This project was funded by NIH grants EY025290, GM127652, and University of Rochester start-up funding.

1. Differentiation of hPSC to RPE
<ol>
	<li>Maintain and passage hPSCs as previously described18. 
	NOTE: All cells (including hPSCs and hPSC-RPEs) are grown in 37 &#176;C at 5% CO2 throughout the duration of the growth and differentiation protocols.</li>
	<li>Prior to differentiation, split confluent hPSCs to pre-coated 6-well plates.
	<ol>
		<li>To coat plates, thaw basement membrane matrix on ice for about 1 h, suspend liquidized matrix in 4 &#176;C DMEM medium at a 1:50 dilution, add 800 &#181;L coating mixture to each well, and incubate for at least 1 h at 37 &#176;C.</li>
		<li>When coating is complete, aspirate the mixture and immediately add 1.5&#8211;2 mL medium into the wells.</li>
		<li>To lift hPSCs from the old culture plates, incubate cells with PBS plus 0.5 mM EDTA for 5 min at room temperature, aspirate the solution, and resuspend cells in small clumps with culture medium.</li>
		<li>Seed cells at ~20% confluency in the new plates. 
		NOTE: Store basement membrane matrix in 100 &#181;L aliquots at -20 &#176;C and use one aliquot to coat a 6-well plate. The time for coating can be extended up to 4 h at 37 &#176;C, or overnight at room temperature. It is important to suspend and seed hPSCs as small clumps of 3&#8211;5 cells rather than single cells.</li>
	</ol>
	</li>
	<li>When cells are grown to full confluency, replace the culture medium with differentiation medium (4 mL/well) (Table 1, without activin-A). Culture for 14 days, changing the medium (4 mL/well) 3x/week. 
	NOTE: The hPSC plate density doubles approximately every 24 h. Significant cell death is expected after the start of the differentiation procedure. Aggregates of dead cells and debris in the wells can be seen by the naked eye during medium changes.</li>
	<li>From day 15 to day 28, supplement differentiation medium with 100 ng/mL human activin-A (Table 1, with human activin-A). Change the medium (4 mL/well) 3x/week.</li>
	<li>Stop activin-A supplementation starting on day 29 (Table 1, without human activin-A). Continue cell culturing in differentiation medium for 8&#8211;10 weeks until pigmented hPSC-RPE clusters appear (Figure 1A-B). 
	NOTE: Clusters of pigmented cells can be seen as small dark dots on a cell culture plate by the naked eye (Figure 1A-B, top). Individual pigmented cells with the signature cobblestone shape can be seen under a 20X microscope (Figure 1A-B, bottom).</li>
</ol>
2. Isolation and Culture of Differentiated RPE Cells
<ol>
	<li>Remove the differentiation medium, wash once with PBS, add 1 mL of 0.05% trypsin plus 1 U/&#181;L of collagenase into each well, and incubate at 37 &#176;C for 20&#8211;30 min. Remove the trypsin/collagenase and gently add 2 mL of pre-warmed RPE medium into each well of the 6-well plate (Table 2). 
	NOTE: Use an inverted microscope to monitor the morphology of the hPSC-RPE cells, which are polygonal before trypsin/collagenase treatment, and become round after sufficient digestion.</li>
	<li>Use a Bunsen burner to pull glass Pasteur pipettes.
	<ol>
		<li>With both hands, hold a long (9 inch) Pasteur pipette at both ends so it is horizontal, and above the Bunsen burner about 2/3 down the length of the thin part of the pipette.</li>
		<li>Once the glass becomes soft from the flame, quickly pull the two ends apart, such that a micro scraper-like tip is formed at the new end of the pipette (Figure 2).</li>
		<li>Repeat several times to create multiple &#34;cell scrapers&#34;. Spray the pulled pipettes with 70% ethanol for sterilization and air-dry them in the cell culture hood.</li>
	</ol>
	</li>
	<li>Under a microscope, use a micro &#34;cell scraper&#34; to gently dissociate pigmented hPSC-RPE clusters from the plate.
	<ol>
		<li>Gently tap (do not scrape) directly on the pigmented cells, which will float up into the culture medium once they dissociate from the bottom of the well.</li>
		<li>Immediately use a 20 &#181;L micropipette to gently suction the dissociated hPSC-RPE cells and transfer them to a pre-coated 6-well plate with RPE medium24.</li>
		<li>Collect ~5&#160;x 103 cells (~10% confluency as observed under a 20X microscope) in each pre-coated well of a 12-well plate by repeating steps 2.3.1&#8211;2.3.2 multiple times, as needed. Bring the final volume of medium to 2&#160;mL. 
		NOTE: To prevent dissociated hPSC-RPE cells from floating away, avoid movement of the plates during the dissociation process such that the dissociated cells are floating in the medium but still remain locally concentrated.</li>
	</ol>
	</li>
	<li>Culture the newly isolated hPSC-RPE cells (P0) on 12-well plates in RPE medium for another 6-8 weeks to allow them to form a pigmented monolayer (Figure 1C).</li>
	<li>Change the medium 3x/week. When changing the medium, leave approximately 1/3 volume of the old medium in each well, and add 2/3 volume of fresh, pre-warmed RPE medium. At this stage, use 2 mL of RPE medium for each well in a 12-well plate (thus, exchange 1.3&#160;mL of medium each time). 
