Work in the lab was supported by postdoctoral fellowship grant to Dr. Anjali Nandal, and Exploratory grant from Maryland Stem Cell Research Fund to BT (TEDCO).

1. Reprogramming Protocol
<ol>
	<li>Generation of human iPSC from primary human pancreatic cells
	<ol>
		<li>Coat a 6-well plate with 1.5 mg/mL cold collagen and allow it to gel at 37 &#176;C for 1 h.</li>
		<li>Plate early passage human primary pancreatic cells in Prigrow III medium (~1 - 1.5 &#215; 105 cells) on a collagen coated 6-well plate on day -2 to achieve approximately 2.5 &#215; 105 cells or at least 60% confluency per well on the day of transduction (day 0). For the first study, plate at least 2 - 3 wells to get one well with desired confluency on day 0. Use at least one well as a control to count the cells.</li>
		<li>On the day of transduction (day 0), warm 1 mL of Prigrow III medium in a water bath for each well to be transduced. Harvest the cells from one control well in 1 mL of 10% FBS medium using 1 mL of trypsin/EDTA for 5 min or till the cells detach to perform a cell count. To make 10% FBS medium, add DMEM, 10% FBS, 1% L-Glutamine, 1% Sodium Pyruvate and 1% non-essential amino acids.</li>
		<li>Count and check the viability of the cells using the cell counter and calculate the volume of each virus needed to reach the target based on the cell number and virus titer. Use multiplicity of infection (MOI) of KOS = 5, hc-Myc = 5 and hKlf4 = 3 for the pancreatic cells.</li>
		<li>Thaw one set of Sendai vector tubes in the -80 &#176;C storage on ice and carefully add the calculated volumes of each of the three Sendai vector tubes to 1 mL of Prigrow III medium, pre-warmed to 37 &#176;C. Pipette gently to mix the solution. One well in a 6-well plate is enough for transducing with Sendai viral vectors to generate reprogrammed cells.</li>
		<li>Aspirate the Prigrow III medium from the cells, and slowly add the reprogramming virus mixture to the wells containing the cells. Incubate the cells overnight in a 37 &#176;C incubator with a humidified atmosphere of 5% CO2.</li>
		<li>Carefully discard the virus mixture on cells and replace with fresh Prigrow III medium the next day, 24 h after transduction. Culture the cells for 5 d and change the medium every alternate day.</li>
		<li>On day 5, prepare MEFs on 0.1% gelatin pre-coated 10 cm culture dishes (1.3 x106 cells/dish). Grow MEFs in T175 flasks till day 4 and then on day 5, expose the cells to 6,000 rads from a &#947;-radiation source before seeding the cells13 or use the commercial MEF source.</li>
		<li>On day 6, use 1 mL of cell detachment solution for 10 min to detach the transduced cells, plate all of them on MEF dishes in Prigrow III medium with rock inhibitor (5 mM stock, 10 &#181;M final) and incubate overnight in a 37 &#176;C incubator with a humidified atmosphere of 5% CO2.</li>
		<li>Change the Prigrow III medium to hESC medium the next day. For making 500 mL of hESC medium, add DMEM/F-12, 20% (v/v) knockout serum replacement (KOSR), 5 ng/mL bFGF; 1 mM l-glutamine; 100 &#181;M nonessential amino acids and 100 &#181;M 2-mercaptoethanol. Change the medium gently every day now.</li>
		<li>Observe the plates regularly for the emergence of cell clumps or colonies indicative of reprogrammed cells. The transformed cells form clonal aggregates with cobblestone morphology and big nucleus and nucleoli. Mark the probable &#39;iPSC&#39; colonies and check them regularly for growth.</li>
		<li>Almost four weeks after transduction as colonies are ready for picking or transfer, prepare 24-well MEF plates by plating 1.3 x 106 cells/gelatin pre-coated plate as before for the transfer of single colonies.
		<ol>
			<li>Prepare matrix membrane (e.g., Matrigel) by aliquoting it based on the dilution factor on the certificate of analysis. Add one aliquot to 25 mL of DMEM/F-12 to coat four 6-well plates (1 mL/well) and incubate at room temperature (RT) for at least 1 h before use. Manually pick 12-24 colonies by using a sterile pipette-tip to aspirate them and transfer onto MEF plates in 500 &#181;L of hESC medium.</li>
			<li>Aspirate the media in the well to gently disrupt or break apart the colonies. Also pick 24-48 colonies on matrix membrane coated 24-well plates. For the membrane coated plates, use mTeSR1 (supplemented with 10 &#181;M rock inhibitor) for 24 h and change every day. Within 6-10 d of picking colonies, they are ready to be transferred to the new 24-well and/or 12-well&#160;membrane plates for clonal expansion.</li>
		</ol>
		</li>
		<li>If needed, detach the colonies on MEFs using 1 mg/mL collagenase for 20 min, wash twice with hESC/mTeSR1 and transfer to&#160;membrane coated 12- or 24-well plates. If there are enough robust colonies growing on the matrix membrane from step 1.1.12, freeze the colonies on MEFs using iPSC freezing media as per the manufacturer&#39;s guidelines.</li>
		<li>Detach the robust colonies plated on the membrane in step 1.1.12 using 500 &#181;L of dispase for 20 min and plate again on matrix membrane coated 12-well plates. Manually scrape off any differentiated cells or contaminating MEF feeder cells to enrich for pluripotent clones on the&#160;membrane.</li>
		<li>Expand the clones after 6-8 d by growing on&#160;membrane coated plates by dissociating with dispase solution as before and plating small cell aggregates on fresh&#160;membrane coated 6- or 12-well plate. Change mTeSR1 medium daily. Characterize the reprogrammed clones by alkaline phosphatase staining, immunostaining for pluripotency markers (OCT3/4 and NANOG), FACS analysis of pluripotent surface markers and differentiation assays. Grow and culture the clones on membrane-coated plates for all the assays including CRISPR editing as explained above unless other coating solution is specifically mentioned.</li>
	</ol>
	</li>
	<li>Characterization of Human iPSCs
	<ol>
		<li>Alkaline Phosphatase staining 
		NOTE: A commercial kit is used here. See the Materials Table.
		<ol>
			<li>Plate putative human iPSCs on 24-well plate and culture for 4-5 d with daily media change.</li>
			<li>To start the staining protocol, aspirate the culture medium and wash the cells with 1 mL of 1x PBS with 0.05% Tween 20 (PBST).</li>
			<li>Add 0.5 mL of fix solution in the kit on the cells and incubate at RT for 2 to 5 min. Aspirate the fix solution and then wash the fixed cells with PBST. Do not allow the wells to dry.</li>
			<li>Add 0.5 mL of freshly prepared AP Substrate Solution per well by mixing solution A, B and C. Incubate the cells in the dark (wrapped with foil or in a dark container) at room temperature for 5 to 15 min. 
			NOTE: Closely monitor the color change and stop the reaction when the color turns bright to avoid non-specific staining.</li>
			<li>Stop the reaction by aspirating the AP Substrate Solution and washing the wells twice with 2 mL of 1x PBS.</li>
			<li>Observe the colonies under the microscope and capture the images at 4X or 10X magnification using any bright field microscope with a camera.</li>
		</ol>
		</li>
		<li>Immunostaining
		<ol>
			<li>Plate human iPSCs in 24-well plate and culture for 4-5 d with daily media change.</li>
			<li>Wash cells once briefly with PBS and fix for 20 min with 4% paraformaldehyde (PFA) in PBS.</li>
			<li>Wash three times for 10 min (3x, 10 min) with PBS at RT.</li>
			<li>Saturate non-specific sites with 10% normal goat or donkey serum (NS, depending on the animal secondary antibody was raised in) in PBS for 40 min at RT. For only intracellular epitopes, include 0.3% TX-100 in PBS (TX/PBS) to permeabilize.</li>
			<li>Wash 3x5 min with PBS at RT.</li>
			<li>Dilute the OCT4, NANOG, TUJ1, NKX2-5 and SOX17 antibodies in a fresh solution of 5% NS in PBS and incubate for 2 h at RT or overnight at 4 &#176;C.</li>
			<li>Wash 3x for 5 min with PBS at RT.</li>
			<li>Dilute appropriate secondary Alexa Fluor 488 or 568 antibodies in 5% NS/PBS and incubate cells for 1 h at RT.</li>
			<li>Wash 3x for 5 min at RT.</li>
			<li>Incubate with DAPI in PBS for 10 min and wash 2x for 5 min at RT. Use a fluorescent microscope to take pictures.</li>
		</ol>
		</li>
		<li>Fluorescence-activated Cell Sorting (FACS)
		<ol>
			<li>Plate human iPSCs in a 6-well plate and culture till the colonies become 80% confluent (at least 105 cells /sample) with daily mTeSR1 medium change.</li>
			<li>On the day of the experiment, isolate the cells and dissociate to a single cell suspension as explained above in the protocol. Wash with 10% FBS medium.</li>
			<li>For surface markers, resuspend in 0.5-1 mL of 10% FBS medium and keep on ice.</li>
			<li>For intracellular markers, resuspend in 1 mL of 4% PFA/PBS and incubate for 10 min at room temperature. Wash with 10% FBS medium. Resuspend in 0.1% TX/PBS and incubate for 15 min on ice. Wash again with 10% FBS medium. Resuspend in 0.5-1 mL of 10% FBS medium and keep on ice.</li>
			<li>Add 50 &#181;L of the cell suspension to 50 &#181;L of 10% FBS medium containing recommended amount of TRA-1-60, TRA-1-81 or SSEA-4 antibodies and incubate for 30 min to 1 h.</li>
			<li>Wash twice with 10% FBS.</li>
