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First published online April 10, 2009
doi: 10.1242/10.1242/dev.029447

Development 136, 1443-1452 (2009)
Published by The Company of Biologists 2009
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Human oligodendrocytes from embryonic stem cells: conserved SHH signaling networks and divergent FGF effects

Bao-Yang Hu*, Zhong-Wei Du*, Xue-Jun Li, Melvin Ayala and Su-Chun Zhang{dagger}

Departments of Anatomy and Neurology, School of Medicine and Public Health, Waisman Center, University of Wisconsin-Madison, 1500 Highland Avenue, Madison, WI 53705, USA.


Figure 1
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  		Fig. 1. SHH-dependent OLIG2 expression for hESC differentiation to OPCs. (A) Experimental paradigm showing differentiation of OLIG2 progenitors and OPCs. (B) OLIG2-expressing progenitors and OLIG2+ PDGFR{alpha}+ OPCs are present in the control, SHH-treated and purmorphamine (Pur)-treated cultures, but rarely in the cyclopamine (Cyclo)-treated cultures at day 35 (5th week) and day 98 (14th week). (C) Time course of OLIG2 expression in response to Pur (1 µM) and SHH (100 ng/ml). *P=0.011, 0.008 and 0.014 at day 18, 23 and 28, respectively. (D) PDGFR{alpha}+ OPCs first appear after 10 weeks of differentiation and reach a plateau at the fifteenth week. (E) Comparative effect of SHH, purmorphamine and cyclopamine on the efficiency of OPC generation at the fifteenth week. *P<0.0001. The percentage PDGFR{alpha} in D and E represents the proportion of PDGFR{alpha}+ cells among total cells. (F) OLIG2 RNAi cells do not express PDGFR{alpha} (arrows), whereas control RNAi cells are positive for PDGFR{alpha} (arrowheads). (G) Quantification of the effect of OLIG2 RNAi on the percentage of PDGFR{alpha}+ cells. Cells infected with the control RNAi virus (GFP+) generated a similar proportion of PDGFR{alpha}-expressing cells to those without viral infection (GFP-), whereas the cells infected with OLIG2 RNAi virus (GFP+) generated fewer PDGFR{alpha}+ OPCs than the non-infected population (GFP-). Ho, Hoechst 33258-stained nuclei. Scale bars: 50 µm.

 

Figure 2
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  		Fig. 2. SHH-dependent induction of pre-OPCs. (A) Experimental design showing three groups treated with or without purmorphamine (Pur) or cyclopamine (Cyclo) from day 24 to 35. (B) OLIG2 and NKX2.2 are expressed at day 24, but not in the same cells. At day 35, OLIG2 and NKX2.2 co-express only in cultures with SHH or purmorphamine. Scale bar: 50 µm.

 

Figure 3
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  		Fig. 3. Requirement of SHH signaling for maintenance of OLIG2 expression. (A) Experimental design showing OLIG2-enriched cells treated with purmorphamine or cyclopamine until day 50. (B) At day 50, an OLIG2+ NKX2.2+ population is present in cultures with or without purmorphamine, but not in cultures with cyclopamine. PDGFR{alpha}+ OPCs appear in cultures with or without purmorphamine, but not in cultures with cyclopamine, at day 100. (C) Quantitative analysis showing the proportion of OLIG2+ NKX2.2+ cells (*P=0.023 between groups with and without purmorphamine; *P=0.0015 between groups without purmorphamine and with cyclopamine) at day 50. (D) Proportion of PDGFR{alpha}+ OPCs at the fourteenth week. *P=0.035 between groups with and without purmorphamine; *P<0.0001 between groups without purmorphamine and with cyclopamine. Scale bar: 50 µm.

 

Figure 4
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  		Fig. 4. FGF2 inhibits motoneuron differentiation and increases pre-OPCs. (A) Experimental design showing that the OLIG2+ cells at day 24 are further cultured for 12 days in the presence of purmorphamine or FGF2 alone, or in a combination of purmorphamine and FGF2 or EGF. (B) FGF2, but not EGF, blocks HB9 expression (column 2). (C) In the presence of purmorphamine, FGF2 but not EGF increases the proportion of OLIG2+ cells by ~50% and decreases the HB9+ population to less than 5%, whereas EGF moderately decreases HB9+ cells. (D) Neither FGF2 nor EGF selectively alters the Ki67+ proportion in OLIG2+ cells. (E) There is a moderate increase in Ki67+ cells in FGF2-treated (for 10 days) cultures. *P<0.05 between the paired groups. Scale bar: 50 µm.

