BMSCs are multipotent stem cells of mesenchymal origin that can promote structural and functional repair of injured and aging organs, and are the most promising seed cells for regenerative medicine. The effectiveness of BMSCs transplantation has been agreed upon, however, the mechanism of cell therapy is still widely discussed. The latest research progress on the mechanism of BMSCs for the treatment of ischemic stroke is reviewed as follows.
1. Effects of BMSCs on cerebral blood perfusion and blood-brain barrier permeability
Cesario et al. transplanted mouse-derived BMSCs into the striatum of ischemic rats and found a significant improvement in cerebral blood flow and blood-brain barrier permeability, which approached normal after 12 d of treatment. The cerebral blood flow was close to normal after 12 d of treatment. Moreover, there was a positive dose-dependent relationship between the amount of transplanted cells and cerebral blood perfusion; the improvement of BBB in the treated rats was significantly better than that in the control group, and the same dose-dependent relationship existed for the improvement of BBB. This finding is very similar to the dose-dependent relationship between dose-behavioral recovery status in the study of Polgar et al. The effectiveness of the treatment is also supported by the increase in vascular diameter and the number of microvascular branches in brain tissue after the application of BMSCs in stroke by Chen et al. However, neovascularization requires a time period, and the increased cerebral blood flow and improved BBB in the early stage of transplantation cannot be explained by neovascularization. Are these results related to contemporaneously elevated neurotrophic factors in the brain? Whether BMSCs-mediated neurotrophic factors rescued the damaged but still viable host vessels needs further study.
2. Cell differentiation and replacement theory
The rational solution of cell transplantation for stroke treatment is to transplant stem cells around the ischemic and injured area and to differentiate and replace the dead neural cells. Basic research has demonstrated that cells can differentiate into neural lineage cells under in vitro induction. However, there is still controversy regarding the survival status and in vivo differentiation of BMSCs after intracerebral transplantation.
The majority of studies have concluded that BMSCs can differentiate into neural lineage cells in vivo, and Lee’s rat autologous BMSCs were found to be alive and distributed in the ipsilateral striatum, hippocampus, and bilateral neocortex even after 4 weeks of transplantation. About 20% and 15% of the cells expressed markers of neurons and astrocytes, respectively. However, the authors did not find BMSCs expressing neuron-specific proteins in an in vitro study using simple medium. Lee also found a tendency for transplanted BMSCs to differentiate into neurons in the cortex and hippocampus and astrocytes in the corpus callosum, suggesting that the microenvironment of autologous BMSCs transplants is more conducive to the cells’ own differentiation. A few studies have shown that in vivo transplanted BMSCs rarely or not at all differentiate into neural lineage cells in vivo. a study by Castro et al. reported that BMSCs were unable to differentiate into neuron-like cells in brain-injured tissue. in Lu’s study, BMSCs were also not found to differentiate into neurons and glial cells in the host CNS. ohta observed that with or without immunosuppression, cells Ohta observed that transplanted cells could be observed at the site of spinal cord injury 1-2 weeks after transplantation with or without immunosuppression, but none of the BMSCs expressed NF, GFAP, O4, and other neuronal cell-specific proteins; almost all of the BMSCs “disappeared” 3 weeks after transplantation, so the functional recovery and improvement after cell transplantation may not be a function of the cells and the injured tissue. However, the disappearance of transplanted cells at least indicates that they are unlikely or have a low probability of being tumorigenic or teratogenic, which indirectly supports the safety of BMSCs transplantation.
It is questionable whether the transplanted cells can effectively repair tissue damage if they fail to differentiate into neural lineage cells or if only a small percentage of them differentiate. For example, nestin is expressed not only in early CNS tissues, but also in other developing cells such as muscle and cardiac muscle; neuron- specific enolase (NSE) is a neuron-specific protein that can also be expressed by non-neuronal tissues such as small cell and non-small cell lung cancer cells. Relying on the expression of specific proteins alone to determine this differentiation is at least not rigorous.
Cogle reported three cases of female hematologic patients who received hematopoietic stem cell transplants from male donors. Analysis of the brain specimens obtained after their death (22d, 63d and 6y after bone marrow transplantation) revealed that the transplanted cells were able to differentiate into neurons, astrocytes and microglia. However, chromosomal analysis did not reveal polyploid cells, only Y chromosome-positive cells, and the conclusion does not support the cell fusion phenomenon. Therefore, the theory that the transplanted cells may have integrated with the host brain and replaced the injured brain tissue to reconstruct neural circuits is not yet reliable.