			<li>Resuspend in 100 &#181;L of 10% FBS medium containing fluorescent secondary antibodies and incubate for 30 min. Wash twice with 10% FBS and resuspend in appropriate volume of 10% FBS. The live cells can be differentiated from dead cells by propidium iodide dye added at &#60;1 &#181;g/mL to estimate cell apoptosis during the process.</li>
		</ol>
		</li>
		<li>Tri-lineage differentiation
		<ol>
			<li>Seed two 6-well plates with 1.5-2 x 106 iPSCs/well in mTeSR1 medium with 10 &#181;M rock inhibitor.</li>
			<li>Allow the cells to grow for 2-3 d with daily mTeSR1 medium change until the wells are 80-90% confluent.</li>
			<li>For ectoderm induction, add neural induction medium (NIM) according to the manufacturer&#39;s instructions in 2-3 wells of the 6-well plates.</li>
			<li>Change the medium to RPMI containing 2% B27 supplement minus insulin and 12 &#181;M CHIR99021 for mesoderm induction in 2-3 wells of the plates14. Add only RPMI containing 2% B27 supplement minus insulin for the next 24 h. Add 5 &#181;M IWP4 to the RPMI medium with 2% B27 supplement minus insulin for the next 24 h. After 72 h, replace the media with RPMI containing 2% B27 supplement leading to the generation of beating cardiomyocytes in 2-3 d.</li>
			<li>For endoderm induction, change the medium in wells of the 6-well plates to MCDB 131 supplemented with 1.5 g/L sodium bicarbonate, 1x Glutamax, 10 mM glucose, 0.5% BSA, 100 ng/mL GDF8, and 5&#181;M of CHIR99021 for 24 h. Culture in the same medium without CHIR99021 for 2-3 d15.</li>
			<li>Follow the immunostaining protocol from step 2 for all the three lineages and check for the expression of relevant markers.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. Genome Editing of Human iPSC Using CRISPR-Cas9
<ol>
	<li>Preparation of Cas9 protein and sgRNA
	<ol>
		<li>Aliquot Cas9 protein into microcentrifuge tubes in sterile conditions and store the aliquots by freezing the tubes at -80 &#176;C.</li>
		<li>Design two targeting guide RNAs per gene based on the software from MIT (http://www.genome-engineering.org/crispr/) or any other CRISPR guide design webtool. Target first or second exon of the gene (based on open reading frame) to generate knockouts. Choose the two guides so that they cut close together (~30-100 bp apart) and we can easily visualize the deletion using the same set of screening primers. Choose the guides from the software output on the basis of higher quality score and lower number of off-target sites. Design screening primers using the program primer blast (https://www.ncbi.nlm.nih.gov/tools/primer-blast/). Screening primers should be at least 100 bp away from the Cas9 cut sites and the PCR product size should preferably be between 400-700 bp for easier amplification by PCR.</li>
		<li>Design the two complementary sgRNA oligo DNAs (19-22 nucleotides in length depending on the guide sequence) with a Bsa1 cut site at each end. The oligo DNAs can be synthesized commercially and annealed to form double-strand DNA. For annealing, add 1 &#181;L each of 100 &#181;M guides, 25 &#181;L of annealing buffer (10 mM Tris, pH7.5-8.0, 50 mM NaCl, 1 mM EDTA) and 23 &#181;L of water. Incubate in a thermocycler programmed to start at 95 &#176;C for 2 min and then gradually cooling to 25 &#176;C over 45 min.</li>
		<li>Clone the 2 &#181;L of resulting fragment into a Bsa1 restriction enzyme digested 1 &#181;L of in-house T7 promoter pCR2.1 vector with a Cas9 binding site using T4 DNA ligase as per the manufacturer&#39;s protocol.</li>
		<li>Sequence the cloned DNA fragments and when fidelity is established, in vitro transcribe the clones using a T7 kit to generate single guide RNAs. Purify the sgRNAs with a commercial kit and elute in RNase-free water. Check the RNA concentration.</li>
	</ol>
	</li>
	<li>Transfection of hiPSCs
	<ol>
		<li>Culture hiPSCs in mTeSR1 medium as described above until the cells are 40-50% confluent.</li>
		<li>Two hours before nucleofection, replace the medium with 2 mL of prewarmed mTeSR1 medium containing 10 &#181;M rock inhibitor.</li>
		<li>One hour later, prepare destination wells for nucleofected cells by aspirating&#160;membrane, prepared as above, from 12-well plate and replacing with prewarmed 1 mL of mTeSR1 medium with 10 &#181;M rock inhibitor. Keep at 37 &#176;C for incubation.</li>
		<li>Prepare nucleofection master mix (scale appropriately depending on the samples) for each sample by adding 16.4 &#181;L of P3 primary cell supplement; 3.6 &#181;L of Supplement 1 from the nucleofector kit; 0.5 &#181;g of Cas9 protein and 0.5 &#181;g of each sgRNA in 22 &#181;L per reaction volume. pMAX GFP vector was also nucleofected as per the manufacturer&#39;s recommendations in cells to roughly estimate the efficiency of iPSC transfection.</li>
		<li>Wash each well with 2 mL of RT PBS after aspirating the medium containing rock inhibitor from the iPSC wells. Then aspirate PBS, add 1 mL of cell detachment solution, and incubate the plate at 37 &#176;C for 10 min.</li>
		<li>Resuspend the cells in 3 mL of mTeSR1 medium and gently pipette up and down to generate a single-cell suspension. Transfer dissociated cells to a 15 mL centrifuge tube containing 5 mL mTeSR1 medium.</li>
		<li>Count cells with cell counter and calculate total volume required for 0.5 x 106 cells/transfection. Place desired quantity of cells in 15-mL centrifuge tube, centrifuge at 200 x g for 5 min at RT and aspirate supernatant.</li>
		<li>Resuspend each unit of 0.5 x 106 cells in 22 &#181;L of the transfection master mix prepared in step 4. Quickly transfer cells into the central chamber of one well of a nucleocuvette strip. Place the strip into a nucleofector device and nucleofect cells using program CB150.</li>
		<li>After nucleofection, quickly add 80 &#181;L of prewarmed mTESR1 medium containing 10 &#181;M rock inhibitor to each well of the nucleofected cells. Gently mix by pipetting up and down.</li>
		<li>Gently transfer cells from the strip to wells of the&#160;membrane pre-coated 12-well plate containing mTeSR1 medium with rock inhibitor prepared in step 3.</li>
		<li>After 1 d, change to fresh mTeSR1 medium without rock inhibitor. Harvest cells 2-3 d after nucleofection for single-cell sorting.</li>
	</ol>
	</li>
	<li>Single-Cell Isolation of targeted hiPSCs
	<ol>
		<li>One day before the sorting, prepare 96-well MEF plates by seeding 2 x 106 cells/gelatin-coated plate in 10% FBS medium. Approximately 70-80% of the clones survive after nucleofection and 2-3 MEF plates can be prepared for each editing experiment.</li>
		<li>Following the overnight incubation, change the medium to hESC medium (as described previously) supplemented with 100 ng/mL bFGF, 1x SMC4 (inhibitors added to the media to enhance single cell viability), and 5 mg/mL fibronectin to promote adhesion.</li>
		<li>Replace the medium on the hiPSCs in the 12-well plate from mTeSR1 to mTeSR1 medium supplemented with 1x SMC4 for at least 2 h before single cell sorting.</li>
		<li>Aspirate the medium from hiPSCs and wash the cells gently with PBS. Add 500 &#181;L of cell detachment solution in each well and incubate at 37 &#176;C for 10 min after aspirating the PBS. Generate the single-cell suspension by adding 1 mL of mTeSR1 (unsupplemented) to each well and pipetting up and down gently several times.</li>
		<li>Place cell suspension in a 15-mL conical tube and centrifuge for 5 min at 200 x g at RT. Aspirate supernatant and resuspend the cells in 1 mL of mTeSR1.</li>
		<li>Sort the cells in single cells using a cell sorter with 100 mm nozzle under sterile conditions with one cell in the individual well of the 96-well plates prepared in step 1.</li>
		<li>Four days after sorting, colony formation should be apparent; at this point replace the culture medium with hESC medium supplemented with 1x SMC4.</li>
		<li>Eight days after sorting, replace medium with hESC medium and culture for 2 d.</li>
		<li>Detach the colonies using 1 mg/mL collagenase for 20 min and transfer them to 24-well membrane coated plates in mTeSR1 with rock inhibitor. Let the single-cell clones grow and extract their genomic DNA after duplicating or expanding them. Use gene specific primers to amplify the target DNA by PCR.</li>
		<li>Use fragment analyzer kit for initial screening of PCR amplified target genomic region of all clones by running them through the gel/dye mix as per the manufacturer&#39;s instructions. Estimate the resulting fragment size in the software PROSize by comparing it with the appropriate ladder, which is run with the samples. Sequence the expected &#39;edited&#39; clones and analyze sequencing data to confirm the editing or deletions.</li>
		<li>For differentiating monoallelic and biallelic clones, amplify the edited sequence with PCR and clone in pJET1.2 vector. Send at least 8-10 clones for sequencing and check the sequence to confirm editing in one or both alleles.</li>
		<li>Once successfully targeted iPSC clones have been identified through genotyping, examine to confirm that they have not lost pluripotency or gained chromosomal abnormalities through the process.</li>
	</ol>
	</li>
</ol>

