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Cellular and Tissue Therapies Branch (CTTB) Site Visit November 18, 2011 Steven R. Bauer, PhD, Chief Brenton McCright, PhD, Senior Investigator Deborah.

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Presentation on theme: "Cellular and Tissue Therapies Branch (CTTB) Site Visit November 18, 2011 Steven R. Bauer, PhD, Chief Brenton McCright, PhD, Senior Investigator Deborah."— Presentation transcript:

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 4

5 Identification and correlation of product characteristics with in vivo and in vitro assays of safety and efficacy ProductCharacteristics PIs: Steve Bauer, Deb Hursh, Brent McCright, Malcolm Moos, Michael Alterman, Raj Puri, Cheng-Hong Wei adipogenesis In vitro immunosuppression hMSC mT-cell Multipotent Stromal Cell Characterization Correlate characteristics with assay outcomes Hursh lab: epigenetics, karyotypes Moos lab: gene expression, qRT-PCR, single cell In vivo model of critical hind limb ischemia In vitro quantitative proliferation and differentiation and differentiation 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 R 2 = 0.93 P5 = 1/126 R 2 =0.86 P7= 1/2444 R 2 =0.96 7

8 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 8 NOD/ShiLtj mice 0% 82.7% 67.7% 44.2%

9 B-lineage defect in Dlk1-/- mice : changes in MSC stroma and osteoblasts? Yin and Li J Clin Invest 5:

10 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

11 Mouse model of Critical Hind Limb Ischemia (HLI) 11 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 10 6 total cells + +

12 12 MSCs incorporate into vascular structures four weeks post-transplantation Transplanted mMSCs are Red One week post-transplantation Four weeks post-transplantation Recipient endothelial cells are Green

13 Arrow = ligation site Tortuous collateral vessel formation can be visualized (arrows). Mouse #4 (+) cellsMouse #7 - PBS only Day +4 Day +12 Day +18 Day +26 MicroCT and Laser Doppler Imaging can be used to monitor angiogenesis 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 Is Chromatin Structure Useful to Predict Quality Attributes for Cell Therapy Products? Turner, BM Cell. 111(3): H3 K4 K9 K27 Me3 Ac Me3 BivalentCommitted Chromatin signatures distinguish regulatory elements/events Potential role for chromatin structure as an early indicator for quality in adult stem cell-based products? Promoters Enhancers Insulators DNA repair 16

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.D., Ph.D Pathway components and regulators Pathway components and regulators ADMP ADMP Frzb Frzb Correct Xenopus ortholog of BMP-7 Correct Xenopus ortholog of BMP-7 CDMP-1, -2, and -3 CDMP-1, -2, and -3 SMOC SMOC Protease control of BMP action Protease control of BMP action Systems level pathway integration Systems level pathway integration “Epigenetic landscape” concept + systems biology modeling suggest ‘all-or-none’, ‘bistable’ controls as Quality Attributes “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 - S Rosinbum, J Mateshaytis, M Moos, 2011, unpublished Increasing abundance Each row is one cell Each column is a specific mRNA CTCTCTCT Heatmapkey GAPDH 21

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

20 Summary SPCs help localize BMP action. SPCs help localize BMP action. HBP1 may be a negative regulator of Wnt signaling important in tissue specification. HBP1 may be a negative regulator of Wnt signaling important in tissue specification. Single cell mRNA profiling can identify previously undetected population heterogeneity. 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. 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


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