Medicina Regenerativa y Pediatría Pasado, Presente y Futuro · Juan R Tejedo Benoit...
Transcript of Medicina Regenerativa y Pediatría Pasado, Presente y Futuro · Juan R Tejedo Benoit...
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Seville (Spain)Cartuja 93- Scientific and Technological Parkwww.cabimer.es
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Colaboradores
CABIMER- Investigadores:
Karim HmadchaAnabel RojasFranz MartínFrancisco J BedoyaJuan R TejedoBenoit Gauthier
-PostdocsChristian Lachaud
MACARENA-SAN LÁZARO
Antonio de la CuestaRafael Ruiz SalmerónInma Pérez Camacho
VALMEManuel RomeroChristian Lachaud
- DoctorandosDaniella PezzollaIrene DelgadoManuel Carrasco
CNIOMario FragaManel Esteller
VIRGEN DEL ROCÍORocio García CarboneroMiguel Marín
VISSUMJorge Alió
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Medicina Regenerativa y Pediatría:
¿Sinónimos?
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- Stem Cells and Regenerative Medicine
- Cellular Medicaments: a Change in the Paradigm
- Cell Therapy in Pediatric - Cell Therapy in Pediatric Diseases
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- Stem Cells and Regenerative Medicine
- Cellular Medicaments: a Change in the Paradigm
- Cell Therapy of Liver Diseases
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Stem Cell ConceptStem Cell Concept
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Células Madre (Troncales)
a. Autorenovaciónb. Diferenciación hacia otros tipos celularesc. Capacidad para colonizar y regenerar un
tejido
Célula progenitora
Célula diferenciada
Aut
oren
ovac
ión
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Utilidad de las
Células Madre
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“Células ESC Humanas”
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Día 2Día 0 Día 1
Día 6-7Día 5Día 3
Methylation Low
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Masa Celular Interna: baja silenciación de genes
(ICM)
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H181
H293
ESI-Sing
Human ES
Human ES
HumanES
H181 Human ES
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Células iPS
Yamanaka (2006) KyotoCell 126: 663-676
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Adult Stem Cells
Mesenquimales- Médula Ósea- Tejido adiposo- Placenta- Endometrio- Pulpa dental, etc
AdMSC Osteogenesis
Mesoteliales
Sustitución delEndotelio corneal
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Modificaciones EpigenéticasModificaciones Epigenéticas
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Goldengate methylation arrays : 1505 sequences / 807 genesGoldengate methylation arrays : 1505 sequences / 807 genes
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hESC Somatic Cancer Genes
A-I - - + Early Differentiation
A-II +/- +/- + Tissue Specification
B-I + - + Early Differentiation (*)
Cancer Hypermethylated Genes
+ - + Differentiation (*)
B-II + +/- + Tissue Specification
(*) Proposed model for“aberrant methylationof cancer genes”
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Epigenetic Drugs
α-actininESCs
Epigenetic DrugsCH3
DNMT
CH3
X
CH3
DNMT
Lineage-specific genes
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- Stem Cells and Regenerative Medicine
- Cellular Medicaments: a Change in the Paradigm in the Paradigm
- Cell Therapy in Pediatric Diseases
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Cell Therapy of Diabetic Complications
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GMP Cell Production Unit: GMP-CABIMER :
- First GMP Unit Certified by AEMPS in Andalucia ( 6th Nov 2009)- Two Production Units- Four Clinical Trials On-going (Diabetic foot and Múltiple Sclerosis)
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Mononuclear Fraction (Ficoll) +
Cytometry CD34+ (CABIMER)
Bone marrow
Intraarterial Infusion 113,7-434,5 106 cells
Angiography
Clinics
MetaMorph (CABIMER)
Cell Transplantation 20(10) 1629-1639 (2011)
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1.- CLINICS (reported bypatients): 0-24 months
1.1 Temperature sensation increase1.2 Pain decreases1.3 Increase walking distance1.4 Gastrocnemius perimeter increases
2.- ABI Increases
Cell Transplantation 20(10) 1629-1639 (2011)
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3.- ULCERS U.TEXAS Classification(Lavery LA et al. J. Foot. Ankle. Surg. 35: 528-31. 1996)
4.- CIL EVOLUTION4.- CIL EVOLUTIONRutherford-Becker Scale (Rutherford RB et al. J. Vasc. Surg. 26:517-38. 1997)
Cell Transplantation 20(10) 1629-1639 (2011)
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5.- ANGIOGRAPHY (Metamorph)
Cell Transplantation 20(10) 1629-1639 (2011)
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UNDERSTANDING “CELLULAR MEDICAMENTS”
Small Molecules(1600 …- XXth century)
Biologicals (mAb , proteins )Biologicals (mAb , proteins )(1922: insulin; mAb 80´s)
Cells(2000 -…)
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USE OF MESENCHYMAL STEM CELLS IN THE TREATMENT OF T HE CRITICAL ISCHAEMIA OF THE LIMBBACKGROUND
UNDERSTANDING “CELLULAR MEDICAMENTS”
1. MSC promote fibrinolysis (Craig K et al, 2007 Antithrombogenicproperties of BM-MSC … PNAS 29: 11915-20)
2. T2 Diabetes Mellitus patients present prothrombotic state (Knudsen EC et al. 2011 J Thromb Haemost and Ay L et al. 2011 Eur J Clin Invest)
.