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Reprogramming of human somatic cells to iPSCs has provided a major boost to the fields of basic biology research, personalized medicine, disease modeling, drug development and regenerative medicine16. Many current and widely used methods of human iPSC generation require the use of virus with the risk of integration into the host genome or episomal vectors with low reprogramming efficiency. Here, we present an efficient method for generating feeder-free iPSCs from human pancreatic cells with Sendai...

The reprogramming of human somatic cells to the pluripotent state by overexpression of reprogramming factors has revolutionized stem cell research with applications in disease modeling, regenerative medicine, and drug development. Several non-viral reprogramming methods are available for delivery of reprogramming factors and generating iPSCs, but the process is labor intensive and not very efficient1. The viral methods, though efficient, are associated with problems of virus integration and tumorigenicity2,3,4. In this manuscript, we report the use of cytoplasmic Sendai virus for delivering reprogramming factors and establishing footprint-free iPSC lines that lack integration of any viral vector sequences into their genomes5. Sendai virus is an RNA virus that is diluted out of cell cytoplasm ~10 passages after infection and produces reprogramming factors in abundance, leading to rapid and efficient reprogramming6,7. The established iPSCs can then be readily transitioned to feeder-free medium&#160;to avoid the use of mouse embryonic fibroblasts (MEFs) as feeder cells8.
In this publication, in addition to outlining the Sendai virus mediated reprogramming, we also describe an improved protocol for editing iPSCs, which has the potential to supply unlimited human cells with desired genetic modifications for research. We have used CRISPR/Cas9 technology for the modification of iPSCs, which is now being used for a wide range of applications including knock-ins and knockouts, large-scale genomic deletions, pooled library screening for gene discovery, genetic engineering of numerous model organisms, and gene therapy9,10,11. This technique involves the formation of complexes of Streptococcus pyogenes-derived Cas9 nuclease and 20-mer guide RNAs that achieve target recognition via base-pairing with genomic target sequence adjacent to 3&#39; nucleotide protospacer adjacent motif (PAM) sequence. The Cas9 nuclease induces a double stranded break ~3 nucleotides from the PAM site, which is subsequently repaired predominantly by non-homologous end joining (NHEJ) pathway leading to insertions or deletions in the open reading frame, and thereby functional knockout of genes12.
Our improved protocol includes the details for culture of human pancreatic cells, their reprogramming on mitotically inactivated mouse embryonic fibroblasts (MEFs) to achieve higher efficiency of reprogramming, subsequent adaptation to feeder-free culture on Matrigel, characterization of established iPSCs, CRISPR guided RNA design and preparation, delivery into iPSCs as RNP complexes, single cell sorting to generate clonal lines of edited iPSCs, easy screening and identification of edits, and characterization of single cell clones. Genomic deletions were efficiently generated in this study by the introduction of Cas9 protein and two CRISPR sgRNA RNP complexes to induce double stranded breaks (DSBs) and deletion of the intervening segment. This method capitalizes on the use of two guides for generating deletions in the open reading frame, high efficiency of NHEJ leading to low number of clones that need to be characterized, and easy preliminary screening of clones by the automated capillary electrophoresis unit, fragment analyzer. These effective genome editing methods to generate human stem cell-based disease models will soon become a standard and routine approach in any stem cell laboratory. Finally, precise genome editing will make it possible to go beyond stem cell disease modeling and potentially could help catalyze cell-based therapies.

