Peripheral nerve injury stem cell therapy
Peripheral nerve injury (PNI) can lead to mitochondrial dysfunction and energy depletion within the affected microenvironment. The objective was to investigate the potential of transplanting mitochondria to reshape the neural regeneration microenvironment. High-purity functional mitochondria with an intact structure were extracted from Human umbilical cord-derived mesenchymal stem cells (hUCMSCs) using the Dounce homogenization combined with ultracentrifugation. Results showed that when hUCMSC-derived mitochondria (hUCMSC-Mitos) were co-cultured with Schwann cells (SCs), they promoted the proliferation, migration, and respiratory capacity of SCs. Acellular nerve allografts (ANAs) have shown promise in nerve regeneration, however, their therapeutic effect is not satisfactory enough. The incorporation of Human umbilical cord-derived mesenchymal stem cells-Mitos within ANAs (referred to as Mito-ANAs) has the potential to remodel the regenerative microenvironment. This approach demonstrated satisfactory outcomes in terms of tissue regeneration and functional recovery. Particularly, they propose for the first time the use of metabolomics and bioenergetic profiling to analyze the energy metabolism microenvironment after PNI. This remodeling occurs through the enhancement of the tricarboxylic acid (TCA) cycle and the regulation of associated metabolites, resulting in increased energy synthesis. Overall, the hUCMSC-Mito-loaded ANAs exhibited high functionality to promote nerve regeneration, providing a novel regenerative strategy based on improving energy metabolism for neural repair 1)
Peripheral nerve injury has remained a substantial clinical complication with no satisfactory treatment options. Despite the great development in the field of microsurgery, some severe types of neural injuries cannot be treated without causing tension to the injured nerve. Thus, current studies have focused on the new approaches for the treatment of peripheral nerve injuries. Stem cells with the ability to differentiate into a variety of cell types have brought a new perspective to this matter 2).
Neural stem cells can not only differentiate into neurons, astrocytes and oligodendrocytes, but can also differentiate into Schwann-like cells, which promote neurite outgrowth around the injury. Transplanted neural stem cells can differentiate into motor neurons that innervate muscles and promote the recovery of neurological function. To promote the repair of peripheral nerve injury, neural stem cells secrete various neurotrophic factors, including brain-derived neurotrophic factor, fibroblast growth factor, nerve growth factor, insulin-like growth factor and hepatocyte growth factor. In addition, neural stem cells also promote regeneration of the axonal myelin sheath, angiogenesis, and immune regulation. It can be concluded that neural stem cells promote the repair of peripheral nerve injury through a variety of ways 3).
Articles on the use of stem cells, Schwann cells, growth factors, collagen, laminin and platelet-rich plasma for peripheral nerve repair were summarized over the course of the review. Based on these studies, it could be concluded that the use of stem cells derived from different sources presents promising results relating to nerve regeneration, because these cells have a capacity for neuronal differentiation, thus demonstrating effective functional results. The use of tubes containing bioactive elements with controlled release also optimizes the nerve repair, thus promoting greater myelination and axonal growth of peripheral nerves. Another promising treatment is the use of platelet-rich plasma, which not only releases growth factors that are important in nerve repair, but also serves as a carrier for exogenous factors, thereby stimulating the proliferation of specific cells for peripheral nerve repair 4).
A literature search of the MEDLINE and Embase databases was performed from inception to April 2012, searching for animal experiments on peripheral nerve reconstruction models in which a nerve conduit was used with and without the support of 3 different types of stem cells. Stem cells were analyzed according to their origin: bone marrow, adipose tissue, and other origins. Included studies had consistent outcome measurements: walking track analysis, muscle mass ratio, and electrophysiology.
Forty-four studies were included in the final analysis. Forest plots of the 3 outcome measurements (walking track analysis, muscle mass ratio, and electrophysiology) showed positive effects of stem cells on the regeneration of peripheral nerves at different time points. Almost all comparisons showed significant differences for all 3 stem cells groups compared with a control group in which stem cells were not used.
