Specification of region-specific neurons including forebrain glutamatergic neurons from human induced pluripotent stem cells

Abstract

Background: Directed differentiation of human induced pluripotent stem cells (hiPSC) into functional, region-specific neural cells is a key step to realizing their therapeutic promise to treat various neural disorders, which awaits detailed elucidation.

Methodology/principal findings: We analyzed neural differentiation from various hiPSC lines generated by others and ourselves. Although heterogeneity in efficiency of neuroepithelial (NE) cell differentiation was observed among different hiPSC lines, the NE differentiation process resembles that from human embryonic stem cells (hESC) in morphology, timing, transcriptional profile, and requirement for FGF signaling. NE cells differentiated from hiPSC, like those from hESC, can also form rostral phenotypes by default, and form the midbrain or spinal progenitors upon caudalization by morphogens. The rostrocaudal neural progenitors can further mature to develop forebrain glutamatergic projection neurons, midbrain dopaminergic neurons, and spinal motor neurons, respectively. Typical ion channels and action potentials were recorded in the hiPSC-derived neurons.

Conclusions/significance: Our results demonstrate that hiPSC, regardless of how they were derived, can differentiate into a spectrum of rostrocaudal neurons with functionality, which supports the considerable value of hiPSC for study and treatment of patient-specific neural disorders.

Figure 1. Comparison of neural induction from hiPSC and hESC.

(A) Morphological changes during neural differentiation from hiPSC. Left to right panels are a hiPSC colony (iPSC - referred to as day 0 of differentiation hereafter), a day-6 EB (d6), day-10 primitive NE cells (d10), and day-17 definitive NE cells (d17). (B) Low-density array for gene expression profile in H9 hESC and TZ1 hiPSC during their neural differentiation. Left to right lanes are day 0, 6, 10 and 17 samples as described in A. Green color refers to low gene expression (high ΔCt value) and red to high gene expression (low ΔCt value). (C) RT-PCR confirmation of the expression patterns of some pluripotent genes and neural differentiation genes. (D) Representative histograms for FACS analysis for ratio of PAX6⁺ cells differentiated from TZ1 and YZ1 at day 10. (E) Bar chart for FACS analysis for ratio of PAX6⁺ cells at three time points of neural differentiation from two hESC lines and four hiPSC lines. Data from multiple biological replicates are presented as mean ± standard deviation. N.A. stands for not available.

Figure 2. Requirement of FGF signaling for neural induction from hiPSC and hESC.

(A) Phase contrast images for EBs at day 8 of neural differentiation from H9 hESC and TZ1 hiPSC treated with 5 µM SU5402 or vehicle (Control) from days 4 through 8. (B) Decline of PAX6⁺ cell ratio detected by FACS at day 10 of neural differentiation from H9 and TZ1 cells treated with SU5402 or vehicle on the last 6 days. (C) The decline of PAX6⁺ cell ratio from B was analyzed statistically. Data are presented as mean ± standard deviation. n = 4. *P<0.05 versus the control group.

Figure 3. Differentiation of hiPSC/hESC-derived NE cells into region-specific neural progenitors.

(A) Schematic for protocols to generate region-specific neural progenitors. (B) RT-PCR analysis for expression of anterior-posterior neural marker genes at days 10 (d10) and 17 (d17) of neural differentiation from H9 hESC and TZ1 and YZ1 hiPSC lines. The day-17 cells were treated with RA (d17R) or FGF8 (d17F) for the last 7 days with untreated cells as a control (d17C). (C & D) Immunostaining for OTX2 and HOXB4 (C) or FOXG1 (D) on neural progenitors differentiated for 25 days from H9, TZ1, and YZ1. Cell nuclei were counterstained with Hoechst 33342. Bar, 50 µm. (E) The same staining on neural progenitors differentiated for 25 days from the 3 cell lines that were treated with RA.

Figure 4. Further differentiation of hiPSC/hESC-derived neural progenitors into region-specific neurons and astrocytes.

(A & B) Immunostaining for the forebrain functional markers TBR1 and MAP2 (A), and CTIP2 and VGLUT1 (B) on cells differentiated for 5 (A) or 6 (B) weeks from H9 hESC and TZ1 and YZ1 hiPSC lines. (C) Immunostaining for the dopaminergic neuron marker TH on cells differentiated from hESC/hiPSC-derived and FGF8/SHH-treated neural progenitors. (D) Immunostaining for the spinal motor neuronal marker HB9 (with βIII-tubulin as a neuronal control marker) on cells differentiated from hESC/hiPSC-derived and RA/SHH-treated neural progenitors. (E) Some cells were positive for S100β (an astrocyte marker) or Synapsin at two months after differentiation from H9 or TZ1 cells. Cell nuclei were counterstained with Hoechst 33342. Bar, 50 µm. (F) Percentage of cells immunostained positive for TBR1, HB9, and TH counted for A, C, and D, respectively.

Figure 5. hiPSC-derived neurons are functional in vitro.

(A) Action potentials (APs) were observed, representative voltage responses to a 20 pA current injection are shown for neurons following 6 weeks of differentiation from H9 and TZ1 cells in the basic neural induction condition without exogenous morphogens. (B) (i) Rapidly activating and inactivating voltage-gated inward currents were elicited by depolarizing to various voltages from a holding potential of −100 mV. (ii) The inward currents were completely blocked by TTX (1 µM). (iii) TTX-sensitive Na⁺ current in H9 and TZ1 cells. (C) (i) Representative traces showing fast inactivating and sustained-outward currents elicited by voltage steps from a holding potential of −100 mV. 4AP (1 mM) eliminated the fast inactivating K⁺ current, and TEA (0.5 mM) blocked the sustained currents. (ii) 4AP-sensitive K⁺ currents. (iii) TEA-sensitive K⁺ currents. Values of the electrophysiological parameters detected in representative neurons differentiated from both H9 and TZ1 cells are shown in Table S3.