Control of obesity and glucose intolerance via building neural stem cells in the hypothalamus

Abstract

Neural stem cells (NSCs) were recently revealed to exist in the hypothalamus of adult mice. Here, following our observation showing that a partial loss of hypothalamic NSCs caused weight gain and glucose intolerance, we studied if NSCs-based cell therapy could be developed to control these disorders. While hypothalamus-implanted NSCs failed to survive in mice with obesity, NF-κB inhibition induced survival and neurogenesis of these cells, leading to effects in counteracting obesity and glucose intolerance. To generate an alternative cell source, we revealed that iPS-derived NSCs were converted into htNSCs by neuropeptide treatment. Of note, obesity condition potentiated the transfer of carotid artery-injected NSCs into the hypothalamus. These iPS-derived cells when engineered with NF-κB inhibition were also effective in reducing obesity and glucose intolerance, and neurogenesis towards POMCergic and GABAergic lineages was accountable. In conclusion, building NSCs in the hypothalamus represents a strategy for controlling obesity and glucose disorders.

Keywords: Glucose tolerance; Hypothalamus; NF-κB; Neural stem cells; Neuropeptide; Obesity; iPS.

Figure 1

Survival of hypothalamic implanted htNSCs engineered with NF-κB inhibition. Cell lines of Con-htNSCs (Con) and ^DNIκBα-htNSCs (IκBα) were generated through lentiviral induction (A), examined for stemness and morphology via GFP and immunostaining of a NSC marker (B), and injected bilaterally in the MBH of HFD-fed vs. chow-fed male C57BL/6 mice (C–E). Images show technical success of delivering cells in the MBH at Day 3 post-injection (C), and longitudinal follow-up of indicated htNSCs in the MBH of HFD-fed mice (D). DAPI staining reveals nuclei of all cells in the sections. Scale bar=50 µm. Curves show longitudinal survival of htNSCs in HFD- vs. chow-fed mice (E). ^*P<0.05, ^**P<0.01 (compared to matched time points in the group of Con, chow), n=4–6 mice per group. Error bars reflect means±s.e.m.

Figure 2

Neurogenesis and metabolic effects of implanting NF-κB-inhibited htNSCs. Dissociated ^DNIκBα-htNSCs (IκBα) vs. Con-htNSCs (Con) were injected in the MBH of chow- vs. HFD-fed C57BL/6 mice, examined for stemness and neurogenesis of htNSCs at ~1 month post-injection via immunostaining of Sox2 (A–C), NeuN (B) and α-MSH (C), and effects of injected htNSCs on metabolic physiology (E–G). Histological data in A–D show MBH sections from HFD-fed mice injected with ^DNIκBα-htNSCs (A–C), and counting of endogenous (white bars) vs. exogenous htNSCs-derived (black, blue, red and green bars) POMC cells (D). Bar=50 µm. Physiological data show body weight (E), food intake (F) and GTT (G) obtained at indicated days (E) or 2–3 months post-implantation (F, G). ^*P<0.05, ^**P<0.01, ^***P<0.001, compared between indicated exogenous cells (D) or compared to HFD-fed controls at matched time points (E, G), n=4 (D) and 5–6 (E–G) per group. Error bars reflect means±s.e.m.

Figure 3

NSCs derived from iPS and conversion into htNSCs phenotype. Model of iPS-derived NSCs (NSCs^iPS) was generated from iPS-derived embryoid bodies (A), verified via neurosphere staining of Sox2 and nestin (B), and assessed for differentiation via Tuj-1, GFAP and O4 immunostaining (C) and programming under neuropeptide treatment (D–F). D–F: NSCs^iPS treated with neuropeptides including α-MSH, CART, NPY, or NPY and BDNF (NPY+B) were examined for induction of htNSCs-specific markers via quantitative PCR (D) and immunostaining (E), or subjected to differentiation followed by measurement of hypothalamic neuropeptide gene expression (F). Vehicle treatment was used to provide baseline levels profiles of various parameters. Native htNSCs under basal condition were included to provide positive controls. E: Bar=50 µm, and Six3- and Vax1-positive cells were counted in lower panels. ^*P<0.05, ^**P<0.01, ^***P<10⁻³, n=4 per group. Error bars reflect means±s.e.m.

Figure 4

Artery delivery of NF-κB-inhibited NSC^iPS and in vivo neurogenesis. Lines of ^DNIκBα-NSC^iPS and Con-NSC^iPS were verified via GFP and immunostaining of a NSC marker (A), and delivered into HFD-fed mice via carotid artery injection (B–E). Brain sections were prepared to analyze the hypothalamic transfer of artery-injected ^DNIκBα-NSC^iPS at ~1 month post-injection (B) and hypothalamic neurogenesis at ~3 months post-injection via immunostaining of NeuN (C) and POMC (D, E). Bar graphs show counting of endogenous cells (white bars) vs. exogenous NSC^iPS-derived cells (black, blue, red and green bars) that expressed POMC (E). ^*P<0.05, ^**P<0.01, ^***P<0.001, (comparisons between the columns of exogenous cells), n=4 mice per group. Error bars reflect means±s.e.m. Scale bar=50 µm (A, B) and 25 µm (C).

Figure 5

Metabolic effects from artery injection of NF-κB-inhibited NSC^iPS. Male C57BL/6 mice were induced to develop dietary obesity through 3-month HFD feeding, and then received intra-carotid artery injection of 2×10^{5 DN}IκBα-NSC^iPS (IκBα^iPS, IκBα) or Con-NSC^iPS (Con^iPS, Con). Mice continued to be maintained on the HFD for the entire experiment. Data show food intake (A), body weight (B), and leptin-induced feeding inhibition in response to an i.c.v. injection of 5 µg leptin (Lep, L) vs. vehicle (Veh, V) following 6-h fasting (C), and blood glucose levels during GTT (D). Data were obtained at the indicated weeks (W) (A, B) or 15 weeks (C) post-surgical recovery. ^*P<0.05, ^**P<0.01, n=5 mice per group (A, B and D), and n=4 mice per group (C). Error bars reflect means±s.e.m.

Figure 6

Neurogenesis in mediating the metabolic effects of NF-κB-inhibited NSC^iPS. (A) NSC^iPS were treated with indicated neuropeptides, and subjected to differentiation followed by mRNA measurement of GABAergic and glutamatergic biomarkers. Native htNSCs under the basal condition were included to provide positive controls. (B–E)  ^DNIκBα-NSC^iPS (IκBα^iPS) and Con-NSC^iPS (Con^iPS, C) received lentiviral induction of POMC shRNA (POMC sh), GAD67 shRNA (GAD sh) or control scramble shRNA (Con sh), injected in the MBH of HFD-fed mice as illustrated (B), and followed up for food intake (C), body weight (D), and GTT (E) at the indicated weeks (C, D) or 3 months (E) post-injection. ^*P<0.05, ^**P<0.01, n=4 (A) and 5–6 (B–E) per group. Error bars reflect means±s.e.m. (F) Proposed model for using NSCs therapy to control obesity and related diseases such as T2D.