Modeling ALS with motor neurons derived from human induced pluripotent stem cells

Journal name:
Nature Neuroscience
Volume:
19,
Pages:
542–553
Year published:
DOI:
doi:10.1038/nn.4273
Received
Accepted
Published online

Abstract

Directing the differentiation of induced pluripotent stem cells into motor neurons has allowed investigators to develop new models of amyotrophic lateral sclerosis (ALS). However, techniques vary between laboratories and the cells do not appear to mature into fully functional adult motor neurons. Here we discuss common developmental principles of both lower and upper motor neuron development that have led to specific derivation techniques. We then suggest how these motor neurons may be matured further either through direct expression or administration of specific factors or coculture approaches with other tissues. Ultimately, through a greater understanding of motor neuron biology, it will be possible to establish more reliable models of ALS. These in turn will have a greater chance of validating new drugs that may be effective for the disease.

At a glance

Figures

  1. Emulating MN developmental signaling in vitro.
    Figure 1: Emulating MN developmental signaling in vitro.

    Developmental stages of human MNs (a) are reproduced in vitro (b) through the use of small molecule and recombinant signaling molecules. (a, panel 1) Blastocyst containing pluripotent stem cells derived from the inner cell mass (blue) is generated in vitro from adult somatic tissue through reprograming into iPSC cultures (b, panel 1). During gastrulation, Wnt-dependent primitive streak formation (a, panel 2) is simulated using the glycogen synthase kinase 3 inhibitor CHIR-99021 (CHIR) (b, 2). Neural ectoderm that emerges during neurulation (a, panel 3) is directed through the use of dual-SMAD inhibitors SB431542 (SB) and LDN193189 (LDN) (b, panel 3). Retinoic acid (RA) (a, panel 4) produced by neighboring somites (not shown) acts as caudalizing molecule that specify a hindbrain and anterior spinal cord fate. The small molecule RA is added (b, panel 4) to caudalize neural ectoderm in vitro. (a, panel 5) SHH is released from the ventral notochord, causing a gradient that induces MN fate in the ventral portion of the spinal cord. (b, panel 5) This is reproduced in vitro with small molecule SHH (smSHH) or recombinant SHH signaling agonists. (a, panel 6) MN progenitors depend on trophic support to connect axon projections to target muscles and develop into functioning LMNs. (b, panel 6) Neurotrophic factors (NTFs) such as GDNF, BDNF, CNTF and others are used in vitro to promote maturation and survival.

  2. Comparison of published LMN differentiation protocols.
    Figure 2: Comparison of published LMN differentiation protocols.

    Twelve iPSC to LMN protocols compared with respect to time (days in vitro). End of experiment (assay) based on last data presented. bFGF, basic fibroblast growth factor; FGF2, fibroblast growth factor 2; BDNF, brain-derived neurotropic factor; GDNF, glial cell line-derived neurotropic factor; CNTF, ciliary neurotropic factor; IGF-1, insulin-like growth factor 1; RA, retinoic acid; RAR, retinoic acid receptor; RXR, retinoid X receptor; SHH, sonic hedgehog; PMN, purmorphamine; EB, embryoid bodies. Yellow columns summarize results, unique MN subtype markers observed and approximate percentage yield of MNs based on reported percentage of cells expressing HB9 or ISL1. B27 and N2 are supplements; KOSR, knockout serum replacement; SAG, Smoothened agonist; DMH1, selective BMP inhibitor; VPA, valproic acid; compound C, dorsomorphin AMP kinase inhibitor; Y27632, Rho-associated kinase inhibitor; iAP, induced action potentials; sAP, spontaneous action potentials; HoxC9, caudal spinal cord associated Hox family member C9; FoxP1 and FoxP2, forkhead box P1 and P2; LHX3, LIM homeobox 3; LHX1/2, LIM homeobox 1 and 2; PHOX2B, paired mesoderm homeobox 2B.

  3. Induced action potentials evolve over time.
    Figure 3: Induced action potentials evolve over time.

    (a) Current injection (100 pA) whole-cell recordings of human iPSC-derived MNs (hPSC-MNs) over time in culture. Example action potential recordings show maturity over time in vitro as depolarization and hyperpolarization events occur more rapidly. (b) More mature hPSC-MNs in vitro display trains of action potentials (arrows) with an abortive event (x). Action potentials displayed as change in membrane voltage (mV) over time (ms).

  4. (a) Coculture of the neuromuscular circuit.
    Figure 4: (a) Coculture of the neuromuscular circuit.

    (b) hPSC-astrocytes; reproduced from Sareen et al.77, Wiley. (c) hPSC-myofibers; reproduced from ref. 79 (T. Hosoyama, T., J.V. McGivern, J.M. Van Dyke, A.D. Ebert and M. Suzuki, Stem Cells Transl. Med. 3, 564–574, 2014; AlphaMed Press). (d) These examples of published iPSC-derived cell types could comprise a conceptualized neuromuscular circuit. Other cell types such as myelinating Schwann cells (gray) and terminal Schwann cells (not shown) have not yet been generated from iPSCs and may be required for functional NMJ formation in vitro. SMI32 is a neurofilament heavy chain epitope; Isl1, Islet-1; GFAP, glial fibrillary acidic protein; SMA, smooth muscle actin.

