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First published online 16 August 2006
doi: 10.1242/dev.02541

Development 133, 3671-3681 (2006)
Published by The Company of Biologists 2006
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Derivation and large-scale expansion of multipotent astroglial neural progenitors from adult human brain

Noah M. Walton1, Benjamin M. Sutter2, Huan-Xin Chen3, Lung-Ji Chang4, Steven N. Roper1,4, Bjorn Scheffler1,* and Dennis A. Steindler1,2,4,5,*,{dagger}

1 Department of Neuroscience, McKnight Brain Institute, University of Florida, Gainesville, FL 32610, USA.
2 Shands Cancer Center, University of Florida, Gainesville, FL 32610, USA.
3 Department of Neurosurgery, University of Florida, Gainesville, FL 32610, USA.
4 Department of Molecular Genetics and Microbiology University of Florida, Gainesville, FL 32610, USA.
5 Program in Stem Cell Biology and Regenerative Medicine, University of Florida, Gainesville, FL 32610, USA.


Figure 1
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  		Fig. 1. Expansion of primary neural cells as a homogenous population of AHNPs. (A) Highly expanded (more than 60 PDs) cells ubiquitously express nestin (red), with a large subset of GFAP+ cells (green). (B) AHNPs express widespread immature neuronal and glial markers, including A2B5 (red) and NG2 (green). (C,D) AHNPs (nestin+, green) express astrotypic markers in a large subset of cells, including S100ß (C, red) and glutamine synthetase (D, green). (E) Voltage-clamp profile of these cells reveal prominent Na+ and K+ channel activity. Data shown for temporal cortex-derived cells. (F) Nestin+ (green) AHNPs proliferated in the presence of BrdU (red) uniformly incorporate thymidine analog. (G) Stereological evaluation of proliferating AHNPs reveals a uniform nestin+ population that frequently co-expresses glial cell markers (GFAP shown). Maintaining these cells in growth medium supplemented with BrdU results in label saturation in AHNPs (BrdU+Nestin+ cells) at a rate of incorporation analagous to previously characterized proliferative dynamics (H). Removal of mitogenic stimuli (GF=EGF+bFGF) results in failure of AHNPs to divide (see Fig. 2F). (I,J) Both hippocampal and temporal cortex-derived AHNPs maintain comparable stable doubling rates and uniform protoplasmic morphologies throughout culture. (K) AHNPs derived from temporal cortex and hippocampus reveals continuous logarithmic expansion throughout culture. Scale bars: 25 µm in A,B,F,J; 50 µm in C; 75 µm in D. Images counterstained with DAPI.

 

Figure 2
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  		Fig. 2. AHNPs avoid immortalizing mutations, and exhibit mitogen- and telomerase-dependent growth. (A) Cultured AHNPs express major growth regulatory proteins longitudinally throughout culture. (B) Karyotyped AHNPs display normal ploidy and have no gross cytogenetic malformations. (C) Following growth arrest by an exogenous TERT inhibitor (EGCG) or growth factor withdrawal cultured cells express SA-ß-Gal. However, only mitogen-withdrawn (-bFGF) cells lose TERT expression when evaluated 7 days later. (D) Physiological (x-irradiation) or chemical inhibitors (apidicolin, EGCG) consistently increase the fraction of cells expressing SA-ß-Gal. (E) Application of reversible growth inhibitors yields a significant reduction in growth rate. AHNPs revert to previous proliferative levels following arrestor washout. (F) Age-matched AHNPs placed in either basic media (N2) or media containing EGF or bFGF only (N2E, N2F) enter irreversible growth arrest compared to defined proliferative conditions (N2EF) and subsequently become unviable. Data shown for temporal cortex derived cells. *P<0.05, Student's t-test. Scale bar: 75 µm in C.

 

Figure 3
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  		Fig. 3. AHNPs maintain viability and assume glial phenotypes upon ventricular transplantation. (A) AHNPs cells injected into the right lateral ventricle of postnatal day 3 mice were detectable with HNA in periventricular tissue adjacent to injection site (*). (B) HNA+ cells (boxed in a) were primarily located within 100 µm of the ventricular wall in the ependymal and subependymal cell layer. (C-E) HNA+ cells (red) integrating into the LV wall display conserved morphology of astrotypic cells, and frequently co-express GFAP (green). (F) Confocal micrograph shows GFAP+ process extension from an AHNP into the subependymal zone. (G,H) Activated dendrimer transfection of Pax6-eGFP of 30 PDs AHNPs 3 days post-transfection. (I) Lentiviral-eGFP transfection of 30 PD AHNPs (20 moi). Scale bars: 40 µm in B; 25 µm in C-E; 8 µm in F; 20 µm in G-I. Images in C-F,H are counterstained with DAPI.

 

Figure 4
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  		Fig. 4. Cortically implanted AHNPs adopt predominantly neuronal fates. (A) Coronal section of engrafted left hemisphere shows ß-III-tubulin+/HNA+ donor cells adjacent to engraftment site. Schematic representation includes two-dimensional proximodistal and lateral distribution of the majority of AHNPs and ectopically migrating cells in two transplanted animals (blue and yellow). (B) Fate analysis indicates few cells adopt an astroglial identity. (C) Integrating AHNPs within the primary engraftment site adopt neuronal morphologies and immunophenotypes. (D) ß-III-tubulin+ cells present within the hippocampus of engrafted animals occasionally displayed HNA (E, from boxed area in d) in CA1 and CA3, where they adopted apparent pyramidal neuron morphologies. (F,G) Single plane confocal image of cortically implanted AHNPs. HNA+ cells form mature neuronal (NeuN+) cell types, which co-exist with endogenous neurons (arrowheads). Scale bars: 200 µm in A,B,D; 50 µm in C,F,G; 100 µm in E.

 

Figure 5
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  		Fig. 5. Differentiation of AHNPs into neuronal cell types. (A) Proliferating cells (30 PDs) assume a compacted morphology immediately after removal of mitogens and addition of dibutyl cAMP, IBMX and NGF. (B) Three days after induction of differentiation, intermediate cells displaying a developmentally intermediate phenotype are appreciated. (C) Five days after induction of differentiation, maturing cells concurrently lose GFAP and continue to strongly express ß-III-tubulin. (D) Seven days after induction of differentiation, newly generated neurons in vitro frequently co-express immature neuron markers, and assume typical bipolar morphologies. (E) Current and voltage clamp analysis of 7-day-old neurons. New neurons exhibit prominent Na+ and K+ channels, and were able to fire elicited action potentials when polarized to -60 mV. (F) ß-III-tubulin neurons generated in the presence of thymidine analog universally incorporate BrdU. Cells generated in this manner display additional type-specific neuronal markers, including PSA-NCAM (G) and neurofilament M (NF-M, H). Scale bars: 75 µm in A; 25 µm in B,H; 100 µm in C,G. Cells counterstained with DAPI.

 

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