Figure 1. Schematic for the STEMdiff™ Motor Neuron Culture System Protocol

Motor neurons can be generated from human pluripotent stem cells (hPSCs) in 14 days. The resulting cells can then be matured for an additional 14 or more days using STEMdiff™ Motor Neuron Maturation Kit.

Figure 2. Pure Population of Motor Neuron Progenitors Are Generated Using the STEMdiff™ Motor Neuron Differentiation Kit

hPSCs from a variety of cell lines were differentiated to motor neuron progenitors using the STEMdiff™ Motor Neuron Differentiation Kit in an ultra-low attachment or an AggreWell™400 plate. (A) hPSCs maintained in mTeSR™1 or mTeSR™ Plus were aggregated to EBs with the STEMdiff™ Motor Neuron Differentiation Kit. On Day 9, EBs were dissociated into single cells and replated for adherent culture.The resulting cultures contain a population of cells expressing motor neuron progenitor marker OLIG2 (red) and are negative for spinal interneuron marker NKX2.2 (green). Nuclei are labeled with Hoechst (blue). (B) The percentage expression of OLIG2 and negative controls NKX2.2, FOXG1, OTX1, and OTX2 in the resulting cultures, derived from 2 hES (H1 and H7) and 2 hiPS (WLS-1C and STiPS-M001) cell lines, were quantified. This differentiation generated OLIG2+ motor neuron progenitors (83.20% ± 2.890%, mean ± SEM; n=4 cell lines, 2 replicates per condition). Numbers are % positive of total Hoechst-positive cells. hPSCs = human pluripotent stem cells; EB = embryoid body

Figure 3. Pure Population of Post-Mitotic Motor Neurons Are Generated Using the STEMdiff™ Motor Neuron Differentiation Kit

Motor neuron progenitors derived from a variety of cell lines were transitioned to post-mitotic motor neurons using the STEMdiff™ Motor Neuron Differentiation Kit. (A) Post-mitotic motor neurons were generated by Day 14 after hPSCs were differentiated with the STEMdiff™ Motor Neuron Differentiation Kit. The resulting cultures contain a population of cells expressing neuronal identity marker βIII-TUB (green), mature motor neuron marker HB9 (red), and (B) ISL1 (red). Nuclei are labeled with Hoechst (blue). (C) The percentage expression of βIII-TUB, ISL1, and HB9 in the resulting cultures, derived from 3 hES (H1, H7, and H9) and 3 hiPS (WLS-1C, STiPS-M001, and STiPS-R038) cell lines, were quantified. This differentiation generated βIII-TUB+ (92.59% ± 1.079%, mean ± SEM; n=6 cell lines, 2 replicates per condition), ISL1+ (55.99% ± 3.723%, mean ± SEM), and HB9+ (65.17% ± 3.514%, mean ± SEM) motor neurons. Numbers are % positive of total Hoechst-positive cells. hPSC = human pluripotent stem cell

Figure 4. STEMdiff™ Motor Neuron System Generates Motor Neurons Patterned to the Cervical Axis

hPSCs from a variety of cell lines were differentiated to motor neuron progenitors using the STEMdiff™ Motor Neuron Differentiation Kit. Expression levels of the resulting cultures, derived from 2 hES (H1 and H7) and 2 hiPS (STiPS-M001 and STiPS-R038) cell lines, were measured by qPCR and hPSC-derived forebrain progenitor cells were used as a control. Analysis of Day 9 motor neuron progenitors showed high expression of cervical AP axis HOXA5 (n = 4) relative to hPSC. hPSCs = human pluripotent stem cells; qPCR = quantitative polymerase chain reaction; AP = anteroposterior

Figure 5. hPSC-Derived Motor Neurons Can Be Matured in STEMdiff™ Motor Neuron Maturation Medium

Motor neuron progenitors derived from a variety of cell lines were matured using the STEMdiff™ Motor Neuron Maturation Kit. (A) Mature motor neurons were generated after hPSCs were cultured with the STEMdiff™ Motor Neuron Differentiation Kit for 14 days and then the STEMdiff™ Motor Neuron Maturation Kit for an additional 14 days. The resulting cultures contain a population of cells expressing neuronal identity marker βIII-TUB (green), mature motor neuron markers HB9 (red), (B) SYNAPSIN (red), and MAP2 (green), as well as (C) cholinergic neuron marker ChAT (green). Nuclei are labeled with Hoechst (blue). (D) The percentage expression of ChAT, HB9, and βIII-TUB in the resulting cultures, derived from 2 hES (H1 and H7) and 2 hiPS (STiPS-R038 and STiPS-M001) cell lines, were quantified. This differentiation generated ChAT+ (65.16% ± 3.737%, mean ± SEM; n=4 cell lines, 2 replicates per condition), HB9+ (79.58% ± 2.570%, mean ± SEM), and βIII-TUB+ (86.56% ± 2.331%, mean ± SEM) motor neurons. Numbers are % positive of total Hoechst-positive cells. hPSC = human pluripotent stem cell

Figure 6. hPSC-Derived Motor Neurons Matured in STEMdiff™ Motor Neuron Maturation Medium Produces Cholinergic Neurons

hPSCs from a variety of cell lines were differentiated to motor neurons using the STEMdiff™ Motor Neuron Differentiation Kit for 14 days and then the STEMdiff™ Motor Neuron Maturation Kit for an additional 14 days prior to analysis. Expression levels of the resulting cultures, derived from 3 hES (H1, H7, and H9) and 3 hiPS (WLS-1C, STiPS-M001, and STiPS-R038) cell lines, were measured by qPCR and hPSC-derived forebrain neurons were used as a control. Analysis of Day 28 mature motor neurons showed higher expression of ChAT (n = 5, P<.0001, Two-way ANOVA with Sidak's multiple comparisons test). hPSCs = human pluripotent stem cells; qPCR = quantitative polymerase chain reaction; ChAT = choline acetyltransferase

Figure 7. hPSC-Derived Motor Neurons Can Be Co-Cultured with hPSC-Derived Myotubes for In Vitro Modeling

hPSCs were differentiated to motor neurons using the STEMdiff™ Motor Neuron Differentiation Kit and Maturation Kit. hPSC-derived myogenic progenitors were generated using the STEMdiff™ Myogenic Progenitor Kit and then induced to differentiate into myotubes using MyoCult™ Differentiation Medium. The resulting co-culture contains a population of cells expressing neuronal identity marker βIII-TUB (red), mature motor neuron marker ISL1 (blue), and AChR (green). Arrows indicate neuromuscular junctions. hPSC = human pluripotent stem cell; AChR = acetylcholine receptor.

Figure 8. STEMdiff™ Motor Neurons Express Expected Motor Neuron Markers

Motor neurons were generated from iPS cell lines STiPS-R038, STiPS-M001, SCTi003-A (all maintained in mTeSR™ Plus), and SCTi003-A (maintained in mTeSR™1). hPSCs were then differentiated with the STEMdiff™ Motor Neuron culture system. RNA from Day 9, Day 14, and mature (in maturation media for 14 days) motor neurons was harvested and subsequently assayed using bulk RNA-seq. Their transcriptomic profiles were also compared to a parent hPSC control. The heat map displays expression levels for select genes associated with hPSCs, motor neuron progenitors, and motor neurons. iPS = induced pluripotent stem; hPSC = human pluripotent stem cell