The authors thank members of the Renee Reijo Pera laboratory for help during development of this protocol and during preparation of this manuscript. This work is supported by California Institute for Regenerative Medicine (CIRM) shared laboratory (CL-00518).

1. Preparation of Culture Media
<ol>
	<li>Prepare mouse embryonic fibroblast (MEF) medium by combining the following: 445 ml DMEM, 50 ml fetal bovine serum (FBS), and 5 ml 100x penicillin/ampicillin stock solution. Keep filter sterilized medium at 4 &#176;C for no more than 14 days.</li>
	<li>Prepare serum-containing hPSC culture medium by combining the following: 385 ml DMEM/F12, 100 ml knockout serum replacement (KSR), 5 ml 100x non-essential amino acid stock solution, 5 ml 100x penicillin/ampicillin stock solution, 5 ml 100x -mercaptoethanol stock solution, and 10 ng/ml bFGF. Keep filter sterilized medium at 4 &#176;C for no more than 10 days.</li>
	<li>Prepare mTeSR1 serum-free medium by combining the following: 400 ml mTeSR1 basal medium, 100 ml 5x supplement, and 5 ml 100x penicillin/ampicillin stock solution. Keep medium at 4 &#176;C for no more than 10 days.</li>
	<li>Prepare 10x collagenase IV stock solution: weigh out 0.5 g collagenase IV power, and dissolve it with 50 ml DMEM/F12 and filter sterilize. Make aliquots and stock them at -20 &#176;C for months.</li>
	<li>Prepare 0.1% gelatin solution: weigh out 0.5 g gelatin (from bovine skin, type B) power and dissolve it with deionized water. Keep autoclave sterilized solution at room temperature for weeks.</li>
	<li>Prepare KSR differentiation medium by combining the following: 410 ml DMEM, 75 ml KSR, 5 ml 100x non-essential amino acid stock solution, 5 ml 100x penicillin/ampicillin stock solution, 5 ml 100x -mercaptoethanol stock solution. Keep filter sterilized medium at 4 &#176;C for no more than 10 days. 
	NOTE: KSR varies from lot to lot, which may affect the differentiation efficiency. It is therefore better to test several batches of KSR to find the best one for differentiation.</li>
	<li>Prepare N2 differentiation medium by combining the following: 98 ml DMEM, 1 ml 100x N2 supplement and 1 ml 100x penicillin/ampicillin stock solution. Keep filter sterilized medium at 4 &#176;C for no more than 10 days.</li>
	<li>Prepare B27 differentiation medium by combining the following: 480 ml neurobasal medium, 10 ml 50x B27 supplement, 5 ml 100x glutamax stock solution, and 5 ml 100x penicillin/ampicillin stock solution. Keep filter sterilized medium at 4 &#176;C for no more than 10 days.</li>
</ol>
2. Culture of hESCs and hiPSCs on MEF Feeder Cells
The H9 hESCs were obtained from WiCell Research Institute and hiPSCs were established in the Reijo Pera laboratory through retrovirus-mediated transduction of Yamanaka factors OCT3/4, SOX2, KLF4, and c-MYC18.
<ol>
	<li>At least one day before cell passage, prepare some gelatin plates by adding 1 ml 0.1% gelatin to 1 well of 6-well plates, and incubate plates at 37 &#176;C for at least 30 min.</li>
	<li>After incubation, aspirate gelatin from plates, and seed irradiation inactivated MEF cells onto the plates at the density of 1.6 x 105 cells/well in MEF medium using a hemocytometer to count the cells. 
	NOTE: Optionally prepare MEF feeders as early as 3 days before cell passage.</li>
	<li>Passage cells one day after plating MEF feeders: aspirate medium from hPSCs, add 1 mg/ml collagenase IV to cells at 1 ml/well, and incubate cells at 37 &#176;C for 1 hr.</li>
	<li>During this incubation, wash MEF feeders once with PBS, and then add warm (37 &#176;C) serum-containing hPSC culture medium at 1.5 ml/well.</li>
	<li>After 1 hr, tap the culture plate a couple of times so that most of the undifferentiated colonies will detach from the plates, while the differentiated colonies remain attached. Carefully collect the floating colonies, and transfer them into 15 ml tube.</li>
	<li>Gently wash the plate once with warm (37 &#176;C) DMEM/F12 and transfer DMEM/F12 into the same 15 ml tube.</li>
	<li>Centrifuge the tubes at 179 x g for 3 min.</li>
	<li>Aspirate medium from the tubes, being careful not to disturb the pellet. Add warm hPSC culture medium, pipette the cells up and down several times to break them into small clusters and mix them well.</li>
	<li>Transfer the cells onto new MEF feeders at 1:3-1:6 ratio.</li>
	<li>Change the medium every day and passage the cells every 5-6 days.</li>
</ol>
3. Preparation of Cells for Differentiation
<ol>
	<li>At least one day before preparation of cells, prepare some Matrigel plates (typically 3 wells of a 6-well plate). Dilute the Matrigel with cold (4 &#176;C) DMEM/F12 at 1:40 and add 1 ml Matrigel per well of 6-well plates. Incubate Matrigel plates at 4 &#176;C overnight. NOTE: One can seal Matrigel plates with Parafilm and stock them at 4 &#176;C for as long as one week.</li>
	<li>Use cells (see Step 2) 4 days after passage. Remove all the differentiated colonies from the plates. Aspirate medium from the culture plate, add 1 ml accutase per well, and incubate cells at 37 &#176;C for 5 min.</li>
	<li>During the incubation, prepare some gelatin plates by adding 1 ml gelatin to 1 well of 6-well plates. Place these plates and the Matrigel-plates prepared one day before in incubator.</li>
	<li>After the 5-min incubation or when cells have become semi-floating, pipette cells up and down several times to make them into single cells.</li>
	<li>Collect single cells into 15 ml tubes, and centrifuge at 258 x g for 3 min.</li>
