BMP inhibition was not required to initiate formation of the rosettes but did influence their morphology: the absence of Noggin led to the formation of defective rosettes and cell flattening. (2001) showed that hESCs in the form of EBs could be directed toward neural precursor cells. (2011). Note: You may coat the plates in advance and store them at 4C, wrapped tightly with Thermo Scientific Parafilm, for up to 2 weeks. The resulting NSC culture was prone to differentiation into midbrain and hindbrain neurons. No. 1283, 137145. Ensure that the cell surfaces are covered uniformly with the antibody solution. The head inducer Cerberus is a multifunctional antagonist of Nodal, BMP and Wnt signals. 17, 30233028. Biotechnol. For example, for transplantation, the cell population must be pure and have well-defined characteristics. 17504), Antibiotic-Antimycotic solution (Cat. Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. 19, 11291133. doi: 10.1038/nmeth.3415, Patani, R., Compston, D. A., Puddifoot, C., Wyllie, D. J., Hardingham, G. E., Allen, N. D., et al. doi: 10.1242/dev.01706, Kaji, K., Norrby, K., Paca, A., Mileikovsky, M., Mohseni, P., and Woltjen, K. (2009). doi: 10.1371/journal.pone.0007327, Pera, E. M., Ikeda, A., Eivers, E., and De Robertis, E. M. (2003). Different NSC lines exhibit different proliferative capacity and differentiation potentials. (1999). doi: 10.1523/jneurosci.1040-07, Bignami, A., and Dahl, D. (1974). doi: 10.1016/j.stem.2008.09.002, Elkabetz, Y., Panagiotakos, G., Al Shamy, G., Socci, N. D., Tabar, V., and Studer, L. (2008). doi: 10.1002/stem.530, Lippmann, E. S., Estevez-Silva, M. C., and Ashton, R. S. (2014). Rev. This review is intended to summarize the knowledge accumulated, to date, by workers in this field. Sci. For this, after EB formation combined with simultaneous neural induction, vascular endothelial growth factor (VEGF) and Wnt7a were added to the differentiating medium. 3D culture, instead, provides a platform for the study of cellcell interactions, the diversity of neural cells, and for investigations of in vivo neurogenesis and the development of neurological diseases (Lancaster et al., 2013; Kim et al., 2015; Gonzalez et al., 2018; Chlebanowska et al., 2020). Neurosci. Dev. Nat. J. Neurosci. Protoc. Comparison of Dual-SMAD inhibition (2D neural induction) with a combination of Dual-SMAD inhibition and SFEB protocols (3D induction) revealed that the 3D induction may promote greater numbers of NSCs with a high level of expression of Pax6 and Nestin, resulting in more forebrain neurons and a gain in neurite outgrowth (Chandrasekaran et al., 2017). Biotechnol. Neuron 23, 247256. Embryonic stem cells transplanted into the inner cell mass of an embryo, as well as intrinsic cells of the inner cell mass, were included in the formation of the neuroectoderm (Gossler et al., 1989). doi: 10.1128/MCB.21.12.3888-3900.2001, Israel, M. A., Yuan, S. H., Bardy, C., Reyna, S. M., Mu, Y., Herrera, C., et al. There are two main types of PSCs: embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). Intrinsic polarity of mammalian neuroepithelial cells. Dev. Repression of BMP-Smad-signaling is necessary for primary neural induction in nave ectoderm during embryogenesis (Pera et al., 2003; Liu and Niswander, 2005). Remove culture medium and gently rinse the cells once with D-PBS, without dislodging the cells. Later still, the neural rosettes begin to lose their epithelial integrity and rosette organization and are characterized by low NSC numbers, increased cellular heterogeneity and a tendency to differentiate into glial cells (Edri et al., 2015). It has been shown that developing neurons express PSA-NCAM during their migration, neurite outgrowth and synaptogenesis. Cell 97, 703716. Neurosci. Since these cells have unlimited proliferative potential, it is possible to maintain them in culture under certain conditions for many years. Neuron 29, 353366. Front. Clonal and population analyses demonstrate that an EGF-responsive mammalian embryonic CNS precursor is a stem cell. Nature 