4.1. Mesenchymal Stem CellsMSCs reach the lesion site through the chemotactic mechanism known as a homing effect. This phenomenon is relevant for therapeutic efficacy not only in the case of the intrathecal and intravenous routes of administration but also in intralesional injection [71]. According to recent studies, many factors are involved in these mechanisms. The SDF-1/CXCR4 (Stromal-cell derived factor-1/CXC chemokine receptor 4) signaling pathway has a significant regulatory role in the homing effect, and its upregulation may improve the migration of MSCs to the injury site [93,94,95]. Inflammation, hypoxia, and ischemia, conditions characterizing the SCI microenvironment, especially in the acute phase, elevate the expression of SDF-1 [96]. Binding SDF-1 (also known as CXCL12) to CXCR4, the surface receptor of MSCs, leads to activation of signaling molecules such as ERK, PI3K, and Akt, attracting MSCs to the lesion site [97]. Other important factors, which stimulate migratory behavior of MSCs, include substance P, aquaporin 1, calcitonin gene-related peptide (CGRP), and a variety of growth factors such as the granulocyte colony-stimulating factor (G-CSF), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), leukemia inhibitory factor (LIF), and hepatocyte growth factor (HGF) [71,72,94,98,99,100,101,102]. Interestingly, substance P impairs the migration of MSCs in response to TGF-β [103]. However, the precise mechanisms determining the homing capacity of MSCs remain unclear. The differentiation potential of MSCs demonstrated by in vitro studies brought great hope for their use in SCI treatment as a cellular replacement for damaged neural cells. In these experiments, MSCs differentiated into neural lineages showed some electrophysiological properties and expressed proteins characteristic of nerve cells [96,104]. However, despite the neuron-like phenotype of differentiated MSCs, these cells were unable to activate action potentials [96]. Moreover, in vivo studies demonstrated a limited differentiation ability of MSCs. Transplanted MSCs did not show specific electrophysiological activity, and their survival number was too small to provide regeneration of damaged structures [71,105,106] Therefore, the differentiation capability of MSCs probably plays a secondary role in functional recovery in patients with SCI. Indeed, data from many studies indicate that benefits provided by SCI therapy rather result from the paracrine and immunomodulatory activity of MSCs than their trans differentiation into the neural cells [107,108].The paracrine effect of MSCs relies on secreting multiple cytokines, growth factors, and other bioactive molecules, which are contained in MSCs’ exosomes and microvesicles [109]. These substances stimulate neuronal and tissue regeneration, reduce glial scarring, enhance angiogenesis, regulate inflammatory processes, and modulate immune responses [109,110]. The secretome of MSCs include the nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), ciliary neurotrophic factor (CNTF), vascular endothelial growth factor (VEGF), insulin-like growth factor-1 (IGF-1), basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF), pigment epithelium-derived factor (PEDF), tissue inhibitor of metalloproteinase-1 (TIMP-1), glia-derived nexin (GDN), interleukin-6 (IL-6), interleukin-8 (IL-8), neurotrophin-1 factor (NT-1), neurotrophin-3 factor (NT-3), galectin-1 (Gal-1), and cystatin C [72,96,111]. Several studies demonstrated that MSCs can exert neuroprotective activities including counteracting nerve degradation and supporting neurogenesis, oligodendrogenesis, remyelination, and axonal growth [72]. The substances secreted by MSCs responsible for those capabilities include BDNF, GDNF, HGF, TIMP-1, NT-1, NT-3, bFGF, and CNTF [71,72]. BDNF, a neurotrophin, is one of the key molecules engaged in neuronal development in CNS [112]. In a spinal cord injury environment, BDNF increases the volume of nerve tissue and decreases the area of the cystic cavity [113]. BDNF achieves a neuroprotective effect probably through activation of the Akt pathways and through its high-affinity tropomyosin-related kinase type B (TrkB.FL) receptor [114]. GDNF has a potentially significant role in the reduction in secondary injury and motor recovery [115,116]. GDNF also demonstrated antioxidative properties by stimulating the enzymes responsible for the neutralization of reactive oxygen species [96]. Moreover, GDNF enhances the survival of grafted MSCs and promotes axonal growth [116,117]. Another growth factor, HGF, through the c-Met receptors, increases axonal growth, promotes angiogenesis, decreases glial scar formation, and inhibits demyelination, blood-brain barrier impairment, and apoptosis [71,118]. Noteworthy, c-Met receptors are overexpressed during the acute phase of spinal cord injury [118]. Furthermore, TIMP-1 secreted by MSCs has demonstrated the capability of oligodendrogenesis stimulation [119]. The glial scar constitutes a barrier that inhibits axonal growth and regeneration after SCI [120]. Transplantation of MSCs in a rat