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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H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+and+in+vivo+analyses+of+human+embryonic+stem+cell-derived+dopamine+neurons.">In vitro and in vivo analyses of human embryonic stem cell-derived dopamine neurons.</a> Journal of Neurochemistry. 92, 1265-1276 (2005).</li><li>Buytaert-Hoefen, K. A., Alvarez, E., Freed, C. 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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.

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			<td height="21" style="height:21px;width:308px;">Name of Material/ Equipment</td>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">penicillin/ampicillin</td>
			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">15140-122</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">DMEM/F12</td>
			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">10565-018</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">KSR</td>
			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">10828-028</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Non-essential amino acid</td>
			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">11140-050</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">b-mercaptoethanol</td>
			<td style="width:184px;">Millipore</td>
			<td style="width:119px;">ES-007-E</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">bFGF</td>
			<td style="width:184px;">R&#38;D Systems</td>
			<td style="width:119px;">233-FB-025</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:308px;">mTesR1</td>
			<td style="width:184px;">STEMCELL Technologies</td>
			<td style="width:119px;">5850</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Collagenase IV</td>
			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">17104-019</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Gelatin</td>
			<td style="width:184px;">Sigma-Aldrich</td>
			<td style="width:119px;">G9391</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">N2 supplement</td>
			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">17502-048</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">B27 supplement</td>
			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">17504-044</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Neurobasal</td>
			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">21103-049</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Glutamax</td>
			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">35050-061</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">PBS</td>
			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">10010-023</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Growth factor reduced matrigel</td>
			<td style="width:184px;">BD Biosciences</td>
			<td style="width:119px;">354230</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:308px;">Accutase</td>
			<td style="width:184px;">MP Biomedicals</td>
			<td style="width:119px;">1000449</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:308px;">Thiazovivin</td>
			<td style="width:184px;">Santa Cruz Biotechnology</td>
			<td style="width:119px;">sc-361380</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">SB431542</td>
			<td style="width:184px;">Tocris Bioscience</td>
			<td style="width:119px;">1614</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">LDN-193189</td>
			<td style="width:184px;">Stemgent</td>
			<td style="width:119px;">04-0074</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">SAG</td>
			<td style="width:184px;">EMD Millipore</td>
			<td style="width:119px;">566660-1MG</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:308px;">Purmorphamine</td>
			<td style="width:184px;">Santa Cruz Biotechnology</td>
			<td style="width:119px;">sc-202785</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">FGF8b</td>
			<td style="width:184px;">R&#38;D Systems</td>
			<td style="width:119px;">423-F8-025</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">CHIR99021</td>
			<td style="width:184px;">Cellagen Technology</td>
			<td style="width:119px;">C2447-2s</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">BDNF</td>
			<td style="width:184px;">R&#38;D Systems</td>
			<td style="width:119px;">248-BD-025</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">GDNF</td>
			<td style="width:184px;">R&#38;D Systems</td>
			<td style="width:119px;">212-GD-010</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">TGF-beta3</td>
			<td style="width:184px;">R&#38;D Systems</td>
			<td style="width:119px;">243-B3-002</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Ascorbic acid</td>
			<td style="width:184px;">Sigma-Aldrich</td>
			<td style="width:119px;">A4034</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">cAMP</td>
			<td style="width:184px;">Sigma-Aldrich</td>
			<td style="width:119px;">D0627</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:308px;">Mouse anti human NESTIN antibody</td>
			<td style="width:184px;">Santa Cruz Biotechnology</td>
			<td style="width:119px;">sc-23927</td>
			<td style="width:167px;">1/1000 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Rabbit anti human OTX2 antibody</td>
			<td style="width:184px;">Millipore</td>
			<td style="width:119px;">AB9566</td>
			<td style="width:167px;">1/2000 dilutiion</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Goat anti human FOXA2 antibody</td>
			<td style="width:184px;">R&#38;D Systems</td>
			<td style="width:119px;">AF2400</td>
			<td style="width:167px;">1/200 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">rabbit anti human LMX1a antibody</td>
			<td style="width:184px;">Millipore</td>
			<td style="width:119px;">AB10533</td>
			<td style="width:167px;">1/1000 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Rabbit anti human TH antibody</td>
			<td style="width:184px;">Pel Freez</td>
			<td style="width:119px;">P40101</td>
			<td style="width:167px;">1/500 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Chicken anti human TH antibody</td>
			<td style="width:184px;">Millipore</td>
			<td style="width:119px;">AB9702</td>
			<td style="width:167px;">1/500 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Mouse anti human TUJ1 antibody</td>
			<td style="width:184px;">Covance</td>
			<td style="width:119px;">MMS-435P</td>
			<td style="width:167px;">1/2000 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Rabbit anti human GIRK2 antibody</td>
			<td style="width:184px;">Abcam</td>
			<td style="width:119px;">ab30738</td>
			<td style="width:167px;">1/300 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Rabbit anti human Calbindin antibody</td>
			<td style="width:184px;">Abcam</td>
			<td style="width:119px;">ab25085</td>
			<td style="width:167px;">1/400 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Centrifuge</td>
			<td style="width:184px;">Eppendorf</td>
			<td style="width:119px;">5804</td>
			<td style="width:167px;"></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 ...

