Glycosphingolipid Synthesis Research Articles

Glycosynthase-Mediated Synthesis of Glycosphingolipids

Glycosphingolipids play crucial roles in virtually every stage of the cell cycle, and their clinical administration has been proposed as a treatment for Alzheimer's, Parkinson's, stroke, and a range of other conditions. However, lack of supply has severely hindered testing of this potential. A novel glycosynthase-based synthetic strategy is demonstrated, involving a mutant of an endoglycoceramidase in which the catalytic nucleophile has been ablated. This mutant efficiently couples a range of glycosyl fluoride donors with a range of sphingosine-based acceptors in yields around 95%. This technology opens the door to large-scale production of glycosphingolipids and, thus, to clinical testing.

D-threo-1-Phenyl-2-decanoylamino-3-morpholino-1-propanol Alters Cellular Cholesterol Homeostasis by Modulating the Endosome Lipid Domains

D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP) is a frequently used inhibitor of glycosphingolipid biosynthesis. However, some interesting characteristics of D-PDMP cannot be explained by the inhibition of glycolipid synthesis alone. In the present study, we showed that d-PDMP inhibits the activation of lysosomal acid lipase by late endosome/lysosome specific lipid, bis(monoacylglycero)phosphate (also called as lysobisphosphatidic acid), through alteration of membrane structure of the lipid. When added to cultured fibroblasts, D-PDMP inhibits the degradation of low-density lipoprotein (LDL) and thus accumulates both cholesterol ester and free cholesterol in late endosomes/lysosomes. This accumulation results in the inhibition of LDL-derived cholesterol esterification and the decrease of cell surface cholesterol. We showed that D-PDMP alters cellular cholesterol homeostasis in a glycosphingolipid-independent manner using L-PDMP, a stereoisomer of D-PDMP, which does not inhibit glycosphingolipid synthesis, and mutant melanoma cell which is defective in glycolipid synthesis. Altering cholesterol homeostasis by D-PDMP explains the unique characteristics of sensitizing multidrug resistant cells by this drug.

A novel soluble mimic of the glycolipid, globotriaosyl ceramide inhibits HIV infection

To determine the effect of a gp120 binding, non-cytotoxic soluble analogue of the glycosphingolipid (GSL), globotriaosyl ceramide (Gb3) on HIV infection in vitro. HIV-1(IIIB) (X4 virus) infection in Jurkat and phytohaemagglutinin (PHA)/interleukin-2 (IL2) activated, peripheral blood mononuclear cells (PBMC), and HIV-1(Ba-L) (R5 virus) infection of PHA activated PBMC in vitro were assessed. We monitored cell surface markers, cell viability, and viral/host cell morphology to eliminate pleiotropic effects. Viral-host cell fusion was measured to further address any inhibitory mechanism. HIV infection was monitored by p24(gag) ELISA. CD4, CCR5, CXCR4 and apoptosis were determined by fluorescent antibody cell sorting. A model fusion system comprising a cell line transfected with either CD4 and CXCR4 or CCR5, cocultured with a cell line expressing gp120 from either X4-, R5-tropic HIV-1 or HIV-2 virions, was used. PHA/IL2 activated PBMC GSL synthesis was monitored by metabolic radiolabelling. AdamantylGb3 blocked X4 and R5 virus infection with a 50% inhibitory concentration of approximately 150 microM. A reverse transcriptase and a protease-resistant X4 HIV-1 strain retained adamantylGb3 sensitivity. AdamantylGb3 had minimal effect on cell viability. Treated Jurkat cells showed a small increase in CCR5/CXCR4 expression and a slight, transient CD4 down-regulation, which was probably not related to the mechanism of inhibition. Electron microscopy showed normal viral and host cell morphology following adamantylGb3 treatment, and viral entry was blocked. AdamantylGb3 was able to prevent virus-host cell fusion irrespective of HIV strain or chemokine receptor preference. These results suggest that adamantylGb3 may provide a new basis for blocking HIV infections, irrespective of HIV envelope/chemokine co-receptor preference or resistance to other therapeutics.

