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How does the body repair itself?

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. How does the body repair itself?. Cohnheim (1867):

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How does the body repair itself?

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  1. One Strategy for Gene/Cell Therapy: Harness the Natural Ability of Adult Stem Cells to Repair TissuesDarwin J. Prockop, M.D., Ph.D.,Director, Center for Gene Therapy,Tulane University Health Sciences Center,New Orleans, LA

  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

  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.

  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 patient’s own cells.

  5. Marrow stromal cells ---expanded ---gene engineered Bone marrow aspirate ---Local anesthesia Therapy of Same Patient Skeletal and Related Disease Diseases of CNS

  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?

  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)

  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?

  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”.

  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.

  11. Easy to Clone & ClonesDifferentiate---without Fusion +/- Myotubes +/- Neural precursors Single-cell clones - - Chondrocytes Osteoblasts Adipocytes

  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.

  13. Expansion of MSCs Beginning with 2 ml of bone marrow aspirate ---can obtain over 0.5 billion MSCs --- 2 weeks.

  14. Effect of density on expansion Up to 2,000-fold in 12 days. (Colter, DiGirolamo, Prockop, 2000).

  15. Several Kinds of Cells 1. Spinde-shaped. 2. Large, flat. 3. Very small, round, “rapidly self- renewing” (RS cells).

  16. 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.

  17. Figure 1 M Cells Counts incorporated and secreted 35S- protein C Cell density per cm2 Day 0 10-12 15-17 Days 5-7 Days MSC cultures labeled with 35S-methioninne. Bars indicated 35S-protein in cells (C) and media (M). Protein synthesis decreases as cultures reach stationary phase.

  18. Autoradiography of conditioned media. Day 5-7 Day 10-12 Day 15-17 185 98 52 31 17 11 6 Autorad C U F L Purified and concentrated 35 kDa extract * Silver Newly-synthesized 35S-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.

  19. Trypsin 16 hrs, SELDI TOF Peptident (TREMBL, SWISSPROT) 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 RK 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 GL E I F Q RC 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 = 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.

  20. Recombinant Dkk-1 Decreases the Lag Period in Cultures of MSCs (duplicate assays) . 10000 12000 +Dkk-1 10000 8000 8000 6000 Cells detected per microtiter well 6000 Control Cells detected per microtiter well 4000 4000 2000 2000 Vehicle 0.01 mg mL-1 Dkk-1 Vehicle 0.1 mg mL-1 Dkk-1 0 20 40 60 80 0 20 40 60 80 Hours of treatment Hours of treatment

  21. Antibody to Dkk-1 peptide---prolongs the lag phase. Cell number Control + AB Days

  22. Wnt inhibitor, Dkk-1 is expressed in rapidly expanding hMSCs Dkk-1: inhibitor of the canonical Wnt pathway Dkk-1 Dkk-1 Wnt LRP 6 Frz Dsh LRP 6 - contact inhibition adherens junctions GSK3 + cadherin P b-catenin b-catenin degradation + Target genes TCF

  23. 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.

  24. C.Gregory et al. JBC 2003. Wnt 5a (-) Dkk-1 (+) Replating at clonal density (4 to 6 Xs) Cells/colony

  25. 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 patient’s own MSC.

  26. The Hierarchy of MSCs-change with culture conditions Pre-RS cell --slowly replicating --long telomeres RS cells -- rapidly replicating “~transitory amplifying” Mature MSC Other (myo-, endo-, hepato-) Osteoblast Neural precursors Chondrocyte Adipocyte Feeder Layer Epithelial

  27. 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.

  28. 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?

  29. Small Airway EpithelIum (SAE) Heat-shocked SAE + GFP+/MSCs a h 12 h 12 h c d j a GFP+ /MSCs 96 h 96 h l (X 20) 120 h 120 h (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.

  30. 2 3 3. How Do MSCs Repair Tissues?Spees et al. PNAS 2003 Example: Epithelial monolayer MSCs in Culture Injury Rapidly self- renewing cells 1.Differentiation. 2. Cell fusion (?). 3. Growth factors (Wnt 5a, VEGF, PDGF, BDNF, NT-3) 1 Mature MSCs (confluent cultures)

  31. 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

  32. Our Approach A disease with -devastating consequences or predictable downhill course. -without any available therapy -reasonable data from animal models.

  33. 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.

  34. 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)

  35. s

  36. Rat Model for Spinal Cord Injury Crush injury in adult rats. Rat MSCs infused 1 week later into the cord. Lift trunk Behavioral test Control 0/10 6.8 +/-0.4 SEM Treated 7/12* 9.2 +/- 0.4 SEM (p=0.013) * 2 took rhythmic steps.

  37. hMSCs Express Neurotrophins (-) (-) NT-3 (-) (-) NGF CNTF BDNF FGF-2 (-) PDGF-C (-)

  38. 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?)

  39. 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).

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