OPTIMIZING MEGAKARYOCYTE POLYPLOIDIZATION INCREASES PROPLATELET FORMATION IN CULTURE MAURO P. AVANZI NYBC PLATELET BIOLOGY LABORATORY
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1 OPTIMIZING MEGAKARYOCYTE POLYPLOIDIZATION INCREASES PROPLATELET FORMATION IN CULTURE MAURO P. AVANZI NYBC PLATELET BIOLOGY LABORATORY
2 LABORATORY EXPANSION OF MEGAKARYOCYTES AND PLATELETS FROM STEM CELLS
3 MEGAKARYOCYTE DEVELOPMENT Reems et al. Trans. Med. Reviews. 2010; 24: 33-43
4 MEGAKARYOCYTE DEVELOPMENT PROPLATELET POLYPLOIDIZATION Reems et al. Trans. Med. Reviews. 2010; 24: 33-43
5 POLYPLOIDIZATION - Increase DNA material without cytokinesis - Ploidy > 8N = Platelet release Mattia et al. Blood. 2002; 99:
6 POLYPLOIDIZATION AGENT NICOTINAMIDE (VIT B3) (Giammona et al. BJH. 2006; 135: ) ZM (AURORA-B INHIBITOR) (Lordier et al. Blood. 2004; 104,4: ) Y27632 (RHO-ROCK INHIBITOR) (Lordier et al. Blood. 2008; 112, 8: ) SU6656 (SRC INHIBITOR) (Gandhi et al. Blood Cells, Mol. and Dis. 2005; 35: 70-74) EFFECT - DECREASE P53 ACTIVITY - INCREASE DNA MATERIAL - INHIBITS MICROTUBULE - MITOTIC SPINDLE FORMATION - INHIBITS ACTIN/MYOSIN - INHIBITS CLEAVAGE FURROW - INHIBITS LYN/FYN - DECREASE MYTOSIS CAPACITY
7 INDUCING MEGAKARYOCYTE POLYPLOIDIZATION Test different reagents to increase megakaryocyte polyploidization Reagents tested: - Nicotinamide (NIC) - Src-inhibitor (SI) - Rho-Rock inhibitor (RRI) - Aurora-B inhibitor (ABI) - Myosin light chain kinase inhibitor (MLCKI) Compare single reagents and combinations
8 INDUCING MEGAKARYOCYTE POLYPLOIDIZATION Methods: Umbilical cord blood derived stem cells (CD34+) Medium: Stemspan Sfem (37ºC; 5% CO 2 ) Cytokines: - SCF 5ng/ml: D1 D8 - TPO 50ng/ml: D1 D11 Polyploidization reagents added on day 8 of culture Ploidy analysis on day 11 with flow cytometry (Propidium Iodide)
9 POLYPLOIDIZATION Results: Comparison between single reagents: CONTROL RRI NIC ABI SI MLCKI Avanzi et al. Transfusion May 21 (Epub ahead of print)
10 POLYPLOIDIZATION Comparison between combinations: Avanzi et al. Transfusion May 21 (Epub ahead of print)
11 SIGNALING PATHWAYS Avanzi et al. Transfusion May 21 (Epub ahead of print)
12 POLYPLOID MEGAKARYOCYTES RRI TREATED UNTREATED RRI TREATED (MICROSCOPE: x 40)
13 POLYPLOID MEGAKARYOCYTES RRI TREATED Avanzi et al. Transfusion May 21 (Epub ahead of print)
14 MEGAKARYOCYTE ULTRA-STRUCTURE RRI TREATED DMS α GRANULE DENSE GRANULE
15 DEMARCATION MEMBRANE SYSTEM (DMS) Stained DMS (Di-8 Anepps) with fluorescent microscopy
16 DEMARCATION MEMBRANE SYSTEM (DMS) Quantified and compared DMS using flow cytometry
17 DEMARCATION MEMBRANE SYSTEM (DMS) High ploidy megakaryocytes have extended DMS
18 PROPLATELET FORMATION (PPF) Patel et al. Journal of Clinical Investigations :
19 STIMULATING PROPLATELET FORMATION AGENT - FLOW / SHEAR STRESS ( Dunois-Larde et al. Blood. 2009;114: ) - FIBRINOGEN ( Balduini et al. Journ of Thromb. and Haemost. 2008;6:1900-7)
20 STIMULATING PROPLATELET FORMATION AGENT FLOW / SHEAR STRESS (Dunois-Larde et al. Blood. 2009;114: ) Experiment: Rocker inside incubator: 15º / 6 rpm (16hs) - FIBRINOGEN (Balduini et al. Journ of Thromb. and Haemost. 2008;6:1900-7) Experiment: Plates coated with Fibrinogen 100ng/ml (16hs)
21 PROPLATELET FORMATION: REAGENTS X STIMULATIONS CTRL SI NIC ABI RRI RRI NIC RRI SI RRI SI NIC NIC SI ABI MLCKI FBNG ROCK FBGN ROCK NONE CTRL=Control; SI=Src-inhibitor; NIC=Nicotinamide; ABI=Aurora-B inhibitor; RRI=Rho-Rock inhibitor; MLCKI=Myosin Light Chain Kinease inhibitor; FBNG=Fibrinogen
