Preparation of Neural Stem Cells and Progenitors: Neuronal Production and Grafting Applications

Abstract

Neural stem cells (NSCs) are a valuable tool for the study of neural development and function as well as an important source of cell transplantation strategies for neural disease. NSCs can be used to study how neurons acquire distinct phenotypes and how the interactions between neurons and glial cells in the developing nervous system shape the structure and function of the CNS. NSCs can also be used for cell replacement therapies following CNS injury targeting astrocytes, oligodendrocytes, and neurons. With the availability of patient-derived induced pluripotent stem cells (iPSCs), neurons prepared from NSCs can be used to elucidate the molecular basis of neurological disorders leading to potential treatments. Although NSCs can be derived from different species and many sources, including embryonic stem cells (ESCs), iPSCs, adult CNS, and direct reprogramming of nonneural cells, isolating primary NSCs directly from fetal tissue is still the most common technique for preparation and study of neurons. Regardless of the source of tissue, similar techniques are used to maintain NSCs in culture and to differentiate NSCs toward mature neural lineages. This chapter will describe specific methods for isolating and characterizing multipotent NSCs and neural precursor cells (NPCs) from embryonic rat CNS tissue (mostly spinal cord) and from human ESCs and iPSCs as well as NPCs prepared by reprogramming. NPCs can be separated into neuronal and glial restricted progenitors (NRP and GRP, respectively) and used to reliably produce neurons or glial cells both in vitro and following transplantation into the adult CNS. This chapter will describe in detail the methods required for the isolation, propagation, storage, and differentiation of NSCs and NPCs isolated from rat and mouse spinal cords for subsequent in vitro or in vivo studies as well as new methods associated with ESCs, iPSCs, and reprogramming.

1. Introduction

1.1. Preface

Neural stem cells (NSCs) derived from the central nervous system (CNS) are a valuable tool for studying neural development and function, disease modeling, and for the design of cellular therapies for neurological disorders [1]. For example, NSCs can be used to study how neurons acquire diverse phenotypes and regional specificity, and how interactions among neurons and glia (e.g., neuron–glia or glia–glia) in the developing nervous system shape the structure and function of the CNS. NSCs can be isolated at early stages of development as multipotent neuroepithelial cells (NEPs) or at later stages as neuronal and glial restricted progenitors (NRPs and GRPs, respectively) for mechanistic studies of differentiation or for transplantation experiments [2]. At this stage of development, NSCs can also be subjected to signaling molecules known to be important in developmental patterning, and thus enriched for specific interneuronal phenotypes. Importantly, the therapeutic potential of NSCs in treating neurological disorders through transplantation has been demonstrated in many preclinical studies using animal models, and led to their translation in some clinical trials [3, 4].

Our ability to manipulate NSCs has facilitated studies of neurological disorders to elucidate the molecular basis of the disease, thus modeling “disease in a dish.” This application has become particularly exciting with induced pluripotent technology, whereby the cells used for these studies can be derived from the patient owns cells [5].

Neural stem cells can be derived from different species and various sources, including embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), developing and adult CNS, and direct reprogramming of nonneural cells [6]. Isolating primary NSCs directly from fetal tissue, which we will emphasize in this chapter, is an important technique with a rich history of efficient protocols for obtaining, culturing, expanding and banking the cells [7]. Whether NSCs are derived from fetal tissue or pluripotent sources, such as ESCs and iPSCs, similar techniques are often used to differentiate NSCs toward mature neural lineages and to maintain NSCs in culture [8]. ESC and iPSC cultures produce neurons only after progressing through a sequence of intermediate cell types with concomitant gradual fate restriction, which include the generation of NSCs. For example, iPSCs have been used to prepare specific types of neurons including dopaminergic, cortical, and motor neurons to study neurodegenerative diseases such as Parkinson’s disease, Alzheimer’s disease, and amyotrophic lateral sclerosis, respectively [9]. In this way, pluripotent stem cell research can follow protocols developed for primary NSC culture. This is particularly evident in the derivation of neurons from NSCs, where the diversity and complexity of neuronal phenotypes can be difficult to replicate in vitro. This chapter will describe specific protocols for isolating fetal tissue [10–12] and subsequently neuronal and glial restricted progenitor cells (NRPs/GRPs) prepared from the embryonic rat and mouse spinal cord tissue [3], a representative enrichment protocol for ventral interneurons, including the V2a interneurons [13], and two protocols for obtaining human CNS neurons either from human ESCs/iPSCs or using a direct reprogramming strategy. Several laboratories have previously shown that these cell types can be used to reliably produce neurons in vitro [14–16], with NRPs/GRPs also reliably producing neurons after transplantation into the adult CNS [2, 15, 17, 18].

1.2. Terminology

Stem cell research exists at the junction of developmental biology, embryology, and cell biology. While this diversity leads to exciting discoveries across multiple areas of research, confusion often arises regarding terminology. Several terms will be briefly defined and discussed for this chapter. Stem cells are defined by their phenotypic potency and capacity for self-renewal. Potency is the range of cells that a particular stem cell can produce and is best tested by clonal analysis. Pluripotent cells can form all the cells of the mature organism, including cells from all three germ layers, and theoretically have unlimited capacity for self-renewal. The term embryonic stem cells (ESCs) specifically refers to pluripotent stem cells derived from the inner cell mass of a developing embryo. Similarly, iPSCs prepared by a variety of methods from a variety of cells are also considered to be pluripotent if authenticated [19]. Multipotent cells can produce multiple types of cells but are generally limited to one germ layer. Both potency and capacity for self-renewal of multipotent cells are more limited than pluripotent cells. The term neural stem cells or NSCs commonly refers to multipotent cells capable of generating neurons, oligodendrocytes and astrocytes, but is often used as a general default term for NSCs or progenitors derived from either embryonic, fetal, or adult CNS. Neural progenitor cells are a class of neural precursor cells (NPCs), that have committed to a more restricted lineage profile than NSCs, and therefore have a limited potency and limited capacity for self-renewal. Specific examples of neural progenitor cells present in the developing CNS include neuronal and glial restricted progenitors (NRPs and GRPs, respectively). The term fetal stem cell (or fetal tissue) is often used to describe stem cells derived from developmental tissue beyond the blastocyst stage (i.e., cells more mature than ESCs) without distinction between embryonic and fetal stages of development. This may be a misnomer, especially when applied to rodent systems that have a relatively short fetal stage (embryonic days 17–21 in rats). However, some human neural stem/progenitor cells derived from aborted tissue can be accurately described as fetal stem cells. Embryonic day is a term that is used to describe the age of developing organisms. For the purposes of harvesting stem cells, the embryonic day is defined as the number of days since mating/fertilization (day 0 is within the first 24 h). However, disagreement exists over how fertilization should be assigned a numerical value. Fertilization is alternatively defined as the beginning of embryonic day 0 (E0) or the beginning of the embryonic day 1 (E1). Likewise, fertilization +12 h is alternatively defined as E0.5 or E1.5. For the purpose of this chapter we will continue to use the nomenclature that we have used in our previous studies with fertilization being defined as the beginning of E0. It is especially important to note, however, that many animal vendors differ in their use of these when selling timed-mated pregnant animals.

1.3. Neural Stem Cells and Neural Progenitors of the Developing Spinal Cord

The developing rodent spinal cord has been well described in the literature, both in terms of isolation of NSC and NPC populations for in vitro studies as well as the use of NSCs, NPCs and fetal spinal cord (FSC) tissue for transplantation studies in vivo [11, 20–22]. At E10.5 the caudal neural tube contains neuroepithelial cells (NEPs), a multipotent NSC population capable of generating neurons, oligodendrocytes and astrocytes. Clonal analysis has confirmed that NEPs of the spinal cord are a common progenitor for the cellular phenotypes found in the adult spinal cord [23], including motor neurons and interneurons [24]. NEPs can be identified by immunocytochemistry for Nestin and Sox2. Although NEPs have a robust capacity to generate multiple neuronal and glial phenotypes in vitro, they show poor survival following transplantation into the adult CNS, likely due to a lack of trophic support in the mature CNS microenvironment [2]. Other sources of multipotent NSCs show better survival but will only produce glial progeny in the spinal cord, despite being able to produce neurons in neurogenic regions of the CNS [25]. Thus, NEPs are a useful source of multiple neuronal phenotypes for in vitro studies but must be predifferentiated to a more mature intermediate stage (e.g., NRPs) prior to use in transplantation studies. The protocols for preparations of NEP have been described in detail in the previous edition of this chapter [26].

