Name: lentiviral mediated gene silencing in human pseudoislet prepared in low attachment plates
Uploaded: 2019-05-14T13:30:00
Description: Watch this Scientific Journal Video about Lentiviral Mediated Gene Silencing in Human Pseudoislet Prepared in Low Attachment Plates at JoVE.com

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 p...

<ol>
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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>CO2 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, ...

It's difficult to modulate the gene expression in human islets, also greater decline of the functioning human islets pose major challenges after modulation of gene expression. By combining the lentivirus mediated SHR8 transduction and the formation of human suitable islets. This protocol allows efficient downregulation of a targeted gene while preserving islet function during prolonged culture.To begin, gently swirl the shipping bottle to keep islets in suspension. Transfer the shipping medium containing islets to a 50 milliliter conical centrifuge tube. Let the tube sit in a biological safety cabinet for 15 minutes so that islets settle to the bottom of the tube.Remove the shipping medium gently using a capillary pipette without disturbing the islet pellet. Re-suspend the islet pellet in CMRL medium with 1%human serum albumin to a concentration of 400 islet equivalent per milliliter. Transfer islets into a non-tissue culture treated dish and culture them at 37 degrees celsius in a 5%carbon dioxide incubator overnight.After overnight culture, transfer human islets into a 15 milliliter conical centrifuge tube, centrifuge at 250 times g for five minutes in a swinging bucket rotor. And aspirate the medium with a capillary pipette without disturbing the islet pellet. Wash the pellet my adding 10 milliliters of PBS to the tube, mix gently and centrifuge at 250 times g for five minutes.After centrifugation, aspirate PBS without disturbing the islet pellet. Re-suspend the pellet in 0.5 milliliters of a pre-warmed proteolytic and collagenolytic enzyme mixture. Pipette five times using a P1000 pipette to mix islets.Incubate at 37 degrees celsius for five minutes. After the incubation, mix pipette petting up and down gently. Check for single cell cloudiness and the number of flakes or undigested islets.Place a 40 micron strainer in a 35 millimeter petri dish and wet the strainer by adding one milliliter of CMRL medium with 10%heat inactivated fetal bovine serum and depressing with a one milliliter syringe plunger. Transfer all the cell suspension on top of the strainer. And collect the pass through in a fresh 15 milliliter tube.Wash the tube used for islet digestion with 0.5 milliliters of fresh CMRL medium to collect leftover cells and pass the wash through the strainer. Combine the pass through in a 15 milliliter tube. Repeat once.To dissociate undigested islets remaining on the strainer, press the strainer with a one milliliter syringe plunger. Collect the pass through again and wash the strainer with fresh plain CMRL 1066 medium to remove all remaining digested islets from the strainer and the dish. Add the pass through to the 15 milliliter tube.Record the total volume of the cell suspension and take a 10 microliter aliquot of cells to count the cell number on a hemocytometer. After centrifuging the cell suspension for five minutes at 200 times g, remove the medium without disturbing the pellet. To produce pseudo-islets using a 96 well ultra low attachment plate, first calculate the total volume of the required one times 10 to the fifth cells per milliliter of the single cell suspension.Then, re-suspend the islet pellet in CMRL medium with 10%heat inactivated fetal bovine serum so that 30 microliters of the cell suspension has 3000 cells. Next, transfer the required volume of the single cell suspension to a fresh 15 milliliter tube. At 250 transduction units per cell of a lentivirus containing SH-RNA targeting a gene of interest or a control.To mix the cell suspension with the virus, pipette gently five times with a P1000 pipette. Then transfer the mixed cells into a 50 milliliter sterile reagent reservoir if using an eight channel pipette. With an eight channel pipette or a P200 pipette, dispense 30 microliters per well of the cell suspension mixed with lentivirus into each well.Next, centrifuge the 96 well plate in a swinging bucket plate centrifuge at 270 times g at room temperature for seven minutes. Culture at 37 degrees celsius in a humidified 5%carbon dioxide incubator overnight. After incubation, remove the 96 well plate from the incubator and place it in the biological safety cabinet.Pipette 100 microliters per islet of 10%high FBSCMRL into a well to prevent drying of islets and continue to culture for one week. Pipette 100 microliters per well of CMRL medium with 10%heat inactivated fetal bovine serum from the reservoir to the pseudo-islets and pipette up and down two to three times gently in the well to lift islets up. Then, aspirate the medium in the well containing a pseudo-islet and eject into a 10 centimeter petri dish.Check the plate under a light microscope to ensure the complete removal of all the pseudo-islets and proceed to downstream experiments. To produce pseudo-islets using a 24 well micro well culture plate, first add 500 microliters per well of an anti-adherence rinsing solution. Centrifuge at 1300 times g for five minutes in a swinging bucket plate centrifuge.Then, observe the plate under a microscope to ensure that air bubbles are removed from micro-wells. In a biological safety cabinet, aspirate the anti-adherence rinsing solution from the wells. Rinse each well with two milliliters of warm plain CMRL 1066 medium once.Then, aspirate the plain CMRL 1066 medium and add 0.5 milliliter per well of warm CMRL with 10%heat inactivated fetal bovine serum to each well planned for use. Determine the total number of cells needed for each well. Re-suspend the single cells in 0.8 milliliters of CMRL medium with 10%heat inactivated fetal bovine serum in a sterile 1.5 milliliter tube.To create one well of pseudo-islet transduced by a lentivirus, add 125 transduction units per cell to the single cell suspension keeping the volume of virus below 0.2 milliliters. Incubate the cell and virus mixture at 37 degrees celsius with occasional gentle mixing for one hour to allow contact of cells with virus before condensation of cells. After one hour, adjust the total volume of the islet cell and virus mixture to one milliliter by adding CMRL medium with 10%heat inactivated fetal bovine serum.If cells form clumps after a one hour incubation disperse into a single cell suspension by gentle and quick pipetting two to three times. Transfer the cell suspension to one well of the 24 well micro-well culture plate. Immediately following pipetting, centrifuge at 100 times g at room temperature for three minutes to capture cells into all micro-wells.Observe under a microscope to verify that cells are evenly distributed in all micro-wells. It's critical to centrifuge the micro-well plate immediately after adding the well mixing the islet single cell suspension into the wells. Sequential changes in morphology of pseudo-islets from 3000 human islet cells created in a 96 well ultra low attachment plate showed monolayer or loose clumps of cells turned into solid aggregates with a smooth round border in one week.In contrast, in a micro-well culture plate, the formation of cell and pseudo-islets from only 500 islet cells is usually visible within four days. The uniform size of pseudo-islets reduces the variation within a test group and allows static incubation using as little as five pseudo-islets per measurement. The Perry Fusion Assay for insulin response showed human pseudo-islets maintained robust first phase insulin secretion in response to glucose after seven days of culture.The introduction of lentivirus targeting human adipose triglyceride lipase or perilipin five genes into a single cell suspension ensures the efficient and homogenous transduction of islet cells and achieves highly efficient downregulation of genes. It is important to remember that the time required for dispersing human islet into single cells is affected by many factors such as:size, distribution, and the donor characteristic. So, it is important to pay close attention to the disappearance of human islets during digestion.Throughout the protocol it is important to handle single cell suspension gently to avoid cell loss. The human pseudo-islets being created can be used for many applications to determine the effect of gene modulation such as:Western Blot, histological study and the measurements of oxygen consumption rates. This protocol allows assess the low of targeted gene in the regulation of islet health and the function in human islets by using common lab ware and simple techniques.The use of human islets may require a prior approval from a local review board. Please consult local review board before initiation of the study. Lentivirus is classified as biosafety level two.Contact biosafety committee before the initiation of the use of the lentivirus.

