Fig 2: Schematic diagram of the NRG1-ERBB4-NRG1 autocrine pathway. Genetic manipulation of ERBB4 into MSC provides irreplaceable partner to ERBB2, the latter of which forms an ERBB2/ERBB4 heterodimer that binds to its ligand NRG1. ERBB4 can bind to NRG1 by forming a homodimer. Activation of the NRG1-ERBB pathway in MSC-ERBB4 enhances cell mobility and anti-apoptosis through the phosphorylation of Akt. Overexpressing ERBB4 in turn regulates the synthesis and secretion of NRG1. The released NRG1 contributes to restore the declined NRG1 level in the infarcted region and support cell growth, dividing and anti-apoptosis of cardiomyocytes. By ERBB4 overexpression in MSCs, we figured out a novel approach that benefits both MSCs and cardiomyocytes, and enable an effective myocardial repair after ischemia

Fig 3: ErbB overactivation caused noninflammatory hypomyelination in Sox10-ErbB2V664E mice. Unless otherwise indicated, quantitative data were presented as mean ± SEM and analyzed by unpaired t test. A, Dox treatment setting for indicated mice and littermate controls. B, Real-time RT-PCR results of ErbB2 expression at indicated day with Dox treatment (dwd). Statistical information for 1 dwd, t(10) = 4.081, p = 0.0022; for 4 dwd, t(4) = 6.37, p = 0.0031. C, Walking speed and percentage of foot slips of Sox10-ErbB2V664E mice and littermate controls at 9 dwd in the grid walking test. n = 4 mice for each group. Statistical information for steps per second, t(6) = 3.504, p = 0.0128; for foot slip percentage, t(6) = 4.429, p = 0.0044. D, Western blotting of indicated proteins in white matter tissues isolated from Sox10-ErbB2V664E (SB) mice in comparison with that from littermate control mice (Ctrl). Activation status of each ErbB receptor or downstream signaling protein was examined by Western blotting with the specific antibody against its phosphorylated form. E, F, Quantitative data of Western blotting results. In E, statistical information for EGFR, t(4) = 0.1983, p = 0.852; for ErbB3, t(4) = 28.34, p < 0.0001; for ErbB4, t(4) = 9.181, p = 0.00078; for Akt, t(4) = 3.380, p = 0.028; for Erk, t(4) = 4.899, p = 0.008. In F, for MBP, t(4) = 48.82, p < 0.0001. G, LFB staining and MBP immunostaining results of coronal sections through the genu of the corpus callosum (CC) in Sox10-ErbB2V664E (SB) and control mice (Ctrl). Black arrows indicate the lower staining intensity of myelin stained in the CC. Statistical information for quantitative data of LFB intensity: the middle part, t(6) = 15.17, p < 0.0001; the lateral part, t(6) = 10.23, p < 0.0001. Statistical information for MBP intensity: CC, t(8) = 5.14, p = 0.0009; CX, t(4) = 4.091, p = 0.015. Statistical information for MBP distribution: CC, t(8) = 4.622, p = 0.0017; CX, t(4) = 0.997, p = 0.375. H, EM images of the CC, optic nerve (ON), and prefrontal cortex (PFC) from Sox10-ErbB2V664E and littermate controls at 9 dwd. Quantitative data were shown for g-ratio analysis of myelinated axons detected by EM. Averaged g-ratio for each mouse were plotted as insets, presented as mean ± SEM, and analyzed by unpaired t test. For CC, t(4) = 3.295, p = 0.0301; for ON, t(4) = 3.775, p = 0.0195; for PFC, t(4) = 1.196, p = 0.298. I, The densities of myelinated axons examined by EM in different brain regions of Sox10-ErbB2V664E (SB) and littermate control mice (Ctrl) at 9 dwd were quantified. Statistical information for CC, t(4) = 0.2773, p = 0.795; for ON, t(4) = 0.1455, p = 0.891. J, K, Astrocytes (GFAP+) and microglia (Iba1+) examined in the subcortical white matter of indicated mice by immunostaining. Cell densities in the CC were quantified, and data were presented as mean ± SEM and analyzed by unpaired t test. In J, t(4) = 0.0501, p = 0.962. In K, t(4) = 1.637, p = 0.178. L, Real-time RT-PCR results of IL-1ß and TNFa transcripts in the CC. Data were presented as mean ± SEM and statistical analysis by unpaired t test revealed no differences. See also Extended Data Figure 4-1 as well as Extended Data Figures 1-1, 1-2.

Fig 4: Overexpression of ERBB4 in MSCs unexpectedly activates a novel NRG1-ERBB4-NRG1 autocrine loop. Overexpressing ERBB4 in MSCs unexpectedly upregulated the synthesize and secretion of its ligand NRG1, detected by conducting western blotting (a) and ELISA (b), respectively. (c) Overexpressing ERBB4 in MSCs upregulated NRG1, but did not alter expression of ERBB2. ERBB3 remained negative before and after ERBB4 overexpressing. (d) Multiple transient transfections using pER4-GFP confirmed that upregulated NRG1 was attributed to exogenous ERBB4 overexpression. (d1) With increased transfection frequency shown by encircular numbers (d1), GFP signal, which indicated transfection efficiency, was escalating accordingly, from 1.2% for the first time (d1) to 32.7% for the fifth time (d1). (d2) The expression of NRG1 was upregulated along with increased transfection efficiency. (e) In human 293FT cells, NRG1 expression was upregulated after ERBB4 transduction. The aforementioned experiments were repeated three times and representative images are shown. 293FTe, 293FT expressing pGFP; 293FT-ERBB4, 293FT expressing pER4-GFP

Fig 5: Exogenous ERBB4 was successfully transduced in MSCs. (a) RT-RCR screening of NRG1-ERBB expression in MSCs. MSC expressed NRG1 and ERBB2, but not ERBB4. (b) MSCs were successfully transduced with pGFP or pER4-GFP (map shown in Supplementary Figure 2), confirmed by using a GFP-positive signal detected under a fluorescent microscope (b1). RT-PCR (b2) and western blotting (b3) confirmed successful manipulation of ERBB4 into MSCs. Elevated p-ERBB4 occurred when additional NRG1 was added. p-ERBB4, phosphorylated ERBB4. (c) Lentiviral-transduced MSCe and MSC-ERBB4 possessed multi-lineage differentiation capacity, confirmed by staining (c1) and lineage specific gene expression (c2). (d) Neither MSCe nor MSC-ERBB4 injection raised malignant formation during 8 weeks of observation, with mouse embryonic stem cells as positive control. mESCs, mouse embryonic stem cells. n=4 for each group