The present report systematically analyzed the different studies that used stem cells as a luminal additive when bridging a large peripheral nerve defect. All 3 different stem cell groups showed a beneficial effect when used in the reconstruction compared with control groups in which stem cells were not used 5).
Maintenance of stem cell viability and differentiation potential in vivo are still major obstacles for translation. Using a made-in-house 96-well vertical electrical stimulation (ES) platform, Du et al., investigated the effects of different stimulating pulse frequency, duration and field direction on human neural crest stem cell (NCSC) differentiation. They observed dendritic morphology with enhanced neuronal differentiation for NCSCs cultured on cathodes subject to 20 Hz, 100μs pulse at a potential gradient of 200 mV/mm. They further evaluated the effect of a novel cell-based therapy featuring optimized pulsatile ES of NCSCs for in vivo transplantation following peripheral nerve regeneration. 15 mm critical-sized sciatic nerve injuries were generated with subsequent surgical repair in sixty athymic nude rats. Injured animals were randomly assigned into five groups (N = 12 per group): blank control, ES, NCSC, NCSC + ES, and autologous nerve graft. The optimized ES was applied immediately after surgical repair for 1 h in ES and NCSC + ES groups. Recovery was assessed by behavioral (CatWalk gait analysis), wet muscle-mass, histomorphometric, and immunohistochemical analyses at either 6 or 12 weeks after surgery (N = 6 per group). Gastrocnemius muscle wet mass measurements in ES + NCSC group were comparable to autologous nerve transplantation and significantly higher than other groups (p < 0.05). Quantitative histomorphometric analysis and catwalk gait analysis showed similar improvements by ES on NCSCs (p < 0.05). A higher number of viable NCSCs was shown via immunochemical analysis, with higher Schwann cell (SC) differentiation in the NCSC + ES group compared to the NCSC group (p < 0.05). Overall, ES on NCSC transplantation significantly enhanced nerve regeneration after injury and repair, and was comparable to autograft treatment. Thus, ES can be a potent alternative to biochemical and physical cues for modulating stem cell survival and differentiation. This novel cell-based intervention presents an effective and safe approach for improved outcomes after peripheral nerve repair 6).
A rat sciatic nerve lesion was performed, and Schwann cells or adipose-derived stem cells, untreated or induced to a “Schwann-cell-like” phenotype (dASC), were injected into the gastrocnemius muscle. Nerves were either repaired immediately or capped to prevent muscle reinnervation. One month later, functionality was measured using a walking track test, and muscle atrophy was assessed by examining muscle weight and histology.
Schwann cells and dASC groups showed significantly better scores on functional tests when compared with injections of growth medium alone. Muscle weight and histology were also significantly improved in these groups.
Cell injections may reduce muscle atrophy and could benefit nerve injury patients 7).
Mesenchymal stem cells (MSCs) support axon regeneration across artificial nerve bridges but their differentiative capacity and ability to promote nerve regeneration remains unclear. In this study, MSCs isolated from bone marrow of Sprague-Dawley rats were characterized by plastic adherence and pluripotency towards mesodermal lineages. Isolated undifferentiated MSCs (uMSCs) were stimulated towards a Schwann cell (SC) phenotype using specific growth factors, and cell marker analysis was performed to verify SC phenotype in vitro. Differentiation resulted in temporally dependent positive immunocytochemical staining for the SC markers, glial fibrillary acidic protein (GFAP), S100, and nerve growth factor receptor (NGFR), with maximal marker expression achieved after 6days of treatment with differentiation media. Quantitative analysis demonstrated that ~50% of differentiated MSCs (dMSCs) have a SC phenotype. Using an indirect co-culture system, we compared the ability of dorsal root ganglion (DRG) cells to extend neurites in indirect contact with uMSCs and dMSCs as compared to SCs. The mean values of the longest length of the DRG neurites were the same for the dMSCs and SCs and significantly higher than the uMSC and DRG mono-culture systems (p < 0.05). In vivo, compared to an empty conduit, dMSC seeded collagen nerve conduits resulted in a greater number of sciatic motoneurons regenerating axons through the conduit into the distal nerve stump. We conclude that bone marrow-derived MSCs differentiate into a SC-phenotype that expresses SC markers transiently and sufficiently to support limited neurite outgrowth in vitro and Axon regeneration equivalent to that of SCs in vitro and in vivo. The nerve autograft remains the most effective conduit for supporting regeneration across nerve gaps 8).