Later in development, at E13.5/E14 in rats E12.5/E13 in mice, the spinal cord contains both neuronal and glial restricted progenitors (NRPs and GRPs, respectively). At this developmental stage, expression of homeodomain transcription factors has already begun to assign specific phenotypic identities to certain interneurons, and can be identified within the embryonic spinal cord using immunohistochemical techniques (Fig. 1). Records of grafting fetal spinal cord (FSC) tissue to treat the injured adult spinal cord can be found as early as 1940 [27], but it was not until the 1980s that researchers began to focus on exploring the potential of these cells as a viable strategy [28], demonstrating the importance of the precise age of FSC tissue at the time of transplantation. When transplanted at this developmental stage (E13–15), the cells survive, grow to fill the lesion, and synapse with the injured host spinal cord [28–31]. Interestingly, even once grafted into the injured adult spinal cord, the surviving FSC cells have the potential to retain their long-term fate, as characterized by donor cell morphology and host-graft innervation [20, 32, 33]. Coincidentally, this age (E13–15) coincides with the expression of transcription factors that push the developmental stage into progenitor-state, rather than true stem cell phenotype. More specifically, E14 rat donor tissue is comprised of approximately 5–10% of NEPs, 30% GRPs and 60% NRPs [34]. In contrast, transplantation of older fetuses (E16–22) resulted in poor graft survival and no detectable functional improvements (for review see [30, 35–37]).

Fig. 1.

The E13.5 rat embryonic spinal cord is host to a variety of spinal interneurons that can be identified with immunohistochemical markers for transcriptional factors such as (a) Chx10 (marker for V2a interneurons), (b) Engrailed-1 (En1; marker for V2b interneurons), (c) Islet-1 (Isl-1, marker for ventral interneurons and a population of dorsal, dI3, interneurons). (d) A schematic showing the distribution of dorsal and ventral transcriptional factors throughout the developing spinal cord. Scale bar is 500 μm

Consequently, NRPs isolated from E13.5/14 tissue will only differentiate into neurons [16] and GRPs will only differentiate into oligodendrocytes and astrocytes [12, 38], thus NRP/GRP cultures are a source for both neurons and glia (Fig. 2a, b, respectively). NRPs can be derived from ESCs, NEPs or directly from the developing spinal cord and retain the ability to generate multiple neurotransmitter phenotypes both in vitro and in vivo [39]. Like NEPs, NRPs/GRPs express the intermediate filament Nestin (Fig. 3b) but NRPs can be specifically identified by cell surface ENCAM expression (Fig. 3c) and GRPs can be specifically identified by cell surface A2B5 expression (Fig. 3d). When transplanted into the intact CNS, NRPs will generate neurons [15] (Fig. 4) with appropriate neurotransmitter identities (Fig. 5), per the local microenvironment [39]. However, when purified NRPs are transplanted into the injured CNS, their survival and differentiation is poor due to the toxicity of the injured spinal cord which lacks the appropriate microenvironment to support NRP development [14]. We have previously demonstrated that combined transplants of NRPs/GRPs into the injured spinal cord produce astrocytes, oligodendrocytes, and neurons, indicating that GRPs are capable of producing a microenvironment that supports NRP survival and differentiation [40]; however, the survival of NRPs/GRPs is compromised when transplanted into a severe injury [17]. NRPs develop into both excitatory and inhibitory neurons in the injured spinal cord when transplanted with GRPs. Neurons derived from NRPs have been shown to form synapses in vitro [16] and in vivo [11, 21], demonstrating that NRPs produce neurons with characteristics of mature, functional neurons.

Fig. 2.

NRP/GRP grown on PLL/Laminin substrate produce high density βIII tubulin⁺ neurons (a) and GFAP⁺ astrocytes (b) when grown in NRP basal media supplemented with 0.5% High concentration Matrigel (BD Biosciences, San Jose, CA). Scale bar is 50 μm

Fig. 3.

Neuroepithelial cells (NEP) grown on fibronectin substrates in the presence of bFGF and CEE express the intermediate filament, nestin (a, red), a neural stem cell marker, throughout the cell body. NRPs/GRPs grown on poly-L-lysine and laminin substrates in the presence of bFGF and NT-3 also express nestin (red, b). However, NRPs also express ENCAM (c) and GRPs also express A₂B₅ (d). ENCAM and A₂B₅ can be used to specifically to identify NRPs and GRPs, respectively, from primary E13.5 spinal cord cultures. Scale bars are 50 μm

Fig. 4.

NRP/GRP derived from AP+ rat embryos survive in the injured cervical spinal cord and produce neurons after differentiation. Immunohistochemistry demonstrates that NRP/GRP express the AP transgene (a, c) and produce NeuN+ neurons (b, c) 4 weeks after grafting. Scale bar is 50 μm

Fig. 5.

NRP/GRP derived from AP+ rat embryos were grafted into the injured cervical spinal cord of adult rats. NRP/GRP survive, fill the lesion and continue to express the AP transgene 6 weeks after grafting in the injured spinal cord (a, d). The NRPs differentiate and express markers for both glutamatergic (glutamate, b) and GABAergic (GAD65/67, e) neurons. Overlay image of (a) and (b) is shown in (c), and overlay image of (d) and (e) is shown in (f). Scale bar is 50 μm

1.4. Preparation of Fetal Spinal Cord (FSC) Tissue from E13.5–14 Rat

Over the past three decades, FSC tissue have been used in transplantation studies designed to repair the injured spinal cord [36] in different stages: acute, subacute, and chronic. Transplanted FSC tissue survives within the lesion area, provides trophic and tropic stimuli to attract host axonal regeneration, forms a trophic bridge for axon growth through the lesion, provides a source of neuronal and glial populations to replace damaged cells, and promotes functional recovery by creating the appropriate relays, reconnecting the injured spinal cord [17, 30, 31, 41, 42]. There are several published methods for preparing FSC tissue for transplantation into hemisection, transection, and contusive spinal cord injuries (SCIs). Here we describe methods for preparing either rat E13.5–14 FSC segments (commonly used for hemi- and transection injuries) or mechanically dissociated FSC tissue (commonly used for contusion injuries; Fig. 6a–c). Although some published studies combine FSC tissue with either matrix gel or growth factors, the protocols described here omit these [12, 17, 22]. Protocols that include an enzymatic dissociation of FSC tissue prior to transplantation with a cocktail containing 4–10 growth factors are described elsewhere [43, 44].

Fig. 6.

Summary schematic of preparation of neural progenitors for transplantation. Spinal cords of E13.5–14 rat or E12.5–13 mouse embryos (a, cross section seen in b and b′) can be dissected out and prepared as fetal spinal cord tissue (FSC, c) for transplantation (d), or can be cultured to produce Neuronal and Glial Restricted Progenitors (NRPs/GRPs; e). These cells can then be frozen (f) and thawed (g) prior to transplantation (d), or cultured from transgenic animals (h) such as the example from a ChxGFP mouse (h′ phase image and h′′ GFP image) for subsequent transplantation or in vitro modeling. Scale bar is 500 μm in b′ and 100 μm in h′′

2. Materials

2.1. Dissection of FSC Tissue

All materials must be sterile.

1. Anesthetic agent.

2. 70% ethanol

3. Surgical scissors.

4. #4, or #5 fine forceps

5. Microscissors.

6. 45° angled forceps

7. DMEM/F12, ice cold.

8. E13.5-E14.0 timed-pregnant rat.

9. Collagenase I/dispase II solution.

10. 24-well culture plate

11. 100 mm × 20 mm cell culture dishes.

