Human Muscle Progenitor Cells Overexpressing Neurotrophic Factors Improve Neuronal Regeneration in a Sciatic Nerve Injury Mouse Model

Abstract

The peripheral nervous system has an intrinsic ability to regenerate after injury. However, this process is slow, incomplete, and often accompanied by disturbing motor and sensory consequences. Sciatic nerve injury (SNI), which is the most common model for studying peripheral nerve injury, is characterized by damage to both motor and sensory fibers. The main goal of this study is to examine the feasibility of administration of human muscle progenitor cells (hMPCs) overexpressing neurotrophic factor (NTF) genes, known to protect peripheral neurons and enhance axon regeneration and functional recovery, to ameliorate motoric and sensory deficits in SNI mouse model. To this end, hMPCs were isolated from a human muscle biopsy, and manipulated to ectopically express brain-derived neurotrophic factor (BDNF), glial-cell-line-derived neurotrophic factor (GDNF), vascular endothelial growth factor (VEGF), and insulin-like growth factor (IGF-1). These hMPC-NTF were transplanted into the gastrocnemius muscle of mice after SNI, and motor and sensory functions of the mice were assessed using the CatWalk XT system and the hot plate test. ELISA analysis showed that genetically manipulated hMPC-NTF express significant amounts of BDNF, GDNF, VEGF, or IGF-1. Transplantation of 3 × 10⁶ hMPC-NTF was shown to improve motor function and gait pattern in mice following SNI surgery, as indicated by the CatWalk XT system 7 days post-surgery. Moreover, using the hot-plate test, performed 6 days after surgery, the treated mice showed less sensory deficits, indicating a palliative effect of the treatment. ELISA analysis following transplantation demonstrated increased NTF expression levels in the gastrocnemius muscle of the treated mice, reinforcing the hypothesis that the observed positive effect was due to the transplantation of the genetically manipulated hMPC-NTF. These results show that genetically modified hMPC can alleviate both motoric and sensory deficits of SNI. The use of hMPC-NTF demonstrates the feasibility of a treatment paradigm, which may lead to rapid, high-quality healing of damaged peripheral nerves due to administration of hMPC. Our approach suggests a possible clinical application for the treatment of peripheral nerve injury.

Keywords: BDNF; GDNF; IGF-1; VEGF; human muscle progenitor cells; neurotrophic factors; peripheral nerve injury; sciatic nerve injury.

FIGURE 1

FACS analysis of CD56 expression in P1 myogenic cells. CD56-positive cells were resolved using light scattering (FS, forward scatter; SS, side scatter). (A) Incubation without antibodies was used as a baseline. (B) Staining for non-specific mouse immunoglobulin G (IgG) isotype fluorescence was used as a control. (C) 92.94% of the isolated cells express the CD56 surface marker related to human myogenic cells according to the peak area shown following staining with anti-human CD56-Phycoerythrin (PE) antibody.

FIGURE 2

FACS analysis of CD56 expression in P3 myogenic cells. CD56-positive cells were resolved using light scattering (FS, forward scatter; SS, side scatter). (A) Incubation without antibodies was used as a baseline. (B) Staining for non-specific mouse immunoglobulin G (IgG) isotype fluorescence was used as a control. (C) 90.19% of the isolated cells express the CD56 surface marker related to human myogenic cells according to the peak area shown following staining with anti-human CD56-PE antibody.

FIGURE 3

Genetically modified human myogenic cells secrete neurotrophic factors (NTFs). Levels of NTFs were measured using ELISA kits 24 and 72 h after the hMPCs were transduced with lentiviruses containing a gene for expressing either (A) BDNF, (B) GDNF, (C) VEGF, or (D) IGF-1. The levels of NTFs secreted from hMPCs harboring the GFP gene were measured as control values. The data are presented as mean ± SEM, ^∗P < 0.05, ^∗∗P < 0.01, one-tailed t-test.

FIGURE 4

Illustration of gait pattern improvement following transplantation of genetically modified hMPCs expressing NTFs after SNI. Representative images of paw prints, acquired using CatWalk XT system, 7 days after SNI without treatment (right) or with 3 × 10⁶ hMPC-NTF treatment (left).

FIGURE 5

Transplantation of genetically modified hMPCs expressing NTFs improved motor function after SNI. Left hind paw maximum tread intensities, at maximum contact, were acquired using the CatWalk XT system on days 3, 7, and 13 post-SNI. These values were compared to those of naïve control mice, whose function was considered 100%. The data are presented as the relative mean function ± SEM of n mice per treatment group. ^∗P < 0.05, ^∗∗P < 0.01, one-tailed t-test.

FIGURE 6

Transplantation of genetically modified hMPCs expressing NTFs improved gait pattern after SNI. Left hind paw print areas were acquired using CatWalk XT system 3, 7, and 13 days post-SNI. These values were compared to those of naïve control mice, whose function was considered 100%. The data are presented as the relative mean function ± SEM of n mice per treatment group. ^∗∗P < 0.01, one-tailed t-test.

FIGURE 7

Transplantation of genetically modified hMPCs expressing NTFs improved sensory deficits after SNI. Nociceptive threshold of the left hind paw was tested by measuring latency of analgesic response in the hot-plate test. These values were compared to those of naïve control mice, whose function was considered 100%. The data are presented as the relative mean response ± SEM of n mice per treatment group, ^∗P < 0.05, one-tailed t-test.

FIGURE 8

In vivo imaging of transplanted cells was correlated with behavioral results. hMPC-GFP cells were tracked after transplantation into the right gastrocnemius muscle using CRI Maestro^TM non-invasive fluorescence imaging system. (A) Schematic illustration of the posterior right limb of a mouse, in which the sciatic nerve and the gastrocnemius muscle are indicated to assist in understanding the following images. (B) A negative control mouse, to which cells have not been transplanted. (C,D) hMPCs marked with green fluorescence protein (GFP) were detected within the gastrocnemius muscle (C) 2 and (D) 5 days after transplantation. (E) Image taken 12 days after transplantation, in which the cells are no longer detectable within the tissue. White arrows in (C–E) mark the transplantation area.