2008

December

Alan Russell

Marina Kameneva, William Wagner, Jörg Gerlach, Mark Yazer

Large Scale Human Placenta Progenitor Cell-Derived Erythrocyte Production – Continuous Red Blood Cell Production

In order to provide a practical and ready supply of safe and effective red blood cells (RBC) for the treatment of battlefield trauma, or diseases, a number of fundamental challenges must be overcome.  These challenges are associated with current small-scale, variable efficiency in vitro cell expansion and erythrogenic differentiation of hematopoietic stem cells (HSC) and include:

• Reducing the quantity of media required to expand and differentiate sufficient HSC in vitro.  Conventional culture systems operate at cell densities of 1x105-1x107 per ml and would require far in excess of the ~1300 liter volume maximally available in the 47cuft DARPA specification to generate 2x1012 RBCs associated with a unit of whole blood.
• Reducing minimum ~21 day period currently required to generate ~ 2x1012 RBCs from conventional HSC numbers (~5x107) derived from umbilical cord blood (hUCB)
• Safely reducing the senescence rate of self-renewing HSC which currently limits the rate and absolute level of HSC expansion.
• Maintaining the pluripotency of the expanded CD34+ population thereby limiting the progressive decrease in pluripotentiality currently observed in expanded HSC populations.
• Demonstrating significant proof of concept and reduction to practice in <36months while assuring that the product will be safe and effective.

To overcome these challenges, a consortium led by Celgene Cellular Therapeutics (CCT) and supported by the University of Pittsburgh/Institute for Transfusion Medicine, Fred Hutchinson Cancer Research Center, The Ohio State University, Loughborough University and The Automation Partnership has been assembled. This consortium will provide all of the necessary skills (cell biology, hematology, cellular therapeutic process development, bioreactor systems, cells separation technology and automation) to ensure effective and safe research, and RBC product development.  The multidisciplinary consortium will develop highly novel and / or unique solutions that include:

• Initiating the process with a high number of cells available by combining HSC derived from umbilical cord blood with human placental perfusate (hPP) that contain highly erythrogenic progenitors and can be obtained in a ready supply post-partum from type O Rh-ve donors.
• Using immobilized Notch ligands to prevent senescence and maintain erythrogenic differentiation potential during multi-log increase in the number of HSC.
• Incorporating selected Celgene proprietary compounds (IMiDs) with the aim of enhancing the expansion of HSC, without a significant loss in multipotentiality, and to enhance erythropoiesis and modulate globin gene expression during differentiation.
• Transient and safe genetic engineering of HSC, in order to modulate cell cycle phase transition times and enhance HSC expansion prior to enrichment of enucleated erythrocytes.
• Development of a semi-fieldable centralized and fieldable automated cell culturing system, to ensure that the footprint of the fieldable unit is <47cubic feet and that battlefield use is fully automated and robust to use by minimally trained personnel. Semi-fieldable processing systems will incorporate large-scale batch methods in central or near-battlefield cell expansion centers. The fieldable bioreactor will be based on current prototype bioreactor that can accurately mimic the bone marrow microenvironment and can culture up to 1x1011 cells in one 800ml cell compartment system, requiring ~2liters of medium in the circuit.
• Development of near-continuous non-invasive sorting systems to enrich progenitors committing to erythrogenic differentiation pathways and to ensure consistency, safety and efficacy of the final product.

Defense Advanced Research Projects Agency

09/01/08 – 05/31/09

November

Edward V. Prochownik

Eric Lagasse, William Saunders, and Youjun Li

Function of a Glycoprotein Ibα, a Subunit of the Von Willebrand’s Factor Receptor as a Transforming Oncoprotein

