Quite amusing stem cells: Muse cells

Stem cells may be the future of therapeutics for stroke due to their regenerative and immunomodulatory capabilities. Major barriers faced when employing stem cells, however, include faulty migration, low cell survival, and diminished proliferation. Multilineage-differentiating stress ensuring (Muse) cells, a subset of mesenchymal stem cells, overcome these barriers. Muse cells aid in neuroregeneration, have immense regenerative potential, and are pluripotent, non-tumorigenic, and immunomodulatory. In stroke specifically, these cells may restore an anti-inflammatory environment, regenerate damaged neurons, and integrate into the neuronal architecture. In fact, Muse cells may be aptly designed to ameliorate neurovascular unit damage following stroke and observed in other neuroinflammatory disorders.

Stem cells and stroke: Stroke is the 5^th leading cause of death in the United States and the number one cause of long-term disability. Despite these devastating outcomes, the available treatments for stroke are imperfect and do not restore ischemic damage. The use of stem cells as a treatment for stroke has been amply described in animal and clinical studies. These studies demonstrate reduced cell death and improved functionality after stroke (Anthony et al., 2022). While there is no consensus on the mechanism underlying stem cells’ regenerative potential, research proposes these cells reduce mechanisms of secondary cell death such as excitotoxicity, oxidative stress, free radical accumulation, mitochondrial dysfunction, impaired neurogenesis, angiogenesis, vasculogenesis, and inflammation (Anthony et al., 2022). Multilineage-differentiating stress ensuring (Muse) cells, a subset of mesenchymal stem cells, offer the immense therapeutic potential to reduce secondary cell death. Originating in the bone marrow, these cells have the potential to migrate and differentiate into nearly all organ systems. The power of Muse cells has been demonstrated in various organ pathologies including stroke, myocardial infarction, aortic aneurysm, lung pathology, chronic kidney disease, liver fibrosis, osteochondral defects, and skin ulcers (Cao et al., 2020; Park et al., 2020). In addition to the anti-inflammatory and neurodegenerative qualities of Muse cells, these cells offer unique qualities such as immense regenerative potential, pluripotency, non-tumorigenicity, and immunomodulatory effects (Park et al., 2021).

Muse cells: a series of impressive qualities: Among the multiple regenerative mechanisms ascribed to Muse cells, their immune-associated action in controlling inflammation appears directly relevant to stroke therapy (Cao et al., 2020; Kuroda et al., 2022; Li et al., 2022). Because Muse cells escape host immune rejection after intravenous implantation, they remain viable over the prolonged post-transplantation period even in the absence of immunosuppression. Moreover, their extended survival suggests that Muse cells can foster acute and chronic anti-inflammatory effects against progressive stroke-induced deleterious inflammation (Kuroda et al., 2022). Yet another advantage, Muse cells do not need to be HLA matched due to endogenous HLA-G expression (Yamashita et al., 2021). Thus, they offer a reduced risk of exacerbated inflammatory rejection responses, a consequence, which could be detrimental in the setting of the stroke where inflammation is already rampant. In tandem with their dampening action against inflammation, Muse cells can also harness a cell replacement mechanism, whereby Muse cells spontaneously differentiate onto site-specific injured cells, leading to tissue regeneration (Yamashita et al., 2021). In fact, Muse cells require a stressed environment for cell activation (Cao et al., 2020). Considering this property, Muse cells can efficiently home to sites of injury and inflammation, where they are able to integrate and modulate inflammation without inflicting unwanted changes in other bodily systems (Park et al., 2020). Additionally, these exogenously implanted Muse cells can enhance the number of circulating endogenous Muse cells, which further boosts the graft-host regenerative process (Yamashita et al., 2021). In a perinatal hypoxic ischemic encephalopathy model, Muse cells can also suppress the aberrant production of damaging glutamate metabolism and microglial activation towards brain repair and cognitive improvement (Suzuki et al., 2021). Conceivably, Muse cells also extend such regulation of glutamate metabolism and reduction of activated microglial cells in the adult stroke condition. Furthermore, Muse cells are triploblastic, meaning they express ectodermal, mesodermal, and endodermal markers following spontaneous differentiation (Uchida et al., 2017). Altogether, these multi-pronged regenerative properties of Muse cells make them appealing donor stem cells for stroke and relevant neurological disorders.

