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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.

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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.
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fig1: 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.

Mentions: The human genome contains 39 HOX genes divided among four clusters and classified into 13 paralogous groups based on sequence homology (Figure 1A). During embryonic stages of body axis elongation, newly formed tissues express HOX genes in sequential, contiguous domains consistent with their order in each cluster, i.e., colinearly. This phenomenon is evolutionarily conserved in bilaterian species and spatially assigns body segment-specific differentiation trajectories to axial progenitors of all three germ layers (Lewis, 1978). During formation of the posterior CNS, progenitors proximal to the node progressively transition from a 3′ to 5′ HOX expression profile as the primitive streak regresses (Iimura and Pourquié, 2006). This process produces nested and overlapping axial domains of HOX expression within the neuroepithelium of hindbrain rhombomeric (HOX1-5) and spinal cord cervical (HOX5-9), thoracic (HOX9-10), and lumbosacral (HOX10-13) vertebral segments (Figure 1A) (Philippidou and Dasen, 2013). The spatial variation of HOX expression along the rostrocaudal (R/C) axis of the posterior CNS diversifies the fates of neuroepithelial progeny and precisely restricts the development of specific neural subtypes to discrete axial positions (Philippidou and Dasen, 2013).


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)

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.
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Related In: Results  -  Collection

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Show All Figures
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fig1: 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.
Mentions: The human genome contains 39 HOX genes divided among four clusters and classified into 13 paralogous groups based on sequence homology (Figure 1A). During embryonic stages of body axis elongation, newly formed tissues express HOX genes in sequential, contiguous domains consistent with their order in each cluster, i.e., colinearly. This phenomenon is evolutionarily conserved in bilaterian species and spatially assigns body segment-specific differentiation trajectories to axial progenitors of all three germ layers (Lewis, 1978). During formation of the posterior CNS, progenitors proximal to the node progressively transition from a 3′ to 5′ HOX expression profile as the primitive streak regresses (Iimura and Pourquié, 2006). This process produces nested and overlapping axial domains of HOX expression within the neuroepithelium of hindbrain rhombomeric (HOX1-5) and spinal cord cervical (HOX5-9), thoracic (HOX9-10), and lumbosacral (HOX10-13) vertebral segments (Figure 1A) (Philippidou and Dasen, 2013). The spatial variation of HOX expression along the rostrocaudal (R/C) axis of the posterior CNS diversifies the fates of neuroepithelial progeny and precisely restricts the development of specific neural subtypes to discrete axial positions (Philippidou and Dasen, 2013).

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