XHP has been supported by National Institute of Health (NIH) grants from National Institute on Aging (NIHK01AG024496) and The Eunice Kennedy Shriver National Institute of Child Health and Human Development (NIHR21HD056530).

1. Solution and Media Preparation
<ol>
	<li>Gelatin coating solution: 0.1% (w/v) gelatin in ddH2O, autoclaved and store at 4&deg;C.</li>
	<li>Matrigel coating solutions. Stock solution: slow thaw Matrigel (10 ml) at 4&deg;C overnight, add 10 ml ice-cold DMEM or DMEM/F12, mix well and aliquot 1 ml/tube underneath sterilized tissue culture hood, store at -20&deg;C. Working solution: slow thaw 1 ml Matrigel aliquot at 4&deg;C for 1-2 hour, transfer to 14 ml chilled DMEM or DMEM/F12 and mix well underneath sterilized tissue culture hood immediately before coating.</li>
	<li>Human laminin coating solution: dilute 1 ml human laminin solution (0.5 mg/ml in Tris Buffered NaCl, store at -80&deg;C, slow thaw at 4&deg;C for 1-2 hour) with ice-cold DMEM or DMEM/F12 to 12.5 ml working solution (40 &mu;g/ml) underneath sterilized tissue culture hood immediately before coating.</li>
	<li>Growth factor stock solutions (500-1000X): dissolve growth factor at 10 &mu;g/ml in sterilized buffer (0.5% BSA, 1.0 mM DTT, 10% glycerol, 1XPBS) and store as 50-100 &mu;l/tube aliquots at -80&deg;C.</li>
	<li>All-trans-retinoic acid working solution (1000X): 10 mM in DMSO, store in aliquots at -80&deg;C.</li>
	<li>HESC media: DMEM/F12 or KO-DMEM (80%), KO serum replacement (20%), L-alanyl-L-gln or L-gln (2 mM), MEM nonessential amino acids (MNAA, 1X), and &beta;-Mercaptoethanol (100 &mu;M), filtered and store at 4&deg;C, supplemented with 20 ng/ml bFGF before use. Or replacing "knock out (KO) serum replacement" with defined components: DMEM/F12 or KO-DMEM (100%), L-alanyl-L-gln or L-gln (2 mM), MNAA (1X), MEM essential amino acids (MEAA, 1X), and &beta;-Mercaptoethanol (100 &mu;M), filtered and store at 4&deg;C, supplemented with bFGF (20 ng/ml), human insulin (20 &mu;g/ml), ascorbic acid (50 &mu;g/ml), human activin A (50 ng/ml), human albumin/Albumax (10 mg/ml), and human transferrin (8 &mu;g/ml) before use.</li>
	<li>NSC media: DMEM/F12 (100%), N-2 supplement (1X), heparin (8 &mu;g/ml).</li>
</ol>
2. Plate Coating
<ol>
	<li>Coat plates with gelatin: add 2.5 ml/well of gelatin solution to 6 well plates and incubate overnight in a 37&deg;C humidified incubator.</li>
	<li>Coat plates with laminin: chill gelatin-coated plates and remove gelatin, add 2.5 ml/well Matrigel or human laminin coating working solution to pre-chilled gelatinized 6-well plates and incubate at 4&deg;C overnight.</li>
</ol>
3. Passaging and Seeding Undifferentiated hESCs under Defined Conditions
<ol>
	<li>Allow hESC colonies grow to 5-7 days old, and take hESC culture plate to dissecting microscope (pre-warm dissecting stage to 37&deg;C) underneath dissecting sterilized hood.</li>
	<li>Select hESC colonies to be split. Morphologically, these colonies should have &gt; 75% undifferentiated hESCs (small compact cells), usually slightly opaque (not white-piled-up cells, not clear-differentiated cells) with defined edge underneath the dissecting microscope. Carefully outline the selected colonies and remove differentiated fibroblast layer surrounding the colony and all differentiated parts (if any) of the colony with the edge of P2 sterile pipette tip or pulled glass capillary. (Please note, pluripotent hESCs are at a transient stage, undergo spontaneous differentiation even under optimal conditions, although preferred, it's difficult to ascertain theses cells are 100% undifferentiated under dissecting microscope, &gt;75% undifferentiated are usually considered as the pass point. The differentiated cells usually will not seed and continue to grow, eliminated in the passaging process)</li>