	<li>Resuspend cells with appropriate amount of serum containing hPSC culture medium, and add Thiazovivin to the final concentration of 2 &#181;M.</li>
	<li>Transfer cells onto gelatin-coated plates at 1:1 ratio, and incubate cells at 37 &#176;C for 30 min to remove MEF feeders.</li>
	<li>Transfer the cells to a 15 ml conical tube, add appropriate serum containing hPSC medium and count the cells with a hemocytometer.</li>
	<li>Centrifuge the cells at 258 x g for 3 min.</li>
	<li>During centrifuge, add Thiazovivin to appropriate mTeSR1 medium to make a concentration of 2 &#181;M.</li>
	<li>After centrifuge, aspirate the medium and add appropriate mTeSR1 medium with Thiazovivin so that there are 1.8 x 105 cells/ml medium.</li>
	<li>Take out the Matrigel plates from the incubator, aspirate the Matrigel and add 2 ml of the mixed cells onto 1 well of the 6-well plates. 
	NOTE: The cell density should be 3.6 x 104 cells/cm2 of surface area.</li>
	<li>Return the plates back to the incubator, and let the cells attach for 24 hr.</li>
	<li>24 hr after plating the cells, aspirate old medium and add 2 ml new mTeSR1 medium per well and let cells grow for another 24 hr before starting the differentiation.</li>
</ol>
Cell Differentiation
<ol>
	<li>Start differentiation 48 hr after replating the cells onto Matrigel plates.</li>
	<li>Aspirate culture medium from the plate, wash cells once with PBS, and add 2 ml of the following differentiation medium per well: KSR differentiation medium supplemented with 10 &#181;M SB431542 and 100 nM LDN-193189. Mark this day as D0 of differentiation.</li>
	<li>Change medium every day from D0 to D20. 
	NOTE: One can prepare medium for use no more than 2 days at a time.</li>
	<li>Use the following medium for D1 and D2: KSR differentiation medium supplemented with 10 &#181;M SB431542, 100 nM LDN-193189, 0.25 &#181;M SAG, 2 &#181;M purmorphamine, and 50 ng/ml FGF8b.</li>
	<li>Use the following medium for D3 and D4: KSR differentiation medium supplemented with 10 &#181;M SB431542, 100 nM LDN-193189, 0.25 &#181;M SAG, 2 &#181;M purmorphamine, 50 ng/ml FGF8b, and 3 &#181;M CHIR99021.</li>
	<li>Use the following medium for D5 and D6: 75% KSR differentiation medium plus 25% N2 differentiation medium supplemented with 100 nM LDN-193189, 0.25 &#181;M SAG, 2 &#181;M purmorphamine, 50 ng/ml FGF8b, and 3 &#181;M CHIR99021.</li>
	<li>Use the following medium for D7 and D8: 50% KSR differentiation medium plus 50% N2 differentiation medium supplemented with 100 nM LDN-193189 and 3 &#181;M CHIR99021.</li>
	<li>Use the following medium for D9 and D10: 25% KSR differentiation medium plus 75% N2 differentiation medium supplemented with 100 nM LDN-193189 and 3 &#181;M CHIR99021.</li>
	<li>Use the following medium for D11 and D12: B27 differentiation medium supplemented with 3 &#181;M CHIR99021, 10 ng/ml BDNF, 10 ng/ml GDNF, 1 ng/ml TGF3, 0.2 mM ascorbic acid and 0.1 mM cAMP.</li>
	<li>Use the following medium for the rest of differentiation: B27 differentiation medium supplemented with 10 ng/ml BDNF, 10 ng/ml GDNF, 1 ng/ml TGF3, 0.2 mM ascorbic acid and 0.1 mM cAMP.</li>
	<li>At about D16 of differentiation, prepare some Poly-L-ornithine/Laminin/Fibronectin plates. Dilute Poly-L-ornithine stock solution with cold (4 &#176;C) PBS to 15 &#181;g/ml, add 1 ml to 1 well of 6-well plates, and incubate the plates at 37 &#176;C overnight.</li>
	<li>Aspirate the Poly-L-ornithine solution, wash the plates 3 times with sterile water and then air dry the plates.</li>
	<li>Dilute laminin stock solution with cold (4 &#176;C) PBS to 1 &#181;g/ml, add 1 ml to one well of 6-well plates, and incubate the plates at 37 &#176;C overnight.</li>
	<li>Aspirate the laminin solution, wash the plates 3 times with sterile water and then air dry the plates.</li>
	<li>Dilute fibronectin stock solution with cold (4 &#176;C) PBS to 2 &#181;g/ml, add 1 ml to 1 well of 6-well plates, and incubate the plates at 37 &#176;C overnight.</li>
	<li>Seal plates with Parafilm and place them at 4 &#176;C for as long as two weeks.</li>
	<li>After 20 days of differentiation, replate the cells to the Poly-L-ornithine/Laminin/Fibronectin plates. Aspirate differentiation medium, add 1 ml warm (37 &#176;C) accutase to 1 well of 6-well plates, and incubate cells at 37 &#176;C for 5 min.</li>
	<li>During the incubation, place the Poly-L-ornithine/Laminin/Fibronectin plates in the incubator.</li>
	<li>After the 5-min incubation or when cells have become semi-floating, gently pipette cells up and down several times to make them into single cells.</li>
	<li>Collect the single cells into 15 ml conical tubes, and add the appropriate amount of neurobasal medium for cell counting, which will vary depending on expansion (after 11 days of differentiation, cells may expand 15-20 fold). Count the cells using a hemocytometer.</li>
	<li>Centrifuge the cells at 258 x g for 3 min. Aspirate supernatant, and resuspend cells with B27 differentiation medium so that the cell concentration is 1 x 106 cells/ml medium.</li>
	<li>Aspirate fibronectin from the plates, wash twice with PBS, and add 2 ml cells to 1 well of 6-well plates. 
	NOTE: The cell density for replating should be 2 x 105 cells/cm2 of surface area.</li>
	<li>Change medium 24 hr later to remove any unattached dead cells.</li>
	<li>Change medium every other day until the desired time points for a given experiment.</li>
</ol>

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The authors declare that they have no competing financial interests.