De-regulation of the CMYC gene and/or its encoded protein, c-Myc, are among the most common molecular abnormalities in human cancers.  c-Myc is a particularly notorious oncoprotein because, in addition to being acutely transforming, it can also mediate genomic instability (GI) at several levels, which contributes to ongoing mutational changes and tumor cell evolution.  A major focus of our laboratory has been the identification of transcriptional targets for c-Myc, which normally functions as a general transcription factor.  Many c-Myc targets have been identified but only a small number have been shown actually to recapitulate the transforming properties of c-Myc itself.  Recently, we have identified a totally unexpected down-stream target of c-Myc, GPIBα, whose encoded proteinGpIbα functions as a subunit of the von Willebrand’s factor receptor (VWFR), previously believed to be expressed only on platelets and megakaryocytes.  In its traditional role, VWFR interacts with von Willebrand’s factor expressed by the vascular sub-endothelium and thus serves to immobilize platelets and allow their aggregation and activation during the initial stages of blood clot formation.  Unexpectedly, we have found that GpIbα is necessary for c-Myc to promote GI and, by itself, is sufficient both for transformation and GI.  In a large panel of normal and tumor cell lines, we have found that GpIbα is expressed at highest levels in the latter cells and in direct proportion to c-Myc.  Now, in collaboration with Dr. Eric Lagasse (McGowan Institute for Regenerative Medicine) and Dr. William Saunders (University of Pittsburgh Department of Biological Sciences), we propose to explore further the mechanism(s) by which GpIbα promotes GI and cellular transformation and to delineate GpIbα’s role in in vivo tumorigenesis.  Thus, in Specific Aim 1, we will define the mechanisms by which GpIbα over-expression leads to GI and transformation (Prochownik).  In Specific Aim 2, we will assess the role of GpIbα in promoting tumorigenesis of established cancer cell lines (Prochownik).  In Specific Aim 3, we will develop an in vivo model of GpIbα-mediated GI and transformation (Lagasse).  Finally, in Specific Aim 4, we will evaluate in detail the nature of GpIbα-mediated GI (Saunders).  Together, these studies will provide new insights into a previously unrecognized and unexpected oncoprotein, namely GpIbα, and will define the mechanisms by which this newly described function differs so dramatically from its traditional role in megakaryocytes and platelets.

Children’s Hospital of Pittsburgh

7/1/08-6/30/09

October

William Federspiel, PhD

Alan Russell, PhD and William Wagner, PhD

Percutaneous Respiratory Assist Catheter

Each year several hundred thousand Americans suffer short term lung failure requiring respiratory support within the intensive care unit. The objective of this proposal is to develop a percutaneous respiratory assist catheter (PRAC) that can be inserted into the venous system to provide supplemental breathing support, independent of the lungs, for patients requiring short-term (~ 4-7 day) respiratory assistance.

The PRAC will be designed for percutaneous insertion into a peripheral vein and placement in the central venous system, where it will be exposed to all the blood returning to the heart. The PRAC will use a rotating impeller within the fiber bundle to generate active mixing of blood to enhance gas exchange. We will also develop novel hollow fiber membranes that incorporate immobilized enzymes that will further accelerate CO2 removal.

The target is a percutaneous assist catheter (20-25 Fr or smaller) that can provide 90-120 ml/min of CO2 removal when used as an adjuvant or replacement to existing therapy for patients with acute lung failure (ARDS, pneumonia) or acute on chronic lung failure (COPD with exacerbation). 

07/08/08 – 05/31/12 (this award); 04/01/02 – 05/31/12 (entire project)

September

Yoram Vodovotz, PhD

Gregory M. Constantine, PhD (Pitt—Mathematics); Steve Chang (CEO of Immunetrics); Drs. Gary Nieman and Kris Maier (SUNY-Syracuse)

