1. Cellular and Tissue Therapies Branch (CTTB) Site Visit November 18, 2011
Steven R. Bauer, PhD, Chief
Brenton McCright, PhD, Senior Investigator
Deborah Hursh, PhD, Senior Investigator
Brian Stultz, MS, Biologist
Malcolm Moos, PhD, MD Senior Investigator
John Terrig Thomas, PhD Staff Scientist 1

2. Cell Therapy Challenges
Inadequate markers predictive of cell state and cell fate
Poor understanding of how cells interact with their microenvironment
Poor understanding of cell fate and survival post transplantation 2

3. CTTB Approaches Complementary Systems
Frogs, Flies, Mouse, and Man
Gene, Protein, Cell, Tissue Interactions
Control of Development
Knowledge and manipulation of growth factor pathways 3

4. Multipotent Stromal Cells (MSCs)
bone
fat
cartilage
stroma
other
Sources:
Bone marrow (MSC)
rare cell
Adipose tissue (ASC)
abundant cell
Others
Clinical Uses:
Cardiac repair
Bone repair
Cartilage repair
Facilitate hematopoietic reconstitution
Immune Diseases
Immunomodulation/
Anti-Inflammatory
This slide has a diagram of a Multipotent Stromal Cell (MCS) surrounded by six arrows pointing to several different tissues or cell types that arise from MSCs, including fat, cartilage, bone, stroma, and other types. 4

5. Identification and correlation of product characteristics with in vivo and in vitro assays of safety and efficacy
In vivo model of
critical hind limb ischemia
In vitro quantitative proliferation
and differentiation
Multipotent Stromal Cell
Characterization
Correlate characteristics with assay outcomes
Hursh lab: epigenetics,
karyotypes
Moos lab: gene expression,
qRT-PCR, single cell
Product
Characteristics
adipogenesis
In vitro immunosuppression
hMSC
mT-cell
PIs: Steve Bauer, Deb Hursh, Brent McCright, Malcolm Moos,
Michael Alterman, Raj Puri, Cheng-Hong Wei 5

6. Improve characterization of MSCs: Steven Bauer, Ph.D.
Quantitative bioassays to measure differentiation/proliferation capacity of MSC
Colony Forming Unit Activity
Cell Size Assessments
Adipogenesis: limiting dilution
In vitro assay to measure immunosuppression capacity of MSCs
Dr. Wei (DCGT)
Role of DLK1 in MSCs
delta-like 1, homology to Notch ligands 6

7. Donor and Passage Effects on MSC Characteristics
Colony forming ability differs between donors; decreases with passage
Cell size and size distribution differs between donors; increases with passage
Limiting dilution assay shows differences in adipogeneic potential between two donors, small cells have more adipogeneic potential
P3 = 1/125
R2 = 0.93
P5 = 1/126
R2 =0.86
P7= 1/2444
R2=0.96 7

8. Immuno-suppression Assay
NOD/ShiLtj mice 0%
82.7%
67.7%
44.2%
Immuno-suppression Assay
Reduce T-cell variability
MSC concentration-dependent
MSC specific
HepG2 hepatocarcinoma
HT-180 epithelial line
Future Plans
Apply to all donors, passages
This slide has two images. The first image is a diagram showing a cell-based assay to detect suppression of CD8 T-cell responses to an antigen called IGRP peptide. The CD8 T-cells are purified from the spleen and lymph nodes of mouse strain designated NOD/8.3. Antigen presenting cells (APCs) are derived from the spleen of a mouse strain designated NOD/ShiLtj. APCs are mixed with the peptide antigen and can then present them to the CD8 T-cells, resulting in activation and proliferation of the T-cells. When human MSCs are added to this mixture of mouse T-cells and APCs, the activation and proliferation of the T-cells can be measured to see if the human MSCs inhibit the T-cell activation and proliferation.
The second image shows histograms based on analysis of mouse T-cell proliferation using the fluorescent dye CFSE. There are four panels. The upper left panel shows that CD8 t-cells by themselves do not proliferate. The upper right-had panel shows that 82.7% of the T-cells proliferated when mixed with the APCs and the peptide. The lower left panel shows that only 67.7% of the T-cells proliferate when there is one human MSC for every ten mouse T-cells. The lower right panel shows that only 44.2% of the T-cells proliferate when there is one human MSC for every five mouse T-cells. 8

9. B-lineage defect in Dlk1-/- mice : changes in MSC stroma and osteoblasts?
The diagram on this slide shows one idea of how different types of cells that come from the bone marrow interact with each other. Part of the diagram shows how MSCs may give rise to chondocytes, osteoblasts, fibroblasts, apipocytes, myocytes, and endothelial cells. Another part of the diagram shows how hematopoietic stem cells (HSCs) give rise to the many types of cells in the blood including T-cells, B-cells, NK cells, Monocytes, macrophages, granulocytes, erhthrocytes, megakaryocytes, and osteoclasts. The middle part of the diagram also shows how osteoblasts, which come from MSCs, may affect the development of B-cell progenitors, which arise HSCs. 31 9
Yin and Li J Clin Invest 5: 1195

10. Developing measures of safety and efficacy for tissue engineered products: Brent McCright, Ph.D.
Mouse model of hind limb ischemia
A pre-clinical model system to evaluate cell and gene therapy products designed to enhance or inhibit vascular repair
Notch signaling as an indicator of Neural Stem Cell differentiation
Improve NSC product characterization and lot release testing
Identify requirements for Notch and Protein Phosphatase 2A signaling during murine heart development
Provide information relevant to cardiac progenitor cell product sources, manufacture, and behavior post-transplantation 10

