Insulin-like Growth Factor-II (IGF-II) and IGF-II Analogs with Enhanced Insulin Receptor-a Binding Affinity Promote Neural Stem Cell Expansion*

Background: IGF-II promotes neural stem cell (NSC) proliferation and self-renewal. Results: IGF-II analogs are useful for elucidating the receptors responsible for NSC expansion. Conclusion: F19A expands NSCs via IR-A. Significance: IGF-II promotes stemness of NSCs via the IR-A and not through activation of either the IGF-1R or the IGF-2R. The objective of this study was to employ genetically engineered IGF-II analogs to establish which receptor(s) mediate the stemness promoting actions of IGF-II on mouse subventricular zone neural precursors. Neural precursors from the subventricular zone were propagated in vitro in culture medium supplemented with IGF-II analogs. Cell growth and identity were analyzed using sphere generation and further analyzed by flow cytometry. F19A, an analog of IGF-II that does not bind the IGF-2R, stimulated an increase in the proportion of neural stem cells (NSCs) while decreasing the proportion of the later stage progenitors at a lower concentration than IGF-II. V43M, which binds to the IGF-2R with high affinity but which has low binding affinity to the IGF-1R and to the A isoform of the insulin receptor (IR-A) failed to promote NSC growth. The positive effects of F19A on NSC growth were unaltered by the addition of a functional blocking antibody to the IGF-1R. Altogether, these data lead to the conclusion that IGF-II promotes stemness of NSCs via the IR-A and not through activation of either the IGF-1R or the IGF-2R.

The insulin-like growth factor (IGF) 3 peptides and their receptors comprise an evolutionarily conserved family of signaling molecules that are essential for normal stem cell proliferation and brain development; however, the individual roles of the ligands are only beginning to be realized. Indeed, the IGF system has been largely overlooked as a critical signaling pathway that is required to propagate neural stem cells (NSCs) in culture. Recent studies have determined IGF-II is involved in NSC proliferation and maintenance in the subventricular zone (SVZ) (1,2).
The most commonly used method to study neural stem and progenitor (NSP) self-renewal, growth, and differentiation is the neurosphere assay (NSA) (3)(4)(5)(6). For this assay, the tissue to be evaluated is microdissected and dissociated into a single cell suspension. When grown in a biochemically defined medium with specific growth factors under non-adherent conditions, self-renewing cells proliferate and form a free-floating colony of cells called a neurosphere. A key aspect of the NSA is that each sphere arises from the proliferation of a single cell. A limitation of this method is that NSCs, progenitors, and lineage-restricted progenitors are all capable of forming self-renewing spheres. Another limitation is that as the spheres grow they become cellularly heterogeneous. Each neurosphere contains a few stem cells with the bulk of the sphere comprised of progenitors and lineage-restricted cells. These limitations have made it difficult to evaluate which population of cells is altered with different treatment conditions in the NSA.
A different approach to the NSA is to exploit the unique molecular identities of the NSPs, enabling cell populations to be quantified by flow cytometry. Buono et al. (7) recently combined four cell surface markers for flow cytometry to reveal the existence of a NSC and seven types of intermediate progenitors.
Not surprisingly, no single marker was sufficient to label the NSCs (7). Nestin, sox2, aldehyde dehydrogenase, and Hoechst exclusion label multipotential progenitors as well as NSCs and did not select for neurosphere-forming cells. Many sets of markers have been used to classify NSCs (for a list see Pastrana et al. (8)). The difficulty of classifying these cells by their molecular identity can be attributed to the complex lineage of the cells and the fact that the cells within the neurosphere are heterogeneous. The assay developed by Buono et al. (7) defined a combination of CD133, CD140a, NG2, and LeX to distinguish multipotential progenitors and NSCs.
