Generation of Murine Sympathoadrenergic Progenitor-Like Cells from Embryonic Stem Cells and Postnatal Adrenal Glands

<div><p>Sympathoadrenergic progenitor cells (SAPs) of the peripheral nervous system (PNS) are important for normal development of the sympathetic PNS and for the genesis of neuroblastoma, the most common and often lethal extracranial solid tumor in childhood. However, it remains difficult to isolate sufficient numbers of SAPs for investigations. We therefore set out to improve generation of SAPs by using two complementary approaches, differentiation from murine embryonic stem cells (ESCs) and isolation from postnatal murine adrenal glands. We provide evidence that selecting for GD2 expression enriches for ESC-derived SAP-like cells and that proliferating SAP-like cells can be isolated from postnatal adrenal glands of mice. These advances may facilitate investigations about the development and malignant transformation of the sympathetic PNS.</p></div

Adrenal spheres contain many SAP-like cells and few sympathetic neurons and glial cells.

<p>(<b>A</b>) <b>A majority of sphere cells express nestin, while none express CD57.</b> Flow cytometric analysis of dissociated adrenal-derived sphere cells stained for nestin (left panel) and CD57 (right panel). Dashed lines define isotype controls and solid lines specific antigen expression. (<b>B</b>) <b>The majority of adrenal-derived sphere cells expresses nestin, BMI1 and MUSASHI1 while few cells express peripherin or GFAP.</b> Immunofluorescence microscopy of spheres allowed to attach overnight on poly-D-lysine/fibronectin-coated glass coverslips in 1% FCS-containing sphere medium, analyzed for co-expression of nestin, peripherin, BMI1, MUSASHI1, GFAP, TH, DBH. Nuclei were counterstained with DAPI. Scale bar equals 100 µm. Quantification of positive cells is shown in the histogram. (<b>C</b>) <b>Adrenal-derived spheres contain a minority of cells with dense core vesicles.</b> Representative electron microscopic image of a cell from an adrenal-derived sphere. Scale bar equals 1 µm. (<b>D</b>) <b>Adrenal-derived spheres harbor many immature appearing cells lacking functional voltage-gated channels and few mature appearing neurons exhibiting electrical activity.</b> Microphotograph showing a neuronal-like cell cluster (*) and an area outside of the cluster (**). Fluorescence change (ΔF) in di-8-ANEPPS stained preparations corresponding to compound action potentials (CAP) from the cluster (*) and lack of CAP in the area outside of it (**), upper right panel. Representative traces of double pulse stimulation, whereby the second CAP was elicited at Δt of 100, 50, 40, 30, 20, 10, and 5 ms, lower left panel. Representative voltage-activated whole-cell inward currents recorded from a cell depolarized in 5 mV steps from –70 to +35 mV for 60 ms, from a holding potential of −120 mV, lower right panel.</p

SAP-like cells can be enriched by sorting for GD2.

<p>(<b>A</b>) <b>A subset of the low-enrichment NCSC-like population expresses GD2.</b> ESC-derived low-enriched NCSC cultures were sorted into GD2⁻ and GD2⁺ fractions. Left upper panel shows the isotype control, right upper panel the GD2-stained population and the lower panels post-sort plots of the GD2⁻ and GD2⁺ fractions. (<b>B</b>) <b>GD2⁺ cells have a markedly higher clonogenicity than GD2</b>⁻<b>cells.</b> GD2-sorted cells were seeded at clonal density and clones were quantified after 7 d of culture in NCSC medium. The means of 3 independent experiments are depicted. Statistical analysis was performed using the t-test. ***, p<0.001. (<b>C</b>) <b>The large majority of GD2⁺ cells are bilineage SAP-like cells.</b> GD2-sorted cells were cultured at clonal density for 7 d in NCSC medium and resultant clones were simultaneously stained for peripherin, GFAP and SMA. A peripherin⁺GFAP⁺ clone is shown, scale bar equals 100 µm. Quantification of the clones is shown in the histogram, the means of three independent experiments (with a total of 219 clones) are depicted. (<b>D</b>) <b>Chromaffin cell-inducing conditions upregulate chromaffin markers in GD2⁺ cells.</b> GD2-sorted cells were differentiated for 6 d on poly-D-lysine/fibronectin-coated coverslips in NCSC medium containing dexamethasone and PMA for chromaffin cell induction. Immunocytochemistry images show GD2⁺ and GD2⁻ cells cultured with or without dexamethasone and PMA, simultaneously stained for TH and peripherin (upper panel), DBH and peripherin (middle panel), and chromogranin A and peripherin (lower panel). Nuclei were counterstained with DAPI. Scale bars equal 100 µm. Quantification of positive cells in the GD2⁺ fraction is shown in the histograms (2954 cells of two independent experiments were analyzed). Statistical analysis was performed using the t-test; n.s., not significant; *, p<0.05; **, p<0.01; ***, p<0.001. (<b>E</b>) <b>Chromaffin cell inducing conditions induce catecholamine production in GD2⁺ cells.</b> HPLC analysis of catecholamine content in lysates of GD2<b>⁺</b> cells differentiated for 6 d in NCSC medium supplemented with or without dexamethasone and PMA. The means of three independent experiments are shown. Statistical analysis was performed using the t-test; n.s., not significant; *, p<0.05.</p