	NOTE: The monolayer of P0&#160;hPSC-RPE&#160;cells may form domes, suggesting that the cells are actively transporting fluids, and are anchored by tight junctions25. Therefore, dome formation is an indicator of mature RPE status.</li>
</ol>
3. RPE Fate Validation by Immunoblotting
<ol>
	<li>Make the hPSC-RPE cell lysate using mammalian protein extraction buffer supplemented with proteinase inhibitor cocktail. Incubate the cell lysate on ice for 30 min before centrifugation at 13,000 x g for 15 min at 4 &#176;C to discard undissolved cell debris. Measure the total protein concentration of lysate.</li>
	<li>Denature 40 &#181;g of total protein in Laemmli SDS sample buffer at 75 &#176;C for 10 min. Separate the protein samples on a 10% Tris-glycine SDS-PAGE gel at a constant voltage of 90 V for 15 min and then 150 V until the dye front reaches the bottom of the gel.</li>
	<li>Transfer the proteins onto a nitrocellulose membrane in pre-cooled Tris-glycine buffer supplemented with 10% methanol at 100 V for 1 h.</li>
	<li>Block the membrane in Tris buffered saline with 0.1% non-ionic detergent (TBST) and 5% non-fat dry milk for 1 h at room temperature.</li>
	<li>Incubate the membrane with primary antibodies diluted in the blocking buffer at 4 &#176;C overnight. Dilute the antibodies targeting RPE specific marker proteins as follows: RPE65, 1:1,000; CRALBP, 1:1,000; BEST1, 1:1,000. Dilute the antibody against &#946;-actin at 1:10,000 to detect &#946;-actin as a loading control.</li>
	<li>Wash the membrane with TBST 3 times, with 5 min for each wash.</li>
	<li>Incubate the membrane with fluorophore conjugated goat anti-mouse IgG secondary antibody diluted 1:10,000 in blocking buffer for 1 h at room temperature.</li>
	<li>Wash the membrane with TBST 4 times, with 10 min for each wash.</li>
	<li>Detect the proteins using an infrared imaging system (Figure 3).</li>
</ol>
4. Checking BEST1 Subcellular Localization by Immunostaining
<ol>
	<li>Wash hPSC-RPE cells with 2 mL of PBS once and fix in 2 mL of 4% paraformaldehyde for 45 min at room temperature.</li>
	<li>Wash with 2 mL of PBS twice, and incubate the cells in 2 mL of PBS with 0.1% non-ionic surfactant and 2% donkey serum for 45 min to block the non-specific binding sites.</li>
	<li>Incubate the cells with BEST1 antibody (1:200 dilution) for 2 h at room temperature.</li>
	<li>Wash the cells with 2 mL of PBS 3 times. Incubate with fluorophore conjugated secondary IgG (1:1,000) for 1 h at room temperature.</li>
	<li>Wash with 2 mL of PBS 3 times to remove unbound secondary antibody.</li>
	<li>Observe stained cells by confocal microscopy (Figure 4).</li>
</ol>
5. Recording Ca2+-dependent Cl- Current in hPSC-RPE by Whole-cell Patch Clamp
<ol>
	<li>24&#8211;72 h before the patch clamp, split a fully confluent well (on a 12-well plate) of mature P0 hPSC-RPE cells (cobblestone-shaped and pigmented). Aspirate the medium, wash once with PBS, and add 1 mL of 0.05% trypsin plus 1 U/&#181;L collagenase into the well. 
	NOTE: Pre-warm the trypsin/collagenase to 37 &#176;C right before use.</li>
	<li>Incubate at 37 &#176;C for 8 min. 
	NOTE: After this step, the hPSC-RPE cells are still adherent and connected.</li>
	<li>Use a 1 mL micropipette to gently wash the cells off the plate, and transfer cells in the digestion mixture to a 15 mL conical tube. Wash the well with 1 mL of pre-warmed trypsin/collagenase to collect the residual cells and combine them into the 15 mL conical tube. 
	NOTE: The hPSC-RPE cells are still in large clumps at this point. Do not try to break the cell clumps by vigorous pipetting. Some clumps may stick to the inner wall of the 1 mL tip.</li>
	<li>Incubate the conical tube in a 37 &#176;C dry bath for 8 min.</li>
	<li>Use a 1 mL micropipette to gently pipette up-and-down 10&#8211;15 times the digestion mix. 
	NOTE: Cell clumps become much smaller but still visible. Avoid vigorous pipetting.</li>
	<li>Repeat the previous two steps. 
	NOTE: Most of the cells should be in single-cell suspension after pipetting. There may still be some tiny cell clumps, which are acceptable. Optional: Incubate the conical tube at 37 &#176;C for an additional 5 min followed by gentle pipetting. The total time in the trypsin/collagenase at 37 &#176;C should not exceed 30 min (8 + 8 + 8 + 5 = 29).</li>
	<li>Add 5 mL of pre-warmed RPE medium to the 15 mL conical tube containing the digested cells. Spin at 200 x g for 5 min to collect the cells. Re-suspend and seed cells on pre-coated 35 mm dishes at 10&#8211;20% confluency (e.g., a 100% confluent well on a 12-well plate to five 35 mm dishes for 10% confluency). 
	NOTE: At all times, avoid vigorous pipetting, which may cause significant cell death as represented by apparent dark cell debris in the supernatant after centrifugation. Well-separated single-cell seeding is essential for patch clamp recording.</li>
	<li>Perform the whole-cell patch clamp as described previously18,26,27,28,29,30,31. Acquire current traces from a family of step potentials (-100 to +100 mV from a holding potential of 0 mV) (Figure 5). 
	NOTE: The recipes of internal and external solutions are described in Table 3 and Table 4, respectively.</li>
</ol>

