Insulin-like Growth Factor II: An Essential Adult Stem Cell Niche Constituent in Brain and Intestine.

Tissue-specific stem cells have unique properties and growth requirements, but a small set of juxtacrine and paracrine signals have been identified that are required across multiple niches. Whereas insulin-like growth factor II (IGF-II) is necessary for prenatal growth, its role in adult stem cell physiology is largely unknown. We show that loss of Igf2 in adult mice resulted in a ∼50% reduction in slowly dividing, label-retaining cells in the two regions of the brain that harbor neural stem cells. Concordantly, induced Igf2 deletion increased newly generated neurons in the olfactory bulb accompanied by hyposmia, and caused impairments in learning and memory and increased anxiety. Induced Igf2 deletion also resulted in rapid loss of stem and progenitor cells in the crypts of Lieberkühn, leading to body-weight loss and lethality and the inability to produce organoids in vitro. These data demonstrate that IGF-II is critical for multiple adult stem cell niches.

Insulin and IGF receptor signalling in neural-stem-cell homeostasis.

Neural stem cells (NSCs) are found in two regions in the adult brain: the subgranular zone (SGZ) in the hippocampal dentate gyrus and the subventricular zone (SVZ) adjacent to the lateral ventricles. Similarly to other somatic stem cells, adult NSCs are found within specialized niches that are organized to facilitate NSC self-renewal. Alterations in stem-cell homeostasis can contribute to the consequences of neurodegenerative diseases, healthy ageing and tissue repair after damage. Insulin and the insulin-like growth factors (IGFs) function in stem-cell homeostasis across species. Studies in the mammalian central nervous system support essential roles for IGF and/or insulin signalling in NSC self-renewal, neurogenesis, cognition and sensory function through distinct ligand-receptor interactions. IGF-II is of particular interest as a result of its production by the choroid plexus and presence in cerebrospinal fluid (CSF). CSF regulates and supports the development, division and migration of cells in the adult brain and is required for NSC maintenance. In this Review, we discuss emerging data on the functions of IGF-II and IGF and/or insulin receptor signalling in the context of NSC regulation in the SVZ and SGZ. We also propose a model for IGF-II in which the choroid plexus is a major component of the NSC niche.

Insulin-Like Growth Factor Receptor Signaling is Necessary for Epidermal Growth Factor Mediated Proliferation of SVZ Neural Precursors in vitro Following Neonatal Hypoxia-Ischemia.

In this study, we assessed the importance of insulin-like growth factor (IGF) and epidermal growth factor (EGF) receptor co-signaling for rat neural precursor (NP) cell proliferation and self-renewal in the context of a developmental brain injury that is associated with cerebral palsy. Consistent with previous studies, we found that there is an increase in the in vitro growth of subventricular zone NPs isolated acutely after cerebral hypoxia-ischemia; however, when cultured in medium that is insufficient to stimulate the IGF type 1 receptor, neurosphere formation and the proliferative capacity of those NPs was severely curtailed. This reduced growth capacity could not be attributed simply to failure to survive. The growth and self-renewal of the NPs could be restored by addition of both IGF-I and IGF-II. Since the size of the neurosphere is predominantly due to cell proliferation we hypothesized that the IGFs were regulating progression through the cell cycle. Analyses of cell cycle progression revealed that IGF-1R activation together with EGFR co-signaling decreased the percentage of cells in G1 and enhanced cell progression into S and G2. This was accompanied by increases in expression of cyclin D1, phosphorylated histone 3, and phosphorylated Rb. Based on these data, we conclude that coordinate signaling between the EGF receptor and the IGF type 1 receptor is necessary for the normal proliferation of NPs as well as for their reactive expansion after injury. These data indicate that manipulations that maintain or amplify IGF signaling in the brain during recovery from developmental brain injuries will enhance the production of new brain cells to improve neurological function in children who are at risk for developing cerebral palsy.

Insulin-like growth factor-II (IGF-II) and IGF-II analogs with enhanced insulin receptor-a binding affinity promote neural stem cell expansion.

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.

Determining mammosphere-forming potential: application of the limiting dilution analysis.

Originally adapted from the neurosphere assay, the nonadherent mammosphere assay has been utilized to assess early progenitor/stem cell frequency in a given population of mammary epithelial cells. This method has also been used to measure the frequency of tumorsphere initiating cells in both primary mammary tumors as well as in tumor cell lines. Although, the mammosphere assay has been used extensively in the mammary gland field, a standard method of quantifying and analyzing sphere growth in this assay has remained undefined. Here, we discuss the use and benefit of using a limiting dilution analysis to quantify sphere-forming frequency in primary mammary epithelial cells grown in nonadherent conditions.

IGF-II promotes stemness of neural restricted precursors.

Insulin-like growth factor (IGF)-I and IGF-II regulate brain development and growth through the IGF type 1 receptor (IGF-1R). Less appreciated is that IGF-II, but not IGF-I, activates a splice variant of the insulin receptor (IR) known as IR-A. We hypothesized that IGF-II exerts distinct effects from IGF-I on neural stem/progenitor cells (NSPs) via its interaction with IR-A. Immunofluorescence revealed high IGF-II in the medial region of the subventricular zone (SVZ) comprising the neural stem cell niche, with IGF-II mRNA predominant in the adjacent choroid plexus. The IGF-1R and the IR isoforms were differentially expressed with IR-A predominant in the medial SVZ, whereas the IGF-1R was more abundant laterally. Similarly, IR-A was more highly expressed by NSPs, whereas the IGF-1R was more highly expressed by lineage restricted cells. In vitro, IGF-II was more potent in promoting NSP expansion than either IGF-I or standard growth medium. Limiting dilution and differentiation assays revealed that IGF-II was superior to IGF-I in promoting stemness. In vivo, NSPs propagated in IGF-II migrated to and took up residence in periventricular niches while IGF-I-treated NSPs predominantly colonized white matter. Knockdown of IR or IGF-1R using shRNAs supported the conclusion that the IGF-1R promotes progenitor proliferation, whereas the IR is important for self-renewal. Q-PCR revealed that IGF-II increased Oct4, Sox1, and FABP7 mRNA levels in NSPs. Our data support the conclusion that IGF-II promotes the self-renewal of neural stem/progenitors via the IR. By contrast, IGF-1R functions as a mitogenic receptor to increase precursor abundance.

Neural stem/progenitor cells participate in the regenerative response to perinatal hypoxia/ischemia.

Perinatal hypoxia/ischemia (H/I) is the leading cause of neurologic injury resulting from birth complications. Recent advances in critical care have dramatically improved the survival rate of infants suffering this insult, but approximately 50% of survivors will develop neurologic sequelae such as cerebral palsy, epilepsy or cognitive deficits. Here we demonstrate that tripotential neural stem/progenitor cells (NSPs) participate in the regenerative response to perinatal H/I as their numbers increase 100% by 3 d and that they alter their intrinsic properties to divide using expansive symmetrical cell divisions. We further show that production of new striatal neurons follows the expansion of NSPs. Increased proliferation within the NSP niche occurs at 2 d after perinatal H/I, and the proliferating cells express nestin. Of those stem-cell related genes that change, the membrane receptors Notch1, gp-130, and the epidermal growth factor receptor, as well as the downstream transcription factor Hes5, which stimulate NSP proliferation and regulate stem cellness are induced before NSP expansion. The mechanisms for the reactive expansion of the NSPs reported here reveal potential therapeutic targets that could be exploited to amplify this response, thus enabling endogenous precursors to restore a normal pattern of brain development after perinatal H/I.