Generating homogenous cortical preplate and deep-layer neurons using a combination of 2D and 3D differentiation cultures

Abstract

Embryonic stem cells (ESCs) can be used to derive different neural subtypes. Current differentiation protocols generate heterogeneous neural subtypes rather than a specific neuronal population. Here, we present a protocol to derive separate two-deep layer cortical neurons from mouse eScs (meScs). mESCs were differentiated into mature Tbr1 or Ctip2-positive neurons using a monolayer-based culture for neural induction and neurosphere-based culture for neural proliferation and expansion. the differentiation protocol relies on SMAD inhibition for neural induction and the use of FGF2 and EGF for proliferation and it is relatively short as mature neurons are generated between differentiation days 12-16. Compared with the monolayer-based differentiation method, mESCs can be directed to generate specific deep-layer cortical neurons rather than heterogeneous cortical neurons that are generated using the monolayer differentiation culture. The early analysis of progenitors using flow cytometry, immunocytochemistry, and qRT-PCR showed high neuralization efficiency. The immunocytochemistry and flow cytometry analyses on differentiation days 12 and 16 showed cultures enriched in Tbr1-and Ctip2-positive neurons, respectively. Conversely, the monolayer differentiation culture derived a mixture of Tbr1 and Ctip2 mature neurons. Our findings suggested that implementing a neurospherebased culture enabled directing neural progenitors to adopt a specific cortical identity. The generated progenitors and neurons can be used for neural-development investigation, drug testing, disease modelling, and examining novel cellular replacement therapy strategies. Embryonic stem cells (ESCs) are an invaluable research tool as they can differentiate into the three germ layers in vitro. The advancement in the differentiation protocols has enabled the derivation of different neural progenitors and mature neurons. These neurons can be used in disease modelling, drug testing, modelling development in a dish, and in establishing novel strategies for cellular replacement therapies. Although the interest has been shifted towards using human embryonic stem cells (hESCs), mouse embryonic stem cells (mESCs) still have a great potential in the field. Essentially, mESCs can be genetically manipulated where their differentiation is

Spatio-temporal dynamics enhance cellular diversity, neuronal function and further maturation of human cerebral organoids

“…Chicken ovalbumin upstream promoter transcription factor integrating protein 2 (CTIP2) and special AT-rich sequence binding protein 2 (SATB2) genes are involved in the genetic control of these neurons, respectively 61 , 62 , where the expressions dictate the spatial separation of cortical layers 63 . As such, deep-layer cortical neuron marker, TBR1 plays a significant role in brain development 64 . CTIP2, SATB2, and TBR1 were upregulated at protein and RNA expression levels in both RCCS and µ-platform organoids mainly on day 120 (Fig.…”

The Effects of Prenatal Exposure to Pregabalin on the Development of Ventral Midbrain Dopaminergic Neurons

“…Freshly isolated primary embryonic mouse E12.5 vmDA neurons were seeded at 6 × 10 4 cells/well in 96-well cell culture plates for 3D cultures. To establish 3D cultures, we used the short peptide IIZK, as previously described [ 11 ]. IIZK was prepared in 1 × DPBS.…”

Comparative Transcriptomic Analysis of Cerebral Organoids and Cortical Neuron Cultures Derived from Human Induced Pluripotent Stem Cells

“…The ability to reprogram somatic cells to induced pluripotent stem cells (iPSCs) has opened new avenues to culture and grow human neuronal cells ex vivo to study human brain development [1]. Human iPSCs can be differentiated to generate a wide range of neuronal and glial cells, including many cell types relevant to disease biology [2][3][4][5][6]. These techniques utilizing iPSC-derived neuronal cultures are being used widely both to study the neurobiology of brain development as well as to study the cellular processes relevant to the disease biology of neuropsychiatric disorders [7][8][9][10][11][12].…”