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The authors have nothing to disclose.

The most important procedure for the disease-in-a-dish approach is to differentiate hPSCs with a disease-causing mutation to the correct cell lineage, which is RPE for BEST1. Thus, after each differentiation experiment, the resulting hPSC-RPE cells should be carefully validated for their mature status by RPE-specific morphologies and protein markers16,17,18. To minimize clonal artifact, multiple hiPSC clones from the sa...

It has been documented that at least five retinal degenerative diseases are caused by genetic mutations in the BEST1 gene1,2,3,4,5,6,7,8, with the number of reported mutations already over 200 and still increasing. These BEST1-associated diseases, also known as bestrophinopathies, cause progressive vision loss and even blindness, and there are currently no effective treatments. The protein product of BEST1, namely BESTROPHIN1 (BEST1), is a Ca2+-activated Cl- channel (CaCC) specifically expressed in the retinal pigment epithelium (RPE) of the eyes5,6,8,9. Importantly, a clinical phenotype of BEST1-associated diseases is the reduced visual response to light stimuli, called light peak (LP) measured in the electrooculogram10,11; LP is believed to be mediated by a CaCC in RPE12,13,14. In order to better comprehend the pathological mechanisms of BEST1 mutations and to work towards potential therapies, it is essential to study mutant BEST1 channels endogenously expressed in human RPE cells.
However, obtaining RPE cells directly from live patients is highly impractical. Although native RPE cells can be harvested from biopsies of human cadavers and fetuses, the difficult accessibility to these sources significantly limits scientific research. Therefore, it is critical to have alternative RPE sources other than human eyes. This call has been answered by recent advancements in stem cell techniques, as functional RPE cells can now be differentiated from human&#160;pluripotent stem cells (hPSCs), including embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs), the latter being generated by reprogramming primary skin fibroblasts from donors16,17,18. Importantly, the self-renewal and pluripotency of hPSCs ensure a reliable source to generate RPEs, while the patient-specificity of hiPSCs and genomic modification potential of hESCs (e.g., by CRISPR) offer a versatile disease-in-a-dish model for desired BEST1 mutations.
hPSC-RPE has several advantages over mice RPE models: 1) BEST1 knockout mice do not display any retinal abnormality19, raising the possibility of different genetic requirement of BEST1 in RPE between mice and humans; 2) only 3% of human RPE cells are binucleate, in contrast to 35% in mice20; 3) hiPSC-RPE potentiates autologous transplantation in clinical treatment of retinal disorders21. Nevertheless, animal models are still indispensable for studying RPE physiology and pathology in a live system, and the oncogenic potential of hiPSC cannot&#160;be overlooked.
The procedure here describes a useful and moderately simple hPSC to RPE differentiation protocol that can be used for research and clinical purposes. This protocol uses nicotinamide (vitamin B3) to augment differentiation of hPSCs to neural tissue, which is further induced to differentiate into RPE by treatment with activin-A. Nicotinamide treatment has been shown to increase the number of pigmented cells (a sign of differentiation into RPE), possibly by attenuating the apoptotic activity of differentiating cells22. The resulting hPSC-RPE cells display the same key markers, cobblestone morphology, and cellular functionality as native human RPE cells22. Thus, in a research setting, the resulting hPSC-RPE cells are suitable for downstream functional analyses including immunoblotting, immunostaining, and whole-cell patch clamp, for which detailed experimental procedures are also provided. Clinically, RPE cells derived from stem cells have shown great potential for transplantation treatment of macular degeneration in both animal studies and human trials23.

