We thank members of 502 laboratory for their technical supports and helpful comments regarding the manuscript. This work was partly supported by the Beijing Municipal Natural Science Foundation (Z200014) and National Key R&#38;D Program of China (2017YFA0105300).

This study was approved by the institutional Ethics Committee of Beijing Tongren Hospital, Capital Medical University. H9 hESCs were obtained from the WiCell Research Institute and genetically engineered to tdTomato-tagged cell line.
1. Generation of human ROs
<ol>
	<li>Culture the hESCs under feeder-free conditions.
	<ol>
		<li>Coat one well of a 6-well plate with 1 mL of 0.1 mg/mL reagent A (Table of Materials) at 37 &#176;C for at least half an hour following the manufacturer&#39;s instructions. Thaw an aliquot of 1x106 hESCs. 
		NOTE: Prepare 3 mL of pre-warmed reagent B (Table of Materials) and transfer the cryopreserved cells (1 mL) into 3 mL of fresh medium. Do not pipette the hESCs to single cells.</li>
		<li>Centrifuge at 200 x g for 5 min and remove the supernatant.</li>
		<li>Seed the cells into the reagent A-coated plate with 2 mL of reagent B and change 2 mL of reagent B daily. Passage cells when reaching approximately 80% confluency (usually around 4 days).</li>
	</ol>
	</li>
	<li>Day 0
	<ol>
		<li>Dissociate hESCs to a single-cell suspension using medium I (Table 1). Prepare medium I by mixing the following: 20% (v/v) KnockOut serum replacement (KSR), 0.1 mM MEM non-essential amino acids solution (NEAA), 1 mM pyruvate,3 &#181;M IWR-1-endo (IWR1e), 30 ng/mL COCO, 100 U/mL penicillin, 100 &#181;g/mL streptomycin (PS), 0.1 mM &#946;-mercaptoethanol, and Glasgow&#39;s Eagle&#39;s minimal essential medium (GMEM). 
		NOTE: Before the start of the dissociation, prepare medium I and transfer 12 mL of medium I with 20 &#181;M Y-27632 into a 10 cm Petri dish, 500 &#181;L of reagent C (Table of Materials) containing 0.05 mg/mL of reagent D (Table of Materials) and 20 &#181;M Y-27632 in a 1.5 mL tube. Perform the above steps in the dark, as component IWR1e in medium I is light-sensitive.</li>
		<li>Wash the hESCs with pre-warmed 1x Dulbecco&#39;s phosphate-buffered saline (DPBS) buffer.</li>
		<li>Add the prepared 500 &#181;L reagent (in the 1.5 mL) tube and incubate the hESCs for 3.5 min at 37 &#176;C and 5% CO2.</li>
		<li>Detach the cells by flicking the side and bottom of the plate for a few seconds and add 500 &#181;L of prepared medium I from the Petri dish into the hESC plate. 
		NOTE: Prepare a 1.5 mL tube with 900 &#181;L of 1x DPBS and use a hemocytometer for cell counting.</li>
		<li>Harvest the cells in a new 1.5 mL tube and pipette the cell suspension up and down and then take out 100 &#181;L from the tube and then add into the tube with 900 &#181;L of DPBS for cell counting.</li>
		<li>Disperse the left 900 &#181;L cell suspension and then dilute the cells with prepared medium I in the Petri dish to 9 x 104 cells/mL. 
		NOTE: The whole volume of the medium is 12 mL; 1.08 x 106 cells are needed in total.</li>
		<li>Add 100 &#181;L of cell suspension to each well of a non-adherent, V-bottom, 96-well plate (Table of Materials). 
		NOTE: Use a multichannel pipette to shorten the time and ensure that each well contains an equivalent cell number. Shake the Petri dish each time before removing 100 &#181;L portions. It is important that the cells are uniformly distributed.</li>
		<li>Lightly spin down the 96-well plate in a low-speed shaker for 5 min and then incubate at 37 &#176;C and 5% CO2. 
		NOTE: Keep the plate in dark. Set the day as day 0.</li>
	</ol>
	</li>
	<li>Day 2
	<ol>
		<li>Add 1% reagent A (Table of Materials). Prepare reagent A (protein concentration of 10 mg/mL) by adding 133.4 &#181;L of reagent A into 2 mL of medium I. 
		NOTE: Maintain reagent A at 4 &#176;C overnight before use to achieve complete and uniform melting. Please note the product information and search the catalog number the and the lot number in the official website of the company to have the protein concentration of reagent A as each bottle of reagent A is at a different protein concentration. If the protein concentration is low, an increased volume of reagent A would be helpful.</li>
		<li>Add 20 &#181;L of prepared reagent A to each well and pipette twice in the center to scatter the dead cells. 