Gangliosides are not essential for influenza virus infection

Sialic acid is known to be an essential part of influenza virus receptors, but the specific identity of the receptor molecules on target cells is still not defined. In particular, the relative roles played by cellular sialylglycoproteins and gangliosides in virus entry into target cells remain unclear. To test whether gangliosides are essential for virus infection, we used the GM-95 mutant cell line of mouse B16 melanoma which lacks synthesis of major glycosphingolipids including gangliosides. We found that GM-95 cells grown in serum-containing medium harboured substantial amounts of ganglioside receptors for influenza virus due to incorporation of serum gangliosides. To obtain ganglioside-free cells, we adapted GM-95 cells to growth in defined serum-free (sf) medium. Ganglioside-free GM-95-sf cells could be infected by avian and human influenza A viruses and produced infectious virus progeny demonstrating that gangliosides were neither absolutely necessary for the early nor for the late stages of the infection. However, sensitivity of the GM-95-sf cells to the viruses was 2-4 times lower than that of the ganglioside-containing parent cell line. Further studies are needed to specify whether this effect was due to the lack of gangliosides, neutral glycosphingolipids, or other effects.

Miglustat: Substrate reduction therapy for glycosphingolipid lysosomal storage disorders

Substrate reduction therapy offers a novel approach to the treatment of lysosomal storage disorders. By reducing the rate of macromolecule synthesis to a level where the residual degradative activity in the cell is sufficient to prevent substrate accumulation, it should be possible to reverse storage and storage-related pathologies. Miglustat is an N-alkylated imino sugar that acts against a number of enzymes involved in processing glycoconjugates, including the ceramide-specific glucosyltransferase, which catalyzes the initial committed step in glycosphingolipid synthesis. Miglustat could therefore be used for substrate reduction therapy in glycosphingolipid lysosomal storage disorders. This article addresses both the preclinical and clinical development of miglustat for treatment of type 1 Gaucher's disease, as well as related neuronopathic glycosphingolipidoses.

Miglustat: Substrate Reduction Therapy for Lysosomal Storage Disorders Associated with Primary Central Nervous System Involvement

Difficulties with delivery of functional enzyme to the brain limit the ability to modify neurologic outcome in patients with neuronopathic forms of the lysosomal storage diseases. In a subset of these disorders, which result from a disruption of glycosphingolipid metabolism, the use of a small molecule inhibitor of substrate precursor synthesis may reduce the amount of brain tissue lipid deposition and lead to amelioration of disease. The efficacy of this approach, termed substrate reduction therapy, has been demonstrated in several animal models; with resultant reduction of ganglioside storage in the brain, delayed onset of symptoms and prolonged survival. This pre-clinical 'proof of therapeutic concept' served as the rationale for proceeding with trials in humans using miglustat; an imino-sugar inhibitor of ceramide-specific glucosytransferase (the catalyst for the first committed step in glycosphingolipid synthesis). The glycosphingolipidoses are rare 'orphan' disorders; the limited number of suitable study subjects and the paucity of information on the natural history of these disorders represent major hurdles in the conduct of clinical trials. As treatment potentially constitutes lifelong administration, there will be a need to identify any potential safety considerations attendant to the use of these agents. With greater understanding of disease mechanism, adjunctive therapies may be identified; offering the prospect of modifying these otherwise relentlessly progressive neurodegenerative diseases.

Lipid Rafts: New Tools and a New Component

Lipid rafts are liquid ordered membrane domains enriched with sphingolipids and cholesterol. After 20 years since the proposal of the original concept, the structure and function of lipid rafts are still obscure. Recently new tools to study lipid rafts have been developed. Lysenin is a sphingomyelin binding protein that specifically recognizes the lipid clusters. Poly(ethyleneglycol)-derivatized cholesterol ether (PEG-Chol) is a non-toxic cholesterol probe. These probes have revealed the heterogeneity of lipid rafts. The heterogeneity of lipid rafts is further supported by the discovery of a new lipid component, phosphatidylglucoside. Metabolic inhibitors are another useful tool. Sulfamisterin is a new addition to the serine palmitoyltransferase inhibitors. Recent findings have uncovered a previously unrecognized activity of a glycosphingolipid synthesis inhibitor, D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP).