2.2. Preparation of FSC Tissue for Transplantation

1. 24-well plate with dissected spinal cord in DMEM/F12 on ice

2. No. 15 scalpel blade.

3. Microscissors.

4. 1.5 Eppendorf tubes

5. P1000 pipette.

6. 1000 μl pipette tips

7. P200 pipette.

8. 200 μl pipette tips

9. 23G, 25.5G, 27G needles

10. Luer lock syringes.

11. Tabletop centrifuge.

2.3. Preparation of Neuronal Restricted Progenitors and Glial Restricted Progenitors from E13.5–14 Rat or from E12.5–13 Mouse Spinal Cord

1. Poly-L-lysine (PLL).

2. Tissue culture (TC) H₂O.

3. 0.22 μm syringe tip filter

4. 60 ml syringe

5. Natural mouse laminin (LAM).

6. Sterile phosphate buffered saline (PBS).

7. 75 cm² and/or 25 cm² flasks.

2.3.1. Preparation of Poly-L-Lysine (PLL) and Laminin (LAM)-Coated Dishes for NRP/GRP Culture

Stock Solutions

1. PLL 10× (150 μg/ml) in TC H₂O (store at (−20 ° C).

2. LAM stocks (−80 ° C).

2.3.2. NRP/GRP Medium

1. DMEM/F12.

2. BSA.

3. B27 (50 × stock solution).

4. Pen/Strep (100× stock solution).

5. N2 supplement (100×).

6. NRP/GRP complete medium.

7. NRP basal medium.

8. bFGF,

9. NT-3.

2.3.3. NRP/GRP Dissection

All materials must be sterile.

1. Anesthetic agent.

2. 70% ethanol

3. 100 mm × 20 mm cell culture dishes

4. DMEM/F12, ice cold.

5. E13.5-E14.0 timed-pregnant rat or E12.5–13 time-pregnant mouse.

6. Surgical scissors.

7. Microscissors.

8. 45° angled forceps

9. #4 or #5 fine forceps

10. Collagenase I/dispase II solution.

11. 24-well culture plate

12. 50 ml conical tube.

2.3.4. NRP/GRP Dissociation

1. 0.05% trypsin–EDTA

2. Soybean trypsin inhibitor.

3. NRP/GRP Basal Medium.

2.3.5. NRP/GRP Culture

1. NRP/GRP complete medium.

2. PLL and Laminin T-75 tissue culture flask, or equivalent.

2.3.6. Differentiation of NRPs/GRPs Toward Neuronal Lineage

1. Basal Medium.

2. Retinoic acid (1 μM).

3. NT-3 (10 ng/ml).

4. 0.5% High concentration Matrigel.

2.3.7. Differentiation of NRPs/GRPs Toward Ventral Lineage

1. Basal Medium.

2. NT-3 (10 ng/ml) to promote neuronal survival.

3. Retinoic acid (10 nM).

4. Purmorphamine (1 μM).

5. N-[N-(3,5-difluorophenacetyl-L-alanyl)]-(S)-phenylglycine-t-butyl-ester (DAPT, 5 μM).

2.3.8. Freezing NRPs/GRPs

1. NRP/GRP Freezing Media (see Table 2).

2. Unplugged glass pipettes for media aspiration.

3. Warmed HBSS.

4. 0.05% trypsin

5. Soybean trypsin inhibitor.

6. Complete Media.

7. Cryovials.

Table 2.

NRP/GRP media

	Concentration
NRP/GRP basal media
DMEM/F12	96%
BSA	1 mg/ml
B-27 (50× stock solution)	2%
Pen/Strep (100× stock solution)	1%
***Pass media through 0.22 μm sterile filter***
N2 supplement (100×)	1%
NRP/GRP complete media
NRP basal media	100%
bFGF	20 ng/ml
NT-3	10 ng/ml

2.3.9. Thawing NRPs/GRPs

1. NRP/GRP Basal Media.

2. NRP/GRP Complete Media.

3. Ice bucket with ice.

4. Plugged glass pipettes and bulb.

5. Unplugged glass pipettes for media aspiration.

6. PLL/LM-coated T75 flasks.

2.4. Characterizing NRP/GRP Cultures In Vitro with Immunocytochemistry

2.4.1. Staining Live Cells

1. Warmed (37 °C) HBSS.

2. Warmed NRP/GRP Complete Media.

3. Phosphate buffered saline (PBS).

4. 4% paraformaldehyde (PFA)

5. 4′,6-diamidino-2-phenylindole (DAPI)

6. Mounting media.

7. Coverslips or SuperFrost slides (if cells are grown on glass coverslips).

2.4.2. Staining Fixed Cells

1. Phosphate buffered saline (PBS).

2. Unplugged glass pipettes for media aspiration.

3. Triton X-100.

4. Dry milk or Blocking Serum.

5. 4′,6-diamidino-2-phenylindole (DAPI)

6. Mounting media.

7. Coverslips or SuperFrost slides.

8. Primary and secondary antibodies (see Table 1).

   Note: A₂B₅, ENCAM, and O4 are IgM antibodies and require specific secondary antibodies.

Table 1.

A partial list of useful antibodies and dilutions

Antigen	Host species	Recommended dilution	Vendor	Product ID
A2B5	Mouse IgM	1:2	DSHB
BIII tubulin	Mouse IgG2a	1:500	Covance	MMS-435P
BIII tubulin	Rabbit	1:1000	Covance	Prb-435P
Nestin	Mouse IgG1	1:1000	BD Pharmingen	556309
GFAP	Mouse IgG1	1:1000	Chemicon	MAB3402
GFAP	Rabbit	1:2000	Chemicon	AB5804
O4	Mouse IgM	1:4	Dr. J Grinspan

Developmental Studies Hybridoma Bank (DSHB)

2.5. Preparation of Human Neurons from Human Embryonic Stem Cells (hESCs), Induced Pluripotent Stem Cells (hiPSCs) and Direct Reprogramming Strategies

2.5.1. Preparation of Dishes and Media for Human Embryonic Stem Cells (hESCs) and Human Induced Pluripotent Cells (hiPSCs) for 2D and 3D Culture

1. Embryonic Stem Cell qualified Matrigel (ES-Matrigel).

2. DMEM/F12 HEPES.

3. Neurobasal medium.

4. mTeSR1 basal medium,

5. STEMdiff Neural Induction Media.

6. Poly-L-ornithine (15 μg/ml).

7. Sterile D-phosphate buffered saline (D-PBS).

8. Tissue culture (TC) ddH₂O.

9. 6-well TC plates.

2.5.2. Thawing hESCs/hiPSCs

1. Matrigel-coated 6-well TC plates.

2. STEMdiff Neural Induction Media.

2.5.3. Monolayer (2D) Differentiation Protocol for hESCs and Human Induced Pluripotent Cells hiPSCs

1. ES-Matrigel-coated 6-well plates.

2. Poly-L-ornithine–coated 6-well plates.

3. Fibronectin- and laminin-coated plates.

4. Accutase.

5. STEMdiff Neural Induction Media.

6. DMEM/F12 with HEPES.

7. Neurobasal Media.

8. SB431542.

9. LDN193189.

10. Y-27632.

11. bFGF,

12. L-ascorbic acid.

13. N2 Supplement.

14. B27 without vitamin A.

15. DAPT.

16. Cyclic AMP (cAMP).

17. Trypan Blue.

2.5.4. Embryoid Body (3D) Differentiation Protocol for hESCs and Human Induced Pluripotent Cells hiPSCs

1. AggreWell plate.

2. ES-Matrigel–coated 6-well plates.

3. Non-ES-Matrigel–coated 6-well plates.

4. PLO-coated 24-well plate with glass coverslips.

5. Anti-Adherence Rinsing Solution.

6. D-PBS.

7. mTeSR1 Media,

8. Dispase.

9. STEMdiff Induction Media.

10. SB431542.

11. LDN193189.

12. Y-27632.

13. Accutase.

14. Trypan Blue.

15. 70 μm cell strainer.

