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

Abstract

An intriguing question in human embryonic stem cell (hESC) biology is whether these pluripotent cells can give rise to stably expandable somatic stem cells, which are still amenable to extrinsic fate instruction. Here, we present a pure population of long-term self-renewing rosette-type hESC-derived neural stem cells (lt-hESNSCs), which exhibit extensive self-renewal, clonogenicity, and stable neurogenesis. Although lt-hESNSCs show a restricted expression of regional transcription factors, they retain responsiveness to instructive cues promoting the induction of distinct subpopulations, such as ventral midbrain and spinal cord fates. Using lt-hESNSCs as a donor source for neural transplantation, we provide direct evidence that hESC-derived neurons can establish synaptic connectivity with the mammalian nervous system. Combining long-term stability, maintenance of rosette-properties and phenotypic plasticity, lt-hESNSCs may serve as useful tool to study mechanisms of human NSC self-renewal, lineage segregation, and functional in vivo integration.

Fig. 1.

Long term self-renewing neural stem cells generated from human embryonic stem cells (lt-hESNSCs). These cells can be continuously propagated as a highly homogenous population forming rosette-like patterns (A, low density; B, high density). In addition to nestin (C), lt-hESNSCs express Sox2 (D), and Sox1 (E). (F) Representative karyotype from lt-hESNSCs (derived from H9.2) in passage 50. Fluorescence in situ hybridization (FISH) studies were performed to screen for trisomy of chromosomes 12 and 17, alterations frequently observed during long-term propagation of undifferentiated hESC (G). RT-PCR analysis reveals high levels of telomerase expression and expression of the neuroepithelial markers Sox1 and Pax6 (H). Upon growth factor withdrawal, lt-hESNSCs give rise to a dominant fraction of neurons expressing beta III-tubulin (I–K), MAP2ab (I and L) and NeuN (I and M). Prolonged differentiation (>2 weeks) promotes differentiation into astrocytes positive for GFAP (I and N) or S100 beta (I) and oligodendrocytes expressing 04 (I and O). Despite continuous passaging, lt-hESNSCs retain a stable neurogenic differentiation potential (I, bar graph depicts percentages of immunoreactive cells in passages 10, 25, 50, and 75 after 28 days of differentiation). The large majority of lt-hESNSC-derived neurons display a GABAergic phenotype, staining positively for GABA (P and Q) and GAD67 (Q). Only occasional neurons exhibit glutamatergic (R) or serotoninergic (S) phenotypes. Immunofluorescence data show representative pictures of lt-hESNSCs (passages 21–51) derived from H9.2. Representative RT-PCR data are from lt-hESNSCs at passages 24–34. (Scale bars, A–E, O, R, and S 50 μm; J–M, and Q: 100 μm; N and P: 200 μm.).

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.)

Fig. 3.

lt-hESNSCs remain responsive to instructive regionalization cues. Schematic representation of the experimental protocol (A). Compared with control cells treated with FGF2/EGF/B27low (B), cells treated with Shh/FGF8 show prominent nuclear immunoreactivity for En1 (C). After growth factor withdrawal, they give rise to large numbers of TH-positive neurons (D), which coexpress DAT (E) but lack GABA (F). Many of the TH expressing neurons coexpress En1 (G). (H and I) Shh/FGF8-mediated induction of midbrain-specific transcripts for En1, Lmx1a, Lmx1b, Pax2, Nurr1, TH, Pitx3, and AADC in proliferating (H) and differentiating (I) lt-hESNSCs. Fetal brain and lt-hESNSCs treated with FGF2/EGF/B27low only were used as controls. (J) SHH/FGF8-mediated induction of TH-positive neurons is stable across the passages. (K and L) Retinoic acid (RA)-exposed lt-hESNSCs show induction of more posterior Hox genes including HoxB1, HoxB4, HoxB6, and HoxC5. On the protein level, numbers of cells with nuclear HoxB4 expression increases from <0.05% to >70% (K). (M) Exposure of RA-treated cells to SHH induces nuclear expression of Olig2 and Nkx2.2 [compared with only single Olig2(+) cells in the control (insert; arrow)]. These cells differentiate into large numbers of Isl1(+) neurons (N), many of which express the motoneuron-specific antigen HB9 (O). This induction of HB9(+) motoneurons is independent of the passage number (P). (Scale bars, B, C, F, K, and O: 25 μm; D: 200 μm; E, G, M, and N: 50 μm.)

Fig. 4.

Functional Integration of lt-hESNSC-derived neurons in vivo. Four months after transplantation lt-hESNSC-derived neurons were found in a variety of brain regions, where they exhibited complex neuronal morphologies. (A) Representative example showing GFP-positive cells in the host striatum. (B) Biocytin-filled neuron (red) in the host thalamus, double labeled with an antibody to human nuclei (green). (C–E) Representative traces from electrophysiological analysis of a lt-hESNSC-derived neuron in the host thalamus, recorded 18 weeks after transplantation into the brain of 1-day-old SCID/beige mice (E). Incorporated neurons exhibited large voltage-dependent inward and complex outward currents, and were able to fire repetitive action potentials. Notably, they also received spontaneous synaptic input (right traces). Immunofluorescence revealed colocalization of biocytin (diffused from the patch pipette into the recorded cell) and EGFP, confirming the donor cell nature of the recorded cells (C and D). (Scale bars: A and B, 30 μm.)