This work was financially supported by National Institutes of Health to Y.I. (R01-DK090490) and American Diabetes Association to Y.I. (1-17-IBS-132). J.A. and Y.I. are supported by the Fraternal Order of Eagles Diabetes Research Center. A.B. is supported by a National Institutes of Health training grant (T32NS45549). Authors utilized human pancreatic islets provided by the NIDDK-funded Integrated Islet Distribution Program (IIDP) at City of Hope (2UC4DK098085).

Prior to commencement of studies, a human subjects research determination was made by the University of Iowa Institutional Review Board, who determined that the study did not meet the criteria for human subjects research. Consult the local review board before the initiation of the study to determine if the source of islets and planned study requires prior approval.
NOTE: Typically, 1,200&#8722;1,400 islet equivalent (IEQ) of human islets are required for the formation of 192 pseudoislets at the size of 3,000 cells/pseudoislets in a 96-well ultra-low attachment plate or 1,200 pseudoislets at the size of 500 cells/pseudoislets in a microwell culture plate. IEQ of islets required varies between different preparations of human islets as donor factors (age, health, weight), isolation efficiency, and culture conditions affect the yield of the single cell suspension. In this protocol, lentivirus containing shRNA targeting a gene of interest is used. The cytomegalovirus (CMV) and human phosphoglycerate kinase (hPGK) promoter based lentiviral vectors are reported to down-regulate gene efficiently in human pseudoislets5,15. The use of lentivirus requires precaution as biohazard16. Contact the local biosafety committee prior to the initiation of the use of lentivirus.
1. Overnight Culture of Human Islets for Recovery After Shipment
<ol>
	<li>Prepare Connaught Medical Research Laboratories 1066 (CMRL-1066) medium supplemented with 1% human serum albumin (HSA) by combining 50 mL of CMRL-1066, 0.5 g of HSA, 0.5 mL of penicillin-streptomycin, and 0.5 mL of 100 mg/mL glutamine (1% HSA CMRL) in biological safety cabinet (BSC) and passing through a 0.2 &#956;m filter for sterilization.</li>
	<li>Gently swirl the shipping bottle to keep islets in suspension. Transfer the shipping medium containing islets to a 50 mL conical centrifuge tube. Let the tube sit in BSC for 15 min so that islets settle to the bottom of the tube.</li>
	<li>Remove shipping medium gently without disturbing the islet pellet using a 10 mL pipette. Re-suspend the islet pellet in 1% HSA CMRL to a concentration of 400 IEQ/mL.</li>
	<li>Transfer islets into a non-tissue culture treated dish. If islets are split into multiple dishes, keep islets evenly suspended in medium by gently swirling before splitting. Culture islets at 37 &#176;C in a 5% CO2 incubator overnight. 
	NOTE: The use of a non-tissue culture treated dish is required to prevent the attachment of islets to the plate.</li>
</ol>
2. Preparation of Single Cell Suspension from Human Islets
<ol>
	<li>Prepare the following: CMRL-1066 medium with 10% heat-inactivated fetal bovine serum (HiFBS), penicillin-streptomycin, and glutamine (10% HiFBS CRML) at room temperature (RT), a 40 &#181;m strainer, a 35 mm Petri dish, a 1 mL tuberculin syringe, and a hemocytometer.</li>
	<li>Transfer human islets after overnight culture into a 15 mL conical centrifuge tube. Centrifuge at 190 x g for 5 min in a swinging-bucket rotor. Aspirate medium with a 5 mL pipette without disturbing the islet pellet.</li>
	<li>Wash the pellet by adding 10 mL of phosphate-buffered saline (PBS) into the tube, mix gently, and centrifuge at 190 x g for 5 min. Aspirate PBS without disturbing the islet pellet.</li>
	<li>Re-suspend the islet pellet in 0.5 mL of a pre-warmed proteolytic and collagenolytic enzyme mixture and pipette 5x using a P1000 pipette to mix islets. Incubate at 37 &#176;C for 5 min. Mix by pipetting up and down gently 1&#8722;5 times. 
	NOTE: Aggressive pipetting will increase cell loss.</li>
	<li>Check for cloudiness (single cells) and the number of flakes (undigested islets). Add 2&#8722;3 min to 37 &#176;C incubation depending on the extent of digestion judged by cloudiness and the number of flakes. Stop digestion when flakes are reduced to ~10% of predigestion and solution is cloudy. 
	NOTE: Time required for digestion differs depending on the islet size distribution of each human islet preparation.</li>
	<li>Place a 40 &#181;m strainer in a 35 mm Petri dish and wet the strainer by adding 1 mL of 10% HiFBS CMRL and pressing with 1 mL syringe plunger. Transfer all the cell suspension on top of the strainer and collect the pass-through in a fresh 15 mL tube.</li>
	<li>Wash the tube used for islet digestion with 0.5 mL of fresh CMRL medium to collect leftover cells and pass the wash through the strainer. Combine the pass-through in a 15 mL tube. Repeat once.</li>
