Bone Marrow Derived Mesenchymal Stem Cells: Biological Characteristics and Perspectives in Clinical Applications

Transcription

1 Bone Marrow Derived Mesenchymal Stem Cells: Biological Characteristics and Perspectives in Clinical Applications

2 Bone marrow haematopoietic stem cells mesenchymal stem cells Stem Cells: Scientific Progress and Future Research Directions. Department of Health and Human Services. June /info/scireport.

3 Bone Marrow Derived Mesenchymal Stem Cells (MSCs) Easily harvested Great proliferative capacity Great differentiation potential

4 Advantages: MSCS: CLINICAL APPLICATION easily harvested and expanded in vitro autologous source no tumorigenic activity no ethic problems Great differentiation capacity observed in vitro : Transdifferentiation towards neural lineages Spontaneous expression of neural markers

5 Human Mesenchymal Stem Cells: Isolation and culture MSCs were isolated from BM samples (San Gerardo Hospital) MSCs OSTEOGENIC DIFFERENTIATION RIGENERAZIONE DEI TESSUTI CRANIOFACCIALI

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7 hmscs: Phenotipic characterization hmsc p3; bar 50µm hmsc present: Fibroblasts-like morphology, round shape, prominent nucleus FACS analysis: positive for CD90, CD105 e HLA-ABC ( 70%) negative CD33, CD34 e HLA-DR ( 5%). Literature description of MSCs

8 hmscs: Differentiation potential OSTEOGENIC differentiation CHONDROGENIC differentiation ADIPOCITIC differentiation ctrl OSmed Alizarin red S Safrarin O Oil red O Immunofluorescence for osteopontin H&E Bar 25µm

9 SEM-EDX: Calcium e Phosphorous colocalization SEM map Ca P map 14 OS medium 28 OS medium 35 OS medium Progressive increase of Ca and P in presence of OS medium Ca e P colocalize presence of Calcium phosphates

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12 Cytogenetic analysis of rat and human MSCs

13 Cytogenetic analysis: rat MSCs Karyotype Normal, female 42,XX Karyotype Normal, female 42,XX

14 Cytogenetics analysis: rat MSCs with numerical abnormalities CROMOSOME 13 TRISOMY (arrows) Karyotype, 43,XX,+13

15 Cytogenetics analysis: rat MSCs with structural abnormalities DERIVATIVE der(2;15) (arrow) Karyotype 42,XX,der(2;15)

16 hmscs do not show alteration until passage 10 46,XX hmsc pass.10

17 Transdifferentiation of MSCs in neurons

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19 Evaluation of rat MSCs neuronal induction protocols : 1) ß-MERCAPTOETHANOL (Woodburry et al, 2000) 2) RETINOIC ACID (RA) + bfgf (Kim et al, 2002) 3) NEUROBASAL MEDIUM + RA + BDNF (Sanchez-Ramos et al, 2002) 4) NEUROBASAL MEDIUM + RA + BDNF + Valproic Acid + Forskolin Morphological analysis Neural markers expression by immunofluorescence experiments

20 Protocol 1 (ß-MERCAPTOETHANOL ) e 2 (RA + RA + bfgf): PI Actin NeuN Merge neuronal-like processes no expression of NeuN

21 Protocol 3 (NBM + RA + BDNF) e 4 (NBM + RA +BDNF+Valproic ACID+Forskolin): Tuj1 NeuN S100 GFAP neuronal-like processe expression of neuronal and glial markers

22 CONCLUSIONS: Protocols 1 and 2 did not induce MSCs transdifferentation in neuronal cells neuron-like processes are probably due to the toxic action of differentiative agents Neurobasal medium, AR e BDNF induce the expression of neuronal marker NeuN and glial markers (GFAP/S100) TRANSDIFFERENTIATION OFTEN DEPENDS ON EXPERIMENTAL CONDITIONS

23 Can MSCs express spontaneously neural markers?

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25 Can undifferentiatied MSCs without any differentiative agent,, express neural markers spontaneously? Correlation with passage? With time in culture?

