Bone Marrow Stroma in Myelodysplastic Syndromes Universidad de Salamanca Prof Mª M Consuelo del Cañizo Hematology Dept. University Hospital, Salamanca SPAIN
Bone marrow stroma in MDS Introduction Mesenchymal stem cells Mesenchymal stem cells in MDS: Morphological and functional data Genomic features
MDS Characteristics -- Hipercellular bone marrow -- Perypheral cytopenias -- Morphological dyshemopoiesis -- Genetic instability -- Transformation to saml Impaired Hematopoiesis
HPC Stromal cells In hematopoiesis both, HPC as well as stromal BM cells are involved
In MDS pathophysyology various mechanisms are involved Growth factors Epigenetic and genetic mechanisms Apoptosis Monocyte Macrophage HSC: clonal expansion Autocrine mechanism TNF-, TGF- IFN-, IL1-b T Lymphocytes: Immunological disregulation MSC Paracrine mechanism Microenvironment Fibronectine Vessels The role of microenvironment is getting a great deal of interest.
Mesenchymal stem cells They constitute 0.001-0.01% 0.01% of the BM cells. They are the BM stromal progenitors They can be easily isolated and expanded They are key cells for hematopoietic maintenance.
BM microenvironment in MDS 1.Capacity to support hematopoiesis: Discordant results 2.Abnormal secretion of chemo- and cytokine : TNFa; IL6; VEGF IFNg IL32 32, SCF; LIF 3.Abnormal cell balance. Increased microvasculature Increased EPC Increased number of osteoblasts Alvi et al Leuk Res 2001 Tauro et al, Leukemia 2002 Campioni et al. Leuk Res 2004 Borojevic et al. Leuk Res 2004 Marcondes et al. PNAS 2008 Working Hypothesis 1. Hematopoiesis is impaired in MDS at both levels, HPC and BM microenvironment 2. Mesenchymal stem cells are the BM stromal progenitors 3. Could MSCs be functionally and/or genomically abnormal?
Patients and controls Patients. N- 76 M/F: 41 / 35 Age: median 72 (34-92) Controls 36 healthy volunteers M/F: 20 / 16 Age : median 60 (37-88) Cytogenetics N Normal 29 Abnormal 47 5q-: 30 +8: 14
MSCs from MDS: In vitro growth Cell growth: 6/32 non confluent cultures Longer time to reach confluence: Controls 15 d.. (11( 11-30) MDS 23,5 d.. (12( 12-90) 32 80,00 p<0.05 MDS Days DiasPase until passage 60,00 40,00 16 2 9 42 45 20,00 Contol 1,00 2,00 SanoSMD MDS Control
Are these features produced by reduced proliferation or increased apoptosis? G0/G1:97.42 % 1600 1200 800 400 0 0 20 40 60 80 100 Channels (FL2-A-FL2-Area ) Número S: 0.6% G2/M: 1.98% A tendency to higher Cell cycle was similar in both groups A tendency to higher apoptosis was shown by MDS MSCs 7AAD 24% 10 0 10 1 10 2 10 3 10 4 FL3-Height -> 7944.005 AnexinaV
CD 105 Phenotypical data Normal expression for: CD90 90, 73, 166, 106, 54, 56 49a, 49b, 34, 45, 19,14.117, 133, HLA-DR Lower expression of CD105 (endoglin) y CD104 (beta integrin). (p<0.05( 0.05) MDS Control Cell purity >98% CD 104 MDS Control 25,00 1,60 20,00 p<0.05 1,40 1,20 p<0.05 cd105fl1 15,00 10,00 4 fl2 0 1,00 1 d c 0,80 5,00 0,60 0,00 0,40 Control donante diagnostico MDS SMD MDS Control donante Control diagnostico MDS SMD MDS MSCs from MDS patients show an abnormal growth capacity as well as an abnormal expression of some surface molecules