A breakthrough for a broken neurovascular unit: Recently, several studies have implicated a critical role of the neurovascular unit (NVU) in neuroinflammatory diseases, including stroke (Wang et al., 2021; Yamashita et al., 2021). The NVU is composed of neurons, endothelial cells, pericytes, microglia, astrocytes, and vascular smooth muscle cells. Together, this powerful barrier protects the central nervous system from outside signaling, such as cytokines or immune cells. When inflammatory mediators damage the NVU, the barrier establishing the central nervous system as an immune-privileged organ system is broken down, unleashing a cyclic inflammatory cascade. Peripheral inflammatory cells flood the central nervous system and worsen already prevalent damage. While stem cells have been theorized to restore the NVU’s integrity following neuroinflammatory damage, major barriers are encountered regarding stem cell migration, survival, and proliferation (Wang et al., 2021). Muse cells may overcome these drawbacks (Figure 1). The survival, immunomodulation, migration, and integration capabilities of Muse cells described thus far suggest these stem cells may have revolutionary abilities to restore NVU impermeability. Furthermore, Muse cells are flexible in their differentiation (Uchida et al., 2017). Thus, these cells may be able to restore the wide array of NVU components, as opposed to solely endothelial or neuronal cells.

Figure 1.

Muse cell mechanisms in stroke therapies.

Unique properties of Muse cells, including endogenous amplification, flexible differentiation, immunomodulation, and migration, may uncover a novel therapy for restoring neurovascular unit impermeability following stroke. Created with Microsoft PowerPoint (Adobe).

In order to aid in NVU reconstruction, Muse cells would have to demonstrate differentiation into neurons, endothelial cells, pericytes, microglia, astrocytes, and vascular smooth muscle cells. Transplanting human BM-Muse cells into immunodeficient mice brains 2 weeks after a lacunar infarction showed differentiation of the cells into neural cells which made ample connections, improved functionality, and demonstrated positive safety outcomes long after graft maturation (reviewed in Li et al. 2022). Muse cells also demonstrate smooth muscle actin and cytokeratin 7 expression, suggesting smooth muscle or vascular endothelial differentiation (Uchida et al., 2017). In this same study, however, microglial and astrocytic differentiation was not observed. A similar lack of GFAP⁺ Muse cell differentiation was observed after Muse cell treatment of ischemic stroke in rats. In this study, neuronal and oligodendrocyte differentiation prevailed, as measured by staining for Neu-N, microtubule associated protein 2 and calbindin, and glutathione S -transferase-π (reviewed in Li et al. 2022). A lack of glial cell differentiation observed by Uchida et al. (2017)’s group may be due to microenvironmental signaling, however, future studies aiming to reestablish the NVU should examine ways to prompt specific Muse cell differentiation regardless of the microenvironment. In an in vitro model of spinal cord injury and an in vivo rat model, Muse cells were shown to differentiate into neurons, astrocytes, and oligodendrocytes, signifying that glial differentiation is feasible under appropriate circumstances (reviewed in Li et al., 2022). Other aspects of the NVU can arise from Muse cells, albeit not in stroke models thus far. A mouse model of hindlimb ischemia treated with Muse cells showed increased microvascular density and higher levels of vascular endothelial growth factor (Hori et al., 2022). Similarly in an aortic aneurysm model, Muse cells showed vascular smooth muscle and endothelial cell differentiation, further supporting that Muse cells could restore NVU integrity (Hosoyama et al., 2018). It may be that different Muse cell lineages prompt various differentiations, and mixed populations of Muse cells may be best for prompting NVU repair following stroke. While BM- and dermal-Muse cells shift towards ectodermal and endodermal lineages, adipose-Muse cells appear to prefer mesodermal differentiation (Ogura et al., 2014). In the context of NVU components, BM- or dermal-Muse cells could be used to restore neurons, endothelial cells, and astrocytes, while adipose-Muse cells could be prompted to become pericytes, microglia, and vascular smooth muscle cells. Ultimately, all components of the NVU could be feasibly differentiated from Muse cells. A greater understanding of microenvironmental signaling is necessary for the optimal use of these triploblastic cells.