Stroke can lead to the conversion of in situ neural stem cells in the subventricular zone into new neurons. However, the vast majority of new neurons will die successively within 1 week after stroke, and the number of viable neurons replacing dead neurons is very small. Therefore, the transplantation of stem cells into the injured area may activate endogenous repair mechanisms in the host brain.
In this way, it seems that the ability of transplanted cells to replace host injured cells is limited and only enhances the possibility of this mechanism rather than simple tissue replacement.
3.Neuropeptides and neurotrophic theory
Neuropeptides and neurotrophic factors are important components of information transmission in neural tissues, and their neural signaling and neurotrophic effects are indispensable for the repair of injured brain tissues. In recent years, their mechanisms have received much attention in the study of BMSCs for the treatment of stroke.
The classical neurotrophic factors include brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), glial cell-derived neurotrophic factor (GDNF), neurotrophic factor 3 (NT-3), neurotrophic factor 4/5 (NT-4/5), hepatocyte growth factor (HGF), platelet-derived BDNF and NT-3 can induce neurite growth and promote the expression of key enzymes for neurotransmitter synthesis; NGF can inhibit apoptosis and promote neuronal regeneration; GDNF can inhibit apoptosis and promote neuronal regeneration. BDNF and NT-3 can induce neurite growth and promote the expression of key enzymes for neurotransmitter synthesis.
HGF is sensitive to environmental changes in the injured brain, and its ability to increase the expression of neurotrophic factors may be the key to recovery of brain function in stroke. The ability of GDNF secretion is regulated by the surrounding microenvironment and self-growth status.
Neurotrophic factors can improve the resistance of host cells, promote the survival, migration and differentiation of endogenous precursor cells, and in addition, BMSCs can play a paracrine role and produce a large number of beneficial cytokines.
Zhang suggested that BDNF could also stimulate the proliferation of oligodendrocytes, which could reduce inflammatory exudation and demyelination of brain tissue. The ELISA study showed that only GDNF, activin A, TGF-β, and TGF-β2 levels were increased after cell transplantation, while other factors were minimally or not measured at all. can still increase the expression of BDNF, while having no effect on NGF.
The secretion of IGF-1 is influenced by changes in extracellular signaling. when Zhang applied BMSCs to treat cerebral ischemia, he found that the secretion of IGF-1 was significantly higher in the treatment group than in the control group.
Transplantation can promote the increase of neurotrophic factor content in the injured brain, however, it cannot be excluded that the host brain’s own cells, especially astrocytes, activate their own neurotrophic factor secretion after cell transplantation, so it is also important to determine the source of the elevated trophic factor, and there are inconsistent findings on various neurotrophic factors, and more in-depth studies on neurotrophic factors are still needed.
BNP, as a neuropeptide, has strong natriuretic and vasodilatory effects like its analogue cardiac natriuretic peptide, which can reduce edema, lower intracranial pressure, and improve cerebral perfusion. pressure. In addition, BNP can increase cerebrospinal fluid reabsorption by decreasing the pressure in the venous plexus within the arachnoid villous granules.Song first discovered that BMSCs can secrete physiologically dose-related BNP in vitro by real-time PCR and radioimmunoassay techniques, and detected that transplanted BMSCs can secrete BNP in the host brain.Therefore, it is possible that when cells are applied via the vascular pathway, they are migration and infiltration into brain tissue before they have already exerted their effective effects, thus promoting functional recovery of the injured brain.
Vascular endothelial growth factor (VEGF): VFGF is a peptide cytokine involved in angiogenesis and development, which has the dual effect of stimulating neovascularization and promoting neural stem cell proliferation, and plays an important pivotal role between the two. Chen co-cultured BMSCs with normal and ischemic brain extracts and found that BMSCs co-cultured with ischemic brain tissue secreted VEGF. 10-d ischemic brain tissue extracts significantly promoted VEGF secretion by BMSCs. The results of this experiment suggest that transplanted BMSCs can promote post-stroke cerebral angiogenesis and facilitate functional recovery.