Inhibition of sphingolipid synthesis affects kinetics but not fidelity of L1/NgCAM transport along direct but not transcytotic axonal pathways

Glycosphingolipids are constituents of lipid rafts which might function in sorting apical and axonal cargoes in the trans-Golgi network. In fact, two GPI-linked proteins, Thy1 and PrP C, require lipid raft lipids for sorting to the axon. It was previously shown that inhibition of glycosphingolipid synthesis by FumonisinB1 (FB1) impairs axon outgrowth but not axon specification, leading to the hypothesis that formation of axonally-targeted vesicles is coupled to sphingolipid synthesis. Since the axonal cell adhesion molecule L1/NgCAM can partition into membrane rafts biochemically, we asked whether correct targeting to the axon is FB1-sensitive, similarly to GPI-linked proteins. We previously showed that cultured hippocampal neurons use more than one trafficking pathway to the axon: a transcytotic pathway and a direct pathway. We show here that reducing raft lipid levels does not disrupt axonal targeting of L1/NgCAM along either pathway. Unexpectedly, FB1 selectively slowed the kinetics of surface expression of a truncated NgCAM using the direct pathway, but not of NgCAM using the transcytotic pathway. Therefore, the formation and/or transport of a subset of axonally-targeted vesicles are coupled to sphingolipid synthesis. Our results yield a mechanism for the axon outgrowth defect observed in FB1.

New Positive Regulators of lin-12 Activity in Caenorhabditis elegans Include the BRE-5/Brainiac Glycosphingolipid Biosynthesis Enzyme

Screens for suppressors of lin-12 hypermorphic alleles in C. elegans have identified core components and modulators of the LIN-12/Notch signaling pathway. Here we describe the recovery of alleles of six new genes from a screen for suppressors of the egg-laying defect associated with elevated lin-12 activity. The molecular identification of one of the new suppressor genes revealed it as bre-5, which had previously been identified in screens for mutations that confer resistance to Bt toxin in C. elegans. bre-5 is the homolog of D. melanogaster brainiac. BRE-5/Brainiac catalyzes a step in the synthesis of glycosphingolipids, components of lipid rafts that are thought to act as platforms for association among certain kinds of membrane-bound proteins. Reducing the activity of several other genes involved in glycosphingolipid biosynthesis also suppresses the effects of constitutive lin-12 activity. Genetic analysis and cell ablation experiments suggest that bre-5 functions prior to ligand-induced ectodomain shedding that activates LIN-12 for signal transduction.

Conditional LoxP‐flanked glucosylceramide synthase allele controlling glycosphingolipid synthesis

Glycosphingolipids are organizational building blocks of plasma membranes that participate in key cellular functions, such as signaling and cell-to-cell interactions. Glucosylceramide synthase--encoded by the Ugcg gene--controls the first committed step in the major pathway of glycosphingolipid synthesis. Global disruption of the Ugcg gene in mice is lethal during gastrulation. We have now established a Ugcg allele flanked by loxP sites (floxed). When cre recombinase was expressed in the nervous system under control of the nestin promoter, the floxed gene underwent recombination, resulting in a substantial reduction of Ugcg expression and of glycosphingolipid ganglio-series levels. The mice deficient in Ugcg expression in the nervous system show a striking loss of Purkinje cells and abnormal neurologic behavior. The floxed Ugcg allele will facilitate analysis of the function of glycosphingolipids in development, physiology, and in diseases such as diabetes and cancer.

Glycosphingolipid Synthesis Inhibitor Represses Cytokine-Induced Activation of the Ras-MAPK Pathway in Embryonic Neural Precursor Cells

Neuronal and glial cells in the central nervous system are generated from common neural precursor cells during development. To evaluate the functions of glycosphingolipids (GSLs) in neural precursor cells, neuroepithelial cells (NECs) were prepared from mouse embryos (E14.5), and the effects of an inhibitor of glucosylceramide synthesis, D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), on NECs was investigated. In PDMP-treated NECs, the expression of GD3, a major ganglioside of NECs, disappeared. We found that basic fibroblast growth factor (bFGF)-induced proliferation and extracellular signal-regulated kinase (ERK) activation were repressed in PDMP-treated NECs. Leukemia inhibitory factor (LIF)-induced ERK activation was also abolished in PDMP-treated NECs, suggesting that PDMP specifically represses the Ras-MAPK pathway. bFGF-induced activation of the Ras-MAPK pathway in NECs is dependent on GSL-enriched microdomains, lipid rafts. The organization of lipid rafts and the distribution of Ras and Grb2-SOS in the microdomains were not affected. However, Ras activation was repressed in PDMP-treated NECs. In PDMP-treated NECs, some neuronal genes were up-regulated and glial genes were down-regulated. These results suggest that GSLs might be involved in the proliferation, survival, signal transduction and differentiation of NECs.

