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TISSUE-SPECIFIC STEM CELLS

Soluble Jagged1 Attenuates Lateral Inhibition, Allowing for the Clonal Expansion of Neural Crest Stem Cells

^aInterdisciplinary Program in Molecular Genetics and Cell Biology, University of Maine, Orono, Maine, USA;
^bCenters for Molecular and Regenerative Medicine, Maine Medical Center Research Institute, Scarborough, Maine, USA

Key Words. Notch • Developmental biology • Adherent cells • Apoptosis • Cellular therapy • Delta1 • Neural crest • Self-renewal

Correspondence: Joseph M. Verdi, Ph.D., Center for Regenerative Medicine, Maine Medical Center Research Institute, 81 Research Drive, Scarborough, Maine 04074, USA. Telephone: 207-885-8200; Fax: 207-885-8179; e-mail: verdij{at}mmc.org

Received on May 1, 2007; accepted for publication on August 21, 2007.

First published online in STEM CELLS EXPRESS  August 30, 2007.

	ABSTRACT

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

 
The activation of Notch signaling in neural crest stem cells (NCSCs) results in the rapid loss of neurogenic potential and differentiation into glia. We now show that the attenuation of endogenous Notch signaling within expanding NCSC clones by the Notch ligand soluble Jagged1 (sJ1), maintains NCSCs in a clonal self-renewing state in vitro without affecting their sensitivity to instructive differentiation signals observed previously during NCSC self-renewal. sJ1 functions as a competitive inhibitor of Notch signaling to modulate endogenous cell-cell communication to levels sufficient to inhibit neural differentiation but insufficient to instruct gliogenic differentiation. Attenuated Notch signaling promotes the induction and nonclassic release of fibroblast growth factor 1 (FGF1). The functions of sJ1 and FGF1 signaling are complementary, as abrogation of FGF signaling diminishes the ability of sJ1 to promote NCSC expansion, yet the secondary NCSCs maintain the dosage sensitivity of the founder. These results validate and build upon previous studies on the role of Notch signaling in stem cell self-renewal and suggest that the differentiation bias or self-renewal potential of NCSCs is intrinsically linked to the level of endogenous Notch signaling. This should provide a unique opportunity for the expansion of NCSCs ex vivo without altering their differentiation bias for clinical cell replacement or transplant strategies in tissue repair.

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

	INTRODUCTION

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

 
During the aging process, the number and responsiveness of quiescent stem cell populations diminish [1]. A major goal of modern biology is to understand the molecular mechanisms that underlie stem cell self-renewal in hopes of facilitating the long-term expansion of stem cell populations ex vivo without quantifiable changes of either their sensitivity to instructive signals or multilineage differentiation potential. Several genes that affect the self-renewal process, including Nanog [2], Nucleostemin [3], Oct4 [4, 5], and Bmi1 [6], have been identified. However, the mechanisms that underlie stem cell self-renewal remain to be determined.

We previously demonstrated that as neural crest stem cells (NCSCs) undergo self-renewal with age or in vitro, quantifiable changes occur to their sensitivity to instructive differentiation signals [7, 8]. In particular, we reported that p75⁺P₀^– NCSCs become progressively more gliogenic and less neurogenic with age [7]. This gradual transition reflects changes in the probability of differentiation to neurons versus glia by individual stem cells in response to instructive signals such as bone morphogenetic protein 2 (BMP2) or Delta-Fc, rather than an asynchronous, all-or-none switch from generating neurons and glia to generating only glia. Thus, multipotential stem cells undergo progressive changes in their differentiation bias while retaining their qualitative properties of self-renewal and multipotency [7, 8]. The mechanism of this change, in part, involves changes in the expression levels of their receptors bone morphogenetic protein receptor 1 (BMPR1) and Notch1 [7, 8]. These changes occur in NCSCs cultured at clonal density in the absence of other cell types and are dependent on local cell-cell interactions within developing colonies that are mediated by Notch signaling.

