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, ...

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