Stem cells and pain

Table 1 Painkillers and numbers needed to treat

Drug	Drug class	NNT	Ref.
Acetaminophen 650 mg + oxycodone 10 mg	NSAID + opioid	2.7	Gaskell et al[178], 2009
Acetaminophen 500 mg + ibuprofen 200 mg	NSAID combination	1.6	Moore and Hersh[179], 2013
Aspirin 1200 mg	NSAID	2.4	Bandolier Extra[180], 2003
Codeine 60 mg	Opioid	16.7	Maxwell and Bateman[181], 2007
Diclofenac 100 mg	NSAID	1.8	Gaskell et al[178], 2009
Ibuprofen 400 mg	NSAID	2.5	Lyngstad et al[182], 2021
Morphine 10 mg (intramuscular)	Opioid	2.9	Bandolier Extra[180], 2003
Naproxen 500 mg	NSAID	2.7	Derry et al[183], 2009
Oxycodone 15 mg	Opioid	4.6	Gaskell et al[178], 2009

NSAIDs: Nonsteroidal anti-inflammatory drugs; NNT: Number needed to treat.

Table 2 Table 2 Articles that used stem cells to treat inflammatory pain (stem cell-based treatment of inflammatory pain or in models where pain is certainly involved, but was not investigated)

Ref.	Stem cell therapy design	Key findings	Pain-related highlights	Route of administration	Number of cells or amount of extracellular vesicles and exosomes
Hsueh et al[69], 2023	iPSC-derived EVs for the treatment of rabbit articular cartilage OA in an in vivo model and an in vitro interleukin (IL)-1β-induced model	Improvement in both in vivo and in vitro models of OA by stimulation of chondrocytes proliferation and decreasing senescence were accompanied by: Decreasing of TNF-α); IL-6; protein 21 (p21); MMP 13; ADAMTS5; and increasing of collagen II	Indirect: Specific pain receptors/pathways weren’t investigated	i.a.	100 μg iPSC-EV
Gao et al[184], 2022	Small EVs from iPSC-derived mesenchymal stem-cells ameliorate tendinopathy pain by inhibiting mast cell activation	The treatment was able to decrease acute pain in tendinopathy, as well as inhibit infiltration of activated mast cells and interactions with nerve fibers in vivo. In the in vitro experiments, the treatment decreased mast cell degranulation and the expression of pro-inflammatory cytokines and genes involved in the hypoxia inducible factor-1 signaling pathway	Pain behavior was measured by the von Frey method. And the weight distribution on the knees by SWB; immunofluorescence staining of tendon sections for tryptase (mast cell marker) and PGP9.5 (nerve fiber marker) was performed to assess the number of activated mast cells and the anatomical interaction between mast cells and nerve fibers. In addition, the SWB and CatWalk test was also. carried out	Local injection (quadriceps tendon)	1 × 109 particles
Yu et al[185], 2022	Intravital imaging and single cell transcriptomic analysis for engraftment of mesenchymal stem-cells in an animal model of interstitial cystitis/bladder pain syndrome	The transplanted cells formed a perivascular-like structure. They were also shown to express cyclin-dependent FOSe kinase-1 which played a key role in modulating the migration, engraftment and anti-inflammatory functions of multipotent MSCs, which determined their therapeutic potency in vivo	In vivo two-photon intravital microscopy and single-cell transcriptome analysis were used to assess the effects of stem cell treatment on interstitial cystitis/bladder pain syndrome	Injected into the outer layer of the anterior wall and dome of the bladder	106
Zhang et al[186], 2022	EVs derived from MSCs alleviate neuroinflammation and mechanical allodynia in interstitial cystitis rats by inhibiting NLRP3 inflammasome activation	SC treatment decreased suprapubic mechanical allodynia and frequent urination in rats with interstitial cystitis. It also decreased glial cell activity as well as neuroinflammation in the spinal cord. Furthermore, the treatment was able to decrease the activation of NLRP3 inflammasomes and the TLR4/NF-κB signaling pathway	Behavioral test (von Frey) was performed to measure allodynia and western blot and immunofluorescence for protein related to inflammation and central sensitization analysis: CD9, CD63, CD81, ALIX, TNF-α, IL-1β, IL-6, IBA-1, GFAP, NLRP3, Caspase-1, IL-18, TLR4, p65 NK-κB, phospho-p65 NK-κB (western blot). NLRP3, neuron-specific nuclear protein, GFAP and OX-42 labeling (immunofluorescence)	i.t.	20 μg