Recently, a bevy of publications has alluded to the necessity of Notch signaling to maintain stem-like progenitors in a nondifferentiated, self-renewing state [9–17]. The most compelling of these is the recent report by Yeo and Chitnis [17] demonstrating that Jagged-mediated Notch signaling maintains proliferating neural progenitors in a stem-like state within the ventral spinal cord. As a whole, these studies on the role of Notch signaling in neural stem cells appear to be in conflict with earlier studies by our group [18] and others [19–25] demonstrating that activated Notch signaling by the Notch ligand soluble Delta1 (sDl) or expression of the Notch-1 intracellular domain (Nicd) promoted the gliogenic differentiation of neural stem cell populations, both in vitro and in vivo. Therefore we prudently re-examined the hypothesis that activation of Notch signaling leads to the permanent loss of neurogenic potential in NCSC and the induction of a gliogenic differentiation program.

Notch signaling plays key roles in normal development through diverse effects on differentiation, survival, and proliferation that are dependent on signal strength and cellular context [26]. Four Notch receptor genes (Notch1–Notch4) and three ligand gene families (Jagged1, Jagged2, Delta1, Delta3, Delta4, and F3/Contactin) have been described [27–29]. These transmembrane ligands activate Notch receptors on neighboring cells, leading to the proteolytic cleavage of Notch that modulates CSL1-dependent transcription [26]. Because Notch signaling is instrumental in the differentiation outcomes of neural stem cells [25] and instructive in NCSCs gliogenic differentiation [18], putative modulators of Notch signaling may be important regulators of NCSC survival, expansion, and differentiation.

Indeed, in this study we demonstrated that the attenuation or reduced activation of endogenous Notch signaling by soluble Jagged1 (sJ1) in vitro inhibits neuronal differentiation, allowing for the expansion of NCSCs. The expansion of stem cells is in part due to the nonclassic release of fibroblast growth factor 1 (FGF1) induced by the suppression of endogenous Notch signaling by sJ1. Importantly, secondary and tertiary stem cell populations generated from founder cells while in the presence of sJ1 maintain their full multipotentiality without a change in sensitivity to instructive differentiation signals. These data suggest a model that integrates the antidifferentiation and survival effects of attenuated but active Notch signaling and the proliferative effects of FGF1 to allow for fibroblast growth factor receptor (FGFR)-dependent cell cycle progression and symmetric cell division, leading to clonal expansion of stem cell populations without an intrinsic shift in instructive factor sensitivity that normally accompanies in vitro self-renewal.

	MATERIALS AND METHODS

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Preparation of Soluble Notch Ligands
The construction of the soluble–notch ligands and the construction of dominant negative Notch1 constructs were previously reported [18, 30]. The purification and quantification of the soluble–notch ligands was performed according to our published methods [7]. In short, confluent cultures of 293T Delta-Fc, Jagged1-Fc, and 293T-Fc cells were refed with Dulbecco's modified Eagle's medium high-glucose medium and allowed to culture for an additional 5 days. The conditioned medium was concentrated by centrifugation in a Centricon Biomax 30 column (Millipore, Stoughton, MA, http://www.millipore.com) to achieve a 50- to 100-fold total net concentration. Prior to use, the concentrated Notch ligand-Fc-containing supernatant was incubated with a 100-fold dilution of anti-human Fc antibody (catalog 109-005-0098; Jackson Immunoresearch Laboratories, West Grove, PA, http://www.jacksonimmuno.com) for 30 minutes at room temperature to cross-link the soluble Fc ligands. The initial concentration and purity of the concentrated ligands were determined by taking a serial dilution of the protein and comparing it with a known amount of bovine serum albumin (BSA) on a 10% SDS–polyacrylamide gel electrophoresis gel and also by standard bicinchoninic acid assay using a serial dilution of BSA to develop the standard curve. In many experiments, three different soluble notch ligands were used to ensure that nothing in the conditioned medium was responsible for the differences observed. The parent constructs were distributed to various researchers upon request and agreement of confidentiality, and their generation of soluble ligand was graciously sent to us for comparison. In sJ1M, sJ1B, and sJ1K, the last letter refers to the laboratory that generated the soluble ligand: M, Maciag; B, Bahtia; K, Kiren laboratories.