<table>
	<tbody>
		<tr>
			<td>Sendai viral vectors - CytoTune-iPS 2.0 Kit</td>
			<td>Invitrogen</td>
			<td>A16517</td>
			<td>Thaw on ice; S No: 1</td>
		</tr>
		<tr>
			<td>Trypsin EDTA</td>
			<td>Gibco Life Tech</td>
			<td>25300-054</td>
			<td>0.05%, 100 ml; S No: 2</td>
		</tr>
		<tr>
			<td>Rock inhibitor (Y-27632)</td>
			<td>Milipore</td>
			<td>SCM075</td>
			<td>Use 10 &#956;M; S No: 3</td>
		</tr>
		<tr>
			<td>DMEM/F-12 medium</td>
			<td>Invitrogen</td>
			<td>11330-032</td>
			<td>S No: 4</td>
		</tr>
		<tr>
			<td>Serum replacement (KSR)</td>
			<td>Gibco</td>
			<td>10828028</td>
			<td>S No: 5</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Invitrogen</td>
			<td>11960069</td>
			<td>1X; S No: 6</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Thermo Scientific</td>
			<td>SH30071.03</td>
			<td>Aliquot; S No: 7</td>
		</tr>
		<tr>
			<td>L-glutamine (Glutamax, 100X), liquid</td>
			<td>Thermo Scientific</td>
			<td>35050061</td>
			<td>1/100; S No: 8</td>
		</tr>
		<tr>
			<td>Non-Essential Amino Acids</td>
			<td>Gibco</td>
			<td>11140-050</td>
			<td>1/100; S No: 9</td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Gibco</td>
			<td>21985023</td>
			<td>55 mM, 1/1,000; S No: 10</td>
		</tr>
		<tr>
			<td>Hausser Hemacytometers</td>
			<td>Hausser Scientific</td>
			<td>02-671-54</td>
			<td>S No: 11</td>
		</tr>
		<tr>
			<td>&#160;0.1% Gelatin Solution</td>
			<td>STEMCELL Technologies</td>
			<td>7903</td>
			<td>Incubate at 37&#186; C for 1 hour; S No: 12</td>
		</tr>
		<tr>
			<td>SSEA-4 antibody</td>
			<td>Santacruz</td>
			<td>sc-21704</td>
			<td>1/100; S No: 13</td>
		</tr>
		<tr>
			<td>TRA-1-81 antibody</td>
			<td>Cell Signaling</td>
			<td>4745S</td>
			<td>1/200; S No: 14</td>
		</tr>
		<tr>
			<td>&#160;OCT4 antibody</td>
			<td>Santa Cruz</td>
			<td>sc-5279</td>
			<td>1/1,000; S No: 15</td>
		</tr>
		<tr>
			<td>Collagen I, Rat Tail</td>
			<td>Life Technologies</td>
			<td>A10483-01</td>
			<td>Keep cold; S No: 16</td>
		</tr>
		<tr>
			<td>Alexa Fluor fluorescent 488/ 568 (secondary antibodies)</td>
			<td>Invitrogen</td>
			<td>A21202/A10042</td>
			<td>1/2,000; S No: 17</td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Hyclone</td>
			<td>SH30028LS</td>
			<td>1X; S No: 18</td>
		</tr>
		<tr>
			<td>100-mm tissue culture dish</td>
			<td>Falcon</td>
			<td>353003</td>
			<td>S No: 20</td>
		</tr>
		<tr>
			<td>96-well tissue culture plate</td>
			<td>Falcon</td>
			<td>353078</td>
			<td>S No: 21</td>
		</tr>
		<tr>
			<td>6-well tissue culture plate</td>
			<td>Falcon</td>
			<td>353046</td>
			<td>S No: 22</td>
		</tr>
		<tr>
			<td>Dissecting scope&#160;</td>
			<td>Nikon</td>
			<td>SMZ745</td>
			<td>S No: 23</td>
		</tr>
		<tr>
			<td>Picking hood</td>
			<td>NuAire</td>
			<td>NU-301</td>
			<td>S No: 24</td>
		</tr>
		<tr>
			<td>15&#160;ml Centrifuge Tube</td>
			<td>Greiner Bio-One</td>
			<td>188271</td>
			<td>S No: 25</td>
		</tr>
		<tr>
			<td>50&#160;ml Centrifuge Tube</td>
			<td>Greiner Bio-One</td>
			<td>227261</td>
			<td>S No: 26</td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Invitrogen</td>
			<td>11360</td>
			<td>S No: 28</td>
		</tr>
		<tr>
			<td>&#946;-mercaptoethanol</td>
			<td>Sigma</td>
			<td>M7522</td>
			<td>S No: 29</td>
		</tr>
		<tr>
			<td>Prigrow III medium</td>
			<td>ABM</td>
			<td>TM003</td>
			<td>S No: 31</td>
		</tr>
		<tr>
			<td>Countess&#8482; Cell Counter</td>
			<td>Invitrogen</td>
			<td>C10227</td>
			<td>S No: 32</td>
		</tr>
		<tr>
			<td>Faxitron X-ray system</td>
			<td>Faxitron</td>
			<td>CellRad</td>
			<td>S No: 33</td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>Innovative cell Technologies</td>
			<td>AT-104</td>
			<td>S No: 34</td>
		</tr>
		<tr>
			<td>Collagenase</td>
			<td>Life Technologies</td>
			<td>17104019</td>
			<td>1mg/ml stock; S No: 35</td>
		</tr>
		<tr>
			<td>Dispase</td>
			<td>STEMCELL Technologies</td>
			<td>7923</td>
			<td>S No: 36</td>
		</tr>
		<tr>
			<td>hESC qualified matrigel</td>
			<td>BD Biosciences</td>
			<td>354277</td>
			<td>To dilute, use cold DMEM/F-12; S No: 37</td>
		</tr>
		<tr>
			<td>bFGF</td>
			<td>R &#38; D</td>
			<td>233-FB</td>
			<td>Stock 10 ug/ml; S No: 38</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>EMS</td>
			<td>15710</td>
			<td>4% stock in PBS; S No: 39</td>
		</tr>
		<tr>
			<td>TRA-1-60</td>
			<td>Santa Cruz</td>
			<td>sc-21705</td>
			<td>1/100; S No: 40</td>
		</tr>
		<tr>
			<td>NANOG</td>