		NOTE: Maintain the reagent A and the 96-well plate under cool conditions and complete all the steps on ice. Place the plate in dark.</li>
	</ol>
	</li>
	<li>Day 2-12
	<ol>
		<li>Clean the bottom of the 96-well plate and incubate at 37 &#176;C and 5% CO2 until day 6. At day 6, change half of the medium by removing 58 &#181;L of the medium from each well and adding 60 &#181;L of medium I. 
		NOTE: Use a multichannel pipette to complete the half medium change. Conduct this step gently to ensure that no cell pellets are removed from the well. Aspirate the medium into a clean 10 cm Petri dish. If there are cell pellets inside, add them back to the 96-well plate.</li>
	</ol>
	</li>
	<li>Day 12- 18
	<ol>
		<li>Change the medium to medium II (Table 1) at day 12. Prepare medium II using 1% (v/v) reagent A by mixing the following: 10% fetal bovine serum (FBS), 0.1 mM NEAA, 1 mM pyruvate,100 U/mL penicillin, 100 &#181;g/mL streptomycin, 0.1 mM &#946;-mercaptoethanol, 100 nM SAG dihydrochloride and GMEM. Store it under cool and dark conditions.</li>
		<li>Harvest the cell pellets in a 15 mL conical tube from the 96-well plate and allow the pellets to settle naturally for 5 min at room temperature. 
		NOTE: Remove the medium and pellets gently under the surface to avoid bubbles.</li>
		<li>Remove the supernatant while taking care of the organoids. Transfer the cell aggregates to a 10 cm suspension dish containing 18 mL of prepared medium II with reagent A. 
		NOTE: Do not add the prepared medium II into the 10 cm dish in advance, as the reagent A should be at 4 &#176;C.</li>
		<li>Incubate at 37 &#176;C and 5% CO2 until day 18.</li>
	</ol>
	</li>
	<li>From day 18 onwards
	<ol>
		<li>Change the medium to medium III (Table 1). Prepare medium III under dark conditions by mixing the following: 10% FBS, 1xsupplement 1, 0.5 &#181;M retinoic acid (RA), 100 &#181;M taurine, 100 U/mL penicillin, 100 &#181;g/mL streptomycin and 1:1 mixture of Dulbecco&#39;s modified eagle medium (DMEM)/nutrient mixture F-12.</li>
		<li>On day 18, when a semitransparent optic vesicle is generated, cut the organoids using a microsurgical knife. 
		NOTE: Cleave the organoid, usually into four pieces, in a Petri dish with medium II. Each piece should grow into an intact optic cup in the following weeks.</li>
		<li>Aggregate all the pellets to the center of the dish. Harvest the cells into a 15 mL conical falcon tube and allow natural settling. Then gently remove the supernatant. 
		&#8203;NOTE: Due to their sizes, organoids can be aggregated at the center by rotating the Petri dish in one direction for 90&#176; on a horizontal plane a few times.</li>
		<li>Suspend and disperse the pellets in two 10 cm Petri dishes, with 18 mL of medium III per dish, and gently transfer the dishes to the incubator to avoid cell aggregation. Continue culturing in medium III at 37 &#176;C and 5% CO2 and change the medium weekly. The CRX expressed after day 45 and until day 120, we could detect the out-segment of the photoreceptor.</li>
	</ol>
	</li>
</ol>
2. Analyzing human ROs
<ol>
	<li>FACS analysis
	<ol>
		<li>Assemble three CRX-tdTomato and three H9 ES-derived organoids from the dishes by using the cut 1 mL tips. Wash the organoids with 1 mL of pre-warmed (room temperature) DPBS.</li>
		<li>Prepare the digestion buffer by mixing 0.25% trypsin-EDTA Solution with 0.05 mg/mL of reagent D.</li>
		<li>Dissociate the organoids into single cells by using the prepared digestion buffer for 8 min at 37 &#176;C. Then add the same volume of DPBS with 10% FBS and 0.05 mg/mL of reagent D to inactivate the reaction.</li>
		<li>Lightly suspend and scatter the cells and then filter using a 100 &#181;m cell strainer and use a Fluorescence-Activated Cell Sorting (FACS) system at 561 nm excitation laser line and 780/60 filter to analyze the CRX-tdTomato positive signals. 
		NOTE: CRX initially expresses at day 45 and increases with the maturation of organoids. Ten thousand cells are used for each test, for each timepoint, at least three repeats are completed.</li>
	</ol>
	</li>
	<li>Fluorescence intensity quantification of ROs
	<ol>
		<li>Prepare the viable organoids randomly for imaging. 
		NOTE: Set the parameters and use the same filters and parameters for all the fluorescence intensity monitors.</li>
		<li>Capture images and analyze the mean fluorescence intensity using suitable software (e.g., ImageJ).</li>