Neural Crest Stem Cell Culture Clonogenic Differentiation Assays
NCSC cultures were generated according to standard methods [31]. Briefly, embryonic day (E) 10 rat neural tubes were dissected above and below the presumptive limb buds, plated on fibronectin, and cultured overnight at 37°C in a humidified 5% CO₂ tissue culture cabinet. The following morning, the neural tube was removed with a tungsten needle, and the explant culture was dissociated with trypsin. The resulting cells were plated on poly D-lysine fibronectin matrix [31, 32] at a density of 50 cells per 60-mm dish. NCSCs were identified by staining the resulting cells with the p75 antibody and an antibody to the myelin protein P₀ [31, 32]. All p75⁺P₀^– were circled using a grease pencil and considered to be putative primary or founder NCSCs. The cells were allowed to grow for 14 days in Morrison medium [32] before being fixed in 4% formaldehyde and stained with antibodies against peripherin (Chemicon, Temecula, CA, http://www.chemicon.com) to detect neurons, smooth muscle actin (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com) to detect myofibroblasts, and glial fibrillary acidic protein (GFAP) (Chemicon) to demarcate Schwann cells in the growing clones. Differentiation profiles of resulting clones were scored based on the appearance of just one phenotypic representative. The number and percentage of clones containing neurons (N) only, Schwann cells (S) only, myofibroblasts (M) only, N + S, N + M, S + M, and N + S + M were scored at day 14 [31, 32]. All clonogenic differentiation assays or self-renewal assay were performed at least three times from fresh NCSCs or NSC isolations.

In washout experiments, NCSCs were treated with sJ1, Dl1-Fc, or nothing for the time described prior to removal of the medium and three successive washes in sterile Hanks' balanced salt solution (HBSS) with Mg²⁺ and Ca²⁺ (Invitrogen, Carlsbad, CA, http://www.invitrogen.com) prior to the addition of fresh medium supplemented with 1 nm BMP2 (R&D Systems Inc., Minneapolis, http://www.rndsystems.com), 7.5 nM Delta-Fc, or standard medium (Morrison medium [18, 32]) to challenge the differentiation potential of the cells.

Retroviral infections retroviral infections were performed as previously described [7, 17]. Virus was generated using the 293GPG packing line and collected in Opti-MEM (Invitrogen). Two milliliters of undiluted virus stock was added to a 60-mm dish for 2 hours in the presence of 3.5 µg/ml polybrene (Sigma-Aldrich) to enhance infection efficiency. Eighteen hours postinfection, non-enhanced green fluorescence protein (non-EGFP) NCSCs were removed from the dish with tungsten needle, and the other cells were allowed to progress.

5-Bromo-2'-Deoxyuridine Incorporation Study
Clonogenic differentiating NCSCs were pulsed for 4 hours with 1 mM 5-bromo-2'-deoxyuridine (BrdU) (BD Biosciences, San Diego, http://www.bdbiosciences.com) prior to washing of the cells and 6 hours of growth in standard medium. Clones were fixed using acid-ethanol for 10 minutes at –20°C. Cells were washed with sterile HBSS at room temperature for 5 minutes prior to permeabilizing the membrane as we described [33]. BrdU was detected by immunohistochemistry using an anti-BrdU antibody (Dako, Glostrup, Denmark, http://www.dako.com) [33].

	RESULTS

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

 
sJ1 Enhances the Self-Renewal of Neural Crest Stem Cells
We previously reported that the transient activation (24 hours) of Notch by soluble Notch ligand delta 1 (sDl1) leads to an irreversible loss of NCSC neurogenic potential and differentiation into glia [18]. Because we know that in other cell systems, the binding of Notch by its two major cognate ligands families (Delta and Jagged) can result in differential biological readouts [34, 35], we assessed whether sJ1 also induces a similar irreversible loss of neurogenic potential or perhaps mediates a distinct phenotypic outcome. Similar to recent work by Yeo and Chitnis [17] and in contrast to sDl1-treated NCSCs, sJ1-treated NCSCs [18] retained neuronal differentiation potential as pronounced as that of untreated control (Table 1). This was true whether Jagged1 was presented as monomer or as a Jagged1-Fc dimer. Although increasing the concentration of sJ1 had little effect on neurogenic potential (data not shown), removal of sJ1 any time during this time frame resulted in differentiation profiles indistinguishable from that of NCSCs differentiating in standard medium (as described in Materials and Methods) or standard medium supplemented with Fc (Table 2). Although this result does not support our previous model, which used Delta-Fc ligand to activate Notch signaling, it is more consistent with the models of Notch signaling in which neurogenic differentiation resumes upon removal or decay of the transient Notch signals [36–38].

