Niche-independent symmetrical self-renewal of a mammalian tissue stem cell - PubMed (original) (raw)

Niche-independent symmetrical self-renewal of a mammalian tissue stem cell

Luciano Conti et al. PLoS Biol. 2005 Sep.

Abstract

Pluripotent mouse embryonic stem (ES) cells multiply in simple monoculture by symmetrical divisions. In vivo, however, stem cells are generally thought to depend on specialised cellular microenvironments and to undergo predominantly asymmetric divisions. Ex vivo expansion of pure populations of tissue stem cells has proven elusive. Neural progenitor cells are propagated in combination with differentiating progeny in floating clusters called neurospheres. The proportion of stem cells in neurospheres is low, however, and they cannot be directly observed or interrogated. Here we demonstrate that the complex neurosphere environment is dispensable for stem cell maintenance, and that the combination of fibroblast growth factor 2 (FGF-2) and epidermal growth factor (EGF) is sufficient for derivation and continuous expansion by symmetrical division of pure cultures of neural stem (NS) cells. NS cells were derived first from mouse ES cells. Neural lineage induction was followed by growth factor addition in basal culture media. In the presence of only EGF and FGF-2, resulting NS cells proliferate continuously, are diploid, and clonogenic. After prolonged expansion, they remain able to differentiate efficiently into neurons and astrocytes in vitro and upon transplantation into the adult brain. Colonies generated from single NS cells all produce neurons upon growth factor withdrawal. NS cells uniformly express morphological, cell biological, and molecular features of radial glia, developmental precursors of neurons and glia. Consistent with this profile, adherent NS cell lines can readily be established from foetal mouse brain. Similar NS cells can be generated from human ES cells and human foetal brain. The extrinsic factors EGF plus FGF-2 are sufficient to sustain pure symmetrical self-renewing divisions of NS cells. The resultant cultures constitute the first known example of tissue-specific stem cells that can be propagated without accompanying differentiation. These homogenous cultures will enable delineation of molecular mechanisms that define a tissue-specific stem cell and allow direct comparison with pluripotent ES cells.

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Figures

Figure 1

Figure 1. Generation of NS Cells from ES Cells

(A) The adherent NS cell culture (LC1) propagated in EGF and FGF-2 (a), shows no expression of neuronal (b) or astrocyte (c) antigens, and uniform expression of the precursor marker RC2 (d) and nestin (not shown). LC1 cells differentiate into GFAP immunopositive astrocytes (e) upon addition of serum and generate TuJ1 immunopositive neurons (f) upon growth factor withdrawal. (B) The proportion of neurons obtained remains greater than 35% of total cells after 115 passages (g). Immunostaining for MAP2 of LC1 differentiation shown at passage 16 (h) and passage 158 (i).

Figure 2

Figure 2. Clonal NS Cells Generated through Sox1 Neural Lineage Selection

(A) Phase image of neural precursors at passage 1 (a) and 5 (c), with (b) and (d) showing corresponding _Sox1_-GFP fluorescence. Image (e) shows a single cell, 1 h after plating in Terasaki well, and (f) shows a phase-contrast image of clonal cell line at passage 20. (B) Differentiation of NS-5 cells into astrocytes (g,h) and neurons (j,k) with loss of nestin immunoreactivity (i,l). (C) These NS-5 cells are immunoreactive for neural precursor cell/radial glia markers (m–o,q,r) and negative for GFAP (p). (D) Clones of NS-5 cells exhibit homogenous expression of BLBP with no immunoreactivity for GFAP in the presence of EGF/FGF (s), and generate neurons upon growth factor withdrawal (t). (E) Metaphase spread of NS-5 (passage 31).

Figure 3

Figure 3. NS Cells Die or Begin to Differentiate in the Absence of EGF

Unlike proliferating cultures in FGF plus EGF (A,C), NS cells on gelatin die by caspase-3-mediated programmed cell death 20 h after removal of EGF (B,D). This death can be overcome if cells are cultured on a laminin substrate in FGF-2 only (F). Under these conditions, cells become slow-dividing and extend longer processes (G,H). Most cells retain RC2 immunoreactivity (H), but a minority begin neuronal differentiation marked by TuJ1 expression (J).

