Developing Ways to Measure Safety and Efficacy for Tissue-engineered Products
Principal Investigator: Brenton McCright, PhD
Office / Division / Lab: OTAT / DCGT / CTTB
The primary purpose of cell and tissue engineering is to fulfill the growing need for new therapies required to treat patients with degenerative diseases. Collections of transplanted cells that are poorly engineered and uncharacterized (i.e., cells whose biological characteristics are not fully identified) may not function reliably in the body after being transplanted. Moreover, such cells might even damage other organ systems of the patient receiving them, or may form tumors. Therefore, our laboratory is studying the key processes that determine how cells mature and contribute to the formation of organs and tissues, primarily using the mouse as a model system. We believe that understanding the evolutionarily conserved signaling pathways involved in the regulation of mammalian organ development and stem cell differentiation is critical to successful cell therapy since these factors are also required for tissue repair and regeneration.
The goal of our work is to identify and understand those molecular signals that might help us to predict how a cell will function after being administered to patients. We are especially interested in understanding the molecular signals that determine whether a cell remains immature and continues to multiply, or stops dividing and matures into a cell that performs a specific function in the body. The molecular signals we study are evolutionarily conserved (present in all animals) and widely used (present in many cell types). We have developed genetically engineered mice in which these molecules can be turned on or off in response to certain stimuli. This allows us to study when these signals are needed during tissue repair and organ development. Our laboratory uses the mouse as a model to study how tissues mature and how biological signals determine the fate of cells in the body because organ development in mice is very similar to that of humans.
In addition to studying the function of key pathways of biochemical signaling that guide the development of organs, we are analyzing the function of these pathways in progenitor cells such as those used for cellular therapies. Progenitor cells are between embryonic stem cells and mature, functional, specific cells as far as their potential to form new tissues is concerned. Progenitors are more mature than embryonic stem cells and therefore limited in the type(s) of cells they can become.
Identifying the biological factors controlling growth and development of organs and tissues is providing data the FDA can use to advise sponsors on how to characterize cell populations or tissue-engineered products administered to patients. The studies are providing tools needed to evaluate potential cell sources, techniques for growing cells, and identifying markers that can predict how cells will behave. It will also help agency scientists develop needed new approaches for quality assessment of novel cell-based therapies with improved ability to predict safety and efficacy over standard methods used for conventional therapies and biologics.
Our research is focused on elucidating the function of key cell-fate-determining factors important in evaluating the safety and efficacy of cellular therapies and tissue engineered products. The research projects are focused on evaluating requirements for specific molecules during embryonic organ development and in mouse models of cellular therapies. Because successful repair and regeneration of damaged tissue will require the same signaling pathways used for embryonic organ formation we expect findings from these two approaches to complement each other.
Regulation of Protein Phosphatase 2A (PP2A) during development and progenitor cell proliferation:
We are phenotypically analyzing mice in which the B56 regulatory subunits of Protein Phosphatase 2A have been inactivated. Our findings demonstrate that mis-regulation of this conserved signaling pathway disrupts the normal expansion and differentiation of cardiac progenitor cells. This suggests that monitoring the expression of these molecules might be useful in evaluating the developmental status of cardiac progenitor cells used in therapies.
The regulation of PP2A activity has also been shown to be critical in the inhibition of cell transformation. In cells derived from mice that have PP2A-B56 regulatory subunits inactivated, we have found that cells do not arrest in mitosis when treated with cell cycling inhibitors. The Spindle Assembly Checkpoint (SAC) is used by proliferating cells to ensure the integrity of chromosome distribution and it is an important mechanism used by cells to control tumorigenesis. The mechanism behind the bypass of the SAC is being further investigated.
Neural stem cell reporters as a tool for identifying optimal growth conditions for cellular products:
Many cellular therapies use progenitor cells isolated from fetal or adult tissues to treat degenerative disease and injury. Progenitor cells such as Neural Stem Cells (NSCs) are used in cellular therapies as undifferentiated cells that take cues from host environment, or can be differentiated in culture into desired cell types prior to injection. In either case, there is a need to expand cells in culture. However, expansion of progenitor cells in culture is limited because primary cells stop proliferating and begin to differentiate after a few passages. NSCs containing Green Fluorescence Protein (GFP) "stemness" reporters to allow high throughput screening of progenitor cell status, will be used to identify growth conditions that maximize expansion. Culture conditions that we plan to vary include; oxygen and serum concentrations, substrates, and growth factors combinations.
After transplantation of cellular therapy products, it is often unclear how long the cells survive and if they migrate away from the injection site. Therefore, Magnetic Resonance Imaging (MRI) contrast agents including Superparamagnetic Iron Oxide Nanoparticles (SPIONs) and Perfluorocarbons (PFCs) are being used or proposed for use in clinical trials to track cellular therapies. We will use MRI to compare the survival and migration of NSCs transplanted into mice. NSC populations with variable amounts of undifferentiated cells as determined by expression of the GFP reporter constructs will be used. The goal is to determine if MRI can be used to detect differences in cellular behavior post-transplantation.
Cell Cycle 2017 Jun 18;16(12):1210-9
PP2A-B56gamma is required for an efficient spindle assembly checkpoint.
Varadkar P, Abbasi F, Takeda K, Dyson JJ, McCright B
Dev Dyn 2014 Jun;243(6):778-90
The protein phosphatase 2A B56gamma regulatory subunit is required for heart development.
Varadkar P, Despres D, Kraman M, Lozier J, Phadke A, Nagaraju K, McCright B
J Immunol 2011 Nov 15;187(10):5114-22
A disintegrin and metalloproteinase 10 regulates antibody production and maintenance of lymphoid architecture.
Chaimowitz NS, Martin RK, Cichy J, Gibb DR, Patil P, Kang DJ, Farnsworth J, Butcher EC, McCright B, Conrad DH
Dev Biol 2010 Jan 15;337(2):386-95
The contribution of Notch1 to nephron segmentation in the developing kidney is revealed in a sensitized Notch2 background and can be augmented by reducing Mint dosage.
Surendran K, Boyle S, Barak H, Kim M, Stomberski C, McCright B, Kopan R
Genesis 2009 Aug;47(8):573-8
Generation of mice that conditionally express the activation domain of Notch2.
Varadkar PA, Kraman M, McCright B
Tissue Eng Part A 2009 Mar;15(3):455-60
Synopsis of the Food and Drug Administration-National Institute of Standards and Technology Co-Sponsored "In Vitro Analyses of Cell/Scaffold Products" Workshop.
McCright B, Dang JM, Hursh DA, Kaplan DS, Ballica R, Benton KA, Plant AL
Dev Dyn 2008 Apr;237(4):1144-52
Notch2 is required for the proliferation of cardiac neural crest-derived smooth muscle cells.
Varadkar P, Kraman M, Despres D, Ma G, Lozier J, McCright B
PLoS One 2008 Mar 26;3(3):e1851
Notch signaling regulates bile duct morphogenesis in mice.
Lozier J, McCright B, Gridley T
Immunity 2007 Jul;27(1):89-99
Direct regulation of gata3 expression determines the T helper differentiation potential of notch.
Amsen D, Antov A, Jankovic D, Sher A, Radtke F, Souabni A, Busslinger M, McCright B, Gridley T, Flavell RA
Genesis 2006 Jan;44(1):29-33
Generation of new Notch2 mutant alleles.
McCright B, Lozier J, Gridley T
FASEB J 2005 Aug;19(10):1311-3
Functional conservation of Notch1 and Notch2 intracellular domains.
Kraman M, McCright B