Our prior studies revealed a role for IGF-II in promoting neural stemness in the NSA (1). However, IGF-II has high affinity to three different receptors: the IGF-2R/mannose 6-phosphate receptor, the IGF type 1 receptor (IGF-1R), and an isoform of the insulin receptor known as the IR-A (9 -11). Our prior findings in neurospheres supported the hypothesis that IGF-II promotes stemness through the insulin receptor; however, the precise makeup of the neurospheres was not examined in that study. Moreover, another study reported IGF-II promotes expansion of neural progenitor cells via activating the IGF-1R (2). Here, we evaluate the effectiveness of three genetically engineered analogs of IGF-II (Table 1), which have different binding affinities for the receptors, to stimulate NSC expansion in culture using both the widely used NSA and the recently established flow cytometry protocol to test the hypothesis that IGF-II promotes neural stemness specifically by activating IR-A (12,13).

EXPERIMENTAL PROCEDURES
Analog Production and Receptor and IGFBP Binding Assays-Production of IGF-II analogs and purification and binding assays using IGF-1R, IR-A, and domains 10 -13 of the IGF2R are described in Delaine et al. (12). IGFBP-2 binding affinities were derived from BIAcore analyses performed as described by Carrick et al. (14).
Neurosphere Propagation and Quantification-The periventricular region of C57Bl/6 pups (P4 -5) was enzymatically dissociated for 15 min at 37°C using 0.25% trypsin/EDTA (Invitrogen) and 100 units of papain (Sigma) in minimal essential medium-HEPES with 250 g/ml of DNase I (Sigma) and 3 mM MgSO 4 . The tissue was triturated in 0.01% trypsin inhibitor (Sigma) in Pro-N medium (16). The cells were plated into wells at 1 ϫ 10 5 cells/ml in medium supplemented with 20 ng/ml recombinant human EGF (PreproTech). Cultures were fed every 2 days by replacing half of the medium with an equal volume of fresh medium. Neurospheres were collected after 8 -9 days in vitro by centrifugation at 200 ϫ g for 5 min. They were dissociated for 5 min at 37°C in Acutase (Millipore) and plated into 12-well plates at 5 ϫ 10 4 cells/ml in Pro-N medium with either no (zero), 4.4 nM, or 4.4 M insulin (Sigma) supplemented with 20 ng/ml EGF and either mouse recombinant IGF-II (R&D Systems Inc) or an IGF-II analog (supplied by B.E. Forbes) at either 30, 15, or 8 nM (8ϫ K D , 4ϫ K D , or 2ϫ K D of IGF-II for IGF-1R). In some experiments, the functional blocking antibody, IMC-A12, a monoclonal antibody against IGF-1R was used (provided by ImClone Systems, a wholly owned subsidiary of Eli Lilly and Co). Neurospheres were quantified as described previously (16). Neurosphere volume was determined from phase contrast images using a Zeiss Axiovision Observer.Z1 (Carl Zeiss) microscope under 10 or 40ϫ magnification. Axiovision software was used to measure neurosphere diameters of a minimum of 50 randomly selected neurospheres per condition.
Flow Cytometry-Secondary spheres were dissociated by incubating them in 0.2 Wünsch unit/ml of Liberase DH (Roche Applied Science) and 250 g of DNase1 (Sigma) in PGM solution (PBS with 1 mM MgCl 2 and 0.6% dextrose) at 37°C for 5 min with gentle shaking. An equal volume of PGM was added, and the spheres were placed onto a shaker (LabLine) at 225 rpm at 37°C for 15 min. After enzymatic digestion, Liberase DH was quenched with 10 ml of PGB (PBS without Mg 2ϩ and Ca 2ϩ with 0.6% dextrose and 2 mg/ml fraction V of BSA (Fisher Scientific, BP1600-100), and cells were collected by centrifugation for 5 min at 200 ϫ g. Cells were dissociated by repeated trituration, collected by centrifugation, counted using ViCell (Beckman Coulter, Miami, FL), and diluted to at least 10 6 cells per 50 l of PGB. All staining was performed in 96 V-bottom plates using 150 l/well. For surface marker analysis, cells were incubated in PGB for 25 min with antibodies against Lewis-X (1:20, LeX/ CD15 FITC, MMA; BD Bioscience), CD133-APC (1:50,13A4; eBioscience), CD140a (1:400, APA5; BioLegend), and NG2 chondroitin sulfate proteoglycan (1:50, AB5320; Millipore). Cells were washed with PGB by centrifugation at 278 ϫ g. Goat anti-rabbit IgG Alexa Fluor 700 (1:100; Invitrogen) was used for NG2. Cells were then incubated in LIVE/DEAD fixable Violet (Invitrogen) for 20 min for dead cell exclusion. Cells were washed with PGB by centrifugation at 278 ϫ g. They were fixed with 1% ultrapure formaldehyde (50000; Polysciences, Inc.) for 20 min, collected by centrifugation for 9 min at 609 ϫ g, resuspended in PBS without Mg 2ϩ and Ca 2ϩ and stored at 4°C for next day analysis. All sample data were collected on the BD LSR II (BD Biosciences Immunocytometry Systems). Matching isotype controls were used for all antibodies, and gates were set based on these isotype controls. Post-acquisition analysis was performed using FlowJoX (Tree Star, Inc., Ashland, OR).