Human pluripotent stem cells (including both hESCs and hiPSCs) can be differentiated in vitro to generate most, if not all, cell types of our body including DA neurons, which has been demonstrated in previous studies19. Here, based on knowledge from embryonic dopaminergic neuron development20 and published protocols from other laboratories14,15, we optimized the culture conditions for the generation of DA neurons from hESCs and hiPSCs. This protocol is efficient, reproducible and we have successfully applied this protocol described here to H1 and H9 hESC lines and to the hiPSC lines from both the PD patients and unaffected controls.
In our protocol, there are three key steps to obtain the high differentiation efficiency: First, there should be no differentiated hPSC colonies in the cells before directed differentiation. As spontaneous differentiation to all three germ layers is often seen in hPSC culture, it is important to remove these differentiated colonies before dissociating these hPSCs into single cells. Second, MEF feeders should be depleted from the dissociated hPSCs, as previous reports have shown that MEF cells can secret factors that promote self-renewal and inhibit differentiation21. For this purpose, incubating the hPSC and MEF cell mixtures on gelatin-coated plates for 30 min should be enough to remove almost all the MEF cells. Third, the single hPSCs should be plated at the appropriate cell density for differentiation. Increasing the cell density will lead to the death of most cells at around D11 of differentiation, while lowering the cell density will result in the detachment and death of cells in the center of the plate in as short as less than 7 days, although cells on the edge will differentiate well (data not shown). If one observes all these three criteria, it is easy to efficiently obtain DA neurons from hPSCs with our stepwise protocol.
The protocol presented here is mainly based on a previous report by L. Studer and colleagues14 with some modifications. First, the differentiation here was started 48 hr after single cell plating when cells reach only about 70% confluence, while in their report, differentiation was started when cells reach full confluence (3 or more days of culture after single cell plating). In our experience, starting differentiation from confluent cells without a cell passage until day 20 leads to severe cell death during the differentiation process. Second, we replace the expensive SHH protein in their study with SAG, a chlorobenzothiophene-containing small molecule agonist for HH pathway22. Therefore, compared with previously published protocols, this one described here is feeder cell and neural rosette independent, and based mainly on small molecules for FP specification; it is thus less expensive and less time- and labor-consuming. Of course, the limitation of this protocol is the use of KSR for the first days of differentiation. KSR varies from lot to lot, which may affect the differentiation efficiency. Therefore, one should test different batches of KSR to get the one that works best in inducing FP precursors after 11 days of differentiation.
In summary, the differentiation protocol descried here should facilitate: (1) the generation of DA neurons from hESCs or hiPSCs of normal persons for cell replacement therapy for PD; (2) the generation of DA neurons from PD patient iPSCs for in vitro modeling and testing potential therapeutic drugs for PD; (3) the study of the genes/signaling pathways regulating human DA neuron development.

Dopaminergic (DA) neurons can be found in several brain regions, including the midbrain, hypothalamus, retina, and olfactory bulbs. The A9 DA neurons in the substantia nigra pars compacta (SNpc) control behavior and movement by projecting to the striatum in the forebrain and forming the extrapyramidal motor system. Degeneration of A9 DA neurons leads to Parkinson&#8217;s disease (PD), which is the second most common human neurodegenerative disorder and currently incurable. Cell replacement therapy is one of the most promising strategies for the treatment of PD1, therefore, there has been a great interest in deriving A9 DA neurons from human pluripotent stem cells (hPSCs), including both human embryonic stem cells (hESCs) and the recent human induced pluripotent stem cells (hiPSCs).
Much research has tried to derive A9 DA neurons from hPSCs using various methods. The earliest reports all generated DA neurons through a neural rosette progenitor stage. By co-culturing with MS52 or PA63-5 stromal cells with or without external growth factors for 2-4 weeks, L. Studer and colleagues and three other groups successfully induced hESCs to produce neural rosettes. They then enriched these rosettes by mechanical dissection or enzymatic digestion for further differentiation. In other reports, researchers generated neural rosettes through embryoid body (EB) floating culture differentiation6-9. Later, researchers established a monolayer based differentiation method10,11, where they plated hESCs and hiPSCs on extra cellular matrix, added different growth factors in the culture to induce the differentiation of hPSCs to DA neurons, which mimics the in vivo embryonic DA neuron development. Although all these studies obtained tyrosine hydroxylase (TH)-expressing cells with some characteristics of DA neurons, the entire differentiation process is time and labor consuming, generally inefficient, and more importantly, the A9 identity of these neurons were not demonstrated in most studies except the one with LMX1a ectopic expression12. Recently, a new floor plate (FP)-based protocol was developed13-16, in which the FP precursors with DA neuron potential were first generated by activation of the sonic hedgehog and canonical Wnt signaling pathways during the early stage of differentiation, and then these FP cells were further specified to DA neurons. Although this protocol is more efficient, there are still some problems; for example, the whole differentiation process takes long time (at least 35 days) and is feeder cell dependent15, or is EB dependent16 or the A9 identity was not demonstrated14.
Here, based on the knowledge from in vivo embryonic DA neuron development and other researchers&#8217; published results, we have optimized the culture conditions for the efficient generation of DA neurons from both hESCs and hiPSCs. We first generated FP precursor cells by activation of the canonical Wnt signaling with small molecule CHIR99021 and sonic hedgehog signaling with small molecules SAG and purmorphamine. These FP cells express FOXA2, LMX1a, CORIN, OTX2 and NESTIN. We then specified these FP cells to DA neurons with growth factors including BDNF, GDNF, etc. The generated DA neurons are of A9 cell type as they are positive for GIRK2 while negative for Calbindin17. This protocol is feeder cell or EB independent, highly efficient and reproducible. Using this protocol, one can derive DA neurons in less than 4 weeks from hESCs or hiPSCs of normal persons for cell transplantation study, or from hiPSCs of PD patients for in vitro modeling of PD or testing potential therapeutic agents for PD.

