Pancreas

November 28, 2007 BME 248: Tissue Engineering

Cole Barthel Adrienne Blount Jason Liu Becca Wilusz

Overview
Background
The Pancreas Diabetes as a disease

Previous research for diabetes

Transplantation Cell therapy Cell sources

Our approach

The Pancreas
Glandular organ located posterior to the stomach in the abdomen Two major functions
Exocrine: production and secretion of digestive enzymes; acinar cells, ductal network Endocrine: regulation of blood sugar; Islets of Langerhans, capillary beds
www.med.umich.edu/1libr/wha/wha_pancreas_art.html

Islets of Langerhans
1-2 million islets in adult humans (2% of the pancreas by weight) Endocrine cell types
cells: insulin, amylin cells: glucagon PP cells: pancreatic polypeptide cells: somatostatin cells: ghrelin

http://www.betacell.org/content/articles/print.php?aid=13

Insulin, Glucose, & Cells

Insulin is produced almost exclusively by cells in the pancreas
Secreted in response to nutrients, hormones, and nervous stimuli

Insulin is central in maintaining plasma glucose levels within the normal physiological range
Fasting blood glucose test: 70-99 mg/dL Random blood glucose test: 70-125 mg/dL

Insulin, Glucose, & Cells

Glucose is the main regulator of insulin gene expression and secretion Once secreted, insulin decreases blood glucose levels by:
Increasing storage of glucose in liver and muscle cells Decreasing glucose synthesis Decreasing glycogen breakdown

http://stemcells.nih.gov/StaticResources/info/scireport/images/figure71.jpg

Insulin, Glucose, & Cells

Other important functions of insulin include:
Promotes fatty acid and triglyceride synthesis in adipose tissue Increasing cellular uptake of circulating amino acids and potassium Arterial muscle tone: forces smooth muscle cells to relax allowing for increased blood flow, especially in micro-arteries

Diabetes Mellitus
Disease characterized by the bodys inability to produce or properly use insulin Classifications:
Type 1: autoimmune destruction of cells results in a progressive failure in insulin production Type 2: insulin resistance and inadequate insulin production Gestational: glucose intolerance during pregnancy Pre-diabetes: higher than normal plasma glucose levels placing an individual at risk for developing type 2 diabetes

Diabetes A Growing Problem

Affects more than 150 million people worldwide 20.8 million Americans are currently affected
14.6 million diagnosed, 6.2 million undiagnosed 54 million classified as pre-diabetic

http://www.diabetesnd.org/whatis.html

Diabetes Complications & Costs

Complications include heart disease, stroke, high blood pressure, blindness, kidney disease, nervous system disease, amputations Contributed to the deaths of over 200,000 people in the US in 2002, making it the sixth leading cause of death Total cost: $132 billion in the US
1 in 4 Medicare dollars goes toward diabetes and its associated complications

Insulin Therapy
Standard technique for managing diabetes
Means of keeping blood glucose levels as close to normal as possible

Requires regular monitoring of blood glucose levels, administration of insulin, and strict dietary control Synthetic human insulin produced via genetic engineering of bacteria and yeast with recombinant DNA
Different kinds based on onset, peak time, and duration of activity

Insulin Therapy
Delivery systems
Injection
Syringes, Pens, Infusers, Jet Injectors Pumps: continuous supply & bolus dose; are not an artificial pancreas

Transdermal Inhaled Oral

Drawbacks
Frequency of blood glucose testing Difficulty in determining appropriate dose Does not protect against effects of acute hypoglycemia or long-term hyperglycemia

Edmonton Protocol
First proposed by A. M. James Shapiro et al. in 2000 Glucocorticoid-free immunosuppresive protocol
Sirolimus, tacrolimus, daclizumab

Islet cells transplanted from multiple donors

10,000 islet equivalents per kilogram injected into the portal vein Compatible with ABO blood type; no HLA matching

International clinical trial

16 of 36 patients were insulin-independent after 1 year; 5 remained insulin-independent at 2 years Shapiro, AM et al. N Eng J Med. 2006. 355(13):1318-30.

Encapsulation Methods
1. Intravascular Implant 2. Macroencapsulation 3. Microencapsulation

Intravascular Implant
A perfusion chamber directly connected to the vasculature Pros:
Good mass transfer Accessible seeding ports

http://www.isletmedical.com/pages/company_competition.htm#istech

Cons:
Thrombogenesis Limited volume of islet tissue
Beck et al. Tissue Engineering. 13(3) 2007.

BioHybrid Technologies Inc. - Artificial Pancreas

Sullivan et al. Science. 252(5006) 1991 Monaco et al. Ann. Surg. 214(3) 1991

Macroencapsulation
Tubular (fiber) or planar (sheet) chambers containing large masses of islet cells Pros:
Extravascular Various sites for implantation Structurally sound

Cons:
Limited diffusion Fibrosis Membrane breakage Immunoprotection

Beck et al. Tissue Engineering. 13(3) 2007.

Cerco Medical - Islet Sheet

Storrs et al. Ann. NY. Acad. Sci. 944. 2001.

http://www.isletmedical.com/pages/company_competition.htm#istech

Microencapsulation
Encapsulate single islets or very small masses Usually spherical, can be cylindrical Pros:
Extravascular High surface to volume ratio Easy to implant

Cons:
Difficult to retrieve Structurally weak Immunoprotection
Beck et al. Tissue Engineering. 13(3) 2007.

Microencapsulation Techniques
Typically involves polyelectrolyte complexation involving a polyanionic gel (e.g. alginate, polyacrylates, and agarose) Polycations are used to form protective layers (e.g. poly-L-lysine, chitosan, poly-L-ornithine, and PEI) Other methods include:
Photopolymerization, micro-machined nanoporous microsystems, crystal gun, and coaxial needles

De Groot et al. J Surg. Res. 121. 2004.

