To demonstrate whether human iPS are able to differentiate down neural lineages to form motor neurons, we generated embryoid bodies (EBs) from human iPS cells (hiPS2) and hESCs (HSF1), as previously described [1
]. The EBs were cultured for one week in hESC media lacking FGF2, and then treated for an additional week with Retinoic Acid (RA; 1 μM) and a Sonic Hedgehog pathway agonist (Purmorphamine, 1.5 μM) [16
]. This method is known to both neuralize and ventralize EBs, as defined by the expression of ventral neural progenitor markers [11
]. Both HSF1 and human iPS cells followed a standard course of development, serially differentiating from pluripotent cells to neural progenitors to fully differentiated motor neurons. As the EB protocol initiates specification in a somewhat stochastic manner, only a proportion of EBs from either HSF1 or iPS cells were specified to be neural, as demonstrated by immunostaining for the neural progenitor markers Brn2, Sox3 and Pax6 (, Fig. S2
hESC and human iPS-derived cells can both generate neural progenitors
We did observe marked differences in the efficiencies by which HSF1 and hiPS cells underwent specification down the neural lineage (Fig. S2
). Because of the well-described variation of differentiation potentials amongst pluripotent stem cell lines [17
], it is unclear whether this finding reflects an inherent difference between embryonic and induced pluripotent stem cells. However, within those EBs that were specified as neural, the percentage of cells expressing neural progenitor markers Brn2, Sox3 and Pax6 was similar whether the EBs were derived from HSF1 or human iPS cells ( and data not shown). These findings suggest that both HSF1 and human iPS-derived cells can be directed to form comparable neural progenitors.
After another week in the presence of RA and Shh pathway agonists, along with neurotrophic factors known to promote motor neuron survival (CNTF 20 ng/ml, BDNF and GDNF, 10 ng/ml each), the EBs were fixed, cryosectioned, and immunostained for Sox3 and the motor neuron progenitor markers Nkx6.1 and Olig2. In the EBs that expressed markers of neural progenitors, the extent of labeling with Nkx6.1 and Olig2 antibodies was similar between HSF1 and human iPS-derived cells (), and the percentage of Sox3+ cells that expressed Olig2 was comparable (59.1% ± 7.07% for HSF1 and 57.6 ± 9.88% for human iPS-derived cells). Further analysis was conducted with a combination of markers known to be specific to differentiated motor neurons. Within EBs that were specified towards a neural fate and expressed markers of motor neuron progenitors (Nkx6.1 and Olig2), a small but significant number of Islet1 and βIII- tubulin double-positive neurons were observed (). The physical limitations of the EB differentiation method precluded detailed functional analysis of these cells, but these results together provide evidence that both HSF1 and human iPS cells can be induced to generate differentiated motor neurons.
The directed differentiation of hESC and human iPS-derived cells recapitulates the stereotypical progression associated with motor neuron formation
To enable a physiological characterization of these iPS-derived motor neurons, we employed another method of directed differentiation using previously described adherent conditions [6
]. Neural rosettes generated from HSF1 and human iPS1, 2 and 18, were mechanically isolated, and then re-plated onto laminin-coated dishes in medium containing RA (1μM) and Shh (200ng/ml). After a week, neurotrophic factors were added (BDNF, CNTF, and GDNF; 10ng/ml each), the Shh concentration was lowered (50ng/ml), and the cells were allowed to differentiate for 3–5 weeks. Both HSF1 and human iPS-derived cells followed the expected course of differentiation, from Nestin-positive neuronal progenitors (), to mature motor neurons (βIII-tubulin, Choline acetyl transferase (ChAT) and Islet1-positive, ). In both HSF1 and human iPS derived βIII-tubulin-positive cells, similar percentage of Islet1-positive cells was detected (28.2% ± 5.7% for HSF1, 33.6% ± 12% for human iPS2)(), suggesting again that once specified to a neuronal fate, human iPS–derived cells and HSF1-derived cells are equally efficient at generating motor neurons in these conditions.
Neurons derived from human iPS cells and hESCs express several motor neuron marrkers
To further demonstrate that this differentiation protocol generates spinal cord neurons, cells from both HSF1 and human iPS cells were also stained with markers of various regions of the spinal cord and a reporter specific for activity of Hb9
), which encodes for a transcription factor specifically expressed by mature motor neurons [6
]. This Hb9-driven GFP reporter was transfected into HSF1 and human iPS derived cells to enable the identification and targeting of motor neurons in which Hb9
was transcriptionally active [6
]. Activity of this reporter tightly correlated with markers characteristic of rostral cervical motor neurons such as Hoxa5 and ChAT in both HSF1 and hiPS-derived cells ()[11
Lastly, to establish the phenotypic maturation of the human iPS-derived neurons we studied their electrophysiological properties. It is well established that the firing of repetitive action potentials in response to current injection is typical of the behavior of adult, vertebrate motor neurons [10
] and that this repetitive firing develops as function of maturation [18
]. The excitability of HSF1 and human iPS-derived motor neurons was assayed by whole cell patch clamping in the current clamp mode. Action potentials were recorded 48 to 62 days after plating. Upon application of current to either hESC or human iPS-derived neurons with Hb9::GFP activity, roughly half responded with single action potentials, while the other half responded with repetitive action potentials (). After recordings were made, the neurons were fixed and analyzed for Hb9::GFP expression and ChAT staining to confirm that those cells that generated a typical motor neuron response to electrical stimulation also possessed cholinergic properties (). Identical results were achieved with motor neurons derived from three independent human iPS cell lines and were indistinguishable from motor neurons derived from HSF1 (Fig. S3
). Together, these results demonstrate the general feasibility of the generating electrophysiologically active motor neurons from human iPS cells.
Neurons derived from human iPS cells and hESCs display mature motor neuron characteristics