PLACENTA UMANA da materiale di scarto biologico.. verso il loro utilizzo per la Terapia Rigenerativa

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1 PLACENTA UMANA da materiale di scarto biologico.. verso il loro utilizzo per la Terapia Rigenerativa Ornella Parolini Centro di Ricerca E.Menni Fondazione Poliambulanza Brescia

2 Amnion Smooth chorion False knot Fetal surface of placenta Umbilical cord 1

3 Why an interest in human placenta? Identify stem cells for cell therapy approaches: Stem cell potential No transplant rejection Placenta may combine these two essential features on the basis of: Embryological origin Immunological characteristics

4 Embryological Origin

5 FETAL MATERNAL TOLERANCE: Pregnancy is a unique event in which a genetically and immunologically foreign fetus survives to full term without rejection by the mother's immune system. Uterus wall trophoblast Fetal membranes Umbilical cord

6 From Placenta:. FRAGMENTS of tissue: Amnioticmembrane Cordfragments CELLS: from amnioticmembrane, chorionicmembrane, cord, Wharton Jelly, cord blood Media derivedfrom culture of placenta derived cells

7 Amniotic derived cells isolation Amnion

8 Reflected fetal membranes Amnion Amnion Chorion Basal Membrane Amnion Epithelium Compact Layer Cellular Layer Spongy Layer 400 x EE Amniotic Epithelial Cell Amniotic Mesenchymal Cell

9 Minimal criteria for defining hamsc Adherence to plastic Formation of fibroblast colony-forming units A specific pattern of surface antigen expression (At passages2-4) Positive (>95%) CD 90 CD 73 CD 105 Negative (<2%) CD 45 CD 34 CD 14 HLA-DR Othermesenchymaland hematopoieticmarkers: CD44+, CD29+,HLA-A,B,C+, CD13+, CD10+, CD49c+, CD49d+, CD49e+, CD54+, CD166+,CD271 low, CD31-, CD133-, Differentiationpotentialtowardoneor more lineages, includingosteogenic, adipogenic, chondrogenic and vascular/endothelial Fetal origin Parolini O. Stem Cells 2008

10 Differentiation potential of hamsc and hcmsc Osteogenic Chondrogenic Control medium Soncini M et al. TERM 2007 Adipogenic

11 IMMUNOMODULATORY FEATURES OF AMNIOTIC DERIVED CELLS

12 A *** cpm B 0 R R+S* R+A* ** ** R: responder (hpbmnc) S*: stimulator (hpbmnc irradiated cells) A: Amnion (irradiated cells) ppbmnc: porcine PBMNC cpm * * * * 0 ppbmc Pig a Pig+hPBMC* ppbmc ppbmc Pig+hA* Pig+hC* ppbmc (CD45 - Gly - ) hpbmc* ha* hc(cd45-glya-)* Transplantation, 2004

13 1. AMSC inhibit lymphocyte proliferation PBMC + CD3/CD28 PBMC + CD3/CD28 + AMSC PBMC+ allo PBMC* + AMSC Transient Inhibition 120 %% proliferation proliferation PBMC+ allo PBMC* R + S1* Magatti M, et al. Stem Cells 2008 R + S1* + AMC Re + S1* Re + S2*

14 In vitro differentiation of monocyte-derived dendritic cells (DC): monocytes GM-CSF + IL-4 [3-5 days] maturation immature-dc (idc) LPS [2 days] mature-dc (mdc)

15 AMSC inhibit monocyte differentiation toward dendritic cells and affect ability of DC to induce T cell proliferation monocyte CD1a CD14 mdc CD1a CD14 [AMSC-mDC*] CD1a CD ** *** ** monocytes idc [idc-amsc] mdc [mdc-amsc] Magatti M, et al. Cell Transplantation 2009

16 Immunological characteristics Amniotic derived cells don t induce T cell proliferation, except when used at a low concentration AM C Amniotic membrane MSC inhibit lymphocyte proliferation The lymphocyte inhibition is mediated by soluble factor(s) T cells The inhibition is reversible Amniotic membrane MSC inhibit the differentiation from monocytes to dendritic cells monocytes DC

17 In Vivo studies: AM derivatives (cells, membranes, conditioned medium..) transplantation Neonatal mice and neonatal swines Mouse model Pulmonary fibrosis in a (bleomycin-induced lung fibrosis) Rat model Myocardial ischemia (Coronary artery ligation) Rat animal model Liver fibrosis (Bile duct ligation)