	<li>Next, dissociate undigested islets remaining on the strainer by pressing the strainer placed in a 35 mm dish with 1 mL syringe plunger. Collect pass-through again and wash the strainer with fresh CMRL-1066 to remove all remaining digested islets from the strainer and the dish. A total ~3 mL of single cell suspension will now be in the 15 mL tube.</li>
	<li>Record the total volume of the cell suspension and take 10 &#181;L aliquot of cells to count the cell number on a hemocytometer.</li>
	<li>Centrifuge the cell suspension for 5 min at 200 x g. Remove medium without disturbing the pellet. Proceed to step 3.1.1 if using a 96-well ultra-low attachment plate or step 3.2.1 if using a 24 well microwell culture plate to reaggregate the cells.</li>
</ol>
3. Pseudoislet Formation and Transduction by Lentivirus
<ol>
	<li>Protocol using a 96-well ultra-low attachment plate
	<ol>
		<li>Determine the desired number of cells per pseudoislet and the number of pseudoislets to create. Typically, 1,000&#8722;3,000 cells are used for each pseudoislet for a 96-well ultra-low attachment plate. For 3,000 cells per pseudoislet, adjust the cell suspension to 1 x 105 cells/mL by resuspending the islet pellet from step 2.10 in 10% HiFBS CMRL so that 30 &#181;L of cell suspension has 3,000 cells. Calculate the total volume of 1 x 105 cells/mL of single cell suspension (mL) required using the following equation: 
		The total volume of 1 x 105 cells/mL of single cell suspension (mL) = (number of cells per pseudoislet) x (number of pseudoislets being made) / 1 x 105. 
		NOTE: Adjust the concentration of the cell suspension based on the desired number of cells per pseudoislet so that 30 &#181;L of cell suspension makes one pseudoislet.</li>
		<li>Transfer the required volume (30 &#181;L x number of pseudoislets being made) of single cell suspension to a fresh 15 mL tube. Add 250 transduction units (TU)/cell of lentivirus containing shRNA targeting a gene of interest or control. 
		CAUTION: Lentivirus is classified as biosafety level 2 and can be integrated into DNA of infected cells. 
		NOTE: Use concentrated lentivirus so that the volume of lentivirus added is minimal. Titer required per cell for efficient gene silencing may differ depending on the lentiviral construct.</li>
		<li>Mix cell suspension with virus by pipetting gently 5x with a P1000 pipette. Transfer mixed cells into a 50 mL sterile reagent reservoir if using an 8-channel pipette.</li>
		<li>Dispense 30 &#181;L per well of single cell suspension mixed with lentivirus into each well using an 8-channel pipette or a P200 pipette depending on the number of the wells.</li>
		<li>Centrifuge the 96-well plate in a swinging-bucket plate centrifuge at 270 x g at RT for 7 min. Check whether cells are gathered in the center of each well. If not, centrifuge again as gathering of all cells in the center of the well is critical for pseudoislet formation. Culture at 37 &#176;C in a humidified 5% CO2 incubator overnight.</li>
		<li>Add 100 &#181;L of pre-warmed 10% HiFBS CMRL per well next morning to avoid drying of cells during subsequent culture. Centrifuge at 270 x g, RT for 7 min. Culture at 37 &#176;C in a 5% CO2 incubator. Pseudoislets will complete formation in 5&#8722;7 days.</li>
		<li>When harvesting pseudoislets, pre-warm the desired volume of 10% HiFBS CMRL (100 &#181;L per islet), prepare one 50 mL sterile reservoir, one sterile 10 cm Petri dish, and one 8-channel pipette in BSC.</li>
		<li>Remove the 96-well plate from the incubator and place in BSC. Pipette 100 &#181;L per islet of 10% HiFBS CMRL into a reservoir.</li>
		<li>Pipette 100 &#181;L per well of 10% HiFBS CMRL from a reservoir to pseudoislets and pipette up and down 2&#8722;3 times gently in the well to lift islets up. Then, aspirate medium in the well containing a pseudoislet and eject into a 10 cm Petri dish. Use of an 8-channel pipette allows transfer of 8 pseudoislets at one time.</li>
		<li>Check the plate under a light microscope to ensure the complete removal of all the pseudoislets. Pseudoislets form firm aggregates and remain aggregated after lifting. The pseudoislets are now ready for downstream experiments.</li>
	</ol>
	</li>
	<li>Protocol using a 24-well microwell culture plate
	<ol>
		<li>Warm up the anti-adherence rinse solution (Table of Materials) to RT for efficient spheroid formation. Also, pre-warm plain CMRL-1066 and 10% HiFBS CMRL.</li>
		<li>Add 500 &#181;L per well of the anti-adherence rinsing solution to each well of the 24-well microwell culture plate to be used for pseudoislets. Centrifuge at 1,300 x g for 5 min in a swinging-bucket plate centrifuge.</li>
		<li>Observe the plate under a microscope to ensure that air bubbles are removed from microwells. If air bubbles are trapped in microwells, centrifuge at 1,300 x g for 5 min again.</li>