<table>
	<tbody>
		<tr>
			<td>Knock-Out (KO) DMEM</td>
			<td>ThermoFisher</td>
			<td>10829018</td>
			<td></td>
		</tr>
		<tr>
			<td>KO serum replacement</td>
			<td>ThermoFisher</td>
			<td>10829028</td>
			<td></td>
		</tr>
		<tr>
			<td>Nonessential amino acids</td>
			<td>ThermoFisher</td>
			<td>11140050</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamine</td>
			<td>ThermoFisher</td>
			<td>35050061</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin</td>
			<td>ThermoFisher</td>
			<td>10378016</td>
			<td></td>
		</tr>
		<tr>
			<td>Nicotinamide</td>
			<td>Sigma-Aldrich</td>
			<td>N0636</td>
			<td></td>
		</tr>
		<tr>
			<td>Human activin-A</td>
			<td>PeproTech</td>
			<td>120-14</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM (a modification)</td>
			<td>Sigma-Aldrich</td>
			<td>M4526</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>VWR</td>
			<td>97068-085</td>
			<td></td>
		</tr>
		<tr>
			<td>N1 supplement</td>
			<td>Sigma-Aldrich</td>
			<td>N6530</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamine-penicillin-streptomycin</td>
			<td>Sigma-Aldrich</td>
			<td>G1146</td>
			<td></td>
		</tr>
		<tr>
			<td>Nonessential amino acids</td>
			<td>Sigma-Aldrich</td>
			<td>M7156</td>
			<td></td>
		</tr>
		<tr>
			<td>Taurine</td>
			<td>Sigma-Aldrich</td>
			<td>T0625</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrocortisone</td>
			<td>Sigma-Aldrich</td>
			<td>H0386</td>
			<td></td>
		</tr>
		<tr>
			<td>Triiodo-thyronin</td>
			<td>Sigma-Aldrich</td>
			<td>T5516</td>
			<td></td>
		</tr>
		<tr>
			<td>mTeSR-1 medium</td>
			<td>Stemcell Technologies</td>
			<td>5850</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Corning</td>
			<td>356230</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase</td>
			<td>Gibco</td>
			<td>17104019</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>VWR</td>
			<td>45000-664</td>
			<td></td>
		</tr>
		<tr>
			<td>M-PER mammalian protein extraction reagent</td>
			<td>Pierce</td>
			<td>78501</td>
			<td></td>
		</tr>
		<tr>
			<td>proteinase inhibitor cocktail</td>
			<td>Sigma-Aldrich</td>
			<td>4693159001</td>
			<td></td>
		</tr>
		<tr>
			<td>RPE65 antibody</td>
			<td>Novus Biologicals</td>
			<td>NB100-355</td>
			<td></td>
		</tr>
		<tr>
			<td>CRALBP antibody</td>
			<td>Abcam</td>
			<td>ab15051</td>
			<td></td>
		</tr>
		<tr>
			<td>BEST1 antibody</td>
			<td>Novus Biologicals</td>
			<td>NB300-164</td>
			<td></td>
		</tr>
		<tr>
			<td>Beta Actin antibody</td>
			<td>ThermoFisher</td>
			<td>MA5-15739</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488-conjugated donkey anti-mouse IgG</td>
			<td>ThermoFisher</td>
			<td>A-21202</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-mouse IgG</td>
			<td>ThermoFisher</td>
			<td>SA5-35521</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Rabbit IgG</td>
			<td>LI-COR Biosciences</td>
			<td>926-68071</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>ThermoFisher</td>
			<td>62249</td>
			<td></td>
		</tr>
		<tr>
			<td>HEKA EPC10 patch clamp amplifier</td>
			<td>Warner Instruments</td>
			<td>895000</td>
			<td></td>
		</tr>
		<tr>
			<td>Patchmaster</td>
			<td>Warner Instruments</td>
			<td>895040</td>
			<td></td>
		</tr>
	</tbody>
</table>

differentiation, maintenance, and analysis of human retinal pigment epithelium cells: a disease-in-a-dish model for best1 mutations

Although over 200 genetic mutations in the human BEST1 gene have been identified and linked to retinal degenerative diseases, the pathological mechanisms remain elusive mainly due to the lack of a good in vivo model for studying BEST1 and its mutations under physiological conditions. BEST1 encodes an ion channel, namely BESTROPHIN1 (BEST1), which functions in retinal pigment epithelium (RPE); however, the extremely limited accessibility to native human RPE cells represents a major challenge for scientific research. This protocol describes how to generate human RPEs bearing BEST1 disease-causing mutations by induced differentiation from human pluripotent stem cells (hPSCs). As hPSCs are self-renewable, this approach allows researchers to have a steady source of hPSC-RPEs for various experimental analyses, such as immunoblotting, immunofluorescence, and patch clamp, and thus provides a very powerful disease-in-a-dish model for BEST1-associated retinal conditions. Notably, this strategy can be applied to study RPE (patho)physiology and other genes of interest natively expressed in RPE.

The most technically challenging step is the manual isolation, which aims to achieve a high purity of a differentiated P0 hPSC-RPE population. After a successful isolation, &#62;90% cells in the P0 population will grow and mature to display signature RPE morphologies (Figure 1C). The existence of a minor portion of non-RPE or immature RPE cells in the P0 population is almost inevitable, but will not interfere with the downstre...