A Cellular Deficiency of Gangliosides Causes Hypersensitivity to Clostridium perfringens Phospholipase C

Clostridium perfringens phospholipase C (Cp-PLC), also called alpha-toxin, is the major virulence factor in the pathogenesis of gas gangrene. Previously, a cellular UDP-Glc deficiency was related with a hypersensitivity to the cytotoxic effect of Cp-PLC. Because UDP-Glc is required in the synthesis of proteoglycans, N-linked glycoproteins, and glycosphingolipids, the role of these gly-coconjugates in the cellular sensitivity to Cp-PLC was studied. The cellular sensitivity to Cp-PLC was significantly enhanced by glycosphingolipid synthesis inhibitors, and a mutant cell line deficient in gangliosides was found to be hypersensitive to Cp-PLC. Gangliosides protected hypersensitive cells from the cytotoxic effect of Cp-PLC and prevented its membrane-disrupting effect on artificial membranes. Removal of sialic acids by C. perfringens sialidase increases the sensitivity of cultured cells to Cp-PLC and intramuscular co-injection of C. perfringens sialidase, and Cp-PLC in mice potentiates the myotoxic effect of the latter. This work demonstrated that a reduction in gangliosides renders cells more susceptible to the membrane damage caused by Cp-PLC and revealed a previously unrecognized synergism between Cp-PLC and C. perfringens sialidase, providing new insights toward understanding the pathogenesis of clostridial myonecrosis.

Miglustat: substrate reduction therapy for glycosphingolipid storage disorders

Miglustat (N-butyldeoxynojirimycin) is a potent inhibitor of ceramide-specific glucosyltransferase, the enzyme that catalyses the first committed step in glycosphingolipid biosynthesis. Inhibition of glycosphingolipid synthesis is a potential strategy for the treatment of a number of lysosomal storage disorders that result in the accumulation of glycosphingolipids within cells, an approach which has been termed substrate-reduction therapy. This article discusses the studies that led to the licensing of miglustat as a treatment for Type 1 Gaucher disease and its current and potential use in this, and other, glycosphingolipid storage disorders.

Glycosphingolipid Accumulation Inhibits Cholesterol Efflux via the ABCA1/Apolipoprotein A-I Pathway: 1-PHENYL-2-DECANOYLAMINO-3-MORPHOLINO-1-PROPANOL IS A NOVEL CHOLESTEROL EFFLUX ACCELERATOR

Cellular glycosphingolipid (GSL) storage is known to promote cholesterol accumulation. Although physical interactions between GSLs and cholesterol are thought to cause intracellular cholesterol "trapping," it is not known whether cholesterol homeostatic mechanisms are also impaired under these conditions. ApoA-I-mediated cholesterol efflux via ABCA1 (ATP-binding cassette transporter A1) is a key regulator of cellular cholesterol balance. Here, we show that apoA-I-mediated cholesterol efflux was inhibited (by up to 53% over 8 h) when fibroblasts were treated with lactosylceramide or the glucocerebrosidase inhibitor conduritol B epoxide. Furthermore, apoA-I-mediated cholesterol efflux from fibroblasts derived from patients with genetic GSL storage diseases (Fabry disease, Sandhoff disease, and GM1 gangliosidosis) was impaired compared with control cells. Conversely, apoA-I-mediated cholesterol efflux from fibroblasts and cholesterol-loaded macrophage foam cells was dose-dependently stimulated (by up to 6-fold over 8 h) by the GSL synthesis inhibitor 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP). Unexpectedly, a structurally unrelated GSL synthesis inhibitor, N-butyldeoxynojirimycin, was unable to stimulate apoA-I-mediated cholesterol efflux despite achieving similar GSL depletion. PDMP was found to up-regulate ABCA1 mRNA and protein expression, thereby identifying a contributing mechanism for the observed acceleration of cholesterol efflux to apoA-I. This study reveals a novel defect in cellular cholesterol homeostasis induced by GSL storage and identifies PDMP as a new agent for enhancing cholesterol efflux via the ABCA1/apoA-I pathway.