Statistics-Data are expressed as means Ϯ S.E. and analyzed using one-way analysis of variance followed by Tukey's post hoc test (Graphpad Prism 4 software).

IGF-2R Stimulation Fails to Promote Neurosphere Growth-
The IGF-2R functions as a scavenger receptor for IGF-II, reducing ligand bioavailability (17). A role for this receptor in IGF-II signaling has been controversial. A recent study suggested that IGF-2R promotes memory consolidation in vivo (18); which is connected to the maintenance of hippocampal neurogenesis. In our prior study defining IGF-II function in NSC self-renewal, we investigated effects of IGF-II through IR or IGF-1R but did not analyze potential IGF-II actions through the IGF-2R (1). Thus, we first tested the F19L analog, which has a phenylalanine substitution at amino acid 19 for leucine and has greater than 6-fold higher binding affinity for IGF-2R than wild type IGF-II while binding the IGF-1R and IR-A with affinities similar to IGF-II (Table 1). Reproducing our earlier studies, primary neurospheres were cultured under standard growth conditions and then dissociated into single cells and grown in control medium (4.4 M insulin), 4.4 nM insulin, or 4.4 nM insulin with IGF-II, F19L, or F19L ϩ A12 antibody (an IGF-1R blocking antibody). Similar to our prior studies, when the level of insulin was reduced to 4.4 nM (to abrogate activation of the IGF-1R), the neurosphere size and number were greatly reduced. Previously, we determined that 15 nM IGF-II produced a modest effect, and 30 nM IGF-II was able to restore neurosphere volume (1). Addition of F19L or IGF-II restored neurosphere number, an indication of self-renewal (Fig. 1). F19L produced the same effect as IGF-II at half of the concentration. The addition of the IGF-1R blocking antibody, A12, did not alter the effectiveness of F19L, indicating its actions are independent of IGF-1R. This is consistent with our previous studies showing the ability of IGF-II to promote NSP self-renewal independent of IGF-1R.
To further address the possibility that IGF-2R binding contributed to IGF-II-induced NSC growth, we used V43M, valine substituted to methionine at amino acid residue 43, an analog of IGF-II that has very low affinity to IGF-1R and to IR-A and high affinity binding to the IGF-2R (0.72-fold compared with wild type IGF-II; Table 1). Few spheres formed when the medium contained V43M (Table 2). In fact, cells cultured in V43M were similar to cells grown without insulin, indicating that IGF-2R stimulation fails to promote the growth of neural precursors.
An IGF-II Analog with Increased Neurosphere-forming Ability-Next, spheres were grown in medium supplemented with a variety of other recombinant IGF-II peptides. The relative affinities of these ligands to the IGF-1R, IGF-2R, and IGFBP2 are provided in Table 1. F19A, similar to F19L, binds with high affinity to IGF-1R and has slightly reduced affinity to the IR. However, F19A, where amino acid 19 has been substituted with alanine, has virtually no binding to the IGF-2R and reduced binding to IGFBP-2. Therefore, we hypothesized that F19A would be effective at a lower concentration than F19L in in situ neurosphere formation as it would be more available to bind the target receptors. We tested this using a concentration of the IGF-II, F19L, and F19A that was equal to 2ϫ the K D of IGF-II to IGF-1R . Only F19A was effective at stimulating neurosphere formation at this concentration, and it produced the greatest number of primary neurospheres (Table 2). F19A produced more spheres than control, or insulin-containing medium, at a lower concentration than either wild-type IGF-II or F19L. As seen in Table 2, F19A enriched the proportion of   Table 3).