Acosta et al (2013) Adipose mesenchymal stromal cells isolated from type 2 diabetic patientsdisplay reduced fibrinolytic activity. Diabetes. 2013 Sep 16. [Epub ahead of print]
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But, in 2010 we observed one adverse effects in
USE OF MESENCHYMAL STEM CELLS IN THE TREATMENT OF T HE CRITICAL ISCHAEMIA OF THE LIMB
UNDERSTANDING “CELLULAR MEDICAMENTS”
But, in 2010 we observed one adverse effects in two patients: MICROTHROMBOSIS that could be reversed by aggressive thrombolytic therapy
Acosta et al (2013) Adipose mesenchymal stromal cells isolated from type 2 diabetic patientsdisplay reduced fibrinolytic activity. Diabetes. 2013 Sep 16. [Epub ahead of print]
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UNDERSTANDING “CELLULAR MEDICAMENTS”
.
Acosta et al (2013) Adipose mesenchymal stromal cells isolated from type 2 diabetic patientsdisplay reduced fibrinolytic activity. Diabetes. 2013 Sep 16. [Epub ahead of print]
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FIBRINOLYSIS
Plasminogen Activators
Plasminogen
tPA
Fibrinolysis and CoagulationFibrinolysis and Coagulation
PlasminXX
PAI-1
Degradation D-dimer
COAGULATION +++
FibrinFibrinogen
Prothrombin + AntithrombinThrombin
tPA: tissue Plasminogen ActivatorPAI-1: Plasminogen Activator Inhibitor
X
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↑PAI-1, ↓tPA, ↓ D-dimer
Control Serum
CIL-NDSerum
CIL-DSerum
AdMSC-C + + ++
Ad-MSC-ND + + ++
AdMSC-D ++ ++ +++
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SC Break the vicious circle and reset the situation
A
Virtouscircle
+SC
B
Blood/TissueFactors
SC ++
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- Stem Cells and Regenerative Medicine
- Cellular Medicaments: a Change in the Paradigm
- Cell Therapy in Pediatric Diseases
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Cell Therapy in Pediatric Diseases
NOT INCLUDED- Hematopoietic stem cell transplantatio n: bone marrow, cord blood
1. Type 1 Diabetes Mellitus1. Type 1 Diabetes Mellitus2. Child Autoimmune Diseases3. Liver Diseases4. Osteogenesis Imperfecta5. Muscular Dystrophies6. Neurological and Neurodegenerative Disorders7. Future
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Type 1 DM
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Qué tienen estas personas en común …
DIABETES
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Transfection
+ Hygromycin
HygroHygro rr
ββββ-geo pGK/Hygro rHIP
e1
differentiationIn vitroHygroHygro rr
Cells Cells
+ NeomycinNeoNeorr
CellsCells
differentiationIn vitro
Diabetes 49: 157-162 (2000)Differentiation 68: 4-5 (2001) Diabetologia 44: 407-415 (2001)Diabetologia 47: 1442-1451 (2004)Stem Cells 24(2): 258-265 (2006)Exp Cell Res 314: 969-974 (2008)
Nº citas: 1.052 (Oct 2013)
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Insulin-producing cells normalize glycaemia in STZ-diabetic mice
Diabetes 49: 157-162 (2000)Diabetologia 47: 1442-1451 (2004)Stem Cells 24(2): 258-265 (2006)Exp Cell Res 314: 969-974 (2008)
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Insulin secreting cellsfromfrom
human ESC and iPSC
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Design of new differentiation protocols(hESC/iPSC)
ES ME DE PG PF PE EP BC
Wnt3a RA Nicotinam.
Wnt
PI3K
Nodal Nodal
PI3K
Shh Notch┴ (*)
(*)
Sirt-1(*)
ES: embryonic stem cells; ME: mesendoderm; DE: definitive endoderm; PG: primitive gut; PF: posterior foregut;
PE: pancreatic endoderm; EP: endocrine precursors; BC: beta cells;
DAPT: N-[N-(3,5-difluorophenacetyl)-L-alanyl-S-phenylglycine t-butyl ester; BTC: betacelulin.
