Protocol for the Standardized Generation of Forward Programmed Cryopreservable Excitatory and Inhibitory Forebrain Neurons

Abstract

Summary This protocol describes a highly standardized pipeline for transcription factor-mediated forward programming of human pluripotent stem cells into highly enriched glutamatergic or GABAergic neurons followed by a cryopreservation step that enables the generation of large quality-controlled batches. This approach is particularly useful for reducing interexperimental variability in the context of collaborative studies across different locations and time points. For complete details on the use and execution of this protocol, please refer to Meijer et al. (2019) and Rhee et al. (2019) .

Comparative analysis of CI- and CIV-containing respiratory supercomplexes at single-cell resolution

“…A possible explanation of this observation is that the incorporation of NDUFS4 in partially superassembled CI occurs at a later stage and is necessary for the stability and optimal functionality of respirasomes as previously shown ( Calvaruso et al., 2012 ; Mimaki et al., 2012 ; Moreno-Lastres et al., 2012 ). To further understand the impact of NDUFS4 KO on CI∗CIV-SCs, we differentiated iPSCs into cortical excitatory neurons by using transcription factor-mediated forward programming ( Peitz et al., 2020 ). Although NDUFS4 KO did not alter iPSC proliferation (data not shown), it severely undermined the survival of immature cortical neurons, with many cells undergoing degeneration and death ( Figure 4 L).…”

“…Sequence-validated clones were further expanded and quality-controlled by virtual karyotyping using SNP arrays and regular mycoplasma testing. Candidate C-14m-s11-NGN2 clones were then differentiated into excitatory neurons following a previously published protocol ( Peitz et al., 2020 ).…”

Comparative profiling of N-respirasomes predicts aberrant mitochondrial bioenergetics at single-cell resolution

“…A possible explanation of this observation is that the incorporation of NDUFS4 in partially superassembled CI occurs at a later stage and is necessary for the stability and optimal functionality of respirasomes as previously shown (Calvaruso et al, 2012;Mimaki et al, 2012;Moreno-Lastres et al, 2012). To further understand the impact of NDUFS4 KO on N-respirasomes, we differentiated iPSCs into cortical excitatory neurons using transcription factor-mediated forward programming (Peitz et al, 2020). While NDUFS4 KO did not alter iPSC proliferation (data not shown), it severely undermined the survival of immature cortical neurons, with many cells undergoing degeneration and death ( Figure 5L).…”

Identification of Adeno-Associated Virus Variants for Gene Transfer Into Human Neural Cell Types by Parallel Capsid Screening

“…Speci cally, we either overexpressed the neurogenic transcription factor NGN2 alone or a combination of ASCL1 plus DLX2. 24 In addition to these neuroectodermal cell types, we performed AAV screening in iPSC-derived microglia (iPSdMiG), since these mesodermal lineage cells are the immune cells of the central nervous system (CNS), and as such relevant for a number of neurological, neuropsychiatric and neurodegenerative diseases.…”

“…All human cell programming-derived neural cell types were generated, cultured and/or expanded using previously described methods: iNSCs 20 , smNPCs 21 , ltNES 22 , RGL-NPCs 23 , as well as forward programmed NGN2-and ASCL1/DLX2-neurons 24 . IPSdMiG were generated via a proprietary protocol from LIFE & BRAIN GmbH (patent application number EP20162230).…”