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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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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.
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fig2: 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.

Mentions: Gouti et al. recently demonstrated that simultaneous induction of Wnt/β-catenin and FGF signaling can differentiate hPSCs to SOX2+/Brachyury(T)+ NMPs, but NMPs persisted for only 3 days before shifting to a mesodermal fate under their treatment regimen (Gouti et al., 2014). We observed a similar trend with simultaneous CHIR and FGF8b treatment inducing uniform Brachyury but also causing a sharp decrease in SOX2 expression (23% ± 0% SOX2+), indicating a mesodermal fate shift (Figure 2A, Route 1). Conversely, pre-treatment with FGF8b prior to CHIR (pre-FGF8b/CHIR) yielded uniform expression of both Brachyury and SOX2 that could be maintained (75%–100% SOX2+/Brachyury+) for 7 days (Figure 2A, Route 2, and Figure 2B). Thus, FGF signaling upstream of Wnt/β-catenin signaling effectively induces a stable NMP identity during hPSC differentiation.


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)

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

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

fig2: 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.
Mentions: Gouti et al. recently demonstrated that simultaneous induction of Wnt/β-catenin and FGF signaling can differentiate hPSCs to SOX2+/Brachyury(T)+ NMPs, but NMPs persisted for only 3 days before shifting to a mesodermal fate under their treatment regimen (Gouti et al., 2014). We observed a similar trend with simultaneous CHIR and FGF8b treatment inducing uniform Brachyury but also causing a sharp decrease in SOX2 expression (23% ± 0% SOX2+), indicating a mesodermal fate shift (Figure 2A, Route 1). Conversely, pre-treatment with FGF8b prior to CHIR (pre-FGF8b/CHIR) yielded uniform expression of both Brachyury and SOX2 that could be maintained (75%–100% SOX2+/Brachyury+) for 7 days (Figure 2A, Route 2, and Figure 2B). Thus, FGF signaling upstream of Wnt/β-catenin signaling effectively induces a stable NMP identity during hPSC differentiation.

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