2.5.5. Preparation of Dishes and Media for Direct Differentiation of Human Somatic Cells (Fibroblasts/Pericytes) into Cortical Glutamatergic Neurons

1. DMEM.

2. Fetal bovine serum (FBS).

3. D-PBS without calcium or magnesium.

4. Laminin.

5. Poly-L-lysine.

6. Gelatin.

7. Tissue culture (TC) ddH₂O.

8. 24-well plate with pretreated plastic or glass coverslips.

2.5.6. Thawing Fibroblasts/Pericytes

1. Gelatin-coated T25 or T75 flasks.

2. DMEM +10% FBS.

2.5.7. Direct Reprogramming of Human Skin Fibroblasts/Pericytes into Cortical Glutamatergic Neurons

1. DMEM.

2. Fetal bovine serum.

3. DMEM/F-12.

4. N2 Supplement.

5. Human Basic FGF.

6. Brain Derived Neurotrophic Factor.

7. Forskolin.

8. Polybrene (hexadimethrine bromide).

9. L-ascorbic acid.

10. Lentiviral particle with desired promoter (Ascl1, Brn2, Myt11, Oligo2, Zic1).

11. 24-well PLL/laminin-coated plate with pretreated plastic/glass coverslips.

3. Methods

3.1. Dissection of FSC Tissu

1. The embryonic dissections are performed in a dissection hood (with laminar flow) and with a dissection microscope.

2. Anesthetize the dam with an appropriate compound, such as Euthasol.

3. When the dam is anesthetized, surgically prepare the skin of the abdomen with 70% ethanol for 30 s.

4. Cut the abdomen with surgical scissors and remove the entire uterus, including both uterine horns.

5. Palpate the uterine horns and count the number of embryos present to best prepare for the dissection (i.e., fill an appropriate number of wells in the 24-well culture plate, prepare Notes to record the crown-to-rump length of each embryo and morphological developmental features of each embryo).

6. Place the entire uterus into a sterile 100 mm × 20 mm cell culture dish filled with DMEM/F12 and place on ice.

7. Separate (cut with surgical scissors) each embryonic sac(one-by-one) from the uterine horn.

8. Rinse each sac in 70% ethanol once and twice in DMEM/F12 and place in a sterile 100 mm × 20 mm cell culture dish.

9. Carefully remove the embryo from the embryonic sac (eitheruse microscissors to cut along the sac, or use #4 forceps to gently rip the sac open) to release the embryo from the sac onto the sterile dish.

10. Transfer the embryo into the lid of a sterile 100 mm × 20 mm cell culture dish filled with DMEM/F12 (see Note 1).

11. Measure the crown-to-rump distance to determine the embryonic age. An E13.5–14 rat embryo should be between 8.5 and 10 mm long, and should have precartilaginous formations within handplates (Altman, P.L. and Katz, D.D., 1962).

12. Using a pair of microscissors (or 45° angled forceps), remove the head at the cisterna magna (see Notes 2 and 3).

13. Place the embryo with the ventral surface down.

14. Firmly hold the embryo down with a pair of sharp #4 or #5 forceps. Place the forceps in an area of the embryo that is not needed as it will be damaged.

15. Remove the skin over the neural tube (i.e., from the dorsal surface) using a pair of microscissors all along the neural axis (see Note 4).

16. After the skin covering the dorsal side of the spinal cord is removed, dissect and clean the spinal cord from the embryo by repeatedly inserting and opening #4 or #5 forceps along the blood vessels running between the spinal cord and meninges, all along the neural axis.

17. If done properly, the spinal cord (and some remaining meninges) will easily separate from the rest of the body, only to be in contact at the lumbar-most part of the cord.

18. After dissecting the cord from the embryo, secure the cord down to the dish by placing forceps (45° angled forceps work well) on the rostral-most part of the cord and carefully strip the meninges from the cord. If done properly, the meninges come off in a single “zipper.”

19. Place cleaned sections of the spinal cord into a well of the24-well plate with ice cold DMEM/F12.

20. The lumbar (less mature) portion of the spinal cord mayrequire the use of collagenase I/dispase II solution to remove the remaining meninges. If so, place the lumbar cord in collagenase I/dispase II for exactly 8 min (see Note 5).

21. Remove cords from collagenase I/dispase II and place into thedissection dish with DMEM/F12.

22. Finish removing the meninges and place the cleaned spinal cord into the same well of the 24-well plate already containing clean spinal cord from the embryo. Remember to keep the 24-well plate on ice at all times (see Note 6).

3.2. Preparation of FSC Tissue for Transplantation

There are two main ways of preparing FSC tissue for transplantation: either as “segments” of tissue blocks or as “dissociated” FSC. For transplanting “segments” of tissue blocks:

1. Cut the FSC tissue into 5–7 small segments (about 1 mm long) using a scalpel blade (No. 15 works well) or microscissors.

2. Keep the tissue blocks in DMEM/F12 in a 24-well plate on ice during the transplantation period.

3. Graft 5–7 embryonic spinal cord tissue blocks into each injured recipient (especially in an acute spinal cord injury setting).

   For transplantation of “dissociated” FSC tissue:

4. Cut the FSC tissue into 5–7 small segments (about 1 mm long) using a scalpel blade (No 15 works well) or microscissors.

5. Using a P1000 pipette set to 800 μl, transfer FSC tissue blocks (approximately 1 mm long) from the 24-well plate into a 1.5 ml tube.

6. Let the segments settle, or use a table centrifuge to spin the tissue down for 5–10 s.

7. Use a P1000 to remove excess media from the centrifuge tube, being careful not to aspirate tissue.

8. Use a P200 set to 180 μl to mechanically dissociate (triturate) the tissue until it looks dissociated by eye.

9. Triturate the tissue with needles of sequentially smaller gauges(23 g, 25.5 g, and 27 g) if necessary to ensure efficient loading into the transplantation syringe (and preventing needle blockage). Take care not to over triturate the cells as they will die.

10. Spin the dissociated FSC tissue down using a table centrifugefor 10–15 s.

11. Carefully remove the supernatant using a P200 pipette, takingcare not to aspirate the cells. Approximately 5 μl of cell suspension is recovered after removal of the supernatant.

12. Load the injection needle and syringe with dissociated FSCtissue and transplant into the injured spinal cord (Fig. 6d). We recommend using a 10-μl pipette tip or a 10 μl Hamilton syringe with a beveled 30 gauge needle (see Note 7).

3.3. Preparation of Neuronal Restricted Progenitors and Glial Restricted Progenitors from E13.5–14 Rat or from E12.5–13 Mouse Spinal Cord

NRPs/GRPs are prepared as a mixed culture of cells that are collectively capable of producing neurons, astrocytes and oligodendrocytes but NRPs will only produce neurons while GRPs will only produce astrocytes and oligodendrocytes. Therefore, NRPs/GRPs are not a true NSC population but represent a necessary intermediate step between multipotent NSCs and mature neural phenotypes. NRPs/GRPs are grown in an adherent culture on dishes coated with poly-L-lysine and laminin. NRPs can be identified by expression of embryonic neural cell adhesion molecule (E-NCAM) and GRPs by expression of A2B5, which can also be used for sorting the different populations. Preparation of NRPs/GRPs from either E13.5–14 rat or E12.5–13 mouse embryos follows a similar procedure, and requires a physical dissection followed by chemical and mechanical dissociation and culturing (Fig. 6e–h). The primary differences between these two dissections are the crown–rump length of the animals: rat embryos at this developmental stage are between 8.5 and 10 mm long, whereas the mouse embryos are between 7.7 and 8.6 mm. One of the biggest advantages of learning how to isolate and culture NRPs and GRPs from embryonic mice, is the increasing availability of various transgenic models that fluorescently tag specific interneuronal populations (Fig. 6h′, h′′ for example). Isolation from transgenic animals may provide a source of enriched populations of interneurons, especially if combined with Fluorescence Activated Cell Sorting (FACS), for transplantation or disease/circuit in vitro models.