26 Passages: P0 P1 P2 P4 P8 P16 P24 P40 Time in culture: days. MARKERS : mesenchymals: osteopontin, osteocalcin, PPARγ2 neuronals: TUJ1, NEUN, NEUROFILAMENT glials: GFAP, S100 NESTIN, progenitor cells marker

27 RESULTS : Nestin is expressed both at earlier and late passages. Tuj1 e GFAP are expressed at least until p16. NeuN is expressed at the earlier passages p0, p1 e p2, and late it decreases The marker expression is strongly reduced or disappeare at p40.

28 NESTIN/TUJ1 NESTIN/GFAP NESTIN/NEUN p p p40 Barra: 50µm

29 Our results suggest the presence of 3 populations: 1) Nestin positive cells. 2) Cells positive for nestin and neural markers. 3) Cells positive only for neural markers. HYPOTHESIS: Nestin positive cells: wide differentiation potential Cells positive for nestin and neural markers: neural committed. Cells positive only for neural markers: earlier stage of neural differentiation. Controversal observations reported in literature may be due to: - experimental conditions - MSCs with different differentiation capacity

30 MSCs effect on post-mitotic sensory DRG neurons from rat embryos Scuteri et al., Brain Research 2006

31 Co-cultures of neurons and MSCs: Neurons Day 0 Day 60 Day 30

32 Co-cultures of neurons and MSCs: after 30 days... neuritic degeneration cellular death MSCs Neurons Neurons primary culture Co-culture of neurons and MSCs MSCs promote long lasting survival of neurons and their differentation AFTER DIRECT CONTACT

33 Analys with Microarray Low Density OligoGEArray specific for Adhesion Molecules and ECM proteins MSCs 30days Co-cultures 30 days Neurons 30days

34 Modyfied molecules: Down-regulated in co-cultures MMP-14 (metalloprotease able to activate MMP pathway) Sparc (antiadhesive protein which activates MMP pathway) Up-regulated in co-cultures Timp-1 (MMP inhibitor)

35 Timp-1: MMP inhibitor Neu 30days Co-cultures Timp-1 is present only in co-cultured neurons Timp-1 MAP-2 Falloidin

36 Sparc: antiadhesive protein able to activate MMP Neu 30days Co-cultures Sparc is present only in neurons alone Falloidin Sparc Map2

37 NNGH: metalloproteases wide range inhibitor % survival ctrl nngh co-colture 20 0 T0 7 gg 14 gg 21 gg 30 gg

38 CONCLUSIONS MSCs promote the long lasting survival of DRG neurons and their differentiation; The effect is not due to neurotrophic factor release but DIRECT CONTACT IS MANDATORY; MSCs induce the inhibition of metalloproteases pathway in neurons

39 MSCs effect on Cisplatintreated Dorsal Root Ganglia Scuteri et al., Neurosci lett. 2008

40 MSCs and DRG treated with Cisplatin (CDDP): CTRL CDDP CDDP is NEUTOXIC it causes a reduction of neurite length with respect to control MSCs does not restore the correct neurite length CDDP+MSCs

41 MSCs and CDDP-treated DRG: viability CDDP2 + MSCs CDDP2 Evaluation of death area MSCs increase the survival of CDDP-treated DRG

42 Si ringraziano: DNTB sezione di Genetica Leda Dalprà Angela Bentivegna Serena Redaelli DNTB- Clinica Odontoiatrica Marco Baldoni Fabrizio Carini Riccardo Monguzzi Alberto Baldini DNTB- Medicina Clinica Enrico Pogliani Daniela Belotti Laboratorio di Terapia Cellulare e Genica S. Verri Andrea Biondi Ettore Biagi Giuseppe Gaipa DNTB sezione di Morfologia Mariarosaria Miloso Elisabetta Donzelli Mario Bossi Virginia Rodriguez-Mendez Arianna Cassetti Dana Foudah Daniele Maggioni Maddalena Ravasi Silvia Pasini Arianna Scuteri Roberta Rigolio Mariele Viganò Pirelli Labs Antonio Zaopo Paolo Mimo Michele Morrone