Watch this Scientific Journal Video about Differentiation, Maintenance, and Analysis of Human Retinal Pigment Epithelium Cells: A Disease-in-a-dish Model for BEST1 Mutations at JoVE.com

Differentiation, Maintenance, and Analysis of Human Retinal Pigment Epithelium Cells: A Disease-in-a-dish Model for BEST1 Mutations

Here we present a protocol to differentiate retinal pigment epithelium (RPE) cells from human pluripotent stem cells bearing patient-derived mutations. The mutant cell lines may be used for functional analyses including immunoblotting, immunofluorescence, and patch clamp. This disease-in-a-dish approach circumvents the difficulty of obtaining native human RPE cells.

Differentiation, Maintenance, and Analysis of Human Retinal Pigment Epithelium Cells: A Disease-in-a-dish Model for BEST1 Mutations

Although over 200 genetic mutations in the human BEST1 gene have been identified and linked to retinal degenerative diseases, the pathological mechanisms ...

differentiation-maintenance-analysis-human-retinal-pigment-epithelium

Universidad de Cartagena UDEC

Research

JoVE Journal

Genetics

6.7K Views.  University of Rochester, School of Medicine and Dentistry. Here we present a protocol to differentiate retinal pigment epithelium (RPE) cells from human pluripotent stem cells bearing patient-derived mutations. The mutant cell lines may be used for functional analyses including immunoblotting, immunofluorescence, and patch clamp. This disease-in-a-dish approach circumvents the difficulty of obtaining native human RPE cells.

Video: Differentiation, Maintenance, and Analysis of Human Retinal Pigment Epithelium Cells: A Disease-in-a-dish Model for BEST1 Mutations

Analysis of Cap-binding Proteins in Human Cells Exposed to Physiological Oxygen Conditions

Translational control is a focal point of gene regulation, especially during periods of cellular stress. Cap-dependent translation via the eIF4F complex is by far the most common pathway to initiate protein synthesis in eukaryotic cells, but stress-specific variations of this complex are now emerging. Purifying cap-binding proteins with an affinity resin composed of Agarose-linked m7GTP (a 5' mRNA cap analog) is a useful tool to identify factors involved in the regulation of translation initiation. Hypoxia (low oxygen) is a cellular stress encountered during fetal development and tumor progression, and is highly dependent on translation regulation. Furthermore, it was recently reported that human adult organs have a lower oxygen content (physioxia 1-9% oxygen) that is closer to hypoxia than the ambient air where cells are routinely cultured. With the ongoing characterization of a hypoxic eIF4F complex (eIF4FH), there is increasing interest in understanding oxygen-dependent translation initiation through the 5' mRNA cap. We have recently developed a human cell culture method to analyze cap-binding proteins that are regulated by oxygen availability. This protocol emphasizes that cell culture and lysis be performed in a hypoxia workstation to eliminate exposure to oxygen. Cells must be incubated for at least 24 hr for the liquid media to equilibrate with the atmosphere within the workstation. To avoid this limitation, pre-conditioned media (de-oxygenated) can be added to cells if shorter time points are required. Certain cap-binding proteins require interactions with a second base or can hydrolyze the m7GTP, therefore some cap interactors may be missed in the purification process. Agarose-linked to enzymatically resistant cap analogs may be substituted in this protocol. This method allows the user to identify novel oxygen-regulated translation factors involved in cap-dependent translation.

Here, we present human cell culture protocols to analyze translation initiation factors that bind the 5' cap of mRNA during physiological oxygen conditions. This method utilizes an Agarose-linked m7GTP cap analog and is suitable to investigate cap-binding factors and their interacting partners.

Translational control is a focal point of gene regulation, especially during periods of cellular stress. Cap-dependent translation via the eIF4F complex ...

Genome Editing and Directed Differentiation of hPSCs for Interrogating Lineage Determinants in Human Pancreatic Development

Interrogating gene function in self-renewing or differentiating human pluripotent stem cells (hPSCs) offers a valuable platform towards understanding human development and dissecting disease mechanisms in a dish. To capitalize on this potential application requires efficient genome-editing tools to generate hPSC mutants in disease-associated genes, as well as in vitro hPSC differentiation protocols to produce disease-relevant cell types that closely recapitulate their in vivo counterparts. An efficient genome-editing platform for hPSCs named iCRISPR has been developed through the TALEN-mediated targeting of a Cas9 expression cassette in the AAVS1 locus. Here, the protocols for the generation of inducible Cas9 hPSC lines using cells cultured in a chemically defined medium and a feeder-free condition are described. Detailed procedures for using the iCRISPR system for gene knockout or precise genetic alterations in hPSCs, either through non-homologous end joining (NHEJ) or via precise nucleotide alterations using a homology-directed repair (HDR) template, respectively, are included. These technical procedures include descriptions of the design, production, and transfection of CRISPR guide RNAs (gRNAs); the measurement of the CRISPR mutation rate by T7E1 or RFLP assays; and the establishment and validation of clonal mutant lines. Finally, we chronicle procedures for hPSC differentiation into glucose-responsive pancreatic &#946;-like cells by mimicking in vivo pancreatic embryonic development. Combining iCRISPR technology with directed hPSC differentiation enables the systematic examination of gene function to further our understanding of pancreatic development and diabetes disease mechanisms.