FBS 0%
Activin A Activin A
FBS 0,2%
Cyclopam. DAPT ??
bFGF
Noggin
Exend-4
BTC
Nitric oxyde (*)
(*)
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1. Developmental Approach1. Developmental Approach
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2. “Small Molecule” ApproachApproach
a. Nitric oxydeb. Sirt-1c. Resveratrol
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Modified from Hess D.T. et al. Nat Rev Mol Cell Biol. 2005 Feb;6(2):150-66.
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Mouse ESC
Human ESC
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Teratome formation by mouse and human ESC cultured in low NO
Tejedo et al (2010) Cell Death Disease
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Mechanism of NO inhibition of Nanog :
- Inhibits MDM2-dependent p53 degradation- Activation of p53 repressor protein by covalent - Activation of p53 repressor protein by covalent
modifications- Binding of p53Ser315 to Nanog promoter region
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NO induces:Resistance to apoptosis and endoderm phenotype
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Calvanese et al (2010) PNAS USA 107:13736-13741
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Day 0 1 2 5 8 11 14 20 22
Activin A [100ng/ml] RA [2 µM]
bFGF [64 ng/ml]
Noggin
[50 ng/ml]
Cyclopamine
[0,25 µM]
Resveratrol [50 µµµµM]
Wnt3a [25ng/ml]
0% 0,2% 2%
RPMI Suspension culture
DMEM-SR 12% + Fib/ITSDefinitive endoderm
Pancreatic commitment
DMEM + SR 12%
MaturationFactors Resveratrol -
- -+ +
+Ins
C-Pep FactorsResveratrol - - +
- + +
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Trends in Immunology (2008) 29: 68-74
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Immunomodulation
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“Immunomodulatory Therapy”: Clinical Trials
Target Drug Year(s)CD3 Teplizuma,
Otexizumab2011
CD20 Rituximab 2009
CD80, CD86 Abatacept 2011
mTOR, IL-R2 IL-2, Rapamicin 2011
GAD65 GAD65-alum 2011
Insulin Insulin 2011
HSP60 DiaPep277 2011
Cow’s Milk Proteins Cow´s milk (prevention)
2010
“Human vs NOD-mice Results”: NOD treatment before insul itis…
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Immunomodulation with MSC
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Mesenchymal stem cells are immunomodulators
1. Block T-cell proliferation,2. Decrease TNFα and INF-γ3. Increase IL-10 and IL-44. Up-regulate Treg4. Up-regulate Treg5. Inhibit NK cells6. Inhibit Monocyte differentiation into DC cells.
Immature DC cells more susceptible to degradationby NK cells, etc
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Yi, Arch Pharm Res 35: 213-221, 2012
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Child Autoimmune Diseases
- Systemic Lupus Erythematosus- Juvenile Idiopathic Arthritis- Graft vs Host Disease- Collagen-induced Arthritis- Rheumatoid Arthritis
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Cell Therapy of Liver Diseases
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Nature 000, 1-4 (2013)
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Ref Disease Dose/Via Results
BM-MNc
(Autologous)
Terai, 2006 Liver cirrosis 5 x 109 Safe
↑Albumin
Lyra, 2007 End Stage Chronic Liver
Disease
100 x 106
Hepatic artery
Safe, Feasible
↑Albumin
↓Bilirrubin
Lyra 2010 Hepatic Cirrosis Hepatic artery ↑Albumin
↓Bilirrubin
Cell Therapy of Liver Diseases (1)
↓Bilirrubin
CD 133+
(Autologous)
Fürst, 2007 PVE+Hepatectomy ↑ Regenera0on
Salama 2010 End-stage liver disease CD34+/CD133+
(G-CSF mobil)
Improve liver function
↑Liver enzymes
Nikeghbalian, 2011 Cirrosis CD133+ vs BM-MNc No adverse effects
Esch, 2012 Hepatectomy ↑ Prolifera0on
Lehwald, 2013 CD133
(HGF/SDF1 mobil)
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Ref Disease Dose/Via Results
CD34+
(Autologous)
Gordon, 2006 Chronic Liver Disease 106 -2 x 108
Portal vein/ Hepatic
artery
Safe
↑Albumin
↓Bilirrubin
Levicar, 2008 Chronic Liver Disease 106 -2 x 108 Safe
Cell Therapy of Liver Diseases (2)
Portal vein/ Hepatic
artery
Pai, 2008 Alcoholic cirrosis
(Chronic Liver disease)
Expanded CD34+ Improvement Bilirrubin & Liver enzymes
Garg, 2012 Acute on Chronic Liver
Failure
G-CSF ↑ Survival
Preclinical
Mintz, 2013 Animal Model
NOD-SCID fibrosis
CD34+ conditioned
medium
Improvement
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Ref Disease Dose/Via Results
BM-MSC
(autologous)
Mohamadnejad, 2007 Liver cirrosis 3 x 107 Improved MELD
Kazahira, 2009 Liver cirrosis 3,5 x 107 Improved MELD, serum creatinine, prothrombin