González-Cubero et al[86], 2022	EV and soluble fractions of adipose tissue-derived MSCs secretome induce inflammatory cytokines modulation in an in vitro model of discogenic pain	There was a decrease in the expression of IL-6, IL-8 and IL-17	Indirect method: The authors measured the regulatory capacity of EVs on the inflammatory molecules IL-1α, IL-1β, IL-6, IL-8, IL-17, nerve growth factor, brain-derived neurotrophic factor, IFN-γ, NF-κB and TNF and MMP-1, MMP-2, MMP-3, MMP-13 and ADAMTS-5	In vitro model	1 × 106
Yang et al[91], 2020	Anti-inflammatory protein TSG-6 secreted by bone marrow MSCs attenuates neuropathic pain by inhibiting the TLR2/MyD88/NF-κB signaling pathway in spinal microglia	Stem cells are capable of secreting TSG-6. This article demonstrated that i.t. administration of this protein leads to a decrease in mechanical allodynia and heat hyperalgesia. In addition to inhibiting neuroinflammation in the spinal cord (IBA-1), the protein administration inhibited the activation of the TLR2/MyD88/NF-κB pathway in the dorsal horn of the ipsilateral spinal cord by the secretion of TSG-6 and reduced the production levels of pro-inflammatory cytokines, such as IL-1β, IL-6 and TNF-α	The activation of the TLR2/MyD88/NF-κB signaling pathway was evaluated by western blot and by immunofluorescence. Mechanical allodynia and heat hyperalgesia were observed by behavioral tests	i.t.	5 × 106
Zhang et al[187], 2019	MSCs exosomes alleviate temporomandibular joint OA by attenuating inflammation and restoring matrix homeostasis	It was observed that the treatment led to repair of the temporomandibular joint, along with a reduction in inflammation and pain. Treatment increased IL-1β-impaired sulfated glycosaminoglycan synthesis and suppressed IL-1β-induced nitric oxide and MMP13 production. These effects were partially abrogated by inhibitors of adenosine receptor, protein kinase B, ERK and adenosine monophosphate activated protein kinase phosphorylation	Mechanical hyper-nociception was assessed using the von Frey microfilament. The expression of inflammatory mediators and other components was measured using quantitative polymerase chain reaction	i.a.	100 μg
Ebbinghaus et al[88], 2018	A promising new approach for the treatment of inflammatory pain: Transfer of stem cell-derived tyrosine hydroxylase-positive cells (mouse model)	It has been demonstrated that the administration of endogenous tyrosine hydroxylase positive cells (iTH+) cells, prior to the induction of antigen-induced arthritis, was not sufficient to suppress the disease. However, the treatment was able to decrease pain behavior evoked by inflammation, largely due to the production of IL-4 induced by iTH+ cells. Furthermore, the treatment was able to reduce the levels of pro-inflammatory molecules, in addition to increasing the number of M2 macrophages in dorsal root ganglia	Inflammatory molecules were quantified, such as: IFN-γ, IL-2, IL-4, IL-6, IL-10, CCL3, CCL5, CXCL1, CXCL2, CXCL10, and CXCL12. Additionally, pain-related behavior tests and IBA-1 and arginase 1 labeling in the dorsal root ganglion via immunofluorescence was performed	i.v.	106
Ichiseki et al[92], 2018	I.a.-injected MSC stimulate anti-inflammatory molecules and inhibits pain related protein and chondrolytic enzymes in a monoiodoacetate-induced rat arthritis model	The treatment was able to inhibit central pain sensitization (decreased expression of CGRP in the spinal cord) and increase the secretion of TSG-6 by stem cells, an anti-inflammatory factor and cartilage protector	For the evaluation of central sensitization, CGRP staining was performed by immunofluorescence. And the histochemical technique was also used for the evidence in the joint of ADAMTS5 and TSG-6	i.a.	5.0 × 106
Fodor and Paulseth[90], 2016	Adipose derived stromal cell injections for pain management of OA in the human knee joint	After 3 mo of treatment, patients showed improvement in WOMAC and VAS scores, which were maintained for 1 yr. ROM and TUG improved until the third month. All patients achieved full activity with decreased knee pain and no infections or adverse effects reported	Patients were evaluated by following scores on the WOMAC, VAS pain scale, ROM, TUG, and magnetic resonance imaging	i.a.	14.1 × 106 nucleated stromal vascular fraction cells per knee