The full dissection should take 1–2 h and we recommend having 2 well-trained technicians prepared to conduct the dissection, as rodent litters may vary between 2 and 15 embryos. Collected tissues should be maintained throughout the dissection in ice-cold, sterile media as outlined.

3.3.1. Preparation of Poly-L-Lysine (PLL) and Laminin (LAM)-Coated Dishes for NRP/GRP Culture

Preparation of PLL/LAM-coated dishes is a two-step process requiring 2 days to complete. The coating should be carried out at 4 °C on a rocker. Coated dishes are stable for 4 weeks after coating.

1. PLL (Day 1).

   1. Dilute stock solution of PLL with TC H₂O to 15 μg/ml.
   2. Filter PLL mixture with 0.22 μm syringe filter and 60 ml syringe.
   3. Coat the bottom of 75cm² flask with 10–15 ml of 15 μg/ml PLL solution.
   4. Place overnight on rocker at 4 °C.
2. LAM (Day 2):

   1. Prepare 15 μg/ml solution of mouse LAM in PBS. Do Not Filter.
   2. Aspirate PLL and wash 2× with PBS.
   3. Add 10–15 ml of 15 μg/ml LAM in PBS to 75cm² flask and coat on rocker overnight at 4 °C.
   4. On the third day, aspirate the LAM solution and rinse the flask once with PBS.
   5. Add 15 ml of HBSS and store at 4 °C for up to 1 month (see Note 8).

3.3.2. NRP/GRP Medium

NRP/GRP media should be prepared under sterile conditions in a tissue culture hood to prevent contamination. The materials required are listed in Table 2 and should be prepared in DMEM/F12 for growth of cells in 5–10% CO₂ environment. NRP/GRP Basal Media can be used for manipulating cells during the dissection and other procedures, but NRP/GRP Complete Media (NRP/GRP Basal + bFGF and NT-3, see Table 2) should be used for culturing cells.

3.3.3. NRP/GRP Dissection

NRPs/GRPs are dissected from E13.5–14 rat or E12.5–13 mouse spinal cords. The dissection protocol is similar to what is used for preparing FSC tissue for transplantation. All materials and surgical instruments are sterile and the dissection of the embryos are performed with a dissection microscope in a dissection hood with laminar flow to prevent contamination.

1. The embryonic dissections are performed in a dissection hood(with laminar air flow) with the aid of a dissection microscope.

2. Anesthetize the dam with an appropriate compound, such as Euthasol.

3. When the dam is anesthetized, surgically prepare the skin of the abdomen with 70% ethanol for 10 s.

4. Cut the abdomen with surgical scissors and remove the entire uterus, including both uterine horns.

5. Palpate the uterine horns and count the number of embryos present to best prepare of the dissection (i.e., fill an appropriate number of wells in the 24-well culture plate, prepare Notes to record the crown-to-rump length of each embryo and morphological developmental features of each embryo).

6. Place the entire uterus into a sterile 100 mm × 20 mm cell culture dish filled with DMEM/F12 and place on ice (see Note 9).

7. Separate (cut with surgical scissors) each embryonic sac(one-by-one) from the uterine horn. Rinse each sac in 70% ethanol once and twice in DMEM/F12 and place into a sterile 100 mm × 20 mm cell culture dish.

8. Carefully remove the embryo from the embryonic sac (eitheruse microscissors to cut along the sac, or use #4 forceps to gently rip the sac open) to release the embryo from the sac onto the sterile dish.

9. Transfer the embryo into the lid of a sterile 100 mm × 20 mm cell culture dish filled with DMEM/F12 (see Note 1).

10. Measure the crown to rump distance to determine the embryonic age. An E13.5–14 rat embryo should be between 8.5 and 10 mm long, whereas a developmentally equivalent mouse embryo (E12.5–13) should be between 7.7 mm and 8.6 mm long, and should have precartilaginous formations within handplates [45].

11. Using a pair of microscissors (or 45° angled forceps), remove the head at the cisterna magna (see Notes 2 and 3).

12. Place the embryo with the ventral surface down.

13. Firmly hold the embryo down with a pair of sharp #4 or #5 forceps. Place the forceps in an area of the embryo that is not needed as it will be damaged.

14. Remove the skin over the neural tube (i.e., from the dorsal surface) using a pair of microscissors all along the neural axis (see Note 4).

15. After the skin covering the dorsal side of the spinal cord is removed, dissect and clean the spinal cord from the embryo by repeatedly inserting and opening #4 or #5 forceps along the blood vessels running between the spinal cord and meninges, all along the neural axis.

16. If done properly, the spinal cord (and some remaining meninges) will easily separate from the rest of the body, only to be in contact at the lumbar-most part of the cord.

17. After dissecting the cord from embryo, secure the cord downto the dish by placing forceps (45° angled forceps work well) on the rostral-most part of the cord and carefully strip the meninges from the cord. If done properly, the meninges come off in a single “zipper.”

18. Place any cleaned sections of the spinal cord into a well of the 24-well plate with ice cold DMEM/F12.

19. The lumbar (less mature) portion of the spinal cord mayrequire the use of collagenase I/dispase II solution to remove the remaining meninges. If so, place the lumbar cord in collagenase I/dispase II for exactly 8 min (see Note 5).

20. Remove the cord from collagenase I/dispase II and place back into the dissection dish with DMEM/F12.

21. Finish removing the meninges and place the cleaned spinal cord into the same well of the 24-well plate already containing clean spinal cord from the embryo. Remember to keep the 24-well plate on ice at all times.

22. After completing all of the spinal cord dissections, use a P1000 pipette set to 800 μl to transfer up to three spinal cords into a 50 ml conical tube with ice cold DMEM/F12.

3.3.4. NRP/GRP Dissociation

1. 1. After transferring up to three spinal cords per 50 ml conical tube, spin the spinal cords down 150–300 × g for 5 min.

2. Decant the supernatant from the 50 ml conical tube containing the cords.

3. Add 1 ml of trypsin-EDTA and incubate for 20 min at 37 °C.

4. Use a sterile cotton-plugged 1000 μl pipette tip, set at 800 μl, to gently triturate cells until chunks of tissue look dissociated by eye.

5. Add equal volume (1 ml) of soybean trypsin inhibitor to the conical vial to quench the trypsin, and gently triturate with 10 ml pipette.

6. Pass the cell suspension through a 40 μm filter into a fresh 50 ml tube.

7. Rinse the filter with another 10 ml NRP Basal Media.

8. Centrifuge the cell suspension 150–300 × g for 5 min to get rid of the trypsin.

9. Carefully decant the supernatant and resuspend in 1 ml of Basal Media. Dilute 10 μl of cell suspension 1:1 in trypan blue and count 10 μl/side with a hemocytometer (see Notes10 and 11).

3.3.5. NRP/GRP Culture

1. Resuspend the pellet with Complete Media (NRP/GRP Basal Medium, supplemented with 30 ng/ml bFGF and 20 ng/ml NT3) in coated flask and place into incubator (37 °C and 5% CO₂).

2. Recommended density.

   1. T-75 flask: 3–5 × 10⁶ cells.
   2. T-25 flask: 5 × 10⁵ cells.
   3. 6-well plate: 20,000 per mm, or 4 × 10⁵ cells/well
   4. 8-well LabTek slide: 2 × 10⁴ cells/well
   5. 24-well plate with PLL/LAM-coated glass coverslips.

3. Feed the cells every other day with fresh NRP/GRP Complete Media.

3.3.6. Differentiation of NRPs/GRPs Toward Neuronal Lineage

Some researchers may want to differentiate NRPs/GRPs toward a neuronal lineage or enrich NRPs/GRPs with either increased populations of NRPs or GRPs. There are many protocols available apart from the one we mention here and ultimately, we recommend the user to decide what works best for specific experiments.

1. Begin with NRPs/GRPs on poly-L-lysine and laminin substrate.

2. Remove the Complete Media and replace with Basal Medium that includes retinoic acid (1 μM) to promote neuronal differentiation and NT-3 (10 ng/ml) to promote neuronal survival.

