Patient specific induced pluripotent stem-cell models for long-qt syndrome
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Luz Eugenia Pérez Jaramillo
3rd Semester
UPB- MEDICINE STUDENT.
Patient-Specific Induced pluripotent Stem-Cell Models
for Long-QT SyndromeAlessandra Moretti, Ph.D.
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Long QT Syndrom
e
Autosomal
dominant disease
Prolonged ventricular
repolarization phase
Risk of sudden cardiac death
due a ventricular
tachyarrhythmia
Characterized
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T: Ventricular repolarization R: Ventricular despolarization
REPOSE
P: Auricular despolarization
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MUTATION IN THE KCNQ1 GENE
Encoding
KvQTI protein (alpha subunity of the Iks Canals)
TIPE I
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STEM CELLS Characterized by the ability to renew
themselves through mitotic cell division and differentiate into a diverse range of specialized cell types.
Embryonic stem cells Adult stem cells
From the inner cell mass of blastocysts
That are found in adult tissues
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INDUCED PLURIPOTENT STEM CELLS
Type of pluripotent stem cell artificially derived from a non- pluripotent cell, typically an adult somatic cell, by inducing a "forced" expression of specific genes.
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(1)Isolated and growth the donant cells.(2)Make a transfection of gene stem cells from
the donor cells by means of viral. (3)Cells were growth with strategies for
growing stem cells using mitotically inactive fibroblasts
(4)A small subset of these transfected cells become induced pluripotent stem cells
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STEM CELLS IMPORTANCE Offers the opportunity to produce large
numbers of patients -pecific Stem cells. If someone has certain cells that don’t
function properly or that have been damaged, then stem cells could be used to replace those damaged cells.
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In this study for example; these showed the capacity of these cells to give rise to functional cardiomyocytes that recapitulate the electrophysiological characteristics of the disorder.
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OBJETIVE Observe induced stem-cell models of
patient-specific with long- QT Syndrome.
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METODOS
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HISTORIA CLINICA Y FENOTIPO GENETICO
HIPERACTIVO
CON DEFICIT DE ATENCION
EN ELECTROCARDIOGRA
MA
INTERVALO QT LARGO
MUTACION EN EL GEN KCNQ1 (CAMBIO DE BASE (569G→A)
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Miembros de sexo Masculino
Miembros de sexo Femenino
Miembros con el SQTL
Miembros no afectados.
Los metodos geneticos mostraron que los miembros de esta familia tenia la misma mutacion heterocigotica, confirmando por esto la herencia autosomica dominante en esta familia.
•El abuelo y la tia han reportado periodos de discinecia y palpitaciones.
Abuelo: 70 años
Papá: 42 años
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GENERACIÓN DE CÉLULAS MADRE PLURIPOTENCIALES DE PACIENTES ESPECÍFICOS
Se cortan y se
ubican en
Cajas de cultivo
Fibroblastos
Infectados
Con una combinación de retrovirus:OCT3/4, SOX2, KLF4, and c-MYC
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DESPUÉS DE 6 DÍAS
Células infectadas
Se sembraron en
Murino embrionarias
Colonia de células madre
HASTA QUE APARECEN
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(Células madre pluripotentes inducidas)
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DIFERENCIACIÓN CARDIACA IN VITRO
3 día- Indujeron las células madres pluripotenciales como cuerpos embrionarios separando las células madre
4 día- El medio fue reemplazado20% suero fetal bovino
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7 día- Los cuerpos embrionarios fueron recubiertos por gelatina.
20-30 días- Las áreas que mostraron excitación espontanea (indicativo de diferenciación cardiaca), fueron micro disecadas y recubiertas por Fibronectina y suero fetal bovino (2%)
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EVALUACIONES DE INMUNOHISTOQUIMICA
Nanog (Abcam), TRA-1-81 (BD Pharmingen), cardiac troponin T (Lab Vision),α-actinin (Sigma–Aldrich), myosin light chain 2a and myosin light chain 2v (Synaptic Systems), KCNQ1 (Abcam),y proteina disulfido isomerasa (Abcam).
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ENSAYOS DE REACCIÓN EN CADENA DE LA POLIMERASA
(PCR)
Para amplificar la región mutada del gen KCNQ1para secuenciación.
Para la evaluación de la expresión de los genes de pluripotencialidad, transgenes retrovirales, y marcadores de linaje celular.
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PROPIEDADES DE LAS CÉLULAS MADRE
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La trasncriptasa inversa (RT)- PCR se utilizo para marcar los fenotipos de miocardiocitos individuales.
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RESULTADOSGeneración de células madre pluripotenciales
-Cambio de base 569G → ACÉLULAS CONTROL CÉLULAS DE SUJETOS CON SQTL
NO se presento SI se presento
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- Presencia de genes pluripotenciales en cada una de las capas embrionarias. (clones de células madre embrionarias)
(PDX1, SOX7, y AFP)
(CD31, DESMIN, ACTA2, SCL, MYL2, y CDH5)
(KTR14, NCAM1)
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VALORACION DEL TIPO DE SINDROME DE QT LARGO
Focos de contratación comenzaron a aparecer después de aprox. 12 días de diferenciación.
Adaptación en pacientes con SQTL
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PAPEL DE R190Q-KCNQ1
Con el uso de RT-PCRSe
observo
KCNQ1
Fueron similares en células control y las de los sujetos con SQTL
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Ubicación del KCNQ1 en la celula
CÉLULAS CONTROL CÉLULAS DE SUJETOS CON SQTL
En la membrana En el interior de la célula junto proteína disulfuro isomerasa
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ANALISIS ELECTROFISIOLOGICO DE LOS CANALES K+
CÉLULAS DE SUJETOS CON SQTL
•Disminución notable en canales de K+•Disminución en canales Iks
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ACCION PROTECTORA DE LOS BETA BLOQUEADORES
OBSERVARON Arritmias
Análisis de efectos de las catecolaminas en la fase ventricular.
ISOPROTERENOL
•Simpaticomimético•Recep. B adrenérgicos
20% el potencial de repolarización (en células control Y NO EN LAS QUE TENIAN SQTL)
EFECTO CRONOTROPICO POSITIVO ( FREC.CARD)
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DISCUSSION
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CONCLUSIONAt present, stem cells are already
used in cell therapies for treatment of some cancer types but this use is still small in the scheme of diseases affecting humans today.
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Nowadays the human genetic disorders can be studied in the physiologic and disease-causing contexts on a patient-specific level.
Their importance ranges from an understanding of the principles behind human development to the cell based therapies addressing those aspects that go awry during development and lead to disease.
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The wide range of cell types and tissues that may develop from stem cells represent a biological system that mimics many of the complex interactions of the cells and tissues of the body, and as such, provides an attractive and valuable screening tool.
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THANKS!!!!