Pettine et al[93], 2016	Treatment of discogenic back pain with autologous bone marrow concentrate injection with minimum two-year follow-up (humans)	Stem cell treatment reduced visual analog scale and Initial Oswestry Disability Index scores. In addition to reducing pain in patients. The treatment proved to be effective for up to 2 yr after the injection	Pain was assessed using scores provided by patients	Intradiscal injection	(0.5-1) × 106
Durand et al[89], 2015	Persistent visceral allodynia in rats exposed to colorectal irradiation is reversed by MSC treatment	Induced a time-dependent reversion of the visceral allodynia and a reduction of the number of anatomical interactions between mast cells and PGP9.51 nerve fibers	Spinal sensitization (was available for labeling of phospho-ERK neurons), colonic neuroplasticity (as increased density of substance P1 nerve fibers); s, visceral sensitivity was evaluated by studying the contraction of the abdominal muscles in response to colorectal distension	i.v.	1.5 × 106
Watanabe et al[87], 2015	Early transplantation of MSC after SCI relieves pain hypersensitivity through suppression of pain-related signaling cascades and reduced inflammatory cell recruitment	The treatment was able to decrease thermal and mechanical hypersensitivity. Improvements in pain were mediated by suppression of PKC-γ and p-CREB expression in dorsal horn neurons. The authors also reported a decrease in the levels of pro-inflammatory cytokines (TNF-α, IL-6), mediators of early secondary vascular pathogenesis (MMP9) and macrophage recruitment factors (CCL2, CCL5 and CXCL10). All in addition to increased levels of a microglial stimulating factor GM-CSF)	Mechanical allodynia and thermal sensitivity were recorded. In addition, immunofluorescence was performed on spinal cord sections, labeling for: PKC-γ or p-CREB, GFAP, cD11B and phospho-protein 38. For immunoblot analysis, components of the mitogen-activated protein kinase family, inflammatory mediators (TNF-α, IL-6, MMP-9), macrophage recruiting factors (CCL2, CCL5, and CXCL10) and GM-CSF (a microglial stimulating factor) were analyzed	Injection into the middle of the contusion site, identified as the middle point of the laminectomy area	2.0 × 105
Emadedin et al[188], 2012	Intra-articular injection of autologous MSC in six patients with knee OA	The treatment was able to improve scores related to pain, the functional status of the knee and the distance covered up to six months after the injection	VAS which is a subjective assessment that represents the patient’s perception of the current pain state with a higher score reflecting more severe pain. Functional status of the knee was assessed by WOMAC OA index. This index evaluates pain, joint stiffness, physical and social function of patients with OA of the knee	i.a.	(20-24) × 106

iPSC: Induced pluripotent stem cells; OA: Osteoarthritis; IL: Interleukin; TNF-α: Tumor necrosis factor alpha; p21: Protein 21; MMP: matrix metalloproteinase; ADAMTS5: A disintegrin and metalloproteinase with thrombospondin motifs 5; i.a.: Intra-articular; EV: Extracellular vesicle; SWB: Static weight bearing; PGP9.5: Protein gene product 9.5; CDK1: Cyclin-dependent FOSe kinase 1; MSC: Mesenchymal stem cells; NLRP3: NOD-like receptor protein 3; i.t.: Intrathecal; TLR: Toll-like receptor; NF-κB: Nuclear factor kappa B; IBA-1: Ionized calcium-binding adapter molecule 1; GFAP: Glial fibrillary acidic protein; CD: Cluster of differentiation; IFN-γ: interferon-gamma; TSG-6: Tumor necrosis factor alpha-stimulated gene 6; MyD88: Myeloid differentiation primary response 88; ERK: Extracellular signal-regulated kinase 1; iTH+: Tyrosine hydroxylase positive cells; CCL: C-C motif chemokine ligand; CXCL: C-X-C motif chemokine ligand; DRG: Dorsal root ganglion; i.v.: Intravenous; CGRP: Calcitonin gene-related peptide; WOMAC: Western Ontario and McMaster Universities Arthritis Index; VAS: Visual Analogue Scale; ROM: Range of motion; TUG: Timed ascent and descent; PKC-γ: Protein kinase C gamma; GM-CSF: Granulocyte-macrophage colony stimulating factor; p-CREB: Phospho cyclic AMP response element binding protein; p-p38: Phospho-protein 38.

