Induced pluripotent stem cell-derived mesenchymal stem cells enhance acellular nerve allografts to promote peripheral nerve regeneration by facilitating angiogenesis

Abstract

JOURNAL/nrgr/04.03/01300535-202605000-00039/figure1/v/2025-10-21T121913Z/r/image-tiff Previous research has demonstrated the feasibility of repairing nerve defects through acellular allogeneic nerve grafting with bone marrow mesenchymal stem cells. However, adult tissue-derived mesenchymal stem cells encounter various obstacles, including limited tissue sources, invasive acquisition methods, cellular heterogeneity, purification challenges, cellular senescence, and diminished pluripotency and proliferation over successive passages. In this study, we used induced pluripotent stem cell-derived mesenchymal stem cells, known for their self-renewal capacity, multilineage differentiation potential, and immunomodulatory characteristics. We used induced pluripotent stem cell-derived mesenchymal stem cells in conjunction with acellular nerve allografts to address a 10 mm-long defect in a rat model of sciatic nerve injury. Our findings reveal that induced pluripotent stem cell-derived mesenchymal stem cells exhibit survival for up to 17 days in a rat model of peripheral nerve injury with acellular nerve allograft transplantation. Furthermore, the combination of acellular nerve allograft and induced pluripotent stem cell-derived mesenchymal stem cells significantly accelerates the regeneration of injured axons and improves behavioral function recovery in rats. Additionally, our in vivo and in vitro experiments indicate that induced pluripotent stem cell-derived mesenchymal stem cells play a pivotal role in promoting neovascularization. Collectively, our results suggest the potential of acellular nerve allografts with induced pluripotent stem cell-derived mesenchymal stem cells to augment nerve regeneration in rats, offering promising therapeutic strategies for clinical translation.

Keywords: Microfil perfusion; acellular nerve allograft; angiogenesis; bioluminescence imaging; conditioned medium; induced pluripotent stem cell–derived mesenchymal stem cells; micro-CT scanning; peripheral nerve injury.

Figure 1

Morphology and characteristics of iMSCs. (A, B) Morphology of iMSCs at passage 4. The cells exhibited spindle-shaped, fibroblast-like morphology. Scale bars: 1 mm in A, 200 μm in B. (C–I) Flow cytometry analysis of iMSC surface antigens: CD34 (0.79%), CD45 (0.54%), HLA-DR (1.23%), CD90 (99.14%), CD73 (99.99%), and CD105 (99.90%). (J) VEGF concentration in iMSC-CM. (K) NGF concentration in iMSC-CM. (L) BDNF concentration in iMSC-CM. Data are presented as the mean ± SD. Enzyme-linked immunosorbent assays were performed in duplicate. *P < 0.05, **P < 0.01 (Student’s t-test). BDNF: Brain-derived neurotrophic factor; Ctrl: control; HLA-DR: human leukocyte antigen DR; iMSC-CM: conditioned medium of induced pluripotent stem cell-derived mesenchymal stem cells; iMSCs: induced pluripotent stem cell-derived mesenchymal stem cells; NGF: neurotrophic growth factor; VEGF: vascular endothelial growth factor.

Figure 2

iMSC-CM promotes angiogenesis in vitro. (A–D) Images of HUVEC tube formation with different conditioned media (CM) at various time points. Scale bars: 200 μm. (E–H) Quantification of tube information assay (violin plots and estimation plots). (E) Number of branch intervals. (F) Total branching length. (G) Number of nodes. (H) Number of meshes. Tube formation assays were repeated four times. *P < 0.05, **P < 0.01 (Student’s t-test). Ctrl: Control (normal Dulbecco’s modified Eagle’s medium); HUVEC: human umbilical vein endothelial cell; iMSC-CM: conditioned medium of induced pluripotent stem cell-derived mesenchymal stem cell.

Figure 3

Survival time of iMSCs in the rat sciatic nerve injury model. (A) Bioluminescence signals at different time points following GFP-Luciferase-iMSC + ANA transplantation. The bioluminescence decreased immediately after cell transplantation and began to decline rapidly by the 8^th day post-transplantation. (B) Quantification of bioluminescence imaging. Data are expressed as mean ± SD (n = 3–10 rats/group). ANA: Acellular nerve allograft; GFP: green fluorescent protein; iMSC: induced pluripotent stem cell-derived mesenchymal stem cell; p: photons; sr: steradian.