   OR

1. Begin with NRPs/GRPs on poly-L-lysine and laminin substrate.

2. Remove the Complete Media and replace with Basal Medium that includes 0.5% High concentration Matrigel (Fig. 3).

3.3.7. Differentiation of NRPs/GRPs Toward Ventral Lineage

1. Begin with NRPs/GRPs on poly-L-lysine and laminin substrate.

2. Remove the Complete Media and replace with Basal Medium that contains survival-promoting and ventral signaling molecules, including NT-3, retinoic acid, purmorphamine, and DAPT (see Notes 12 and 13).

3.3.8. Freezing NRPs/GRPs

1. Check the cells to make sure they look healthy and are about 80% confluent.

2. Carefully remove the media from T-75 flask.

3. Rinse 2× with warmed HBSS to remove any dead or floating cells.

4. Add 3.5–4 ml of 0.05% trypsin-EDTA and incubate at 37 °C for 3–5 min until cells are dislodged.

5. Once the cells have lifted, quench the trypsin by adding an equal amount of soybean trypsin inhibitor.

6. Pipet gently up and down to break up clumps of cells.

7. Collect the cells into a 50 ml conical tube.

8. Rinse the flask with 5–7 ml of Complete Media and collect remaining cells into the same 50 ml conical tube.

9. Centrifuge the cells at 150–200 × g for 8–9 min.

10. While waiting, prepare the freezing medium and keep on ice.

11. Carefully decant the supernatant into a fresh 50 ml tube.

12. Gently resuspend the pellet in 2 ml (or more depending on cell yield and desired freezing concentration) of freezing media (see Table 3). One confluent T-75 flask usually yields 3 ml suspension.

13. Count the cells by diluting 10 μl of the cell suspension with 10 μl trypan blue and loading into a hemocytometer.

14. Aliquot 1 ml of cell suspension into each 2 ml cryovial (as perdesired density).

15. Place the cells into −80 °C overnight.

16. Transfer to liquid nitrogen the following day.

Table 3.

NRP/GRP freezing medium

NRP/GRP freezing media	Concentration
NRP/GRP basal	80%
DMSO	10%
CEE	10%
NT-3	20 ng/ml
bFGF	30 ng/ml

3.3.9. Thawing NRPs/GRPs

1. Prior to thawing cells warm (37 °C) 10 ml of NRP basal media and 15 ml of NRP complete media.

2. Take cryovial from liquid nitrogen and bury it in bucket of wet ice.

3. Rapidly thaw aliquot in 37 °C water bath until cells just begin to thaw.

4. Add 1 ml warmed media drop by drop to cryovial.

5. Add the 2 ml suspension drop by drop to 10 ml warmed Basal Media.

6. Rinse the cryovial once with 1 ml warmed Complete Media and collect in the same 15 ml conical tube as the cells.

7. Spin the 15 ml conical tube at 150–300 × g for 5 min.

8. Carefully decant/aspirate the supernatant without disturbing the pellet.

9. Resuspend cells in 15 ml NRP/GRP complete media and platein a PLL/LM coated T-75 tissue culture flask.

3.4. Characterizing NRP/GRP Cultures In Vitro with Immunocytochemistry

NRPs/GRPs can be easily identified in culture with immunocytochemistry. NRPs express Nestin and ENCAM and GRPs express Nestin and A2B5. ENCAM and A2B5 are cell surface molecules and should be stained using live cell staining. The other markers, as well as markers for mature neuronal phenotypes (such as BIII tubulin), subpopulations of interneurons (Chx10, En1, Isl1, etc.), markers for differentiated glial cells (such as GFAP and RIP) can be performed on fixed cells. It is important to note that although here we describe characterization of cells using immunocytochemistry, molecular genetics (such as real-time polymerase chain reaction (qPCR), RNA sequencing (RNA-Seq)), and electrophysiological techniques (voltage and patch clamp cellular recording), are also commonly used to characterize cellular phenotypes, especially of mature populations.

3.4.1. Staining Live Cells

1. Begin with NRPs/GRPs cultured on PLL/LM coated glass coverslips (see 5.5 NPR/GRP culture) and carefully decant or aspirate the media.

2. Wash the cells with 37 °C HBSS 2×.

3. Add 37 °C Complete Media with primary antibody (see Table1).

4. Incubate at 37 °C and 5% CO₂ for 30 min.

5. Carefully aspirate the media (or use a Kimwipes^® to wick away the media).

6. Wash the cells with warm HBSS 2.×

7. Add warmed Complete Media with secondary antibody (1:400).

8. Incubate at 37 C and 5% CO₂ for 30 min (make sure this is done in the dark for fluorescent secondary antibodies).

9. Wash the cells with 37 C HBSS 2×.

10. Carefully add 4% PFA for 10 min for fix the cells onto the coverslip.

11. Wash the cells 3× with PBS.

    1. Option 1: counterstain with DAPI (1:1000), coat with aqueous coverslipping media and coverslip.
    2. Option 2: Store in PBS at 4 °C for additional staining at a later time.
    3. Option 3: perform another stain as described in the following section.

3.4.2. Staining Fixed Cells

1. After fixation with 4% PFA, wash with PBS for 5 min 3×.

2. Block for 30 min in 5% Dry Milk or 5% Normal Serum at room temperature.

3. Treat with 0.2% Triton in PBS if needed (especially if staining for nuclear markers).

4. Add primary antibody in PBS with 2% Milk or Serum.

5. Incubate for 30 min at room temperature.

6. Wash the primaries off with PBS for 5 min 3×.

7. Add secondary antibody (1:400; see Table 1) in PBS with 2% Milk or Serum.

8. Incubate for 30 min at room temperature.

9. Wash the secondary antibodies off with PBS for 5 min 3.

10. Counterstain with DAPI (1:1000; see Table 1), coat with aqueous coverslipping media and add coverslip.

3.5. Preparation of Human Neurons from Human Embryonic Stem Cells (hESCs), Induced Pluripotent Stem Cells (hiPSCs) and Direct Reprogramming Strategies

Although studies that utilize CNS cells from nonhuman species are crucial for discoveries that drive scientific progress in basic science and preclinical settings, the best cell type to study human disease are human cells. However, obtaining neurons directly from human CNS for disease modeling and tissue transplantation remains a challenge. Advances in differentiation protocols for pluripotent stem cells, such as human embryonic stem cells (hESCs; Fig. 7a, b) and induced pluripotent stem cells (hiPSCs), or direct reprogramming strategies from human somatic cells, such as human skin fibroblasts and human pericytes have greatly propelled the availability of commercialized human CNS cell lines (Fig. 7c, d). It is important to note that differentiation protocols for both approaches are usually modified by different research groups to suit their experimental needs. On average, however, generation of neurons from ESC/iPSC usually results in identifiable, mature neurons, but takes a relatively long time when compared to direct reprogramming protocols, which often results in less mature neurons but on a shorter timescale. Both approaches are described in the following section and example images can be found in Fig. 7.

Fig. 7.

Example phase images of 2D (a) and 3D (b) human induced pluripotent stem cell cultures (hiPSCs) derived from human fibroblasts. Embryoid bodies (EBs) such as the one shown in (b) can be cultured up to 600–800 μm in diameter. Directly reprogrammed neurons from astrocytes can be identified with neuronal markers such as Tau1 (c, red), SMI312 (d, red), and NeuN (d, green). Scale bars are 100 μm

3.5.1. Preparation of Dishes and Media for Human Embryonic Stem Cells (hESCs) and Human Induced Pluripotent Cells (hiPSCs) for 2D and 3D Culture

1. Non-ES (used for neural rosette differentiation) and ES-Matrigel coated wells (Used for thawing and Day 0 to initially plate hESCs/hiPSCs after reaching confluency).

