Human Cortical Neural Stem Cells Expressing Insulin-Like Growth Factor-I: A Novel Cellular Therapy for Alzheimer's Disease

Abstract

Alzheimer's disease (AD) is the most prevalent age-related neurodegenerative disorder and a leading cause of dementia. Current treatment fails to modify underlying disease pathologies and very little progress has been made to develop effective drug treatments. Cellular therapies impact disease by multiple mechanisms, providing increased efficacy compared with traditional single-target approaches. In amyotrophic lateral sclerosis, we have shown that transplanted spinal neural stem cells (NSCs) integrate into the spinal cord, form synapses with the host, improve inflammation, and reduce disease-associated pathologies. Our current goal is to develop a similar "best in class" cellular therapy for AD. Here, we characterize a novel human cortex-derived NSC line modified to express insulin-like growth factor-I (IGF-I), HK532-IGF-I. Because IGF-I promotes neurogenesis and synaptogenesis in vivo, this enhanced NSC line offers additional environmental enrichment, enhanced neuroprotection, and a multifaceted approach to treating complex AD pathologies. We show that autocrine IGF-I production does not impact the cell secretome or normal cellular functions, including proliferation, migration, or maintenance of progenitor status. However, HK532-IGF-I cells preferentially differentiate into gamma-aminobutyric acid-ergic neurons, a subtype dysregulated in AD; produce increased vascular endothelial growth factor levels; and display an increased neuroprotective capacity in vitro. We also demonstrate that HK532-IGF-I cells survive peri-hippocampal transplantation in a murine AD model and exhibit long-term persistence in targeted brain areas. In conclusion, we believe that harnessing the benefits of cellular and IGF-I therapies together will provide the optimal therapeutic benefit to patients, and our findings support further preclinical development of HK532-IGF-I cells into a disease-modifying intervention for AD.

Keywords: Alzheimer’s disease; Cellular therapy; Insulin-like growth factor-I; Neural stem cell; Neurodegeneration; Stem cell transplantation.

Figure 1.

IGF-I production, growth factor profile, and signaling in HK532 and HK532-IGF-I cells. (A): Production of IGF-I in HK532 and HK532-IGF-I throughout early differentiation. Representative immunocytochemistry images of D7 HK532 (B) and HK532-IGF-I (C) cells labeled with 4′,6-diamidino-2-phenylindole (DAPI) (blue) and IGF-IR (green). (D): BDNF, GDNF, and VEGF production in undifferentiated HK532 and HK532-IGF-I cells (D0) and throughout early differentiation (D3, D5, and D7). Growth factor production is expressed as fold change relative to parental HK532 cells. (E): Western blot analysis of IGF-I signaling in undifferentiated and differentiated (D7) HK532 and HK532-IGF-I cells. Cells were treated with an inhibitor panel of LY, U, or NVP for 1 hour, followed by IGF-I treatment for 30 minutes. All blots were probed with pIGF-IR, IGF-IR, pERK, ERK, pAKT, and AKT. β-actin was used as a loading control. Data are presented as mean + SD or are representative images of at least three independent experiments. Scale bar = 50 μm. ∗, p < .05. Abbreviations: BDNF, brain-derived neurotrophic factor; D0 (D3, D7), day 0 (day 3, day 7); GDNF, glial cell line-derived neurotrophic factor; IGF-I, insulin-like growth factor-I; IGF-IR, insulin-like growth factor-I receptor; LY, LY294002; NVP, NVPAEW541; U, U0126; VEGF, vascular endothelial growth factor.

Figure 2.

Induced insulin-like growth factor-I (IGF-I) expression does not affect HK532 cell proliferation and migration. (A): Quantification of the percent of EdU-positive cells at D0, D3, and D7 in HK532 and HK532-IGF-I cultures. (B–E): Representative immunocytochemistry images of D0 and D7 HK532 and HK532-IGF-I cells labeled with 4′,6-diamidino-2-phenylindole (DAPI) (blue) and EdU (green). (F–G): Quantification of absorbance of migrated HK532 and HK532-IGF-I cells at D0 and D7. Data are presented as mean + SD or are representative images of at least three independent experiments. Scale bar = 200 µm. ∗, p < .05. Abbreviations: D0 (D3, D7), day 0 (day 3, day 7); EdU, 5′-ethynyl-2’-deoxyuridine.