directed-dopaminergic-neuron-differentiation-from-human-pluripotent

Nanyang Technological University

Research

JoVE Journal

Neuroscience

16.4K 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

Generation of Neural Stem Cells from Discarded Human Fetal Cortical Tissue

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ultimately give rise to intermediate progenitors and later, the various neuronal and glial cell subtypes that form the cerebral cortex. The capacity to generate and expand human NSCs (so called neurospheres) from discarded normal fetal tissue provides a means with which to directly study the functional aspects of normal human NSC development 1-5. This approach can also be directed toward the generation of NSCs from known neurological disorders, thereby affording the opportunity to identify disease processes that alter progenitor proliferation, migration and differentiation 6-9. We have focused on identifying pathological mechanisms in human Down syndrome NSCs that might contribute to the accelerated Alzheimer's disease phenotype 10,11. Neither in vivo nor in vitro mouse models can replicate the identical repertoire of genes located on human chromosome 21. 
Here we use a simple and reliable method to isolate Down syndrome NSCs from aborted human fetal cortices and grow them in culture. The methodology provides specific aspects of harvesting the tissue, dissection with limited anatomical landmarks, cell sorting, plating and passaging of human NSCs. We also provide some basic protocols for inducing differentiation of human NSCs into more selective cell subtypes.

A simple and reliable method on isolation and culture of neural stem cells from discarded human fetal cortical tissue is described. Cultures derived from known human neurological disorders can be used for characterization of pathological cellular and molecular processes, as well as provide a platform to assess pharmacological efficacy.

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ...

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 ...

The Specification of Telencephalic Glutamatergic Neurons from Human Pluripotent Stem Cells

Here, a stepwise procedure for efficiently generating telencephalic glutamatergic neurons from human pluripotent stem cells (PSCs) has been described. The differentiation process is initiated by breaking the human PSCs into clumps which round up to form aggregates when the cells are placed in a suspension culture. The aggregates are then grown in hESC medium from days 1-4 to allow for spontaneous differentiation. During this time, the cells have the capacity to become any of the three germ layers. From days 5-8, the cells are placed in a neural induction medium to push them into the neural lineage. Around day 8, the cells are allowed to attach onto 6 well plates and differentiate during which time the neuroepithelial cells form. These neuroepithelial cells can be isolated at day 17. The cells can then be kept as neurospheres until they are ready to be plated onto coverslips. Using a basic medium without any caudalizing factors, neuroepithelial cells are specified into telencephalic precursors, which can then be further differentiated into dorsal telencephalic progenitors and glutamatergic neurons efficiently. Overall, our system provides a tool to generate human glutamatergic neurons for researchers to study the development of these neurons and the diseases which affect them.

This procedure yields telencephalic neurons by going through checkpoints which are similar to those observed during human development. The cells are allowed to spontaneously differentiate, are exposed to factors which push them towards the neural lineage, are isolated, and are plated onto coverslips to allow for terminal differentiation and maturation.

Here, a stepwise procedure for efficiently generating telencephalic glutamatergic neurons from human pluripotent stem cells (PSCs) has been described. The ...

A cGMP-applicable Expansion Method for Aggregates of Human Neural Stem and Progenitor Cells Derived From Pluripotent Stem Cells or Fetal Brain Tissue

A cell expansion technique to amass large numbers of cells from a single specimen for research experiments and clinical trials would greatly benefit the stem cell community. Many current expansion methods are laborious and costly, and those involving complete dissociation may cause several stem and progenitor cell types to undergo differentiation or early senescence. To overcome these problems, we have developed an automated mechanical passaging method referred to as &#8220;chopping&#8221; that is simple and inexpensive. This technique avoids chemical or enzymatic dissociation into single cells and instead allows for the large-scale expansion of suspended, spheroid cultures that maintain constant cell/cell contact. The chopping method has primarily been used for fetal brain-derived neural progenitor cells or neurospheres, and has recently been published for use with neural stem cells derived from embryonic and induced pluripotent stem cells. The procedure involves seeding neurospheres onto a tissue culture Petri dish and subsequently passing a sharp, sterile blade through the cells effectively automating the tedious process of manually mechanically dissociating each sphere. Suspending cells in culture provides a favorable surface area-to-volume ratio; as over 500,000 cells can be grown within a single neurosphere of less than 0.5 mm in diameter. In one T175 flask, over 50 million cells can grow in suspension cultures compared to only 15 million in adherent cultures. Importantly, the chopping procedure has been used under current good manufacturing practice (cGMP), permitting mass quantity production of clinical-grade cell products.