Flow Cytometry Reveals IGF-II and F19A Promote Expansion of NSCs-The nervous system, similar to the hematopoietic system, contains a variety of precursors at different stages of developmental restriction. Although the neurosphere assay is a widely used technique for propagating and evaluating NSC, it fails to distinguish the stem cells from neural progenitor cells. Buono et al. (7) recently combined four cell surface markers for flow cytometry to reveal the existence of a NSC and seven types of intermediate progenitors. Moreover, each of these eight types of precursors was competent to form a neurosphere (7). Therefore, an increase in neurosphere number does not distinguish NSCs from intermediate progenitors. We used the same set of cell markers, CD133, LeX, CD140a, and NG2 used by Buono et al. (7) (Table 4) to establish which precursors were present within the neurospheres grown under standard NSA conditions versus those grown in a biochemically defined medium containing IGF-II or the F19A analog. Neurospheres were cultured in control medium or in medium with reduced insulin (4.4 nM insulin) or in medium with reduced insulin and supplemented with IGF-II (8ϫ K D ) or F19A (2ϫ K D ). The concentration of each ligand was selected to be the lowest concentration at which neurosphere number was restored to the numbers observed in control medium. Cells were grown for 6 days in vitro and then dissociated for flow cytometric analyses. Gates were set for forward and side scatter and only live cells, based on live/dead dye staining, were analyzed (Fig. 2). NSCs were defined as NG2/CD140a double negative and CD133/LeX double positive. These flow cytometric analyses revealed that F19A and IGF-II treatment almost tripled the proportion of NSCs while nearly reducing by 2-fold the multipotential progenitors (MPs) termed "PDGF FGF-responsive MPs" (Fig. 2). Other progenitor populations such as MP1, MP2, and MP4 were not affected. Thus, F19A and IGF-II promote the expansion of early NSCs and decrease the number of later multipotential progenitors such as PDGF FGF responsive MPs.
Modified Ligand, F19A, Is Biologically Active-Having established that F19A and IGF-II promote the expansion of early NSCs via IR-A, we wanted to determine whether the modified ligand activates the IR signaling pathway. Because NSCs express IR-A, IR-B, and IGF-1R (1), we chose to use a cell line that expresses IR-A in high copy number and does not express IR-B or IGF-1R, enabling us to selectively examine the activation of IR-A via F19A. We performed Western blots to examine IR phosphorylation and activation of downstream signaling P-AKT (Ser-473) and P-S6 (Fig. 3). Fibroblasts engineered to express only the IR-A form of the IR were treated with IGF-II or F19A for 20 min. This time point was chosen based on time course activation studies of IGF-II on this cell type (15). IGF-II and F19A produced similar activation of signaling pathways downstream of the IR-A indicating that F19A is stimulating IR-A.

