Conditioned medium derived from mesenchymal stem cells and spinal cord injury: A review of the current therapeutic capacities

Abstract

Spinal cord injury (SCI) is a debilitating condition of the nervous system that imposes considerable challenges for subjects, such as bladder and bowel incontinence and infections. The standard therapeutic strategy is methylprednisolone utilization accompanied by surgical decompression. However, achieving an effective therapy with the minimum side effects for SCI is still a puzzle. Nowadays, mesenchymal stem cell (MSC) therapy has received much consideration in scientific communities in light of its pharmacological and therapeutic properties, for instance, anti-inflammatory, regenerative, analgesic, and immunomodulatory influences. Despite the mentioned advantages for MSCs, their tumorigenic potential is a limiting agent for its wide therapeutic application. Recent documents show that the use of conditioned medium (CM) derived from MSCs can largely solve these problems. CM encompasses neuroprotective growth factors and cytokines, such as stem cell factor (SCF), vascular endothelial growth factor (VEGF), and glial cell line-derived neurotrophic factor (GDNF). The persuasive evidence from experimental studies revealed that CM originating from MSCs can have a considerable role in the amelioration of SCI. Hence, in the current papers, we will review and summarize evidence indicating the anti-SCI mechanisms of MSC-derived CM by relying the current experimental data.

Introduction

Spinal cord injury (SCI) is described as a debilitating disease related to the central nervous system that gives rise to motor and cognitive impairments and imposes considerable pressure on the patients and their families (Li et al., 2024, Pourkhodadad et al., 2019). Based on reports, the global incidence rate of SCI is between 10.4 and 83 cases annually per one million population (Grijalva-Otero and Doncel-Pérez, 2024). SCI is grouped into two stages, including primary and secondary injuries (Ju and Dong, 2024). SCI patients can suffer from bladder and bowel incontinence and some health problems, like infections, bedsores, cramps, and muscle atrophy (Gong et al., 2023). SCI has restricted clinical diagnosis and complex treatment (Zhang et al., 2023). Magnetic resonance imaging (MRI), X-ray computed tomography (CT), and neurophysiological and lumbar puncture cerebrospinal fluid assessment are aiding tools for SCI diagnosis (Nasser et al., 2016). The routine clinical approach for SCI treatment is high-dose administration of methylprednisolone along with surgical decompression. Unfortunately, using these therapeutic methods is accompanied by some challenges. The barriers to methylprednisolone therapy at high doses include low accumulation efficiency and bioavailability and damage to other body systems. Also, surgical techniques, despite improving spinal stability and decreasing compression for nerve reconstruction, have not provided a promising landscape for nerve repair yet (Gong et al., 2023). Ergo, there is a substantial need to find a novel and efficient therapeutic strategy against SCI. Recently, stem cell therapy has created a new chance to treat different pathological problems, such as neurological diseases (Chang et al., 2024), cardiovascular disorders (Tominaga et al., 2024), malignancies (Tavakoli et al., 2022), metabolic impairments (Chen et al., 2024), autoimmune ailments (Ruiz et al., 2023), etc. Stem cells are known for their potential for self-renewal, proliferation, and differentiation (Bahmanpour et al., 2019). In conjunction with the differentiation capability of stem cells, four stem cell types have been introduced, comprising unipotent, multipotent, pluripotent, and totipotent stem cells. Among these, multipotent stem cells, such as mesenchymal stem cells (MSCs), possess a more restricted potential in comparison with pluripotent and totipotent stem cells and are able to differentiate into various cell types of a specific lineage or tissue type (ArefNezhad et al., 2023, Hashemitabar et al., 2021). Several sources have been offered for obtaining MSCs, like bone marrow, adipose tissue, umbilical cord, fetal lung, dental tissue, and brain, and each one possesses its challenges and opportunities (Arefnezhad et al., 2024, Arefnezhad et al., 2024). Nowadays, MSCs have acquired much interest in medicine thanks to their anti-inflammatory (Kalantari et al., 2024), regenerative (Han et al., 2019), analgesic (Almahasneh et al., 2023), and immunomodulatory (Wang et al., 2018) influences. However, MSCs are not able to be engrafted into the selected tissue, and their tumorigenic capacity is still a big challenge (Mousavi et al., 2023). One of the suggested approaches to conquer these problems is harnessing conditioned medium (CM) derived from MSCs (Rezaei-Tazangi et al., 2020). CM comprises several growth factors and cytokines, some of which are involved in neuroprotection, such as stem cell factor (SCF), vascular endothelial growth factor (VEGF), and glial cell line-derived neurotrophic factor (GDNF) (Rezaei-Tazangi et al., 2021). New reports obtained from experimental studies have shown that MSCs-originated CM has a striking role in ameliorating SCI (Arefnezhad et al., 2024, Arefnezhad et al., 2024). Hence, this review aimed to debate the anti-SCI effects of MSC-derived CM with a mechanistic focus.