448, 313317. doi: 10.1002/jnr.22547, Munji, R. N., Choe, Y., Li, G., Siegenthaler, J. Cell Stem Cell 13, 706719. Neural differentiation medium requires supplementation of Neurobasal medium with B-27 Serum-Free Supplement and GlutaMAX-I. There are many different protocols that are able to provide the generation of rosettes of neural progenitors. Induction of midbrain dopaminergic neurons from ES cells by stromal cell-derived inducing activity. (2003). Nat. (2005). Human cerebral cortex development from pluripotent stem cells to functional excitatory synapses. This protocol showed an 80% efficiency of hESC and hiPSC differentiation into Pax6-positive NSCs. (2002). Biol. doi: 10.1371/journal.pone.0213374, Buc-Caron, M. H. (1995). doi: 10.1523/JNEUROSCI.3314-07.2008, Boncinelli, E., Gulisano, M., Spada, F., and Broccoli, V. (1995). Such cultures came to form rosette-like patterns. 21:694. doi: 10.3390/ijms21030694. Nodal antagonists in the anterior visceral endoderm prevent the formation of multiple primitive streaks. doi: 10.1016/0896-6273(95)90265-1, Wilson, P. A., and Hemmati-Brivanlou, A. Defined human pluripotent stem cell culture enables highly efficient neuroepithelium derivation without small molecule inhibitors. Neuron 14, 11811188. Neurogenesis in the adult hippocampus. The most convenient and acceptable source of NSCs is pluripotent stem cells (embryonic stem cells or induced pluripotent stem cells). Natl. There is a similarity between pNSCs and NSC-populations generated via the Dual-SMAD inhibition protocol, however, the second type represents a heterogeneous neural population of primitive neuroepithelial and polarized rosette-forming cells, the proliferative and differentiation potentials of which cannot be maintained in culture. After dissociation of the SFEBq culture the neural progenitors migrated to the adhesion substrate and differentiated into cortical-type neurons. Based on the 2D culture system, a protocol with a high efficiency (>95%) of NSC generation from hiPSCs in a serum-free culture medium, with low cell density plating, and without any morphogens or factors (FGF-2, BMP-inhibitors and others) was developed (Fedorova et al., 2019). (2009). 6, 2127. Dynamic and cell-specific DACH1 expression in human neocortical and striatal development. Sci. Genes Cells 10, 10011014. The next historical step was to obtain NSCs from ESCs through neural rosettes. (2015). Front. Astrocyte-specific protein and radial glia in the cerebral cortex of newborn rat. Nat. Neural stem cells from the SGZ are called Type I cells and can generate proliferating IPCs, called Type 2 cells, similar to the type B cells of the SVZ. If desired, add 1 mL of Antibiotic-Antimycotic solution per 100 mL of medium. They exposed EBs from hESCs to SB431542 (TGF-/Activin/Nodal inhibitor), Dorsomorphin (BMP inhibitor), CHIR99021 (GSK3 inhibitor), and purmorphamine (PMA; Reinhardt et al., 2013). Three-dimensional neural cultures can reproduce the intercellular interactions, cytoarchitecture, and the complete variety of cell types, and, therefore, more accurately reflect the morphogen gradients. Nat. Oblique radial glial divisions in the developing mouse neocortex induce self-renewing progenitors outside the germinal zone that resemble primate outer subventricular zone progenitors. (2018) used a model of AD based on cerebral organoids from patients iPSCs, induced with the DUAL SMAD inhibition protocol. doi: 10.1073/pnas.0808387106, Kowalczyk, T., Pontious, A., Englund, C., Daza, R. A., Bedogni, F., Hodge, R., et al. Additionally, there are the radial glial cells (RGCs). doi: 10.1111/j.1365-2443.2005.00893.x, Mahairaki, V., Ryu, J., Peters, A., Chang, Q., Li, T., Park, T. S., et al. Forebrain-specific promoter/enhancer D6 derived from the mouse Dach1 gene controls expression in neural stem cells. Astrocyte differentiation medium is stable for 4 weeks when stored in the dark at 28C. With this approach, it is easiest to obtain a homogeneous population of identical NSCs. 17, 330341. Biol. Cell 91, 127138. Neural induction of