<table><tbody><tr><td>DMEM</td><td>Life Technologies</td><td>10569-010</td><td></td></tr><tr><td>FBS</td><td>Life Technologies</td><td>26140</td><td></td></tr><tr><td>penicillin/ampicillin</td><td>Life Technologies</td><td>15140-122</td><td></td></tr><tr><td>DMEM/F12</td><td>Life Technologies</td><td>10565-018</td><td></td></tr><tr><td>KSR</td><td>Life Technologies</td><td>10828-028</td><td></td></tr><tr><td>Non-essential amino acid</td><td>Life Technologies</td><td>11140-050</td><td></td></tr><tr><td>b-mercaptoethanol</td><td>Millipore</td><td>ES-007-E</td><td></td></tr><tr><td>bFGF</td><td>R&amp;amp;D Systems</td><td>233-FB-025</td><td></td></tr><tr><td>mTesR1</td><td>STEMCELL Technologies</td><td>5850</td><td></td></tr><tr><td>Collagenase IV</td><td>Life Technologies</td><td>17104-019</td><td></td></tr><tr><td>Gelatin</td><td>Sigma-Aldrich</td><td>G9391</td><td></td></tr><tr><td>N2 supplement</td><td>Life Technologies</td><td>17502-048</td><td></td></tr><tr><td>B27 supplement</td><td>Life Technologies</td><td>17504-044</td><td></td></tr><tr><td>Neurobasal</td><td>Life Technologies</td><td>21103-049</td><td></td></tr><tr><td>Glutamax</td><td>Life Technologies</td><td>35050-061</td><td></td></tr><tr><td>PBS</td><td>Life Technologies</td><td>10010-023</td><td></td></tr><tr><td>Growth factor reduced matrigel</td><td>BD Biosciences</td><td>354230</td><td></td></tr><tr><td>Accutase</td><td>MP Biomedicals</td><td>1000449</td><td></td></tr><tr><td>Thiazovivin</td><td>Santa Cruz Biotechnology</td><td>sc-361380</td><td></td></tr><tr><td>SB431542</td><td>Tocris Bioscience</td><td>1614</td><td></td></tr><tr><td>LDN-193189</td><td>Stemgent</td><td>04-0074</td><td></td></tr><tr><td>SAG</td><td>EMD Millipore</td><td>566660-1MG</td><td></td></tr><tr><td>Purmorphamine</td><td>Santa Cruz Biotechnology</td><td>sc-202785</td><td></td></tr><tr><td>FGF8b</td><td>R&amp;amp;D Systems</td><td>423-F8-025</td><td></td></tr><tr><td>CHIR99021</td><td>Cellagen Technology</td><td>C2447-2s</td><td></td></tr><tr><td>BDNF</td><td>R&amp;amp;D Systems</td><td>248-BD-025</td><td></td></tr><tr><td>GDNF</td><td>R&amp;amp;D Systems</td><td>212-GD-010</td><td></td></tr><tr><td>TGF-beta3</td><td>R&amp;amp;D Systems</td><td>243-B3-002</td><td></td></tr><tr><td>Ascorbic acid</td><td>Sigma-Aldrich</td><td>A4034</td><td></td></tr><tr><td>cAMP</td><td>Sigma-Aldrich</td><td>D0627</td><td></td></tr><tr><td>Mouse anti human NESTIN antibody</td><td>Santa Cruz Biotechnology</td><td>sc-23927</td><td>1/1,000 dilution</td></tr><tr><td>Rabbit anti human OTX2 antibody</td><td>Millipore</td><td>AB9566</td><td>1/2,000 dilutiion</td></tr><tr><td>Goat anti human FOXA2 antibody</td><td>R&amp;amp;D Systems</td><td>AF2400</td><td>1/200 dilution</td></tr><tr><td>rabbit anti human LMX1a antibody</td><td>Millipore</td><td>AB10533</td><td>1/1,000 dilution</td></tr><tr><td>Rabbit anti human TH antibody</td><td>Pel Freez</td><td>P40101</td><td>1/500 dilution</td></tr><tr><td>Chicken anti human TH antibody</td><td>Millipore</td><td>AB9702</td><td>1/500 dilution</td></tr><tr><td>Mouse anti human TUJ1 antibody</td><td>Covance</td><td>MMS-435P</td><td>1/,2000 dilution</td></tr><tr><td>Rabbit anti human GIRK2 antibody</td><td>Abcam</td><td>ab30738</td><td>1/300 dilution</td></tr><tr><td>Rabbit anti human Calbindin antibody</td><td>Abcam</td><td>ab25085</td><td>1/400 dilution</td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5804</td><td></td></tr></tbody></table>