This protocol describes a novel mechanical chopping method that allows the expansion of spherical neural stem and progenitor cell aggregates without dissociation to a single cell suspension.&#160; Maintaining cell/cell contact allows rapid and stable growth for over 40 passages.

A cell expansion technique to amass large numbers of cells from a single specimen for research experiments and clinical trials would greatly benefit the ...

Feeder-free Derivation of Neural Crest Progenitor Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a source of transplantable cells for regenerative applications after disease or accidents. Neural crest (NC) cells are the precursors for a large variety of adult somatic cells, such as cells from the peripheral nervous system and glia, melanocytes and mesenchymal cells. They are a valuable source of cells to study aspects of human embryonic development, including cell fate specification and migration. Further differentiation of NC progenitor cells into terminally differentiated cell types offers the possibility to model human diseases in vitro, investigate disease mechanisms and generate cells for regenerative medicine. This article presents the adaptation of a currently available in vitro differentiation protocol for the derivation of NC cells from hPSCs. This new protocol requires 18 days of differentiation, is feeder-free, easily scalable and highly reproducible among human embryonic stem cell (hESC) lines as well as human induced pluripotent stem cell (hiPSC) lines. Both old and new protocols yield NC cells of equal identity.

Neural crest (NC) cells derived from human pluripotent stem cells (hPSC) have great potential for modeling human development and disease and for cell replacement therapies. Here, a feeder-free adaptation of the currently widely used in vitro differentiation protocol for the derivation of NC cells from hPSCs is presented.

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a ...

Post-differentiation Replating of Human Pluripotent Stem Cell-derived Neurons for High-content Screening of Neuritogenesis and Synapse Maturation

Neurons differentiated in two-dimensional culture from human pluripotent stem-cell-derived neural progenitor cells (NPCs) represent a powerful model system to explore disease mechanisms and carry out high content screening (HCS) to interrogate compound libraries or identify gene mutation phenotypes. However, with human cells the transition from NPC to functional, mature neuron requires several weeks. Synapses typically start to form after 3 weeks of differentiation in monolayer culture, and several neuron-specific proteins, for example the later expressing pan-neuronal marker NeuN, or the layer 5/6 cerebral cortical neuron marker CTIP2, begin to express around 4-5 weeks post-differentiation. This lengthy differentiation time can be incompatible with optimal culture conditions used for small volume, multi-well HCS platforms. Among the many challenges are the need for well-adhered, uniformly distributed cells with minimal cell clustering, and culture procedures that foster long-term viability and functional synapse maturation. One approach is to differentiate neurons in a large volume format, then replate them at a later time point in HCS-compatible multi-wells. Some main challenges when using this replating approach concern reproducibility and cell viability, due to the stressful disruption of the dendritic and axonal network. Here we demonstrate a detailed and reliable procedure for enzymatically resuspending human induced pluripotent stem cell (hiPSC)-derived neurons after their differentiation for 4-8 weeks in a large-volume format, transferring them to 384-well microtiter plates, and culturing them for a further 1-3 weeks with excellent cell survival. This replating of human neurons not only allows the study of synapse assembly and maturation within two weeks from replating, but also enables studies of neurite regeneration and growth cone characteristics. We provide examples of scalable assays for neuritogenesis and synaptogenesis using a 384-well platform.

This protocol describes a detailed procedure for resuspending and culturing human stem cell derived neurons that were previously differentiated from neural progenitors in vitro for multiple weeks. The procedure facilitates imaging-based assays of neurites, synapses, and late-expressing neuronal markers in a format compatible with light microscopy and high-content screening.

Neurons differentiated in two-dimensional culture from human pluripotent stem-cell-derived neural progenitor cells (NPCs) represent a powerful model ...

Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro

The neural differentiation of mouse embryonic stem cells (mESCs) is a potential tool for elucidating the key mechanisms involved in neurogenesis and potentially aid in regenerative medicine. Here, we established an efficient and low cost method for neuronal differentiation from mESCs in vitro, using the strategy of combinatorial screening. Under the conditions defined here, the 2-day embryoid body formation + 6-day retinoic acid induction protocol permits fast and efficient differentiation from mESCs into neural precursor cells (NPCs), as seen by the formation of well-stacked and neurite-like A2lox and 129 derivatives that are Nestin positive. The healthy state of embryoid bodies and the timepoint at which retinoic acid (RA) is applied, as well as the RA concentrations, are critical in the process. In the subsequent differentiation from NPCs into neurons, N2B27 medium II (supplemented by Neurobasal medium) could better support the long term maintenance and maturation of neuronal cells. The presented method is highly efficiency, low cost and easy to operate, and can be a powerful tool for neurobiology and developmental biology research.

Here, we established a low cost and easy to operate method that directs fast and efficient differentiation from embryonic stem cells into neurons. This method is suitable for popularization among laboratories and can be a useful tool for neurological research.

The neural differentiation of mouse embryonic stem cells (mESCs) is a potential tool for elucidating the key mechanisms involved in neurogenesis and ...

Derivation, Expansion, Cryopreservation and Characterization of Brain Microvascular Endothelial Cells from Human Induced Pluripotent Stem Cells

Brain microvascular endothelial cells (BMECs) can be differentiated from human induced pluripotent stem cells (iPSCs) to develop ex vivo cellular models for studying blood-brain barrier (BBB) function. This modified protocol provides detailed steps to derive, expand, and cryopreserve BMECs from human iPSCs using a different donor and reagents than those reported in previous protocols. iPSCs are treated with essential 6 medium for 4 days, followed by 2 days of human endothelial serum-free culture medium supplemented with basic fibroblast growth factor, retinoic acid, and B27 supplement. At day 6, cells are sub-cultured onto a collagen/fibronectin matrix for 2 days. Immunocytochemistry is performed at day 8 for BMEC marker analysis using CLDN5, OCLN, TJP1, PECAM1, and SLC2A1. Western blotting is performed to confirm BMEC marker expression, and absence of SOX17, an endodermal marker. Angiogenic potential is demonstrated with a sprouting assay. Trans-endothelial electrical resistance (TEER) is measured using chopstick electrodes and voltohmmeter starting at day 7. Efflux transporter activity for ATP binding cassette subfamily B member 1 and ATP binding cassette subfamily C member 1 is measured using a multi-plate reader at day 8. Successful derivation of BMECs is confirmed by the presence of relevant cell markers, low levels of SOX17, angiogenic potential, transporter activity, and TEER values ~2000 &#937; x cm2. BMECs are expanded until day 10 before passaging onto freshly coated collagen/fibronectin plates or cryopreserved. This protocol demonstrates that iPSC-derived BMECs can be expanded and passaged at least once. However, lower TEER values and poorer localization of BMEC markers was observed after cryopreservation. BMECs can be utilized in co-culture experiments with other cell types (neurons, glia, pericytes), in three-dimensional brain models (organ-chip and hydrogel), for vascularization of brain organoids, and for studying BBB dysfunction in neuropsychiatric disorders.

This protocol details an adapted method to derive, expand, and cryopreserve brain microvascular endothelial cells obtained by differentiating human induced pluripotent stem cells, and to study blood brain barrier properties in an ex vivo model.

Brain microvascular endothelial cells (BMECs) can be differentiated from human induced pluripotent stem cells (iPSCs) to develop ex vivo cellular models ...

Generation of Human Neurons and Oligodendrocytes from Pluripotent Stem Cells for Modeling Neuron-Oligodendrocyte Interactions

In Alzheimer&#8217;s disease (AD) and other neurodegenerative disorders, oligodendroglial failure is a common early pathological feature, but how it contributes to disease development and progression, particularly in the gray matter of the brain, remains largely unknown. The dysfunction of oligodendrocyte lineage cells is hallmarked by deficiencies in myelination and impaired self-renewal of oligodendrocyte precursor cells (OPCs). These two defects are caused at least in part by the disruption of interactions between neuron and oligodendrocytes along the buildup of pathology. OPCs give rise to myelinating oligodendrocytes during CNS development. In the mature brain cortex, OPCs are the major proliferative cells (comprising ~5% of total brain cells) and control new myelin formation in a neural activity-dependent manner. Such neuron-to-oligodendrocyte communications are significantly understudied, especially in the context of neurodegenerative conditions such as AD, due to the lack of appropriate tools. In recent years, our group and others have made significant progress to improve currently available protocols to generate functional neurons and oligodendrocytes individually from human pluripotent stem cells. In this manuscript, we describe our optimized procedures, including the establishment of a co-culture system to model the neuron-oligodendrocyte connections. Our illustrative results suggest an unexpected contribution from OPCs/oligodendrocytes to the brain amyloidosis and synapse integrity and highlight the utility of this methodology for AD research. This reductionist approach is a powerful tool to dissect the specific hetero-cellular interactions out of the inherent complexity inside the brain. The protocols we describe here are expected to facilitate future studies on oligodendroglial defects in the pathogenesis of neurodegeneration.