		<li>Aspirate the anti-adherence rinsing solution from the wells in a BSC. Rinse each well with 2 mL of warm plain CMRL-1066 once. Aspirate CMRL-1066. Add 0.5 mL/well of warm 10% HiFBS CMRL to each well planned for use. Wells are now ready for loading dispersed human islet cells prepared in step 2.10.</li>
		<li>Determine the total number of cells needed for each well. One well of 24-well microwell culture plate contains 1,200 microwells and forms 1,200 pseudoislets. 500 cells per pseudoislet x 1200 microwells = 6 x 105 cells. Resuspend single cells from step 2.10 to 6 x 105 cells in 0.8 mL of 10% HiFBS CMRL in a sterile 1.5 mL tube. 
		NOTE: The maximum volume per well is 2 mL. Volume of cell suspension should not exceed 1.5 mL. The protocol describes steps for creating one well of pseudoislet transduced by lentivirus. Scale up depending on the number of wells to be made for each lentivirus.</li>
		<li>For viral transduction, add 125 TU/cell to the single cell suspension. Keep the volume of virus below 0.2 mL. Incubate the cell and virus mixture at 37 &#176;C with occasional gentle mixing for 1 h to allow contact of cells with virus before condensation of cells in step 3.2.8. 
		NOTE: Use concentrated lentivirus so that the volume of lentivirus added is minimum. Lower number of TU per cell is used for Protocol 2 compared with Protocol 1 as the total volume of medium per cell number is less. However, titer required per cell for efficient gene silencing may differ depending on the lentiviral construct and needs optimization. 
		CAUTION: Lentivirus is classified as biosafety level 2 and can be integrated into DNA of infected cells.</li>
		<li>After 1 h, adjust the total volume of the islet cell and virus mixture to 1 mL by adding 10% HiFBS CMRL. If cells form clump after 1 h incubation, disperse into single cell suspension by gentle and quick pipetting 2&#8722;3 times. The pipetting is very important for even distribution of cells across microwells. Transfer cell suspension to one well of the 24-well microwell culture plate.</li>
		<li>Immediately following pipetting, centrifuge at 100 x g for 3 min at RT to capture cells into all microwells. Observe under a microscope to verify that cells are evenly distributed in all microwells.</li>
		<li>Culture the microwell culture plate at 37 &#176;C in a 5% CO2 incubator. Pseudoislets will form in 24&#8722;48 h. Pseudoislets can be cultured without medium change for up to 7 days.</li>
		<li>When changing the medium for culture beyond 7 days, replace 50%&#8722;75% of medium for each medium change as follows. Slowly remove 0.5&#8722;1 mL of medium using a P1000 pipette from each well. Add 0.5&#8722;1 mL of fresh 10% HiFBS CMRL slowly by placing a tip to the wall of the well to avoid dislodging pseudoislets from the microwell culture plate.</li>
		<li>To prepare for harvesting pseudoislets, warm up the 10% HiFBS CMRL medium. Pseudoislets tend to float up in serum free CMRL-1066 making it hard to pick them.</li>
		<li>Aspirate 0.5 mL of medium from well using a 1 mL pipette and dispense media forcefully back to plate surface to lift up pseudoislets from the microwell culture plate.</li>
		<li>Gently aspirate dislodged pseudoislets using the 1 mL pipette and transfer islets into a non-tissue culture treated 6-well plate. Pass through a small 37 &#181;m reversible strainer placed on a 15 mL conical tube. Pseudoislets will remain on the filter; any unincorporated single cells will flow through. 
		NOTE: To avoid loss of smaller size pseudoislets through strainer, the strainer may be omitted.</li>
		<li>Dispense 1 mL of 10% HiFBS CMRL across the entire surface of the well to dislodge any remaining pseudoislets, aspirate dislodged pseudoislets, and pass through the strainer. Repeat 3x to ensure complete collection of all pseudoislets from wells.</li>
		<li>Observe the microwell culture plate under an invert microscope to ensure that all pseudoislets are collected. Repeat the wash as in step 3.2.14 if pseudoislets remain.</li>
	</ol>
	</li>
</ol>
4. RNA Extraction for Evaluation of Gene Silencing Efficiency
<ol>
	<li>Pick pseudoislets into the RNase free PBS in a 1.5 mL microcentrifuge tube and centrifuge at 300 x g for 3 min at 4 &#176;C. Remove PBS without disturbing the islet pellet and wash once with PBS followed by centrifugation at 300 x g for 3 min at 4 &#176;C.</li>
	<li>Aspirate most of PBS using a P1000 pipette. Then, change to a P10 pipette to remove the rest of PBS without disturbing the islet pellet.</li>
	<li>Add 0.5 mL of guanidinium thiocyanate RNA extraction reagent (Table of Materials) per tube. Homogenize the pseudoislets using a motor-driven pestle for 2&#8722;3 times. The homogenate in the guanidinium thiocyanate RNA extraction reagent can now be stored at -80 &#176;C or processed for RNA purification. 
	NOTE: 24 of the 3,000 cell-pseudoislets formed in a 96 well plate or 48 of the 500 cell-pseudoislets formed in a microwell culture plate are sufficient to obtain 0.5&#8722;1 &#181;g of RNA.</li>
</ol>

