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First published online August 9, 2007
Stem Cells Vol. 25 No. 11 November 2007, pp. 2864 -2873
doi:10.1634/stemcells.2007-0308; www.StemCells.com
© 2007 AlphaMed Press

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TISSUE-SPECIFIC STEM CELLS

Unique Glycerophospholipid Signature in Retinal Stem Cells Correlates with Enzymatic Functions of Diverse Long-Chain Acyl-CoA Synthetases

Jianxue Lia, Zheng Cuib, Sheyun Zhaoc, Richard L. Sidmana

aDepartment of Neurology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, USA;
bDepartment of Pathology, Wake Forest University School of Medicine, Winston-Salem, North Carolina, USA;
cFlow Cytometry Facility, Hospital for Sick Children, University of Toronto, Toronto, Ontario, Canada

Key Words. Retinal stem cells • Glycerophospholipids • Long-chain acyl-CoA synthetases • Membrane fluidity Polyunsaturated fatty acids

Correspondence: Jianxue Li, M.D., Ph.D., 77 Avenue Louis Pasteur, Harvard Institute of Medicine 838, Boston, Massachusetts 02115, USA. Telephone: 1-617-667-0868; Fax: 1-617-667-0810; e-mail: jli7{at}caregroup.harvard.edu; or Richard L. Sidman, M.D., 77 Avenue Louis Pasteur, Harvard Institute of Medicine 855A, Boston, Massachusetts 02115, USA. Telephone: 1-508-341-6552; Fax: 1-617-667-0810; e-mail: richard_sidman{at}hms.harvard.edu

Received on April 25, 2007; accepted for publication on July 31, 2007.

First published online in STEM CELLS EXPRESS  August 9, 2007.

   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Summary
 Disclosure of Potential...
 Acknowledgments
 References
 
Lipidomics is an emerging research field that comprehensively characterizes lipid molecular species and their metabolic regulation and biological roles. We performed the first lipidomics study on glycerophospholipids (GPLs) in adult mammalian retinal stem cells (RSCs) and non-RSC control cells. A unique GPL signature identified by electrospray ionization tandem mass spectrometry showed new prominent peaks of 16:0 (sn-1)-18:0 (sn-2) or 16:0–16:0 saturated fatty acids, instead of 18:0–20:4 or 18:0–22:6 polyunsaturated essential fatty acids, at 720 m/z of phosphatidylethanolamine, 764 m/z of phosphatidylserine, and 809 m/z of phosphatidylinositol in RSCs (sphere colony RSCs and enriched RSCs), but not in non-RSCs (retinal cells, ciliary cells, sphere colony-derived retinal cells, and nonretinal cells). To seek whether the GPL signature was associated with long-chain acyl-CoA synthetase (LACS), a potential modulator of fatty acid profiles in de novo GPL synthesis, we analyzed gene expression, catabolic activity, substrate selectivity, and inhibitor sensitivity of diverse LACSs. LACSs in RSCs mediated less utilization by GPLs of polyunsaturated essential fatty acids, including arachidonic acid (20:4 [n-6], a second messenger in cell signaling), which was accompanied by lower plasma membrane fluidity in proliferating RSCs compared with differentiated non-RSCs. These novel findings suggest that LACS-associated GPL signature and cell membrane fluidity may participate in regulating proliferation versus differentiation in RSCs and, perhaps, other types of stem cells.

Disclosure of potential conflicts of interest is found at the end of this article.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Summary
 Disclosure of Potential...
 Acknowledgments
 References
 
Lipidomics, a new term defined as the systematic decoding of lipidomes in biosystems, is an emerging research field that extensively characterizes lipid molecular species and their metabolic regulation and biological roles [1–5]. Glycerophospholipids (GPLs), mainly including phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), and phosphatidylinositol (PI), are major lipids in cell membranes. Although all GPL molecules contain the same glycerol backbone, the diversity of head groups, acyl chains, and degree of unsaturation can produce hundreds of different lipid species within a given cell. These varied structures not only allow for a variety of physical and chemical membrane properties such as permeability, fluidity, and curvature, but they also provide substrate pools for production of second messengers in cellular signaling events, either to modulate cell proliferation and differentiation or to mediate inflammation, cancer, and neurodegeneration [6–9]. Lysophosphatidic acid, for example, a key derivate of GPL catabolism, induces clonal generation of mouse neurospheres via proliferation of neural progenitors [10] and promotes proliferation of rat embryonic neural stem cells and their differentiation to cholinergic neurons [11].

The explosion of information in genomics and proteomics is not yet matched by a corresponding surge of knowledge in lipid research, largely because of both the complexity of lipids and the lack of powerful tools for their analysis [1, 2]. This imbalance is also seen in stem cell biology. Large-scale systematic gene expression analysis recently disclosed specific properties and commitments of stem cells in general [12, 13], and for adult neural stem cells in particular [14–16], allowing a shift from expression profiling to functional studies on molecular mechanisms regulating stem cell self-renewal and differentiation [17, 18]. New powerful studies of function involve proteomics and lipidomics [19]. Proteomes of mouse embryonic stem cells [20] and a human neural stem cell line [21] show stem cell differentiation-associated changes in large-scale protein profiles [22, 23], but so far we have not found published lipidomic analyses of stem cells.

Retinal stem cells (RSCs) have been identified in adult mouse [24], rat [25], and human [26] eyes. When cultured under conditions that promote formation of neurosphere colonies, cells from mouse ciliary marginal tissue, but not from retina, can produce sphere colonies at a low rate of <0.1%, suggesting the existence of a small population of RSCs among the primary ciliary cells. The sphere colony RSCs can be passaged serially to form new sphere colonies (self-renewal) or to differentiate into each of the various retinal cell types (multipotential) [24, 27]. Although it is not yet clear whether adult RSCs are progenitors of neural stem cells in general, as suggested [25], or are a subtype specific to the neural retina and retinal pigment epithelium [26], it is evident that they possess the defining stem cell properties of proliferation, self-renewal, and multipotentiality that make them suitable for lipidomics study.

In the present study, we established for the first time, by an advanced technique of electrospray ionization tandem mass spectrometry (ESI-MS/MS), unique signature of individual GPLs in adult mouse RSCs in proliferation phase. We also analyzed long-chain acyl-CoA synthetase (LACS) regulation of the signature and found lower plasma membrane fluidity in proliferating RSCs compared with differentiated non-RSCs. The GPL properties uncovered in RSCs might extrapolate to other neural stem cells or even to stem cells in general.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Summary
 Disclosure of Potential...
 Acknowledgments
 References
 
Preparation of Retinal and Ciliary Cells
The cells were prepared as described [24, 27]. Briefly, adult (10-week-old) male albino CD1 mouse eyes were washed in artificial cerebrospinal fluid and then rinsed with 70% ethanol for 20 seconds. Retinal and ciliary marginal tissues were separately dissected out. The ciliary marginal tissue was treated with dispase (Gibco, Grand Island, NY, http://www.invitrogen.com) at room temperature for 10 minutes and then with a mixture of trypsin, hyaluronidase, and kynurenic acid (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com) at 37°C for another 10 minutes to facilitate the separation of ciliary cells from the underlying basement membrane. The ciliary cells were transferred into Dulbecco's modified Eagle's medium (DMEM)/F12 (Gibco) containing trypsin inhibitor (1 mg/ml ovomucoid; Sigma) and triturated to yield a suspension of single cells.