SCI model demonstrated reduced glial scar formation and increased axonal regeneration [121]. In this phenomenon, the paracrine activity of MSCs also plays a significant role. Indeed, transplantation of human UCMSCs overexpressing bFGF to a mouse SCI model improved neural regeneration and glial scarring through the activation of the PI3K-Akt-GSK-3β pathway [122]. Moreover, reduction in the levels of TGF-β through HGF secretion by MSCs also suppressed glial scar formation [123]. Furthermore, MSCs can inhibit the TGF-β/Smads signaling pathway in astrocytes, which is also involved in glial scar formation [124]. The modulation of astrogliosis via the matrix metalloproteinase-2/signal transducer and activator of transcription 3 (MMP-2/STAT3) signaling pathway is the other important mechanism responsible for suppressing glial scarring by MSCs [71,125]. Inhibiting glial scar formation is beneficial for neural repair in subacute and chronic SCI. However, in the acute phase of SCI, the suppression of glial scarring may increase the spread of various inflammatory cells and toxic molecules from the lesion site [126]. A study on the SCI rat model showed that MSCs decreased glial scarring in a chronic stage of SCI and increased the formation of glial scar in the early stage, but this observation should be confirmed in further studies [127].Angiogenesis induction at the lesion site is an especially important capability in supporting spinal cord injury healing [128,129]. This phenomenon is carried out through secretion by MSCs with the molecules such as VEGF, PDGF, bFGF, HGF, IGF-1, GDNF, BDNF, TIMP, IL-6, and IL-8, which are responsible for creating new vasculature from pre-existing vessels [72,96,111]. Angiogenesis stimulation facilitates axonal regeneration, improves ischemia, and hypoxia, and prevents accumulation of inflammatory molecules at the injury site [96,128]. The immune reactions after SCI are thought to be one of the most significant secondary injury factors [130]. At the lesion site, transplanted MSCs exert immunoregulative function through suppression of the inflammatory response, inhibition of T cells, and reprogramming of the microglia phenotype [71]. Studies showed that MSCs reduce levels of inflammatory cytokines including TNFα, IL-1β, IL-2, IL-4, IL-6, and IL-12 at the injury site [131]. In these phenomena, paracrine activity of MSCs also has substantial relevance and includes cytokines and trophic factors such as CNTF, TNF-beta1, neurotrophin 3 factor (NT-3), IL-18 binding protein, and interleukins (IL-13, IL-10, IL-12p70, IL-17E, IL-27) secreted by MSCs [72]. Moreover, MSCs transplanted into the lesion site maintain MHC-I, Sca1, and CD29 expression levels on their surface and additionally boost their expression of MHC-II and CD45, which means that MSCs adopt the immune cell-like phenotype in response to the SCI microenvironment [132]. Probably, interferon-gamma (IFNγ) present in a SCI environment is mainly responsible for the induction of MHC-II expression by MSCs [38,132]. Moreover, exposure to IFNγ and TNF-α triggers anti-inflammatory properties in MSCs through induction of indoleamine 2,3-dioxygenase (IDO1), IL-4, IL-10, CD274, and PD-L1 expression [96]. MSCs may also inhibit the proliferation and activation of T cells through the promotion of p27Kip1 expression and decreasing of the cyclin D2 expression, which results in the arrest of the cell cycle at the G1 phase [133]. This process is mediated by many molecules including TGF-β1, PGE2, HGF, IDO1, and NO [134]. MSCs may also inhibit Th1 and Th17, while at the same time promoting the formation of Treg and Th2 cells [135]. Furthermore, MSCs inhibit neurotoxic A1 astrocytes probably through inhibiting the nuclear translocation of phosphorylated nuclear factor kappa B (NfκB) pathway p65 subunit [136]. The inflammatory reaction is inhibited by MSCs also by increasing the M2 polarization of macrophages and decreasing the M1 macrophage polarization [137,138]. M1 mainly produces pro-inflammatory cytokines including TNF-α, IFN-γ, IL-1β, IL-6, IL-12, and IL-23, whereas M2 releases immunosuppressive molecules such as IL-4, IL-10, IL-13, and TGF-β promoting tissue repair [139,140,141]. IL-10 secreted by MSCs is considered one of the key factors responsible for the transformation of the macrophage phenotype through activation of the JAK/STAT3 signaling in macrophages [123]. 