directed dopaminergic neuron differentiation from human pluripotent stem cells

Dopaminergic (DA) neurons in the substantia nigra pars compacta (also known as A9 DA neurons) are the specific cell type that is lost in Parkinson&#8217;s disease (PD). There is great interest in deriving A9 DA neurons from human pluripotent stem cells (hPSCs) for regenerative cell replacement therapy for PD. During neural development, A9 DA neurons originate from the floor plate (FP) precursors located at the ventral midline of the central nervous system. Here, we optimized the culture conditions for the stepwise differentiation of hPSCs to A9 DA neurons, which mimics embryonic DA neuron development. In our protocol, we first describe the efficient generation of FP precursor cells from hPSCs using a small molecule method, and then convert the FP cells to A9 DA neurons, which could be maintained in vitro for several months. This efficient, repeatable and controllable protocol works well in human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) from normal persons and PD patients, in which one could derive A9 DA neurons to perform in vitro disease modeling and drug screening and in vivo cell transplantation therapy for PD.

An overview of the differentiation protocol is shown in Figure 1.&#160;The efficiency of the differentiation protocol presented here relies upon the status of the starting cells. Therefore, it is critical to make sure that, first one removes all the differentiated colonies before dissociating hPSCs into single cells for differentiation, and second, one depletes most, if not all, the MEF feeder cells by incubating the cells on gelatin-coated plates for 30 min, and third, one plates the hPSC single cells at the appropriate density onto Matrigel plate. As illustrated in Figure 2, replated hPSC single cells will expand as clusters during the 48 hr before differentiation, forming around 70% confluence in the plates. Then after differentiation, these cells reach full confluence in as short as 3-4 days, and starting from around D16-D17, these confluent cells start to make some spaces in the plates, and some axons/neurites could already be observed in these spaces and under the cell layers. After being replated at D20, the differentiated cells attached and grew as small clumps. Then during the next 5 days, much more intensive and morphologically intact axons/neurites come out from the clumps, and axons/neurites from different clumps form some connections. During long-term culture, neurons in one clump generate more extensions, get more mature and have more connections with neurons in neighboring clumps.
After 11 days of differentiation, hPSCs could be efficiently converted to FP precursors, and as illustrated in Figure 3, almost all the cells express the FP precursor cell markers NESTIN and FOXA2, and over 90% of the cells express another three markers CORIN, OTX2, and LMX1a. Then after more days of differentiation, the FP precursor cells are specified to DA neurons as they express TUJ1 and TH (Figure 4A). These DA neurons are of A9 identity as they express GIRK2 while are negative for Calbindin (Figure 4B). Immunostaining experiments also show that there are no GFAP+ astrocytes and very few GABAergic neurons (Figure 4C), indicating that the differentiation is exclusively specified toward the DA neuron lineage.
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/51737/51737fig1highres.jpg" width="500" /> 
Figure 1. Overview of the differentiation protocol, with growth factors/small molecules and culture medium used for each day. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51737/51737fig1highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" fo:content-width="5in" src="/files/ftp_upload/51737/51737fig2highres.jpg" width="500" /> 
Figure 2. Morphological changes during differentiation. H9 hESCs were plated as single cells in the appropriate cell density as described, and 48 hr after that, these cells reach about 70% confluence in the plate as colonies (D0). During the first a few days of differentiation, the cells expand rapidly, reaching over 90% confluence after 24 hr of differentiation (D1), and then become confluence after 48 more hr (D3). However, most of these cells still exhibit typical hESC morphology with high nucleus to cytoplasm ratio (D3). As differentiation goes on, cells expand more and gradually lose hESC morphology. At the end of D11 of differentiation, there are more than one cell layers in many parts of the plate, as evidenced by darker color of cells in some areas than the others (D11). Starting from about D17 to D20, some cells died, leaving some spaces in the plate, and some neuron projections could already be observed in these spaces and sometimes under the cell layer (D20). After cell replating at the end of D20 to make more spaces for cells to grow and differentiation, cells aggregate as small clusters, a lot more axons/neurites emerge from these clusters, and axons/neurites from different clusters form some connections in as short as 4-5 days (D25). Scale bar, 200 &#956;m.
<img alt="Figure 3" fo:content-width="5in" src="/files/ftp_upload/51737/51737fig3highres.jpg" width="500" /> 
Figure 3. Immunostaining for FP markers at early stage of differentiation. H9 hESCs were differentiated for 11 days using the protocol described here. Cells were then fixed with 4% paraformaldehyde, permeabilized with Triton X-100, blocked with donkey serum and then stained for FP precursor cell markers. As shown here, almost all the cells express FOXA2 and NESTIN, and over 90% of the cells express LMX1a, CORIN, and OTX2, indicating a very high efficiency conversion from hESCs to FP cells. Scale bar, 200 &#956;m.
<img alt="Figure 4" fo:content-width="5in" src="/files/ftp_upload/51737/51737fig4highres.jpg" width="500" /> 
Figure 4. Immunostaining for DA neuron markers at late stages of differentiation. (A) H9 hESCs were differentiated for 25 days, and cells were subject to immunostaining for the pan-neuron marker TUJ1 and the commonly used DA neuron marker TH. As shown here, although there are more TUJ1 positive cells than TH positive cells, all the TH-expressing cells residue in the TUJ1-expressing cells, and the TH-expressing cells represent at least half of all the cells. (B). The H9 hESCs were further differentiated for another 25 days (totally 50 days) and then cells were subject to immunostaining. The results showed that there is higher percentage of TH-expressing cells than that at d25. Almost all of these TH-expressing cells also express GIRK2 while most of them are negative for Calbindin, indicating the A9 subtype identity of the generated DA neurons, which is the cell type that is lost in PD. (C) Immunostaining for cells at D25 of differentiation showed that there are no cells expressing GFAP, and very few cells express GABA. Scale bar, 200 &#956;m.