Novel Class of Glycosphingolipids Involved in Male Fertility

Mice require testicular glycosphingolipids (GSLs) for proper spermatogenesis. Mutant mice strains deficient in specific genes encoding biosynthetic enzymes of the GSL pathway including Galgt1 (encoding GM2 synthase) and Siat9 (encoding GM3 synthase) have been established lacking various overlapping subsets of GSLs. Although male Galgt1-/- mice are infertile, male Siat9-/- mice are fertile. Interestingly, GSLs thought to be essential for male spermatogenesis are not synthesized in either of these mice strains. Hence, these GSLs cannot account for the different phenotypes. A novel class of GSLs was observed composed of eight fucosylated molecules present in fertile but not in infertile mutant mice. These GSLs contain polyunsaturated very long chain fatty acid residues in their ceramide moieties. GSLs of this class are expressed differentially in testicular germ cells. More importantly, the neutral subset of this new GSL class strictly correlates with male fertility. These data implicate polyunsaturated, fucosylated GSLs as essential for spermatogenesis and male mouse fertility.

Quantitative electron microscopy and fluorescence spectroscopy of the membrane distribution of influenza hemagglutinin

Although lipid-dependent protein clustering in biomembranes mediates numerous functions, there is little consensus among membrane models on cluster organization or size. Here, we use influenza viral envelope protein hemagglutinin (HA0) to test the hypothesis that clustering results from proteins partitioning into preexisting, fluid-ordered “raft” domains, wherein they have a random distribution. Japan HA0 expressed in fibroblasts was visualized by electron microscopy using immunogold labeling and probed by fluorescence resonance energy transfer (FRET). Labeled HA coincided with electron-dense, often noncircular membrane patches. Poisson and K-test (Ripley, B.D. 1977. J. R. Stat. Soc. Ser. B. 39:172–212) analyses reveal clustering on accessible length scales (20–900 nm). Membrane treatments with methyl-β-cyclodextrin and glycosphingolipid synthesis inhibitors did not abolish clusters but did alter their pattern, especially at the shortest lengths, as was corroborated by changes in FRET efficiency. The magnitude and density dependence of the measured FRET efficiency also indicated a nonrandom distribution on molecular length scales (∼6–7 nm). This work rules out the tested hypothesis for HA over the accessible length scales, yet shows clearly how the spatial distribution of HA depends on lipid composition.

A sensitive and reproducible assay to measure the activity of glucosylceramide synthase and lactosylceramide synthase using HPLC and fluorescent substrates

Glucosylceramide synthase (GlcT) and lactosylceramide synthase (GalT) are key enzymes for the synthesis of major glycosphingolipids of vertebrates. In this article, we report a new reliable method to determine GlcT and GalT activities using the fluorescent acceptor substrates C6-4-nitrobenzo-2-oxa-1,3-diazole (NBD)-ceramide and C6-NBD-glucosylceramide, respectively, and a normal-phase high-performance liquid chromatography (HPLC). The reaction products, C6-NBD-glucosylceramide for GlcT and C6-NBD-lactosylceramide for GalT, could be separated from the corresponding acceptor substrates within 6 min under the conditions used. Reaction products were able to be detected quantitatively at concentrations ranging from 50 fmol to 50 pmol, making it possible to determine both activities using the lysate from 1 × 10 4 cultured CHOP cells (Chinese hamster ovary cells expressing polyoma LT antigen) and one zebrafish embryo. This method was used successfully to evaluate the degree of knockdown of GlcT and GalT during zebrafish embryogenesis after injection of the morpholino-oligo-based antisense into one- to four-cell embryos. These results indicate that the fluorescence-based HPLC method is a highly sensitive, rapid, and reproducible assay for determining GlcT and GalT activities and is useful for evaluating the activities in gene knockdown experiments.