Figure 4

Effect of co-graft of ANA and iMSCs on motor nerve recovery in rats with sciatic nerve injury. (A) Schematic of the timeline for CatWalk gait and electrophysiological analysis of the rat model (created with Biorender.com). (B) Representative 2D footprints of each group. Red indicates the right hind (RH); green, the left hind (LH); blue, right front (RF); and yellow, the left front (LF). (C) Representative 3D paw pressure changes of the hind limbs in each group. X-axis represents the time when limbs hit the ground, and Y-axis represents the pressure of the limbs on the ground. (D) The trend of SFI changes over time. (E) Swing times of RH in each group. (F) Ratio of mean intensity of the 15 most intense pixels (experimental side/normal side). The statistical results of D-F are shown in Additional Table 2. (G) Representative waveforms of CMAP. (H) The ratio of latency of CMAP (injured side/normal side). (I) The ratio of peak amplitude of CMAP (injured side/normal side). Data are expressed as mean ± SD (n = 4–8 rats/group). *P < 0.05, **P < 0.01; #P < 0.05, ##P < 0.01, ###P < 0.001, vs. 1 week; †P < 0.05, ††P < 0.01, †††P < 0.001, vs. 4 weeks; &P < 0.05, vs. 8 weeks (one-way analysis of variance followed by Tukey’s post hoc test). ANA: Acellular nerve allograft; CMAP: compound muscle action potential; iMSCs: induced pluripotent stem cell-derived mesenchymal stem cells; ns: not significant.

Figure 5

Effect of co-graft of ANA and iMSCs on the target muscles of the regenerated nerves in rats with sciatic nerve injury after 12 weeks. (A) General view of the gastrocnemius muscle in each group. The left side is the healthy side, and the right side is the operated side. (B) Representative image of Masson trichromatic staining of rat gastrocnemius. Scale bars: 10 mm in A, and 200 μm in B. (C) The wet weight ratio of the gastrocnemius muscle (injured side/normal side) in each group. The raw data is shown in Additional Table 3. (D) The mean cross-sectional area of the gastrocnemius muscle fiber. Data are expressed as mean ± SD (n = 5–6 rats/group). *P < 0.05, **P < 0.01 (one-way analysis of variance followed by Tukey’s post hoc test). ANA: Acellular nerve allograft; iMSCs: induced pluripotent stem cell-derived mesenchymal stem cells; ns: not significant.

Figure 6

Effect of co-graft of ANA and iMSCs on the vascularization around regenerated nerves in rats with sciatic nerve injury after 12 weeks. (A) Illustration of Microfil methods. (B) Morphological images of Microfil-perfused nerves. White arrow: Microfil-perfused microvessel. (C) 3D reconstruction image of Microfil-perfused nerves. (D) Immunofluorescence staining of the regenerated tissues. NF200 (a marker for neurofilament, green), S100 (a marker for SCs, red), and nuclei (DAPI, blue). White arrow: suture knots. Scale bars: 500 μm in B, 1000 μm in C, and 500 μm in D. (E) Number of nerve fibers at the midpoint of allografts. (F) Surface area of vessels of Microfil-perfused nerves. (G) Volume of vessels of Microfil-perfused nerves. Data are expressed as mean ± SD (n = 5–6 rats/group). *P < 0.05, **P < 0.01, ***P < 0.001 (one-way analysis of variance followed by Tukey’s post hoc test). 3D: Three-dimensional; ANA: acellular nerve allograft; CT: computed tomography; DAPI: 4′,6-diamidino-2-phenylindol; iMSCs: induced pluripotent stem cell-derived mesenchymal stem cells; NF200: neurofilament protein 200.

Figure 7

Effect of co-graft of ANA and iMSCs on regenerated myelin in rats with sciatic nerve injury after 12 weeks. (A) Toluidine blue staining of the distal nerve graft. (B) Transmission electron microscope images of myelin sheath in the distal nerve graft. (C) Enlarged transmission electron microscopy images of red boxes in B. Scale bars: 20 μm in A, 5 μm in B and 2 μm in C. (D) The average number per unit area of the myelin sheath. (E) The average diameter of the myelin sheath. (F) The average thickness of the myelin sheath. Data are expressed as mean ± SD (n = 3 rats/group). *P < 0.05, **P < 0.01 (one-way analysis of variance followed by Tukey’s post hoc test). ANA: Acellular nerve allograft; iMSCs: induced pluripotent stem cell-derived mesenchymal stem cells; ns: not significant.