Watch this Scientific Journal Video about Directed Dopaminergic Neuron Differentiation from Human Pluripotent Stem Cells at JoVE.com

Directed Dopaminergic Neuron Differentiation from Human Pluripotent Stem Cells

We, based on knowledge from developmental biology and published research, developed an optimized protocol to efficiently generate A9 midbrain dopaminergic neurons from both human embryonic stem cells and human induced pluripotent stem cells, which would be useful for disease modeling and cell replacement therapy for Parkinson&#8217;s disease.

Dopaminergic (DA) neurons in the substantia nigra pars compacta  (also known as A9 DA neurons) are the specific cell type that is lost in ...

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Nanyang Technological University

Research

JoVE Journal

Neuroscience

16.5K Views.  Stanford University School of Medicine. We, based on knowledge from developmental biology and published research, developed an optimized protocol to efficiently generate A9 midbrain dopaminergic neurons from both human embryonic stem cells and human induced pluripotent stem cells, which would be useful for disease modeling and cell replacement therapy for Parkinson’s disease.

Video: Directed Dopaminergic Neuron Differentiation from Human Pluripotent Stem Cells

Rapid Neuronal Differentiation of Induced Pluripotent Stem Cells for Measuring Network Activity on Micro-electrode Arrays

Neurons derived from human induced Pluripotent Stem Cells (hiPSCs) provide a promising new tool for studying neurological disorders. In the past decade, many protocols for differentiating hiPSCs into neurons have been developed. However, these protocols are often slow with high variability, low reproducibility, and low efficiency. In addition, the neurons obtained with these protocols are often immature and lack adequate functional activity both at the single-cell and network levels unless the neurons are cultured for several months. Partially due to these limitations, the functional properties of hiPSC-derived neuronal networks are still not well characterized. Here, we adapt a recently published protocol that describes production of human neurons from hiPSCs by forced expression of the transcription factor neurogenin-212. This protocol is rapid (yielding mature neurons within 3 weeks) and efficient, with nearly 100% conversion efficiency of transduced cells (&#62;95% of DAPI-positive cells are MAP2 positive). Furthermore, the protocol yields a homogeneous population of excitatory neurons that would allow the investigation of cell-type specific contributions to neurological disorders. We modified the original protocol by generating stably transduced hiPSC cells, giving us explicit control over the total number of neurons. These cells are then used to generate hiPSC-derived neuronal networks on micro-electrode arrays. In this way, the spontaneous electrophysiological activity of hiPSC-derived neuronal networks can be measured and characterized, while retaining interexperimental consistency in terms of cell density. The presented protocol is broadly applicable, especially for mechanistic and pharmacological studies on human neuronal networks.

We modify and implement a previously published protocol describing the rapid, reproducible, and efficient differentiation of human&#160;induced&#160;Pluripotent Stem Cells (hiPSCs) into excitatory cortical neurons12. Specifically, our modification allows for control of neuronal cell density and use on micro-electrode arrays to measure electrophysiological properties at the network level.

Neurons derived from human induced Pluripotent Stem Cells (hiPSCs) provide a promising new tool for studying neurological disorders. In the past decade, ...

Developmental Biology

Efficient Derivation of Human Neuronal Progenitors and Neurons from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system (CNS) structure and circuitry in today's healthcare industry. Cell-based therapies hold great promise to restore the lost nerve tissue and function for CNS disorders. However, cell therapies based on CNS-derived neural stem cells have encountered supply restriction and difficulty to use in the clinical setting due to their limited expansion ability in culture and failing plasticity after extensive passaging1-3. Despite some beneficial outcomes, the CNS-derived human neural stem cells (hNSCs) appear to exert their therapeutic effects primarily by their non-neuronal progenies through producing trophic and neuroprotective molecules to rescue the endogenous cells1-3. Alternatively, pluripotent human embryonic stem cells (hESCs) proffer cures for a wide range of neurological disorders by supplying the diversity of human neuronal cell types in the developing CNS for regeneration1,4-7. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity7-10. In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic11-13. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules14 (please see a schematic in Fig. 1). Retinoic acid (RA) does not induce neuronal differentiation of undifferentiated hESCs maintained on feeders1, 14. And unlike mouse ESCs, treating hESC-differentiated embryoid bodies (EBs) only slightly increases the low yield of neurons1, 14, 15. However, after screening a variety of small molecules and growth factors, we found that such defined conditions rendered retinoic acid (RA) sufficient to induce the specification of neuroectoderm direct from pluripotent hESCs that further progressed to neuroblasts that generated human neuronal progenitors and neurons in the developing CNS with high efficiency (Fig. 2). We defined conditions for induction of neuroblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human neuronal cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of neuroblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human neuronal progenitors and neuronal cell types in the developing CNS for neural repair.

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system ...