		<li>Import the images and then convert to 8-bit using Image | Type.</li>
		<li>Adjust the threshold by using the default parameters by Image | Adjust | Threshold.</li>
		<li>Determine the measured area and then evaluate the gray value by using Analyze | Measure. 
		NOTE: The mean values in the result panel are the mean fluorescence intensity of the measured area.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Xie, 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=Chromatin+accessibility+analysis+reveals+regulatory+dynamics+of+developing+human+retina+and+hiPSC-derived+retinal+organoids.">Chromatin accessibility analysis reveals regulatory dynamics of developing human retina and hiPSC-derived retinal organoids.</a> Science Advances. 6 (6), 5247 (2020).</li><li>Lu, Y. F., 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=Single-Cell+Analysis+of+Human+Retina+Identifies+Evolutionarily+Conserved+and+Species-Specific+Mechanisms+Controlling+Development.">Single-Cell Analysis of Human Retina Identifies Evolutionarily Conserved and Species-Specific Mechanisms Controlling Development.</a> Developmental Cell. 53 (4), 473-491 (2020).</li><li>Cowan, C. S., 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=Cell+Types+of+the+Human+Retina+and+Its+Organoids+at+Single-Cell+Resolution.">Cell Types of the Human Retina and Its Organoids at Single-Cell Resolution.</a> Cell. 182 (6), 1623-1640 (2020).</li><li>Jin, Z. B., 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=Stemming+retinal+regeneration+with+pluripotent+stem+cells.">Stemming retinal regeneration with pluripotent stem cells.</a> Progress in Retinal and Eye Research. 69, 38-56 (2019).</li><li>Nakano, T., 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=Self-formation+of+optic+cups+and+storable+stratified+neural+retina+from+human+ESCs.">Self-formation of optic cups and storable stratified neural retina from human ESCs.</a> Cell Stem Cell. 10 (6), 771-785 (2012).</li><li>Pan, D., 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=COCO+enhances+the+efficiency+of+photoreceptor+precursor+differentiation+in+early+human+embryonic+stem+cell-derived+retinal+organoids.">COCO enhances the efficiency of photoreceptor precursor differentiation in early human embryonic stem cell-derived retinal organoids.</a> Stem Cell Research and Therapy. 11 (1), 366 (2020).</li><li>Zhou, S., 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=Differentiation+of+human+embryonic+stem+cells+into+cone+photoreceptors+through+simultaneous+inhibition+of+BMP,+TGFbeta+and+Wnt+signaling.">Differentiation of human embryonic stem cells into cone photoreceptors through simultaneous inhibition of BMP, TGFbeta and Wnt signaling.</a> Development. 142 (19), 3294-3306 (2015).</li><li>Deng, W. L., 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=Gene+Correction+Reverses+Ciliopathy+and+Photoreceptor+Loss+in+iPSC-Derived+Retinal+Organoids+from+Retinitis+Pigmentosa+Patients.">Gene Correction Reverses Ciliopathy and Photoreceptor Loss in iPSC-Derived Retinal Organoids from Retinitis Pigmentosa Patients.</a> Stem Cell Reports. 10 (4), 1267-1281 (2018).</li><li>Gao, M. L., 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=Patient-Specific+Retinal+Organoids+Recapitulate+Disease+Features+of+Late-Onset+Retinitis+Pigmentosa.">Patient-Specific Retinal Organoids Recapitulate Disease Features of Late-Onset Retinitis Pigmentosa.</a> Frontiers in Cell and Developmental Biology. 8, 128 (2020).</li><li>Zhang, C. J., Ma, Y., Jin, Z. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+road+to+restore+vision+with+photoreceptor+regeneration.">The road to restore vision with photoreceptor regeneration.</a> Experimental Eye Research. 202, 108283 (2020).</li><li>Reichman, S., 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=From+confluent+human+iPS+cells+to+self-forming+neural+retina+and+retinal+pigmented+epithelium.">From confluent human iPS cells to self-forming neural retina and retinal pigmented epithelium.</a> Proceedings of the National Academy of Sciences of the U. S. A. 111 (23), 8518-8523 (2014).</li><li>Kuwahara, A., 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=Generation+of+a+ciliary+margin-like+stem+cell+niche+from+self-organizing+human+retinal+tissue.">Generation of a ciliary margin-like stem cell niche from self-organizing human retinal tissue.</a> Nature Communications. 6, 6286 (2015).</li></ol>