Musing about the future: While relatively novel, having first been reported in 2010, Muse cells have elicited well-deserved excitement amongst the scientific community (Li et al., 2022). Being widely accessible, low risk, migratory, and immensely adaptable, Muse cells appear to be an ideal cell-based therapy. In stroke specifically, these cells may restore an anti-inflammatory environment, regenerate damaged neurons, and integrate into the neuronal architecture. While these microenvironmental changes are enthusing, Muse cells importantly demonstrate an impressive functional recovery in mice models of intracerebral hemorrhagic and ischemic stroke as well as prompting future clinical studies (Reviewed in Li et al., 2022). Also encouraging are the few clinical trials currently underway for using Muse cells to treat myocardial infarction (JAPIC ID: JapicCTI-195067; JAPIC ID: JapicCTI-183834), spinal cord injury (JAPIC ID: JapicCTI-194841), stroke (JAPIC ID: JapicCTI-184103), epidermolysis bullosa (JAPIC ID: JapicCTI-184563), and neonatal hypoxic ischemic encephalopathy (JRCT ID: jRCT2043190112) (Park et al., 2020). Excitingly, the properties of Muse cells prompt future study regarding these cells’ abilities to restore the damaged NVU seen in stroke and a plethora of other neuroinflammatory disorders. Not only would recovery of the NVU be beneficial for stroke pathology, but it could drastically improve the prognosis for several other neuroinflammatory diseases, such as amyotrophic lateral sclerosis, multiple sclerosis, traumatic brain injury, Parkinson’s disease, and Alzheimer’s disease.

Subsequent studies analyzing specific microenvironmental elements which prompt neuronal, endothelial, pericyte, microglial, astrocytic, and vascular smooth muscle differentiation must be established to optimize the restoration of the NVU using in vitro modified Muse cells implanted into in vivo models. It is apparent from aforementioned studies that Muse cells have the potential to differentiate into the various components of the NVU; however, many of these studies were performed with non-neurological pathologies. With a greater understanding of which elements of the microenvironment encourage different differentiation, Muse cells may be manipulated to restore each component of the NVU. Furthermore, careful vis-à-vis comparisons of Muse cells with other stem cells, such as mesenchymal stem cells, induced pluripotent stem cells, and embryonic stem cells, may further reveal its advantages and limitations, allowing further optimization of cell dose, delivery and timing of transplantation in stroke towards their improved safety and efficacy in the clinic.

Conclusion: The concept of NVU disruption following stroke may revolutionize treatment initiatives for reducing the detrimental inflammation seen following stroke. While other cell-based therapies have speculated NVU restoration, Muse cells, with their immense differentiation possibilities, may be ideal for replenishing the various cell types contributing to the NVU. Further study regarding how to modulate Muse cell differentiation is necessary prior to clinical efforts to rebuild a deteriorated NVU following a stroke.

We acknowledge the contributions of brgfx and pikisuperstar who designed the vectors downloaded from freepik.com and used for the figure.

CVB was funded by the National Institutes of Health (NIH) R01NS090962, NIH R01NS102395, and NIH R21NS109575. Additionally, CVB was funded and received royalties and stock options from Astellas, Asterias, Sanbio, Athersys, KMPHC, and International Stem Cell Corporation and has also received consultant compensation from Chiesi Farmaceutici. CVB also declares patents and patent applications related to stem cell therapy.