Spinal cord injury and pathogenesis

As mentioned above, SCI is categorized into primary and secondary stages (Fig. 1). The primary stage pertains to edema, local hemorrhage, and the destruction of nerves and the glial membrane. The secondary one is known as a pathologic occurrence with continuous damage following primary injury, such as hypoxia, ischemia, oxidative stress, inflammation, and cell death processes (i.e., apoptosis, ferroptosis, and necroptosis), which in turn exaggerate tissue dysfunction (Bakar et al., 2013, Xia et al., 2022; Shen and Cai, 2023; Anjum et al., 2020). Following SCI, hypoxia, ischemia, reactive oxygen species (ROS) formation, and excitotoxicity lead to the production of cellular debris, whose toxicity is possible (Wanner et al., 2013). Several processes are triggered to eliminate this cellular debris in order to exert protective roles in normal cells against damage. The primary responders to cellular debris are astrocytes and microglia that serve as phagocytic elements and induce different cytokines, growth factors, and blood-borne inflammatory factors to eradicate toxic agents (Anjum et al., 2020). Upon SCI, naïve astrocytes turn into reactive astrocytes that form inhibitory molecules, like laminin and proteoglycans, in the extracellular space. Thereafter, reactive astrocytes are attracted into the site encompassing the lesion center and result in the formation of the glial scar. The glial scar restricts regenerative processes and serves as a biochemical and physical obstacle to axonal regeneration (Saxena et al., 2011, Silver and Miller, 2004).

Fig. 1.

Some pathogenic mechanisms related to spinal cord injury during the primary and secondary stages.

Inflammatory reactions are one of the important mechanisms of the secondary stage of SCI. Under these conditions, astrocytes and microglia/macrophages are activated, and immune mediators (neutrophils and macrophages) are recruited from the blood flow to the injured region (Liu et al., 2021). Astrocytes and microglia/macrophages have a central role in the secretion of some pro-inflammatory cytokines, for example, interleukin (IL)-1, tumor necrosis factor-α (TNF-α), and IL-6 (Lukacova et al., 2021). Also, microglia/macrophages and neutrophils by ROS formation stimulate the secretion of pro-inflammatory mediators (cytokines), comprising TNFα, interferon (IFN)-γ, IL-2 and proteases and potentiates the apoptosis of oligodendrocytes (Domingues et al., 2016). Oligodendrocyte apoptosis confers axon demyelination and gives rise to a defect in axonal stability and functionality since each oligodendrocyte myelinates multiple axons (Domingues et al., 2016, Almad et al., 2011). Also, oligodendrocyte demyelination instigates expression of Fas-receptor releasing caspases-3 and caspase-8, key mediators of apoptosis (Liu et al., 2018, Sobrido-Cameán and Barreiro-Iglesias, 2018). In addition to apoptosis, recent evidence showed that other types of cell death, especially ferroptosis, have a role in SCI pathogenesis (Dong et al., 2023). Ferroptosis is defined by iron-dependent elevation of lipid peroxidation (Li et al., 2020). Regarding this matter, a bioinformatic study showed the upregulation of four hub genes pertaining to ferroptosis induction, including CYBB, HIF-1α, HMOX1, and TLR4, following SCI (Dong et al., 2023). Traumatic damage to neuronal cell membrane causes integrity disruption, resulting in elevation of permeability for some ions. Importantly, the transmission of calcium ions into the intracellular space gives rise to triggering apoptotic signals, disturbing mitochondrial activity, and triggering proteases (Quadri et al., 2020). Increased intracellular Ca²⁺ leads to the secretion of glutamate, a well-known excitatory neurotransmitter in the central nervous system (Shen et al., 2022). After traumatic SCI, glutamate at the extracellular levels increases to neuro-toxic levels at the vicinity of the injury area because of disrupted uptake and excess release (Slater et al., 2022). Traumatic injury to the spinal cord also stimulates considerable hemorrhage all over the gray matter, causing hemorrhagic necrosis and consequently central myelomalacia (Spitzbarth et al., 2020).

