In Vitro Generation of Posterior Motor Neurons from Human Pluripotent Stem Cells

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The ability to generate in-vivo spinal cord motor neurons from human pluripotent stem cells would be a major milestone in motor neuron-based diseases such as ALS. enter image description here

A key step in the design of human pluripotent stem cells differentiation strategies aiming to produce in-vitro motor neurons involves induction of the appropriate anteroposterior (A-P) axial identity, an important factor influencing motor neuron subtype specification, functionality, and disease vulnerability.

The anterior grey column contains motor neurons that affect the skeletal muscles while the posterior grey column receives information regarding touch and sensation. The anterior grey column is the column where the cell bodies of alpha motor neurons are located.

In-vitro generation of neural progenitors from human pluripotent stem cells holds a great promise for the development of cell-therapy-based approaches and the study of the specification of lineages and hence has attracted a considerable amount of research interest.

The protocols reported in literature generally are based on a multistep process that includes multiple neural induction, differentiation and maturation phases. This multistep process last weeks.

Scientists have previously described the generation of neural crest populations corresponding to various levels along the anteroposterior (A-P) axis from human pluripotent stem cells, including vagal neural crest (Frith et al., 2018). Yet stem cell derived motor neurons are often functionally immature.

Neural crest is a temporary group of cells unique to vertebrates that arise from the embryonic ectoderm germ layer, and in turn give rise to a diverse cell lineage—including melanocytes, craniofacial cartilage and bone, smooth muscle, peripheral and enteric neurons and glia. The ectoderm is the outermost layer of the three primary germ layers formed in early embryonic development.

To date, differentiation strategies have either implemented human pluripotent stem cells in the presence of EGF, FGF signals (Li et al., 2018; Workman et al., 2017) or employed a monolayer differentiation approach that relies on transforming growth factor β (TGF-β) signaling suppression, bone morphogenetic protein (BMP) signaling regulation and WNT pathway stimulation to generate an neural crest like population (Barber et al., 2019; Lau et al., 2019).

Patterning of the in vitro derived neural crest to a vagal axial identity is routinely achieved by retinoic acid (RA) addition while further commitment has been mediated by co-culture with intestinal/colonic organoids (Lau et al., 2019), gut tissue explants (Li et al., 2018), or further differentiation following culture in neurotrophic medium (Barber et al., 2019; Lau et al., 2019).

An efficient method of directed differentiation, generates progenitors from human pluripotent stem cells via the combined WNT signaling stimulation and TFG-β pathway inhibition together with precise levels of bone morphogenetic protein signaling.

Most current protocols for induction of motor neurons from human pluripotent stem cells produce predominantly cells of a mixed hindbrain/cervical axial identity marked by expression of Hox paralogous group (PG) members 1-5, but are inefficient in generating high numbers of more posterior thoracic/lumbosacral Hox PG(8-13)+ spinal cord motor neurons.

Here, the authors describe a protocol for efficient generation of thoracic spinal cord cells and motor neurons from human pluripotent stem cells. This step-wise protocol relies on the initial generation of a neuromesodermal-potent axial progenitor population, which is differentiated first to produce posterior ventral spinal cord progenitors and subsequently to produce posterior motor neurons exhibiting a predominantly thoracic axial identity.

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