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Motor Neuron Differentiation from Pluripotent Stem Cells and Other Intermediate Proliferative Precursors that can be Discriminated by Lineage Specific Reporters

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Abstract

We have used a four stage protocol to generate spinal motor neurons (MNs) from human embryonic stem cells (ESCs) and human induced pluripotent stem cells (iPSCs). These stages include the pluripotent stem cell (PSC) stage, neural stem cell (NSC) stage, OLIG2 expressing motor neuron precursor (MNP) stage, and HB9 expressing mature-MN stage. To optimize the differentiation protocol reporter lines marking the NSC and MNP stages were used. The NSC stage is a pro-proliferative precursor stage at which cells can be directed to differentiate to other neural types like cortical neurons also, in addition to MNs; thus, NSCs can be expanded and stored for future differentiation to different neural types thereby, shortening the differentiation interval as compared to the complete process of differentiation from ESCs or iPSCs. Additionally, we find that OLIG2 positive cells at the MNP stage can be cryopreserved and then recovered to continue the process of MN differentiation, thereby providing a highly stable and reproducible technique for bulk differentiation. MNPs were differentiated to MNs expressing the marker HB9 demonstrating that mature-MNs can be generated with this protocol.

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Abbreviations

PSC:

Pluripotent stem cell

iPSC:

Induced pluripotent stem cell

hESC:

Human embryonic stem cell

NSC:

Neural stem cell

MNP:

Motor neuron precursor

MN:

Motor neuron

CNS:

Central nervous system

O-L plate:

Ornithine-laminin coated plate

bFGF:

Basic fibroblast growth factor

BMP:

Bone morphogenetic protein

RA:

Retinoic acid

SHH:

Sonic hedgehog

BDNF:

Brain derived neurotrophic factor

GDNF:

Glial cell line derived neurotrophic factor

HD:

Homeodomain

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Conflict of Interest

The work in this manuscript was funded by the NIH Common Fund, National Institutes of Health, Bethesda, USA. The authors declare no potential conflicts of interest.

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  1. Laboratory of Stem Cell Biology, National Institutes of Health (NIH), Bethesda, MD, USA

    Balendu Shekhar Jha, Mahendra Rao & Nasir Malik

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  1. Balendu Shekhar Jha
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  2. Mahendra Rao
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  3. Nasir Malik
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Correspondence to Nasir Malik.

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Jha, B.S., Rao, M. & Malik, N. Motor Neuron Differentiation from Pluripotent Stem Cells and Other Intermediate Proliferative Precursors that can be Discriminated by Lineage Specific Reporters. Stem Cell Rev and Rep 11, 194–204 (2015). https://doi.org/10.1007/s12015-014-9541-0

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