	<li>Remove the old media containing floating detached differentiated cells by aspirating. Wash with hESC media (without bFGF) once. Add 3 ml/well fresh hESC media containing 20 ng/ml bFGF.</li>
	<li>Cut the undifferentiated hESC colonies into small pieces, and detach with sterile pipette tip or pulled glass capillary.</li>
	<li>Pool the media containing detached colony pieces together in a 50 ml conical tube. Wash the plate once with 1 ml/well hESC media containing 20 ng/ml bFGF and pool together.</li>
	<li>Aspirate the Matrigel or human laminin solution from the coated fresh plates. Aliquot 4 ml/well hESC media containing colony pieces to a 6-well plate. Gently transfer the plate to incubator without shaking and allow colony pieces seed overnight without disturbing in a humidified 37&deg;C incubator with an atmosphere of 5% CO2.</li>
</ol>
4. Neural Induction of hESCs under Defined Culture System with Retinoic Acid
<ol>
	<li>At day 3 after seeding, remove most of old media from each well of the plate and leave enough media to allow hESC colonies to be submerged (never allow hESCs to dry out). Replace with 4 ml/well fresh hESC media containing 20 ng/ml bFGF and 10 &mu;M retinoic acid.</li>
	<li>Replace old media with fresh hESC media containing 20 ng/ml bFGF and 10 &mu;M retinoic acid every other day, and allow neural-induced hESC colonies grow to day 7 or 8. All the cells within the colony will undergo morphology changes to large differentiated cells that will continue to multiply. The colonies will increase in size to cover the plate by day 7 or 8, and cells will begin to pile up in some areas of the colonies.</li>
</ol>
5. Continuing Neuronal Differentiation in Suspension Culture
<ol>
	<li>Take hESC culture plate to dissecting microscope (pre-warm dissecting stage to 37&deg;C) underneath dissecting sterilized hood. Carefully outline the colonies and remove fibroblast layer surrounding the colony (differentiated cells migrated out of the colony) with the edge of P2 sterile pipette tip or pulled glass capillary.</li>
	<li>Remove the old media containing floating detached fibroblast cells by aspirating. Wash with hESC media (without bFGF) once. Add 3 ml/well fresh hESC media (without bFGF).</li>
	<li>Cut the neural-induced hESC colonies into small pieces, and detach with sterile pipette tip or pulled glass capillary.</li>
	<li>Pool the media containing detached colony pieces together in a 50 ml conical tube. Wash the plate once with 1 ml/well hESC media (without bFGF) and pool together.</li>
	<li>Aliquot 4 ml/well serum-free hESC media containing colony pieces to a 6-well ultralow attachment plate and incubate in a 37&deg;C humidified incubator to allow floating cellular clusters (neuroblasts) to form for 4-5 days.</li>
</ol>
6. Neuronal Phenotype Maturation in Adhesive Culture
<ol>
	<li>Pool the media containing floating neuroblasts together in a 50 ml conical tube. Centrifuge at 1400 rpm for 5 min. Aspirate the old media as much as you can and add equal amount of fresh NSC media containing VEGF (20 ng/ml), NT-3 (10 ng/ml), and BDNF (10 ng/ml).</li>
	<li>Pipette up and down to mix floating neuroblasts and aliquot 4 ml/well to 6-well plates. The neuroblasts can also be seeded in a laminin/collagen (Matrigel) or human laminin polymerized 3-dimensional matrix in serum-free NSC media containing VEGF (20 ng/ml), NT-3 (10 ng/ml), and BDNF (10 ng/ml). Transfer the plates to a 37&deg;C humidified incubator to allow neuroblasts to attach overnight.</li>
	<li>Replace NSC media containing VEGF (20 ng/ml), NT-3 (10 ng/ml), and BDNF (10 ng/ml) every other day. Extensive networks of neurite-bearing neuronal cells and pigmented cells will begin to appear within 2 weeks of continuous cultivation and increase in numbers with time, and could be sustained for over 3 months.</li>