Protocols to generate hPSC mutant lines using the iCRISPR platform and to differentiate hPSCs into glucose-responsive &#946;-like cells are described. Combining genome editing technology with hPSC-directed differentiation provides a powerful platform for the systematic analysis of the role of lineage determinants in human development and disease progression.

Interrogating gene function in self-renewing or differentiating human pluripotent stem cells (hPSCs) offers a valuable platform towards understanding ...

Immunostaining for DNA Modifications: Computational Analysis of Confocal Images

For several decades, 5-methylcytosine (5mC) has been thought to be the only DNA modification with a functional significance in metazoans. The discovery of enzymatic oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) as well as detection of N6-methyladenine (6mA) in the DNA of multicellular organisms provided additional degrees of complexity to the epigenetic research. According to a growing body of experimental evidence, these novel DNA modifications may play specific roles in different cellular and developmental processes. Importantly, as some of these marks (e. g. 5hmC, 5fC and 5caC) exhibit tissue- and developmental stage-specific occurrence in vertebrates, immunochemistry represents an important tool allowing assessment of spatial distribution of DNA modifications in different biological contexts. Here the methods for computational analysis of DNA modifications visualized by immunostaining followed by confocal microscopy are described. Specifically, the generation of 2.5 dimension (2.5D) signal intensity plots, signal intensity profiles, quantification of staining intensity in multiple cells and determination of signal colocalization coefficients are shown. Collectively, these techniques may be operational in evaluating the levels and localization of these DNA modifications in the nucleus, contributing to elucidating their biological roles in metazoans.

Newly discovered oxidized forms of 5-methylcytosine (oxi-mCs), 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) may represent distinct DNA modifications with unique functional roles. Here a semi-quantitative workflow for visualization of oxi-mCs' spatial distribution, signal intensity profiling and colocalization is described.

For several decades, 5-methylcytosine (5mC) has been thought to be the only DNA modification with a functional significance in metazoans. The discovery of ...

Highly Efficient Gene Disruption of Murine and Human Hematopoietic Progenitor Cells by CRISPR/Cas9

Advances in the hematopoietic stem cell (HSCs) field have been aided by methods to genetically engineer primary progenitor cells as well as animal models. Complete gene ablation in HSCs required the generation of knockout mice from which HSCs could be isolated, and gene ablation in primary human HSCs was not possible. Viral transduction could be used for knock-down approaches, but these suffered from variable efficacy. In general, genetic manipulation of human and mouse hematopoietic cells was hampered by low efficiencies and extensive time and cost commitments. Recently, CRISPR/Cas9 has dramatically expanded the ability to engineer the DNA of mammalian cells. However, the application of CRISPR/Cas9 to hematopoietic cells has been challenging, mainly due to their low transfection efficiencies, the toxicity of plasmid-based approaches and the slow turnaround time of virus-based protocols.
A rapid method to perform CRISPR/Cas9-mediated gene editing in murine and human hematopoietic stem and progenitor cells with knockout efficiencies of up to 90% is provided in this article. This approach utilizes a ribonucleoprotein (RNP) delivery strategy with a streamlined three-day workflow. The use of Cas9-sgRNA RNP allows for a hit-and-run approach, introducing no exogenous DNA sequences in the genome of edited cells and reducing off-target effects. The RNP-based method is fast and straightforward: it does not require cloning of sgRNAs, virus preparation or specific sgRNA chemical modification. With this protocol, scientists should be able to successfully generate knockouts of a gene of interest in primary hematopoietic cells within a week, including downtimes for oligonucleotide synthesis. This approach will allow a much broader group of users to adapt this protocol for their needs.

A protocol for fast CRISPR/Cas9-mediated gene disruption in mouse and human primary hematopoietic cells is described in this article. Cas9-sgRNA ribonucleoproteins are introduced via electroporation with sgRNAs generated through in vitro transcription and commercial Cas9. High editing efficiencies are achieved with limited time and financial cost.

Advances in the hematopoietic stem cell (HSCs) field have been aided by methods to genetically engineer primary progenitor cells as well as animal models. ...