Table 3 Articles that used stem cells for neuropathic pain treatment (stem cell-based treatment of neuropathic pain)

Ref.	Stem cell therapy design	Key findings	Pain-related highlights	Delivery route	Number of cells or amount of extracellular vesicles and exosomes
Gao et al[189], 2023	Huc-MSCs-derived exosomes attenuate neuropathic pain by inhibiting activation of the TLR2/MyD88/NF-κB signaling pathway in the spinal microglia by targeting radical S-adenosyl methionine domain containing 2	Huc-MSCs-derived decreased protein levels of TLR2, MyD88, and p-p65 that were significantly upregulated in the CCI group in model rats	The protein levels of TLR2, MyD88, p65, and p-p65 were examined by western blotting	i.t.	5 μg
Miyano et al[190], 2022	I.v. administration of human MSCs derived from adipose tissue and umbilical cord improves neuropathic pain via suppression of neuronal damage and anti-inflammatory actions in rats	Both the mechanical threshold and the differences in weight bearing of the right and left hind paws improved significantly. In addition, the authors also reported a decrease in the ATF-3 and IBA-1 in DRG. The authors also reported that the treatment significantly improved the partial sciatic nerve ligation-induced decrease in the level of myelin basic protein in the sciatic nerve	Was performed by von Frey and dynamic weight bearing. Also, the authors did I against ATF-3, IBA-1, myelin basic protein, NeuN, neurofilament (NF) 200	i.v.	5 × 106
González-Cubero et al[191], 2022	Application of adipose-derived MSCs in an in vivo model of peripheral nerve damage	Rat sciatic nerve damage models both ex vivo, on TNF-induced Schwann cells, and in vivo using biomaterial implants containing TNF. Upregulation of c-Jun and downregulation of early growth response protein 2 myelin-associated transcription factors were induced by TNF-related damage, but the addition of ASCs or ASC-conditioned medium (secretome) were able to revert the profile	qPCR, western blot, and confocal microscopy were chosen to quantify nerve healing-related protein expression and production in vivo and ex vivo. The sciatic functional index was calculated to assess nerve regeneration, but no pain-specific mechanisms were investigated	Sciatic nerve	ex vivo 0.5 × 106 cells; in vivo 4 × 106 ASCs
An et al[192], 2022	Immortalized bone MSCs with inducible galanin expression produce controllable pain relief in neuropathic rats	hTERT-BMSCs/Tet-on/GAL cells were able to induce controllable pain relief by spared nerve injury of sciatic nerve under the transcriptional control of doxycycline	To determine the analgesic efficacy acted through GalR1, GalR2, and phospho-protein kinase Mζ expression levels in spinal dorsal horn were analyzed by western blot assay	Subarachnoid space	106
Lee et al[193], 2022	MSCs spheroids alleviate neuropathic pain by modulating chronic inflammatory response genes	The authors report a decrease in mechanical allodynia, related to a decrease in TNF-α and IFN-γ levels. In addition to a smaller number of cells marked with cluster of differentiation (CD) 68 in the region	The von Frey test was performed to assess mechanical allodynia, while immunofluorescence was used to observe changes in CD68 and IBA-1 levels. TNF-α and IFN-γ levels were assessed by the ELISA assay	Intramuscular	106
Chen et al[194], 2022	Synergic Effect of early administration of probiotics and adipose-derived MSCs on alleviating inflammation-induced chronic neuropathic pain in rodents	The authors demonstrate that treatment with stem cells alone can reduce thermal hyperalgesia and mechanical allodynia, with the potentiated effects after combined treatment with probiotics. Interestingly, they found a reverse correlation between protein expressions of inflammatory (phospho-NF-κB, IL-1β, TNF-α and MMP-9), apoptotic (cleaved-caspase-3, cleaved-PARP), oxidative-stress (NOX-1, NOX-2), deoxyribonucleic acid (DNA)-damaged (γ-H2AX) and MAPK-family (p-P38, p-JNK, p-ERK 1/2) biomarkers as well as the protein levels of voltage-gated sodium channels (Nav.) 1.3, Nav.1.8, and Nav.1.9 in L4-L5 in DRG to the pain-behavior results obtained by thermal hyperalgesia and mechanical allodynia testing, characterizing a set of “pain-connived cells” presenting the following profiles: Nav1.8+/peripherin+, p-ERK+/peripherin+, p-p38+/peripherin+ and p-p38+/NF200+. Mainly by suppressing inflammation and oxidative stress, the combination of probiotic and ASCs therapy was found superior for alleviating CCI-induced neuropathic pain	To observe pain-related behavior alterations, Hargreaves and von Frey tests were applied. Immunofluorescence was performed for p-p38; NF200; peripherin, 53BP1, β3 Tubulin analysis. Western blot was chosen to identify alteration of p-NF-kB, IL-1ß, TNF-α, MMP-9, NOX-1, NOX-2, caspase 3, cleaved-PARP, γ-H2AX, p-ERK1/2, p-JNK, p-p38, Nav.1.3, Nav.1.8, Nav.1.9 and immunoglobulin G	i.v.	3.0 × 105