Mathematical Modeling of Inflammation in ARDS

Trauma and systemic infection elicit an acute inflammatory response.  Inflammation involves complex interactions among leukocytes, their products (cytokines, free radicals, and proteases), and the tissue damage/dysfunction that ensues. This multiple organ dysfunction often manifests as septic shock and severe lung dysfunction, referred to collectively as the acute respiratory distress syndrome (ARDS), and contributes to the 215,000 annual deaths in the U.S. from sepsis. The complexity of this process has stymied the progress towards immunomodulatory ARDS therapeutics. We have developed a mathematical model of these elements in order to unravel this complex interplay in various settings of acute inflammation, and have calibrated distinct variants of this model with data from mice, rats, swine, and humans (University of Pittsburgh Inflammatory Analyte/Modeling Component). Our modeling platform has been used to gain both basic and translational insights, the latter including simulated (in silico) clinical trials. In conjunction with these efforts, we developed a sepsis + gut ischemia/reperfusion (Sepsis+I/R) porcine model that mimics the pathogenesis of human septic shock and ARDS (Upstate Medical University ARDS Animal Model Component). We hypothesize that mathematical analysis of the complex biochemical and physiologic data generated in our Sepsis+I/R model will enable us to isolate key therapeutic targets and to test novel therapeutics; one such agent is the modified tetracycline COL-3. Our Specific Aims are: 1) to develop a robust mathematical model describing Sepsis+I/R-induced shock and ARDS in swine, its pathologic consequences, and possible therapies, 2) to utilize COL-3 as a tool to further calibrate the mathematical model and 3) to demonstrate that NE, MMP-2 and MMP-9 are critical components in Sepsis+I/R-induced septic shock and ARDS pathogenesis. Our calibrated mathematical model will be used to conduct in silico clinical trials and establish a platform for the rational development of novel ARDS therapeutics. The in silico trials will be validated in animal experiments. The proposed translational studies will develop a robust mathematical model capable of describing the complex pathogenesis of sepsis-induced ARDS and identify target molecules whose modulation would significantly improve clinical outcome. 

NIH (National Heart, Lung and Blood Institute)

7/1/08-6/30/11

August

Bradley Keller, MD

Engineered Early Embryonic Cardiac Tissue

We have developed an Engineered Early Embryonic Cardiac Tissue, termed EEECT, using embryonic cardiac cells isolated during the period of primary morphogenesis in order to investigate the regulation of embryonic CM proliferation and differentiation and to generate tissues with optimal properties for cardiac repair. Our EEECT construct uses a simple cylindrical geometry which is reproducible, scalable, and preserves the unique proliferative and contractile properties of developing myocardium.  Using EEECT we can investigate the regulation of CM proliferation and maturation within a functioning in vitro 3D environment.  EEECT proliferation and force production increases in response to cyclic mechanical stretch. With prolonged culture EEECT acquires a post-natal myocardial phenotype (reduced proliferation, increased calcium and β-adrenergic sensitivity, and increased force production). Preliminary data show that cylindrical EEECT can be implanted onto recipient injured adult myocardium as part of a cardiac repair/recovery strategy. Implanted EEECT survive, proliferate, and functionally contribute to recipient cardiac functional recovery.  

Specific Aim 1: Define molecular pathways that regulate the EEECT CM proliferation. We hypothesize that (1) EEECT CM proliferation is regulated by interactions between integrin-linked kinase (ILK), p38 mitogen-activated protein kinase (p38 MAPK), and Akt; (2) cyclic mechanical strain stimulates cell proliferation via ILK, p38MAPK, and Akt; and (3) Thyroid hormone triggers CM within EEECT to shift from an immature proliferative to a post-natal hypertrophic growth phenotype.
    
Specific Aim 2: Determine the fate of EGFP+ EEECT following implantation onto injured adult rat EGFP- myocardium and the contribution of EEECT to recipient myocardial function and remodeling.  We hypothesize that following implantation (1) EEECT undergo limited cell death followed by significant CM proliferation; (2), become vascularized by vessels composed predominantly of recipient endothelial cells; (3) positively contribute to the diastolic and systolic functional recovery of recipient infarcted myocardium.

Significance. Our experimental strategy translates insights gained from investigating in vivo embryonic myocardium and in vitro Engineered Early Embryonic Cardiac Tissue (EEECT) towards the long term goal of developing a functioning engineered cardiac graft that optimizes post-implantation cell survival, proliferation, and sustainable functional recovery of injured myocardium. 