Single Droplet Digital Polymerase Chain Reaction for Comprehensive and Simultaneous Detection of Mutations in Hotspot Regions

Droplet digital polymerase chain reaction (ddPCR) is a highly sensitive quantitative polymerase chain reaction (PCR) method based on sample fractionation into thousands of nano-sized water-in-oil individual reactions. Recently, ddPCR has become one of the most accurate and sensitive tools for circulating tumor DNA (ctDNA) detection. One of the major limitations of the standard ddPCR technique is the restricted number of mutations that can be screened per reaction, as specific hydrolysis probes recognizing each possible allelic version are required. An alternative methodology, the drop-off ddPCR, increases throughput, since it requires only a single pair of probes to detect and quantify potentially all genetic alterations in the targeted region. Drop-off ddPCR displays comparable sensitivity to conventional ddPCR assays with the advantage of detecting a greater number of mutations in a single reaction. It is cost-effective, conserves precious sample material, and can also be used as a discovery tool when mutations are not known a priori.

Here, we present a protocol to accurately quantify multiple genetic alterations of a target region in a single reaction using drop-off ddPCR and a unique pair of hydrolysis probes.

Droplet digital polymerase chain reaction (ddPCR) is a highly sensitive quantitative polymerase chain reaction (PCR) method based on sample fractionation ...

Endogenous Protein Tagging in Human Induced Pluripotent Stem Cells Using CRISPR/Cas9

A protocol is presented for generating human induced pluripotent stem cells (hiPSCs) that express endogenous proteins fused to in-frame&#160;N- or C-terminal fluorescent tags. The prokaryotic CRISPR/Cas9 system (clustered regularly interspaced short palindromic repeats/CRISPR-associated 9) may be used to introduce large exogenous sequences into genomic loci via homology directed repair (HDR). To achieve the desired knock-in, this protocol employs the ribonucleoprotein (RNP)-based approach where wild type Streptococcus pyogenes Cas9 protein, synthetic 2-part guide RNA (gRNA), and a donor template plasmid are delivered to the cells via electroporation. Putatively edited cells expressing the fluorescently tagged proteins are enriched by fluorescence activated cell sorting (FACS). Clonal lines are then generated and can be analyzed for precise editing outcomes. By introducing the fluorescent tag at the genomic locus of the gene of interest, the resulting subcellular localization and dynamics of the fusion protein can be studied under endogenous regulatory control, a key improvement over conventional overexpression systems. The use of hiPSCs as a model system for gene tagging provides the opportunity to study the tagged proteins in diploid, nontransformed cells. Since hiPSCs can be differentiated into multiple cell types, this approach provides the opportunity to create and study tagged proteins in a variety of isogenic cellular contexts.

Described here is a protocol for tagging endogenously expressed proteins with fluorescent tags in human induced pluripotent stem cells using CRISPR/Cas9. Putatively edited cells are enriched by fluorescence activated cell sorting and clonal cell lines are generated.

A protocol is presented for generating human induced pluripotent stem cells (hiPSCs) that express endogenous proteins fused to in-frame&#160;N- or ...

CRISPR-mediated Genome Editing of the Human Fungal Pathogen Candida albicans

This method describes the efficient CRISPR mediated genome editing of the diploid human fungal pathogen Candida albicans. CRISPR-mediated genome editing in C. albicans requires Cas9, guide RNA, and repair template. A plasmid expressing a yeast codon optimized Cas9 (CaCas9) has been generated. Guide sequences directly upstream from a PAM site (NGG) are cloned into the Cas9 expression vector. A repair template is then made by primer extension in vitro. Cotransformation of the repair template and vector into C. albicans leads to genome editing. Depending on the repair template used, the investigator can introduce nucleotide changes, insertions, or deletions. As C. albicans is a diploid, mutations are made in both alleles of a gene, provided that the A and B alleles do not harbor SNPs that interfere with guide targeting or repair template incorporation. Multimember gene families can be edited in parallel if suitable conserved sequences exist in all family members. The C. albicans CRISPR system described is flanked by FRT sites and encodes flippase. Upon induction of flippase, the antibiotic marker (CaCas9) and guide RNA are removed from the genome. This allows the investigator to perform subsequent edits to the genome. C. albicans CRISPR is a powerful fungal genetic engineering tool, and minor alterations to the described protocols permit the modification of other fungal species including C. glabrata, N. castellii, and S. cerevisiae.

CRISPR-mediated Genome Editing of the Human Fungal Pathogen Candida albicans

Efficient genome engineering of Candida albicans is critical to understanding the pathogenesis and development of therapeutics. Here, we described a protocol to quickly and accurately edit the C. albicans genome using CRISPR. The protocol allows investigators to introduce a wide variety of genetic modifications including point mutations, insertions, and deletions.

This method describes the efficient CRISPR mediated genome editing of the diploid human fungal pathogen Candida albicans. CRISPR-mediated genome editing ...

