Human Islet and Adenovirus Core (located at Mt. Sinai)

Leadership

Overview

The first mission of the HIAC is to provide key advice, methods, technology and infrastructure to assist investigators in the use of human islets for research, with the goal of furthering understanding of normal and pathophysiologic islet cell growth and function. Although rodents unequivocally continue to provide new important insights into islet biology and pathobiology, it is of utmost importance to translate the advances and discoveries made in rodent beta cells and cell lines to human islet investigation and therapies. Conversely, when important advances are made in human islet cells, mechanistic studies are often best performed in isolated mouse or rat islets, or in insulinoma cell lines. HIAC personnel have extensive expertise in the preparation, methodology, handling and use of both human and rodent islets, and all relevant insulinoma and alpha cell lines. Thus, while the focus is on human islets, the HIAC also has provided, and will continue to provide, isolation of rodent islets, access to rodent insulinoma cell lines, and specific experimental analyses such as assays for insulin secretion, islet bioenergetics, beta cell proliferation, mass and survival, as well as human islet transplantation into immunodeficient mice, with subsequent functional analyses.The second mission of the HIAC is to generate and make available to the ES-DRC community reagents and tools including adenovirus or lentivirus viral vectors for gene delivery of cDNAs and shRNAs of interest to beta cells and other islet cell types to study beta cell regeneration, differentiation, survival and function. These reagents are also available for use in the transduction of non-islet cell types and tissues.

Objectives

  1. To assist new islet investigators in accessing human and rodent islets and related cell lines for use in investigator-driven studies.
  2. To train students, postdoctoral fellows, investigators and technical staff in the design and use of molecular, cellular and physiologic approaches to human and rodent islet biology and pathophysiology.
  3. To provide pure populations of live human beta cells using fluorescence-activated cell sorting (FACS) and/or to facilitate the use of specialized protocols, adenovirus/lentivirus and transduction methods for gene delivery to rodent and human beta cells and islets that enhance investigator-initiated research.
  4. To conduct specialized assays for the determination of insulin secretion, islet bioenergetics and beta cell differentiation, proliferation, survival and mass in vitro and in vivo using syngeneic, allotransplant or xenotransplant of rodent/human islets into immunocompromised euglycemic or diabetic mouse models.
  5. To assist investigators with study design and data interpretation to advance experimental approaches focused on the molecular and physiologic basis of human islet cell function and dysfunction in Types 1 and 2 diabetes.

Services Provided

The services of this Core are available to investigators new to diabetes research, as well as to investigators working on diabetes-related projects that can be enriched and extended by the use of the expertise and facilities of this Core.

  1. Isolation and preparation of human and rodent islets/beta cells and cell lines for investigator-initiated research, which is augmented by the availability of specific islet/beta cell biological assays for the study of beta cell growth, regeneration, survival, immunobiology and function.
  2. Generation of specific viral vectors (adenovirus and lentivirus) for gene delivery of cDNAs and shRNAs of interest to beta cells and other islet cell types. In addition, we will provide viral vectors for transduction of other cell types and tissues under investigation for non-beta cell/islet investigators.

All these services are available to investigators new to diabetes research, as well as to investigators working on diabetes-related projects that can be enriched and extended by the use of the expertise and facilities of this core.

Service ES-DRC members Non-ES-DRC members
Training in Human Islet Procurement and Use $30/training episode or hr. $70/training episode or hr.
aIslet culture $40/islet prep $80/islet prep
bIslet cell dispersion and culture $50/islet prep $100/islet prep
cIslet cell proliferation/survival assays (Brdu/Ki67/PHH3 or TUNEL/Caspase3) $100/assay $200/assay
cIn Vitro Islet cell number (Beads+Flow cytometer) $300/assay $600/assay
cIn Vitro Islet Function Assay (GSIS in static incubation/GSIS by perifusion/Mitochondrial Function, Seahorse Analysis) $100/assay
$300/assay
$300/assay
$200/assay
$600/assay
$600/assay
dIslet Xenotransplant (not incl. mouse purchase, human islets and per diems) $75/mouse transplant $150/mouse transplant
eIslet Xenograft Assessment (assay-dependent) $75-300/assay $150-600/assay
fRodent Islet Isolation (not incl. animal purchase and per diems) $70-100/animal $140-200/animal
gHuman beta cell FACS sort (user provides islets) $500/sort $1000/sort
hAdenovirus Library Access: Existing Virus (10µl aliquot/108-109 pfu/µl) see below $50/virus $100/virus
Custom Adenovirus Prep (100µl aliquot/108-109 pfu/µl) $600/virus $1500/virus
Custom Lentivirus Prep $600/virus $1500/virus
a,bHuman Islet Adenoviral or Lentiviral Transduction $50/Transdux $100/Transdux
cAssessment of Human Islet Adenoviral Efficiency $50/assay $100/assay
Other service Negotiable Negotiable

aCost is for culturing islets in one 6-well plate for one preparation. If more plates are required the discount will apply.