22 PROPLATELET FORMATION CONTROL TREATED
23 HOW TO COUNT PROPLATELETS?
24 GRID COUNTING: PROPLATELET SEGMENTS
25 GRID COUNTING: PROPLATELET SEGMENTS
26 PROPLATELET FORMATION: REAGENTS X STIMULATIONS CTRL SI NIC ABI RRI RRI NIC RRI SI RRI SI NIC NIC SI ABI MLCKI FBNG ROCK FBGN ROCK NONE Average number of PPF segments/treatment CTRL=Control; SI=Src-inhibitor; NIC=Nicotinamide; ABI=Aurora-B inhibitor; RRI=Rho-Rock inhibitor; MLCKI=Myosin Light Chain Kinase inhibitor; FBNG=Fibrinogen
27 PROPLATELET FORMATION: REAGENTS X STIMULATIONS CTRL SI NIC ABI RRI RRI NIC RRI SI RRI SI NIC NIC SI ABI MLCKI FBNG ROCK FBGN ROCK NONE Average number of PPF segments/treatment CTRL=Control; SI=Src-inhibitor; NIC=Nicotinamide; ABI=Aurora-B inhibitor; RRI=Rho-Rock inhibitor; MLCKI=Myosin Light Chain Kinase inhibitor; FBNG=Fibrinogen
28 PROPLATELET FORMATION: REAGENTS X STIMULATIONS CTRL SI NIC ABI RRI RRI NIC RRI SI RRI SI NIC NIC SI ABI MLCKI FBNG ROCK FBGN ROCK NONE Average number of PPF segments/treatment CTRL=Control; SI=Src-inhibitor; NIC=Nicotinamide; ABI=Aurora-B inhibitor; RRI=Rho-Rock inhibitor; MLCKI=Myosin Light Chain Kinase inhibitor; FBNG=Fibrinogen
29 PROPLATELET FORMATION (PPF) SEGMENTS
30 PROPLATELET FORMATION (PPF) SEGMENTS
31 PROPLATELET FORMATION: COMPARING DIFFERENT STIMULATIONS
32 PROPLATELET FORMATION STRUCTURE (αiibβiii) RRI+NIC
33 PROPLATELET FORMATION STRUCTURE (αiibβiii) RRI+NIC
34 PROPLATELET FORMATION CYTOSKELETON (β TUBULIN) RRI+NIC
35 SUMMARY Rho-Rock inhibition resulted in the highest final ploidy High level of polyploidization correlated with extended DMS High ploidy megakaryocytes released more PPF segments NIC increased PPF in RRI treated megakaryocytes Association of NIC+RRI released the highest number of PPF No additional effect of shear stress or fibrinogen in PPF
36 ACKNOWLEDGMENT NYBC PLATELET BIOLOGY LABORATORY - W. Beau Mitchell - Amanda Chen - Jennifer Davila - Francine Goldberg NYBC / LINDSLEY F. KIMBALL RESEARCH INSTITUTE - Mohandas Narla - Wu He - Vijay Nandy - Aaron Bell NYBC / NATIONAL CORD BLOOD PROGRAM WEILL-CORNELL MEDICAL COLLEGE (Cellular Therapy Laboratory) - Melissa Cushing - Ellery Moore-Mendez F.M. KIRBY FOUNDATION
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