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First published online 4 August 2004
doi: 10.1242/dev.01295

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Insulin acts as a myogenic differentiation signal for neural stem cells with multilineage differentiation potential

Mahmud Bani-Yaghoub^1,*, Stephen E. Kendall², Daniel P. Moore², Stephen Bellum², Rebecca A. Cowling², George N. Nikopoulos², Chris J. Kubu^1,, Calvin Vary³ and Joseph M. Verdi^1,2,

¹ The John P. Roberts Research Institute, 100 Perth Drive, London, ON, N6A 5K8, Canada
² Center for Regenerative Medicine, Maine Medical Center Research Institute, 81 Research Drive, Scarborough, ME 04074, USA
³ Center for Molecular Medicine, Maine Medical Center Research Institute, 81 Research Drive, Scarborough, ME 04074, USA

View larger version (95K): [in a new window]   Fig. 1. Single neural stem cells can differentiate into neurons, glia and myocytes. A single retrovirally labeled E14 CD31- CD35- NSC was expanded in suspension in MB-media for 6 days then transferred to an adherent surface and allowed to differentiate. Differentiated progeny were identified by immunostaining 14 days post-plating. (A-C) Epifluorescent images of an expanding EGFP+ NSC. The resulting sphere was dissociated and grown as an adherent culture for 14 days to complete the differentiation process. (D) Presented are epifluorescent images of EGFP+ cells (green) and sk-MHC immunoreactive cells (red). Arrowheads indicate a phenotypic EGFP+ neuron in a cluster of EGFP+ sk-MHC+ myocytes. Scale bar: 80 µm. (E-H) The observed multilineage differentiation covered many combinations of differentiation possibilities. (E,F) Confocal z-sections of differentiated clones containing BAG5+ cardiac myocytes (red) and MAP2 immunoreactive neurons (green); (G,H) BAG5 cardiac myocytes (red) and GFAP+ glial cells (green). To further verify that differentiated clones arose from a single EGFP+ cell, Southern analysis was performed on random differentiated progeny; (I) Southern blot. Clone 15-1 is the founder clone and clone 15-2 represents the secondary clone arising from the primary clone 15-1. Controls 1 and 2 are mixtures of two or more individual clones.

View larger version (53K): [in a new window]   Fig. 2. Neural stem cells can generate skeletal and cardiac myocytes. In order to demonstrate the potential of NSCs, CD31- CD35- EGFP+ cortical progenitor cells were expanded for 6 days in MB-media prior to plating on an adherent surface to complete differentiation. By both reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemical analyses, differentiated progeny expressed markers of myogenic differentiation. (A) RT-PCR was performed to assess the steady state levels of MyoD expression of skeletal muscle actin and cardiac actin mRNAs at 7 and 18 days of MLNSC differentiation. A photomicrograph of an ethidium bromide stained agarose gel displaying the amplification products for reactions using mRNA isolated from cultures at day 7 and day 18 of differentiation is shown. (B-D) Time course of sk-MHC immunoreactivity within differentiating clones 9 days (B), 11 days (C) and 18 days (D) post-plating is presented. (E) Immunofluorescent images for cardiac-specific MHC and the gap junction protein, Cnx43, immunoreactivity at day 18 after plating. NSCs gave rise to cardiac myocyte immunoreactive progeny. Scale bar: 150 µm.

View larger version (24K): [in a new window]   Fig. 3. Survival of MLNSC in insulin correlates with insulin-induced Akt activation. Cell recovery and BrdU assays were performed on cultures of CD31- CD35- EGFP+ neurospheres grown in MB-media, MB-media minus insulin or MB-media plus 100 µM quercetin. (A) Sphere recovery was determined by counting the number of spheres daily and normalizing to the number of spheres grown in complete MB-media. Presented is the mean±s.d. for three experiments from distinct cellular isolations. (B) Proliferation is unaltered in BM-media. BrdU incorporation assays were conducted on neurospheres grown in MB-media, MB-media minus insulin and MB-media plus 100 µm quercetin. Little change in BrdU incorporation was observed whether BrdU was delivered for 4 (B) or 8 (C) hours. Presented is the mean±s.d. of three experiments normalized to neurospheres grown in MB-media containing insulin. (D) Insulin-induced survival correlates with the activation of Akt. Western analysis of neurospheres cultured in MB-media minus insulin (lane A), in MB-media plus 100 µM quercetin (lane B) and in MB-media (lanes C,D). (E) Caspase 3 is activated in neurospheres the absence of insulin signaling. Western analysis of procaspase 3 and activated caspase 3 in MB-media (lanes A,B) or MB-media plus 100 µM quercetin (lane C).