</ol>
7. Representative Results:
Retinoic acid (RA) is rendered sufficient to induce hESCs maintained in the defined culture system to transition from pluripotency exclusively to a neuroectodermal phenotype (Fig. 2A). Upon exposure of undifferentiated hESCs to RA, all the cells within the colony will undergo morphology changes to large differentiated cells that cease expressing pluripotency-associated markers, as indicated by Oct-4, and begin expressing various neuroectoderm-associated markers, such as HNK1, AP2, and TrkC (Fig. 2B). These large differentiated cells will continue to multiply and the colonies will increase in size, proceeding spontaneously to express the early neuronal marker &beta;-III-tubulin (Fig. 2B). The more mature neuronal marker Map-2 will begin to appear in areas of the colonies where cells have piled up (Fig. 2B). Coincident with the appearance of the neuroectodermal cells and neuronal differentiation, the neuronal specific transcriptional factor Nurr1, implicated in dopaminergic neuronal differentiation and activation of the tyrosine hydroxylase (TH) gene16, will translocate to the nucleus (Fig. 2B). After detached, the RA-treated hESCs will form floating cellular clusters (neuroblasts) in a suspension culture to continue the neural differentiation process. Upon removal of bFGF and after permitting the neuroblasts to attach to a tissue culture plate or seeded in a laminin/collagen polymerized 3-dimensional matrix in a serum-free defined medium, &beta;-III-tubulin- and Map-2-expressing, neurite-bearing cells and pigmented cells will begin to appear with a drastic increase in efficiency as compared to spontaneous multi-lineage differentiation of hESCs without treatment over the same time period, and could be sustained for over 3 months (Fig. 2C).
<img alt="Figure 1" src="/files/ftp_upload/3273/3273fig1.jpg" /> 
Figure 1 A schematic of well-controlled efficient induction of human pluripotent stem cells exclusively to a particular clinically-relevant lineage by simple provision of small molecules.
<img alt="Figure 2" src="/files/ftp_upload/3273/3273fig2.jpg" /> 
Figure 2 Retinoic acid induces neural lineage specification and neuronal progression direct of pluripotency under defined conditions. (A) Schematic depicting of the protocol time line of directed neuronal differentiation of hESCs. (B) Upon exposure of undifferentiated hESCs to retinoic acid (RA) under the defined culture system, large differentiated Oct-4 (red) negative cells within the colony began to emerge, as compared to mock-treated (DMSO) hESCs as the control. RA-induced differentiated Oct-4-negative cells began to express HNK-1 (red), AP2 (red), TrkC (green), and then &beta;-III-tubulin (red), consistent with early neuroectodermal differentiation. These cells continued to mature ultimately expressing the neuronal marker Map-2 (green), usually in areas where cells began to pile up. Coincident with the appearance of the neuroectodermal cells and neuronal differentiation, the neuronal specific transcriptional factor Nurr1 (green), implicated in dopaminergic neuronal differentiation and activation of the tyrosine hydroxylase (TH) gene, translocated to the nucleus. All cells are shown by DAPI staining of their nuclei (blue). (C) RA treatment induces differentiation towards a neuronal lineage with high efficiency as assessed by extensive networks of neurite-bearing cells expressing &beta;-III-tubulin (red) and Map-2 (green, shown in a 3-dimentional matrix). Arrows indicate pigmented cells typical of those in the CNS. All cells are shown by DAPI staining of their nuclei (blue) in the insets. Scale bars: 0.1 mm.