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The authors have nothing to disclose.

Here, a detailed protocol to generate human pseudoislets that are transduced by lentivirus using a 96-well ultra-low attachment plate or a microwell culture plate is presented. Pseudoislets have been reported to demonstrate morphology and secretory functions similar to native human islets and can be cultured for prolonged time in vitro5,11,18. Unlike native human islets that show a wide variation in size, pseudoislets are relati...

The loss of functional beta cell mass is the central pathology for both type 1 and type 2 diabetes1. While beta cells are the producers of insulin in pancreatic islets, communication between beta cells and non-beta cells plays a critical role in the regulation of insulin secretion2. In addition, dysregulation of glucagon secretion contributes to hyperglycemia in diabetes3. Thus, there is strong interest to modulate gene expression of cells within pancreatic islets to address the mechanism behind the development of islet dysfunction in diabetes. A variety of approaches including transgenic mice are available to modulate gene expression of mouse islets. However, human and mouse islets show distinct innervation, cell distribution, ratio of beta to alpha cells, and response to secretagogues4. Therefore, direct assessment of gene function in human islets is extremely important for understanding the pathophysiology of human pancreatic islets.
Adenoviral vector is the most widely used viral vector to transduce pancreatic islets in vitro due to the high efficiency of transduction in non-dividing cells. However, adenovirus does not penetrate to the core of islets efficiently, especially in human islets5, and is cytotoxic at high doses6. Comparatively, lentiviral vector is less cytotoxic and delivers exogenous genes permanently into the chromosome of post-mitotic cells, making it a widely tested vehicle for gene therapy7. However, the ability of the lentivirus to penetrate the core of intact human islets is also limited, thus requiring partial dispersion by enzymatic digestion to increase the transduction efficiency8. The caveat with the dispersion of intact human islets is the interruption of cell-cell and cell-matrix communication, which compromises the dynamic regulation of insulin secretion critical for the maintenance of glucose homeostasis in humans9. Thus, it has been challenging to assess the impact of gene modulation on the dynamic regulation of islet function in a model of human islets.
It has been known that dispersed islet cells from human and rodent islets autonomously reaggregate into islet-like structures called &#8220;pseudoislets&#8221;. Pseudoislets show beta and non-beta cell distribution similar to native islets10,11. Additionally, after long-term culture, native islets progressively lose robust first phase insulin secretion5,10,11,12. Yet, pseudoislets demonstrated better preservation of first phase insulin secretion in response to glucose compared with native islets after the same culture period5. In addition to having better preservation of insulin secretion, size-controlled reaggregation of human islet cells in low attachment plates11 provides a window of opportunity to introduce lentivirus vectors prior to their reaggregation into pseudoislets. Several studies have demonstrated the utility of pseudoislets combined with lentiviral mediated transduction. Caton et al.13 reported that the introduction of the green fluorescent protein (GFP) expressing lentivirus had little effect on insulin secretion while achieving homogenous expression of GFP in rat pseudoislets compared with non-infected control. They also demonstrated the specific effect of different connexins on insulin secretion by overexpressing connexins 32, 36, and 43 via lentivirus13. Human pseudoislets prepared with a commercially available 96-well ultra-low attachment plate demonstrated that lentiviral-mediated overexpression of transcription factor SIX3 improves insulin secretion assessed by static incubation14. Recently, human pseudoislets prepared with a 96-well ultra-low attachment plate were used to downregulate glucokinase via lentiviral short hairpin RNA (shRNA) as a proof of principle to show that glucose-stimulated insulin secretion is reduced, while KCl-stimulated insulin secretion was preserved5. The study also demonstrated that human pseudoislets are similar to native islets in gene expression and secretory profiles, further supporting the utility of human pseudoislets to dissect the regulation of islet function5. Although perifusion was not performed, a bioengineered microwell culture plate that recently became commercially available, was also reported to be compatible for lentiviral transduction and produced human pseudoislets that exhibited excellent insulin secretion in vitro and in vivo after transplantation11. Collectively, human pseudoislet formation combined with lentiviral transduction is a simple and efficient approach to investigate human islet pathophysiology, providing a valuable tool to perform mechanistic studies in human islets.
In the current report, a protocol to form human pseudoislets transduced with lentivirus using two commercially available platforms, a 96-well ultra-low attachment plate and a microwell culture plate is presented. Both achieve efficient modulation of gene expression and create human pseudoislets that are compatible for downstream assessments including static incubation and perifusion.&#160;