View larger version (10K): [in a new window]   Fig. 4. Myogenic differentiation potential is correlated with insulin receptor expression. In an attempt to correlate the level of insulin signaling with myogenic differentiation potential, fluorescence activated cell sorting (FACS) was used to identify subpopulations of NSCs, based on IR expression. (A) FACS analysis showing two subpopulation of NSC based on IR expression. (B) To further correlate IR expression with myogenic potential, neurospheres were generated from EGFP-labeled IRhigh and IRlow cells and allowed to expand and differentiate in MB-media. Presented is the mean±s.d. of three experiments demonstrating that myogenic potential correlates with insulin receptor expression. Note that few multilineage clones were established from IR negative founders. *P<0.05; **P<0.01. (C) IRlow NSC arise from IRhigh cells during expansion. E14 IRhigh cortical progenitor cells were isolated and a fraction frozen down (control) for comparison to the remainder that was expanded for 5 days, to assess the changes of IR expression during expansion. After 5 days of expansion in MB-media, the cells were resorted to examine the IR expression profile. The re-distribution of IR+ cells now includes IRlow-expressing and IRneg cells. R1, IRlow population; R2, IRhigh population.

View larger version (23K): [in a new window]   Fig. 5. Insulin is a dose-sensitive myogenic-instructive differentiation signal. The myogenic potential of MLNSC was determined as a function of insulin concentration. (A) The dose sensitivity of CD31- CD35- NSCs grown in MB-media to produced multilineage clones was determined. (B) The differentiation of NSCs into specific myogenic derivatives is also dose sensitive. Presented is the mean±s.d. of three experiments demonstrating that, as the concentration of insulin is increased, skeletal myogenic (sk-MHC+) differentiation is favored relative to cardiomyocytes (cardiac MHC and troponin immunoreactivity and the visual appearance of beating). (C,D) Increasing insulin signaling favors skeletal muscle differentiation. Presented is the mean±s.d. of three experiments using IRlow (C) and IRhigh (D) fractionated NSCs. Cardiomyocyte differentiation is favored at low concentrations or when the number of receptors is reduced, whereas increased insulin signaling favored skeletal muscle formation.

View larger version (57K): [in a new window]   Fig. 6. Neural stem cells can give rise to functional cardiac myocytes. NSCs were plated at clonal density and allowed to differentiate in MB-media supplemented with 20 ng/ml bFGF on days 1 and 2 of expansion. NSC-derived cardiomyocytes were tested for functionality via mechanical coupling and responses to mediators of pace making activity. (A) Merged images of dye transfer in NSC-derived cardiomyocytes. N, neuron; S, skeletal muscle. (B) A single cardiomyocyte was preloaded with calcein-AM and diI (yellow), and parachuted over a cluster of NSC-derived cardiomyocytes. Calcein passes from the donor cell to other cardiomyocyte in the cluster. DiI, which is not transferable via gap junctions, was used to trace the location of the donor cell. (C) Presented is the beat rate of MLNSC-derived cardiomyocytes in response to sympathetic (10 µM epinephrine) and parasympathetic (2 µm acetylcholine) stimulation.

View larger version (112K): [in a new window]   Fig. 7. Neural stem cells engraft and differentiate in damaged host tissue. Clusters of EGFP+ differentiating NSCs grown in MB-media were injected into physically damaged hearts and allowed to engraft. Epifluorescent images of the damaged heart before (A) and after (B) injection are shown. (C) EGFP+ NSCs engraft into damaged myocardium and differentiate. Immunofluorescent images of cardiac myosin heavy chain immunoreactive cells and EGFP+ injected cells are presented. In the merged image, the EGFP+-injected cells are also cardiac MHC-positive. (D) Scanning z sections through the injured heart further demonstrate that the EGFP+ cells have initiated myocardial differentiation as measured by the appearance of cardiac myosin expression. Scale bar: 10 µm.