			<td>ReproCELL</td>
			<td>RCAB0004P-F</td>
			<td>1/100; S No: 41</td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma</td>
			<td>P9416-100ML</td>
			<td>S No: 42</td>
		</tr>
		<tr>
			<td>Alkaline Phosphatase kit</td>
			<td>Stemgent</td>
			<td>00-0055</td>
			<td>S No: 43</td>
		</tr>
		<tr>
			<td>Cas9 protein</td>
			<td>PNA Bio</td>
			<td>CP01-50</td>
			<td>Thaw and aliquot; S No: 44</td>
		</tr>
		<tr>
			<td>Goat or donkey serum</td>
			<td>Sigma</td>
			<td>D9663/G9023</td>
			<td>S No: 45</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>X100-100ML</td>
			<td>S No: 46</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Thermo Scientific</td>
			<td>D1306</td>
			<td>S No: 47</td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>Sigma</td>
			<td>9285-100ML</td>
			<td>S No: 48</td>
		</tr>
		<tr>
			<td>NaCL</td>
			<td>Sigma</td>
			<td>S7653-250G</td>
			<td>S No: 49</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma</td>
			<td>BP2482-500</td>
			<td>S No: 50</td>
		</tr>
		<tr>
			<td>T4 DNA ligase</td>
			<td>NEB</td>
			<td>M0202T</td>
			<td>S No: 51</td>
		</tr>
		<tr>
			<td>Mega Shortscript T7 kit</td>
			<td>Thermo Scientific</td>
			<td>AM1354</td>
			<td>S No: 52</td>
		</tr>
		<tr>
			<td>Mega Clear kit</td>
			<td>Thermo Scientific</td>
			<td>AM1908</td>
			<td>S No: 53</td>
		</tr>
		<tr>
			<td>SMC4</td>
			<td>BD Biosciences</td>
			<td>354357</td>
			<td>S No: 54</td>
		</tr>
		<tr>
			<td>Fibronectin</td>
			<td>STEMCELL Technologies</td>
			<td>7159</td>
			<td>S No: 55</td>
		</tr>
		<tr>
			<td>CloneJET cloning kit</td>
			<td>Thermo Scientific</td>
			<td>K1232</td>
			<td>S No: 56</td>
		</tr>
		<tr>
			<td>Fragment analyzerTM</td>
			<td>Advanced Analytical</td>
			<td></td>
			<td>S No: 57</td>
		</tr>
		<tr>
			<td>mTeSR1 medium kit</td>
			<td>STEMCELL Technologies</td>
			<td>5850</td>
			<td>Warm to room temperature; S No: 58</td>
		</tr>
		<tr>
			<td>Freezing medium mFreSR&#8482;</td>
			<td>STEMCELL Technologies</td>
			<td>5855</td>
			<td>S No: 59</td>
		</tr>
		<tr>
			<td>Freezing medium CryoStor&#174;</td>
			<td>STEMCELL Technologies</td>
			<td>7930</td>
			<td>S No: 60</td>
		</tr>
		<tr>
			<td>MEFs</td>
			<td>Globalstem</td>
			<td>GSC-6301G</td>
			<td>S No: 61</td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Invitrogen</td>
			<td>25030081</td>
			<td>S No: 62</td>
		</tr>
		<tr>
			<td>Human pancreatic cells</td>
			<td>ABM</td>
			<td>T0159</td>
			<td>S No: 63</td>
		</tr>
		<tr>
			<td>STEMdiff&#8482; Neural Induction Medium</td>
			<td>Stemcell Technologies</td>
			<td>5835</td>
			<td>S No: 64</td>
		</tr>
		<tr>
			<td>RPMI</td>
			<td>Thermofisher</td>
			<td>11875-093</td>
			<td>S No: 65</td>
		</tr>
		<tr>
			<td>2% B27-insulin</td>
			<td>Thermofisher</td>
			<td>A1895601</td>
			<td>S No: 66</td>
		</tr>
		<tr>
			<td>CHIR99021</td>
			<td>Stemcell Technologies</td>
			<td>72052</td>
			<td>S No: 67</td>
		</tr>
		<tr>
			<td>IWP4</td>
			<td>Stemcell Technologies</td>
			<td>72552</td>
			<td>S No: 68</td>
		</tr>
		<tr>
			<td>2% B27</td>
			<td>Thermofisher</td>
			<td>17504044</td>
			<td>S No: 69</td>
		</tr>
		<tr>
			<td>MCDB 131</td>
			<td>Life Technologies</td>
			<td>10372019</td>
			<td>S No: 70</td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>Sigma-Aldrich</td>
			<td>S8761-100ML</td>
			<td>S No: 71</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G8270-100G</td>
			<td>S No: 72</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Proliant</td>
			<td>68700</td>
			<td>S No: 73</td>
		</tr>
		<tr>
			<td>GDF8</td>
			<td>Pepro-Tech</td>
			<td>120-00</td>
			<td>S No: 74</td>
		</tr>
		<tr>
			<td>TUJ1 antibody</td>
			<td>EMD Milipore</td>
			<td>AB9354</td>
			<td>S No: 75</td>
		</tr>
		<tr>
			<td>NKX2-5 antibody</td>
			<td>Santa Cruz</td>
			<td>Sc-14033</td>
			<td>S No: 76</td>
		</tr>
		<tr>
			<td>SOX17 antibody</td>
			<td>R &#38; D systems</td>
			<td>AF1924</td>
			<td>S No: 77</td>
		</tr>
		<tr>
			<td>Propidium iodide</td>
			<td>Thermo Scientific</td>
			<td>P3566</td>
			<td>S No: 78</td>
		</tr>
		<tr>
			<td>Amaxa 4D-nucleofector&#8482;</td>
			<td>Lonza</td>
			<td>AAF-1002</td>
			<td>S No: 79</td>
		</tr>
		<tr>
			<td>FACSAria II (cell sorter)</td>
			<td>BD biosciences</td>
			<td>SORP UV</td>
			<td>S No: 80</td>
		</tr>
	</tbody>
</table>

efficient generation and editing of feeder-free ipscs from human pancreatic cells using the crispr-cas9 system