DISCUSSION
Stem cells throughout the body are found within specialized niches. The NSC niche is complex and is elegantly organized to favor specific cell-cell interactions, as well as access to the cerebral microvasculature, extracellular matrix components, meninges, and cerebral spinal fluid (CSF). Whereas the lineage of NSCs within the SVZ as well as the generation and migration of NSP progeny has been an area of intense focus, the niche microenvironment has only recently been studied in detail. The notion that the CSF and IGFs, in particular, play an integral role in the NSC niche has only garnered attention recently (1,2,19). The CSF stimulates the growth of neurospheres in an IGF-IIdependent manner. In fact, IGF-II in the CSF binds to primary cilia protruding from the wall of the lateral ventricles in vivo (2). Our previous study showed that IGF-II could increase the num-    FEBRUARY 21, 2014 • VOLUME 289 • NUMBER 8

JOURNAL OF BIOLOGICAL CHEMISTRY 4629
ber of cultured postnatal SVZ neurospheres and promoted self-renewal based on differentiation, Q-PCR gene profiling, limiting dilution analysis and transplantation studies (1). Additionally, we demonstrated that IR-A was more highly expressed than IGF-1R on the medial SVZ as opposed to lateral SVZ by laser capture microdissection and Q-PCR. IR-A was also the most highly expressed isoform on neurospheres and, as cells became more lineage restricted, IR-A decreased. Thus, this work supported the idea that the in vitro effects of IGF-II were via IR-A and independent of IGF-1R. Taken together, these studies strongly support a role for IGF-II in the neural stem cell niche. Here, we have shown that in vitro simulation of SVZderived neurospheres with IGF-II or a modified form of IGF-II, F19A increases the proportion of NSCs as quantified using a new flow cytometry analysis and that these actions are independent of the IGF-2R and IGF-1R. In extending our analyses of stemness genes activated by IGF-II, here we show that IGF-II promotes the expression of the Id2 gene, which has been shown to facilitate self-renewal and proliferation of NSCs (20). Studies of the precursors in the other neurogenic region of the brain suggest that IGF-II promotes the maintenance of hippocampal NSCs in the subgranular zone (19). FACs sorted, microdissected hippocampal cells expressing GFP driven off a sox2 promoter were used in a microarray analysis to identify genes that were induced in these putative stem cells; a gene identified from this study was IGF-II. The importance of IGF-II was further evaluated in the hippocampus using lentiviral shRNA knockdown of IGF-II expression, which resulted in decreased hippocampal precursor proliferation (19). Other papers support a role for IGF-II and neurogenesis in the SVZ (1,2) Together, these studies lend support for a role for IGF-II in the promotion of neurogenesis in both SVZ and subgranular zone NSCs.
IGF-II consists of four domains, B, C, A, and D (the B and A domains are similar to the corresponding domains in insulin) (13). These domains contain residues that are important in binding IGF-1R and IR. The IR (and the structurally similar IGF-1R) is a disulfide-linked homodimer composed of two ␤ and two ␣ subunits. Although a recent structure of a receptor fragment in complex with insulin has been published, the structure of the entire complex has not been solved (21). Various studies and other structural information have led to a model for how ligands bind to these receptors (22,23). Ligand binds to the IR in a 1:1 stoichiometry despite the existence of two binding pockets within each receptor. Each binding pocket comprises a high and a low affinity binding site (sites 1 and 2, respectively), including residues from both monomers. Binding of ligand results in cross-linking of the two receptor halves leading to a structural change and subsequent activation of the tyrosine kinase domain. The residues on IGF-II important for binding the IR site 1 include Val-43, Phe-26, and Tyr-27, whereas residues Glu-12, Phe-19, Leu-53, and Glu-57 contact site 2 (13). In this study, we used modified forms of IGF-II that contained mutations at these sites of interaction. F19A has high affinity for the IR and IGF-1R. V43M on the other hand has significantly reduced affinities for both the IGF-1R and IR. The signaling efficacy of these analogs has yet to be evaluated; therefore, we have interpreted their effects based on the affinities of these analogs to their receptors.
