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One Strategy for Gene/Cell Therapy: Harness the Natural Ability of Adult Stem Cells to Repair Tissues Darwin J. Prockop, M.D., Ph.D., Director, Center for Gene Therapy, Tulane University Health Sciences Center, New Orleans, LA Slide 2 How does the body repair itself? Cohnheim (1867): ---probably by cells that come from the blood stream. More recent hypothesis ---probably first by the stem-like cells found in many tissues. ---probably after these are exhausted ---by stem cells from the bone arrow Slide 3 How well does the repair system work? We are not at the top of the tree of evolution: Hydra, planaria, etc. ---complete organism from a few cells. Salamanders and related ---complete limbs or tails Mice and rats ---difficult to produce permanent damage in most organs. Slide 4 One Strategy for Gene/Cell Therapy Use stem-like cells from bone marrow ---to make man more like a rodent or perhaps salamander. Our approach ---large numbers of a patients own cells. Slide 5 Bone marrow aspirate ---Local anesthesia Skeletal and Related Disease Diseases of CNS Marrow stromal cells ---expanded ---gene engineered Therapy of Same Patient Slide 6 Questions 1. Are we certain that marrow contains stem cells for non-hematopoietic tissues? 2. If so, which cells? 3. If so, how do the cells repair tissues? Slide 7 Questions 1. Are we certain marrow contains stem cells for non-hematopoietic tissues? Conclusive date now from a. differentiation of cells in culture. b. trials in animals. b. patients with bone marrow and organ transplants. (see Prockop et al. PNAS 100(1): 11917- 23, 2003) Slide 8 2. Which Cells from Marrow? 1. Hematopoietic stem cells (CD 34* or CD 133*)? 2. Side population cells ? 3. Multipotential adult progenitor cells ? 4. Mesenchymal stem/marrow stromal cells(MSCs)? OR some still unidentified cell? Slide 9 One Candidate for Reparative Cell from Bone Marrow Isolated by ---plating bone marrow on plastic. Most common names: ---marrow stromal cells ---used as feeder layers for HSCs. ---mesenchymal stem cells ---differentiate into most cell types. COMPROMISE: MSCs. Slide 10 Friedenstein, 1976 Adherent cells (MSCs): ---1:10,000 to 100,000 of nucleated cells. ---can grow rapidly. ---differentiate into bone, fat, cartilage in vitro & in vivo. Slide 11 Easy to Clone & Clones Differentiate ---without Fusion - - Single-cell clones Adipocytes Osteoblasts Chondrocytes +/- Myotubes +/- Neural precursors Slide 12 Appeal of MSCs for Therapy 1. Relatively easy to ---isolate and expand. ---obtain from the same patient. 2. Multipotential for differentiation. 3. Do not form tumors. 4. Tendency to home to sites of tissue injury and repair the tissue. Slide 13 Expansion of MSCs Beginning with 2 ml of bone marrow aspirate ---can obtain over 0.5 billion MSCs --- 2 weeks. Slide 14 Effect of density on expansion Up to 2,000-fold in 12 days. (Colter, DiGirolamo, Prockop, 2000). Slide 15 Several Kinds of Cells 1. Spinde-shaped. 2. Large, flat. 3. Very small, round, rapidly self- renewing (RS cells). Slide 16 Slide 17 Effect of density on expansion But why --- the lag period? --- the stationary phase before the cultures are confluent? Hypothesis ---need to synthesize & secrete a growth stimulant. Slide 18 Days Days 5-7 10-1215-17 Figure 1 Day 0 Counts incorporated and secreted Cell density per cm 2 Cells 35 S- protein C M MSC cultures labeled with 35 S-methioninne. Bars indicated 35 S-protein in cells (C) and media (M). Protein synthesis decreases as cultures reach stationary phase. Slide 19 Autoradiography of conditioned media. C U F L * Silver 6 11 17 31 52 98 185 Day 5-7Day 10-12Day 15-17 Autorad Purified and concentrated 35 kDa extract Newly-synthesized 35 S-proteins secreted by cells are primarily fibronectin (F), laminin (L) and an unidentified protein (*). The unidentified protein was purified from the large amount of fetal calf proteins in the medium. Slide 20 M M A L G A A G A T R V F V A M V A A A L G G H P L L G V S A T L N S V L N S N A I K N L P P P L G G A A G H P G S A V S A A P G I L Y P G G N K Y Q T I D N Y Q P Y P C A E D E E C G T D E Y C A S P T R G G D A G V Q I C L A C R K R R K R C M R H A M C C P G N Y C K N G I C V S S D Q N H F R G E I E E T I T E S F G N D H S T L D G Y S R R T T L S S K M Y H T K G Q E G S V C L R S S D C A S G L C C A R H F W S K I C K P V L K E G Q V C T K H R R K G S H G L E I F Q R C Y C G E G L S C R I Q K D H H Q A S N S S R L H T C Q R H Peptident (TREMBL, SWISSPROT) Trypsin 16 hrs, SELDI TOF = Dickkopf-1, inhibitor of Wnt signaling. Seven tryptic peptides from the 35 kDa protein had the same molecular weights as seven peptides from a previously identified inhibitor of Wnt signaling. Slide 21 . 