Deterministic HOX patterning in human pluripotent stem cell-derived neuroectoderm

Abstract

Colinear HOX expression during hindbrain and spinal cord development diversifies and assigns regional neural phenotypes to discrete rhombomeric and vertebral domains. Despite the precision of HOX patterning in vivo, in vitro approaches for differentiating human pluripotent stem cells (hPSCs) to posterior neural fates coarsely pattern HOX expression thereby generating cultures broadly specified to hindbrain or spinal cord regions. Here, we demonstrate that successive activation of fibroblast growth factor, Wnt/β-catenin, and growth differentiation factor signaling during hPSC differentiation generates stable, homogenous SOX2(+)/Brachyury(+) neuromesoderm that exhibits progressive, full colinear HOX activation over 7 days. Switching to retinoic acid treatment at any point during this process halts colinear HOX activation and transitions the neuromesoderm into SOX2(+)/PAX6(+) neuroectoderm with predictable, discrete HOX gene/protein profiles that can be further differentiated into region-specific cells, e.g., motor neurons. This fully defined approach significantly expands capabilities to derive regional neural phenotypes from diverse hindbrain and spinal cord domains.

Graphical abstract

Figure 1

Wnt/β-Catenin and FGF Signaling Synergistically Coordinate HOX Activation during hPSC Differentiation (A) HOX paralog expression in the hindbrain and spinal cord color-coded to the region where its expression is first detected (modified from a previous report; Philippidou and Dasen, 2013). (B) Timeline of H9 hESC differentiation corresponding to (C)–(E). (C) RT-PCR analysis of cultures at days 2–5. (D) qPCR analysis at day 5. ^∗p < 0.005; ^∗∗p < 0.001. (E) OTX2 and SOX2 expression at day 5 after RA or CHIR treatment. Scale bars, 100 μm. Adjacent images are the same field. (F) qPCR analysis at day 2 using the H9 ishcat2 line with or without doxycycline treatment. ^∗p < 0.02; ^∗∗p < 0.005. (G) qPCR analysis at day 5 using the H9 ishcat2 line with or without doxycycline treatment. ^∗p < 0.02; ^∗∗p < 0.0001. All qPCR data are presented as mean ± SD calculated from independent biological triplicates. H9 data are normalized to the condition yielding maximum expression and ishcat2 data are normalized to each doxycycline-free condition. Statistical significance was calculated using the Student’s unpaired t test.

Figure 2

Colinear HOX Expression in hPSC-Derived NMPs (A) Analysis of the NMP phenotype by flow cytometry and immunocytochemistry. Gray histogram, immunoglobulin G (IgG) control; red histogram, label of interest. Scale bars, 100 μm. (B) Optimized NMP propagation scheme. The addition of CHIR is denoted as t = 0 hr in all panels. Purity of NMPs was assessed by flow cytometry in the presence or absence of GDF11 (data are presented as mean ± SD). qPCR analysis of HOXD10 and HOXD11 was conducted in the presence or absence of GDF11 (expression normalized to the time point of maximum expression). (C) Schematic for HOX induction in NMPs. “t” indicates time under Wnt, FGF, and GDF signaling. (D) qPCR analysis of colinear HOX expression normalized to the time point of maximum expression. GDF11 was included according to (B). qPCR data in all panels are presented as mean ± SD calculated from technical duplicates. Full profiles for all analyzed genes can be found in Figure S2A. For all flow cytometry data, a minimum of two biological replicates was used to calculate mean ± SD.

Figure 3

RA Induces a Neuroectodermal Fate and Halts Colinear HOX Activation (A) NMPs exposed to RA or CHIR acquire a neuroectodermal or mesodermal fate as assessed by flow cytometry at day 6 (gray histogram, IgG control; red histogram, antigen of interest). (B) Schematic for transition from NMPs to neuroectoderm by RA treatment. “t” indicates time under Wnt, FGF, and GDF signaling prior to RA treatment, which halts HOX progression to yield a defined rostrocaudal identity. (C and D) Representative hindbrain (C) and spinal cord (D) cultures assessed by qPCR, immunocytochemistry, and flow cytometry. For all plots, flow cytometry data are presented as mean ± SD from biological duplicates, and qPCR data are mean ± SD from technical duplicates normalized to the time point of maximum expression for each gene. For hindbrain cultures, HOXB1 and HOXB4 were quantified by immunocytochemistry relative to DAPI⁺ nuclei (technical triplicates per time point, minimum 10,000 cells counted, and relative HOX expression patterns were qualitatively verified across biological duplicates), while SOX2 and PAX6 were quantified by flow cytometry. For spinal cord cultures, HOXD10 was quantified by immunocytochemistry relative to DAPI⁺ nuclei (technical duplicates, >2,000 cells counted), while SOX2, PAX6, and HOXB4 were quantified by flow cytometry. Scale bars, 100 μm. For (D), dorsomorphin was included with GDF11 and throughout RA treatment (further details in Figure S3). (E) Mass spectrometry comparison of HOX profiles in cervical, thoracic, and lumbar neuroectoderm cultures. Cervical differentiation: 1 day FGF8b, 2 days FGF8b/CHIR, 4 days RA; thoracic differentiation: 1 day FGF8b, 6 days FGF8b/CHIR, 4 days RA; lumbar differentiation: 1 day FGF8b, 4 days FGF8b/CHIR, 2 days FGF8b/CHIR/GDF11, 4 days RA. Biological triplicates (neuroepithelium isolated individually from three separate culture wells) were used to calculate statistical significance between cervical, thoracic, and lumbar samples. Data are presented as mean ± SD and statistical significance was calculated using the Student’s unpaired t test. ^∗p < 0.01; ^∗∗p < 0.002; ^∗∗∗p < 0.0001.

Figure 4

Derivation of Region-Specific MNs (A) Neuronal maturation from various hindbrain locales. NMPs were propagated for 4, 12, or 24 hr before conversion to ventralized neuroectoderm and differentiation to neurons. DAPI (blue) is overlaid in most images. Scale bars, 20 μm. Quantified data are presented as mean ± SD (four technical replicates, >100 HB9⁺ cells counted per sample). (B) Evaluation of FOXP1 columnar identity in ISL1⁺ and HB9⁺ MNs. Cervical differentiation (C): 1 day FGF8b, 2 days FGF8b/CHIR; thoracic differentiation (T): 1 day FGF8b, 5 days FGF8b/CHIR; lumbar differentiation (L): 1 day FGF8b, 4 days FGF8b/CHIR, 2 days FGF8b/CHIR/GDF11; prior to en bloc passage and further differentiation as indicated. NTFs, neurotrophic factors (described in Experimental Procedures). For plotted data, open circles are percentages from individual fields and bars indicate the mean. Statistical significance was calculated using the Student’s unpaired t test (three to five fields were counted across three biological replicates for each condition, minimum 2,500 cells counted). n.s., p > 0.02; ^∗∗p < 0.000001. Scale bars, 100 μm. (C) Neuronal maturation from cervical, thoracic, and lumbar patterned neuroectoderm corresponding to the NMP state described in (B). DAPI is overlaid in all images. Scale bars, 20 μm. RT-PCR (50 cycles) demonstrates maintenance of regional identity.