Proliferation and Differentiation of Sphere Colony RSCs
The ciliary cells were cultured in DMEM/F12 supplemented with 10 ng/ml basic fibroblast growth factor (bFGF), 2 µg/ml heparin, and 1x B27 (Gibco) (sphere colony-forming medium) for 7 days to form sphere colony RSCs. Some sphere colony RSCs were enzymatically dissociated and replated to check their ability to form subsequent sphere colonies. Other sphere colony RSCs were directly transferred into laminin-coated wells in DMEM/F12 containing 1% fetal bovine serum (FBS) supplemented with or without bFGF (differentiation medium). The medium was changed twice per week. After 3 weeks, sphere colony-derived retinal (with bFGF) and nonretinal (without bFGF) cells were identified by morphology and immunocytochemistry [24, 27].

RSCs Enriched from Ciliary Cells by Fluorescence-Activated Cell Sorting
Ciliary cells were incubated with fluorescein isothiocyanate (FITC)-conjugated antibody against CD34 and phycoerythrin-conjugated antibody against Sca1 (Santa Cruz Biotechnology, Santa Cruz, CA, http://www.scbt.com) in phosphate-buffered saline (PBS) containing 0.1% sodium azide (a preservative against bacteria and fungi) and 1% FBS at 4°C for 30 minutes. After washing with PBS thoroughly, the cells were aseptically fractionated by fluorescence-activated cell sorting (FACS) (FACStar Plus; Spectron Corporation, Burlington, WA, http://www.spectroncorp.com) based on cell size and surface antigens. Various fractions were separately cultured to identify the enriched RSC population. These cells grew well in culture, similar to those without sodium azide treatment, and were less likely to become contaminated.

Immunocytochemistry and Histology
Sphere colony RSCs and sphere colony-derived cells were fixed with 4% paraformaldehyde in PBS for 10 minutes, rinsed with PBS, and blocked with 3% goat serum and 0.3% Triton X-100 in PBS for 30 minutes. The samples were then incubated at 4°C overnight with primary antibody against nestin (undifferentiated cells; BD Biosciences, San Diego, http://www.bdbiosciences.com), Rho1D4 (photoreceptors; Abcam, Cambridge, MA, http://www.abcam.com), PKC{alpha} (bipolar cells; Chemicon, Temecula, CA, http://www.chemicon.com), calbindin (horizontal cells; Sigma), Pax6 (amacrine cells; Chemicon), tubulin βIII (retinal ganglion cells; Sigma), or glial fibrillary acidic protein (Müller glia; Chemicon). After washing with PBS, the samples were incubated with FITC- or Cy3-conjugated secondary antibody (Chemicon); 4,6-diamidino-2-phenylindole in mount medium (Vector Laboratories, Burlingame, CA, http://www.vectorlabs.com) was used to visualize cell nuclei. Some samples were stained with hematoxylin/eosin or Giemsa for histology or embedded in Epon 812 for transmission electron microscopy.

Lipid Extraction and Organic Phosphorus Determination
Tissue or cell samples were sonicated in ice-cold methanol. Ten percent of each suspension was used for protein and DNA assay; the remainder was centrifuged at 4,000 rpm for 5 minutes and separated into two phases [28, 29]. For organic phosphorus detection, a small aliquot of the lower phase was dried under N2, dissolved in perchloric acid, and digested at 180°C until clear. After adding ammonium molybdate and ascorbic acid, the sample was heated in boiling water bath for 10 minutes and cooled on ice. Phosphorus (P) level was determined at 890 nm.

Thin Layer Chromatography and Gas Chromatography
Individual GPLs in total cellular lipid extract were separated and purified by thin layer chromatography (TLC) on a silica gel G plate (Analtech, Newark, DE, http://www.analtech.com) with a mobile phase of chloroform/triethylamine/ethanol/water (30:30:34:8 by volume); PC, PE, PS, and PI standards (Sigma) were used to identify the corresponding bands with .09, .54, .30, and .42 retention factor (Rf) values, respectively [29]. Individual GPL levels were expressed as concentrations (nmol P/µg DNA) and proportions (% of total GPLs). For fatty acid detection [30], powders from the bands were methylated by alkaline methanolysis, mixed with saturated NaOH in chloroform/methanol (2:1 vol/vol), stirred for 10 minutes, added with HCl, and centrifuged at 3,000g for 5 minutes. The lower phase was dried under N2 and resuspended in 20 µl of hexane, 1 µl of which was injected into a gas chromatography (GC) apparatus (Hewlett-Packard 5880A; Hewlett-Packard, Palo Alto, CA, http://www.hp.com). Fatty acid methyl esters were identified by retention times, and their peak areas were quantified by an automatic integrator. Fatty acid amounts were expressed as concentrations (pmol/µg DNA) and proportions (% of total fatty acids).

ESI-MS/MS
GPLs (2–5 nmol/ml) were analyzed with a Micromass Quattro II triple quadrupole mass spectrometer (Waters Corporation, Milford, MA, http://www.waters.com). Data were acquired with MassLynx NT 3.5 TM software. PC, PE, and PS were measured in the positive ion mode using collision at 15–40 V, in which PC profile was discriminated by precursor ion scanning at m/z +184 [31], and PE and PS profiles were detected by scanning for neutral losses of 141 Da and 185 Da, respectively [32]. PI profile was detected by precursor ion scanning for m/z –241 in the negative ion mode [33] at collision energy of 50 V. Fatty acid profiles in individual GPL species were determined in negative ion mode by product ion analysis [31].

Reverse Transcription-Polymerase Chain Reaction
Total RNA from each cell sample was isolated, transcribed, and amplified with RNA purification, reverse transcription (RT), and polymerase chain reaction (PCR) systems (Invitrogen, Carlsbad, CA, http://www.invitrogen.com). Mouse LACS3 mRNA expression was detected with a forward primer, 5'-AGGCAGGGTGTGACCATAAG-3', and a reverse primer, 5'-TCTCCAAGACCTCAGCCACT-3'. LACS1 mRNA was detected with a forward primer, 5'-CTCCAACCTGGTCTGAGTTCC-3', and a reverse primer, 5'-CTGCAATATCTGAGGGCAGTG-3'. Glyceraldehyde-3-phosphate dehydrogenase mRNA was detected as a positive control in the same cDNA preparation using a forward primer, 5'-ACTGGCGTCTTCACCACCAT-3', and a reverse primer, 5'-TCCACCACCCTGTTGCTGTA-3'.

Subcellular Fraction Preparation
Dissociated cells from sphere colonies or monolayer cultures were collected, sonicated in ice-cold PBS containing 0.25 M sucrose, and centrifuged at 900g for 10 minutes at 4°C to yield a nuclear fraction pellet. The supernatant was subjected to successive centrifugations at 15,000g for 20 minutes and at 230,000g for 60 minutes to yield mitochondrial (including peroxisomes and lysosomes) and microsomal fractions, respectively. The last supernatant was the soluble cytoplasm.