2000 4000 6000 8000 10000 12000 2000 4000 6000 8000 10000 020406080 020406080 Hours of treatment Cells detected per microtiter well Vehicle 0.01 g mL -1 Dkk-1 Vehicle 0.1 g mL -1 Dkk-1 Recombinant Dkk-1 Decreases the Lag Period in Cultures of MSCs (duplicate assays) +Dkk-1 Control Slide 22 Antibody to Dkk-1 peptide ---prolongs the lag phase. Cell number Days Control + AB Slide 23 Dkk-1: inhibitor of the canonical Wnt pathway Wnt inhibitor, Dkk-1 is expressed in rapidly expanding hMSCs degradation cadherin contact inhibition adherens junctions GSK3 -catenin P - Wnt Dkk-1 LRP 6 Frz Dsh Target genes + TCF + Dkk-1 Slide 24 The unusual growth kinetics of MSCs in culture: a. Cells plated at low density remain in a lag phase ---until they synthesize and secrete a burst of Dkk-1, an inhibitor of Wnt signaling. b. They pass into a stationary phase ---when synthesis of Dkk-1 stops --- they express Wnt-5a, a ligand for positive Wnt signaling. c. On replating the cells the processes is repeated ---for 4 to 6 passages and until the cells pproach senescence. Slide 25 Wnt 5a (-) Dkk-1 (+) Replating at clonal density (4 to 6 Xs) Cells/colony C.Gregory et al. JBC 2003. Slide 26 Dickkopf-1 (Dkk-1) Inhibitor of the canonical Wnt signaling pathway. In most systems inhibition of Wnt stops cell growth. One possibility: Use to culture MSCs. Another: Therapy with Dkk-1 might expand a patients own MSC. Slide 27 Pre-RS cell --slowly replicating --long telomeres RS cells -- rapidly replicating ~transitory amplifying Mature MSC Feeder Layer Osteoblast Chondrocyte Adipocyte Epithelial Neural precursors Other (myo-, endo-, hepato-) The Hierarchy of MSCs- change with culture conditions Slide 28 Question: Can MSCs be expanded for clinical trials? Can produce up to 0.5 billion cells in 2 wks from 2 ml bone marrow. But culture conditions are critical. Variability a serious problem in the field ---Tulane Center just awarded $4.3 million NIH grant to provide standardized preparations to other investigators. Slide 29 Questions 1. Are we certain that marrow contains stem cells for non-hematopoietic tissues? 2. If so, which cells? 3. If so, how do the cells repair tissues? Slide 30 a c a d h j l 12 h 96 h 120 h 12 h 96 h 120 h Small Airway EpithelIum (SAE) GFP+ /MSCs ( X 20) Heat-shocked SAE + GFP+/MSCs (X 40) After the monolayer of epithelial cells is damaged by heat shock, the GFP-labeled MSCs repair the damage by entering the monolayer and assuming the broad flat morphology of epithelial cells. Slide 31 3. How Do MSCs Repair Tissues? Spees et al. PNAS 2003 3 2 1 Rapidly self- renewing cells Mature MSCs (confluent cultures) Example: Epithelial monolayer Injury 1.Differentiation. 2. Cell fusion (?). 3. Growth factors (Wnt 5a, VEGF, PDGF, BDNF, NT-3) MSCs in Culture Slide 32 What are the disease targets? A curious situation ---at the moment it is difficult to exclude any disease ---heart, liver, kidney, metabolic, central nervous system Slide 33 Our Approach A disease with -devastating consequences or predictable downhill course. -without any available therapy -reasonable data from animal models. Slide 34 One Target: Spinal Cord Injury Over 3,000 people in US permanently paralyzed. Healthcare costs up $500,000 per year. No effective therapy. Four labs have promising results in rat models. Several clinical trials with IV therapy with MSCs have not shown toxicity. Slide 35 Rat Model for Spinal Injury MSCs improve ---motor function ---regeneration of axons ---by providing milieu for regeneration ---as they do for hematopoietic stem cells in the marrow. (Hoffstetter et al. PNAS 2002) Slide 36 s Slide 37 Rat Model for Spinal Cord Injury Crush injury in adult rats. Rat MSCs infused 1 week later into the cord. Lift trunkBehavioral test Control 0/10 6.8 +/-0.4 SEM Treated 7/12* 9.2 +/- 0.4 SEM (p=0.013) * 2 took rhythmic steps. Slide 38 hMSCs Express Neurotrophins BDNF NGF NT-3 CNTF FGF-2PDGF-C (-) Slide 39 Our Trial with Spinal Cord Injury Not the first --- but we hope the best ---with carefully standardized MSCs from the patient to be treated. ---international committee to oversee. ---probably multicenter trial. (A useful step toward more common CNS diseases?) Slide 40 For recent review and references see: Prockop, D. J., C. A. Gregory and J. L. Spees. One strategy for cell and gene therapy: Harnessing the power of adult stem cells to repair tissues. Proceedings of the National Academy of Sciences 100 (1): 11917-11923 (2003).