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The authors declare competing interests. XHP is the founder of Xcelthera. XHP and EYS have intellectual properties related to hESCs.

One of the major challenges for both developmental study and clinical translation has been how to channel the broad differentiation potential of pluripotent human stem cells to a desired phenotype efficiently and predictably. Although such cells can differentiate spontaneously in vitro into cells of all germ layers by going through a multi-lineage aggregate stage, only a small fraction of cells pursue a given lineage1,4. In those hESC-derived aggregates, the simultaneous appearance of a substantial amount of widely divergent undesired cell types that may reside in three embryonic germ layers often makes the emergence of desired phenotypes not only inefficient, but uncontrollable and unreliable as well. Although cardiac and neural lineages have been derived in previous reports, however, the inefficiency in generating specialized cells through germ-layer-induction of pluripotent cells and the high risk of tumorigenicity following transplantation have hindered further clinical translation.
The hESC lines initially were derived and maintained in co-culture with growth-arrested mouse embryonic fibroblasts (MEFs)4. Although several human feeder, feeder-free, and chemically-formulated culture systems have been developed for hESCs11-13, the elements necessary and sufficient for sustaining the self-renewal of human pluripotent cells remain unsolved. These exogenous feeder cells and biological reagents help maintain the long-term stable growth of undifferentiated hESCs whereas mask the ability of pluripotent cells to respond to developmental signals. Maintaining undifferentiated hESCs in a defined biologics-free culture system that allows faithful expansion and controllable direct differentiation is one of the keys to their therapeutic utility and potential. RA was not sufficient to induce neuronal differentiation of undifferentiated hESCs maintained under previously-reported conditions containing exogenous feeder cells. Although neural lineages appear at a relatively early stage in hESC differentiation, treating hESC-differentiated multi-lineage aggregates (embryoid bodies) with RA only slightly increased the low yield of neurons1, 14, 15. In order to achieve uniformly conversion of human pluripotent stem cells to a particular lineage, we employed a defined culture system capable of insuring the proliferation of undifferentiated hESCs to identify conditions for well-controlled efficient induction of pluripotent hESCs exclusively to a particular clinically-relevant lineage by simple provision of small molecules (Fig. 1, Fig. 2). Future studies will reveal genetic and epigenetic control molecules in human CNS development as alternatives, which may pave the way for small molecule-mediated direct control and modulation of hESC pluripotent fate when deriving clinically-relevant lineages for regenerative therapies. Without RA treatment, 1-5% hESCs will undergo spontaneous differentiation into neurons1, 14, 15. With RA treatment, we have been able to generate &gt; 95% embryonic neuronal progenitors and neurons from hESCs maintained under a define culture in a process that might emulate human embryonic development14. Recently, known neural-fate determining genes have been used to transdifferentiate mouse fibroblasts into adult neural progenitors and neurons with a low efficiency ranging 0.5-8%17, 18. However, reprogrammed somatic cells have historically been associated with abnormal gene expression and accelerated senescence with impaired therapeutic utility19-21. Finally, the protocol we established here is limited to pluripotent hESCs derived from the inner cell mass (ICM) or epiblast of the human blastocyst4, may not apply to other pluripotent cells, including animal-originated ESCs, ESCs derived from earlier morula (eight-cell)-stage embryos22, and artificially reprogrammed cells23.

<table border="1">
<tbody>
<tr>
<th>Name of the reagent			</th>
<th>Company</th>
<th>Catalogue number</th>
<th>Comments (optional)</th>
</tr>
<tr>
<td>Gelatin		</td>
<td>Sigma</td>
<td>G1890</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Matrigel			</td>
<td>BD bioscience</td>
<td>356231</td>
<td>Growth factor reduced</td>
</tr>
<tr>
<td>Human laminin		</td>
<td>Sigma</td>
<td>L6274</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>all-trans-Retinoic acid		</td>
<td>Sigma</td>
<td>R2625</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>DMEM/F12		</td>
<td>Invitrogen</td>
<td>10565018</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>DMEM		</td>
<td>Invitrogen</td>
<td>31053036</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>DMEM-KO		</td>
<td>Invitrogen</td>
<td>10829018</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Knock-out serum replacement		</td>
<td>Invitrogen</td>
<td>10828028</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>MEM nonessential amino acid solution 
(MNAA, 100X)		
</td>
<td>Invitrogen</td>
<td>11140050</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>MEM 
amino acids solution
(MEAA, 100X)		
</td>
<td>Invitrogen</td>
<td>11130050</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>&beta;-Mercaptoethanol		</td>
<td>Invitrogen</td>
<td>21985023</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Albumax		</td>
<td>Invitrogen</td>
<td>11020021</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Ascorbic acid		</td>
<td>Sigma</td>
<td>A4403</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Human transferrin		</td>
<td>Sigma</td>
<td>T8158</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Human bFGF		</td>
<td>PeproTech</td>
<td>AF-100-18B</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Human insulin 		</td>
<td>Invitrogen</td>
<td>12585014</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Human activin A		</td>
<td>PeproTech</td>
<td>120-14E</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Human BDNF		</td>
<td>PeproTech</td>
<td>AF-450-02</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Human VEGF		</td>
<td>PeproTech</td>
<td>AF-100-20</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Human NT-3		</td>
<td>PeproTech</td>
<td>450-03</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Heparin		</td>
<td>Sigma</td>
<td>H5284</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>N-2 supplement (100X)		</td>
<td>Invitrogen</td>
<td>17502048</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>6-well ultralow attachment plate	</td>
<td>Corning	</td>
<td>3471</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>6-well plate	</td>
<td>Corning	</td>
<td>3516</td>
<td>&nbsp;</td>
</tr>
</tbody>
</table>

