Progress and challenges in directing the differentiation of human iPSCs into spinal motor neurons

Abstract

Motor neuron (MN) diseases, including amyotrophic lateral sclerosis, progressive bulbar palsy, primary lateral sclerosis and spinal muscular atrophy, cause progressive paralysis and, in many cases, death. A better understanding of the molecular mechanisms of pathogenesis is urgently needed to identify more effective therapies. However, studying MNs has been extremely difficult because they are inaccessible in the spinal cord. Induced pluripotent stem cells (iPSCs) can generate a theoretically limitless number of MNs from a specific patient, making them powerful tools for studying MN diseases. However, to reach their potential, iPSCs need to be directed to efficiently differentiate into functional MNs. Here, we review the reported differentiation protocols for spinal MNs, including induction with small molecules, expression of lineage-specific transcription factors, 2-dimensional and 3-dimensional cultures, as well as the implementation of microfluidics devices and co-cultures with other cell types, including skeletal muscle. We will summarize the advantages and disadvantages of each strategy. In addition, we will provide insights into how to address some of the remaining challenges, including reproducibly obtaining mature and aged MNs.

Keywords: assembloids; directed differentiation of pluripotent stem cells; iPS cells; induced pluripotent stem cells; motor neurons; neurodegenerative diseasaes; organoids.

FIGURE 1

MN development in vivo and its recapitulation in vitro using small molecules. (A) Timeline of MN development in vivo. (B) Patterning along the dorsoventral axis is regulated by BMP and SHH produced by the roof and floor plates, respectively, as well as RA from adjacent somites. (C) Patterning of the rostrocaudal axis is regulated by WNT, FGF, GDF11 and RA. (D) Timeline showing how small molecules can be used to recapitulate developmental factors that specify MN development. Created with BioRender.com.

FIGURE 2

Transcription factors regulating MN development in vivo and their use in differentiating iPSCs into MNs in vitro. (A) Transcription factors expressed in the MN progenitors (pMN) and post-mitotic MNs (MN) in the developing neural tube. (B) Representative protocol for using expression of specific transcription factors to direct the differentiation of iPSCs into MNs. Created with BioRender.com.

FIGURE 3

Comparison between mature MNs and aged MNs. “Aged” MNs show considerable differences compared to “young” MNs, including changes in mitochondria, synaptic function, integrity in the nuclear membrane integrity as well as increased DNA damage and reduced telomere length. Created with BioRender.com.