Embryonic and induced pluripotent stem cells can self-renew and differentiate into multiple cell types of the body. The pluripotent cells are thus coveted for research in regenerative medicine and are currently in clinical trials for eye diseases, diabetes, heart diseases, and other disorders. The potential to differentiate into specialized cell types coupled with the recent advances in genome editing technologies including the CRISPR/Cas system have provided additional opportunities for tailoring the genome of iPSC for varied applications including disease modeling, gene therapy, and biasing pathways of differentiation, to name a few. Among the available editing technologies, the CRISPR/Cas9 from Streptococcus pyogenes has emerged as a tool of choice for site-specific editing of the eukaryotic genome. The CRISPRs are easily accessible, inexpensive, and highly efficient in engineering targeted edits. The system requires a Cas9 nuclease and a guide sequence (20-mer) specific to the genomic target abutting a 3-nucleotide &#34;NGG&#34; protospacer-adjacent-motif (PAM) for targeting Cas9 to the desired genomic locus, alongside a universal Cas9 binding tracer RNA (together called single guide RNA or sgRNA). Here we present a step-by-step protocol for efficient generation of feeder-independent and footprint-free iPSC and describe methodologies for genome editing of iPSC using the Cas9 ribonucleoprotein (RNP) complexes. The genome editing protocol is effective and can be easily multiplexed by pre-complexing sgRNAs for more than one target with the Cas9 protein and simultaneously delivering into the cells. Finally, we describe a simplified approach for identification and characterization of iPSCs with desired edits. Taken together, the outlined strategies are expected to streamline generation and editing of iPSC for manifold applications.

In this publication, we have followed a simple but efficient protocol for the generation of iPSC from human pancreatic cells using integration or footprint-free Sendai virus vectors. Figure 1A shows a schematic representation of this reprogramming protocol. The human pancreatic cells were purchased commercially, cultured in Prigrow III medium and transduced with Sendai virus as explained above. Transduced spindle shaped pancreatic cells did not show any morph...

Watch this Scientific Journal Video about Efficient Generation and Editing of Feeder-free IPSCs from Human Pancreatic Cells Using the CRISPR-Cas9 System at JoVE.com

Efficient Generation and Editing of Feeder-free IPSCs from Human Pancreatic Cells Using the CRISPR-Cas9 System

This protocol describes in detail the generation of footprint-free induced pluripotent stem cells (iPSCs) from human pancreatic cells in feeder-free conditions, followed by editing using CRISPR/Cas9 ribonucleoproteins and characterization of the modified single-cell clones.

Embryonic and induced pluripotent stem cells can self-renew and differentiate into multiple cell types of the body. The pluripotent cells are thus coveted ...

efficient-generation-editing-feeder-free-ipscs-from-human-pancreatic

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Bioengineering

10.0K Views.  University of Maryland. This protocol describes in detail the generation of footprint-free induced pluripotent stem cells (iPSCs) from human pancreatic cells in feeder-free conditions, followed by editing using CRISPR/Cas9 ribonucleoproteins and characterization of the modified single-cell clones.

Video: Efficient Generation and Editing of Feeder-free IPSCs from Human Pancreatic Cells Using the CRISPR-Cas9 System

Therapeutic Gene Delivery and Transfection in Human Pancreatic Cancer Cells using Epidermal Growth Factor Receptor-targeted Gelatin Nanoparticles

More than 32,000 patients are diagnosed with pancreatic cancer in the United States per year and the disease is associated with very high mortality 1. Urgent need exists to develop novel clinically-translatable therapeutic strategies that can improve on the dismal survival statistics of pancreatic cancer patients. Although gene therapy in cancer has shown a tremendous promise, the major challenge is in the development of safe and effective delivery system, which can lead to sustained transgene expression. 
Gelatin is one of the most versatile natural biopolymer, widely used in food and pharmaceutical products. Previous studies from our laboratory have shown that type B gelatin could physical encapsulate DNA, which preserved the supercoiled structure of the plasmid and improved transfection efficiency upon intracellular delivery. By thiolation of gelatin, the sulfhydryl groups could be introduced into the polymer and would form disulfide bond within nanoparticles, which stabilizes the whole complex and once disulfide bond is broken due to the presence of glutathione in cytosol, payload would be released 2-5. Poly(ethylene glycol) (PEG)-modified GENS, when administered into the systemic circulation, provides long-circulation times and preferentially targets to the tumor mass due to the hyper-permeability of the neovasculature by the enhanced permeability and retention effect 6. Studies have shown over-expression of the epidermal growth factor receptor (EGFR) on Panc-1 human pancreatic adenocarcinoma cells 7. In order to actively target pancreatic cancer cell line, EGFR specific peptide was conjugated on the particle surface through a PEG spacer.8
Most anti-tumor gene therapies are focused on administration of the tumor suppressor genes, such as wild-type p53 (wt-p53), to restore the pro-apoptotic function in the cells 9. The p53 mechanism functions as a critical signaling pathway in cell growth, which regulates apoptosis, cell cycle arrest, metabolism and other processes 10. In pancreatic cancer, most cells have mutations in p53 protein, causing the loss of apoptotic activity. With the introduction of wt-p53, the apoptosis could be repaired and further triggers cell death in cancer cells 11. 
Based on the above rationale, we have designed EGFR targeting peptide-modified thiolated gelatin nanoparticles for wt-p53 gene delivery and evaluated delivery efficiency and transfection in Panc-1 cells.

Type B gelatin-based engineered nanovectors system (GENS) was developed for systemic gene delivery and transfection in the treatment of pancreatic cancer. By modification with epidermal growth factor receptor (EGFR) specific peptide on the surface of nanparticles, they could target on EGFR receptor and release plasmid under reducing environment, such as high intracellular glutathione concentrations.

More than 32,000 patients are diagnosed with pancreatic cancer in the United States per year and the disease is associated with very high mortality 1. ...