Conditioned medium from bone marrow mesenchymal stem cells

Among different types of MSCs, the majority of scientific attempts have been dedicated to the potential of CM derived from bone marrow mesenchymal stem cells (BMMSCs) in SCI improvement. According to the recent study of Wang et al. (2023), administration of CM derived from BMMSCs alleviates SCI in rats by decreasing the expression of galectin-3, as a subgroup of the galectin family, and NLR family pyrin domain containing 3 (NLRP3), ameliorating inflammatory reactions, activating macrophage/microglia M2 polarization, and inhibiting macrophage/microglia M1 polarization (Wang et al., 2023). The attenuation of Gal-3, a key biomarker for SCI, leads to a reduction of the secretion of inflammation-elevating cytokines and blockage of NLRP3 inflammasome actuation in colonic macrophages (Ren et al., 2019). Macrophages/microglia are the main mediators of inflammatory occurrences after SCI (Kong and Gao, 2017). Macrophages/microglia (M1) are induced by Th1 cytokines (e.g., IFN-γ and TNF-α) and form pro-inflammatory cytokines considerably, including TNF-α, IL-1β, IL-6, and IL-23, which prime axonal degeneration and neurotoxicity (Fu et al., 2022). On the contrary, macrophages/microglia (M2) are stimulated by Th2 cytokines, like IL-13 and IL-4. These factors release IL-4, transforming growth factor-β (TGF-β), and IL-10 in order to inhibit inflammation, instigate angiogenesis, and potentiate tissue regeneration (Fu et al., 2022). Tsai and co-workers explored the advantages of CM originating from BMMSCs on neuron-glial cultures of the spinal cord and animal models of the disease (Tsai et al., 2018). Application of the CM in these cultures led to remarked elevation of oligodendroglial numbers and neuronal connection, exhibiting the promoting effects of CM obtained from BMMSCs on cell survival. Moreover, the CM could ameliorate cell damage conferred by oxygen-glucose deprivation. In vivo results addressed that the intravenous utilization of BMMSCs-derived CM considerably enhanced functional recovery from this injury and axon density in the lesion region in the treatment group in comparison with SCI rats. Interestingly, a few days post SCI induction, BMMSCs-derived CM overexpressed autophagy-associated proteins (p60 and LC3 II) and the protein expression of HSP70 and Olig-2 in SCI rats (Tsai et al., 2018). A failure in autophagy flux is related to neuronal cell death in neurodegenerative disorders; however, its exact role and mechanism in these diseases, like SCI (Liu et al., 2015). HSP70 belongs to the heat shock proteins family and exerts as a protective chaperone protein against neuroinflammatory conditions, particularly SCI (Xu et al., 2021). Oligo-2 is defined as a transcription factor serving as a key function in oligodendrocyte differentiation (Drake et al., 2023). In a different approach, Cizkova and colleagues scrutinized the outcomes of intrathecal usage of CM obtained from rat bone marrow stromal cells in adult rats with SCI (Cizkova et al., 2018). Initially, the biological features of prepared CM for axon outgrowth and forming neurosphere-like structures were observed following in vitro tests on primary cultures of dorsal root ganglion. Next, SCI was established in adult male Wistar rats by the modified balloon compression method. Subsequently, SCI rats took BMMSCs-derived CM (30 µL four times) at days 1, 5, 9, and 13 post-SCI induction. The analyzed data showed that this CM-based therapy decreased inflammation, increased GAP-43 expression, elevated remnant spinal cord tissue, and potentiated motor function recovery compared with the SCI group receiving a vehicle (30 µL DMEM). In addition, reduced levels of pro-inflammatory cytokines, such as IL-2, TNF-α, and IL-6, were other outcomes of this study to prove the anti-inflammatory potential of this mesenchymal treatment (Cizkova et al., 2018). Another experimental investigation pointed out that CM extracted from animal bone marrow protects cerebellar granule neurons from apoptosis, triggers macrophages, and exerts a pro-angiogenic role in vitro (Cantinieaux et al., 2013). Also, the results of motor skill assessments using the Basso, Beattie, and Bresnahan (BBB) open-field test in rats with contusion SCI reflected motor recovery improvement. This therapeutic aspect of CM was supported by histopathological evaluations indicating decreased lesion extension in damaged spinal cord in vivo (Cantinieaux et al., 2013).