LACS Activity Assay
The isotopic assay of LACS activity relies on both solubility of nonreacted free fatty acids (substrates) and insolubility of long-chain acyl-CoA esters (products) in heptane [34, 35]. Final reaction mixture contained 150 mM Tris-HCl (pH 8.0), 10 mM MgCl2, 8 mM ATP, 200 µM Coenzyme A, 1 mM 2-mercaptoethanol, 0.1% Triton X-100, 20 nmol [3H]arachidonic acid or [3H]oleic acid (5 nCi/nmol; New England Nuclear, Waltham, MA, http://las.perkinelmer.com), and 200 µg of protein from a subcellular fraction as a source of enzyme. After incubation with or without competitive fatty acids (Sigma) or inhibitor triacsin C (BIOMOL International LP, Plymouth Meeting, PA, http://www.biomol.com) at 37°C for 0–40 minutes, the reaction was stopped with a mixture of isopropanol, heptane, and sulfuric acid and vortexed vigorously. The organic phase was discarded; the aqueous phase was extracted twice with heptane containing palmitic acid and counted for [3H]arachidonoyl-CoA or [3H]oleoyl-CoA levels by liquid scintillation spectrometry (Beckman Coulter, Fullerton, CA, http://www.beckmancoulter.com). A blank sample was treated identically, except that enzyme addition and incubation at 37°C were omitted. The products of LACSs were also verified by TLC [36]. More than 80% of the radioactivity was cochromatographed with arachidonoyl-CoA or oleoyl-CoA standard (Rf = .30).

Fatty Acid Incorporation into GPLs
Cells in cultures were labeled with 0.5 µCi/ml [3H]arachidonic acid (10 µM) for 1 day. GPLs extracted from the cells were separated by TLC, and the radioactivity in individual GPLs was measured as above.

Membrane Fluidity Measurement
Cell suspensions were incubated in either 1,6-diphenyl-1,3,5-hexatriane (DPH; 2 nmol/ml) or 1-anilino-8-naphthalene sulfonate (ANS; 33 nmol/ml) (Sigma) at 37°C for 2 hours. The degree of steady-state fluorescence polarization was measured with a spectrofluorometer (SLM model 4800) equipped with Glan Thompson prism polarizers in the T-optical format. The lower the fluorescence polarization value, the higher the degree of membrane fluidity [37].

Protein and DNA Assay
Protein was detected with the bicinchoninic acid assay (PerkinElmer Life and Analytical Sciences, Waltham, MA, http://www.perkinelmer.com) [38]. DNA was measured with the Hoechst fluorescent spectrometric method (Hoefer, San Francisco, http://www.hoeferinc.com) [39] with calf thymus DNA as the standard.

Statistical Analysis
Student's t test, two-way variance analysis, and multiple factor analysis were applied for comparisons between groups. Metabolic kinetics and regression analysis was performed following linear and nonlinear models. Data were expressed as means ± SD, and differences between groups were considered significant at p < .05 or very significant at p < .01.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Summary
 Disclosure of Potential...
 Acknowledgments
 References
 
Characterization of RSCs and Non-RSCs
Cells from adult CD1 mouse eyes were divided into two groups: (a) RSCs and (b) non-RSCs. The RSCs were subgrouped into: (ai) sphere colony RSCs (SC-RSC) that were in proliferation phase and derived from cultured ciliary cells in sphere colony-forming medium for 7 days and (aii) FACS-enriched RSCs (E-RSC) that were 15–20 µm in diameter and CD34–/Sca1– (see below), enriched by twice FACS from uncultured ciliary cells. The non-RSCs were subgrouped into: (bi) retinal cells (RC) that were dissected directly from uncultured retinal tissue without enzymatic digestion; (bii) ciliary cells (CC) that were dissected from uncultured ciliary marginal tissue and dissociated with dispase, trypsin, and hyaluronidase treatment; (biii) sphere colony-derived retinal cells (SC-RC) that were differentiated from sphere colony RSCs in differentiation medium with bFGF for 3 weeks; and (biv) sphere colony-derived nonretinal cells (SC-non-RC) that were distinguished from sphere colony RSCs in differentiation medium without bFGF for 3 weeks. Therefore, we can make comparisons between two groups of cells: (a) the RSC group, which included SC-RSC and E-RSC, and (b) the non-RSC group, which included RC, CC, SC-RC, and SC-non-RC. All six kinds of cells could be prepared from the same mouse eyes.

Ciliary cells cultured in sphere colony-forming medium had three fates: most of them (>95%, non-RSCs) died soon after plating, a small portion of them (~5%, non-RSCs) attached to the culture dish and differentiated quickly, and a few of them (0.1%, RSCs) proliferated and, within one week, formed floating sphere colonies of 200–300 µm in diameter (Fig. 1A). Sphere colony cells were verified as RSCs by their self-renewal and multipotential properties; they were passaged in sphere colony-forming medium serially to form new sphere colonies and differentiated in differentiation medium longer than 3 weeks into either sphere colony-derived retinal cells in the presence of bFGF or sphere colony-derived nonretinal cells in the absence of bFGF (Fig. 1A).


Figure 1
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  		Figure 1. Adult mouse retinal stem cells (RSCs). (A): Sphere colony RSC proliferation and differentiation. (Aa): A sphere colony composed of RSCs was produced from ciliary cells cultured in sphere colony-forming medium for 1 week. (Ab, Ac): Sphere colony-derived retinal cells in differentiation medium with basic fibroblast growth factor (bFGF) for more than 3 weeks. (Ad): Sphere colony-derived nonretinal cells in differentiation medium without bFGF for more than 3 weeks. Scale bar = 100 µm. (B): Fluorescence immunostaining of enriched RSCs. (Ba): Undifferentiated RSCs in a sphere colony produced from fluorescence-activated cell sorting (FACS)-sorted ciliary cells in sphere-forming medium were stained with neural stem cell marker (nestin+). (Bb–Bg): Differentiated retinal cells from the above sphere colony were stained with various cell markers for photoreceptor cell (Rho1D4+) (Bb), bipolar cell (PKC{alpha}+) (Bc), horizontal cell (calbindin+) (Bd), amacrine cell (Pax6+) (Be), retinal ganglion cell (tubulin βIII) (Bf), and Müller cell (glial fibrillary acidic protein+) (Bg). Cell nuclei were counterstained with 4,6-diamidino-2-phenylindole (Bb–Bg). (C): RSC morphological feature. (Ca): Giemsa staining showed that FACS-sorted "large cells" were indeed single large cells, not clusters of small cells. (Cb–Cd): Transmission electron microscopy showed large nuclei with extensive euchromatin and prominent nucleoli in an enriched RSC (Cb), a sphere colony RSC (Cc), and a corresponding cell in ciliary marginal tissue in vivo (Cd). Scale bars = 10 µm. Abbreviation: N, nuclei.

 
RSCs were also enriched from uncultured ciliary cells. First, the ciliary cell population was sorted by FACS, based on cell size and the presence of cell surface markers (CD34 and Sca1), into several cell fractions. Then, these cell fractions were cultured separately in sphere colony-forming medium. Finally, the sphere colony-forming cells were verified as RSCs by cell passaging and differentiation assays (Fig. 1B). The FACS procedure itself did not change cell viability but did reduce sphere colony size; B27 supplement could restore the sphere colony diameter close to 200 µm (data not shown).