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.

Watch this Scientific Journal Video about Efficient Derivation of Human Neuronal Progenitors and Neurons from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction at JoVE.com

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

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 ...

efficient-derivation-human-neuronal-progenitors-neurons-from

Research

JoVE Journal

Neuroscience

15.2K Views.  San Diego Regenerative Medicine Institute. 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.

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

DiOLISTIC Labeling of Neurons from Rodent and Non-human Primate Brain Slices

DiOLISTIC staining uses the gene gun to introduce fluorescent dyes, such as DiI, into neurons of brain slices (Gan et al., 2009; O'Brien and Lummis, 2007; Gan et al., 2000). Here we provide a detailed description of each step required together with exemplary images of good and bad outcomes that will help when setting up the technique. In our experience, a few steps proved critical for the successful application of DiOLISTICS. These considerations include the quality of the DiI-coated bullets, the extent of fixative exposure, and the concentration of detergent used in the incubation solutions. Tips and solutions for common problems are provided. This is a versatile labeling technique that can be applied to multiple animal species at a wide range of ages. Unlike other fluorescent labeling techniques that are limited to preparations from young animals or restricted to mice because they rely on the expression of a fluorescent transgene, DiOLISTIC labeling can be applied to animals of all ages, species and genotypes and it can be used in combination with immunostaining to identify a specific subpopulation of cells. Here we demonstrate the use of DiOLISTICS to label neurons in brain slices from adult mice and adult non-human primates with the purpose of quantifying dendrite branching and dendritic spine morphology.

We demonstrate the use of the gene gun to introduce fluorescent dyes, such as DiI, into neurons in brain slices from rodents and non-human primates of different ages. In this particular case, we use adult mice (3-6 months old) and adult cynomologus monkeys (9-15 years old). This technique, originally described by the laboratory of Dr. Lichtman (Gan et al., 2000), is well suited for the study of dendritic branching and dendritic spine morphology and can be combined with traditional immunostaining, if detergents are kept at a low concentration.

DiOLISTIC staining uses the gene gun to introduce fluorescent dyes, such as DiI, into neurons of brain slices (Gan et al., 2009; O'Brien and Lummis, 2007; ...

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 ...

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 ...

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.

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 ...

Conversion of Human Induced Pluripotent Stem Cells (iPSCs) into Functional Spinal and Cranial Motor Neurons Using PiggyBac Vectors

We describe here a method to obtain functional spinal and cranial motor neurons from human induced pluripotent stem cells (iPSCs). Direct conversion into motor neuron is obtained by ectopic expression of alternative modules of transcription factors, namely Ngn2, Isl1 and Lhx3 (NIL) or Ngn2, Isl1 and Phox2a (NIP). NIL and NIP specify, respectively, spinal and cranial motor neuron identity. Our protocol starts with the generation of modified iPSC lines in which NIL or NIP are stably integrated in the genome via a piggyBac transposon vector. Expression of the transgenes is then induced by doxycycline and leads, in 5 days, to the conversion of iPSCs into MN progenitors. Subsequent maturation, for 7 days, leads to homogeneous populations of spinal or cranial MNs. Our method holds several advantages over previous protocols: it is extremely rapid and simplified; it does not require viral infection or further MN isolation; it allows generating different MN subpopulations (spinal and cranial) with a remarkable degree of maturation, as demonstrated by the ability to fire trains of action potentials. Moreover, a large number of motor neurons can be obtained without purification from mixed populations. iPSC-derived spinal and cranial motor neurons can be used for in vitro modeling of Amyotrophic Lateral Sclerosis and other neurodegenerative diseases of the motor neuron. Homogeneous motor neuron populations might represent an important resource for cell type specific drug screenings.