IGF-2R or cation independent mannose 6-phosphate receptor consists of a large extracellular domain and a very small cytoplasmic tail that displays no intrinsic kinase activity (17,24). IGF-II binds to IGF-2R, via an interaction at Phe-19 on IGF-II (25). There are many recent reports implying that IGF-II activates downstream signaling pathways such as MAPK via IGF-2R; however, it is more likely that IGF-2R interacts with other ligands and subsequent receptors rather than directly activating these signaling moieties (26). Recently, hippocampal memory retention and fear extinction were reported to be dependent on IGF-II and, in part, on IGF-2R. Agis-Balboa et al. (27) found that IGF-II blocking also inhibited contextual fear extinction, which is hippocampal-dependent. They reported that blocking IGR-1R using the IR antagonist JB1 negated the  effects of IGF-II on fear extinction. However, it is important to note that JB1 cross reacts with IR tyrosine kinase; therefore, an effect via IR-A cannot be ruled out in this study (28). Agis-Balboa et al. (27) did not see a block in extinction when using an antibody against IGF-2R, which is in contradiction to Chen et al. (18). Chen et al. (18) found that IGF-II administration into the hippocampus enhanced memory retention and prevented extinction in inhibitory avoidance training. Additionally, they found that blocking IGF-2R abolished the memory enhancement effect of IGF-II. Memory formation in the hippocampus is dependent on neurogenesis (reviewed in Zhao et al. (29)). In our hands, IGF-2R stimulation alone, achieved using V43M, failed to support neurosphere growth. Although IGF-2R may play a role in modulating memory enhancement, the exact nature of this role remains elusive as evidence that it acts directly as a signaling receptor is lacking.
IGF-II acts via the IR-A in several different cell types. IGF-II via IR-A has been found to regulate glycogen synthesis in fetal hepatocytes (30). In fibroblasts that express only IR-A, IGF-II, and insulin display differential signaling (15). IGF-II is a more potent activator of p70S6 kinase than insulin. Additionally IGF-II results in prolonged ERK activation whereas insulin has prolonged AKT activation (15). Furthermore, differential gene expression and a unique set of phosphorylated proteins was found depending on whether IR-A was activated via IGF-II or insulin (31,32). Integrin ␣5, ICAM, and acidic nuclear phosphoprotein 32, a protein involved in embryogenesis and is switched off in differentiated cells, are more regulated by IGF-II than insulin. Endocytic sorting of IR-A differs depending on whether it is activated by insulin or IGF-II. Stimulation with insulin results in the degradation of IR-A and IRS-1, whereas IGF-II protects IR-A and IRS-1 from degradation, which may be responsible for the prolonged activation observed in p70S6 kinase and ERK (33). This evidence supports the role for different ligands, IGF-II, or insulin in this case, producing various effects via the same receptor, IR-A. Here, we have shown that IGF-II via IR-A promotes NSCs in culture.
Given our findings that F19A, an IGF-II analog, is more potent than IGF-II to support and expand NSCs in vitro, it would be interesting to determine its effects after injury. There are numerous consequences to the central nervous system when the homeostasis of CSF is disrupted, such as with aging or after injury. The CSF regulates and supports the development, division and migration of cells. In fact, the presence of modulated, and flowing CSF is required for stem cell maintenance (34). If CSF flow is disrupted by injury or disease, neurons cannot migrate properly (35), and there is a reduction in the clearance of toxins from the brain and reduced nutrient content (34,36). Certain growth factors, such as IGF-II, are tightly controlled and modulated after brain injuries, similar to cortical trauma, and are essential for wound healing, which helps to bring the environment back to homeostasis (37). Infusions of various growth factors, including FGF, EGF, NGF, VEGF, GDNF, and BDNF, after different injury models have improved outcome after injury due to either neurogenesis or neuroprotection (as reviewed in Johanson et al. (38)). The CSF has been hypothesized to modulate the environment after injury. We have shown that IGF-II is an integral player in NSC self-re-newal; however, altered growth factors with restricted interactions such as F19A are more potent and therefore may be a viable option for therapeutics.
In conclusion, this study demonstrates that a genetically engineered form of IGF-II, F19A, stimulates the expansion of NSCs in culture. F19A appeared to be more potent, as it does not bind IGF-2R and only poorly binds the IGFBPs, thus effectively increasing the local concentration compared with IGF-II or F19L. The action of IGF-II and analogs is independent from IGF-2R and IGF-1R, indicating that its effects are via IR-A. The neurosphere assay demonstrated that the analogs produce an increased number of spheres and that a greater percentage of cells in the population are able to give rise to spheres. Flow cytometry analyses revealed an expansion of the NSCs specifically when cultured in IGF-II or F19A, making F19A a useful analog for culturing NSCs.