Adrenal-derived sphere cells can be induced <i>in vitro</i> towards neuronal and chromaffin lineage.

<p>(<b>A</b>) <b>Neuronal and chromaffin differentiation of spheres.</b> 7 d old spheres adhering to poly-D-lysine/fibronectin-coated coverslips were differentiated for 6 d in differentiation medium containing ATRA and ascorbic acid (for neuronal differentiation) or dexamethasone and PMA (for chromaffin cell induction). Immunocytochemistry images show spheres cultured with or without specific factors as indicated and stained for β3-Tubulin, peripherin, TH, DBH and CHGA. Nuclei were counterstained with DAPI. Scale bars equal 100 µm. Quantification of positive cells is shown in histograms (1994 cells of two independent experiments were analyzed). Statistical analysis was performed using the t-test. *, p<0.05; **, p<0.01; ***, p<0.001. (<b>B</b>) <b>Chromaffin cell inducing conditions upregulate catecholamines in spheres.</b> HPLC analysis of catecholamine content in lysates of spheres differentiated in adherence for 6 d in differentiation medium supplemented with or without dexamethasone and PMA. The means of three independent experiments are shown. Statistical analysis was performed using the t-test; n.s., not significant; *, p<0.05.</p

Strategies and selection of neural progenitor cells.

<p>(<b>A</b>) <b>Strategies to generate NCSCs and SAPs.</b> Two strategies were used, derivation of NCSC-like cells and SAP-like cells from murine embryonic stem cells (ESCs) and isolation of SAP-like cells from postnatal adrenal glands of mice. For derivation from ESCs, germ layer specification was induced by forming embryoid bodies (EBs) from mouse embryonic stem cells (mESCs). NPCs were differentiated from EBs by serum deprivation and mitogen addition. By applying culture conditions supportive for neural crest stem cells, a low-enriched NCSC-like population was generated. Sorting this population for CD57⁺ cells led to a population of higher-enriched NCSC-like cells while sorting for GD2⁺ cells generated a fraction of highly-enriched SAP-like cells. For isolation of SAP-like cells from postnatal adrenal glands, dissociated adrenal cells are grown as spheres in serum-free medium. (<b>B</b>) <b>NPC-lineage selection from ESCs generates cells expressing markers of NCSCs and SAPs.</b> EBs generated from murine ES cells were subjected to NPC-lineage selection. Specific mRNA levels of genes associated with NCSCs, SAPs and sympathetic neurons were analyzed by qRT-PCR and are shown in relation to TATA-Box binding protein-1 (TBP1). RNA was isolated on d 4 of EB differentiation and d 7 of NPC culture. The experiment was repeated twice with similar results.</p

Postnatal mouse adrenal glands contain sphere-forming cells expressing markers of NCSCs and SAPs.

<p>Mouse adrenals were dissociated to single cells and cultured using differential plating in low-attachment plastic dishes with serum-free medium containing bFGF and EGF. (<b>A</b>) <b>Dissociated postnatal mouse adrenal glands form spheres in non-adherent serum-free conditions.</b> Phase contrast image of spheres derived from the adrenal glands of a 2 d old mouse growing in suspension culture. Scale bar equals 200 µm. (<b>B</b>) <b>Early spheres contain proliferating cells.</b> BrdU was added to the culture medium of spheres at different time of culture. 24 h later BrdU was detected by immunofluorescence microscopy. Micrograph of a sphere cultured for 7 d stained with anti-BrdU and DAPI is shown in the left panel, scale bar equals 50 µm. Quantification of BrdU-incorporating sphere cells is depicted in the right panel. The means of two independent experiments are shown (15 wells/time-point). Statistical analysis was performed using the t-test. ***, p<0.001. (<b>C</b>) <b>Frequency of sphere forming cells decreases with age of mice.</b> 1.2×10⁵ cells/ml derived from adrenal glands of mice of increasing age were seeded in non-adherent serum-free condition. Phase contrast images of spheres are shown, scale bar equals 200 µm. Sphere-forming frequency was calculated as frequency of spheres formed per number of cells seeded (histogram). The means of three independent experiments are shown. Statistical analysis was performed using the t-test. ***, p<0.001. (<b>D</b>) <b>Sphere cells express markers of SAPs and NCSCs.</b> RT-PCR analysis was performed on RNA isolated from adrenal-derived spheres.</p

Adrenal-derived sphere cells integrate <i>in situ</i> where they downregulate expression of progenitor markers but do not differentiate to chromaffin cells.