PSCs may be conducted in 2D conditions (monolayer and coculture) or in 3D conditions (embryoid bodies). Development 135, 24892503. doi: 10.1016/s0092-8674(02)00835-8, Williams, B. P., and Price, J. Neurochem. On reaching a certain size, the spontaneously differentiated organoids gain increased heterogeneity, where there are regions resembling various brain tissues: cerebral cortex, choroid plexus, retina, and meninges. Gilbert, S. F. (2010). Sci. doi: 10.1038/embor.2012.12, Ham, O., Jin, Y. Induced pluripotent stem cell-derived NSCs and their differentiated derivates can have applications in various fields from the investigation and modeling of neurological diseases and drug screening to the treatment of neurodegenerative diseases. Natl. Increased susceptibility to A toxicity in neuronal cultures derived from familial Alzheimers disease (PSEN1-A246E) induced pluripotent stem cells. However, the subsequent development of this area, and the approaches to obtaining NSCs have been repeatedly improved. However, rosettes obtained with the spin EB method exhibited typical features: neuroepithelial cell morphology, and expression of the markers of neural progenitors: Sox1, Sox2, Pax6, Dach1, and NCAM. 22, 152165. If desired, mount using 3 drops of ProLong Gold antifade reagent per slide and seal with the cover slip. Once the solution has dissolved, the tubes cool on ice. Dyn. The issue is that brain organoids are able to generate all types of cells of the neuroectodermal line, including various types of neurons, astrocytes, and oligodendrocytes, but are not able to form endothelial cells. doi: 10.1523/JNEUROSCI.4773-10.2011, Simons, B. D., and Clevers, H. (2011). No. Such neural cultures can be maintained for extended periods (up to 2 years) (Eiraku et al., 2011; Lancaster et al., 2013). Proc. (2003). Neuron 28, 3140. No. After culturing the NSSs on an adhesive substrate with mitogens, the NSCs migrated onto the substrate and formed a monolayer culture that could be differentiated into neurons (Nakayama et al., 2004). For larger volumes, increase the component amounts proportionally: If desired, add 1 mL of Antibiotic-Antimycotic solution per 100 mL of complete medium. Development 139, 39383949. The germinal matrices in the developing dentate gyrus are composed of neuronal progenitors at distinct differentiation stages. Table 2. PHG0314), Gibco Geltrex Reduced Growth Factor Basement Membrane Matrix (Cat. Sox2 is critical for maintaining the self-renewal and pluripotency of ESCs. However, not all the ESCs differentiate into neuronal precursors under the described conditions, a proportion of the cells differentiate into non-neural cells while 1015% of the cells remain in an ESC state (Ying et al., 2003). Since cerebral organoids can be maintained in culture for a very long time, the accumulation of A aggregates resembling plaques can be observed in such a model (Lancaster et al., 2013; Gonzalez et al., 2018). Because of difficulties of the animal modeling of human neurological diseases, in vitro models have an advantage and can be used for pathology investigation and for testing drug treatment approaches. Natl. 6:6500. doi: 10.1038/ncomms7500, Eiraku, M., Takata, N., Ishibashi, H., Kawada, M., Sakakura, E., Okuda, S., et al. doi: 10.1038/nprot.2015.065, Kirkeby, A., Grealish, S., Wolf, D. A., Nelander, J., Wood, J., Lundblad, M., et al. doi: 10.1016/s0896-6273(00)00083-0, Kawasaki, H., Suemori, H., Mizuseki, K., Watanabe, K., Urano, F., Ichinose, H., et al. (2008). (2011). Fibroblast growth factor-2 is also involved in the neural induction of PSCs in vitro (Kunath et al., 2007; Stavridis et al., 2007). An in vivo model of functional and vascularized human brain organoids. Aliquot 2 mL in 15-mL tubes, freeze the tubes on dry ice, and store them at 20C. A rosette-type, self-renewing human ES cell-derived neural stem cell with potential for in vitro instruction and synaptic integration. Dual role of brain factor-1 in regulating growth and patterning of the cerebral hemispheres. Dev. Exposure of hESCs to CHIR99021, SB431542, Compound E and hLIF in a serum-free culture medium for 7 days induced their differentiation into primitive neuroepithelium. (2013) set themselves the task of obtaining NSCs that were able to differentiate not only into the neurons of the forebrain but also into other neural tube and neural crest lineages. Cell 126, 663676. Remove the vessel from the incubator and store it until use. U.S.A. 88, 21992203. However, they differ from RGCs in their lower proliferation rate (Simons and Clevers, 2011). 