18 Bleomycin fibrosis induced in a mouse model 34 C57BL/6 mice BLEO ALLO-TRANSPLANTATION AMNION CELLS C57BL/6 mice BLEO BALB/c mice Balb/c GFP mice C57BL/6 mice BLEO XENO-TRANSPLANTATION From HUMAN PLACENTA C57BL/6 mice BLEO (Cargnoni A. et al. Cell Transplantation 2009

19 Damaged cells secrete Bleomycin growth intratracheal factors and chemokines which Pulmonary alveolar stimulate Bleomycin inflammatory is a chemotherapeutic injectioncell recruitment agent that ( thickening produces alveolar of structure interalveolar epithelial cell spaces) damage through DNA strand breakageby Reactive Oxygen Radicals. Type I Pneumocytes Myofibroblasts are the major responsible of extracellular matrix deposition leading to Inflammatory cells produce pro-fibrotic cytokines (TGF-β, IL-13) which stimulate fibroblast migration, formation of patchy pulmonary fibrosis ( alveolar occlusion) proliferation and fibroblast activation to myofibroblasts (α-sma positive cells) Capillaries Collagen deposition Fibroblasts Type II Pneumocytes Alveolar Macrophages BLEO Bronchial Cells

20 Bronchi * Alveolar Spaces Vessels * * * interstitium * *

21 * * Alveolar Spaces * interstitium * * * Bronchi Vessels

22 Bronchi Vessels interstitium

23 Semiquantitative Modified Hagood System for Fibrosis Determination (Hagood et al., 2005) Fibrosis Extent 1: 0-25% 2: 26-50% 3: 51-75% 4: 76%-100% X Alveolar Obliteration (1-2-3)* + Fibroblast proliferation (1-2-3)* + Collagen Deposition (1-2-3)* = Fibrosis Score 400X 400X 400X 400X 400X * 1=Mild 2=Moderate 3=Severe Centro di Ricerca E. Menni

24 100 Fibrosis extent (% of area involved) Allo-transplantation 5 day 14 n=19 4 n=8 * 3 n=7 ** 2 * 1 (score units) n=3 n=3 Fibrosis severity 0 Bleo Bleo+Cells Cells IP route IT route IP route IT route 1 Cargnoni et al, Cell Transplantation 2009

25 100 Fibrosis extent (% of area involved) 5 day Xeno-transplantation n=19 ** * n=8 n=7 Fibrosis severity (score units) 0 Bleo Bleo+Cells Cells IP route IT route IP route IT route n=4 n=3 Cargnoni et al, Cell Transplantation 2009

26 Xeno-transplantation a1 a2 a3 a4 Intra-peritoneal delivery Intra-tracheal delivery Allo-transplantation Cargnoni et al, Cell Transplantation 2009

27 Which disease animal models have we studied? Pulmonary fibrosis (Cargnoni A. et al.; Cell Transplantation, 2009) Myocardial ischemia (Cargnoni A. et al.; Cell Transplantation 2009) Amniotic membrane-derived cell transplantation significantly reduced bleomycin-induced lung fibrosis Amniotic membrane application significantly improved cardiac functions in ischemic rat hearts for at least 2 months post-injury Liver fibrosis (Sant`Anna Barros L. et al.; Cell Transplantation 2010) Amniotic membrane application significantly reduced liver fibrosis induced in rats by BDL

28 General considerations from our data: In all disease models studied, placenta-derived cells and amniotic membrane exerted anti-fibrotic effects No appreciable cell engraftment was observed in any of the target organs of cell transplantation /amniotic membrane application Soluble factors, rather than cells per sé, may represent the true actors

29 Could conditioned medium (CM) derived from amniotic cell culture replace placenta-derived cell therapy or amniotic membrane application? Bleomycin Bleomycin + Control Medium Bleomycin + AM-Conditioned Medium

30

31

32 REGENERATION versus REPAIR In vivo studies demonstrate mainly the ability of amniotic cells/amniotic membrane to modify NEW WAY the TO environment CONSIDER CELL and exert THERAPY? paracrine effects that improve local surrounding tissue, favouring repair by the host cells

33 IPLASS aims to become a worldwide networking platform to promote Research on all aspects concerning knowledge, experimentation and clinical use of placenta-derived cells Committee Members: Ornella Parolini (ITALY) President Cesar Borlongan (USA) Vice President Marco Evangelista (Australia) Secretary Heinz Redl (Austria) Bing Liu (China) Steffen Zeisberger (Switzerland) Susanne Wolbank (Austria) Stephen Strom (USA) Sankar Venkatachalam (India) Marta Magatti (Italy) Ursula Manuelpillai (Australia)

34 Placenta, The Tree of Life

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