Limits...
Endogenous WNT signaling regulates hPSC-derived neural progenitor cell heterogeneity and specifies their regional identity.

Moya N, Cutts J, Gaasterland T, Willert K, Brafman DA - Stem Cell Reports (2014)

Bottom Line: However, current protocols for differentiating NPCs toward neuronal lineages result in a mixture of neurons from various regions of the CNS.In this study, we determined that endogenous WNT signaling is a primary contributor to the heterogeneity observed in NPC cultures and neuronal differentiation.Furthermore, exogenous manipulation of WNT signaling during neural differentiation, through either activation or inhibition, reduces this heterogeneity in NPC cultures, thereby promoting the formation of regionally homogeneous NPC and neuronal cultures.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Medicine, Stem Cell Program, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0695, USA.

Show MeSH

Related in: MedlinePlus

Analysis of Neurons Derived from CHIR- and IWP2-Treated and Untreated NPC Cultures(A) IF of mature neuronal markers B3T in neuronal cultures differentiated from 500 nM CHIR- and 1,000 nM IWP2-treated and untreated NPC cultures derived from H9 hPSCs (scale bar, 100 μm).(B) IF of cortical-, forebrain-, midbrain-, hindbrain-, and spinal-cord-related neuronal genes in neurons differentiated from 500 nM CHIR- and 1,000 nM IWP2-treated and untreated NPC cultures derived from H9 hPSCs (scale bar, 100 μm).(C) Expression of cortical-, forebrain-, midbrain-, hindbrain-, and spinal-cord-related neuronal genes in neurons differentiated from 500 nM CHIR- and 1,000 nM IWP2-treated and untreated NPC cultures derived from H9, HUES9, HES3, and RiPSC hPSCs (mean ± SEM, n = 4 independent experiments). Populations were compared with neurons differentiated from untreated (N) NPCs using Student’s t test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4264562&req=5

fig5: Analysis of Neurons Derived from CHIR- and IWP2-Treated and Untreated NPC Cultures(A) IF of mature neuronal markers B3T in neuronal cultures differentiated from 500 nM CHIR- and 1,000 nM IWP2-treated and untreated NPC cultures derived from H9 hPSCs (scale bar, 100 μm).(B) IF of cortical-, forebrain-, midbrain-, hindbrain-, and spinal-cord-related neuronal genes in neurons differentiated from 500 nM CHIR- and 1,000 nM IWP2-treated and untreated NPC cultures derived from H9 hPSCs (scale bar, 100 μm).(C) Expression of cortical-, forebrain-, midbrain-, hindbrain-, and spinal-cord-related neuronal genes in neurons differentiated from 500 nM CHIR- and 1,000 nM IWP2-treated and untreated NPC cultures derived from H9, HUES9, HES3, and RiPSC hPSCs (mean ± SEM, n = 4 independent experiments). Populations were compared with neurons differentiated from untreated (N) NPCs using Student’s t test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Mentions: We subsequently investigated whether NPCs generated with IWP2 or CHIR treatment retained their regional phenotype upon differentiation to neurons. IWP2- and CHIR-treated NPCs were expanded for four passages and then subjected to the neuronal differentiation protocol. IF (Figure 5A) and gene expression (Figure S5G) revealed that IWP2- and CHIR-treated NPCs generated similar numbers of B3T+ neurons compared with untreated NPCs. However, neurons generated from IWP2-treated NPCs expressed higher levels of the forebrain- and cortical-related neuronal markers FOXG1, TBR1, SATB2, CTIP2, EMX1, CUX1, and OTX2 compared with neurons generated from CHIR-treated or -untreated NPCs (Figures 5B and 5C). On the other hand, neurons generated from CHIR-treated NPCs expressed higher levels of the hindbrain- and spinal-cord-specific markers HOXA2, HOXB4, HOXB6, and MNX1 (Figures 5B and 5C). Together, our results indicate that NPCs retain their regional identity over multiple passages and manifest that identity in differentiated neuron cultures.