Retinal organoid differentiation is a desirable method for the generation of ample functional retinal cells. The RO is a composite of different retinal cells, such as ganglion cells, bipolar cells, and photoreceptors, generated by pluripotent stems cells toward the neural retina4,5,8,9. Although confluent ROs could be harvested, it is time-consuming, which may require long culturing periods (up...

Pluripotent stem cells (PSCs) are characterized by their self-renewal and ability to differentiate into all kinds of somatic cells. Thus, organoids derived from PSCs have become an important resource in regenerative medicine research. Retinal degeneration is characterized by the loss of photoreceptors (rods and cones) and retinal pigment epithelium. Retinal cell replacement could be an encouraging treatment for this disease. However, it is not feasible to obtain human retinas for disease research and therapy. Therefore, retinal organoids (ROs) derived from PSCs, which effectively and successfully recapitulate multi-layered native retinal cells, are beneficial for basic and translational research1,2,3. Our research focuses on RO differentiation to provide sufficient and quality cells for studying retinal degeneration4.
Methods for differentiating ROs are continuously emerging, with three-dimensional (3D) suspension differentiation pioneered by the Sasai laboratory in 20125. We introduced the CRX-tdTomato tag in the human embryonic stem cells (hESCs) to specifically track the photoreceptor precursor cells and modified the method with the addition of COCO, a multifunctional antagonist of the Wnt, TGF-&#946;, and BMP pathways6. COCO has been shown to efficiently improve the differentiation efficiency of photoreceptor precursors and cones6,7.
Altogether, by modifying the classical differentiation method, we have developed an accessible protocol to harvest abundant photoreceptor precursors and cones from human ROs for analyzing the retinal disease associated with the photoreceptors through laboratory investigations and for further clinical application/transplantation.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2-mercaptoethanol</td>
			<td>Life Technologies</td>
			<td>21985-023</td>
			<td></td>
		</tr>
		<tr>
			<td>COCO</td>
			<td>R&#38;D Systems</td>
			<td>3047-CC-050</td>
			<td>DAN Domain family of BMP antagonists</td>
		</tr>
		<tr>
			<td>DMEM/F-12</td>
			<td>Gibco</td>
			<td>10565-042</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Gibco</td>
			<td>C141905005BT</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Thermo</td>
			<td>15575020</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS), Qualified for Human Embryonic Stem Cells</td>
			<td>Biological Industry</td>
			<td>04-002-1A</td>
			<td></td>
		</tr>
		<tr>
			<td>GMEM</td>
			<td>Gibco</td>
			<td>11710-035</td>
			<td></td>
		</tr>
		<tr>
			<td>KnockOut Serum Replacement-Multi-Species</td>
			<td>Gibco</td>
			<td>A3181502</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM Non-essential Amino Acid Solution (100X)</td>
			<td>sigma</td>
			<td>M7145</td>
			<td></td>
		</tr>
		<tr>
			<td>Pen Strep</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Primesurface 96 V-plate</td>
			<td>Sbio</td>
			<td>MS9096SZ</td>
			<td>Cell aggregation in 1.2.7</td>
		</tr>
		<tr>
			<td>Pyruvate</td>
			<td>Sigma</td>
			<td>S8636</td>
			<td></td>
		</tr>
		<tr>
			<td>Reagent A</td>
			<td>BD</td>
			<td>356231</td>
			<td>Matrigel in 1.1.1</td>
		</tr>
		<tr>
			<td>Reagent B</td>
			<td>StemCell</td>
			<td>5990</td>
			<td>mTeSR- E8 , PSCs basal medium in 1.1.2</td>
		</tr>
		<tr>
			<td>Reagent C</td>
			<td>Gibco</td>
			<td>12563-011</td>
			<td>TrypLE Express in 1.2</td>
		</tr>
		<tr>
			<td>Reagent D</td>
			<td>Roche</td>
			<td>11284932001</td>
			<td>DNase I , in 1.2</td>
		</tr>
		<tr>
			<td>Retinoic acid</td>
			<td>Sigma</td>
			<td>R2625-100MG</td>
			<td></td>
		</tr>
		<tr>
			<td>SAG</td>
			<td>Enzo Life Science</td>
			<td>ALX-270-426-M001</td>
			<td></td>
		</tr>
		<tr>
			<td>Supplement 1</td>
			<td>Life Technologies</td>
			<td>17502-048</td>
			<td>N-2 Supplement (100X), Liquid, supplemet in medum III</td>
		</tr>
		<tr>
			<td>Taurine</td>
			<td>Sigma</td>
			<td>T-8691-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.25%), phenol red</td>
			<td>Gibco</td>
			<td>25200056</td>
			<td>organoids dissociation in 2.1.3</td>
		</tr>
		<tr>
			<td>Wnt Antagonist I, IWR-1-endo - Calbiochem</td>
			<td>Sigma</td>
			<td>681669</td>
			<td>Wnt inhibitor</td>
		</tr>
		<tr>
			<td>Y-27632 2HCl</td>
			<td>Selleck</td>
			<td>S1049</td>
			<td></td>
		</tr>
	</tbody>
</table>