<table><tbody><tr><td>Anti-adherence rinsing solution</td><td>Stemcell technologies</td><td>7919</td><td></td></tr><tr><td>Biological safety cabinet</td><td>Thermo Scientific</td><td>1300 Series Type A2</td><td></td></tr><tr><td>Cell strainer, 40 micrometer</td><td>Corning</td><td>431750</td><td></td></tr><tr><td>CMRL-1066</td><td>ThermoFisher</td><td>11530037</td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; incubator</td><td>Thermo Scientific</td><td>Heracell VIOS 160i</td><td></td></tr><tr><td>Conical centrifuge tube, 15 mL</td><td>VWR</td><td>89039-666</td><td></td></tr><tr><td>Conical centrifuge tube, 50 mL</td><td>VWR</td><td>89039-658</td><td></td></tr><tr><td>Fetal bovine serum</td><td>ThermoFisher</td><td>26140079</td><td></td></tr><tr><td>Guanidinium thiocyanate RNA extraction reagent</td><td>ThermoFisher</td><td>15596026</td><td>Trizol</td></tr><tr><td>Glutamine</td><td>ThermoFisher</td><td>25030164</td><td></td></tr><tr><td>Hemocytometer</td><td>Marien Feld</td><td>Neubauer-Improved Bright line</td><td></td></tr><tr><td>Human serum albumin</td><td>Sigma</td><td>A1653</td><td></td></tr><tr><td>Inverted microscope</td><td>Fisher brand</td><td>11-350-119</td><td></td></tr><tr><td>Microcentrifuge</td><td>Beckman Coulter</td><td>Microfuge 20</td><td></td></tr><tr><td>Microcentrifuge tube, 1.5 mL</td><td>USA Scientific</td><td>1615-5500</td><td></td></tr><tr><td>Microwell culture plate</td><td>Stemcell technologies</td><td>34411</td><td>Aggrewell 400, 24 well</td></tr><tr><td>Motor-driven pestle</td><td>GAMUT</td><td>#399X644</td><td></td></tr><tr><td>Non-tissue culture treated dish, 10 cm</td><td>Fisher Scientific</td><td>FB0875713</td><td></td></tr><tr><td>PBS</td><td>ThermoFisher</td><td>14190250</td><td></td></tr><tr><td>Penicillin-streptomycin</td><td>ThermoFisher</td><td>10378016</td><td></td></tr><tr><td>Petri dish, 35 mm</td><td>Celltreat</td><td>229638</td><td></td></tr><tr><td>Pipette, 5 mL</td><td>DOT Scientific,</td><td>667205B</td><td></td></tr><tr><td>Pipette, 8-channel</td><td>VWR</td><td>#613-5253</td><td></td></tr><tr><td>Pipette, 10 mL</td><td>VWR</td><td>667210B</td><td></td></tr><tr><td>Pipette, P10</td><td>Denville</td><td>UEZ-P-10</td><td></td></tr><tr><td>Pipette, P200</td><td>Denville</td><td>UEZ-P-200</td><td></td></tr><tr><td>Pipette, P1000</td><td>Denville</td><td>UEZ-P-1000</td><td></td></tr><tr><td>Proteolytic and collagenolytic enzyme mixture</td><td>Sigma</td><td>A6965</td><td>Accutase</td></tr><tr><td>Reagent reservoir, 50 mL</td><td>VWR</td><td>89094-680</td><td></td></tr><tr><td>Reversible strainer, 37 micrometer</td><td>Stemcell technologies</td><td>27251</td><td></td></tr><tr><td>Swing bucket plate centrifuge</td><td>Beckman Coulter</td><td>Allegra X-14R</td><td></td></tr><tr><td>Swing bucket rotor</td><td>Beckman Coulter</td><td>SX4750A</td><td></td></tr><tr><td>Tuberculin syringe, 1 mL</td><td>BD</td><td>309659</td><td></td></tr><tr><td>Ultra low attachment microplate, 96 well</td><td>Corning</td><td>4515</td><td></td></tr></tbody></table>

lentiviral mediated gene silencing in human pseudoislet prepared in low attachment plates

Various genetic tools are available to modulate genes in pancreatic islets of rodents to dissect function of islet genes for diabetes research. However, the data obtained from rodent islets are often not fully reproduced in or applicable to human islets due to well-known differences in islet structure and function between the species. Currently, techniques that are available to manipulate gene expression of human islets are very limited. Introduction of transgene into intact islets by adenovirus, plasmid, and oligonucleotides often suffers from low efficiency and high toxicity. Low efficiency is especially problematic in gene downregulation studies in intact islets, which require high efficiency. It has been known that enzymatically-dispersed islet cells reaggregate in culture forming spheroids termed pseudoislets. Size-controlled reaggregation of human islet cells creates pseudoislets that maintain dynamic first phase insulin secretion after prolonged culture and provide a window to efficiently introduce lentiviral short hairpin RNA (shRNA) with low toxicity. Here, a detailed protocol for the creation of human pseudoislets after lentiviral transduction using two commercially available multiwell plates is described. The protocol can be easily performed and allows for efficient downregulation of genes and assessment of dynamism of insulin secretion using human islet cells. Thus, human pseudoislets with lentiviral mediated gene modulation provide a powerful and versatile model to assess gene function within human islet cells.

Figure 1 illustrates key steps in the production of pseudoislets using a 96-well ultra-low attachment plate and a microwell culture plate. Figure 2a shows sequential changes in morphology during the formation of pseudoislets from 3 x 103 human islet cells in a 96-well ultra-low attachment plate. Monolayer or loose clumps of cells observed in day 1 changed into solid aggregates with a smooth, round border by day 5 to 7 ...

Watch this Scientific Journal Video about Lentiviral Mediated Gene Silencing in Human Pseudoislet Prepared in Low Attachment Plates at JoVE.com

Lentiviral Mediated Gene Silencing in Human Pseudoislet Prepared in Low Attachment Plates

A protocol to create gene modified human pseudoislets from dispersed human islet cells that are transduced by lentivirus carrying short hairpin RNA (shRNA) is presented. This protocol utilizes readily available enzyme and culture vessels, can be performed easily, and produces genetically modified human pseudoislets suitable for functional and morphological studies.

Various genetic tools are available to modulate genes in pancreatic islets of rodents to dissect function of islet genes for diabetes research. However, ...

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Research

JoVE Journal

Genetics

5.4K Views.  University of Iowa. Het is moeilijk om de genexpressie in menselijke eilandjes te moduleren, ook een grotere afname van de functionerende menselijke eilandjes vormen grote uitdagingen na modulatie van genexpressie. Door het lentivirus te combineren bemiddelde SHR8 transductie en de vorming van menselijke geschikte eilandjes. Dit protocol maakt een efficiënte downregulatie van een gericht gen mogelijk met behoud van de eilandjesfunctie tijdens langdurige kweek.Om te beginnen, voorzichtig wervelen de schee...