View larger version (14K): [in this window] [in a new window]   Figure 1. sJ1 does not enhance the proliferation of nondifferentiated neural crest stem cells (NCSCs). Shown is BrdU analysis of developing NCSC cultures. BrdU (1 mM) was added for 4 hours prior to removal and washing of the cells. Standard medium was replaced, and the cells were allowed to continue for an addition 2 hours prior to fixation and immunohistological staining for BrdU. Presented are the means ± SD of three experiments demonstrating that sJ1 does not increase the overall production of nondifferentiated progeny (right axis) but does change the percentage of nondifferentiating cells taking up BrdU. *, 0.05 between sJ1 and standard medium. Abbreviations: BrdU, 5-bromo-2'-deoxyuridine; GFAP, glial fibrillary acidic protein; sD1, soluble Delta1; sJ1, soluble Jagged1.

Because differentiated progeny in the expanding sJ1-treated NCSC clones were not selectively undergoing cell death, we inquired as to the nature of the cells within these clones. Staining the clones at day 10, 53% ± 17% of the cells per clone (n = 10 clones examined) expressed p75 and another marker of neural stem cells, nestin [31]. To confirm that sJ1 treatment promoted the undifferentiated expansion of p75⁺ NCSC, we directly examined the multipotentiality of p75⁺ cells at day 10 by subcloning (Table 3). Founder NCSCs expanded in sJ1 for 10 days displayed extensive self-renewal as evidenced by the identification of numerous p75⁺ cells that generated multipotential secondary clones upon differentiation by the removal of sJ1. On average, 244 ± 76 (p .05) p75⁺ secondary stem cells gave rise to multipotential clones from founder clones expanded in sJ1 medium (n = 8 dissociated cones examined) compared with 123 ± 53 and 96 ± 52 multipotential cells in standard medium (n = 7 dissociated clones examine) and Fc-containing medium (n = 7 dissociated clones examined), respectively. As previously demonstrated by Morrison et al. [18], few if any multipotential cells remained in cultures treated with sDl1 (9 ± 5). NCSCs expanded in sJ1 also displayed a significant increase (p .05) in multilineage differentiation (neurons, Schwann cells, and myofibroblasts) potential compared with secondary NCSCs originating from control, sDl1-treated, and Fc-treated cultures. Of secondary NCSCs originating from sJ1-treated founders, 77% ± 10% were tripotent, compared with subclones arising from Fc (51% ± 8%), standard (59% ± 9%), or sDl1 (0%) conditions. These data support the previous observations that sDl1 promotes gliogenic differentiation. Furthermore, these results demonstrate that, in contrast to sDl1, sJ1 promotes the undifferentiated expansion of NCSCs.

View larger version (20K): [in this window] [in a new window]   Figure 2. NCSCs expanded in sJ1 retain dosage sensitivity to instructive differentiation signals. NCSC clones were expanded for 10 days in sJ1 prior to dissociation in trypsin. Secondary stem cells (p75+) were challenged with the instructive differentiation factor 1 nM BMP2 (neuronal) or 7.5 nM Delta-Fc (glial). (A): Presented are the means ± SD of three experiments comparing the production of neuron- and glia-only clones in BMP2 and Delta-Fc, respectively. In both graphs, the red line refers to freshly isolated NCSCs; the black line refers to differentiation profile of secondary stem cells originating from primary clones generated without supplementation; and the blue line refers to secondary stem cells originating from primary clones expanded in sJ1. (B): NCSCs retain dosage sensitivity of the founder for upwards of 60 days in culture. Founder NCSCs were dissociated after 10 days of expansion, and secondary stem cells were generated. These were allowed to grow for 10 days before repeating the process to produce tertiary stem cells. The pattern was repeated every 10 days for 60 days, at which point cells were removed from sJ1 medium, treated with standard medium, and allowed to differentiate in the presence of instructive neurogenic signals. (C, D): Quantitative PCR for the levels of mRNA for known genes whose expression are associated with either gliogenic or neurogenic differentiation bias of NCSCs after the time shown in the presence of NA or 7.5 sJ1. Shown are Notch mRNA (C) and the levels of Numb (red), BMPR1 (green), and BMP2 (blue) mRNA (D). Presented in all panels are the means ± SD of at least three experiments taken from distinct NSCS preparations. *, p .05 by Student's two-tailed t test comparing levels after 14 days without supplementation to levels in the presence of sJ1. Abbreviations: BMP, bone morphogenetic protein; BMPR, bone morphogenetic protein receptor; NA, no supplementation; NCSC, neural crest stem cells; sJ1, soluble Jagged1.