Cell size-based FACS (first sorting) of whole ciliary cell population produced a fraction of large cells (15–20 µm) that was 100-fold enriched in sphere colony formation (Fig. 2A). Cell surface marker-based FACS (second sorting) of the large-cell fraction produced a CD34–/Sca1– fraction that was 250-fold enriched in sphere colony formation (Fig. 2B). Thus, 25% of the twice-sorted cells in the 15–20-µm diameter CD34–/Sca1– fraction were the enriched RSCs.


Figure 2
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  		Figure 2. Retinal stem cell (RSC) enrichment. (A): Among ciliary cells (Aa), the rate of sphere colony formation was found to be <0.1% (<0.1% RSC rate). A cell fraction within the rectangle in (Aa) was first-sorted by fluorescence-activated cell sorting (FACS), based on cell size, into cell fractions represented by the two rectangles in (Ab). Small-cell fraction (left rectangle in [Ab]) had <1% RSC rate, and large (15–20 µm diameter) cell fraction (right rectangle in [Ab]) had <10% RSC rate. (B): The small- and large-cell fractions within the two rectangles in (Ab) were secondarily sorted by FACS, based on cell surface markers, into a small-cell (CD34–/Sca1–) fraction (rectangle in [Ba]) that had <3% RSC rate and a large-cell (15–20 µm diameter and also CD34–/Sca1–) fraction (rectangle in [Bb]) that had ~25% RSC rate.

 
It should be noted that the data presented here were obtained from CD1 albino mice. With the same FACS strategy, we failed to enrich sphere colony-forming cells from the ciliary cell population of C57BL/6J mouse eyes, probably due to interfering pigmentation.

Light microscopy confirmed that the large fraction was indeed composed of single large cells rather than clusters of small cells (Fig. 1C). Transmission electron microscopy showed that three kinds of cells—the enriched RSCs, the sphere colony RSCs, and the relevant cells in ciliary marginal tissue in vivo—had large nuclei with extensive euchromatin and prominent nucleoli (Fig. 1C), a common feature of adult retinal stem/progenitor cells [40].

Unique GPL Signatures of Fatty Acid Profiles in RSCs Compared with Non-RSCs
To detect GPL levels and individual GPL proportions, total GPLs were extracted from RSCs and non-RSCs. PC, PE, PS, and PI were separated by one-dimensional TLC, and phosphorus in each sample was determined with dipalmitoyl PC as standard. Total GPLs per cell (nmol P/µg DNA) in sphere colony-derived retinal cells and sphere colony-derived nonretinal cells were significantly higher than those in sphere colony RSCs and enriched RSCs (p < .05, n = 4), whereas the proportions (% of total GPLs) of individual GPLs were similar among the six kinds of RSCs and non-RSCs (Fig. 3A).


Figure 3
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  		Figure 3. GPL content and structure. (A): GPL levels and proportions. Total GPLs were extracted from RSCs (SC-RSCs and E-RSCs) and non-RSCs (RCs, CCs, SC-RCs, and SC-non-RCs). Individual GPLs, including PC, PE, PS, and PI, were purified by thin layer chromatography. Phosphorus in each sample was determined with dipalmitoyl PC as standard. The total GPL levels per cell in SC-RCs and SC-non-RCs were much higher than those in RSCs (p < .05, n = 4), whereas the proportions of individual GPLs were similar between RSCs and non-RSCs. (B): GPL structures. In non-RSCs, the major PE or PI with 18:0–20:4 (stearic acid-arachidonic acid) was 1-octadecanoyl, 2-(5(Z),8(Z),11(Z),14(Z)-eicosatetraenoyl)-sn-glycero-3-phosphoethanolamine or -phosphoinositol. In RSCs, the major PE or PS with 16:0–18:0 (palmitic acid-stearic acid) was 1-hexadecanoyl, 2-octadecanoyl-sn-glycero-3-phosphoethanolamine or -phosphoserine. Abbreviations: CC, ciliary cell; E-RSC, enriched-retinal stem cell; GPL, glycerophospholipid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PS, phosphatidylserine; RC, retinal cell; RSC, retinal stem cell; SC-non-RC, sphere colony-derived nonretinal cell; SC-RC, sphere colony-derived retinal cell; SC-RSC, sphere colony-retinal stem cell.

 
To explore fatty acid profiles, the purified individual GPLs and the total GPLs were analyzed by GC and ESI-MS/MS, respectively. In PC, content (pmol/µg DNA) and percent composition (% of total fatty acids) of various fatty acids were similar among different kinds of cells, but in PE, PS, and PI, fatty acid profiles differed significantly between RSCs and non-RSCs. GC data indicated that sphere colony RSCs and enriched RSCs contained much fewer very long-chain polyunsaturated (20:4 and 22:6) fatty acids than did any of the four kinds of non-RSCs (p < .05, n = 3). ESI-MS/MS data showed that, although RSCs and non-RSCs shared a similar fatty acid profile in PC, they had different signatures in PE, PS, and PI. Remarkably, both sphere colony RSCs and enriched RSCs displayed a new high peak composed of saturated fatty acids at 720 m/z of PE, 764 m/z of PS, and 809 m/z of PI, which was absent in all non-RSCs (Fig. 4). In addition, in sphere colony RSCs, the predominant fatty acids were 16:0 (sn-1)-18:1 (sn-2) for PC, 16:0–18:0 for PE and PS, and 16:0–16:0 for PI, whereas in non-RSCs of ciliary cells and sphere colony-derived retinal cells, they were 16:0–18:1 for PC, 18:0–20:4 for PE and PI, and 18:0–22:6 for PS (Fig. 3B; Table 1). The fact that the sn-2 position in PE, PS, or PI of sphere colony RSCs was occupied by saturated fatty acids was not due to lack of polyunsaturated fatty acids in the culture medium, because the F12 and B27 supplements and the dead ciliary cells in the cultures provided ample essential unsaturated fatty acids.


Figure 4
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  		Figure 4. Glycerophospholipid (GPL) signature. Fatty acid profiles in individual GPLs extracted from six kinds of cells were analyzed by electrospray ionization tandem mass spectrometry. PC signatures were similar among various cells. Signatures of PE, PS, and PC were distinctly different between retinal stem cells (RSCs) (SC-RSCs and E-RSCs) and non-RSCs (RCs, CCs, SC-RCs, and SC-non-RCs). Unique peaks (*) composed of 16:0–18:0 or 16:0–16:0 saturated fatty acids appeared in PE (720 m/z), PS (764 m/z), and PI (809 m/z) of SC-RSCs and E-RSCs but not in four kinds of non-RSCs. Abbreviations: CC, ciliary cell; E-RSC, enriched-retinal stem cell; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PS, phosphatidylserine; RC, retinal cell; SC-non-RC, sphere colony-derived nonretinal cell; SC-RC, sphere colony-derived retinal cell; SC-RSC, sphere colony-retinal stem cell.

 


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  		Table 1. Relative intensity of three pairs of prominent fatty acids in GPLs of RSCs and non-RSCs

 
Lower Activation of Polyunsaturated Fatty Acids by LACSs in Proliferating RSCs than in Differentiated Non-RSCs
Next, we explored what may regulate unique GPL signatures in RSCs. LACS3, mainly existing in the central nervous system, is an important isoform of five types of LACSs (structurally classified) and activates special polyunsaturated long-chain fatty acids such as 20:4 and 20:5 [41]. To seek a possible relationship between LACS3 and fatty acid profiles in individual GPLs, LACS3 expression was measured by RT-PCR in RSCs and non-RSCs. LACS3 mRNA in sphere colony RSCs was obviously lower than that in ciliary cells and sphere colony-derived retinal cells (Fig. 5A), a finding consistent with the smaller amount of polyunsaturated fatty acids in most individual GPLs in sphere colony RSCs than in ciliary cells and sphere colony-derived retinal cells (Table 1). By contrast, the expression of LACS1 that mainly activates saturated or monounsaturated fatty acids did not differ between RSCs and non-RSCs (data not shown).