Video: Lentiviral Mediated Gene Silencing in Human Pseudoislet Prepared in Low Attachment Plates

Assessing Replication and Beta Cell Function in Adenovirally-transduced Isolated Rodent Islets

Glucose homeostasis is primarily controlled by the endocrine hormones insulin and glucagon, secreted from the pancreatic beta and alpha cells, respectively. Functional beta cell mass is determined by the anatomical beta cell mass as well as the ability of the beta cells to respond to a nutrient load. A loss of functional beta cell mass is central to both major forms of diabetes 1-3. Whereas the declining functional beta cell mass results from an autoimmune attack in type 1 diabetes, in type 2 diabetes, this decrement develops from both an inability of beta cells to secrete insulin appropriately and the destruction of beta cells from a cadre of mechanisms. Thus, efforts to restore functional beta cell mass are paramount to the better treatment of and potential cures for diabetes.
Efforts are underway to identify molecular pathways that can be exploited to stimulate the replication and enhance the function of beta cells. Ideally, therapeutic targets would improve both beta cell growth and function. Perhaps more important though is to identify whether a strategy that stimulates beta cell growth comes at the cost of impairing beta cell function (such as with some oncogenes) and vice versa.
By systematically suppressing or overexpressing the expression of target genes in isolated rat islets, one can identify potential therapeutic targets for increasing functional beta cell mass 4-6. Adenoviral vectors can be employed to efficiently overexpress or knockdown proteins in isolated rat islets 4,7-15. Here, we present a method to manipulate gene expression utilizing adenoviral transduction and assess islet replication and beta cell function in isolated rat islets (Figure 1). This method has been used previously to identify novel targets that modulate beta cell replication or function 5,6,8,9,16,17.

This protocol allows one to identify factors that modulate functional beta cell mass to find potential therapeutic targets for the treatment of diabetes. The protocol consists of a streamlined method to assess islet replication and beta cell function in isolated rat islets following manipulation of gene expression with adenoviruses.

Glucose homeostasis is primarily controlled by the endocrine hormones insulin and glucagon, secreted from the pancreatic beta and alpha cells, ...

Medicine

Lentiviral Vector Platform for the Efficient Delivery of Epigenome-editing Tools into Human Induced Pluripotent Stem Cell-derived Disease Models

The use of hiPSC-derived cells represents a valuable approach to study human neurodegenerative diseases. Here, we describe an optimized protocol for the differentiation of hiPSCs derived from a patient with the triplication of the alpha-synuclein gene (SNCA) locus into Parkinson&#8217;s disease (PD)-relevant dopaminergic neuronal populations. Accumulating evidence has shown that high levels of SNCA are causative for the development of PD. Recognizing the unmet need to establish novel therapeutic approaches for PD, especially those targeting the regulation of SNCA expression, we recently developed a CRISPR/dCas9-DNA-methylation-based system to epigenetically modulate SNCA transcription by enriching methylation levels at the SNCA intron 1 regulatory region. To deliver the system, consisting of a dead (deactivated) version of Cas9 (dCas9) fused with the catalytic domain of the DNA methyltransferase enzyme 3A (DNMT3A), a lentiviral vector is used. This system is applied to cells with the triplication of the SNCA locus and reduces the SNCA-mRNA and protein levels by about 30% through the targeted DNA methylation of SNCA intron 1. The fine-tuned downregulation of the SNCA levels rescues disease-related cellular phenotypes. In the current protocol, we aim to describe a step-by-step procedure for differentiating hiPSCs into neural progenitor cells (NPCs) and the establishment and validation of pyrosequencing assays for the evaluation of the methylation profile in the SNCA intron 1. To outline in more detail the lentivirus-CRISPR/dCas9 system used in these experiments, this protocol describes how to produce, purify, and concentrate lentiviral vectors and to highlight their suitability for epigenome- and genome-editing applications using hiPSCs and NPCs. The protocol is easily adaptable and can be used to produce high titer lentiviruses for in vitro and in vivo applications.

Targeted DNA epigenome editing represents a powerful therapeutic approach. This protocol describes the production, purification, and concentration of all-in-one lentiviral vectors harboring the CRISPR-dCas9-DNMT3A transgene for epigenome-editing applications in human induced pluripotent stem cell (hiPSC)-derived neurons.

The use of hiPSC-derived cells represents a valuable approach to study human neurodegenerative diseases. Here, we describe an optimized protocol for the ...

Neo-Islet Formation in Liver of Diabetic Mice by Helper-dependent Adenoviral Vector-Mediated Gene Transfer

Type 1 diabetes is caused by T cell-mediated autoimmune destruction of insulin-producing cells in the pancreas. Until now insulin replacement is still the major therapy, because islet transplantation has been limited by donor availability and by the need for long-term immunosuppression. Induced islet neogenesis by gene transfer of Neuogenin3 (Ngn3), the islet lineage-defining specific transcription factor and Betacellulin (Btc), an islet growth factor has the potential to cure type 1 diabetes.
Adenoviral vectors (Ads) are highly efficient gene transfer vector; however, early generation Ads have several disadvantages for in vivo use. Helper-dependent Ads (HDAds) are the most advanced Ads that were developed to improve the safety profile of early generation of Ads and to prolong transgene expression1. They lack chronic toxicity because they lack viral coding sequences2-5 and retain only Ad cis elements necessary for vector replication and packaging. This allows cloning of up to 36 kb genes.
In this protocol, we describe the method to generate HDAd-Ngn3 and HDAd-Btc and to deliver these vectors into STZ-induced diabetic mice. Our results show that co-injection of HDAd-Ngn3 and HDAd-Btc induces 'neo islets' in the liver and reverses hyperglycemia in diabetic mice.

