Human Islet and Adenovirus Core (located at Mt. Sinai)
Leadership
-
Co-Core Director
Location: Icahn School of Medicine at Mount Sinai
sarah.stanley@mssm.edu -
Co-Core Director
Location: Icahn School of Medicine at Mount Sinai
donald.scott@mssm.edu -
Operations Manager
Location: Icahn School of Medicine at Mount Sinai
peng.wang@mssm.edu
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
- To assist new islet investigators in accessing human and rodent islets and related cell lines for use in investigator-driven studies.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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 | ||||