Zhang et al[195], 2021	Lncenc1 is identified as a novel regulator in neuropathic pain by interacting with EZH2 and downregulating the expression of Bai1 in mouse microglia	Virgin embryonic stem cells express Lncenc1, which can activate microglia in DRG and induce the production of TNF-α, IL-1β, and MCP-1. Lncenc1 silencing reduced mechanical and thermal hyperalgesia, as well as lower levels of pro-inflammatory cytokines	The mechanical withdrawal threshold was measured by von Frey filaments and thermal hyperalgesia via hot plate assay. Immunofluorescence was performed to analyze OX-42, western blot to assess EZH2, suppressor of zeste 12, embryonic ectoderm development, BAI1, tri-methylation of histone 3 lysine 27 (H3K27me3), H3K27ac, total histone H3, glyceraldehyde-3-phosphate dehydrogenase and OX-42. RT-qPCR was performed to identify expression alterations on Lncenc1, EZH2, BAI1, OX-42, inflammatory factors TNF-α, IL-1β and chemokine MCP-1. TNF-α, IL-1β and MCP-1 protein changes were assessed by ELISA	Not informed	Not informed
Masoodifar et al[161], 2021	Effect of the conditioned medium of MSCs on the expression levels of P2X4 and P2X7 purinergic receptors in the spinal cord of rats with neuropathic pain	Animals treated with the conditioned medium (stem cells secretome) showed a reduction in mechanical and thermal hyperalgesia. A decrease in the expression of P2X4 and P2X7 receptors was related to the interaction of neurons and glial cells in neuropathic pain	The von Frey and hot plate tests were applied to measure mechanical and thermal hyperalgesia, respectively. In addition, qPCR was performed to measure the expression of P2X4 and P2X7 receptors	i.p.	1 × 105
Kotb et al[196], 2021	Preemptive stem cells ameliorate neuropathic pain in rats: A central component of preemptive analgesia	MSCs-treatment increased allodynia, mechanical hyperalgesia, and thermal hyperalgesia thresholds. Stem cells were able to reach the cerebral cortex, as the CCI group had few stem cells expressing PCNA, CD117 and nestin in the cerebral cortex. The treated group had numerous CD117-, nestin-, PCNA-positive stem cells recently proliferated in the cerebral cortex. Together, the results indicate a potential central analgesic effect of i.v. MSC-treatment	To evaluate pain behavior, von Frey, Randall and Selitto, and hot plate tests were performed. Immunohistochemical analyses of GFAP, PCNA and nestin were also performed	i.v.	1 × 106
Zhang et al[197], 2021	Therapeutic effects of peripherally administered neural crest stem cells on pain and spinal cord changes after sciatic nerve transection	The treatment was able to induce thermal and mechanical analgesia, possibly by decreasing the expression of TRPV1, cFOS, p-ERK, ERK, iNOS and NF-κB, p65 and increasing BDNF and GAP-43 in the spinal cord	To assess mechanical allodynia, the authors used the BEM-404 device (similar to the von Frey). For thermal withdrawal latency, they use Hargreaves. In the western blot, they had the following targets: BDNF, cFOS, GAP-43, p-ERK, ERK 1/2, TRPV1 and iNOS. Immunofluorescence: IBA-1, GFAP and CGRP were assessed	Local injection	2 × 106
Yang et al[91], 2020	Anti-inflammatory protein TSG-6 secreted by BMSCs attenuates neuropathic pain by inhibiting the TLR2/MyD88/NF-κB signaling pathway in spinal microglia	I.t. administration of TSG-6 secreted from stem cells decreases mechanical allodynia and thermal hyperalgesia, inhibiting IBA-1 and the activation of the TLR2/MyD88/NF-κB pathway in the dorsal horn of the ipsilateral spinal cord. Levels of pro-inflammatory cytokines, such as IL-1β, IL-6 and TNF-α, were also reduced	The activation of the TLR2/MyD88/NF-κB signaling pathway was evaluated by western blot and immunofluorescence, while allodynia and hyperalgesia were assessed by the behavioral tests Dynamic Plantar Aesthesiometer, Hargreaves and rotarod system	i.t.	5 × 106
Jwa et al[198], 2020	ASCs alleviate cold allodynia in a rat spinal nerve ligation model of neuropathic pain	ASCs or ASC-derived culture medium decreased neuropathic pain behaviors in a rat model with L5 spinal nerve ligation	Mechanical and cold allodynia were assessed by von Frey filaments and acetone assay, respectively. Mechanisms were not assessed	Intrathecal or injection into the right retro-orbital sinus	106