4.2. Neural Stem CellsNSCs are self-renewing, multipotent cells that can give rise to neurons, astrocytes, and oligodendrocytes. They can be observed in states of dormancy and mitotic activation, depending on the parameters of their environment. Neural Stem Cells tend to express low levels of extracellular matrix receptors in their dormant state, but, when they become mitotically active, receptors such as integrin- α6β1, syndecan-1, and Lutheran have a much higher expression [142]. As for outside components, a family of proteins known as BMP (bone morphogenic proteins) plays a role in the proliferation and differentiation of NCS. LRP2, a receptor for BMP4 for example, is theorized to be crucial in their proliferation, as research shows that in mice without this receptor, neural progenitors cease to proliferate. When BMP secretion inhibitors’ overexpression was tested, specifically the Noggin, NSC enhanced their proliferation of progenitors and shifted SVZ lineage progression from mature astrocytes to transit amplifying cells and oligodendrocyte precursors. Noggin also promoted the differentiation of both oligodendrocytes and neurons, which was inhibited by BMP4 [143]. Other molecules that have been shown to upregulate NSCs’ proliferation in the subependymal zones such as Ansomin-1 binding to FGFR1, as well as induce their migration [144]. A crucial part of NSCs’ research is finding novel molecules that orient them in their environment and allow them to connect into more complex chains, such is the case with Epherin-A and B signaling pathways. Research finds that especially EphA4 suppression causes the population of neuroblasts and astrocytes to become loosely aligned and chaotic, often migrating into neighboring structures [145]. NSCs and progenitor cells descended from them express Wnt receptor FZD1 playing a similar role, as the knockout of FZD1 was prioved to cause astroglial differentiation with increased migration of adult-born neurons but also a shutdown of new neuron differentiation [146]. Neurotransmitters abundant in the regions of the NSC residency also play a major role in shaping stem cells. The best-described example of regulating neurogenesis, particularly in the SEZ region is gamma-aminobutyric acid. GABAergic neurons were proven to control NSC populations by maintaining their status of quiescence in the hippocampus [147]. Neurogenesis stemming from choline acetylase was explored in rodent SVZs where a stroke was experimentally induced; a population of ChAT-positive neurons was found to have participated in the proliferation of NSCs and their homing to zones damaged by the stroke, resulting in better recovery [148].

A neurotransmitter that induces NSCs’ activity is norepinephrine via the β3 adrenergic receptors.

Ghrelin administration was proven to induce cellular proliferation of hippocampal NSC via such pathways as ERK1 and 2, as well as PI3K, and Janus kinase 2 [149]. Melatonin was proven to facilitate fetal bovine serum-induced neural differentiation of NSCs without affecting the astroglial differentiation [150]. 4.3. Hematopoietic Stem CellsHSCs as multipotent stem cells can differentiate into all types of blood cells and lymphoid lineages [151]. Transplanted into the SCI microenvironment, HSCs exert their therapeutic activity through differentiation and releasing numerous cytokines and neurotrophic factors. The differentiation capacity of HSCs at the SCI microenvironment includes transforming into astrocytes, neuroprotective glia, and oligodendrocytes [152]. In a recent in vitro study, human umbilical cord blood-derived CD133+ HSCs after exposure to the mixture of sonic hedgehog, BDNF, B27, and retinoic acid demonstrated increased expression of Isl-1, AchE, SMI-32, and Nestin, which are markers specific for motor neurons [153]. That suggests the potential of HSCs for differentiation into motor neuron-like cells.Preclinical studies showed that a plethora of growth factors and cytokines could be released by HSCs including VEGF, thrombopoietin, neurotrophin-3 (NT-3), mitogen-activated protein kinase-1 (MEK-1), angiopoietin-1, IL-11, and colony-stimulating factor I (CSF-I) [89,154,155]. An animal study by Xiong et al. demonstrated that the administered in the chronic phase of SCI HSCs increased expression levels of NT-3 and MEK-1 suggesting that HSCs exert their neuroregenerative properties trough release mainly of these two factors [154]. The signaling pathways that involve MEK-1 and NT-3 play important roles in neuroprotection and are significantly downregulated after SCI, which indicates that HSCs restore proper MEK-1 and NT-3 levels [156,157]. Moreover, inhibition of astrogliosis, enhancement of 5-HT-positive fibers, and oligogenesis promotion after HSCs’ administration were also observed [154]. Suppressing astrogliosis inhibits the formation of a glial scar at the lesion site. As above mentioned, the benefits coming from inhibition or promotion of glial scarring may vary regarding the phase of SCI. Therefore, inhibition of astrogliosis at the chronic stage of SCI unleashes regenerating axons from suppressive effects of inhibitory molecules and fibrotic scarring [154], whereas, during the acute phase of SCI, promotion of astrogliosis may be beneficial due to the protective role of the glial scar against the inflammatory environment of acute SCI [154]. On the other hand, stimulation of oligogliosis regenerates demyelinated axons, and enhancement of 5-HT fibers extends their lateral branches, which enhances neural improvement [154].

An exact molecular mechanism of action through which HSCs exert their neuroregenerative properties in the treatment of SCI remains not thoroughly investigated; thus, further studies are needed to unveil other molecular interactions involved in their activity.