RESUMO
Neurons differentiated from induced pluripotent stem cells (iPSCs) typically show regular spiking and synaptic activity but lack more complex network activity critical for brain development, such as periodic depolarizations including simultaneous involvement of glutamatergic and GABAergic neurotransmission. We generated human iPSC-derived neurons exhibiting spontaneous oscillatory activity after cultivation of up to 6 months, which resembles early oscillations observed in rodent neurons. This behavior was found in neurons generated using a more "native" embryoid body protocol, in contrast to a "fast" protocol based on NGN2 overexpression. A comparison with published data indicates that EB-derived neurons reach the maturity of neurons of the third trimester and NGN2-derived neurons of the second trimester of human gestation. Co-culturing NGN2-derived neurons with astrocytes only led to a partial compensation and did not reliably induce complex network activity. Our data will help selection of the appropriate iPSC differentiation assay to address specific questions related to neurodevelopmental disorders.
Assuntos
Diferenciação Celular , Sistema Nervoso/crescimento & desenvolvimento , Neurônios/citologia , Sinapses/metabolismo , Proliferação de Células , Fenômenos Eletrofisiológicos , Corpos Embrioides/citologia , Humanos , Modelos Biológicos , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismoRESUMO
De novo mutations in the KCNA2 gene, encoding the voltage-gated potassium channel KV1.2, have been identified to cause early-onset developmental and epileptic encephalopathies (DEE). KV1.2 channels conduct delayed-rectifier type K+ currents and play a crucial role in action potential repolarization. In this study we reprogrammed fibroblasts from a 6-months-old male patient with DEE carrying a de novo point mutation (c.1120Aâ¯>â¯G, p.Thr374Ala) in KCNA2 to induced pluripotent stem cells. Their pluripotency was verified by the capability to differentiate into all three germ layers and the expression of several pluripotency markers on RNA and protein levels.
Assuntos
Epilepsia/patologia , Células-Tronco Pluripotentes Induzidas/citologia , Canal de Potássio Kv1.2/genética , Diferenciação Celular , Linhagem Celular , Reprogramação Celular , Epilepsia/genética , Fibroblastos/citologia , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Lactente , Masculino , Polimorfismo de Nucleotídeo ÚnicoRESUMO
Developmental and epileptic encephalopathies (DEE) can be caused by mutations in the KCNA2 gene, coding for the voltage-gated K+ channel Kv1.2. This ion channel belongs to the delayed rectifier class of potassium channels and plays a role during the repolarization phase of an action potential. In this study we reprogrammed fibroblasts from a 30-year-old male patient with DDE carrying a point mutation (c.890Gâ¯>â¯A, p.Arg297Gln) in KCNA2 to induced pluripotent stem cells. Pluripotency state of the cells was verified by the capability to differentiate into all three germ layers and the expression of several pluripotency markers on RNA and protein levels.
Assuntos
Diferenciação Celular , Epilepsia/genética , Fibroblastos/patologia , Células-Tronco Pluripotentes Induzidas/patologia , Canal de Potássio Kv1.2/genética , Mutação , Transtornos do Neurodesenvolvimento/genética , Adulto , Células Cultivadas , Reprogramação Celular , Epilepsia/patologia , Fibroblastos/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Masculino , Transtornos do Neurodesenvolvimento/patologia , FenótipoRESUMO
Mutations in the KCNA2 gene, coding for the voltage-gated K+ channel Kv1.2, can cause developmental and epileptic encephalopathies. Kv1.2 channels play an important role in the repolarization phase of an action potential in nerve cells. Here, we reprogrammed human skin fibroblasts from a 13-year-old male patient with developmental and epileptic encephalopathy carrying a point mutation (c.982T>G, p.Leu328Val) in KCNA2 to human induced pluripotent stem cells (iPSCs) (HIHDNEi001-A). The cells maintained a normal karyotype and their pluripotency state was verified by the expression and staining of several pluripotency markers and capability to differentiate into all three germ layers.
