Limits...
Deterministic HOX patterning in human pluripotent stem cell-derived neuroectoderm.

Lippmann ES, Williams CE, Ruhl DA, Estevez-Silva MC, Chapman ER, Coon JJ, Ashton RS - Stem Cell Reports (2015)

Bottom Line: 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.This fully defined approach significantly expands capabilities to derive regional neural phenotypes from diverse hindbrain and spinal cord domains.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering, University of Wisconsin, Madison, WI 53706, USA; Wisconsin Institute for Discovery, University of Wisconsin, Madison, WI 53706, USA.

Show MeSH

Related in: MedlinePlus

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.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4400649&req=5

fig4: 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.

Mentions: We used MN differentiation to evaluate whether the regional specification imparted by our HOX patterning approach yielded characteristic differences in the neuroectoderm progeny. First, we verified that MNs derived from neuroectoderm of discrete hindbrain domains likewise exhibited distinct HOX signatures. Hindbrain neuromesoderm was propagated for 4, 12, and 24 hr before conversion to ventralized neuroectoderm using RA, sonic hedgehog (SHH) and purmorphamine (PM, a sonic hedgehog agonist) exposure and further differentiation to MNs using media containing RA and neurotrophic factors (NTFs). MN differentiation was verified by the presence of cells co-expressing βIII-tubulin/ISL1 and SMI-32 reactive non-phosphorylated neurofilament heavy chain with ISL1 or synapsin (Amoroso et al., 2013; Li et al., 2005) (Figure 4A). Immunocytochemical analysis of HB9+ MNs generated from 4 hr neuromesoderm revealed no co-expression of HOXB1 or HOXB4. However, HB9+ MNs generated from 12 hr neuromesoderm predominantly co-expressed HOXB1 but not HOXB4, and those generated from 24 hr neuromesoderm predominantly co-expressed both HOXB1 and HOXB4 (Figure 4A). Also, within all cultures, many ISL1+ cells co-expressed PHOX2B indicative of hindbrain identity (Maury et al., 2014; Pattyn et al., 2000). Thus, MNs derived from hindbrain neuroectoderm with discrete Hox gene and protein profiles (Figure 3C) likewise express distinct HOX profiles, although the precise regional identity of these MNs based on their HOX signatures is currently unclear.


Deterministic HOX patterning in human pluripotent stem cell-derived neuroectoderm.

Lippmann ES, Williams CE, Ruhl DA, Estevez-Silva MC, Chapman ER, Coon JJ, Ashton RS - Stem Cell Reports (2015)

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.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4400649&req=5

fig4: 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.
Mentions: We used MN differentiation to evaluate whether the regional specification imparted by our HOX patterning approach yielded characteristic differences in the neuroectoderm progeny. First, we verified that MNs derived from neuroectoderm of discrete hindbrain domains likewise exhibited distinct HOX signatures. Hindbrain neuromesoderm was propagated for 4, 12, and 24 hr before conversion to ventralized neuroectoderm using RA, sonic hedgehog (SHH) and purmorphamine (PM, a sonic hedgehog agonist) exposure and further differentiation to MNs using media containing RA and neurotrophic factors (NTFs). MN differentiation was verified by the presence of cells co-expressing βIII-tubulin/ISL1 and SMI-32 reactive non-phosphorylated neurofilament heavy chain with ISL1 or synapsin (Amoroso et al., 2013; Li et al., 2005) (Figure 4A). Immunocytochemical analysis of HB9+ MNs generated from 4 hr neuromesoderm revealed no co-expression of HOXB1 or HOXB4. However, HB9+ MNs generated from 12 hr neuromesoderm predominantly co-expressed HOXB1 but not HOXB4, and those generated from 24 hr neuromesoderm predominantly co-expressed both HOXB1 and HOXB4 (Figure 4A). Also, within all cultures, many ISL1+ cells co-expressed PHOX2B indicative of hindbrain identity (Maury et al., 2014; Pattyn et al., 2000). Thus, MNs derived from hindbrain neuroectoderm with discrete Hox gene and protein profiles (Figure 3C) likewise express distinct HOX profiles, although the precise regional identity of these MNs based on their HOX signatures is currently unclear.

Bottom Line: 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.This fully defined approach significantly expands capabilities to derive regional neural phenotypes from diverse hindbrain and spinal cord domains.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering, University of Wisconsin, Madison, WI 53706, USA; Wisconsin Institute for Discovery, University of Wisconsin, Madison, WI 53706, USA.

Show MeSH
Related in: MedlinePlus