Endogenous WNT signaling regulates hPSC-derived neural progenitor cell heterogeneity and specifies their regional identity.

Moya N, Cutts J, Gaasterland T, Willert K, Brafman DA - Stem Cell Reports (2014)

Analysis of Neurons Derived from CHIR- and IWP2-Treated and Untreated NPC Cultures(A) IF of mature neuronal markers B3T in neuronal cultures differentiated from 500 nM CHIR- and 1,000 nM IWP2-treated and untreated NPC cultures derived from H9 hPSCs (scale bar, 100 μm).(B) IF of cortical-, forebrain-, midbrain-, hindbrain-, and spinal-cord-related neuronal genes in neurons differentiated from 500 nM CHIR- and 1,000 nM IWP2-treated and untreated NPC cultures derived from H9 hPSCs (scale bar, 100 μm).(C) Expression of cortical-, forebrain-, midbrain-, hindbrain-, and spinal-cord-related neuronal genes in neurons differentiated from 500 nM CHIR- and 1,000 nM IWP2-treated and untreated NPC cultures derived from H9, HUES9, HES3, and RiPSC hPSCs (mean ± SEM, n = 4 independent experiments). Populations were compared with neurons differentiated from untreated (N) NPCs using Student’s t test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4264562&req=5

fig5: Analysis of Neurons Derived from CHIR- and IWP2-Treated and Untreated NPC Cultures(A) IF of mature neuronal markers B3T in neuronal cultures differentiated from 500 nM CHIR- and 1,000 nM IWP2-treated and untreated NPC cultures derived from H9 hPSCs (scale bar, 100 μm).(B) IF of cortical-, forebrain-, midbrain-, hindbrain-, and spinal-cord-related neuronal genes in neurons differentiated from 500 nM CHIR- and 1,000 nM IWP2-treated and untreated NPC cultures derived from H9 hPSCs (scale bar, 100 μm).(C) Expression of cortical-, forebrain-, midbrain-, hindbrain-, and spinal-cord-related neuronal genes in neurons differentiated from 500 nM CHIR- and 1,000 nM IWP2-treated and untreated NPC cultures derived from H9, HUES9, HES3, and RiPSC hPSCs (mean ± SEM, n = 4 independent experiments). Populations were compared with neurons differentiated from untreated (N) NPCs using Student’s t test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.
Mentions: We subsequently investigated whether NPCs generated with IWP2 or CHIR treatment retained their regional phenotype upon differentiation to neurons. IWP2- and CHIR-treated NPCs were expanded for four passages and then subjected to the neuronal differentiation protocol. IF (Figure 5A) and gene expression (Figure S5G) revealed that IWP2- and CHIR-treated NPCs generated similar numbers of B3T+ neurons compared with untreated NPCs. However, neurons generated from IWP2-treated NPCs expressed higher levels of the forebrain- and cortical-related neuronal markers FOXG1, TBR1, SATB2, CTIP2, EMX1, CUX1, and OTX2 compared with neurons generated from CHIR-treated or -untreated NPCs (Figures 5B and 5C). On the other hand, neurons generated from CHIR-treated NPCs expressed higher levels of the hindbrain- and spinal-cord-specific markers HOXA2, HOXB4, HOXB6, and MNX1 (Figures 5B and 5C). Together, our results indicate that NPCs retain their regional identity over multiple passages and manifest that identity in differentiated neuron cultures.

Bottom Line: However, current protocols for differentiating NPCs toward neuronal lineages result in a mixture of neurons from various regions of the CNS.In this study, we determined that endogenous WNT signaling is a primary contributor to the heterogeneity observed in NPC cultures and neuronal differentiation.Furthermore, exogenous manipulation of WNT signaling during neural differentiation, through either activation or inhibition, reduces this heterogeneity in NPC cultures, thereby promoting the formation of regionally homogeneous NPC and neuronal cultures.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Medicine, Stem Cell Program, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0695, USA.

Show MeSH
Related in: MedlinePlus