Digital PCR-based Competitive Index for High-throughput Analysis of Fitness in Salmonella

A competitive index is a common method used to assess bacterial fitness and/or virulence. The utility of this approach is exemplified by its ease to perform and its ability to standardize the fitness of many strains to a wild-type organism. The technique is limited, however, by available phenotypic markers and the number of strains that can be assessed simultaneously, creating the need for a great number of replicate experiments. Concurrent with large numbers of experiments, the labor and material costs for quantifying bacteria based on phenotypic markers are not insignificant. To overcome these negative aspects while retaining the positive aspects, we have developed a molecular-based approach to directly quantify microorganisms after engineering genetic markers onto bacterial chromosomes. Unique, 25 base pair DNA barcodes were inserted at an innocuous locus on the chromosome of wild-type and mutant strains of Salmonella. In vitro competition experiments were performed using inocula consisting of pooled strains. Following the competition, the absolute numbers of each strain were quantified using digital PCR and the competitive indices for each strain were calculated from those values. Our data indicate that this approach to quantifying Salmonella is extremely sensitive, accurate, and precise for detecting both highly abundant (high fitness) and rare (low fitness) microorganisms. Additionally, this technique is easily adaptable to nearly any organism with chromosomes capable of modification, as well as to various experimental designs that require absolute quantification of microorganisms.

Digital PCR-based Competitive Index for High-throughput Analysis of Fitness in Salmonella

This molecular-based approach for determining bacterial fitness facilitates precise and accurate detection of microorganisms using unique genomic DNA barcodes that are quantified via digital PCR. The protocol describes calculating the competitive index for Salmonella strains; however, the technology is readily adaptable to protocols requiring absolute quantification of any genetically-malleable organism.

A competitive index is a common method used to assess bacterial fitness and/or virulence. The utility of this approach is exemplified by its ease to ...

Single-cell RNA Sequencing and Analysis of Human Pancreatic Islets

Pancreatic islets comprise of endocrine cells with distinctive hormone expression patterns. The endocrine cells show functional differences in response to normal and pathological conditions. The goal of this protocol is to generate high-quality, large-scale transcriptome data of each endocrine cell type with the use of a droplet-based microfluidic single-cell RNA sequencing technology. Such data can be utilized to build the gene expression profile of each endocrine cell type in normal or specific conditions. The process requires careful handling, accurate measurement, and rigorous quality control. In this protocol, we describe detailed steps for human pancreatic islets dissociation, sequencing, and data analysis. The representative results of about 20,000 human single islet cells demonstrate the successful application of the protocol.

Here, we present a protocol to generate high-quality, large-scale transcriptome data of single cells from isolated human pancreatic islets using a droplet-based microfluidic single-cell RNA sequencing technology.

Pancreatic islets comprise of endocrine cells with distinctive hormone expression patterns. The endocrine cells show functional differences in response to ...

Identifying Mutations by High Resolution Melting in a TILLING Population of Rice

Target Induced Local Lesions In Genomes (TILLING) is a strategy of reverse genetics for the high-throughput screening of induced mutations. However, the TILLING system has less applicability for insertion/deletion (Indel) detection and traditional TILLING needs more complex steps, like CEL I nuclease digestion and gel electrophoresis. To improve the throughput and selection efficiency, and to make the screening of both Indels and single base substitions (SBSs) possible, a new high-resolution melting (HRM)-based TILLING system is developed. Here, we present a detailed HRM-TILLING protocol and show its application in mutation screening. This method can analyze the mutations of PCR amplicons by measuring the denaturation of double-stranded DNA at high temperatures. HRM analysis is directly performed post-PCR without additional processing. Moreover, a simple, safe and fast (SSF) DNA extraction method is integrated with HRM-TILLING to identify both Indels and SBSs. Its simplicity, robustness and high throughput make it potentially useful for mutation scanning in rice and other crops.

In this article, we present the protocol that is described as high-resolution melting analysis (HRM)-based Target Induced Local Lesions In Genomes (TILLING). This method utilizes fluorescence changes during the melting of the DNA duplex and is suitable for high-throughput screening of both insertion/deletion (Indel) and single base substition (SBS).

Target Induced Local Lesions In Genomes (TILLING) is a strategy of reverse genetics for the high-throughput screening of induced mutations. However, the ...

Differentiation, Maintenance, and Analysis of Human Retinal Pigment Epithelium Cells: A Disease-in-a-dish Model for BEST1 Mutations

Summary

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Chapters in this video

Analysis of Cap-binding Proteins in Human Cells Exposed to Physiological Oxygen Conditions

Genome Editing and Directed Differentiation of hPSCs for Interrogating Lineage Determinants in Human Pancreatic Development

Immunostaining for DNA Modifications: Computational Analysis of Confocal Images

Highly Efficient Gene Disruption of Murine and Human Hematopoietic Progenitor Cells by CRISPR/Cas9

Single Droplet Digital Polymerase Chain Reaction for Comprehensive and Simultaneous Detection of Mutations in Hotspot Regions

Endogenous Protein Tagging in Human Induced Pluripotent Stem Cells Using CRISPR/Cas9

CRISPR-mediated Genome Editing of the Human Fungal Pathogen Candida albicans

Digital PCR-based Competitive Index for High-throughput Analysis of Fitness in Salmonella

Single-cell RNA Sequencing and Analysis of Human Pancreatic Islets

Identifying Mutations by High Resolution Melting in a TILLING Population of Rice