sJ1 Antagonizes Endogenous Notch Signaling
To begin to elucidate the mechanism by which sJ1 leads to NCSC self-renewal, we returned to our clonal in vitro analyses to assess the expression of downstream effectors of Notch signaling in expanding rat E10.5 NCSCs clones upon sJ1 exposure to better define the molecular differences initiated by sJ1 and sDl1. We established NCSC clones and allowed them to reach 4–8 cells in size, and used quantitative real-time polymerase chain reaction (PCR) to examine the activation of the downstream Notch effector genes, Hes1 and Hey, daily for 7 days while in the presence of sJ1 or sDl1 that was refreshed daily. The expression of Hes1 message in NCSC was approximately sevenfold greater with sDl1 than with sJ1 after the initial 24 hours of ligand exposure (Fig. 3A, 3B). Unlike sDl1, the level of Hes1 mRNA expression in sJ1-treated cells was less than the levels of untreated clones, suggesting that sJ1 suppressed active endogenous Notch signaling within expanding NCSCs clones and indicating that sJ1 may act as an antagonist or modulator of Notch-mediated events in NCSCs (Fig. 3B). Over the first 7 days of NCSC clonogenic differentiation, other Notch effector genes were induced in sDl1 (Fig. 3A) and attenuated in sJ1-treated cultures (Fig. 3B). Hey1 (green line) and Deltex (blue line) mRNAs were induced to their maximum levels within 36 hours in sDl1-treated NCSCs, concurrent with the loss of neurogenic potential and onset of gliogenic differentiation. Conversely, NCSCs treated with sJ1 demonstrated a suppression of Hes1, Hey1, and Deltex messages compared with untreated and expanding NCSCs clones that was maintained for several days. The levels of the three effector genes reached the levels comparable to levels reached in clones grown without supplementation by day 7.

View larger version (26K): [in this window] [in a new window]   Figure 3. sJ1 leads to a slow accumulation of Notch target genes. Real-time polymerase chain reaction (PCR) levels of Notch effectors genes during neural crest stem cell (NCSC) expansion. (A, B): Quantitative real-time PCR measurements of NCSCs in culture for the expression of Hes1, Deltex, and Hey1 mRNAs in 5 nM sDl1 (A) and 7.5 nM sJ1 (B). (C) sJ1 is a competitive inhibitor of sDl1 function. Clonogenic differentiation assays of NCSCs cultured in 5 nM sDl1 for 24 hours with increasing concentrations of sJ1. After 24 hours, medium was washed out and cells were challenged with 1 nM BMP2 of the neurogenic instructive factor. After an additional 14 days, clones were fixed and immunohistochemistry was used to determine the differentiation profiles. Dark gray columns indicate the percentage of neuron-only clones; light gray columns indicate the percentage of neuron-containing clones. (D): The retention of neurogenic potential correlated with a decrease in Hes1 mRNA expression. Quantitative real-time PCR was used to determine the expression of Hes1 mRNA in developing neural crest clones 72 hours after addition of 5 nM -Fc and increasing concentrations of sJ1. (A–D): Means ± SD of at least three experiments taken from distinct NSCS preparations. Abbreviations: BMP, bone morphogenetic protein; sDl1, soluble Notch ligand Delta1; sJ1, soluble Jagged1.

To elucidate whether sJ1 antagonized sDl1 binding or initiated an alternative signaling pathway, we overexpressed the Nicd in E10 rat NCSCs, treated the cells with 10-fold greater sJ1 than required to block sDl1 actions, and tested the cells for the ability to respond to respond to BMP2 24 hours later. Most if not all Nicd-expressing cells were incompetent to respond to BMP and began to initiate gliogenic differentiation as measured by the appearance of GFAP immunoreactivity within 36 hours in culture. These experiments demonstrate that sJ1 functions as a competitive inhibitor of Notch activation and does not induce a novel signal transduction pathway that inhibits or overrides normal Notch signaling events.