Chemotactic factors: Inflammatory mediators and cytokines such as IL-8, intracellular adhesion molecule 1 (ICAM-1), and chemotactic factors such as macrophage inflammatory protein 1 (MIP-1) and monocyte chemotactic protein 1 (MCP-1) are released after ischemic injury in brain tissue. BMSCs have the characteristics of inflammatory cells and target the injured tissue like inflammatory cells. BMSCs selectively migrate to the injured tissue by responding to cytokines, and exert their therapeutic effects through neurotrophic factors and neuropeptides at the site of injury.
4. Effects of BMSCs on intercellular synaptic structures and their signaling
Can a cell become a functional cell unit after transplantation? How does the transplanted cell establish a signaling connection with the host cell? There are no findings in the literature that can confirm the existence of a morphological corollary between the transplanted cells and the host cells.
In normal brain tissue, astrocytes play an important role in neuroprotection, not only by providing energy material to neurons, but also by forming a network of intercellular pathways through gap junctions between astrocytes, mainly composed of gap junction protein 43 (Cx43), which regulate potassium ion and neurotransmitter concentrations through gap junction astrocytes. Gao showed that human BMSCs produce soluble factors that improve gap junction-mediated intercellular connections between astrocytes, and that the expression of Cx43 is consistent with this improvement, and that BMSCs improve astrocyte connections by upregulating Cx43 levels.Li transplanted BMSCs in rats 7 d after ischemia and found that the axonal marker GAP-43 was significantly higher in the treated group , and a reduction in ischemic scarring, which the investigators attributed to the responsiveness of glial cells in the ischemic area to the grafts that could promote axonal regeneration.
Synaptic structures are an important way of transmitting and signaling between cells, and synaptophysin is a specific polysaccharide protein on the membrane of synaptic vesicles. Synaptophysin causes neurotransmitter release, information transmission and processing by binding to Ca2 +. Wang found a significant increase in synaptophysin in the BMSCs-treated group compared with the control group by immunohistochemistry, indirectly demonstrating the presence of synaptogenesis.
Applying BMSCs to treat cerebral ischemia in rats, a significant increase in the thickness of axons and myelin sheaths in the ischemic marginal zone, as well as a significant increase in the area of the corpus callosum and the number of white matter bundles in the striatum, and the protrusions of astrocytes were remodeled and oriented to the ischemic zone were found in the treated rats. The authors concluded that BMSCs treatment of stroke could promote oligodendrocyte and astrocyte responsiveness associated with neurological recovery, enhancement and axonal remodeling in rats.
5. Role of BMSCs transgenic vectors
Naokado used herpes simplex virus as a vector to transfer fibroblast growth factor-2 (bFGF-2) into BMSCs and transplanted transgenic cells 24 hours after rat brain infarction, which showed a significant increase in the secretion of FGF-2 in the treated group, a significant reduction in the volume of rat brain infarction, and an improvement in behavioral function. The results showed that the secretion of FGF-2 was significantly increased in the treated group, and the volume of brain infarction was significantly reduced and the improvement of behavioral function was more obvious.
After transplantation of BDNF and reporter gene GFP into BMSCs into the animal model of spinal cord injury, the cells not only could highly express BDNF in the host, but also could promote the growth of host axons at the site of injury, while the grafts could increase the secretion of NGF and NT-3 in the host.
6. Outlook
Biological therapy is an emerging means of disease treatment after drug therapy. Hematopoietic stem cells have been used in clinical treatment of hematological diseases for more than 40 years, while the clinical application of non-hematopoietic stem cells has been fluctuating several times. In recent years, stem cell therapy has been revitalized due to increased awareness and the discovery of multipotential BMSCs. They have shown an exciting prospect of application.
Numerous research data have confirmed the exact role of BMSCs in the treatment of cerebral ischemia, and no significant toxicity or side effects have been reported yet, but the long-term safety of cell therapy still needs further verification. The focus and difficulty of current basic research is the cell therapy mechanism of BMSCs, such as: can the cells survive and proliferate indefinitely for a long time after transplantation? Are the transplanted cells and their progeny a functional cell population in vivo? Is there a structural link between the transplanted cells and the host cells? What is the function of this structural linkage, etc.? What nutritional factors are ultimately at work in the recently promoted neurotrophic theory? Is it the transplanted cell or the host that plays the main role in this nutritional support? Therefore, objectively speaking, the road of BMSCs for the treatment of cerebral ischemia to the clinic is still winding, although the future is bright. We still need more basic and preclinical studies to elucidate the mechanisms of cell therapy.