07/01/08 – 05/31/12

July

G. Bard Ermentrout, PhD, Beatrice Riviere, PhD, Jonathan Rubin, PhD, David Swigon, PhD, and Ivan Yotov, PhD

Research Training Group Award

Will provide resources to develop training programs for mathematics students to work with physicians and biologists to help resolve complicated medical problems through mathematics.  A variety of computer models will be produced based on differential equations to create immune system models to plot the various chemical and physical changes that occur as the body battles influenza, inflammation, sepsis, necrosis, and wounds.  The researchers hope to be able to plot and pinpoint the origin of uncontrollable inflammation and infection that can occur as complications following surgery.

National Science Foundation ($1.8 million) and the University of Pittsburgh School of Arts and Sciences

Total of $2.5 million

June

1. In vivo and in vitro evaluation of porcine dermal product for pelvic floor and body wall (hernia) reconstruction. ($90,000)
2. Manufacturing process review and modification. ($96,000)
3. In vitro characterization of porcine dermis ECM and products in development. ($118,000)
4. Evaluation of gel form of porcine dermal matrix. ($60,130)

May

Gplb-alpha deregulation and genomic instability in stem cells.  The objective of this project is to generate mice with overexpression of Gplb-alpha in hematopoietic stem cells and their progeny.

$62,575 Annual

April

The University of Pittsburgh’s McGowan Institute for Regenerative Medicine and the Institute for Regenerative Medicine at Wake Forest University Baptist Medical Center have been selected as co-leaders of a national $85 million program to use the science of regenerative medicine to develop new treatments for wounded soldiers.

A new federally funded institution – the Armed Forces Institute of Regenerative Medicine (AFIRM) – will be made up of the U.S. Army Institute of Surgical Research and consortia involving the McGowan-Wake Forest team and another led by Rutgers and the Cleveland Clinic. Each group was awarded $42.5 million. The Wake Forest-McGowan team includes collaborators from 15 other institutions.

AFIRM will be co-directed by Alan J. Russell, Ph.D., director of the McGowan Institute for Regenerative Medicine, and Anthony Atala, M.D., director of the Wake Forest Institute for Regenerative Medicine. The massive project will be dedicated to repairing battlefield injuries through the use of regenerative medicine, science that takes advantage of the body’s natural healing powers to restore or replace damaged tissue and organs. Therapies developed by AFIRM also will benefit people in the civilian population with burns or severe trauma due to illness or injury.
The McGowan and Wake Forest team has committed to develop clinical therapies over the next five years that will focus on:

• Burn repair
• Wound healing without scarring
• Craniofacial reconstruction
• Limb reconstruction, regeneration or transplantation

Compartment syndrome, a condition related to inflammation after surgery or injury that can lead to increased pressure, impaired blood flow, nerve damage and muscle death.

$42.5 million

March

Addressing the needs of new approaches for anti-cancer therapies by combining stem cell biology, cancer biology and bioengineering. Our central hypothesis is that cancer stem cells are initiating and sustaining the growth of ovarian cancer. In consequence, the identification of the cancer stem cells represents a major step forward in the elucidation of ovarian cancer hierarchy and could hold the key to understanding the origin and maintenance of ovarian cancer, the relapses and possibly the metastases in advanced cases. Another problem facing cancer cell biology is the access of in vitro culture models for research and study of cancer development and its pathophysiology. Here we propose to adopt bioreactors used for bioartificial livers (BAL) to provide tumor cells with a 3-D perfusion culture instrument that recapitulate vasculature and microenvironment.

$111,375

February

January

Harvey Borovetz, PhD

Levitronix Phase II SBIR

Development of a magnetically levitated, bearingless pump for a pediatric ventricular assist device.

9/30/07-6/30/10

Year 1 direct = $383,458
Year 1 indirect = $98,658
Year 1 total = $482,116
Total award for the entire project period: ~$1.68 million