Notch Signaling Is Required for NCSC Viability
Attenuation of Notch signaling via sJ1 seemed to promote the undifferentiated expansion of multipotent NCSCs. In an attempt to resolve this paradox, we attempted to abrogate Notch signaling of NCSCs in vitro by overexpressing a dominant negative Notch1 receptor [40] or the dominant negative suppressor of hairless construct (Su(H)^DMB) that we used previously [18] in NCSCs by retroviral induction. Initially, we examined the level of Hes1 mRNA in green fluorescent protein (GFP), Su(H)^DMB, and dominant negative Notch1-transduced cultures as one potential readout for Notch activation. NCSCs expressing either mutant displayed significantly repressed levels of Hes1 mRNA compared with sJ1-treated, GFP-infected NCSCs (Fig. 4A). This demonstrates that these mutants were more efficacious at lowering Hes1 transcription and potentially overall Notch signaling than is sJ1. Clonogenic differentiation assays were conducted to determine whether enrichment in NCSCs after 7 days would result. This observation, that NCSCs transduced with dominant negative Notch1 or Su(H)^DMB showed a dramatically impaired viability during expansion and clonogenic differentiation compared with β-galactosidase- or EGFP-transduced NCSC, is consistent with the observations put forth by Guentchev and McKay [11]. By day 3, a significant reduction in clone viability was apparent (Fig. 4B). On average 55%, or 16.8 ± 3.7 of 30.2 ± 5.4, Su(H)^DMB founders and 41%, or 14.2 ± 5.8 of 34.8 ± 7.9, dominant negative (dn) Notch1 founders survived compared with 98% (44 of 45) of p75⁺ E10 rat NCSCs or 91% (44 of 49) GFP-infected E10 rat NCSCs treated with sJ1.

View larger version (17K): [in this window] [in a new window]   Figure 4. Notch signaling is required for NSC survival. (A): Quantitative real-time PCR of developing p75+ neural crest stem cells (NCSCs) expressing dnNOTCH1 or dominant negative suppressor of hairless for Hes1 mRNA levels. Presented are the means ± SD of four experiments performed in quadruplicate, demonstrating that relative to standard medium without supplementation, the levels of Hes1 mRNA are reduced in sJ1 and further reduced in cells expressing constructs designed to abrogate Notch signaling. (B): The reduction of Notch signaling correlates with a loss of cell viability. Individual NCSCs infected with dnNotch1 or dnSu(H)DMB virus were plated on fibronectin-laminin dishes and allowed to grow for 6 days. The number of clones established was counted by hand. Presented are the means ± SD of three experiments (starting with 50 clones) of surviving clones per day. Abbreviations: dnNOTCH1, dominant negative Notch1; No add, no supplementation to standard Morrison medium; sJ1, soluble Jagged1.

FGF Signaling Is Required for the Robust Expansion of NCSCs
Because sJ1 induces the transcription and nonclassic release of FGF1 [40], we inquired whether NCSCs also induced and released FGF1 when exposed to sJ1. FGF1 mRNA was not detectable after 35 cycles of PCR prior to sJ1 addition; however, within 12 hours, the earliest time point tested, FGF1 mRNA was detected in three separate expanding NCSC clones (Fig. 5). Because sJ1 could induce FGF1 mRNA production, we determined whether FGF1 was being released from NCSCs in an autocrine fashion to promote NCSC expansion. A dominant negative FGFR1 (dnFGFR1) construct was expressed in NCSCs, and clonogenic differentiation assays were conducted in the presence of sJ1 or in standard medium without further supplementation. NCSC clones expressing dnFGFR1 demonstrated a dramatic reduction in their response to sJ1 (Table 4) in their ability to produce secondary stem cells at day 7, but they demonstrated only a small decrease in their ability to generate differentiation outcomes of the secondary stem cells in standard medium supplemented with 2 ng/ml basic FGF.