<p>Pre-implantation adrenal-derived spheres attached to glass coverslips (left column) and 3 week post-implantation rat adrenal gland prior implanted with CFSE-labeled sphere cells (middle and right columns) were stained for nestin, BMI1, peripherin and TH. Stainings were visualized by immunohistochemistry. In the middle and right columns, fluorescent images (CFSE, green) are overlaid on immunohistochemistry images. Images in the right column are magnifications of images shown in the middle column. Scale bars are 200 µm (left and middle columns) and 50 µm (right column).</p

ESC-derived NCSC-like cells are enriched by sorting for expression of CD57.

<p>ESC-derived NPCs were cultured in medium containing chicken embryonic extract, retinoic acid, bFGF and IGF-1 under hypoxic conditions to generate low-enriched NCSC-like cells. (<b>A</b>) <b>A subset of low-enriched NCSC-like cells expresses CD57 and can be isolated by FACS.</b> Low-enriched NCSC-like cells were used. Left upper panel shows isotype control, right upper panel unsorted CD57-stained cells and lower panels post-sort plots of the CD57⁻ and CD57⁺ fractions. (<b>B</b>) <b>Stronger expression of some NCSC and SAP markers in CD57⁺ cells.</b> RNA isolated from unsorted, CD57⁻ and CD57⁺ cells was analyzed by RT-PCR to detect markers of CD57 biosynthesis, pluripotent cells, NCSCs, SAPs and sympathetic neurons. As positive control for TrkA and peripherin RT-PCR, cDNA from Neuro2a (a mouse NB) was used, while for TrkB mouse brain cDNA was employed. (<b>C</b>) <b>CD57⁺ cells have more trilineage NCSC potential as compared to the CD57</b>⁻<b>fraction.</b> CD57⁻sorted cells were cultured at clonal density in NCSC medium and resultant clones were simultaneously stained for NF160, GFAP, SMA and DAPI. A trilineage clone is shown. Scale bar equals 100 µm. The percentages of clones with cells of all three lineages, two lineages, one lineage or none of the lineage (“other”) were quantified. The means of three independent experiments (with a total of 183 clones) are depicted. Statistical analysis was performed using the t-test. *, p = 0.019. (<b>D</b>) <b>Clones from CD57⁺ cells do not contain cells expressing peripherin protein.</b> Clones (left panel) and neuro2a NB cells used as positive control (right panel) were stained for peripherin. Nuclei were counterstained with DAPI. Scale bar equals 100 µm.</p

Enhanced formation of stroma-poor neuroblastoma by CD57^high U-NB1 cells.

<p>(<b>A</b>) <b>Enhanced tumor formation of CD57^high U-NB1 cells.</b> CD57^high and CD57^low tumor cells were isolated by FACS from one primary orthotopic tumor. 1×10⁴ cells were surgically transplanted into the left adrenal glands of immunodeficient mice (8 animals per group). The development of tumors was monitored by palpation and verified by necropsy when mice had to be killed because of excessively large tumors or when the experiment was terminated at 90 days. The log rank test was used for statistical analysis. The experiment was repeated twice, with similar results. (<b>B</b>) <b>Orthotopic U-NB1 tumors are stroma-poor.</b> Tumors were formalin-fixed, paraffin-embedded and HE-stained. Scale bar equals 200 µm.</p

CD57^high but not CD57^low U-NB1 and SK-N-BE(2)-C cells are responsive to inducers of neuronal or glial lineage attributes.

<p>CD57^low and CD57^high U-NB1 cells (<b>A</b>) or CD57^low and CD57^high SK-N-BE(2)-C cells (B) were isolated by FACS. Cell fractions were plated in clonal density onto collagen-coated cover slips. Cells were treated over a ten day period with ATRA or with NRG1–ß1 for 14 days in medium supplemented with 2% FBS, followed by double–immunofluorescence staining for peripherin (green) and GFAP (red). Nuclei were stained with DAPI (blue). (<b>C</b>) Frequency of peripherin/GFAP double-negative clones upon forced differentiation as in (A) and (B). The asterisks denote p<0.05 using Student's t-test. Scale bars correspond to 50 µm. All experiments were repeated at least three times, with similar results.</p