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

Summary

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.

Abstract

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.

Introduction

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....

Protocol

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, 1,200−1,400 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/pseudoislet....

Results

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 .......

Discussion

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 vitro^5,11,18. Unlike native human islets that show a wide variation in size, pseudoislets are relati.......

Materials

Name	Company	Catalog Number	Comments
Anti-adherence rinsing solution	Stemcell technologies	7919
Biological safety cabinet	Thermo Scientific	1300 Series Type A2
cell strainer, 40 micrometer	Corning	431750
CMRL-1066	ThermoFisher	11530037
CO2 incubator	Thermo Scientific	Heracell VIOS 160i
conical centrifuge tube, 15 mL	VWR	89039-666
conical centrifuge tube, 50 mL	VWR	89039-658
fetal bovine serum	ThermoFisher	26140079
guanidinium thiocyanate RNA extraction reagent	ThermoFisher	15596026	Trizol
glutamine	ThermoFisher	25030164
Hemocytometer	Marien Feld	Neubauer-Improved Bright line
Human serum albumin	Sigma	A1653
inverted microscope	Fisher brand	11-350-119
microcentrifuge	Beckman Coulter	Microfuge 20
microcentrifuge tube, 1.5 mL	USA Scientific	1615-5500
microwell culture plate	Stemcell technologies	34411	Aggrewell 400, 24 well
motor-driven pestle	GAMUT	#399X644
non-tissue culture treated dish, 10 cm	Fisher Scientific	FB0875713
PBS	ThermoFisher	14190250
Penicillin-streptomycin	ThermoFisher	10378016
Petri dish, 35 mm	Celltreat	229638
pipette, 5 mL	DOT Scientific,	667205B
pipette, 8-channel	VWR	#613-5253
pipette, 10 mL	VWR	667210B
pipette, P10	Denville	UEZ-P-10
pipette, P200	Denville	UEZ-P-200
pipette, P1000	Denville	UEZ-P-1000
proteolytic and collagenolytic enzyme mixture	Sigma	A6965	Accutase
reagent reservoir, 50 mL	VWR	89094-680
reversible strainer, 37 micrometer	Stemcell technologies	27251
swing bucket plate centrifuge	Beckman Coulter	Allegra X-14R
swing bucket rotor	Beckman Coulter	SX4750A
tuberculin syringe, 1 mL	BD	309659
ultra low attachment microplate, 96 well	Corning	4515