MSCs shows genomic changes?: Analysis using CGH-arrays Expanded MSCs (N=13): 100% showed genomic abnormalities 19p13.3 13.3 38% 11q13.1 13.1 31% 20q13.33 31% 1q31.2 23% 3q11.2 23% 4q22.1 23% Genomic gains were more frequent 7p22 23% 7q21.11 23% 17q25.1 23% 19p13.11 13.11 23% 19q13.33 13.33 23% 21q22.3 22.3 23% Genomic losses 7q11.23 23% BAC RPCI11 11-440G2: 1q31.1212 Genomic changes are not polymorphisms FISH analysis using the same BAC Expanded MSCs show genomic changes CGH Arrays also performed: T- lymphocytes: they are not constitutional changes. HPC: they are not present in HPC
MSC-MDS CGH arrays: Unsupervised analysis Cluster I N WHO 1 RAEB-2 1 RCMD-RS 1 umds Cytogenetics +8 Normal Normal N 5 1 WHO MDS 5q- RA Cluster II Cytogenetics 5q- 4% 5q- 2 RAEB-e Normal 1 1 RARS RAEB-1 +8 5q- Marrow morphological features compatible with 5q- syndrome MSCS from 5q- syndrome are different from those from other MDS
MSC-MDS enrichment by cell sorting Fresh bone marrow samples + Fraction BM Lysis CD45/CD73/ CD34/CD271 Sorter - Fraction Cell purity >99% CD73 CD45 45-/CD34- CD73 CD45 45-/CD34- CD73 CD45 45-/CD34- CD271 Genomic analysis Frequent genomic gains: 1q31, 10q26 y 20q13 MSC are already abnormal before the culture process CD271 CD271 FISH confirmed BAC RP11 11-132L1111 7p13 López-Villar et al, Leukemia 2009
Genes differently expressed by 5q- CGH arrays Unsupervised analysis Chr Genes 1p36.33 TNFRSF18;TNFRSF4 2p25.1 ID2 2p25.1 7p22.1 PDGF ; IEF2AK1 7p22.3 NUDT1 8q24.21 11q12.2 MS4A3 11q13.1 FGF4; PRDX5 16p13.3 IL-32; TRAF7 16q23.1 16q24.3 17p11.2 RASD1 17q25.1 17q25.1 CD300A 17q25.3 EPR1; BIRC5 19p13.11 GDF15 19p13.3 19p13.3 19q13.2 19q13.41 KLK6-14 Angiogenesis Thrombopoiesis Apoptosis Hb synthesis 20q13 RASSF2A 21q22.3 21q22.3
RT-PCR PCR: Overexpression in 5q- Thrombopoiesis Angiogenesis p>0.05 p>0.05 p=0.032 p=0.027 p=0.010 p>0.05 FGF4 Controls 5q- Non5q- Controls 5q- Non 5q- p=0.012 p>0.05 TNF p=0.007 p>0.05 p=0.010 p=0.032 IL32 PDGF Apoptosis Controls 5q- Non 5q- Controls 5q- Non 5q-
5q- Pathophysyology Mir145, mir146: Thrombocytosis Mir145, mir146 SPARC: Clonal advantage for HPC? H P C Platelets Adhesion molecules cytokines Erythrocytes Altered stromal function Adhesion? Cytokines? BM stromal cell RPS14: Erythroid expansion Apoptosis J ar d Apoptosis
5q- Pathophysyology Mir145, mir146: Thrombocytosis Mir145, mir146 SPARC: Clonal advantage for HPC? H P C Platelets Adhesion molecules FGF4 IL32 Erythrocytes Altered stromal function Adhesion? Cytokines? BM stromal cell RPS14: Erythroid expansion Apoptosis Apoptosis
Deletion of Dicer1 specifically in mouse osteoprogenitors disrupts the integrity of haematopoiesis. Therefore, perturbation of specific mesenchymal subsets of stromal cells can disorder differentiation, proliferation and apoptosis of heterologous cells, and disrupt tissue homeostasis. Furthermore, primary stromal dysfunction can result in secondary neoplastic disease, supporting the concept of niche-induced induced oncogenesis.
Universidad de Salamanca