   1. Dilute ES (Non-ES)-Matrigel 1:50 in DMEM/F12 with HEPES.
   2. Pipet about 3 ml of ES (Non-ES)-Matrigel into each well (enough to cover the bottom of the well).
   3. Incubate at 37 °C for 1–2 h.
   4. Aspirate the coating media before plating the cells.
2. Poly-L-ornithine (PLO)-coated wells (Used at Days 8–11 for2D culture and Day 19 for 3D culture).

   1. Dilute PLO in D-PBS (final concentration 15 μg/ml).
   2. Pipet enough to fully cover the bottom of vessel.
   3. Incubate overnight at 37 °C.
   4. Aspirate the coating media the next day.
   5. Wash the vessel with ddH₂O prior to use.
3. Fibronectin- and laminin-coated wells (Used at Day 12 for 2Dculture and Day 20 for 3D Culture).

   1. Dilute the fibronectin (10 μg/ml) and laminin (10 μg/ml) in D-PBS.
   2. Pipet enough to fully cover the bottom of the well.
   3. Incubate for 4 h at 37 °C.
   4. Aspirate the coating media and wash with ddH₂O prior to use.

3.5.2. Thawing hESCs/hiPSCs

1. Prior to thawing cells, warm (37 °C) 10 ml of mTeSR1 media.

2. Take cryovial from Liquid Nitrogen and bury it in bucket of wet ice.

3. Thaw aliquot in 37 °C water bath until cells just begin to thaw.

4. Add 1 ml warmed media drop by drop to cryovial.

5. Add the 1 ml suspension drop by drop to 8 ml warmed mTeSR1media.

6. Rinse the cryovial once with 1 ml warmed mTeSR1 Media.

7. Spin the 15 ml conical at 150–300 × g for 5 min.

8. Carefully decant the supernatant without disturbing the pellet.

9. Resuspend cells in 3 ml of mTeSR1 Media and plate into a ES-Matrigel coated 6well plate.

3.5.3. Monolayer (2D) Differentiation Protocol for hESCs and Human Induced Pluripotent Cells hiPSCs

1. After thawing hESCs/hiPSCs, allow them to reach 80% confluency.

2. Day 0.

   1. Pipet 1 ml of Accutase into each 6-well plate with 80% confluent cells to digest the colonies into single cell suspension.
   2. After the cells lift off, collect them into a 15 ml conical.
   3. Rinse the well 2× with STEMdiff Neural Induction Media and collect cells into the same 15 ml conical.
   4. Centrifuge for 5 min at 300 × g.
   5. Resuspend the cells in 1 ml of STEMdiff neural induction media.
   6. Dilute a 10 μl suspension of cells in 10 μl of Trypan Blue and count cells with hemocytometer after mixing.
   7. Plate cells at desired density (recommended concentration is 50,000–200,000/cm²) on Non-ES-Matrigel–coated 6-well plates (adjust well numbers according to the scale of your experiments) in STEMdiff neural induction media + SB431542 (10 μM) + LDN193189 (250 nM) + Y-27632 (10 μM) + bFGF (20 ng/ml) + L-ascorbic acid (200 μM).
3. Days 1–4.

   1. Carefully aspirate the media and replace with STEMdiff neural induction media + SB431542 (10 μM) + LDN193189 (250 nM) + L-ascorbic acid (200 μM). Change the media every day.
4. Days 5–7.

   1. Carefully aspirate the media and replace with STEMdiff neural induction media + N2/B27 NeuroStem Media (1× DMEM/F12 + 1 Neurobasal media, supplemented with 1 × N2 supplement and 1 × B27 without Vitamin A) + L-ascorbic acid (200 μM).
   2. On day 5, it is 3/4 STEMdiff neural induction media +1/4 N2/B27 NeuroStem Media; on day 6 it is 1/2 STEMdiff neural induction media +1/2 N2/B27 NeuroStem Media. On day 7, it is 1/4 STEMdiff neural induction media +3/4 N2/B27 NeuroStem Media.
5. Days 8–11:

   1. Carefully aspirate the media and replace with N2/B27 NeuroStem Media+ DAPT (10 μM) + L-ascorbic acid (200 μM) + cAMP (500 μM). Change media every day.
6. Day 12:

   1. Carefully aspirate the media and rinse twice with warmed D-PBS.
   2. After rinsing, lift the cells with 1 ml of Accutase and digest for 5 min at 37 °C.
   3. Collect the cells into a 15 ml conical tube and rinse 2× with D-PBS to collect remaining cells.
   4. Centrifuge for 5 min at 300 × g.
   5. Resuspend the cells in N2/B27 Neural Media (1× DMEM/F12 + 1× Neurobasal media, supplemented with 1× N2 supplement and 1× B27 with Vitamin A) + Y-27632 (10 μM) + DAPT (10 μM) + L-ascorbic acid (200 μM) + cAMP (500 μM).
   6. If large cell aggregates are present after digestion and resuspension, use a cell strainers (45 μm) to separate the cells.
   7. Plate the cells at desired density (recommended concentration is 50,000–200,000/cm²) on the PLO-, fibronectin-, and laminin-coated wells (glass/plastic coverslips can be used if they are the final platform for your experiments.)
7. Days 13 and 18.

   1. Carefully aspirate the media and replace with N2/B27 Neural Media + DAPT (10 mM) + L-ascorbic acid (200 μM) + cAMP (500 μM). Change media every day.
8. Day 21 onward.

   1. Carefully aspirate the media and replace with Astrocyte Conditioned N2 Media (DMEM/F12 with 1 × N2 Supplement, conditioned in monolayer purified astrocytic cultures for 48 h) + L-ascorbic acid (200 μM) + cAMP (500 μM) + BDNF (20 ng/ml) every 3 days to keep the neurons healthy.

3.5.4. Embryoid Body (3D) Differentiation Protocol for hESCs and Human Induced Pluripotent Cells hiPSCs

1. Prepare the AggreWell plate by pipetting 500 μl of Anti-Adherence Rinsing Solution to each well.

2. Centrifuge the plate for 5 min at 2000 × g.

3. Make sure there are no bubbles in the wells.

4. Carefully aspirate the Rinsing Solution.

5. Pipet 500 μl of mTeSR1 + Y-27632 (10 μM) into each well.

6. Centrifuge the plate at 2000 × g for 5 min to get rid of any bubbles.

7. Keep in the incubator until use.

1. Day 0.

   1. Begin with cells that have reached 80% confluency on ES-Matrigel–coated 6-well plate after thawing (see Subheading 3.5.2).
   2. Rinse hESCs/hiPSCs twice with warmed D-PBS.
   3. Pipet 1 ml of Accutase into each well to digest the colonies into single cell suspension.
   4. Collect the cells into a 15 ml conical tube. Rinse the wells twice with D-PBS and collect into 15 ml conical tube.
   5. Centrifuge for 5 min at 300 × g.
   6. Resuspend the cells in mTeSR1 media + Y-27632 (10 μM) .
   7. Count the cells using a hemocytometer by diluting 10 μl of cells in 10 μl Trypan Blue in a 0.6 ml Eppendorf Tube.
   8. Plate the cells in a 500 μl solution with total of 3 × 10⁶ cells/ml (10,000 cells/microwell) into AggreWell.
   9. Pipet up and down to evenly disperse cells.
   10. Centrifuge the cells in the AggreWell plate for 3 min at 100 × g.
2. Days 1 to 4.

   1. On day 1, carefully aspirate the media and replace with 3/4 mTeSR1 Media and 1/4 STEMdiff neural induction media + SB431542 (10 μM) + LDN193189 (250 nM); on day 2 carefully aspirate the media and replace with 1/2 mTeSR1 Media and 1/2 STEMdiff neural induction media + SB431542 (10 μM) + LDN193189 (250 nM); on day 3, carefully aspirate the media and replace with 1/4 mTeSR1 Media and 3/4 STEMdiff neural induction media + SB431542 (10 μM) + LDN193189 (250 nM); on day 4, carefully aspirate the media and replace with 100% STEMdiff neural induction media + SB431542 (10 μM) + LDN193189 (250 nM).
3. Day 5.