Figure 5
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  		Figure 5. LACS feature. (A): LACS3 mRNA expression detected by reverse transcription-polymerase chain reaction was much lower in RSCs (SC-RSCs) than in non-RSCs (CCs and SC-RCs). (B): AS activity in subcellular fractions. Protein in nuclei, mitochondria, microsomes, or cytoplasm fraction prepared from non-RSCs (SC-RCs) (Ba) or RSCs (SC-RSCs) (Bb) by successive centrifugation was used as enzyme source. AS activity was expressed as the level of [3H]arachidonoyl-CoA formed in the standard reaction mixture at 5, 10, 20, and 40 minutes; n = 4–6. (C): Oleoyl-CoA synthetase (OS) activity in subcellular fractions. Protein enzyme source was prepared as the above. OS activity was expressed as the level of [3H]oleoyl-CoA; n = 4–6. (D): Substrate specificity of AS. Microsomal protein from non-RSCs (SC-RCs) and RSCs (SC-RSCs) was used as enzyme source. An additional 10 nmol of nonisotopic fatty acid (palmitic acid [16:0], oleic acid [18:1], or arachidonic acid [20:4]) was added to the standard reaction mixture. AS activities in different treatments were expressed as percentage of control that was not added with nonisotopic fatty acid; n = 3. (E): Dose-dependent inhibition of AS activity. Microsomal protein from non-RSCs (SC-RCs) and RSCs (SC-RSCs) was used as enzyme source. Triacsin C (2–67 µM) as inhibitor was added to the standard reaction mixture. AS activities were expressed as percentage of control that was not added with the inhibitor; n = 3. (F): Incorporation of exogenous polyunsaturated fatty acid into glycerophospholipids (GPLs). Non-RSCs (SC-RCs) and RSCs (SC-RSCs) were exposed to [3H]arachidonic acid for 1 day. Isotopic arachidonic acid-labeled GPLs were purified by thin layer chromatography, and radioactivities were measured in PC, PE, PS, and PI. Incorporation of exogenous fatty acid was expressed as the level of [3H]arachidonic acid (pmol/µg DNA) in individual GPLs; n = 4. Values represent means ± SD. Comparison between treatments or cells, * p < .05, ** p < .01. Abbreviations: AS, arachidonoyl-CoA synthetase; CC, ciliary cell; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; LACS, long-chain acyl-CoA synthetase; min, minutes; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PS, phosphatidylserine; RSC, retinal stem cell; SC-RC, sphere colony-derived retinal cell; SC-RSC, sphere colony-retinal stem cell.

 
To detect activation of polyunsaturated fatty acids in sphere colony RSCs (proliferating RSCs) and sphere colony-derived retinal cells (differentiated non-RSCs), we used a set of enzymatic dynamics assays to analyze enzymatic features of two types of LACSs (functionally classified): (a) arachidonoyl-CoA synthetase (AS), which is predominantly for polyunsaturated fatty acids and basically represents LACS3 activity; and (b) oleoyl-CoA synthetase (OS), which is mainly for saturated or monounsaturated fatty acids and reflects general activities of other LACS members, including LACS1. In various organelles of both RSCs and non-RSCs, AS (Fig. 5B) and OS (Fig. 5C) activities in decreasing order were in microsomes, cytoplasm, mitochondria, and nuclei. In microsomes of non-RSCs, AS activity (89.8 nmol/10 minutes per mg protein) (Fig. 5Ba) was much higher than OS activity (11.7 nmol/10 minutes per mg protein) (Fig. 5Ca), and the AS/OS ratio = 7.7. In microsomes of RSCs, AS activity (51.1 nmol/10 minutes per mg protein) (Fig. 5Bb) was significantly lower than in non-RSCs (Fig. 5Ba) (p < .01), whereas OS activity (12.4 nmol/10 minutes per mg protein) (Fig. 5Cb) was similar to that of non-RSCs (Fig. 5Ca), resulting in a lower AS/OS ratio of 4.1. These results indicated that AS activated certain polyunsaturated fatty acids less in proliferating RSCs than in differentiated non-RSCs.

Less Utilization of Arachidonic Acid by GPLs in Proliferating RSCs than in Differentiated Non-RSCs
Diverse enzymatic properties of LACSs were assumed to result in GPL heterogeneity among different kinds of cells. We performed substrate competitive experiments with nonisotopic major fatty acids, including palmitic acid (16:0), oleic acid (18:1), and arachidonic acid (20:4), in microsomes of sphere colony RSCs and sphere colony-derived retinal cells. In non-RSCs, only arachidonic acid among the three tested fatty acids inhibited competitively AS activities by approximately 47% (Fig. 5D), with an apparent Ki value of 49 µM that approximated its Km (44 µM). In RSCs, all three fatty acids (palmitic, oleic, and arachidonic acids) inhibited AS activities by approximately 22%, 25%, and 35%, respectively (Fig. 5D), with apparent Ki values of 85 µM, 74 µM, and 51 µM. These data indicated that AS in non-RSCs mainly utilized polyunsaturated fatty acids (represented by arachidonic acid) as a specific substrate, whereas "AS" in RSCs might reflect a mix of different LACSs that utilized polyunsaturated, monounsaturated, and saturated fatty acids. These results of substrate competitive experiments were further reinforced by an inhibitory experiment with triacsin C (2–67 µM), a selective inhibitor of certain LACSs that activate saturated and monounsaturated fatty acids for de novo GPL synthesis [29, 42]. AS activity in sphere colony-derived retinal cells was not obviously influenced by triacsin C, whereas "AS" activity in sphere colony RSCs was inhibited dose-dependently by this agent with an IC50 value of 38 µM (Fig. 5E).

To check utilization of polyunsaturated fatty acids by GPLs, sphere colony RSCs and sphere colony-derived retinal cells were treated with isotopic arachidonic acid, and its incorporation into individual GPLs of the treated cells was measured. Levels of isotopic arachidonic acid in PC were similar between RSCs and non-RSCs, whereas levels in PE, PS, and PI were much lower in proliferating RSCs than in differentiated non-RSCs (p < .05, n = 4) (Fig. 5F). Taken together, these findings suggested that LACS-associated utilization of some polyunsaturated fatty acids was associated with RSC proliferation and differentiation.

GPL Signature-Related Membrane Fluidity in RSCs and Non-RSCs
Fatty acid profiles determined the GPL signatures, which, in turn, could affect biophysical properties of cell plasma membranes. To measure membrane fluidity in different kinds of cells, two fluorescent probes, DPH and ANS, were separately added to cell suspensions to label hydrophobic cores and polar head regions, respectively, within the plasma membranes of RSCs and non-RSCs. DPH-induced fluorescence polarization in hydrophobic cores of the lipid bilayer was significantly higher in RSCs than in non-RSCs (p < .05) (Fig. 6). ANS-induced fluorescence polarization in polar head regions of the lipid bilayer did not differ between RSCs and non-RSCs (data not shown). These results suggested that RSCs, with less polyunsaturated fatty acids in their GPLs, had lower membrane fluidity than non-RSCs.


