TY - JOUR

T1 - Rapid generation of regionally specified CNS neurons by sequential patterning and conversion of human induced pluripotent stem cells

AU - Chen, Muwan

AU - Maimaitili, Muyesier

AU - Habekost, Mette

AU - Gill, Katherine P.

AU - Mermet-Joret, Noëmie

AU - Nabavi, Sadegh

AU - Febbraro, Fabia

AU - Denham, Mark

PY - 2020/10

Y1 - 2020/10

N2 - The differentiation of patient-specific induced pluripotent stem cells (iPSCs) into specific neuronal subtypes has been exploited as an approach for modeling a variety of neurological disorders. However, achieving a highly pure population of neurons is challenging when using directed differentiation methods, especially for neuronal subtypes generated by complex and protracted protocols. In this study, we efficiently produced highly pure populations of regionally specified CNS neurons by using a modified NGN2-Puromycin direct conversion protocol. The protocol is amenable across a range of iPSC lines, with more than 95% of cells at day 21 positive for the neuronal marker MAP2. We found that conversion from pluripotent stem cells resulted in neurons from the central and peripheral nervous system; however, by incorporating a short CNS patterning step, we eliminated these peripheral neurons. Furthermore, we used the patterning step to control the rostral-caudal identity. This approach of sequential patterning and conversion produced pure populations of forebrain neurons, when patterned with SMAD inhibitors. Additionally, when SMAD inhibitors and WNT agonists were applied, the approach produced anterior hindbrain excitatory neurons and resulted in a neuronal population containing VSX2/SHOX2 V2a interneurons. Overall, this sequential patterning and conversion protocol can be used for the production of a variety of CNS excitatory neurons from patient-derived iPSCs, and is a highly versatile system for investigating early disease events for a range of neurological disorders including Alzheimer's disease, motor neurons disease and spinal cord injury.

AB - The differentiation of patient-specific induced pluripotent stem cells (iPSCs) into specific neuronal subtypes has been exploited as an approach for modeling a variety of neurological disorders. However, achieving a highly pure population of neurons is challenging when using directed differentiation methods, especially for neuronal subtypes generated by complex and protracted protocols. In this study, we efficiently produced highly pure populations of regionally specified CNS neurons by using a modified NGN2-Puromycin direct conversion protocol. The protocol is amenable across a range of iPSC lines, with more than 95% of cells at day 21 positive for the neuronal marker MAP2. We found that conversion from pluripotent stem cells resulted in neurons from the central and peripheral nervous system; however, by incorporating a short CNS patterning step, we eliminated these peripheral neurons. Furthermore, we used the patterning step to control the rostral-caudal identity. This approach of sequential patterning and conversion produced pure populations of forebrain neurons, when patterned with SMAD inhibitors. Additionally, when SMAD inhibitors and WNT agonists were applied, the approach produced anterior hindbrain excitatory neurons and resulted in a neuronal population containing VSX2/SHOX2 V2a interneurons. Overall, this sequential patterning and conversion protocol can be used for the production of a variety of CNS excitatory neurons from patient-derived iPSCs, and is a highly versatile system for investigating early disease events for a range of neurological disorders including Alzheimer's disease, motor neurons disease and spinal cord injury.

KW - Cortical neurons

KW - Direct conversion

KW - Induced pluripotent stem cells

KW - Patterning

KW - V2a interneuron

UR - http://www.scopus.com/inward/record.url?scp=85089221143&partnerID=8YFLogxK

U2 - 10.1016/j.scr.2020.101945

DO - 10.1016/j.scr.2020.101945

M3 - Journal article

C2 - 32791483

AN - SCOPUS:85089221143

VL - 48

JO - Stem Cell Research

JF - Stem Cell Research

SN - 1873-5061

M1 - 101945

ER -