directed induction of retinal organoids from human pluripotent stem cells

Retinal cell transplantation is a promising therapeutic approach, which could restore the retinal architecture and stabilize or even improve the visual capabilities to the degenerated retina. Nevertheless, progress in cell replacement therapy presently faces the challenges of requiring an off-the-shelf source of high quality and standardized human retinas. Therefore, an easy and stable protocol is needed for the experiments. Here, we develop an optimized protocol, based on a self-organizing method with the use of exogenous molecules and reagent A as well as manual excision to generate the three-dimensional human retina organoids (RO). The human Pluripotent Stem Cells (PSCs)-derived RO expresses specific markers for photoreceptors. With the addition of COCO, a multifunctional antagonist, the differentiation efficiency of photoreceptor precursors and cones is significantly increased. The efficient use of this system, which has the benefits of cell lines and primary cells, and without the sourcing issues associated with the latter, could produce confluent retinal cells, especially photoreceptors. Thus, the differentiation of PSCs to RO provides an optimal and biorelevant platform for disease modelling, drug screening and cell transplantation.

The schematic illustration depicts the differentiation protocol to improve precursor cells with COCO (Figure 1). From PSC to ROs, numerous details could cause result variations. It is recommended to record every step and even the catalog number and lot number of every medium to track the entire procedure.
Herein, we provide bright field images for days 6, 12, 18, and 45 (Figure 2). On day 6, the organoids are usually around 600 &#181;...

Watch this Scientific Journal Video about Directed Induction of Retinal Organoids from Human Pluripotent Stem Cells at JoVE.com

Directed Induction of Retinal Organoids from Human Pluripotent Stem Cells

Using a self-organizing method, we develop a protocol with the addition of COCO that could significantly increase the generation of photoreceptors.

Retinal cell transplantation is a promising therapeutic approach, which could restore the retinal architecture and stabilize or even improve the visual ...

directed-induction-retinal-organoids-from-human-pluripotent-stem

Research

JoVE Journal

Medicine

2.8K Views.  Beijing Tongren Hospital, Capital Medical University, Beijing Ophthalmology & Visual Science Key Laboratory. Using a self-organizing method, we develop a protocol with the addition of COCO that could significantly increase the generation of photoreceptors.