109, 2841. It is considered that this type of NSC culture has limited differentiation potential, although some reports have shown that the NSCs obtained in this way still have a fairly wide potential (Yan et al., 2013). They also found that the addition of N2 and B27 supplements to serum-free culture media increased the efficiency of neural differentiation. 27, 275280. Cell Stem Cell 3, 519532. Mol. For larger volumes, increase the component amounts proportionally. In 2007, the same approach, but with the application of a selective Rho-associated kinase (ROCK) inhibitor, was applied to hESCs (Watanabe et al., 2007). Approaches using neural differentiation of PSCs in 2D cultures have several limitations. Persistent expression of stabilized beta-catenin delays maturation of radial glial cells into intermediate progenitors. Self-organizing optic-cup morphogenesis in three-dimensional culture. Cell Neurosci. U.S.A. 106, 32253230. doi: 10.1073/pnas.0308600100, Hegarty, S. V., OKeeffe, G. W., and Sullivan, A. M. (2013). Neural induction was achieved by prolonged hiPSC-derived EB cultivation under serum-free conditions (63 days) followed by dissociation and amplification as hiNSCs neurospheres, originating from individual cells (Rosati et al., 2018). In cultures from all three models, neurons expressing MAP2 and III-tubulin were detected, while having specific AD-like phenotypes. 2D induction usually takes less time than 3D induction via EBs, is easier to perform and has a greater efficiency (Yan et al., 2013). Cell 70, 829840. Clinical-scale purification of pluripotent stem cell derivatives for cell-based therapies. The authors showed that low-density ESCs have an autonomous tendency for neural differentiation that decreases with increasing cell density and cellular interactions. Small-molecule inhibitors of bone morphogenic protein and activin/nodal signals promote highly efficient neural induction from human pluripotent stem cells. The most popular models are those based on the differentiation of patient-derived iPSCs into NSCs and their differentiated derivates (Israel et al., 2012; Mehta et al., 2018) or into brain organoids (Gonzalez et al., 2018; Virlogeux et al., 2018; Chlebanowska et al., 2020). Their approach made it possible to obtain neuroepithelial cells from hESCs and hiPSCs with high efficiency and without any EB cultivation stage or cocultures with stromal cells. (2001) obtained floating spheres (neurospheres) of primitive NSCs from single ESCs of mice (mESCs), on chemically defined serum-free medium, feeder layer-free, in low-density culture conditions, in the presence of leukemia inhibitory factor (LIF) and FGF-2. This is why the protocol has been termed Dual-SMAD-inhibition. doi: 10.1371/journal.pone.0017540, Zhang, S.-C., Wernig, M., Duncan, I. D., Brustle, O., and Thomson, J. Radial glial cells are also capable of generating glial precursors, but this occurs mainly in the late stages of embryonic neurogenesis after the main wave of neuron production (Malatesta et al., 2000, 2003). doi: 10.1002/cne.21669, Okita, K., Ichisaka, T., and Yamanaka, S. (2007). Genet. The gradients of various morphogens determine neuroaxis formation. Compound E is a small molecule, an inhibitor of -secretase, which is involved in Notch-signaling (Li et al., 2011). In addition to NSCs, such populations could include undifferentiated iPSCs and their random derivatives plus intermediate differentiation variants of NSCs, as well as neurons and glia cells.
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