Research

JoVE Journal

Genetics

siRNA Transfection and EMSA Analyses on Freshly Isolated Human Villous Cytotrophoblasts

Human primary villous cytotrophoblasts are a very useful source of primary cells to study placental functions and regulatory mechanisms, and to comprehend ...

Parallel Measurement of Circadian Clock Gene Expression and Hormone Secretion in Human Primary Cell Cultures

Circadian clocks are functional in all light-sensitive organisms, allowing for an adaptation to the external world by anticipating daily environmental ...

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative ...

Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells

The CRISPR/Cas9 genome engineering system has revolutionized biology by allowing for precise genome editing with little effort. Guided by a single guide ...

Chromatin Immunoprecipitation (ChIP) Protocol for Low-abundance Embryonic Samples

Chromatin Immunoprecipitation ChIP Protocol for Low-abundance Embryonic Samples

Chromatin immunoprecipitation (ChIP) is a widely-used technique for mapping the localization of post-translationally modified histones, histone variants, ...

A Standard Methodology to Examine On-site Mutagenicity As a Function of Point Mutation Repair Catalyzed by CRISPR/Cas9 and SsODN in Human Cells

Combinatorial gene editing using CRISPR/Cas9 and single-stranded oligonucleotides is an effective strategy for the correction of single-base point ...

CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy

CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy

As a promising genome editing platform, the CRISPR/Cas9 system has great potential for efficient genetic manipulation, especially for targeted integration ...

A Protocol for the Production of Integrase-deficient Lentiviral Vectors for CRISPR/Cas9-mediated Gene Knockout in Dividing Cells

Lentiviral vectors are an ideal choice for delivering gene-editing components to cells due to their capacity for stably transducing a broad range of cells ...

CRISPR-Mediated Reorganization of Chromatin Loop Structure

Recent studies have clearly shown that long-range, three-dimensional chromatin looping interactions play a significant role in the regulation of gene ...

Lentiviral Mediated Production of Transgenic Mice: A Simple and Highly Efficient Method for Direct Study of Founders

For almost 40 years, pronuclear DNA injection represents the standard method to generate transgenic mice with random integration of transgenes. Such a ...