Gama et al[164], 2018	Conditioned medium of BMSCs as a therapeutic approach to neuropathic pain: A preclinical evaluation	The animals showed improvement in thermal hyperalgesia and mechanical allodynia. They also showed reduced levels of IL-1β, TNF-α and IL-6 and increased IL-10 in the spinal cord and sciatic nerve	To evaluate thermal hyperalgesia, the Hargreaves test was performed, and von Frey mechanical allodynia. To evaluate the motor function test, the rotarod test was performed. Using the ELISA method, TNF-α, IL-1β, IL-6 and IL-10 were quantified	i.v.	106
Lin et al[165], 2017	Autologous ASCs reduce burn-induced neuropathic pain in a rat model	There was no difference between the groups regarding thermal hyperalgesia, whereas in mechanical allodynia, the treated group presented analgesia from the 3rd wk of the first treatment. Western blot analyses revealed a decrease in p-Akt/Akt and Bax/Bcl-2 and levels of LC3B-II and Beclin 1 in the spinal cord, suggesting that the treatment also decreased apoptosis and autophagy. This effect was accompanied by a reduction in COX-2, iNOS and nNOS. The treated group also showed lower expression of p-JNK (an inflammatory marker), TUNEL (apoptosis marker), phospho-NFκB (inflammatory marker) and increased p-IκB (an inhibitor of NFκB activation)	Immunofluorescence were performed to analyze p-IκB; NeuN, GFAP, phospho-NFκB and p-JNK; and western blot for COX-2, iNOS, nNOS, Akt/protein kinase B, p-Akt, B-cell lymphoma 2, Bcl-2-associated X protein, β-actin, LC3B and Beclin 1	Subcutaneous into the scar tissue of the right hind paw	106
Vaquero et al[199], 2018	I.t. administration of autologous bone marrow stromal cells improves neuropathic pain in patients with SCI	Treatment with mesenchymal stromal cells for human chronic SCI: Pain scores demonstrated a continuous decrease in neuropathic pain from the first month until the 10th	Intensity of neuropathic pain was evaluated by standard numerical rating scale (visual analogue scale) from 0 to 10. Mechanisms were not assessed	i.t.	106
Sun et al[200], 2017	I.t. administration of hBMSCs genetically modified with human proenkephalin gene decrease nociceptive pain in neuropathic rats	hBMSCs engineered with human proenkephalin gene were used on sciatic nerve (CCI)-induced model to reduce neuropathic pain in rats	Mechanical withdrawal threshold (von Frey filaments) and paw thermal withdrawal assays were used to assess the changes in pain-related behavior. Levels of Leu-enkephalin, a neurotransmitter that activates opioid receptors and is released by hBMSCs were found augmented via ELISA assay in genetically modified BMSCs compared to secretions released by naıve BMSCs	i.t.	6 × 106
Fischer et al[201], 2017	Inhibition of neuropathic hyperalgesia by i.t. BMSCs is associated with alteration of multiple soluble factors in cerebrospinal fluid	BMSCs decrease the levels of intracellular adhesion molecule 1, IL-1β, hepatocyte growth factor), IL-10, and Nope protein relacionated by Tibial nerve injury	Antibody array analysis was performed and the levels of cytokines and other soluble factors in cerebrospinal fluid samples was measured	i.t.	2.5 × 105
Xie et al[202], 2017	Active nerve regeneration with failed target reinnervation drives persistent neuropathic pain	Semaphorin 3A, an inhibitory axonal guidance molecule, reduces functional regeneration, spontaneous activity, and pain behaviors when applied to the injury site in vivo. Silencing of the upregulated GAP43 with interfering RNA injected into the axotomized sensory ganglion reduced pain behaviors	Behavior assays: von Frey filaments acetone cold sensitivity, dynamic tactile allodynia with a wisp of cotton across the plantar surface of the hindpaws, and spontaneous guarding behavior score. Immunohistochemistry for GAP43 tracer methods to assess anatomical nerve regeneration	Injury site
Brini et al[203], 2017	Therapeutic effect of human ASCs and their secretome in experimental diabetic pain	Treatments with both human ASC and their secretome were able to reverse mechanical, thermal allodynia and thermal hyperalgesia inducing high IL-1β, IL-6 and TNF-α and low IL-10 levels, restoring cytokine balance, Th1/Th2 balance and preventing skin innervation loss in neuropathic STZ-diabetic mice model	Mechanical allodynia was tested using the Dynamic Plantar aesthesiometer, a drop (50 μL) of acetone was placed in the middle of the plantar surface of the hind paw to evaluate cold allodynia and the hot-plate test was used to assess thermal hyperalgesia. Immunohistochemistry and ELISA were performed for cytokines assessment	i.v.	1 × 106