View larger version (30K): [in this window] [in a new window]   Figure 5. The roles of FGF and Notch signaling are complementary in stem cell self-renewal. (A): Polymerase chain reaction analysis of neural crest stem cells (NCSCs) treated with sJ1 or sDl1 for 12, 24, or 48 hours. Amplicons (25 cycles) were separated on 1% agarose Tris acetate EDTA gel and visualized and photographed using ethidium bromide under UV light. Presented is a typical gel after 48 hours of treatment. (B): Clonogenic differentiation assays of NCSCs expressing a dnFGFR1 and treated with sJ1. Shown are dose-response curves of clonogenic differentiation outcomes (the production of neuron- or glia-only clones in BMP2 and Delta-Fc, respectively). Note that although the number of secondary subclones is less in cells expressing dnFGFR1, the sensitivity to instructive differentiation factors does not change. Primary indicates freshly isolated NCSCs. (C): Sliding scale of Notch signaling level. Low Notch activation results in inhibition of neuronal differentiation, promotion of survival, and self-renewal; moderate levels of Notch signaling result in loss of neurogenic potential; and high levels of Notch signaling result in gliogenesis. (D, E): Model of Notch signaling in NCSCs. High levels of Notch signaling, either by Jagged1 or Delta1 binding of the Notch receptors, result in cleavage of the Nicd and translocation to the nucleus, where it binds other activators, presumably MaM, which is highly expressed in NCSCs, resulting in CSL-dependent elevated Hes1 and Hey1 transcription. Soluble Jagged-Notch interaction also results in cleavage of the Nicd and translocation to the nucleus, resulting in attenuated and delayed Hes1 and Hey1 transcription. CSL-dependent transcription also leads to immediately elevated (large arrow) transcription of FGF1, which is coupled with the nonclassic release of FGF1 and promotion of FGFR-mediated expansion of NCSCs. Abbreviations: BMP, bone morphogenetic protein; dnFGFR1, dominant negative fibroblast growth factor receptor 1; FGF, fibroblast growth factor; FGFR, fibroblast growth factor receptor; MaM, Mastermind; Nicd, Notch-1 intracellular domain; No add, secondary NCSCs derived from primary clones grown in standard Morrison medium; sDl1, soluble Notch ligand Delta1; sJ1, soluble Jagged1.

	DISCUSSION

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

 
Stem cells persist throughout adult life in numerous tissues, including the peripheral and central nervous systems. This raises the intriguing possibility that common mechanisms may regulate self-renewal in various stem cell populations. Despite a number of genes being identified that foster self-renewal, little is known about the mechanism that regulates this event. In this study, we present evidence for the involvement of FGF and attenuation of endogenous Notch signaling in the process of stem cell self-renewal. Our model (Fig. 5C) for the role of Notch signaling by sJ1 is similar to a model put forth recently by Jadhav et al. [12]. The hallmark of our model is that the biological readouts of neural stem cells are based on the activation level of Notch, with low Notch activation requisite for survival and enhanced signaling that is sufficient to inhibit neural differentiation, whereas increasing the activity leads to a permanent loss of neurogenic potential and ultimately gliogenic differentiation. This is parsimonious with our previous work and the current bevy of notch papers concerning Notch's role in stem cell self-renewal.

Notch Signaling Strength Underlies the Transition from Self-renewal to Differentiation
Because dnFGFR1 expression abrogates the ability of sJ1 to facilitate self-renewal, and FGF1 release is dependent upon the repression of classic Notch signaling [30], we show that sDl1 enhances Notch activity in NCSC populations, whereas the nontransmembrane form of Jagged1 suppresses Notch signaling in these cells. Indeed, RT-PCR analyses of Notch downstream effectors demonstrate a suppression of the steady-state levels of Hey1 and Deltex transcripts in sJ1- but not sDl1-treated NCSC populations, and these results agree with the differences mediated by sJ1 and sDl1 to induce self-renewal or gliogenesis, respectively. It is highly significant that sDl1 increases Notch activation, leading to loss of neurogenic potential and gliogenic differentiation, whereas suppression of these same downstream targets by sJ1 inhibits differentiation, maintains multipotentiality, and initiates the robust FGF signaling-associated expansion of neural stem cells. Thus, the levels of Notch signaling and the presence of sJ1 may be key elements in regulating the undifferentiated expansion of multipotential neural stem cells, without concomitant changes to instructive differentiation factor sensitivity.