Zimmermann H. et al. J Materi. Sci. 16. 2005.

Objective
We propose to design an intravascular implant that: 1. Sustains beta cell viability 2. Produces a quicker response to glucose stimulation 3. Reduces the risk of thrombogenesis 4. Modulates the host immune response

Beta Cell Sources

Lock et al. 2007

Engineering Beta Cells

Isolation and expansion of hMSCs
Obtain bone marrow from donor Expand cells in vitro

Generating retroviral vector

Def: plasmid which relies on reverse transcription of RNA to cDNA Pancreas sample taken from donor to obtain Mesenchymal stem cells stained with haematoxylon and eosin. Y. Lu, Z. Wang, M. Zhu , Annals of Clinical & Laboratory Science. 2006 human insulin gene RNA cDNA and amplified through RT-PCR

Engineering Beta Cells

Retroviral vector expanded
The insulin DNA extracted from the PCR is combined with retroviral vector Vector is transfected into a bacteria where it is able to expand

BIO. Biology in Perspecitve. Biotechnology Industry Organization 1990

Engineered Beta Cells

Virus production
Packaging cells were then transfected with the recombinant plasmid These cells burst and release a virus in its media supernatant

MSC transfection

Other Utilized Cell Types

Pancreatic islet endothelial cells
Surrounds the engineered beta cells High level of fenestration, allows the outflow of insulin Produces an extracellular matrix needed for the support of beta cells

M. Hermann, R. Margreiter, P. Hengster, J. Cell. Mol. Med. 2007

Other Utilized Cell Types

MSCs (mesenchymal stem cells)
Protection against autoimmune insulitis

Human Umbilical Vein Endothelial Cells

Surrounds the mesenchymal stem cells Lines the shunt to prevent thrombosis

MSCs as an Immunosuppression Strategy

MSCs have been shown to modulate the function of T lymphocytes, B cells, dendritic cells, and natural killer cells in vitro and act as immune suppressors in vivo
Exact mechanism is unknown

Generally accepted that

Soluble growth factors play a major role MSCs need to be simulated to become immunosuppressive

Stagg, J & Galpieau J. Handb Exp Pharmacol. 2007; (180):45-66.

MSCs as an Immunosuppression Strategy

In vivo success for tissue grafts
Intravenous injection of MSCs prolongs survival of allogenic skin grafts in baboons (Bartholomew et al., 2002) Successful treatment of severe, steroid refractory graft-versushost disease in 6 of 8 human patients with no adverse side effects (Ringden et al., 2006) In a murine model, injection of MSCs originating from the receiver dramatically improves the long-term allograft success of hematopoietic stem cells as compared to donor MSCs (Nauta et
al., 2006)

MSCs as an Immunosuppression Strategy

Differentiation-independent success in vivo
Infusion of MSCs confers a protective effect on injured neurons in neurological diseases (Chopp & Li 2002, Muller et al. 2006) Infusion of MSCs enhanced recovery of renal function in an acute renal failure model (Togel et al. 2005)

Intravenous injection of MSCs into diabetic NOD/scid mice resulted in an increased number of pancreatic islets and insulin-producing cells (Lee et al. 2006)

MSCs as an Immunosuppression Strategy

Advantages
Local suppression of the immune system in the area of the graft Eliminate the need for immune suppression drugs
Edmonton protocol immune suppression agents (Sirolimus and Tacrilimus) drastically reduce cell proliferation in rats

Disadvantages
Risk of tumorgenicity as a result of spontaneous transformation with in vitro expansion Potential for MSC differentiation within the graft

Cell Encapsulation Methods

cells

Basal lamina-type ECM

Cylindrical microcapsules

Mesenchymal Stem Cells (MSCs)

Collagen I

Cylindrical microcapsules

Cell Encapsulation Methods

Cylindrical microcapsules with cells

Pancreatic islet endothelial cells

Cell-coated cylindrical microcapsules

Cylindrical microcapsules with MSCs

Human umbilical vein endothelial cells

Cell-coated cylindrical microcapsules

Cell Encapsulation Methods

Perfusion of media through interstitial spaces

Experimental Models of Immunoprotection of Islet Transplantation

Gray, DWR . Ann NY Acad Sci. 2001 Nov; 944:226-239.

Test for insulin production from our differentiated cells

An ELISA (Enzyme Linked Immunosorbent Assay) able to detect insulin will be used to test for insulin presence.

microvet.arizona.edu

Test for glucose sensing abilities of differentiated cells

RT-PCR technique will be used to test for expression of GLUT-2 and Gk Stimulate cells with high glucose concentrations and measure secreted insulin Record insulin response time

MSC considerations
Examine proliferation and migration of encapsulated MSCs MLR (mixed lymphocyte reaction) will be run in vitro to determine if the immune suppressive properties are present with encapsulation Test for genetic markers specific for MSC differentiation via western blotting

Test for microcapsule permeability with cell coating

Capsules are added to a polymer solution with multiple sized molecules. After incubation a liquid chromatograph is used to test for remaining molecules Repeat insulin release and glucose stimulation with pancreatic islet endothelial cell coating

Thrombogenesis
Perfuse whole blood through construct Measure percent platelet loss to quantify the antithrombogenic capabilities of the HUVEC coating
http://cache.eb.com/eb/image?id=98328&rendTypeId=4

In Vivo Methods
Shunt hepatic portal vein Monitor blood glucose over time Canine model
STZ (streptozotocin) induced diabetes Autoimmune diabetes

Large primate model with autoimmune diabetes

http://www.clevelandclinic.org/health/health-info/pictures/tipspost.gif

Questions?