Given their documented ability to differentiate into Schwann cells (SCs) in vitro, we hypothesized that skin-derived precursor cells (SKPs) could represent a clinically-relevant source of transplantable cells that would enhance nerve regeneration following peripheral nerve injury. In this study, we examined the potential for SKP-derived Schwann cells (SKP-SCs) or nerve-derived SCs to improve nerve regeneration across a 12 mm gap created in the sciatic nerve of Lewis rats bridged by a freeze-thawed nerve graft. Immunohistology after 4 weeks showed survival of both cell types and early regeneration in SKP seeded grafts was comparable to those seeded with SCs. Histomorphometrical and electrophysiological measurements of cell-treated nerve segments after 8 weeks survival all showed significant improvement as compared to diluent controls. A possible mechanistic explanation for the observed results of improved regenerative outcomes lies in SKP-SCs' ability to secrete bioactive neurotrophins. We therefore conclude that SKPs represent an easily accessible, autologous source of stem cells for transplantation therapies which act as functional Schwann cells and show great promise in improving regeneration following nerve injury 9).
The optimal source of stem cells for regenerative medicine is a major question. Embryonic stem (ES) cells have shown promise for pluripotency but have ethical issues and potential to form teratomas. Pluripotent stem cells have been produced from skin cells by either viral-, plasmid- or transposon-mediated gene transfer. These stem cells have been termed induced pluripotent stem cells or iPS cells. iPS cells may also have malignant potential and are inefficiently produced. Embryonic stem cells may not be suited for individualized therapy, since they can undergo immunologic rejection. To address these fundamental problems, our group is developing hair follicle pluripotent stem (hfPS) cells. Our previous studies have shown that mouse hfPS cells can differentiate to neurons, glial cells in vitro, and other cell types, and can promote nerve and spinal cord regeneration in vivo. hfPS cells are located above the hair follicle bulge in what we have termed the hfPS cell area (hfPSA) and are nestin positive and keratin 15 (K-15) negative. Human hfPS cells can also differentiate into neurons, glia, keratinocytes, smooth muscle cells, and melanocytes in vitro. In the present study, human hfPS cells were transplanted in the severed sciatic nerve of the mouse where they differentiated into glial fibrillary-acidic-protein (GFAP)-positive Schwann cells and promoted the recovery of pre-existing axons, leading to nerve generation. The regenerated nerve recovered function and, upon electrical stimulation, contracted the gastrocnemius muscle. The hfPS cells can be readily isolated from the human scalp, thereby providing an accessible, autologous and safe source of stem cells for regenerative medicine that have important advantages over ES or iPS cells 10).
Tohill et al., examined the differentiation of marrow stromal cells (MSCs) in culture and during nerve regeneration. MSCs from adult rats were exposed to glial growth factor (GGF) to stimulate glial differentiation. Subsequently differentiated MSCs were retrovirally labelled with green fluorescent protein and transplanted into 1 cm nerve conduits in the rat sciatic nerve. Fifteen days post-operatively the conduits were examined for axonal and Schwann cell regeneration and MSC integration. In vitro, MSCs exposed to GGF expressed S100 and glial fibrillary acidic protein. Following transplantation, MSCs maintained S100 expression and enhanced nerve regeneration, with significant Schwann cell regeneration compared to control (2.7 +/- 0.21 vs. 2.05 +/- .21 mm; P < 0.05). MSCs not exposed to GGF prior to transplantation expressed S100 in vivo indicating glial differentiation in response to local cytokines and growth factors 11).