Conditioned medium from other sources of mesenchymal stem cells

The anti-SCI potential of CM derived from other mesenchymal sources, including Wharton’s jelly, adipose tissue, and dental pulp, has been scrutinized (Table 1). It has been shown that intrathecal transplantation of human Wharton’s jelly-derived mesenchymal stromal cells (WJ-MSCs) and their CM (for 21 days) leads to the promotion of sensory (plantar reflex) and motor (BBB and BW test) function in rats underwent balloon compression lesion. These two mesenchymal sources could also increase the amount of white matter and spared gray and promote the expression of axonal growth-related genes (FGF-2 and GAP-43). However, only CM derived from WJ-MSCs was able to diminish reactive astrocyte number and enhance axonal sprouting in the lesion regions (Chudickova et al., 2019). Reactive astrocytes following SCI constitute a glial scar that forms axonal growth suppressors and limits axonal repair (Okada et al., 2018). Another preclinical investigation inspected the neuroprotective potential of CM extracted from rat adipose tissue-derived mesenchymal stem cells (ADMSCs) on the viability of astrocytes and neurons (originated form at brain cortex) subjected to CM prepared from SCI (caudal, rostral, and lesion portion). Finally, the findings outlined that glial cells (astrocytes) are not influenced by this cell-based method; however, the population of cortex neurons was regulated following treatment with ADMSCs, possibly by secreting trophic and neuroprotective factors (Szekiova et al., 2018). Asadi-Golshan et al. explored the effects of intrathecal injection of CM, derived from dental pulp stem cells, encapsulated with collagen hydrogel on SCI rats using evaluating histological parameters by stereological methods. In the end, the results revealed that the administration of this biomaterial increased the overall number of oligodendrocytes and neurons and the overall volume of conserved gray matter and white matter. In contrast, the length and volume of the lesion region were mitigated. By relying on these outcomes, the authors concluded that this CM has a preventive role in tissue loss (gray matter and white matter) and a protective role for neurons and glial cells (oligodendrocytes) (Asadi-Golshan et al., 2021). Analogously, Liu et al. (2024) have examined the intraperitoneal injection of CM prepared from human dental pulp stem cells on animals with SCI induced by laminectomy and downfalling a weight on the subjected spinal cord. Finally, the CM could effectually improve motor and sensory function (Fig. 2) in SCI rats. In addition, authors found that CM obtained from the mentioned mesenchymal source suppressed the overexpressed GSDMD, IL-1β, caspase-1, NLRP3, microglial pyroptosis landmarks, in BV2 cells (a kind of microglial cells acquired form C57/BL6 murine) following LPS treatment. This research team has declared that the used CM by the mediation of microglial pyroptosis attenuation is able to exert a reparative role after SCI (Liu et al., 2024). The study of Semita et al. (2023) showed that in rats with spinal cord injury, treatment with human neural stem cells-secretome improved locomotor recovery and increased neurogenesis (nestin, BDNF, and GDNF), neuroangiogenesis (VEGF), anti-apoptotic (Bcl-2), anti-inflammatory (IL-10 and TGF-β), but decreased pro-inflammatory (NF-κB, MMP9, TNF-α), F2-Isoprostanes, and spinal cord lesion size (Semita et al., 2023).