Figure 6
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  		Figure 6. Membrane fluidity. Retinal stem cells (RSCs) (SC-RSCs and E-RSCs) and non-RSCs (RCs, CCs, SC-RCs, and SC-non-RCs) in cell suspension were labeled with a fluorescent probe, 1,6-diphenyl-1,3,5-hexatriane. The degree of steady-state fluorescence polarization was acquired, and values represent means ± SD, n = 5. Comparison between RSCs and non-RSCs, * p < .05. The fluorescence polarization value correlates inversely with the degree of membrane fluidity. Abbreviations: CC, ciliary cell; E-RSC, enriched-retinal stem cell; RC, retinal cell; SC-non-RC, sphere colony-derived nonretinal cell; SC-RC, sphere colony-derived retinal cell; SC-RSC, sphere colony-retinal stem cell.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Summary
 Disclosure of Potential...
 Acknowledgments
 References
 
Proliferating RSCs in a given cultured sphere colony (Fig. 1) might represent a clone derived from a single RSC [24] among >1,000 initial ciliary cells, although the clonal character of such spheres has been questioned recently [43]. To avoid possible influence of the culture procedure itself on experimental results, we also tested uncultured RSCs that had been enriched by FACS from uncultured ciliary cells and had a 25% rate of sphere colony formation (Figs. 1, 2). Sphere colony RSCs and enriched RSCs showed similar (but not identical) individual GPL proportions and had lower GPL levels than sphere colony-derived retinal and nonretinal cells in differentiation phase (Fig. 3A). GPL signatures of RSCs showed a unique peak of saturated fatty acids (16:0–18:0 or 16:0–16:0), whereas those of non-RSCs contained predominantly polyunsaturated fatty acids (18:0–20:4 or 18:0–20:6) (Figs. 3B, 4; Table 1). LACSs with varied gene expression, enzymatic activity, substrate specificity, and inhibitor sensitivity regulated activation and utilization of polyunsaturated fatty acids in RSCs and non-RSCs (Fig. 5). Both GPL signatures and LACS enzymatic features may further affect membrane fluidity (Fig. 6).

PC is usually the most abundant GPL and the key building block of membrane bilayers. Although PE is also a key component of membrane bilayers, it has a smaller head group to hydrogen bond through its ionizable amine group and acts as a "chaperone" during membrane protein assembly to guide the folding path for associated proteins. PS may comprise 10–20 mole percentage of total GPLs in plasma membrane and endoplasmic reticulum, with the greatest concentration in central nervous system myelin. The fatty acid composition of PS varies from tissue to tissue, which influences protein kinase C activation, blood coagulation, and apoptosis regulation. PI, usually less than PC, PE, and PS in membranes, functions as a participant in essential metabolic processes and as a primary source of arachidonic acid and diacylglycerol, which serve as signaling molecules regulating activity of a group of related enzymes known collectively as protein kinase C, which in turn regulate many key cellular functions, including proliferation, differentiation, metabolism, and apoptosis.

Individual GPL signatures in retinal cells, which are a subset of central nervous system cells, are similar to those of brain tissue [44]. The present lipidomic analyses of RSCs and non-RSCs establish for the first time that unique signatures of GPLs may be used to identify different cell types. For example, a high peak at 764 m/z of PE might distinguish neural cells (retinal cells and sphere colony-derived retinal cells) from non-neural cells (ciliary cells and sphere colony-derived nonretinal cells); a high peak at 836 m/z of PS might distinguish noncultured cells (retinal cells and ciliary cells) from cultured cells (sphere colony-derived retinal and nonretinal cells); and a high peak at 885 m/z of PI was shared by all non-RSCs. The finding of greatest interest, however, was that new unique predominant peaks, composed of 16:0–18:0 or 16:0–16:0 fatty acids, at 720 m/z of PE, 764 m/z of PS, and 809 m/z of PI, appeared in proliferating RSCs (sphere colony RSCs and enriched RSCs) but not in differentiated non-RSCs, suggesting that the proliferation and differentiation of RSCs might be associated with, or even mediated in part by, fatty acid profiles in individual GPLs. Additional structural features, such as complex N-glycan number and branching, might also affect cell proliferation and differentiation [9, 45].

The composition of various fatty acids with different carbon chain lengths, unsaturated bond numbers, and positions determines GPL signatures. In mature cells, PE, PS, and PI contain higher proportions of arachidonic acid than PC, and this polyunsaturated fatty acid is concentrated in position sn-2, with saturated fatty acids most abundant in position sn-1. Activated metabolites of free polyunsaturated fatty acids degraded from GPLs play vital roles in many cellular processes such as cell proliferation and differentiation. Lipoxygenase derivatives of arachidonic acid act as second messengers to promote progenitor cell proliferation and suppress cell inflammation after corneal wounding [46], to induce proliferation of human cord blood hematopoietic stem cells and exert antiapoptotic effects on these cells [47], to regulate granulocyte-macrophage progenitor proliferation/differentiation and thus promote granulopoiesis [48], and to accelerate CD34+ cell differentiation toward erythroid lineage [49]. Therefore, diverse activation and utilization of arachidonic acid by RSCs and non-RSCs probably represented one of the molecular mechanisms by which GPL signature modulated RSC proliferation and/or differentiation.

GPL signatures of fatty acid profiles also influence biophysical properties of cell plasma membrane, which further link to cell proliferation and differentiation [50]. The term "fluidity" (the reciprocal of viscosity) is loosely used to describe the extent of disorder and the molecular motion within a lipid bilayer [51]. Membranes composed predominantly of saturated fatty acids tend to be relatively rigid, whereas polyunsaturated fatty acids with multiple, restrictive double bonds promote lipid disorder and increase membrane fluidity [37, 52, 53], which might further alter other features of cell plasma membrane, such as perception [51], reception [54], permeabilization [55], and transportation [56]. Therefore, GPLs with more saturated fatty acids resulted in relatively low membrane fluidity, a probable factor in maintaining sphere colony RSCs in the proliferation phase.

What regulates the unique GPL signature in RSCs? Although LACSs activate fatty acids into acyl-CoAs, their key roles in disposal of intracellular long-chain fatty acids into pathways of β-oxidation or glycerol lipid synthesis were largely ignored until five LACS isoforms were recently identified in rodents and humans [57–59]. The differential expression of LACS mRNAs in different tissues and under varying nutritional regimens, their separate locations in different subcellular membranes, and the various responses of oxidative and synthetic pathways to specific inhibitors now suggest that the fate of a particular acyl-CoA may depend on a given LACS that catalyzes its synthesis [41]. LACS3, unlike the other four LACS isoforms, was cloned from a brain library [60], and its mRNA is expressed predominantly in brain and neural cell lines [58, 60, 61]; its fatty acid substrates are mainly 20:4 and 20:5 [41], and it may be associated with brain development [57]. Based on these features, we assumed that AS analysis might correspond to LACS3, which utilizes mainly polyunsaturated fatty acids. Different AS features between RSCs and non-RSCs might characterize stem cells in different states, such as proliferation and differentiation. Low activation of polyunsaturated fatty acids by AS in RSCs could further lead to less incorporation of these fatty acids into GPLs. These data, including the expression of LACS3 mRNA, the enzymatic activity, substrate preference, and inhibitor sensitivity of AS, and the incorporation of polyunsaturated fatty acids into GPLs, collectively support our hypothesis that heterogeneous LACSs could regulate GPL fatty acid signatures and resultant differences in membrane fluidity, which might, in turn, influence RSC proliferation and differentiation.