We describe hepatic neo-islet formation in STZ (streptozotocin)-induced diabetic mice by gene transfer of Neurogenin3 (Ngn3) and Betacellulin (Btc) using helper-dependent adenoviral vector (HDAd) and the reversal of hyperglycemia. Our method takes advantages of helper-dependent adenoviral vectors with their highly efficient in vivo transduction and the long lasting gene expression.

Type 1 diabetes is caused by T cell-mediated autoimmune destruction of insulin-producing cells in the pancreas. Until now insulin replacement is still the ...

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 ...

Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

Type 2 diabetes is a chronic disease affecting 382 million people in 2013, and is expected to rise to 592 million by 2035 1. During the past 2 decades, the role of beta-cell dysfunction in type 2 diabetes has been clearly established 2. Research progress has required methods for the isolation of pancreatic islets. The protocol of the islet isolation presented here shares many common steps with protocols from other groups, with some modifications to improve the yield and quality of isolated islets from both the wild type and diabetic Leprdb (db/db) mice. A live-cell 2-photon imaging method is then presented that can be used to investigate the control of insulin secretion within islets.

Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

We present here a protocol for the isolation of islets from the mouse model of type 2 diabetes, Leprdb and details of a live-cell assay for measurement of insulin secretion from intact islets that utilizes 2 photon microscopy.

Type 2 diabetes is a chronic disease affecting 382 million people in 2013, and is expected to rise to 592 million by 2035 1. During the past 2 decades, ...

Lentiviral Vector-mediated Gene Therapy of Hepatocytes Ex Vivo for Autologous Transplantation in Swine

Gene therapy is an ideal choice to cure many inborn errors of metabolism of the liver. Ex-vivo, lentiviral vectors have been used successfully in the treatment of many hematopoietic diseases in humans, as their use offers stable transgene expression due to the vector&#39;s ability to integrate into the host genome. This method demonstrates the application of ex vivo gene therapy of hepatocytes to a large animal model of hereditary tyrosinemia type I. This process consists of 1) isolation of primary hepatocytes from the autologous donor/recipient animal, 2) ex vivo gene delivery via hepatocyte transduction with a lentiviral vector, and 3) autologous transplant of corrected hepatocytes via portal vein injection. Success of the method generally relies upon efficient and sterile removal of the liver resection, careful handling of the excised specimen for isolation of viable hepatocytes sufficient for re-engrafting, high-percentage transduction of the isolated cells, and aseptic surgical procedures throughout to prevent infection. Technical failure at any of these steps will result in low yield of viable transduced hepatocytes for autologous transplant or infection of the donor/recipient animal. The pig model of human type 1 hereditary tyrosinemia (HT-1) chosen for this approach is uniquely amenable to such a method, as even a small percentage of engraftment of corrected cells will lead to repopulation of the liver with healthy cells based on a powerful selective advantage over native-diseased hepatocytes. Although this growth selection will not be true for all indications, this approach is a foundation for expansion into other indications and allows for manipulation of this environment to address additional diseases, both within the liver and beyond, while controlling for exposure to viral vector and opportunity for off-target toxicity and tumorigenicity.

Lentiviral Vector-mediated Gene Therapy of Hepatocytes Ex Vivo for Autologous Transplantation in Swine

This protocol is intended to describe porcine hepatocyte isolation and ex vivo gene delivery to cure models of metabolic diseases via autologous cell transplantation. Although this particular model enjoys unique advantages that favor successful therapy, the application is a relevant foundation to address additional diseases and indications.

Gene therapy is an ideal choice to cure many inborn errors of metabolism of the liver. Ex-vivo, lentiviral vectors have been used successfully in the ...

Visualization of Endogenous Mitophagy Complexes In Situ in Human Pancreatic Beta Cells Utilizing Proximity Ligation Assay

Mitophagy is an essential mitochondrial quality control pathway, which is crucial for pancreatic islet beta cell bioenergetics to fuel glucose-stimulated insulin release. Assessment of mitophagy is challenging and often requires genetic reporters or multiple complementary techniques not easily utilized in tissue samples, such as primary human pancreatic islets. Here we demonstrate a robust approach to visualize and quantify formation of key endogenous mitophagy complexes in primary human pancreatic islets. Utilizing the sensitive proximity ligation assay technique to detect interaction of the mitophagy regulators NRDP1 and USP8, we are able to specifically quantify formation of essential mitophagy complexes in situ. By coupling this approach to counterstaining for the transcription factor PDX1, we can quantify mitophagy complexes, and the factors that can impair mitophagy, specifically within beta cells. The methodology we describe overcomes the need for large quantities of cellular extracts required for other protein-protein interaction studies, such as immunoprecipitation (IP) or mass spectrometry, and is ideal for precious human islet&#160;samples generally not available in sufficient quantities for these approaches. Further, this methodology obviates the need for flow sorting techniques to purify beta cells from a heterogeneous islet population for downstream protein applications. Thus, we describe a valuable protocol for visualization of mitophagy highly compatible for use in heterogeneous and limited cell populations.

This protocol outlines a method for quantitative analysis of mitophagy protein complex formation specifically in beta cells from primary human islet samples. This technique thus allows analysis of mitophagy from limited biological material, which are crucial in precious human pancreatic beta cell samples.