Video: Directed Induction of Retinal Organoids from Human Pluripotent Stem Cells

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly isolated from human tissues and serially propagated in immunodeficient mice, typically through antibody labeling of subpopulations of cells and fractionation by flow cytometry. However, the unique clinical features of prostate cancer have considerably limited the study of prostate CSCs from fresh human tumor samples. We recently reported the isolation of prostate CSCs directly from human tissues by virtue of their HLA class I (HLAI)-negative phenotype. Prostate cancer cells are harvested from surgical specimens and mechanically dissociated. A cell suspension is generated and labeled with fluorescently conjugated HLAI and stromal antibodies. Subpopulations of HLAI-negative cells are finally isolated using a flow cytometer. The principal limitation of this protocol is the frequently microscopic and multifocal nature of primary cancer in prostatectomy specimens. Nonetheless, isolated live prostate CSCs are suitable for molecular characterization and functional validation by transplantation in immunodeficient mice.

The isolation of cancer stem cells (CSCs) directly from human tissues is requisite for their biological characterization. This manuscript describes a methodology for the isolation of prostate CSCs from human tissues, while also providing tips on troubleshooting difficult steps.

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly ...

Enrichment for Chemoresistant Ovarian Cancer Stem Cells from Human Cell Lines

Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, invasive, and chemoresistant tumor cells. Therefore, CSCs are an attractive population of cells to target therapeutically. CSCs are predicted to contribute to a number of types of malignancies including those in the blood, brain, lung, gastrointestinal tract, prostate, and ovary. Isolating and enriching a tumor cell population for CSCs will enable researchers to study the properties, genetics, and therapeutic response of CSCs. We generated a protocol that reproducibly enriches for ovarian cancer CSCs from ovarian cancer cell lines (SKOV3 and OVCA429). Cell lines are treated with 20 &#181;M cisplatin for 3 days. Surviving cells are isolated and cultured in a serum-free stem cell media containing cytokines and growth factors. We demonstrate an enrichment of these purified CSCs by analyzing the isolated cells for known stem cell markers Oct4, Nanog, and Prom1 (CD133) and cell surface expression of CD177 and CD133. The CSCs exhibit increased chemoresistance. This method for isolation of CSCs is a useful tool for studying the role of CSCs in chemoresistance and tumor relapse.

Cancer stem cells (CSCs) are&#160;implicated in tumor relapse due to chemoresistance. We have optimized a protocol for selection and expansion of CSCs from ovarian cancer cell lines. By treating cells with the chemotherapeutic cisplatin and culturing&#160;in a stem cell promoting media we enrich for non-adherent CSC cultures.

Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, ...

Generation of Induced Pluripotent Stem Cells from Muscular Dystrophy Patients: Efficient Integration-free Reprogramming of Urine Derived Cells

Dystrophic cardiomyopathy is a poorly understood consequence of muscular dystrophy. Generating induced Pluripotent Stem Cells (iPSCs) from patients with muscular dystrophy is an invaluable cellular source for in&#160;vitro disease model systems and can be used for drug screening studies. Patient-derived urine cells have been used in successful reprogramming into induced pluripotent stem cells in order to model dystrophic cardiomyopathy1. Addressing the safety concerns of integrating vector systems, we present a protocol using a non-integrating Sendai virus vector for transduction of Yamanaka factors into urine cells collected from patients with muscular dystrophy. This protocol generates fully reprogrammed clones within 2&#8211;3 weeks. The pluripotent cells are vector-free by passage-13. These dystrophic iPSCs can be differentiated into cardiomyocytes and used either to study disease mechanisms or for drug screening.

This protocol entails detailed procedures for isolation of urine derived cells from muscular dystrophy patients; their efficient and rapid reprogramming through Sendai virus transduction.