complex
Amer, End stage liver failure 2 x 107 cells (pretreated
HGF: liver committed)
Improved MELD
Preclinical
Cell Therapy of Liver Diseases (3)
Preclinical
Tanimoto, 2012 Murine Liver cirrosis 5 x 105 ↓ Liver Fibrosis
↑ MMP-9;
↓ αSMA, ↓TNFα, ↓TGFβ
LIVER CELLS
(Heterologous)
Progenitors
(MSC-like)
PROMETHERA
(Belgium )
Sokal,
Crigler-Najjar
Urea Cycle deficiencies
HepaStem (adult liver
progenitor cells)
Hepatocytes
(isolated/cultured)
Castell, 2103 Inherited Liver dis (
Crigler-Najjar, )
Liver failure
etc
Hepatocyte (from donor
livers)
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� Muchos Ensayos Fase I-II
Conclusiones:
EFECTOS BENEFICIOSOS, pero
� No ensayos Fase II y Fase III
� La mayoría no se publican
� Mecanismo desconocido
� No hay una hipótesis directriz
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� Muchos Ensayos Fase I-II
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�Están muy conservados en distintas especies
�Se unen a la secuencia GATA de los promotores de los genes a a los que activa
Familia de factores de transcripciónGATA
GATA 1, 2, 3 Hematopoiesis
GATA 4, 5, 6 Se expresan en:- EMBRIÓN: mesodermo y endodermo- ADULTO: corazón, pulmones, hígado y páncreas
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Mutaciones en genes GATA asociados a defectos cardíacos en
humanos
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Factores GATA
Función de los factores GATA en la formación del páncreas
Factores GATA
Progenitores pancreáticos
ProliferaciónDiferenciación
Identidad
Control GATA4/GATA6 KO
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GATA4 se expresa en lascélulas estrelladas hepáticas (mesodermo), pero no en los hepatocitos (endodermo)
Hepatocito
Célula hepática estrellada
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Inactivación de GATA4
(específicamente) en las células estrelladas del hígadoproduce hígado hipoplásico y cirrótico
GATA4 -/-HígadoHígadohipoplasico y cirrótico
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Ausencia de GATA4 :
1. Activación de células estrelladashepáticas
2. Deposición de proteínas de matrizextracelular (colágeno IV, laminina)
Activación de Células EstrelladasHSCs
Deposición de proteínas de matriz extracelular
Ctrl G4KOCélulas EstrelladasHSCs extracelular
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F0-F1
Los niveles de expresión de GATA4 en célulasestrelladas humanas disminuye durante la fibrosis y
cirrosis
GATA
Sirius Red GATA4
F3-F4
fibro
ss10 X 40 X
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La inducción de la expresión de GATA4 en células estrelladas activadas
revierte o atenúa la progresión de la fibrosis?
Célula estrellada quiescente Célula estrellada activada
Lesión
Célula estrellada quiescente(GATA4 +)
Célula estrellada activada (produccion de laminina, colágeno, etc…)
(GATA4 -)
Sobreexpressión de GATA4
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Sobreexpresión de GATA4 en línea célular estrellada hum ana(activada) disminuye/revierte el fenotipo activado
GATA4+/Laminina-
GATA4- /Laminina+
GATA4+/Laminina-
GATA4- /Laminina+
GATA4; LAMININA; DAPI
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¿ Como podemos sobreexpresar GATA4 en la
Célula Estrellada Humana Célula Estrellada Humana
para revertir el fenotipo activado (fibrosis-cirrosis?
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BUSQUEDA DE COLABORACIÓN:
¿ Tienen algun caso de
�Fibrosis Neonatal asociada a un cuadro.� pancreático?� pancreático?� cardíaco?
1. Biopsia hepática y GATA 42. Posible aproximación terapéutica para la disminución de la
Fibrosis
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Osteogenesis Imperfecta
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Muscular Dystrophies
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CELL THERAPY APPROACHES
- BM-MNc (CTrial)- Fetal Skeletal Muscle Progenitors- Mesoangioblasts (CTrial)
- Fibrosis: MSC (matrixmetalloproteinase)
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Neurological and Neurodegenerative Disorders
1. Lisosomal Storage Diseases: Phase I-IIa (Safe/Feasible)- Neuronal Lipofuscinosis (HuNSC + ImmunosuppressionJ Neurosurg Pediat 11(6) 643-652)
2. Cerebral Paralsy- Cord Blood (less immunogenic)
3. Etc
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Futuro
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Laboratorio Terapia Celular DMUPO Marzo 2004
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