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells (hESCs) and Induced Pluripotent Stem Cells (iPSCs)

We present a method for deriving natural killer (NK) cells from undifferentiated hESCs and iPSCs using a feeder-free approach. This method gives rise to high levels of NK cells after 4 weeks culture and can undergo further 2-log expansion with artificial antigen presenting cells. hESC- and iPSC-derived NK cells developed in this system have a mature phenotype and function. The production of large numbers of genetically modifiable NK cells is applicable for both basic mechanistic as well as anti-tumor studies. Expression of firefly luciferase in hESC-derived NK cells allows a non-invasive approach to follow NK cell engraftment, distribution, and function. We also describe a dual-imaging scheme that allows separate monitoring of two different cell populations to more distinctly characterize their interactions in vivo. This method of derivation, expansion, and dual in vivo imaging provides a reliable approach for producing NK cells and their evaluation which is necessary to improve current NK cell adoptive therapies.

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells hESCs and Induced Pluripotent Stem Cells iPSCs

This protocol describes the development, expansion, and in vivo imaging of NK cells derived from hESCs and iPSCs.

We present a method for deriving natural killer (NK) cells from undifferentiated hESCs and iPSCs using a feeder-free approach. This method gives rise to ...

Production of Genetically Engineered Golden Syrian Hamsters by Pronuclear Injection of the CRISPR/Cas9 Complex

The pronuclear (PN) injection technique was first established in mice to introduce foreign genetic materials into the pronuclei of one-cell stage embryos. The introduced genetic material may integrate into the embryonic genome and generate transgenic animals with foreign genetic information following transfer of the injected embryos to foster mothers. Following the success in mice, PN injection has been applied successfully in many other animal species. Recently, PN injection has been successfully employed to introduce reagents with gene-modifying activities, such as the CRISPR/Cas9 system, to achieve site-specific genetic modifications in several laboratory and farm animal species. In addition to mastering the special set of microinjection skills to produce genetically modified animals by PN injection, researchers must understand the reproduction physiology and behavior of the target species, because each species presents unique challenges. For example, golden Syrian hamster embryos have unique handling requirements in vitro such that PN injection techniques were not possible in this species until recent breakthroughs by our group. With our species-modified PN injection protocol, we have succeeded in producing several gene knockout (KO) and knockin (KI) hamsters, which have been used successfully to model human diseases. Here we describe the PN injection procedure for delivering the CRISPR/Cas9 complex to the zygotes of the hamster, the embryo handling conditions, embryo transfer procedures, and husbandry required to produce genetically modified hamsters.

Pronuclear (PN) injection of the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein-9 nuclease (CRISPR/Cas9) system is a highly efficient method for producing genetically engineered golden Syrian hamsters. Herein, we describe the detailed PN injection protocol for the production of gene knockout hamsters with the CRISPR/Cas9 system.

The pronuclear (PN) injection technique was first established in mice to introduce foreign genetic materials into the pronuclei of one-cell stage embryos. ...

Microinjection of Western Corn Rootworm, Diabrotica virgifera virgifera, Embryos for Germline Transformation, or CRISPR/Cas9 Genome Editing

The western corn rootworm (WCR) is an important pest of corn and is well known for its ability to rapidly adapt to pest management strategies. Although RNA interference (RNAi) has proved to be a powerful tool for studying WCR biology, it has its limitations. Specifically, RNAi itself is transient (i.e. does not result in long-term Mendelian inheritance of the associated phenotype), and it requires knowing the DNA sequence of the target gene. The latter can be limiting if the phenotype of interest is controlled by poorly conserved, or even novel genes, because identifying useful targets would be challenging, if not impossible. Therefore, the number of tools in WCR&#39;s genomic toolbox should be expanded by the development of methods that could be used to create stable mutant strains and enable sequence-independent surveys of the WCR genome. Herein, we detail the methods used to collect and microinject precellular WCR embryos with nucleic acids. While the protocols described herein are aimed at the creation of transgenic WCR, CRISPR/Cas9-genome editing could also be performed using the same protocols, with the only difference being the composition of the solution injected into the embryos.

Microinjection of Western Corn Rootworm, Diabrotica virgifera virgifera, Embryos for Germline Transformation, or CRISPR/Cas9 Genome Editing

Here we present protocols for collecting and microinjecting precellular western corn rootworm embryos for the purpose of performing functional-genomic assays such as germline transformation and CRISPR/Cas9-genome editing.

The western corn rootworm (WCR) is an important pest of corn and is well known for its ability to rapidly adapt to pest management strategies. Although ...

Generation of Cationic Nanoliposomes for the Efficient Delivery of In Vitro Transcribed Messenger RNA

The development of messenger RNA (mRNA)-based therapeutics for the treatment of various diseases becomes more and more important because of the positive properties of in vitro transcribed (IVT) mRNA. With the help of IVT mRNA, the de novo synthesis of a desired protein can be induced without changing the physiological state of the target cell. Moreover, protein biosynthesis can be precisely controlled due to the transient effect of IVT mRNA.
For the efficient transfection of cells, nanoliposomes (NLps) may represent a safe and efficient delivery vehicle for therapeutic mRNA. This study describes a protocol to generate safe and efficient cationic NLps consisting of DC-cholesterol and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) as a delivery vector for IVT mRNA. NLps having a defined size, a homogeneous distribution, and a high complexation capacity, and can be produced using the dry-film method. Moreover, we present different test systems to analyze their complexation and transfection efficacies using synthetic enhanced green fluorescent protein (eGFP) mRNA, as well as their effect on cell viability. Overall, the presented protocol provides an effective and safe approach for mRNA complexation, which may advance and improve the administration of therapeutic mRNA.

Generation of Cationic Nanoliposomes for the Efficient Delivery of In Vitro Transcribed Messenger RNA

Here we describe a protocol for the generation of cationic nanoliposomes, which is based on the dry-film method and can be used for the safe and efficient delivery of in vitro transcribed messenger RNA.

The development of messenger RNA (mRNA)-based therapeutics for the treatment of various diseases becomes more and more important because of the positive ...

Isolation of Adult Human Dermal Fibroblasts from Abdominal Skin and Generation of Induced Pluripotent Stem Cells Using a Non-Integrating Method

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable diseases, for the reconstitution and regeneration of injured tissues and for the development of new drugs. Despite all the advantages related to the use of iPSCs, such as the low risk of rejection, the lessened ethical issues, and the possibility to obtain them from both young and old patients without any difference in their reprogramming potential, problems to overcome are still numerous. In fact, cell reprogramming conducted with viral and integrating viruses can cause infections and the introduction of required genes can induce a genomic instability of the recipient cell, impairing their use in clinic. In particular, there are many concerns about the use of c-Myc gene, well-known from several studies for its mutation-inducing activity. Fibroblasts have emerged as the suitable cell population for cellular reprogramming as they are easy to isolate and culture and are harvested by a minimally invasive skin punch biopsy. The protocol described here provides a detailed step-by-step description of the whole procedure, from sample processing to obtain cell cultures, choice of reagents and supplies, cleaning and preparation, to cell reprogramming by the means of a commercial non-modified RNAs (NM-RNAs)-based reprogramming kit. The chosen reprogramming kit allows an effective reprogramming of human dermal fibroblast to iPSCs and small colonies can be seen as early as 24 h after the first transfection, even with modifications with the respect to the standard datasheet. The reprogramming procedure used in this protocol offers the advantage of a safe reprogramming, without the risk of infections caused by viral vector-based methods, reduces the cellular defense mechanisms, and allows the generation of xeno-free iPSCs, all critical features that are mandatory for further clinical applications.