Watanabe et al[87], 2015	Early transplantation of MSCs after SCI relieves pain hypersensitivity through suppression of pain-related signaling cascades and reduced inflammatory cell recruitment	BMSC improved SCI model via: Down of protein kinase C-γ and phosphocyclic AMP response element binding protein on DRG neurons, both of which are upregulated in association with at-level allodynia after contusion spinal cord. Decreased activation of MAPK signaling in injured spinal cord by p-p38 and p-ERK1/2 decrease. Decreasing macrophage recruitment through. Down TNF-α, IL-6, MMP-9, CCL2, CCL5, and C-X-C motif chemokine ligand 10. Decreased microglia stimulation factor, granulocyte-macrophage colony stimulating factor, platelet-derived growth factor receptor α	For behavioral and sensory testing, the Basso Mouse Locomotor Scale, the Dynamic Plantar Aesthesiometer (allodynia), and the Plantar Test Apparatus (thermal sensitivity) were assessed. immunohistochemistry, flow cytometry and immunoblot assays were performed to determine protein levels	BMSCs were injected into the middle of the contusion site, identified as the middle point of the laminectomy area	2 × 105
Zhang et al[204], 2014	I.t. administration of MSCs reduces the ROS and pain behavior in neuropathic rats	I.t. rat MSCs injection reduced pain response and ROS production in the dorsal horn of neuropathic rats induced by spinal nerve L5 ligation model	Mechanical sensitivity was assessed using von Frey filaments and production of ROS via dihydroethidium fluorescent staining	i.t.	105
Liu et al[205], 2014	MSCs inhibit lipopolysaccharide-induced inflammatory responses of BV2 microglial cells through TSG-6	Anti-inflammatory effects of MSCs and TSG-6 in an in vitro LPS-induced BV2 microglial activation model inhibiting NF-κB and MAPK pathways. MSCs can modulate microglia activation through TSG-6 and TSG-6 attenuates the inflammatory cascade in activated microglia	RT-qPCR, western blot, electrophoretic mobility shift assay, immunofluorescence and laser-scanning confocal microscopy techniques were used	In vitro	1.0 × 105 LPS-activated MSCs
Vicker et al[206], 2014	A preliminary report on stem cell therapy for neuropathic pain in humans	Treatment led to a reduction in stem cell treatment pain intensity scores in 7/9 patients (two with marginal improvement and five subjects with good to excellent pain reduction). Five of these positive responders also reduced their need for gabapentin medication	Patients were assessed for: Change in pain intensity and the secondary outcome was any reduction in daily consumption of anti-neuropathic medication	Perineural, directly in the center or source of pain, and in the adjacent pain field of the affected branches of the trigeminal nerve	Number of cells not reported, but extracted from 100-200 g of patient tissue
Tao et al[154], 2013	Role of NRG1/ErbB signaling in stem cell therapy for SCI-induced chronic neuropathic pain	The treatment induces remyelination in the injured spinal cord and reduces SCI-injury-induced chronic neuropathic pain. In addition to increasing levels of NRG1 and ErbB4 slightly reduced by SCI. Also, the author related that Stem cells differentiated into oligodendrocytes	To evaluate mechanical allodynia, the von Frey filament test was applied. Immunofluorescence for NG2, APC-CC1, GFAP, NeuN, and western blot for NRG1 and ErbB4 levels assessment	i.t.	106
Xu et al[207], 2013	I.t. transplantation of NSCs appears to alleviate neuropathic pain in rats through release of GDNF	The treatment was able to cause thermal and mechanical analgesia. Accompanied by an increase in GDNF in the DRG and spinal cord. The authors also suspected that these changes occurred due to the transformation of stem cells into astrocytes in the spinal cord	To evaluate the mechanical withdrawal threshold, the Electric von Frey test was used. For thermal withdrawal latency, a method with a high-intensity projection lamp bulb was used. For immunofluorescence: Nestin; βIII-tubulin; GFAP. For ELISA: BDNF and GDNF	i.t.	106
Choi et al[208], 2013	Core-shell nanoparticle controlled human adipose tissue-derived stem cells neurogenesis for neuropathic pain therapy	Treatment activated biochemical functions of Dicer, Oct4, Sox2, Nanog, and glutathione peroxidase 3 improving stem cells self-renewal and differentiation abilities	von Frey and Hargreaves behavior tests were performed to assess mechanical and thermal hyperalgesia changes, respectively. Immunofluorescence, western blot and RT-qPCR techniques were used to study alterations in protein production/expression/localization	i.t.	Unspecified