Dystrophic cardiomyopathy is a poorly understood consequence of muscular dystrophy. Generating induced Pluripotent Stem Cells (iPSCs) from patients with ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Rapid and Efficient Generation of Recombinant Human Pluripotent Stem Cells by Recombinase-mediated Cassette Exchange in the AAVS1 Locus

Even with the revolution of gene-targeting technologies led by CRISPR-Cas9, genetic modification of human pluripotent stem cells&#160;(hPSCs)&#160;is still time consuming. Comparative studies that use recombinant lines with transgenes integrated into safe harbor loci could benefit from approaches that use site-specific targeted recombinases, like Cre or FLPe, which are more rapid and less prone to off-target effects. Such methods have been described, although they do not significantly outperform gene targeting in most aspects. Using Zinc-finger nucleases, we previously created a master cell line in the AAVS1 locus of hPSCs&#160;that contains a GFP-Hygromycin-tk expressing cassette, flanked by heterotypic FRT sequences. Here, we describe the procedures to perform FLPe recombinase-mediated cassette exchange (RMCE) using this line. The master cell line is transfected with a RMCE donor vector, which contains a promoterless Puromycin resistance, and with FLPe recombinase. Application of both a positive (Puromycin) and negative (FIAU) selection program leads to the selection of RMCE without random integrations. RMCE generates fully characterized pluripotent polyclonal transgenic lines in 15 d&#160;with 100% efficiency. Despite the recently described limitations of the AAVS1 locus, the ease of the system paves the way for hPSC transgenesis in isogenic settings, is necessary for comparative studies, and enables semi-high-throughput genetic screens for gain/loss of function analysis that would otherwise be highly time consuming.

Rapid and Efficient Generation of Recombinant Human Pluripotent Stem Cells by Recombinase-mediated Cassette Exchange in the AAVS1 Locus

Here we report a rapid and efficient gene editing method based on RMCE in the AAVS1 locus of human Pluripotent Stem Cells (hPSCs) that improves upon previously described systems. Using this technique, isogenic lines can be rapidly and reliably generated for proper comparative studies, facilitating transgenesis-mediated research with hPSCs.

Even with the revolution of gene-targeting technologies led by CRISPR-Cas9, genetic modification of human pluripotent stem cells&#160;(hPSCs)&#160;is ...

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant CFTR channel in subjects with cystic fibrosis (CF). Identification of subjects that may benefit from these drugs is challenging because of the extensive heterogeneity of CFTR mutations, as well as other unknown factors that contribute to individual drug efficacy. Here, we describe a simple and relatively rapid assay for measuring individual CFTR function and response to CFTR modulators in vitro. Three dimensional (3D) epithelial organoids are grown from rectal biopsies in standard organoid medium. Once established, the organoids can be bio-banked for future analysis. For the assay, 30-80 organoids are seeded in 96-well plates in basement membrane matrix and are then exposed to drugs. One day later, the organoids are stained with calcein green, and forskolin-induced swelling is monitored by confocal live cell microscopy at 37 &#176;C. Forskolin-induced swelling is fully CFTR-dependent and is sufficiently sensitive and precise to allow for discrimination between the drug responses of individuals with different and even identical CFTR mutations. In vitro swell responses correlate with the clinical response to therapy. This assay provides a cost-effective approach for the identification of drug-responsive individuals, independent of their CFTR mutations. It may also be instrumental in the development of future CFTR modulators.

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

This protocol describes an assay for measuring CFTR function and CFTR modulator responses in cultured tissue from subjects with cystic fibrosis (CF). Biopsy-derived intestinal organoids swell in a cAMP-driven fashion, a response that is defective (or strongly reduced) in CF organoids and can be restored by exposure to CFTR modulators.

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant ...

Ex Vivo Expansion of Hematopoietic Stem Cells from Human Umbilical Cord Blood-derived CD34+ Cells Using Valproic Acid

Umbilical cord blood (UCB) units provide an alternative source of human hematopoietic stem cells (HSCs) for patients who require allogeneic bone marrow transplantation. While UCB has several unique advantages, the limited numbers of HSCs within each UCB unit limits their use in regenerative medicine and HSC transplantation in adults. Efficient expansion of functional human HSCs can be achieved by ex vivo culturing of CD34+ cells isolated from UCBs and treated with a deacetylase inhibitor, valproic acid (VPA). The protocol detailed here describes the culture conditions and methodology to rapidly isolate CD34+ cells and expand to a high degree a pool of primitive HSCs. The expanded HSCs are capable of establishing both short-term and long-term engraftment and are able to give rise to all types of differentiated hematopoietic cells. This method also holds potential for clinical application in autologous HSC gene therapy and provides an attractive approach to overcome the loss of functional HSCs associated with gene editing.