 

Figure 5
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  		Fig. 5. FGF2 inhibits the transition of pre-OPCs to OPCs by blocking co-expression of OLIG2 and NKX2.2. (A) Experimental design showing that the gliogenic OLIG2 progenitors at day 35 are treated with or without FGF2 and then examined at day 50 or day 98. (B) At day 50 (left column), the majority of the cells co-express OLIG2 and NKX2.2, whereas few double-positive cells are present in cultures with FGF2. At day 98, cultures without FGF2 (upper row) contain mostly PDGFR{alpha}+ cells with few cells positive for βIII-tubulin or GFAP. In the presence of FGF2, the majority of cells are positive for βIII-tubulin or GFAP (lower row). Scale bar: 100 µm. (C) A higher proportion of cells expressing OPC markers (percentage PDGFR{alpha}+, SOX10+ or NG2+ cells among total cells) is generated in the absence of FGF2 at day 98. (D) The population of PDGFR{alpha}+, βIII-tubulin+ and GFAP+ cells in the presence or absence of FGF2 at day 98. (E) FGF2 treatment results in a lower proportion of OLIG2+ NKX2.2+ cells at day 50. *P=0.048, **P=0.0018; n=3. (F) FACS analysis at day 50 shows a slight decrease (10%) in the OLIG2+ population and a leftward shift (indicating decreased expression level in individual cells) for the group with FGF2 (blue) compared with that without FGF2 (red). (G) Quantitative RT-PCR analysis at day 50 showing that FGF2 decreases SHH expression by half and increases the transcription of GLI2 and GLI3.

 

Figure 6
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  		Fig. 6 Maturation and myelination of hESC-derived OPCs. (A) Summary of procedures for directed differentiation of OPCs from hESCs. (B) Bipolar cells are present surrounding the progenitor cluster 3 days after plating. (C) Dissociated OPCs exhibit a bipolar morphology. (D-G) The OPCs co-express OLIG2+ NKX2.2+ (D), SOX10+ PDGFR{alpha}+ (E), NKX2.2+ PDGFR{alpha}+ (F) and OLIG2+ NG2+ (G). (H,I) Six weeks following OPC differentiation, cells exhibit multipolar and web-like processes and express O4. (J) After an additional month, some cells express MBP. (K) Three months after transplantation, the grafted cells, as revealed by hNA and MBP, preferentially localize in the corpus callosum (CC, outlined). (L) All of the human cells are positive for OLIG2. (M) The human cells exhibit multiple MBP+ processes. (N) Confocal analysis shows adjacent expression of MBP+ human cell fibers with NF+ axons in the corpus callosum. (O) The hNA+ cells are negative for GFAP. (P) Electron micrograph showing a typical oligogodendrocyte surrounded by many myelin profiles in the grafted brain. (Q) Higher magnification from P showing compact myelin sheaths in the grafted brain with identifiable major dense lines (inset). (R) Electron micrograph showing thin non-compact sheaths wrapping around axons in the corpus callusom of a shiverer mouse without transplant. Magnification: P, 6000x; Q,R, 20,000x; Q inset, 60,000x. Scale bars: 50 µm in B-I,K-O; 10 µm in J.

 

Figure 7
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  		Fig. 7. Regulation of OPC specification from hESCs by SHH and FGF2. Human ESCs (hESCs) are differentiated to neuroepithelial (NE) cells without exogenous growth factors (1), then to OLIG2-expressing ventral spinal progenitors (2) and OLIG2+ NKX2.2+ pre-OPCs (3) before becoming SOX10+ PDGFR{alpha}+ OPCs (4). The induction of OLIG2 progenitors (2) and pre-OPCs (3) is largely dependent upon exogenous SHH (thick arrows). SHH produced in the culture (thin arrow) is sufficient for the transition of pre-OPCs to OPCs (4). FGF2 increases the pre-OPCs during the neurogenic phase through inhibition of motoneuron differentiation (3), but blocks the transition of pre-OPCs to OPCs by inhibiting the SHH-dependent co-expression of OLIG2 and NKX2.2 in pre-OPCs (4).

 

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© The Company of Biologists Ltd 2009