Sign In

A subscription to JoVE is required to view this content. Sign in or start your free trial.

In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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 p.......

Representative 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 vitro5,11,18. Unlike native human islets that show a wide variation in size, pseudoislets are relati.......

Acknowledgements

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).

....

Materials

NameCompanyCatalog NumberComments
Anti-adherence rinsing solutionStemcell technologies7919
Biological safety cabinetThermo Scientific1300 Series Type A2
cell strainer, 40 micrometerCorning431750
CMRL-1066ThermoFisher11530037
CO2 incubatorThermo ScientificHeracell VIOS 160i
conical centrifuge tube, 15 mLVWR89039-666
conical centrifuge tube, 50 mLVWR89039-658
fetal bovine serumThermoFisher26140079
guanidinium thiocyanate RNA extraction reagentThermoFisher15596026Trizol
glutamineThermoFisher25030164
HemocytometerMarien FeldNeubauer-Improved Bright line
Human serum albuminSigmaA1653
inverted microscopeFisher brand11-350-119
microcentrifugeBeckman CoulterMicrofuge 20
microcentrifuge tube, 1.5 mLUSA Scientific1615-5500
microwell culture plateStemcell technologies34411Aggrewell 400, 24 well
motor-driven pestleGAMUT#399X644
non-tissue culture treated dish, 10 cmFisher ScientificFB0875713
PBSThermoFisher14190250
Penicillin-streptomycinThermoFisher10378016
Petri dish, 35 mmCelltreat229638
pipette, 5 mLDOT Scientific,667205B
pipette, 8-channelVWR#613-5253
pipette, 10 mLVWR667210B
pipette, P10DenvilleUEZ-P-10
pipette, P200DenvilleUEZ-P-200
pipette, P1000DenvilleUEZ-P-1000
proteolytic and collagenolytic enzyme mixtureSigmaA6965Accutase
reagent reservoir, 50 mLVWR89094-680
reversible strainer, 37 micrometerStemcell technologies27251
swing bucket plate centrifugeBeckman CoulterAllegra X-14R
swing bucket rotorBeckman CoulterSX4750A
tuberculin syringe, 1 mLBD309659
ultra low attachment microplate, 96 wellCorning4515

References

  1. Chen, C., Cohrs, C. M., Stertmann, J., Bozsak, R., Speier, S. Human beta cell mass and function in diabetes: Recent advances in knowledge and technologies to understand disease pathogenesis. Molecular Metabolism. 6 (9), 943-957 (2017).
  2. Hong, H., Jo, J., Sin, S. J.

Explore More Articles

LentiviralGene SilencingHuman PseudoisletLow Attachment PlatesIslet FunctionGene Expression ModulationSHR8 TransductionProteolytic EnzymeCollagenolytic EnzymeCell DissociationCMRL MediumFetal Bovine Serum

This article has been published

Video Coming Soon

JoVE Logo

Privacy

Terms of Use

Policies

Research

Education

ABOUT JoVE

Copyright © 2024 MyJoVE Corporation. All rights reserved