bCost is for dispersing islets in one 24-well plate for one preparation. If more plates are required the discount will apply.

cPrice for a single service, i.e. one in vitro cell proliferation, number, survival, transfection efficiency, GSIS >or mitochondrial function analysis experiment with three experimental conditions tested. There is a 20% discount per assay. If additional assays are requested, for example five GSIS experiments the charge is $400/$1300.

dHuman islet transplant in one immunodeficient mouse. For additional transplanted mice there is a 20% discount per additional mouse. Weekly blood glucose measurements are included in the price. If mice need to be treated, the following cost applies:

  • Tail vein injection: $8/mouse
  • Intraperitoneal (ip) injection: $6/mouse
  • Subcutaneous (sc) injection: $6/mouse
  • Osmotic pump implant: $45/mouse
  • Additional personnel time: $25/hr

eHuman islet transplant assessment assays are:

  • Islet graft harvest, fixation, embedding, sectioning and staining for insulin in three sections and beta cell area analysis: $100
  • Islet graft harvest, fixation, embedding, sectioning and analysis of beta cell proliferation/death: $200
  • IPGTT/OGTT, intraperitoneal or oral glucose tolerance test: $75 performed at MSSM
  • hyperglycemic clamp and plasma insulin levels see APC and BARC Fee Schedule.

fRodent islet isolation will be performed in mice ($70/mouse) or rats ($100/rat) using histopaque gradient. If islet handpicking is requested after purification, a $10 fee per animal will be charged. Any additional mouse or rat islet isolation will have the 20% discount.

gCost of human beta cell sorting is per preparation and for two treatments, control and experimental. If no treatments are requested, cost will be half.

h

CMV Promoter shRNA Tet-Inducble
GFP PKB/Akt-WT hCdk4 hp14arf p14 SMAD1  
eGFP PKB/Akt-CA hCdk6 hp16 p16 SMAD2 CMV TTA (tet transactivator)
LacZ PKB/Akt-KD hCyclinD1 hp18 p18 SMAD3 TRE-hCdk4
LacZ-GFP PI3K hCyclinD2 hp19 p19 SMAD4 TRE-hCdk6
PDGFRA PTEN hCyclinD3 hp21 p21 SMAD5 TRE-CyclinD1
PDGFRB STAT5 hCdk1 hp27 p27 SMAD7 TRE-CyclinD3
cMet JAK2 hCdk2 hp57 p57 SMAD8 TRE-cMyc
PRLR PKCζ-WT hCyclinA hp53 pRb KDM5B RIP1-Driven
PTHrP PKCζ-CA hCyclinE 4mtD3cpv p107 KDM6A RIP-LacZ
HGF PKCζ-KD hBmi1 long TmfSF4-mcherry p130 KDM6B RIP-ZsGreen
PL NFaT2 hEzh2 Bach2 GC lats1 RIP-mCherry
Betacellulin mCherry-NFATc3 hcMyc Tag-BFP DYRK1A lats2 RIP-TTA
VEGF ICUE3 hcMyc-S62A BNC2 DYRK1B Dlk1 RIP-Medial Go-D1
Pericam D4ER hcMyc-T58A ZRANB DYRK2 HDAC6 RIP-Trans Go-D1
GC Dlk1 B-catenin-y33 GAS7 DYRK3 HDAC8 RIP-VSVG-GFP
TagBFP DYRK1A ISL1 VIVT DYRK4 LacZ RIP-GJD2
Cre DYRK1B Mybl2 ChREBP GSK3ß p300 IRX1-cherry-rip1-zsgreen
GSK3α DYRK2 Kdr Glucokinase GLP-1R Creb3 Tm4sf4-cherry-rip1-zsgreen
FOXM1 DYRK3 SST2 mcherry Keap1 CREBBP CLK1 RIP-CyclinD1
SMAD6 DYRK4 SST5 mcherry Nrf2 Scramble CLK2 RIP-Cdk4
Sp1       Men1 CLK4  

Hours: 9:00 AM - 5:00 PM
Locations: Icahn School of Medicine at Mount Sinai
Berg Laboratory Building Floor 5th Floor
Room AB5-10 1428 Madison Avenue,
New York, NY 10029
Phone: Adolfo Garcia-Ocana, PhD, 212.241.9793
Email: adolfo.garcia-ocana@mssm.edu