The neuron-glial interactions in neurodegeneration are not well understood due to inadequate tools and methods. Here, we describe optimized protocols to obtain induced neurons, oligodendrocyte precursor cells, and oligodendrocytes from human pluripotent stem cells and provide examples of the values of these methods in understanding cell-type-specific contributions in Alzheimer&#8217;s disease.

In Alzheimer&#8217;s disease (AD) and other neurodegenerative disorders, oligodendroglial failure is a common early pathological feature, but how it ...

Quantifying Levels of Dopaminergic Neuron Morphological Alteration and Degeneration in Caenorhabditis elegans

Dopamine neuron loss is involved in the pathology of Parkinson&#39;s Disease (PD), a highly prevalent neurodegenerative disorder affecting over 10 million people worldwide. Since many details about PD etiology remain unknown, studies investigating genetic and environmental contributors to PD are needed to discover methods of prevention, management, and treatment. Proper characterization of dopaminergic neuronal loss may be relevant not only to PD research, but to other increasingly prevalent neurodegenerative disorders.
There are established genetic and chemical models of dopaminergic neurodegeneration in the Caenorhabditis elegans model system, with easy visualization of neurobiology supported by the nematodes&#39; transparency and invariant neuronal architecture. In particular, hermaphroditic C. elegans&#39; dopaminergic neuron morphological changes can be visualized using strains with fluorescent reporters driven by cell-specific promotors such as the dat-1 dopamine transporter gene, which is expressed exclusively in their eight dopaminergic neurons.
With the capabilities of this model system and the appropriate technology, many laboratories have studied dopaminergic neurodegeneration. However, there is little consistency in the way the data is analyzed and much of the present literature uses binary scoring analyses that capture the presence of degeneration but not the full details of the progression of neuron loss. Here, we introduce a universal scoring system to assess morphological changes and degeneration in C. elegans&#39; cephalic neuron dendrites. This seven-point scale allows for analysis across a full range of dendrite morphology, ranging from healthy neurons to complete dendrite loss, and considering morphological details including kinks, branching, blebs, and breaks. With this scoring system, researchers can quantify subtle age-related changes as well as more dramatic chemical-induced changes. Finally, we provide a practice set of images with commentary that can be used to train, calibrate, and assess the scoring consistency of researchers new to this method. This should improve within- and between- laboratory consistency, increasing rigor and reproducibility.

Quantifying Levels of Dopaminergic Neuron Morphological Alteration and Degeneration in Caenorhabditis elegans

In this article, we showcase how to use a seven-point scoring system to consistently quantify changes to dopaminergic neuron dendrite morphology in C. elegans. This system is intended for analyses of dopaminergic neurodegeneration assays utilizing genetic, chemical, and age-based models of neurodegenerative disorders.

Dopamine neuron loss is involved in the pathology of Parkinson&#39;s Disease (PD), a highly prevalent neurodegenerative disorder affecting over 10 million ...

Directed Dopaminergic Neuron Differentiation from Human Pluripotent Stem Cells

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Chapters in this video

Generation of Neural Stem Cells from Discarded Human Fetal Cortical Tissue

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

The Specification of Telencephalic Glutamatergic Neurons from Human Pluripotent Stem Cells

A cGMP-applicable Expansion Method for Aggregates of Human Neural Stem and Progenitor Cells Derived From Pluripotent Stem Cells or Fetal Brain Tissue

Feeder-free Derivation of Neural Crest Progenitor Cells from Human Pluripotent Stem Cells

Post-differentiation Replating of Human Pluripotent Stem Cell-derived Neurons for High-content Screening of Neuritogenesis and Synapse Maturation

Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro

Derivation, Expansion, Cryopreservation and Characterization of Brain Microvascular Endothelial Cells from Human Induced Pluripotent Stem Cells

Generation of Human Neurons and Oligodendrocytes from Pluripotent Stem Cells for Modeling Neuron-Oligodendrocyte Interactions

Quantifying Levels of Dopaminergic Neuron Morphological Alteration and Degeneration in Caenorhabditis elegans