Franchi et al[137], 2012	I.v. NSCs abolish nociceptive hypersensitivity and trigger nerve regeneration in experimental neuropathy	NSCs administration in CCI mouse model significantly decreased proinflammatory (IL-1β, IL-6), activated anti inflammatory (IL-10) cytokines in the sciatic nerve, and reduced spinal cord Fos expression in laminae I-VI	Thermal hyperalgesia was tested according to the Hargreaves using a Plantar Test Apparatus, while mechanical allodynia was assessed using the Dynamic Plantar Aesthesiometer. Immunohistochemistry, immunofluorescence, and qPCR plus ELISA assays were performed for Fos and GFPI; substance P and CGRP; and IL-1β, IL-6 and IL-10, respectively	i.v.	106
Sacerdote et al[209], 2013	Systemic aAdministration of human ASCs reverts nociceptive hypersensitivity in an experimental model of neuropathy	Human ASCs were able to completely revert neuropathic pain symptoms in a murine CCI model by: IL-1β decreased and IL-10 increased in the lesioned nerve. Restored normal iNOS expression	Thermal hyperalgesia was tested according to the Hargreaves, while mechanical allodynia was assessed using the Dynamic Plantar Aesthesiometer (von Frey filament)	i.v.	1 × 106, 3 × 106 and 6 × 106
Choi et al[73], 2011	Anti-inflammatory protein TSG-6 secreted by activated MSCs attenuates zymosan-induced mouse peritonitis by decreasing TLR2/NF-κB signaling in resident macrophages	TSG-6 interacts through the CD44 receptor on resident macrophages to decrease zymosan/TLR2-mediated nuclear translocation of the NF-κB	RT-qPCR, ELISA, NF-κB translocation assays and isolation of resident macrophage RNA was performed	i.p.	1.6 × 106
Siniscalco et al[210], 2011	Long-lasting effects of human MSCs systemic administration on pain-like behaviors, cellular, and biomolecular modifications in neuropathic mice	The treatment was able to reduce pain-like behaviors such as mechanical allodynia and thermal hyperalgesia. In addition to reducing IL-1β and IL-17 levels and increasing IL-10 in the spinal cord and reducing labeling for alternatively activated macrophages (CD106)	For behavior analysis, the following tests were applied: von Frey filaments, Rotarod and Hargreaves. Immunofluorescence: CD73; IL-1β; IL-17; CD4; GFAP; IBA-1; western blot: IL-1β, IL-17, IL-10 e CD106	i.v.	2 × 106

Huc-MSCs: Human umbilical cord mesenchymal stem cells; TLR2: Toll-like receptor 2; MyD88: Myeloid differentiation primary response 88; NF-κB: Nuclear factor kappa B; Rsad2: Radical S-adenosyl methionine domain containing 2; p-p65: Phospho-protein 65; CCI: Chronic constriction injury; i.t.: Intrathecal; i.v.: Intravenous; MSCs: Mesenchymal stem cells; ATF-3: Activating transcription factor 3; IBA-1: Ionized calcium-binding adapter molecule 1; DRG: Dorsal root ganglion; MBP: Myelin basic protein; NeuN: Neuron-specific nuclear protein; NF: Neurofilament; TNF: Tumor necrosis factor; ASCs: Adipose tissue derived-mesenchymal stem cells; RT-qPCR: Real time quantitative polymerase chain reaction; hTERT-BMSCs/Tet-on/GAL: Rafted human telomerase reverse transcriptase-immortalized bone marrow mesenchymal stromal cells with inducible galanin expression; GalR: Galanin receptor; SDH: Spinal dorsal horn; IFN-γ: Interferon gamma; CD: Cluster of differentiation; ELISA: Enzyme-linked immunosorbent assay; i.m.: Intramuscular; IL: Interleukin; MMP: Matrix metalloproteinase; PARP: Poly ADP-ribose polymerase; NOX: NADPH oxidase; MAPK: Mitogen-activated protein kinase; p-JNK: Phosphorylated Jun N-terminal kinase, p-ERK: Phospho-extracellular signal-regulated kinase; Nav: Voltage-gated sodium channels; LncRNA: Long-chain noncoding ribonucleic acid; Lncenc1: Long-chain noncoding RNA embryonic stem cells expressed 1; EZH2: Enhancer of zeste homologue 2; MCP-1: Monocyte chemoattractant protein-1; SUZ12: Suppressor of zeste 12; EED: Embryonic ectoderm development; BAI1: Brain-specific angiogenesis inhibitor 1; H3K27me3: Tri-methylation of histone 3 lysine 27; P2X4: P2X purinoceptor 4; i.p.: Intraperitoneal; PCNA: Proliferating cell nuclear antigen; GFAP: Glial fibrillary acidic protein; TRPV1: Transient receptor potential cation channel subfamily vanilloid 1; iNOS: Inducible nitric oxide synthase; p65: Protein 65; BDNF: Brain-derived neurotrophic factor; GAP-43: Growth-associated protein 43; CGRP: Calcitonin gene-related peptide; TSG-6: Tumor necrosis factor-α-stimulated gene 6 protein; BMSC: Bone marrow mesenchymal stem cells; p-Akt: Phospho protein kinase B; LC3B: Light chain-3B; COX-2: Cyclooxygenase 2; nNOS: Neural nitric oxide synthase; hBMSCs: Human bone marrow stem cells; CSF: Cerebrospinal fluid; Th1: Type 1 helper; SCI: Spinal cord injury; p-CREB: Phosphocyclic AMP response element binding protein; CCL: C-C motif chemokine ligand; CXCL: C-X-C motif chemokine ligand; GM-CSF: Granulocyte-macropgahe colony stimulating factor; PDGFR-α: Platelet-derived growth factor receptor α; ROS: Reactive oxygen species; LPS: Lipopolysaccharides; NRG1: Neuregulin-1; GDNF: Glial cell derived neurotrophic factor; NSC: Neural stem cells.