   SUMMARY
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Summary
Disclosure of Potential...
 Acknowledgments
 References
 
This lipidomics study reports for the first time a unique signature of GPLs in proliferating RSCs, its regulation by LACSs, and its effects on plasma membrane fluidity. However, further work will be required to identify whether this novel finding can be extrapolated to other neural stem cells or possibly to stem cells in general.


   DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Summary
 Disclosure of Potential...
Acknowledgments
 References
 
The authors indicate no potential conflicts of interest.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Summary
 Disclosure of Potential...
 Acknowledgments
References
 
We thank Drs. Roderick R. McInnes, Richard J. Wurtman, and Derek van der Kooy for expert advice and Brenda Coles, Carol Watkins, Michael Samuel, and Lili Yu for technical assistance. This work was supported in part by grants from the Canada Foundation of Fragile X Mental Retardation and the William Randolph Hearst Fund (to J.L.), the American Cancer Society (to Z.C.), and the Nancy Lurie Marks Family Foundation (to R.L.S.).


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Summary
 Disclosure of Potential...
 Acknowledgments
 References
 

  1. Wenk MR. The emerging field of lipidomics. Nat Rev Drug Discov 2005;4:594–610.[CrossRef][Medline]

  2. Adibhatla RM, Hatcher JF, Dempsey RJ. Lipids and lipidomics in brain injury and diseases. AAPS J 2006;8:E314–E321.[CrossRef][Medline]

  3. Serhan CN, Hong S, Lu Y. Lipid mediator informatics-lipidomics: Novel pathways in mapping resolution. AAPS J 2006;8:E284–E897.[CrossRef][Medline]

  4. German JB, Gillies LA, Smilowitz JT et al. Lipidomics and lipid profiling in metabolomics. Curr Opin Lipidol 2007;18:66–71.[Medline]

  5. Wiest MM, Watkins SM. Biomarker discovery using high-dimensional lipid analysis. Curr Opin Lipidol 2007;18:181–186.[Medline]

  6. Forrester JS, Milne SB, Ivanova PT et al. Computational lipidomics: A multiplexed analysis of dynamic changes in membrane lipid composition during signal transduction. Mol Pharmacol 2004;65:813–821.[Abstract/Free Full Text]

  7. van Meer G. Cellular lipidomics. EMBO J 2005;24:3159–3165.[CrossRef][Medline]

  8. Wada K, Arita M, Nakajima A et al. Leukotriene B4 and lipoxin A4 are regulatory signals for neural stem cell proliferation and differentiation. FASEB J 2006;20:1785–1792.[Abstract/Free Full Text]

  9. Lau KS, Partridge EA, Grigorian A et al. Complex N-glycan number and degree of branching cooperate to regulate cell proliferation and differentiation. Cell 2007;129:123–134.[CrossRef][Medline]

  10. Svetlov SI, Ignatova TN, Wang KK et al. Lysophosphatidic acid induces clonal generation of mouse neurospheres via proliferation of Sca-1- and AC133-positive neural progenitors. Stem Cells Dev 2004;13:685–693.[CrossRef][Medline]

  11. Cui HL, Qiao JT. Promotive action of lysophosphatidic acid on proliferation of rat embryonic neural stem cells and their differentiation to cholinergic neurons in vitro. Sheng Li Xue Bao 2006;58:547–555.[Medline]

  12. Ivanova NB, Dimos JT, Schaniel C et al. A stem cell molecular signature. Science 2002;298:601–604.[Abstract/Free Full Text]

  13. Zhao Y, Clark J, Ding S. Genomic studies in stem cell systems. Curr Opin Mol Ther 2005;7:543–550.[Medline]

  14. Parker MA, Anderson JK, Corliss DA et al. Expression profile of an operationally-defined neural stem cell clone. Exp Neurol 2005;194:320–332.[CrossRef][Medline]

  15. Shin S, Rao MS. Large-scale analysis of neural stem cells and progenitor cells. Neurodegener Dis 2006;3:106–111.[CrossRef][Medline]

  16. Williams C, Wirta V, Meletis K et al. Catalog of gene expression in adult neural stem cells and their in vivo microenvironment. Exp Cell Res 2006;312:1798–1812.[CrossRef][Medline]

  17. Aiba K, Carter MG, Matoba R et al. Genomic approaches to early embryogenesis and stem cell biology. Semin Reprod Med 2006;24:330–339.[CrossRef][Medline]

  18. de Haan G, Gerrits A, Bystrykh L. Modern genome-wide genetic approaches to reveal intrinsic properties of stem cells. Curr Opin Hematol 2006;13:249–253.[Medline]

  19. Ile KE, Schaaf G, Bankaitis VA. Phosphatidylinositol transfer proteins and cellular nanoreactors for lipid signaling. Nat Chem Biol 2006;2:576–583.[CrossRef][Medline]

  20. Wang D, Gao L. Proteomic analysis of neural differentiation of mouse embryonic stem cells. Proteomics 2005;5:4414–4426.[CrossRef][Medline]

  21. Hoffrogge R, Beyer S, Volker U et al. 2-DE proteomic profiling of neuronal stem cells. Neurodegener Dis 2006;3:112–121.[CrossRef][Medline]

  22. Van Hoof D, Mummery CL, Heck AJ et al. Embryonic stem cell proteomics. Expert Rev Proteomics 2006;3:427–437.[CrossRef][Medline]

  23. Vodicka P, Skalnikova H, Kovarova H. The characterization of stem cell proteomes. Curr Opin Mol Ther 2006;8:232–239.[Medline]

  24. Tropepe V, Coles BL, Chiasson BJ et al. Retinal stem cells in the adult mammalian eye. Science 2000;287:2032–2036.[Abstract/Free Full Text]

  25. Ahmad I, Tang L, Pham H. Identification of neural progenitors in the adult mammalian eye. Biochem Biophys Res Commun 2000;270:517–521.[CrossRef][Medline]

  26. Coles BL, Angenieux B, Inoue T et al. Facile isolation and the characterization of human retinal stem cells. Proc Natl Acad Sci U S A 2004;101:15772–15777.[Abstract/Free Full Text]

  27. Coles BL, Horsford DJ, McInnes RR et al. Loss of retinal progenitor cells leads to an increase in the retinal stem cell population in vivo. Eur J Neurosci 2006;23:75–82.[CrossRef][Medline]

  28. Van Veldhoven PP, Bell RM. Effect of harvesting methods, growth conditions and growth phase on diacylglycerol levels in cultured human adherent cells. Biochim Biophys Acta 1988;959:185–196.[Medline]

  29. Li J, Wurtman RJ. Nerve growth factor stimulates diacylglycerol de novo synthesis and phosphatidylinositol hydrolysis in pheochromocytoma cells. Brain Res 1998;803:44–53.[CrossRef][Medline]

  30. Tacconi M, Wurtman RJ. Phosphatidylcholine produced in rat synaptosomes by N-methylation is enriched in polyunsaturated fatty acids. Proc Natl Acad Sci U S A 1985;82:4828–4831.[Abstract/Free Full Text]