Ex Vivo Expansion of Hematopoietic Stem Cells from Human Umbilical Cord Blood-derived CD34+ Cells Using Valproic Acid

Here, we describe the ex vivo expansion of hematopoietic stem cells from CD34+ cells derived from umbilical cord blood treated with a combination of a cytokine cocktail and VPA. This method leads to a significant degree of ex vivo expansion of primitive HSCs for either clinical or laboratory applications.

Umbilical cord blood (UCB) units provide an alternative source of human hematopoietic stem cells (HSCs) for patients who require allogeneic bone marrow ...

Induction and Analysis of Oxidative Stress in Sleeping Beauty Transposon-Transfected Human Retinal Pigment Epithelial Cells

Oxidative stress plays a critical role in several degenerative diseases, including age-related macular degeneration (AMD), a pathology that affects ~30 million patients worldwide. It leads to a decrease in retinal pigment epithelium (RPE)-synthesized neuroprotective factors, e.g., pigment epithelium-derived factor (PEDF) and granulocyte-macrophage colony-stimulating factor (GM-CSF), followed by the loss of RPE cells, and eventually photoreceptor and retinal ganglion cell (RGC) death. We hypothesize that the reconstitution of the neuroprotective and neurogenic retinal environment by the subretinal transplantation of transfected RPE cells overexpressing PEDF and GM-CSF has the potential to prevent retinal degeneration by mitigating the effects of oxidative stress, inhibiting inflammation, and supporting cell survival. Using the Sleeping Beauty transposon system (SB100X) human RPE cells have been transfected with the PEDF and GM-CSF genes and shown stable gene integration, long-term gene expression, and protein secretion using qPCR, western blot, ELISA, and immunofluorescence. To confirm the functionality and the potency of the PEDF and GM-CSF secreted by the transfected RPE cells, we have developed an in vitro assay to quantify the reduction of H2O2-induced oxidative stress on RPE cells in culture. Cell protection was evaluated by analyzing cell morphology, density, intracellular level of glutathione, UCP2 gene expression, and cell viability. Both, transfected RPE cells overexpressing PEDF and/or GM-CSF and cells non-transfected but pretreated with PEDF and/or GM-CSF (commercially available or purified from transfected cells) showed significant antioxidant cell protection compared to non-treated controls. The present H2O2-model is a simple and effective approach to evaluate the antioxidant effect of factors that may be effective to treat AMD or similar neurodegenerative diseases.

Induction and Analysis of Oxidative Stress in Sleeping Beauty Transposon-Transfected Human Retinal Pigment Epithelial Cells

We present a protocol for the development and use ofan oxidative stress-model by treating retinal pigment epithelial cells with H2O2, analyzing cell morphology, viability, density, glutathione, and UCP-2 level. It is a useful model to investigate the antioxidant effect of proteins secreted by transposon-transfected cells to treat neuroretinal degeneration.

Oxidative stress plays a critical role in several degenerative diseases, including age-related macular degeneration (AMD), a pathology that affects ~30 ...

Retinal Organoid Induction System for Derivation of 3D Retinal Tissues from Human Pluripotent Stem Cells

Retinal degenerative diseases are the main causes of irreversible blindness without effective treatment. Pluripotent stem cells that have the potential to differentiate into all types of retinal cells, even mini-retinal tissues, hold huge promises for patients with these diseases and many opportunities in disease modeling and drug screening. However, the induction process from hPSCs to retinal cells is complicated and time-consuming. Here, we describe an optimized retinal induction protocol to generate retinal tissues with high reproducibility and efficiency, suitable for various human pluripotent stem cells. This protocol is performed without the addition of retinoic acid, which benefits the enrichment of cone photoreceptors. The advantage of this protocol is the quantification of EB size and plating density to significantly enhance the efficiency and repeatability of retinal induction. With this method, all major retinal cells sequentially appear and recapitulate the main steps of retinal development. It will facilitate downstream applications, such as disease modeling and cell therapy.

Here we describe an optimized retinal organoid induction system, which is suitable for various human pluripotent stem cell lines to generate retinal tissues with high reproducibility and efficiency.

Retinal degenerative diseases are the main causes of irreversible blindness without effective treatment. Pluripotent stem cells that have the potential to ...