   1. Use a wide-bore tip to resuspend the embryoid bodies (EBs) that formed in each of the wells of the AggreWell plate by vigorously pipetting up and down.
   2. Transfer the EBs into the 70 μm cell strainer to let single cells pass through, catching the EBs.
   3. Invert the EBs over a 50 ml conical tube and dislodge the EBs with STEMdiff neural induction media + SB431542 (10 μM) + LDN193189 (250 nM).
   4. Transfer the EBs to the ES-Matrigel–coated 6-well plate in the STEMdiff neural induction media + SB431542 (10 μM) + LDN193189 (250 nM)with Y-27632 (10 μM).
4. Days 6–11.

   1. Carefully change the media to N2/B27 NeuroStem Media + SB431542 (10 μM) + LDN193189 (250 nM) + L-ascorbic acid (200 μM). Change media every other day.
   2. This should result in neural rosettes.
5. Day 12.

   1. Carefully aspirate the media from the cells and rinse twice with warmed D-PBS.
   2. Add 1 ml dispase (iU/ml) into the well for 5–8 min. Aspirate the dispase and rinse twice with DMEM/F12 media (2 ml each time). Add 2 ml N2/B27 NeuroStem media.
   3. Pick the rosettes from the cultures by using pipette tips (usually 200p) under the microscope.
   4. Collect them into a 15 ml conical tube.
   5. Resuspend and titrate (3 times) the rosettes in 1 ml of N2/B27 NeuroStem Media + Y-27632 (10 μM) + SB431542 (10 μM) + LDN193189 (250 nM) + L-ascorbic acid (200 μM).
   6. Plate the rosettes on non-ES-Matrigel–coated 6-well plate (plate one well of the isolated rosettes into one well of the non-ES–coated well).
6. Days 13–18.

   1. Carefully aspirate the media from the wells and replace the media every other day with N2/B27 Neural Media + SB431542 (10 μM) + LDN193189 (250 nM) + L-ascorbic acid (200 μM).
7. Day 19.

   1. Carefully aspirate the media from the wells and rinse cells with warmed D-PBS.
   2. Pipet 1 ml of Accutase to digest the cultured cells into single cell suspension.
   3. Collect the cells into a 15 ml conical tube.
   4. Centrifuge the cells for 5 min at 300 × g.
   5. Resuspend the cells in 1 ml N2/B27 Neural Media + Y-27632 (10 μM) + L-ascorbic acid (200 μM) + cAMP (500 μM).
   6. If large cell aggregates are present after digestion and resuspension, use a cell strainers (45 μm) to separate the cells.
   7. Plate the cells at desired density (recommended concentration is 50,000–200,000/cm²) on the PLO-, fibronectin-, and laminin-coated wells (glass/plastic coverslips can be used if they are the final platform for your experiments.)
8. Day 20–25.

   1. Carefully aspirate the media and replace the media with N2/B27 Neural Media + DAPT (10 μM) + L-ascorbic acid (200 μM) + cAMP (500 μM). Change media every day.
9. Day 25 onward.

   1. Replace the media with Astrocyte Conditioned N2 Media + L-ascorbic acid (200 μM) + cAMP (500 μM) + BDNF (20 μg/ml) until the cells are used for experiments.

3.5.5. Preparation of Dishes and Media for Direct Differentiation of Human Somatic Cells (Fibroblasts/Pericytes) into Cortical Glutamatergic Neurons

1. Gelatin-Coated T25 or T75 Flasks

   1. Use 0.1% gelatin solution to coat the vessels.
   2. Pipet enough to fully cover the bottom of each flask.
   3. Let dry overnight.
2. Poly-L-lysine (PLL)- and laminin-coated wells.

   1. Dilute PLL in 1PBS (final concentration 1 mg/ml).
   2. Pipet enough to fully cover the bottom of each well.
   3. Incubate overnight at room temperature.
   4. Aspirate the coating media the next day.
   5. Wash the vessel with ddH2O, three times for 10 min each wash.
   6. Fill each well with ddH2O and place in incubator overnight (the plate can be in the incubator for up to 1 week).
   7. Dilute laminin in D-PBS (final concentration 0.1 mg/ml) and coat the wells for 4 h at 37 °C.
   8. Aspirate the coating solution prior to use.
3. Viral Infection Media.

   1. Combine DMEM with 10% FBS and 8 μg/μl of polybrene with no antibiotics.

3.5.6. Thawing Fibroblasts/Pericytes

1. Warm 10 ml of DMEM +10% FBS.

2. Take cryovial from liquid nitrogen and bury it in bucket of wet ice.

3. Thaw aliquot in 37 °C water bath until cells just begin to thaw.

4. Add 1 ml warmed media drop by drop to cryovial.

5. Add the 2 ml suspension drop by drop to 10 ml warmed media.

6. Rinse the cryovial once with 1 ml warmed DMEM +10% FBS.

7. Spin the 15 ml conical at 300 × g for 5 min.

8. Carefully decant the supernatant without disturbing the pellet.

9. Resuspend cells in 3 ml of DMEM +10% FBS and plate into thegelatin-coated T25 or T75 flask.

3.5.7. Direct Reprogramming of Human Skin Fibroblasts/Pericytes into Cortical Glutamatergic Neurons

1. Day 0.

   1. After thawing human fibroblasts/pericytes, allow them to reach 80% confluency.
   2. Pipet 1 ml of Accutase into T25 flask or 3 ml into T75 flask to digest the cells into single cell suspension.
   3. After the cells lift off, collect them into a 15 ml conical.
   4. Rinse the well 2× with DMEM +10% FBS and collect cells into the same 15 ml conical.
   5. Centrifuge for 5 min at 300 × g.
   6. Resuspend the cells in 1 ml of DMEM +10% FBS.
   7. Dilute a 10 μl suspension of cells in 10 μl of Trypan Blue and count cells with hemocytometer after mixing.
   8. Plate cells at desired density (recommended concentration is 20,000/cm²) in DMEM+10%FBS onto PLL + laminin–coated 24-well plate.
2. Day 1.

   1. Aspirate the media from the 24-well plate with seeded human fibroblasts/pericytes.
   2. Replace with Viral Infection Media (DMEM +10% FBS and 8 μg/μl of polybrene with no antibiotics).
   3. Pipet lentiviral particles encoding Ascl1, Brn2, Myt1l, Olig2, and/or Zic1, the total MOI of the viral combination should be around 5.
   4. Agitate the plate to ensure the virus is well mixed and incubate.
3. Days 2 and 3.

   1. Carefully aspirate the media and replace with fresh DMEM +10% FBS + forskolin (10 mg/ml) + L-ascorbic acid (0.4 mM/ml).
4. Days 4–18.

   1. Carefully aspirate the media from each well and wash the cells with N2 media [DMEM/F-12 + N2 Supplement (1)] twice.
   2. Replace media with glia-culture conditioned N2 media + forskolin (10 mg/ml) + L-L-ascorbic acid (0.4 mM/ml) + bFGF (20 ng/ml).
   3. Make sure to replenish with fresh media every other day for 2 weeks.
5. Day 19 onward.

   1. Carefully aspirate the media and replace with glia-culture conditioned N2 media (DMEM/F-12 + N2 supplement) + forskolin (10 mg/ml) + L-ascorbic acid (0.4 mM/ml) + BDNF (20 ng/ml).
   2. Make sure to change the media every other day until the cells obtain a neuronal morphology.
   3. Neuronal phenotypes should be identifiable after day 21 (see Fig. 7c, d).

3.6. Summary

Neural stem cells and neural progenitor cells can be a useful source of neurons for in vitro and in vivo studies of neural function. Both NSC, such as neuroepithelial cells (NEP), and NPC, such as neuronal and glial restricted progenitors (NRP/GRP), can be directly isolated from the developing mammalian spinal cord or from pluripotent sources. The techniques discussed here should provide the ability to dissociate, culture, freeze, thaw, and differentiate NRPs/GRPs toward mature neuronal phenotypes as well as provide a starting point for generating neurons from human embryonic stem cells, induced pluripotent stem cells or direct reprogramming.