A rosette-type, self-renewing human ES cell-derived neural stem cell with potential for in vitro instruction and synaptic integration - PubMed (original) (raw)

Fig. 2.

lt-hESNSCs express transcription factors compatible with an anterior ventral hindbrain fate and maintain rosette-properties. lt-hESNSCs cultured for >15 passages exhibit a posterior identity corresponding to an anterior hindbrain location. (A) Telencephalic markers FoxG1, Emx1, Emx2, Otx2 Gsh2, and Nkx2.1 are not detectable by RT-PCR. Instead, lt-hESNSCs show prominent expression of the anterior hindbrain markers Gbx2, HoxA2, HoxB2, and Krox20. More posterior markers (e.g., HoxB6) are absent. In the dorso-ventral axis, lt-hESNSCs express markers compatible with a ventral hindbrain localization (B, D–F), that is, Irx3, Pax6 (B and D), Nkx6.1 (B and F) and Nkx2.2 (B and E); note lacking expression of the dorsal marker Pax7. Differentiated neurons express transcription factors found in V0–V3 interneurons and pMN-derived motoneurons (C, G–K) including Lim1/2 (G), En1 (H), Lim3 (I), Isl1 (J), and HB9 (K). Neuronal identity was confirmed by costaining for beta III-tubulin (green, G–K). (L) Rosettes generated from plated EBs and lt-hESNSCs (passage 48) express NSC markers at comparable levels. (N) lt-hESNSCs and rosettes share expression of PLZF, DACH1, MMRN1, PLAGL1, NF2F1, DMTR3, and LMO3, genes previously described as “rosette-specific”. (M) lt-hESNSCs do not express PMP2, AQP4, SPARCL1, S100beta, or HOP, genes proposed to be characteristic of FGF/EGF-expanded cells with limited patterning potential. (O) Some rosette-specific genes were no longer expressed in lt-hESNSCs, including FAM70A, EVI1, ZNF312 LIX1, and RSPO3. (P and Q) lt-hESNSCs in passage 22 (P) or passage 76 (Q) show nuclear expression of PLZF, a columnar growth pattern and apical nuclei as well as ZO-1 expression, which was particularly accentuated at the apical and lateral surface of clustered lt-hESNSCs. At high density, lt-hESNSCs form typical rosette-like structures with pronounced central expression of ZO-1 (R). (S) lt-hESNSCs show homogeneous nuclear expression of DACH1 [cells in (R) and (S) are from passage 52]. (Scale bars, D, E, G–K, and S: 50 μm; F: 100 μm; P–R: 25 μm.)