  5. Classification of diverse neocortical projection neurons.
    Figure 5: Classification of diverse neocortical projection neurons.

    Neocortical projection neurons can be subdivided into broad classes, types and subtypes largely based on their axonal projections. (Adapted from Greig et al.83, Nature Publishing Group). Illustrations are of the mouse brain (adapted with permission from original artwork by M. Galazo).

  6. Cell-extrinsic and cell-intrinsic factors regulate the development of corticofugal projection neurons in sequential, 'nested' stages of differentiation.
    Figure 6: Cell-extrinsic and cell-intrinsic factors regulate the development of corticofugal projection neurons in sequential, 'nested' stages of differentiation.

    (1) 'Default' neural and rostral differentiation occurs by repression of alternative signaling pathways induced by multiple morphogens (for example, Noggin inhibits BMP signaling during neural plate formation at ~E3.5–E6.5 in mice; cortical progenitors require low or absent expression of caudalizing retinoids (RA) and ventralizing SHH at ~E6.5–E8.5). (2) The dorsal aspect of the telencephalon is called the pallium; it gives rise to the neocortex. In contrast, the ventral telencephalon is called the subpallium. The delineation of these two telencephalic progenitor domains occurs between ~E8.5 and ~E10.5. (3) During corticogenesis, beginning at ~E10.5 in mice, diverse cortical projection neuron classes, types and subtypes are sequentially generated from cortical progenitors. These projection neurons become refined with continued maturation through postnatal ages. (4) Early stages of corticofugal projection neuron (CFuPN) differentiation are largely mediated by cell-extrinsic factors, whereas later stages are largely mediated by cell-intrinsic factors. (5) Following SHH-mediated dorsal–ventral patterning of the telencephalon, cortical and ventral identities are reinforced by transcriptional regulation (Pax6 and Sox6 in the pallium; Gsh2 in the ventral areas). (6) Early cortical progenitors give rise to more definitive (neo)cortical progenitors, which generate projection neuron subtypes at ~E10.5. CFuPNs populate the deep layers of the cortex. Later-born callosal projection neurons (CPNs) populate both deep and superficial layers of cortex. Molecular distinction of CPN and CFuPN occurs with continued maturation (represented by transition from yellow, dual-marker expression to red or green single-marker expression). (7) Nested expression of distinct transcriptional regulators at distinct developmental stages promotes stepwise CFuPN and thus CSMN/UMN differentiation. SCPN, subcerebral projection neuron; CThPN, corticothalamic projection neuron; CSMN, corticospinal motor neuron; CTPN, corticotectal projection neuron; P, postnatal day.

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Author information

  1. These authors contributed equally to this work.

    • Lucie I Bruijn,
    • Siddharthan Chandran,
    • Kevin Eggan,
    • Ritchie Ho,
    • Joseph R Klim,
    • Matt R Livesey,
    • Emily Lowry,
    • Jeffrey D Macklis,
    • David Rushton,
    • Cameron Sadegh,
    • Dhruv Sareen,
    • Hynek Wichterle &
    • Su-Chun Zhang

Affiliations

  1. Board of Governors Regenerative Medicine Institute, Cedars-Sinai Medical Center, Los Angeles, California, USA.

    • Samuel Sances,
    • Ritchie Ho,
    • David Rushton,
    • Dhruv Sareen &
    • Clive N Svendsen
  2. ALS Association Headquarters, Washington, DC, USA.

    • Lucie I Bruijn
  3. Centre for Clinical Brain Sciences, University of Edinburgh, Edinburgh, UK.

    • Siddharthan Chandran &
    • Matt R Livesey
  4. Harvard Stem Cell Institute, Harvard University, Cambridge, Massachusetts, USA.

    • Kevin Eggan,
    • Jeffrey D Macklis &
    • Cameron Sadegh
  5. Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, Massachusetts, USA.

    • Joseph R Klim,
    • Jeffrey D Macklis &
    • Cameron Sadegh
  6. Centre for Integrative Physiology, University of Edinburgh, UK.

    • Matt R Livesey
  7. Department of Pathology and Cell Biology, Columbia University, New York, New York, USA.

    • Emily Lowry &
    • Hynek Wichterle
  8. Center for Brain Science, Harvard University, Cambridge, Massachusetts, USA.

    • Jeffrey D Macklis &
    • Cameron Sadegh
  9. Waisman Center, University of Wisconsin, Madison, Wisconsin, USA.

    • Su-Chun Zhang

Competing financial interests

The authors declare no competing financial interests.

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