Mitophagy is an essential mitochondrial quality control pathway, which is crucial for pancreatic islet beta cell bioenergetics to fuel glucose-stimulated ...

Lentiviral Vector Preparation for In Vivo Gene Modulation 

Lentiviral Vector Preparation for Efficient Gene and MicroRNA Modulation of Peritoneal Cavity Tissue&#45;Resident Macrophages In Vivo in Mice

Peritoneal tissue-resident macrophages have broad functions in the maintenance of homeostasis and are involved in pathologies within local and neighboring tissues. Their functions are dictated by microenvironmental cues; thus, it is essential to investigate their behavior in an in vivo physiological niche. Currently, specific peritoneal macrophage-targeting methodologies employ whole-mouse transgenic models. Here, a protocol for effective in vivo modulation of mRNA and small RNA species (e.g., microRNA) expression in peritoneal macrophages using lentivirus particles is described. Lentivirus preparations were made in HEK293T cells and purified on a single sucrose layer. In vivo validation of lentivirus effectivity following intraperitoneal injection revealed predominant infection of macrophages restricted to local tissue. Targeting of peritoneal macrophages was successful during homeostasis and thioglycolate-induced peritonitis. The limitations of the protocol, including low-level inflammation induced by intraperitoneal delivery of lentivirus and time restrictions for potential experiments, are discussed. Overall, this study presents a quick and accessible protocol for the rapid assessment of gene function in peritoneal macrophages in vivo.

Author Spotlight: Exploring Macrophage Immunometabolism Through Lentiviral Vector&#45;Mediated Gene Manipulation

We demonstrate a step-by-step protocol for the investigation of gene function in peritoneal tissue-resident macrophages in vivo, using lentiviral vectors.

Peritoneal tissue-resident macrophages have broad functions in the maintenance of homeostasis and are involved in pathologies within local and neighboring ...

Immunology and Infection

A Protocol for Lentiviral Transduction and Downstream Analysis of Intestinal Organoids

Intestinal crypt-villus structures termed organoids, can be kept in sustained culture three dimensionally when supplemented with the appropriate growth factors. Since organoids are highly similar to the original tissue in terms of homeostatic stem cell differentiation, cell polarity and presence of all terminally differentiated cell types known to the adult intestinal epithelium, they serve as an essential resource in experimental research on the epithelium. The possibility to express transgenes or interfering RNA using lentiviral or retroviral vectors in organoids has increased opportunities for functional analysis of the intestinal epithelium and intestinal stem cells, surpassing traditional mouse transgenics in speed and cost. In the current video protocol we show how to utilize transduction of small intestinal organoids with lentiviral vectors illustrated by use of doxycylin inducible transgenes, or IPTG inducible short hairpin RNA for overexpression or gene knockdown. Furthermore, considering organoid culture yields minute cell counts that may even be reduced by experimental treatment, we explain how to process organoids for downstream analysis aimed at quantitative RT-PCR, RNA-microarray and immunohistochemistry. Techniques that enable transgene expression and gene knock down in intestinal organoids contribute to the research potential that these intestinal epithelial structures hold, establishing organoid culture as a new standard in cell culture.

In this video protocol we give a step by step explanation of lentiviral transduction in organoids of primary intestinal epithelium and of processing and downstream analysis of these cultures by quantitative RT-PCR, RNA-microarray and immunohistochemistry.

Intestinal crypt-villus structures termed organoids, can be kept in sustained culture three dimensionally when supplemented with the appropriate growth ...

Biology

Lentiviral Mediated Delivery of shRNAs to hESCs and NPCs Using Low-cost Cationic Polymer Polyethylenimine (PEI)

The current protocol describes the use of lentiviral particles for the delivery of short hairpin RNAs (shRNAs) to both human embryonic stem cells (hESCs) as well as neural progenitor cells (NPCs) derived from hESCs at high efficiency. Lentiviral particles were generated by co-transfecting HEK293T cells using entry vectors (carrying shRNAs) along with packaging plasmids (pAX and pMD2.G) using the low-cost cationic polymer polyethylenimine (PEI). Viral particles were concentrated using ultracentrifugation, which resulted in average titers above 5 x 107. Both hESCs and NPCs could be infected at high efficiencies using these lentiviral particles, as shown by puromycin selection and stable expression in hESCs, as well as transient GFP expression in NPCs. Furthermore, western blot analysis showed a significant reduction in the expression of genes targeted by shRNAs. In addition, the cells retained their pluripotency as well as differentiation potential, as evidenced by their subsequent differentiation into different lineages of CNS. The current protocol deals with the delivery of shRNAs; however, the same approach could be used for the ectopic expression of cDNAs for overexpression studies.

Lentiviral Mediated Delivery of shRNAs to hESCs and NPCs Using Low-cost Cationic Polymer Polyethylenimine PEI

Using the low-cost cationic polymer polyethylenimine (PEI), we produced lentiviral particles for stable expression of shRNAs in H9 human embryonic stem cells (hESCs) and transiently transduced H9-derived neural progenitor cells (NPCs) at high efficiency.

The current protocol describes the use of lentiviral particles for the delivery of short hairpin RNAs (shRNAs) to both human embryonic stem cells (hESCs) ...

Lentiviral Mediated Gene Silencing in Human Pseudoislet Prepared in Low Attachment Plates

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