Conversion of Human Induced Pluripotent Stem Cells iPSCs into Functional Spinal and Cranial Motor Neurons Using PiggyBac Vectors

This protocol allows rapid and efficient conversion of induced pluripotent stem cells into motor neurons with a spinal or cranial identity, by ectopic expression of transcription factors from inducible piggyBac vectors.

We describe here a method to obtain functional spinal and cranial motor neurons from human induced pluripotent stem cells (iPSCs). Direct conversion into ...

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 ...

Subretinal Transplantation of Human Embryonic Stem Cell-Derived Retinal Tissue in a Feline Large Animal Model

Retinal degenerative (RD) conditions associated with photoreceptor loss such as age-related macular degeneration (AMD), retinitis pigmentosa (RP) and Leber Congenital Amaurosis (LCA) cause progressive and debilitating vision loss. There is an unmet need for therapies that can restore vision once photoreceptors have been lost. Transplantation of human pluripotent stem cell (hPSC)-derived retinal tissue (organoids) into the subretinal space of an eye with advanced RD brings retinal tissue sheets with thousands of healthy mutation-free photoreceptors and has a potential to treat most/all blinding diseases associated with photoreceptor degeneration with one approved protocol. Transplantation of fetal retinal tissue into the subretinal space of animal models and people with advanced RD has been developed successfully but cannot be used as a routine therapy due to ethical concerns and limited tissue supply. Large eye inherited retinal degeneration (IRD) animal models are valuable for developing vision restoration therapies utilizing advanced surgical approaches to transplant retinal cells/tissue into the subretinal space. The similarities in globe size, and photoreceptor distribution (e.g., presence of macula-like region area centralis) and availability of IRD models closely recapitulating human IRD would facilitate rapid translation of a promising therapy to the clinic. Presented here is a surgical technique of transplanting hPSC-derived retinal tissue into the subretinal space of a large animal model allowing assessment of this promising approach in animal models.

Presented here is a surgical technique for transplanting human pluripotent stem cell (hPSC)-derived retinal tissue into the subretinal space of a large animal model.

Retinal degenerative (RD) conditions associated with photoreceptor loss such as age-related macular degeneration (AMD), retinitis pigmentosa (RP) and ...

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 ...

Human Microglia-like Cells: Differentiation from Induced Pluripotent Stem Cells and In Vitro Live-cell Phagocytosis Assay using Human Synaptosomes

Microglia are the resident immune cells of myeloid origin that maintain homeostasis in the brain microenvironment and have become a key player in multiple neurological diseases. Studying human microglia in health and disease represents a challenge due to the extremely limited supply of human cells. Induced pluripotent stem cells (iPSCs) derived from human individuals can be used to circumvent this barrier. Here, it is demonstrated how to differentiate human iPSCs into microglia-like cells (iMGs) for in vitro experimentation. These iMGs exhibit the expected and physiological properties of microglia, including microglia-like morphology, expression of proper markers, and active phagocytosis. Additionally, documentation for isolating and labeling synaptosome substrates derived from human iPSC-derived lower motor neurons (i3LMNs) is provided. A live-cell, longitudinal imaging assay is used to monitor engulfment of human synaptosomes labeled with a pH-sensitive dye, allowing for investigations of iMG's phagocytic capacity. The protocols described herein are broadly applicable to different fields that are investigating human microglia biology and the contribution of microglia to disease.

Human Microglia-like Cells: Differentiation from Induced Pluripotent Stem Cells and In Vitro Live-cell Phagocytosis Assay using Human Synaptosomes

This protocol describes the differentiation process of human induced pluripotent stem cells (iPSCs) into microglia-like cells for in vitro experimentation. We also include a detailed procedure for generating human synaptosomes from iPSC-derived lower motor neurons that can be used as a substrate for in vitro phagocytosis assays using live-cell imaging systems.

Microglia are the resident immune cells of myeloid origin that maintain homeostasis in the brain microenvironment and have become a key player in multiple ...