Figure 3.

Induced insulin-like growth factor-I (IGF-I) expression does not affect maintenance of progenitor status or neurite outgrowth during differentiation. (A, B): Representative immunocytochemistry (ICC) image of D0 HK532 and HK532-IGF-I cells labeled with 4′,6-diamidino-2-phenylindole (DAPI) (blue) and Nestin (red). (C): Quantification of Nestin-positive D0 HK532 and HK532-IGF-I cells. (D, E): Representative ICC image of D7 HK532 and HK532-IGF-I cells labeled with DAPI (blue) and TUJ1 (red). (F): Quantification of the neural index measurement (μm² per cell). Data are presented as mean + SD or are representative images of at least three independent experiments. Scale bar = 200 µm. ∗, p < .05. Abbreviation: D7, day 7.

Figure 4.

Terminal phenotype of HK532 and HK532-IGF-I cells. (A, B): Representative immunocytochemistry (ICC) images of D7 HK532 and HK532-IGF-I cells labeled with 4′,6-diamidino-2-phenylindole (DAPI) (blue) and GAD65 (green). (C, D): Representative ICC image of D7 cells labeled with DAPI (blue) and VGLUT (red). (E): Quantification of GAD65-positive gamma-aminobutyric acid (GABA)ergic neurons in HK532 and HK532-IGF-I cells. HK532-IGF-I cells preferentially differentiate into GABAergic neurons (∗, p < .05 vs. HK532). (F): Quantification of VGLUT-positive glutamatergic neurons in HK532 and HK532-IGF-I cells. Data are presented as representative images or mean + SD of at least three independent experiments. Scale bar = 200 µm. Abbreviations: D7, day 7; GAD, glutamic acid decarboxylase; VGLUT, vesicular glutamate transporter.

Figure 5.

HK532-IGF-I cells are neuroprotective in vitro. (A): Quantification of apoptosis and cleaved caspase-3 (CC3) activation in response to Aβ toxicity in primary CNs and undifferentiated and differentiated (D7) HK532 and HK532-IGF-I cells. Both HK532 cell lines were more resistant than CNs, and HK532-IGF-I cells displayed significantly increased resistance, which was negated when insulin-like growth factor-I (IGF-I) signaling is blocked by addition of NVP. (B–G): Representative immunocytochemistry images of primary CNs labeled with DAPI and CC3, where conditions included (B) control CNs with no Aβ treatment, (C) CNs with Aβ treatment, (D) CNs with Aβ treatment cocultured with HK532 cells, (E) CNs with Aβ treatment cocultured with HK532-IGF-I cells, (F) CNs with Aβ treatment cocultured with HK532 cells plus NVP, and (G) CNs with Aβ treatment cocultured with HK532-IGF-I cells plus NVP. (H): Quantification of Aβ-mediated apoptosis and CC3 activation in CN/HK532 cocultures. HK532-IGF-I cells exhibited an increased neuroprotective capacity (∗, p < .05 vs. HK532), which was reversed with addition of NVP. Data are presented as mean + SD or representative images of at least three independent experiments. Scale bar = 200 µm. ∗, p < .05; ∗∗, p < .0005. Abbreviations: Aβ, amyloid β; CC3, cleaved caspase-3; CN, cortical neuron; NVP, NVPAEW541.

Figure 6.

HK532-IGF-I cells survive grafting into APP/PS1 mice with Alzheimer’s disease (AD) and wild-type (WT) mice. Representative images of HK532-IGF-I cells in the hippocampal area of APP/PS1 AD (A–F) and WT (G–L) mice 10 weeks following transplantation to the fimbria fornix. H&E staining shows the transplanted target area (square) in AD (A) and WT (G) mice. Immunofluorescent DAPI, HuNu, and DCX labeling of human early neural precursor cells in the hippocampal area of AD (B–F) and WT animals (H–L). Data are presented as representative images (A–F: APP/PS1, n = 4; G–L: WT, n = 4). (A, G): Scale bar = 200 µm. (B, H): ×10 scale bar = 200 µm. (C–F, I–L): ×60 scale bar = 50 µm. Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; DCX, Doublecortin; H&E, hematoxylin and eosin; HuNu, human nuclei.