Generation of Induced Neural Stem Cells from Peripheral Mononuclear Cells and Differentiation Toward Dopaminergic Neuron Precursors for Transplantation Studies

Parkinson&#39;s disease (PD) is caused by degeneration of dopaminergic (DA) neurons at the substantia nigra pars compacta (SNpc) in the ventral mesencephalon (VM). Cell replacement therapy holds great promise for treatment of PD. Recently, induced neural stem cells (iNSCs) have emerged as a potential candidate for cell replacement therapy due to the reduced risk of tumor formation and the plasticity to give rise to region-specific neurons and glia. iNSCs can be reprogrammed from autologous somatic cellular sources, such as fibroblasts, peripheral blood mononuclear cells (PBMNCs) and various other types of cells. Compared with other types of somatic cells, PBMNCs are an appealing starter cell type because of the ease to access and expand in culture. Sendai virus (SeV), an RNA non-integrative virus, encoding reprogramming factors including human OCT3/4, SOX2, KLF4 and c-MYC, has a negative-sense, single-stranded, non-segmented genome that does not integrate into host genome, but only replicates in the cytoplasm of infected cells, offering an efficient and safe vehicle for reprogramming. In this study, we describe a protocol in which iNSCs are obtained by reprogramming PBMNCs, and differentiated into specialized VM DA neurons by a two-stage method. Then DA precursors are transplanted into unilaterally 6-hyroxydopamine (6-OHDA)-lesioned PD mouse models to evaluate the safety and efficacy for treatment of PD. This method provides a platform to investigate the functions and therapeutic effects of patient-specific DA neural cells in vitro and in vivo.

The protocol presents the reprogramming of peripheral blood mononuclear cells to induce neural stem cells by Sendai virus infection, differentiation of iNSCs into dopaminergic neurons, transplantation of DA precursors into the unilaterally-lesioned Parkinson&#39;s disease mouse models, and evaluation of the safety and efficacy of iNSC-derived DA precursors for PD treatment.

Parkinson&#39;s disease (PD) is caused by degeneration of dopaminergic (DA) neurons at the substantia nigra pars compacta (SNpc) in the ventral ...

Efficient Differentiation of Postganglionic Sympathetic Neurons using Human Pluripotent Stem Cells under Feeder-free and Chemically Defined Culture Conditions

Human pluripotent stem cells (hPSCs) have become a powerful tool for disease modeling and the study of human embryonic development in vitro. We previously presented a differentiation protocol for the derivation of autonomic neurons with sympathetic character that has been applied to patients with autonomic neuropathy. However, the protocol was built on Knock Out Serum Replacement (KSR) and feeder-based culture conditions, and to ensure high differentiation efficiency, cell sorting was necessary. These factors cause high variability, high cost, and low reproducibility. Moreover, mature sympathetic properties, including electrical activity, have not been verified. Here, we present an optimized protocol where PSC culture and differentiation are performed in feeder-free and chemically defined culture conditions. Genetic markers identifying trunk neural crest are identified. Further differentiation into postganglionic sympathetic neurons is achieved after 20 days without the need for cell sorting. Electrophysiological recording further shows the functional neuron identity. Firing detected from our differentiated neurons can be enhanced by nicotine and suppressed by the adrenergic receptor antagonist propranolol. Intermediate sympathetic neural progenitors in this protocol can be maintained as neural spheroids for up to 2 weeks, which allows expansion of the cultures. In sum, our updated sympathetic neuron differentiation protocol shows high differentiation efficiency, better reproducibility, more flexibility, and better neural maturation compared to the previous version. This protocol will provide researchers with the cells necessary to study human disorders that affect the autonomic nervous system.

In this protocol, we describe a stable, highly efficient differentiation strategy for the generation of postganglionic sympathetic neurons from human pluripotent stem cells. This model will make neurons available for the use of studies of multiple autonomic disorders.

Human pluripotent stem cells (hPSCs) have become a powerful tool for disease modeling and the study of human embryonic development in vitro. We previously ...

Directed Differentiation of Hemogenic Endothelial Cells from Human Pluripotent Stem Cells

Blood vessels are ubiquitously distributed within all tissues of the body and perform diverse functions. Thus, derivation of mature vascular endothelial cells, which line blood vessel lumens, from human pluripotent stem cells is crucial for a multitude of tissue engineering and regeneration applications. In vivo, primordial endothelial cells are derived from the mesodermal lineage and are specified toward specific subtypes, including arterial, venous, capillary, hemogenic, and lymphatic. Hemogenic endothelial cells are of particular interest because, during development, they give rise to hematopoietic stem and progenitor cells, which then generate all blood lineages throughout life. Thus, creating a system to generate hemogenic endothelial cells in vitro would provide an opportunity to study endothelial-to-hematopoietic transition, and may lead to ex vivo production of human blood products and reduced reliance on human donors. While several protocols exist for the derivation of progenitor and primordial endothelial cells, generation of well-characterized hemogenic endothelial cells from human stem cells has not been described. Here, a method for the derivation of hemogenic endothelial cells from human embryonic stem cells in approximately 1 week is presented: a differentiation protocol with primitive streak cells formed in response to GSK3&#946; inhibitor (CHIR99021), then mesoderm lineage induction mediated by bFGF, followed by primordial endothelial cell development promoted by BMP4 and VEGF-A, and finally hemogenic endothelial cell specification induced by retinoic acid. This protocol yields a well-defined population of hemogenic endothelial cells that can be used to further understand their molecular regulation and endothelial-to-hematopoietic transition, which has the potential to be applied to downstream therapeutic applications.

Presented here is a simple protocol for the directed differentiation of hemogenic endothelial cells from human pluripotent stem cells in approximately 1 week.

Blood vessels are ubiquitously distributed within all tissues of the body and perform diverse functions. Thus, derivation of mature vascular endothelial ...