Table 1.

The improving effects of conditioned medium derived from mesenchymal stem cells on spinal cord injury.

Mesenchymal source	Molecular target (s)	Effect/mechanism (s)	Model	Refs.
Animal bone marrow	VEGF	Protecting neurons from apoptosis, triggering macrophages, inducing angiogenesis, and promoting motor recovery	In vivo and in vitro	(Cantinieaux et al., 2013)
Animal bone marrow	HSP70, Olig-2, LC3 II, and p62	Enhancing functional recovery, elevating axon density in the lesion region, activating autophagy, and potentiating cell survival	In vivo and in vitro	(Tsai et al., 2018)
Animal bone marrow	-	Improving locomotor behavior and elevating axon density	In vivo	(Kanekiyo et al., 2018)
Human bone marrow	NLRP3 and Gal-3	Ameliorating inflammatory reaction, repressing M1 microglia/macrophage polarization, and activating M2 microglia/macrophage polarization	In vivo and in vitro	(Wang et al., 2023)
Animal bone marrow	GAP-43	Improving motor function recovery, decreasing inflammation, and elevating spared spinal cord tissue	In vivo and in vitro	(Cizkova et al., 2018)
Animal bone marrow	-	Improving locomotor recovery, enhancing bladder function, and partially ameliorating joint movement	In vivo	(Vikartovska et al., 2020)
Human Wharton Jelly	IRF5, MRC1, NFκB, FGF-2, and GAP-43	Increasing white matter and spared gray amount, promoting axonal growth, improving axonal sprouting, and decreasing the number of reactive astrocytes	In vivo and in vitro	(Chudickova et al., 2019)
Human dental pulp	IL-1β, caspase-1, GSDMD, and NLRP3	Enhancing motor and sensory function, attenuating microglial pyroptosis, potentiating myeline and axonal regeneration, and repressing glial scar formation	In vivo and in vitro	(Liu et al., 2024)
Dental pulp	-	Increasing the total volume of preserved gray matter and white matter and the total number of oligodendrocytes and neurons and decreasing the length and volume of lesion site	In vivo	(Asadi-Golshan et al., 2021)
Human umbilical cord blood-derived mesenchymal stem cell	-	Attenuating inflammation and decreasing apoptotic cell numbers, astrogliosis, and oxidative stress	In vivo	(Yeng et al., 2016)
Umbilical cord matrix cells and bone marrow mesenchymal stromal cells	-	Decreasing acute vascular pathology, particularly functional vascularity and lesion volume	In vivo	(Vawda et al., 2020)
Human adipose tissue	-	Improving neurite growth and motor recovery	In vivo and in vitro	(Pinho et al., 2022)

Fig. 2.

The beneficial effects of mesenchymal conditioned medium derived from bone marrow, umbilical cord, adipose tissue, and dental pulp on spinal cord injury.