Figure 4

Figure 4. Phenotype and Electrical Activity of NS Cell–Derived Neurons

(A) This LC1 NS cell–derived neuron at 27 d of differentiation displays mature morphology and expresses GAD67. (B) Expression of MAP2/synaptophysin after 7 d of differentiation. (C) Superimposed inward and outward current tracings obtained at different membrane potentials (between −70 and +40 mV from a holding potential of −90 mV), from NS cell–derived neurons after differentiation for 6 (i), 20 (ii), and 30 d (iii). (D) Superimposed voltage responses obtained following injection of depolarising rectangular current pulses in the same three cells (i–iii) by switching from voltage- to current-clamp immediately after current recordings shown in (c) were obtained. The dashed line represents a voltage level of −60 mV. (E)Average Na+ currents elicited at −20 mV from cells cultured in differentiating medium for increasing times as indicated by labels. Bars indicate SE. (F) Superimposed inward currents elicited at −40 mV and 0 mV in 10 mM Ba2+ and in the presence of TTX; the holding potential was −90 mV. (G) Current/voltage relationship from the same cell as in (F).

Figure 5

Figure 5. ES Cell–Derived or Forebrain-Derived NS Cells are Similar to Radial Glia

NS cells were derived from independent ES cell lines (CGR8, E14Tg2a) or primary cortical (Cor-1) and striatal (Str-1) tissue. (A) RT-PCR of stem cell/radial glia markers. (B) RT-PCR for pan-neural and region-specific transcriptional regulators. (C) Double immunostaining for Pax6 and Pax6/RC2 (a,b), Olig2 and Olig2/RC2 (c,d) and Olig2/Pax6 (f). DAPI only for Olig2/Pax6 (e). (D) The ES cell–derived line (CGR8-NS) and foetal cell–derived line (Cor-1) are indistinguishable from LC1 by morphology and NS cell/radial glial marker immunoreactivity (g,h,k,l), and can each differentiate into neurons (i,m) and astrocytes (j,n). (E) The ability of Cor-1 to generate neurons (TuJ1+) is retained after 16 passages, more than 30 generations (p,o).

Figure 6

Figure 6. NS Cells Incorporate and Differentiate within the Adult Brain

(A–H) Confocal images of LC1 NS cells, lentivirally transduced with enhanced GFP, 4 wk post-grafting into hippocampus (A,B) or striatum (C–H). (B) and (D) show higher magnification of the insets in panels (A) and (C), respectively. Examples of enhanced GFP grafted NS cells (green) showing co-expression (yellow) of the neuronal markers TuJ (E, red) or MAP-2 (F, red), astroglial marker GFAP (G, red), neural progenitor marker nestin (H, red). (I) Quantitative analysis of graft-derived neuronal (MAP2), astroglial (GFAP), progenitor (Nestin), and proliferating (Ki67) cells, 4 wk after transplantation into adult mouse striatum. Data are means (± standard deviation) of at least 500 enhanced GFP+ cells from five independent animals. DG, Dentate Gyrus; ST, Striatum. Scale bars: A,C, 100 μm; B, D, E, 40 μm.; F–H, 20 μm.

Figure 7

Figure 7. Human ES Cell or Foetal-Derived NS Cells

(A) Derivation from human ES cells: human ES cell primary culture (a), differentiation of human ES cells into neural-rosette structures (b), passage 9 in NS expansion medium (c), individual cells exhibit radial glial morphology (d), and immunostaining for NS cell/radial glia markers (e–h). (B) Derivation from human foetal forebrain: floating clusters (i) generated from cortex, attachment and outgrowth (j), passage 5 in NS expansion medium (k), radial glia morphology (l), and NS cell/radial glial markers (m–p). (C) Differentiation of human foetal NS cells: TuJ1 positive neuronal cells generated by sequential growth factor withdrawal (q), and GFAP positive astrocytes induced by exposure to serum (r).

References

    1. Schofield R. The relationship between the spleen colony-forming cell and the hemopoietic stem cell. Blood Cells. 1978;4:7–25. - PubMed
    1. Watt FM, Hogan BL. Out of Eden: Stem cells and their niches. Science. 2000;287:1427–1430. - PubMed
    1. Spradling A, Drummond-Barbosa D, Kai T. Stem cells find their niche. Nature. 2001;414:98–104. - PubMed
    1. Doetsch F. A niche for adult neural stem cells. Curr Opin Genet Dev. 2003;13:543–550. - PubMed
    1. Potten CS, Loeffler M. Stem cells: Attributes, cycles, spirals, pitfalls and uncertainties. Lessons for and from the crypt. Development. 1990;110:1001–1020. - PubMed

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