Rapid and Efficient Generation of Neurons from Human Pluripotent Stem Cells in a Multititre Plate Format

Existing protocols for the generation of neurons from human pluripotent stem cells (hPSCs) are often tedious in that they are multistep procedures involving the isolation and expansion of neural precursor cells, prior to terminal differentiation. In comparison to these time-consuming approaches, we have recently found that combined inhibition of three signaling pathways, TGF&beta;/SMAD2, BMP/SMAD1, and FGF/ERK, promotes rapid induction of neuroectoderm from hPSCs, followed by immediate differentiation into functional neurons. Here, we have adapted our procedure to a novel multititre plate format, to further enhance its reproducibility and to make it compatible with mid-throughput applications. It comprises four days of neuroectoderm formation in floating spheres (embryoid bodies), followed by a further four days of differentiation into neurons under adherent conditions. Most cells obtained with this protocol appear to be bipolar sensory neurons. Moreover, the procedure is highly efficient, does not require particular expert skills, and is based on a simple chemically defined medium with cost-efficient small molecules. Due to these features, the procedure may serve as a useful platform for further functional investigation as well as for cell-based screening approaches requiring human sensory neurons or neurons of any type.

Protocols for neuronal differentiation of pluripotent human stem cells (hPSCs) are often time-consuming and require substantial cell culture skills. Here, we have adapted a small molecule-based differentiation procedure to a multititre plate format, allowing simple, rapid, and efficient generation of human neurons in a controlled manner.

Existing protocols for the generation of neurons from human pluripotent stem cells (hPSCs) are often tedious in that they are  multistep procedures ...

Biology

Automated Production of Human Induced Pluripotent Stem Cell-Derived Cortical and Dopaminergic Neurons with Integrated Live-Cell Monitoring

Manual culture and differentiation protocols for human induced pluripotent stem cells (hiPSC) are difficult to standardize, show high variability and are prone to spontaneous differentiation into unwanted cell types. The methods are labor-intensive and are not easily amenable to large-scale experiments. To overcome these limitations, we developed an automated cell culture system coupled to a high-throughput imaging system and implemented protocols for maintaining multiple hiPSC lines in parallel and neuronal differentiation. We describe the automation of a short-term differentiation protocol using Neurogenin-2 (NGN2) over-expression to produce hiPSC-derived cortical neurons within 6&#8210;8 days, and the implementation of a long-term differentiation protocol to generate hiPSC-derived midbrain dopaminergic (mDA) neurons within 65 days. Also, we applied the NGN2 approach to a small molecule-derived neural precursor cells (smNPC)&#160;transduced with GFP lentivirus and established a live-cell automated neurite outgrowth assay. We present an automated system with protocols suitable for routine hiPSC culture and differentiation into cortical and dopaminergic neurons. Our platform is suitable for long term hands-free culture and high-content/high-throughput hiPSC-based compound, RNAi and CRISPR/Cas9 screenings to identify novel disease mechanisms and drug targets.

We show the automation of human induced pluripotent stem cell (hiPSC) cultures and neuronal differentiations compatible with automated imaging and analysis.

Manual culture and differentiation protocols for human induced pluripotent stem cells (hiPSC) are difficult to standardize, show high variability and are ...

Generation of Dopaminergic Neuronal Organoids from Human Embryonic Stem Cells

Source: Cosset, E. et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/59682">Human Neural Organoids for Studying Brain Cancer and Neurodegenerative Diseases.</a> J. Vis. Exp. (2019)
This video demonstrates the differentiation of human embryonic stem cells (hESCs) into dopaminergic neurons, to provide a model for studying Parkinson&#39;s disease. The hESCs are cultured in a neural induction medium in a microwell plate to form 3D neurospheres. These are then cultured in a neural induction medium to promote the differentiation into dopaminergic progenitor neurons. The final maturation of the neurons occurs in a culture insert under air-liquid interface conditions, resulting in 3D organoids consisting of mature dopaminergic neurons.

1. hESC-derived dopaminergic organoids for Parkinson&#39;s Disease (PD) studies

	Day 0: Amplify hESCs in 2D culture up to 60% confluency (day 0), then ...

JoVE Encyclopedia of Experiments

Neuropathology

Neurodegenerative Diseases

Differentiating Non-Transgenic and Transgenic Human Pluripotent Stem Cells into Neural Progenitor Cells

Source: Cvetkovic, C., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/58034">Synaptic Microcircuit Modeling with 3D Cocultures of Astrocytes and Neurons from Human Pluripotent Stem Cells.</a> J. Vis. Exp. (2018).
This video demonstrates the generation of neural progenitor cells from non-transgenic and transgenic human pluripotent stem cells (hPSCs). hPSCs are differentiated into neural progenitor cells using a neuronal differentiation medium. While differentiation is induced in non-transgenic cells using small molecules, the transgenic hPSCs are differentiated by adding an antibiotic that induces the promoter for a neuronal differentiation-specific transcription factor.

Ethic approvals were obtained when performing the experiments.
1. Cell Culture and Reagent Preparation

	Prepare coated plates for cell culture.
	
		...

Neuronal Culture Techniques

Advanced Neuronal Models

Human Pluripotent Stem Cell Differentiation in Neurons Using Engineered Lentiviruses

Source: Assetta, B., et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/61778">Generation of Human Neurons and Oligodendrocytes from Pluripotent Stem Cells for Modeling Neuron-Oligodendrocyte Interactions.</a> J. Vis. Exp. (2020).
This video demonstrates the process of inducing human pluripotent stem cells to differentiate into neurons by transfecting them with engineered lentiviruses carrying a neurogenic transcription factor gene under the control of&#160; tetracycline-responsive element, followed by treatment with a tetracycline derivative and maturation medium.

1. Human neuron induction from human pluripotent stem cells

	Lentivirus preparation (~5 days, detailed protocol as described previously)
	
		Plate ~1 ...

Directed Dopaminergic Neuron Differentiation from Human Pluripotent Stem Cells

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