Acellular secretome therapies hold great translational potential and have several advantages over conventional cell therapies including the mitigation of the risk of immune rejection, decreased risk of tumorigenesis and ability to cryopreserve treatments without need to consider the issues of maintaining cell viability. The MSC secretome contains a number of molecules which may have immunomodulatory effects on immune cells such as activated microglia and infiltrated macrophages after SCI. Stem cell secretome therapies polarised macrophages towards a more regulatory M2-like phenotype. Angiogenesis may also contribute to observed improvements in recovery. Mesenchymal stem cells and neural stem cells secrete VEGF which is a potent promoter of angiogenesis. One included study showed that administration of NSC-derived exosomes transfected VEGF-A significantly enhanced angiogenesis and locomotor recovery, compared with exosomes in which VEGF-A was knocked down. Briefly, stem cell secretome may have great potential as a therapy for spinal cord injury and neuroprotection is the key mechanism of action (Cunningham et al., 2020).

The study of Wan et al. (2021) confirmed that mesenchymal stem cell secretome is rich in extracellular vesicles and soluble molecules such as growth factors and neurotrophic factors, and it plays prominent place in reducing cell apoptosis, regulating immune response, inhibiting scar formation, promoting nerve regeneration and angiogenesis. Also, mesenchymal stem cell secretome can promote nerve regeneration and function recovery after spinal cord injury, and it avoids the disadvantages of cell transplantation (Wan et al., 2021).

Some studies have provided evidence that intravenous administration of neural progenitor/stem cell-derived EVs significantly reduced neuronal apoptosis, microglia activation and neuroinflammation, thereby promoting functional recovery after SCI in rodents. Another report suggested that intrathecal administration of the subventricular zone-derived EVs resulted in remarkable motor recovery by suppressing the formation of the NLRP3 inflammasome complex. Furthermore, EVs were able to modulate the activity of target cells by interacting directly with the cell and to induce angiogenesis, thus decreasing the vascular damage (P Pajer et al., 2021).

The study of Assunção-Silva et al. (2024) demonstrated positive impacts of ASC secretome on their functional recovery which were correlated with histopathological markers of regeneration. Furthermore, in our mice study, secretome induced white matter preservation together with modulation of the local and peripheral inflammatory response. Altogether, these results demonstrate the neuroregenerative and potential for inflammatory modulation of ASC secretome suggesting it as a good candidate for cell-free therapeutic strategies for SCI (Assunção-Silva et al., 2024).

Conclusion

SCI is known by remarked damage, high incidence, hindered clinical diagnosis, and challenging treatment. Among these, MSCs have provided a promising prospect for treating different diseases like neurological problems. However, some barriers (e.g., tumorigenic potential and low ability to effectively engraftment to targeted tissue) have led to the encouragement of researchers to find an optimum method for better use of MSCs in the medical arena. In this conjunction, many studies have implicated the superiority of CM originating from MSCs to intact MSCs. The anti-SCI capacity of CM prepared from MSCs extracted from various sources, including bone marrow, adipose tissue, and Wharton’s jelly, has been examined. The results of preclinical investigations showed that reparative effects of the CM are exerted by several cellular and molecular mechanisms, for example, decreasing inflammatory factors (e.g., IL-2, TNF-α, and IL-6), stimulating macrophage/microglia M2 polarization, repressing macrophage/microglia M1 polarization, promoting motor and sensory function, attenuating microglial pyroptosis, enhancing the expression of axonal growth-associated genes (FGF-2 and GAP-43), and elevating the overall number of oligodendrocytes. Thus, this MSC-based strategy may herald bright futures for patents with SCI in clinical trial studies.

Ethics approval

Ethical issues (including plagiarism, data fabrication, double publication) have been completely observed by the authors.

Informed consent

Not applicable.

Funding

This work was financially supported by Baqiyatallah University of Medical Sciences.

CRediT authorship contribution statement

Kaka Gholam Reza: Writing – review & editing, Project administration. Modarresi Farrokh: Writing – original draft.

Conflicts of Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.