  31. DeLong CJ, Baker PR, Samuel M et al. Molecular species composition of rat liver phospholipids by ESI-MS/MS: The effect of chromatography. J Lipid Res 2001;42:1959–1968.[Abstract/Free Full Text]

  32. Brugger B, Erben G, Sandhoff R et al. Quantitative analysis of biological membrane lipids at the low picomole level by nano-electrospray ionization tandem mass spectrometry. Proc Natl Acad Sci U S A 1997;94:2339–2344.[Abstract/Free Full Text]

  33. Heeley E, Wright S, Wilton D et al. Molecular species of acidic phospholipids in human lung surfactant. Biochem Soc Trans 1998;26:S227.[Medline]

  34. Wilson DB, Prescott SM, Majerus PW. Discovery of an arachidonoyl coenzyme A synthetase in human platelets. J Biol Chem 1982;257:3510–3515.[Abstract/Free Full Text]

  35. Li J, Wurtman RJ. Mechanisms whereby nerve growth factor increases diacylglycerol levels in differentiating PC12 cells. Brain Res 1999;818:252–259.[CrossRef][Medline]

  36. Mishina M, Kamiryo T, Tashiro S et al. Subcellular localization of two long-chain acyl-coenzyme-A synthetases in Candida lipolytica. Eur J Biochem 1978;89:321–328.[CrossRef][Medline]

  37. Stubbs CD, Smith AD. The modification of mammalian membrane polyunsaturated fatty acid composition in relation to membrane fluidity and function. Biochim Biophys Acta 1984;779:89–137.[Medline]

  38. Smith PK, Krohn RI, Hermanson GT et al. Measurement of protein using bicinchoninic acid. Anal Biochem 1985;150:76–85.[CrossRef][Medline]

  39. Labarca C, Paigen K. A simple, rapid, and sensitive DNA assay procedure. Anal Biochem 1980;102:344–352.[CrossRef][Medline]

  40. Sheedlo HJ, Brun-Zinkernagel AM, Oakford LX et al. Rat retinal progenitor cells and a retinal pigment epithelial factor. Brain Res Dev Brain Res 2001;127:185–187.[CrossRef][Medline]

  41. Coleman RA, Lewin TM, Van Horn CG et al. Do long-chain acyl-CoA synthetases regulate fatty acid entry into synthetic versus degradative pathways? J Nutr 2002;132:2123–2126.[Abstract/Free Full Text]

  42. Igal RA, Wang P, Coleman RA. Triacsin C blocks de novo synthesis of glycerolipids and cholesterol esters but not recycling of fatty acid into phospholipid: Evidence for functionally separate pools of acyl-CoA. Biochem J 1997;324:529–534.[Medline]

  43. Singec I, Knoth R, Meyer RP et al. Defining the actual sensitivity and specificity of the neurosphere assay in stem cell biology. Nat Methods 2006;3:801–806.[CrossRef][Medline]

  44. Hicks AM, DeLong CJ, Thomas MJ et al. Unique molecular signatures of glycerophospholipid species in different rat tissues analyzed by tandem mass spectrometry. Biochim Biophys Acta 2006;1761:1022–1029.[Medline]

  45. Stanley P. A method to the madness of N-glycan complexity? Cell 2007;129:27–29.[CrossRef][Medline]

  46. Bazan HE. Cellular and molecular events in corneal wound healing: Significance of lipid signaling. Exp Eye Res 2005;80:453–463.[CrossRef][Medline]

  47. Chung JW, Kim GY, Mun YC et al. Leukotriene B4 pathway regulates the fate of the hematopoietic stem cells. Exp Mol Med 2005;37:45–50.[Medline]

  48. Pasquale D, Chikkappa G. Lipoxygenase products regulate proliferation of granulocyte-macrophage progenitors. Exp Hematol 1993;21:1361–1365.[Medline]

  49. Desplat V, Ivanovic Z, Dupuis F. Effects of lipoxygenase metabolites of arachidonic acid on the growth of human blood CD34(+) progenitors. Blood Cells Mol Dis 2000;26:427–436.[CrossRef][Medline]

  50. Balint E, Grimley PM, Gan Y et al. Plasma membrane biophysical properties linked to the antiproliferative effect of interferon-alpha. Acta Microbiol Immunol Hung 2005;52:407–432.[CrossRef][Medline]

  51. Murata N, Los DA. Membrane fluidity and temperature perception. Plant Physiol 1997;115:875–879.[Medline]

  52. van Blitterswijk WJ, van der Meer BW, Hilkmann H. Quantitative contributions of cholesterol and the individual classes of phospholipids and their degree of fatty acyl (un)saturation to membrane fluidity measured by fluorescence polarization. Biochemistry 1987;26:1746–1756.[CrossRef][Medline]

  53. Bayer AS, Prasad R, Chandra J et al. In vitro resistance of Staphylococcus aureus to thrombin-induced platelet microbicidal protein is associated with alterations in cytoplasmic membrane fluidity. Infect Immun 2000;68:3548–3553.[Abstract/Free Full Text]

  54. Berlin E, Hannah JS, Yamane K et al. Fatty acid modification of membrane fluidity in Chinese hamster ovary (TR715–19) cells. Int J Biochem Cell Biol 1996;28:1131–1139.[CrossRef][Medline]

  55. Xiong YQ, Mukhopadhyay K, Yeaman MR et al. Functional interrelationships between cell membrane and cell wall in antimicrobial peptide-mediated killing of Staphylococcus aureus. Antimicrob Agents Chemother 2005;49:3114–3121.[Abstract/Free Full Text]

  56. Bookstein C, Musch MW, Dudeja PK et al. Inverse relationship between membrane lipid fluidity and activity of Na+/H+ exchangers, NHE1 and NHE3, in transfected fibroblasts. J Membr Biol 1997;160:183–192.[CrossRef][Medline]

  57. Pei Z, Fraisl P, Berger J et al. Mouse very long-chain Acyl-CoA synthetase 3/fatty acid transport protein 3 catalyzes fatty acid activation but not fatty acid transport in MA-10 cells. J Biol Chem 2004;279:54454–54462.[Abstract/Free Full Text]

  58. Van Horn CG, Caviglia JM, Li LO et al. Characterization of recombinant long-chain rat acyl-CoA synthetase isoforms 3 and 6: Identification of a novel variant of isoform 6. Biochemistry 2005;44:1635–1642.[CrossRef][Medline]

  59. Durgan DJ, Smith JK, Hotze MA et al. Distinct transcriptional regulation of long-chain acyl-CoA synthetase isoforms and cytosolic thioesterase 1 in the rodent heart by fatty acids and insulin. Am J Physiol Heart Circ Physiol 2006;290:H2480–H2497.[Abstract/Free Full Text]

  60. Fujino T, Kang MJ, Suzuki H et al. Molecular characterization and expression of rat acyl-CoA synthetase 3. J Biol Chem 1996;271:16748–16752.[Abstract/Free Full Text]

  61. Fujino T, Man-Jong K, Minekura H et al. Alternative translation initiation generates acyl-CoA synthetase 3 isoforms with heterogeneous amino termini. J Biochem (Tokyo) 1997;122:212–216.[Abstract/Free Full Text]





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