Cell reprogramming requires the introduction of key genes, which regulate and maintain the pluripotent cell state. The protocol described enables the formation of induced pluripotent stem cells (iPSCs) colonies from human dermal fibroblasts without viral/integrating methods but using non-modified RNAs (NM-RNAs) combined with immune evasion factors reducing cellular defense mechanisms.

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable ...

Scalable Generation of Mature Cerebellar Organoids from Human Pluripotent Stem Cells and Characterization by Immunostaining

The cerebellum plays a critical role in the maintenance of balance and motor coordination, and a functional defect in different cerebellar neurons can trigger cerebellar dysfunction. Most of the current knowledge about disease-related neuronal phenotypes is based on postmortem tissues, which makes understanding of disease progression and development difficult. Animal models and immortalized cell lines have also been used as models for neurodegenerative disorders. However, they do not fully recapitulate human disease. Human induced pluripotent stem cells (iPSCs) have great potential for disease modeling and provide a valuable source for regenerative approaches. In recent years, the generation of cerebral organoids from patient-derived iPSCs improved the prospects for neurodegenerative disease modeling. However, protocols that produce large numbers of organoids and a high yield of mature neurons in 3D culture systems are lacking. The protocol presented is a new approach for reproducible and scalable generation of human iPSC-derived organoids under chemically-defined conditions using scalable single-use bioreactors, in which organoids acquire cerebellar identity. The generated organoids are characterized by the expression of specific markers at both mRNA and protein level. The analysis of specific groups of proteins allows the detection of different cerebellar cell populations, whose localization is important for the evaluation of organoid structure. Organoid cryosectioning and further immunostaining of organoid slices are used to evaluate the presence of specific cerebellar cell populations and their spatial organization.

This protocol describes a dynamic culture system to produce controlled size aggregates of human pluripotent stem cells and further stimulate differentiation in cerebellar organoids under chemically-defined and feeder-free conditions using a single-use bioreactor.

The cerebellum plays a critical role in the maintenance of balance and motor coordination, and a functional defect in different cerebellar neurons can ...

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically carried out in low throughput. Rapid and ongoing expansion of induced pluripotent stem cell (iPSC) technology presents a new standard in cardiovascular research and alternate methods are now necessary to increase throughput of electrophysiological data at a single cell level. VF2.1Cl is a recently derived voltage sensitive dye which provides a rapid single channel, high magnitude response to fluctuations in membrane potential. It possesses kinetics superior to those of other existing voltage indicators and makes available functional data equivalent to that of traditional microelectrode techniques. Here, we demonstrate simplified, non-invasive action potential characterization in externally paced human iPSC derived cardiomyocytes using a modular and highly affordable photometry system.

Here we describe optical acquisition and characterization of action potentials from induced pluripotent stem cell derived cardiomyocytes using a high-speed modular photometry system.

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically ...

Microinjection of Corn Planthopper, Peregrinus maidis, Embryos for CRISPR/Cas9 Genome Editing

The corn planthopper, Peregrinus maidis, is&#160;a pest of maize and a vector of several maize viruses. Previously published methods describe the triggering of RNA interference (RNAi) in P. maidis through microinjection of double-stranded RNAs (dsRNAs) into nymphs and adults. Despite the power of RNAi, phenotypes generated via this technique are transient and lack long-term Mendelian inheritance. Therefore, the P. maidis toolbox needs to be expanded to include functional genomic tools that would enable the production of stable mutant strains, opening the door for researchers to bring new control methods to bear on this economically important pest. However, unlike the dsRNAs used for RNAi, the components used in CRISPR/Cas9-based genome editing and germline transformation do not easily cross cell membranes. As a result, plasmid DNAs, RNAs, and/or proteins must be microinjected into embryos before the embryo cellularizes, making the timing of injection a critical factor for success. To that end, an agarose-based egg-lay method was developed to allow embryos to be harvested from P. maidis females at relatively short intervals. Herein are provided detailed protocols for collecting and microinjecting precellular P. maidis embryos with CRISPR components (Cas9 nuclease that has been complexed with guide RNAs), and results of Cas9-based gene knockout of a P. maidis eye-color gene, white, are presented. Although these protocols describe CRISPR/Cas9-genome editing in P. maidis, they can also be used for producing transgenic P. maidis via germline transformation by simply changing the composition of the injection solution.

Microinjection of Corn Planthopper, Peregrinus maidis, Embryos for CRISPR/Cas9 Genome Editing

Herein are protocols for collecting and microinjecting precellular corn planthopper embryos for the purpose of modifying their genome via CRISPR/Cas9-based genome editing or for the addition of marked transposable elements through germline transformation.

The corn planthopper, Peregrinus maidis, is&#160;a pest of maize and a vector of several maize viruses. Previously published methods describe the ...

In vitro Induction of Human Dental Pulp Stem Cells Toward Pancreatic Lineages

As of 2000, the success of pancreatic islet transplantation using the Edmonton protocol to treat type I diabetes mellitus still faced some obstacles. These include the limited number of cadaveric pancreas donors and the long-term use of immunosuppressants. Mesenchymal stem cells (MSCs) have been considered to be a potential candidate as an alternative source of islet-like cell generation. Our previous reports have successfully illustrated the establishment of induction protocols for differentiating human dental pulp stem cells (hDPSCs) to insulin-producing cells (IPCs). However, the induction efficiency varied greatly. In this paper, we demonstrate the comparison of hDPSCs pancreatic induction efficiency via integrative (microenvironmental and genetic manipulation) and non-integrative (microenvironmental manipulation) induction protocols for delivering hDPSC-derived IPCs (hDPSC-IPCs). The results suggest distinct induction efficiency for both the induction approaches in terms of 3-dimensional colony structure, yield, pancreatic mRNA markers, and functional property upon multi-dosage glucose challenge. These findings will support the future establishment of a clinically applicable IPCs and pancreatic lineage production platform.

In vitro Induction of Human Dental Pulp Stem Cells Toward Pancreatic Lineages

This protocol presents a comparison between two different induction protocols for differentiating human dental pulp stem cells (hDPSCs) toward pancreatic lineages in vitro: the integrative protocol and the non-integrative protocol. The integrative protocol generates more insulin producing cells (IPCs).

As of 2000, the success of pancreatic islet transplantation using the Edmonton protocol to treat type I diabetes mellitus still faced some obstacles. ...