Substrate reduction reduces gangliosides in postnatal cerebrum-brainstem and cerebellum in GM1 gangliosidosis mice

II3NeuAc-GgOse4Cer (GM1) gangliosidosis is an incurable lysosomal storage disease caused by a deficiency in acid beta-galactosidase (beta-gal), resulting in the accumulation of ganglioside GM1 and its asialo derivative GgOse4Cer (GA1) in the central nervous system, primarily in the brain. In this study, we investigated the effects of N-butyldeoxygalacto-nojirimycin (N B-DGJ), an imino sugar that inhibits ganglioside biosynthesis, in normal C57BL/6J mice and in beta-gal knockout (beta-gal-/-) mice from postnatal day 9 (p-9) to p-15. This is a period of active cerebellar development and central nervous system (CNS) myelinogenesis in the mouse and would be comparable to late-stage embryonic and early neonatal development in humans. N B-DGJ significantly reduced total ganglioside and GM1 content in cerebrum-brainstem (C-BS) and in cerebellum of normal and beta-gal-/- mice. N B-DGJ had no adverse effects on body weight or C-BS/cerebellar weight, water content, or thickness of the external cerebellar granule cell layer. Sphingomyelin was increased in C-BS and cerebellum, but no changes were found for cerebroside (a myelin-enriched glycosphingolipid), neutral phospholipids, or GA1 in the treated mice. Our findings indicate that the effects of N B-DGJ in the postnatal CNS are largely specific to gangliosides and suggest that N B-DGJ may be an effective early intervention therapy for GM1 gangliosidosis and other ganglioside storage disorders.

Sensitive assay for hormone-sensitive lipase using NBD-labeled monoacylglycerol to detect low activities in rat adipocytes

The recent finding that p-nitrobenzofurazan (NBD)-FA is incorporated into and released from the acylglycerols of isolated rat adipocytes in an insulin-sensitive manner [G. Muller, H. Jordan, C. Jung, H. Kleine, and S. Petry. 2003. Biochimie. 85: 1245-1246] suggests that NBD-FA-labeled acylglycerols are cleaved by rat adipocyte hormone-sensitive lipase (HSL) in vivo. In the present study, we developed a continuous, sensitive in vitro lipase assay using a monoacylglycerol (MAG) containing NBD (NBD-MAG). NBD-MAG was found to provide an efficient substrate for rat adipocyte and human recombinant HSL. Ultrasonic treatment applied in the presence of phospholipids leads to the incorporation of NBD-MAG into the phospholipid liposomes and to a concomitant change of its spectrophotometric properties. The enzymatic release of NBD-FA and its dissociation from the carrier liposomes is accompanied by the recovery of the original spectrophotometric characteristics. The rate of lipolysis was monitored by measuring the increase in optical density at 481 nm, which was found to be linear with time and linearly proportional to the amount of lipase added. To assess the specific activity of recombinant HSL, we determined the molar extinction coefficient of NBD-FA under the assay conditions. This convenient assay procedure based on NBD-MAG should facilitate the search for small molecule HSL inhibitors.

Roles of lipid rafts in integrin-dependent adhesion and gp130 signalling pathway in mouse embryonic neural precursor cells.

Neuronal and glial cells organizing the central nervous system are generated from common neural precursor cells present in the neuroepithelium during development. We tried to clarify functions of a cell surface microdomain, lipid raft, in neuroepithelial cells (NECs). NECs are suggested to adhere to fibronectin substratum dependently on integrin molecules. We found that beta1 integrin, a component of fibronectin receptors, was distributed in lipid rafts. Methyl-beta-cyclodextrin (MBCD), an inhibitor of lipid raft formation, inhibited the integrin-fibronectin interaction-dependent adhesion of NECs. However, inhibition of synthesis of glycosphingolipids (GSL), components of lipid rafts, did not affect NEC adhesion. Leukaemia inhibitory factor (LIF), an interleukin 6 type cytokine, induces astrocyte differentiation of NECs via activation of a transcription factor STAT3. We detected gp130, JAK1 and Ras but not STAT3 and ERK2 molecules in lipid rafts of NECs. Disruption of lipid rafts by MBCD inhibited LIF-induced ERK activation but not STAT3 activation. It is thus suggested that LIF-downstream molecules have differential lipid raft-dependency in terms of activation upon LIF-stimulation. In this study, we found functions of lipid rafts in cell adhesion and signal transduction in NECs. This is the first report that characterized functions of lipid rafts in embryonic neural precursor cells.