11. Mouse model of Critical Hind Limb Ischemia (HLI)+
The femoral artery is ligated in two locations between the femoral triangle and the popliteal artery – (+)
Intramuscular injections of MSCs one day after ligation into six locations
2 x 106 total cells 11

12. MSCs incorporate into vascular structures four weeks post-transplantation
Transplanted mMSCs are Red
One week post-transplantation
The pictures on this slide were taken through a microscope on tissues around and including blood vessels. The mouse was injected with MSCs and there location around blood vessels was tracked using red fluoresence for the MSCs, and green fluoresence for the endothelial cells lining the blood vessels. Pictures were taken of tissues one and four weeks after injection of MSCs.
Recipient endothelial cells are Green 12

13. MicroCT and Laser Doppler Imaging can be used to monitor angiogenesis
Arrow = ligation site
Tortuous collateral vessel formation can be visualized (arrows).
Mouse #4 (+) cells
Mouse #7 - PBS only
Day +4
Day +12
Day +18
Day +26
On the left, this slide shows two x-ray images from a microCT experiment. A contrast dye was injected into the mouse blood system, and microCT images show the blood vessels in the injured and non-injured leg of a mouse (upper left). A red arrow shows the site of the injury. The lower left panel shows where new small vessels were formed near the injury site. These vessels have a characteristic tortuous path which is shown by two red arrows in the lower image. The images on the right show the result of lased doppler analysis on the injured and non-injured legs of a mouse. Red shows areas of high blood flow, orange, yellow, and green show lower amounts of blood flow, and blue shows minimal blood flow. 13

14. Developing Markers of Safety and Efficacy of Cell-Based Products: Deborah Hursh
Project 1- Bone Morphogenetic Protein
(BMP) Signaling
Genetic interaction screens to identify proteins that interact with the TGF-β/BMP pathway in adult head formation
Genetic analysis of BMP action during head morphogenesis
Role of BMP signaling in cell viability
Project 2- chromosomal and chromatin stability in cultured Multipotent Stromal Cells (MSCs)
Karyotype analysis of primary MSCs from multiple donors over extended passages
Analysis of histone modifications at promoters of functionally important genes in primary MSC populations 14

15. Chromatin signatures distinguish regulatory elements/events
Is Chromatin Structure Useful to Predict Quality Attributes for Cell Therapy Products?
Promoters
Enhancers
Insulators
DNA repair
Chromatin signatures distinguish regulatory elements/events H3 K4 K9
K27
Me3 Ac
Bivalent
Committed
This slide shows how histones can be modified and bind to DNA around elements that regulate gene expression. Modifications shown in red are associated with activation of gene expression, while modifcations shown in blue are associated with supression of gene expression. The illustration on the right shows how some histones are thought to carry both activating and supressing histone modifications at the same DNA control element. Such modifcations are termed bivalent and are thought to represent genes that are ready to respond to signals to differentiate into more mature cell types. When differentiation occurs, the supressive modifications can be removed and gene expression can be activated at this type of gene.
Potential role for chromatin structure as an early indicator for quality in adult stem cell-based products? 16
Turner, BM Cell. 111(3): 15

16. Summary
Dynamic chromatin structures mirror physiological roles of genes and may be useful early markers of cell fate
Chromatin marks may precede changes in transcription or protein expression and may reflect cell potential. 18

17. Cell Fate Decision Controls as Quality Attributes Malcolm Moos Jr. , M
Cell Fate Decision Controls as Quality Attributes Malcolm Moos Jr., M.D., Ph.D
Pathway components and regulators
ADMP
Frzb
Correct Xenopus ortholog of BMP-7
CDMP-1, -2, and -3
SMOC
Protease control of BMP action
Systems level pathway integration
“Epigenetic landscape” concept + systems biology modeling suggest ‘all-or-none’, ‘bistable’ controls as Quality Attributes S R 19

18. MSCs—Single cell PCR all cells CD29+, 44+, 105+, 166+, 45-, 14-, 34-
Increasing abundance
Each row is one cell
Each column is a specific mRNA CT
Heatmap
key
GAPDH
This diagram shows results from analysis of RNA expression of many genes in many single cells. Each column shows the expression of one particular gene, each row shows the expression of that particular gene in a particular cell. Black indicates no expression could be detected and lighter colors indicate more expression, with orange being the highest level of expression. 21
S Rosinbum, J Mateshaytis, M Moos, 2011, unpublished

19. Hypothesis: Clinical benefit will depend on the number of cells in the correct, discrete state
Response
control
Nothing
active
Marginal effect
Maximal
Stimulus 22

20. Summary SPCs help localize BMP action.
HBP1 may be a negative regulator of Wnt signaling important in tissue specification.
Single cell mRNA profiling can identify previously undetected population heterogeneity.
Convergence of experimental biology, systems biology/control theory, and analytical technology may allow increasingly effective product manufacture and testing strategies. 23

21. CTTB Research: Addressing Cell Therapy Challenges
Complementary approaches
Cell-Cell interactions
Genetic interaction screens
Protein-Protein interactions
Organogenesis 24

22. Significance for Cell Therapy
Findings may reveal cell product quality attributes that lead to:
Improved characterization of cell-based therapies
Methods to monitor manufacturing differently
Ability to choose donors differently
Scientific basis for:
policy development
guidance for sponsors 25