FGF1 Release and Attenuation of Notch Signaling Lead to Maximum Clonal Self-Renewal
It is generally accepted that as stem cells transition through the self-renewal process, they appear qualitatively similar in vivo and in vitro, maintaining their basic properties of self-renewal and multilineage differentiation by their abilities to respond to instructive differentiation signals [18]. No abrupt restriction in self-renewal potential of NSCs is observed in culture or in vivo; rather, there is a bias in the differentiation outcome mediated through changes in responsiveness to instructive differentiation factors. This shift in bias is more likely to occur through cell-cell communication than through extrinsic signals derived from either the extracellular matrix or surrounding tissues, where instructive differentiation signals originate. Thus, stimulation of endogenous classic Notch signaling may bias NCSC populations toward a gliogenic differentiation program while maintaining a permissive response to non-neuronal instructive signals [18]. In contrast, the generation of sJ1 either resets or represses this outcome. In clonal neural crest assays, sJ1 allows for the expansion of trifatent stem cells at the expense of bipotential and unipotential differentiation. Instructive differentiation factors such as BMP2 or Delta1, which restrict the self-renewal potential of NCSC, may do so by either eliminating or overriding the sJ1 signal. Because our data suggest that the activity of sDl1 in NCSC is dominant to that of sJ1, we suggest that as stem cells self-renew, a slight shift in factor responsiveness occurs. As this process continues, each subsequent stem cell is slightly different, until a particular phenotypic outcome is lost because of the lowering of its ability to respond to a particular instructive differentiation factor. Thus, expanding stem cell populations may maintain their self-renewal potential when analyzed in vitro, where one can manipulate the concentrations of ligand, but this potential may be lost in vivo. However, the process of stem cell self-renewal may be enhanced through the repression of classic Notch signaling during the serial passage of the stem cell populations in culture [7] or as a result of the generation of sJ1 by yet to be identified proteases in the cellular milieu. Preliminary evidence demonstrates that thrombin cleaves Jagged1 on the cell surface, and the addition of thrombin to NCSC cultures phenocopies the differentiation profile of sJ1 addition. The addition of thrombin, like the addition of sJ1, leads to a dampened CSL-mediated Notch activation [30, 40]. Thrombin is absent during neural crest development and differentiation; this observation is potentially significant for the expansion of NSCs and other stem cell populations after injury because thrombin is produced at the site of injury.

It has been demonstrated that the total abrogation of Notch signaling results in a loss in the maintenance of NSC populations [41]. Because we suggested that the activation of Notch signaling enables a similar event by instructing the differentiation of NCSC or NSC in general into glia [18], we also suggest that a threshold of endogenous Notch signaling is paramount for NSC maintenance. Furthermore, the highest levels of Notch signaling may result in the loss of neurogenic potential and differentiation into glia. This is in contrast to attenuated Notch signaling that may result in enhanced cell survival and cell proliferation by enabling the nonclassic release of FGF1 by the target stem cell. It is widely thought that Notch activation occurs in trans from two adjacent cells. In this study, we cannot rule out the possibility that in NCSCs or NSCs that cis activation of Notch or cis attenuation of Notch by soluble ligands is occurring. Thus, it is of clinical interest that we demonstrate that the repression of Notch signaling by sJ1 enables the long-term expansion of adult stem cell populations without altering their responsiveness to instructive differentiation signals, multipotentiality, or self-renewal potential.

	CONCLUSIONS

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

 
Notch signaling via sJ1 in neural stem cells serves at least two independent roles during self-renewal. A major role is to inhibit neural differentiation while allowing for the induction and release of FGF1. The other role is serving as an internal meter of CSL-dependent transcriptional activity. At low levels, neural stem cells survive and proliferate, whereas increasing the level leads to the loss of neurogenic differentiation potential and eventually the onset of gliogenic differentiation.

	DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST

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The authors indicate no potential conflicts of interest.

	ACKNOWLEDGMENTS

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This work is dedicated to the memory of Thomas Maciag, scientist, mentor, and friend. We thank G. Weinmaster (UCLA) for Dl1-Fc and M. Bhatia (McMaster University) for sJ1. This work was supported in part by NIH Grant RR 18789, Medical Research Council of Canada, and Alzheimer's Society of Canada (to J.M.V.); NIH Grants HL 32348 and HL 35627 (to I.P.); and NIH Grant RR 15555 (to R.F.). G.N.N. is supported by a predoctoral fellowship from the American Heart Association. M.D. is currently affiliated with the Life and Health Science Research Institute, University of Minho, Bzaga, Portugal. C.J.K. is currently affiliated with USB Pharmaceuticals, Cleveland, OH.

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