Stem cell therapy as a promising approach for ischemic stroke treatment

Ischemia as the most common type of stroke is the main cause of death and disability in the world. However, there are few therapeutic approaches to treat ischemic stroke. The common approach to the treatment of ischemia includes surgery-cum-chemical drugs. Surgery and chemical drugs are used to remove blood clots to prevent the deterioration of the nervous system. Given the surgical hazards and the challenges associated with chemical drugs, these cannot be considered safe approaches to the treatment of brain ischemia. Besides surgery-cum-chemical drugs, different types of stem cells including mesenchymal stem cells and neurological stem cells have been considered to treat ischemic stroke. Therapeutic approaches utilizing stem cells to treat strokes are promising because of their neuroprotective and regenerative benefits. However, the mechanisms by which the transplanted stem cells perform their precisely actions are unknown. The purpose of this study is to critically review stem cell-based therapeutic approaches for ischemia along with related challenges.


Introduction
Stroke as a multi-factorial disorder is the second most common cause of mortality and the major cause of long-term disability.One-third of stroke victims suffer from functional and neurological disorders following the incident (Boehme et al., 2017).Stroke is the result of obstruction or rupture of blood vessels, and almost more than 80% of all stroke cases are ischemic, which are usually caused by blockage of the middle cerebral artery (Kuriakose and Xiao, 2020;Tajalli-Nezhad et al., 2019;Vahidinia et al., 2021).Researchers have demonstrated that risk factors such as age, gender, hypertension, smoking, unhealthy diet, physical inactivity, diabetes, excessive alcohol and drug use, heart diseases as well as genetic factors are all associated with the risk of ischemic stroke (Boehme et al., 2017;Nejati et al., 2018).The more common approaches for the treatment of brain ischemia include surgery-cum-chemical drugs (Behdarvandy et al., 2020;Zhu et al., 2018).
The main purpose of treating brain ischemia with chemical and surgical drugs is to remove blood clots to prevent the deterioration of the nervous system (Nozohouri et al., 2020;Tameh et al., 2018).Given the surgical hazards, it is not generally regarded as a safe option in the treatment of brain ischemia.In addition to surgery, the use of FAD-approved chemical drugs can be effective for the treatment of ischemic stroke (Nozohouri et al., 2020;Vahidinia et al., 2020).Medicinal options for the treatment of ischemic stroke include tissue plasminogen activator (tPA), aspirin, warfarin, clopidogrel and statins (Tremonti and Thieben, 2021).Despite the efficiency of chemical drugs in the treatment of ischemic stroke, some restrictions are considered for their application.For instance, they should be initiated in patients with clinical standards shortly after the development of ischemic stroke (Brzica et al., 2017;Khassafi et al., 2022;Vahidinia et al., 2022).There are also challenges such as the potential of drug-drug interactions, difficulty getting the drug through blood-brain barrier, gastric bleeding, etc. in the course of treatment of ischemic stroke with chemical drugs (Aleksic et al., 2019;Hochain et al., 2000;Pardridge, 2001).While antiplatelet treatment with aspirin diminishes the risk of early stroke recurrence within 48 h following its onset, it does not the ability to treat the previously formed stroke.Newer antiplatelet agents alone or combined with aspirin, demonstrated promising results for prevention of premature recurrence of stoke, though clinical tests are still underway (Bansal et al., 2013).
Numerous studies have been dedicated to evaluate the therapeutic potential of various types of stem cells including mesenchymal stem cells, inducible pluripotent stem cells, embryonic stem cells, and neural stem cells as a therapeutic option for the treatment of ischemic stroke (Marei et al., 2018).Stem cell-based therapies for stroke are promising solutions due to their neuroprotective and regenerative potential (Mahla, 2016).The aim of stem cell-based therapies for the treatment of stroke is neurological regeneration, diminished loss of neurons and neuronal protection in the acute phase of stroke to limit the spread of injury (F.Wang et al., 2018).The results of stroke treatment with stem cells are interesting, but many have provided contradictory results.Besides, the underlying mechanisms by that transplanted stem/precursor cells perform their actions are largely unknown (Misra et al., 2012).The purpose of this study is to describe the stem cell-based therapeutic approaches for ischemia along with the challenges facing them.

Common therapies for ischemic stroke: surgical procedures, pharmacological treatments, and their associated challenges and complications
In recent years, physicians have resorted to chemical drugs and surgery to treat ischemic stroke.The main goal of ischemic stroke treatment with the above-mentioned methods is to prevent blood clots from advancement in order to prevent the deterioration of the nervous system (Barahimi et al., 2021;Nozohouri et al., 2020).The aim of endovascular thrombectomy is to be the preferred therapeutic strategy for the treatment of acute ischemic stroke induced by the occlusion of large cerebral arteries, mainly in cases with high clot burden or in the presence of contraindications of intravenous thrombolysis (Berkhemer et al., 2015).Prompt cannulation of occluded vessels is critical for obtaining a good clinical outcome (Khatri et al., 2009).Carotid arteries are most often accessible through femoral puncture and catheterization of aortic arch within minutes.However, there are some conditions in which this approach becomes impossible or time-consuming with negative impacts on clinical outcomes (Levy et al., 2002).
Considering the risks of surgery, it cannot be considered as a safe approach for the treatment of ischemic stroke.Besides surgery, application of chemical drugs approved by FAD can be effective in the treatment of ischemic stroke (Nozohouri et al., 2020).Regarding enhancement of rehabilitation following stroke, numerous drugs have demonstrated promising results in small clinical trials, however, none of them have been assessed in large phase III clinical trials or approved by US Clinical Supervision.Some agents such as tenecteplase, edaravone, and minocycline may be approved for universal use in the future (Kikuchi et al., 2014).
Drug treatment for stroke can be divided into categories of specific stroke treatment and stroke prevention (Gilman, 2006).Available drug options for the treatment of ischemic stroke include tissue plasminogen activator and antiplatelet agents.The major role of medical treatment of stroke is to control the patient's blood pressure and intracranial pressure (Hinkle and Guanci, 2007).Currently, some drugs are thought to act on neurotransmitters or neuromodulators in the central nervous system, while others appear to act at peripheral neuromuscular sites.In addition, each drug agent has the potential for having side effects that should be considered before prescribing (Gallichio, 2004).The development of agents with the potential to diminish brain damage subsequent to the development of ischemic stroke requires novel and diverse approaches based on a better understanding of underlying pathophysiologic mechanisms of ischemic stroke.Furthermore, the future therapeutic strategies for ischemic stroke are possibly combined therapy rather than monotherapy.Besides, additional approaches for testing and applying neurovascular protectors should also be considered (Kikuchi et al., 2014).
Despite the efficacy of pharmacotherapy in the treatment of ischemic stroke, several limitations have been reported regarding their use.For example, they should be prescribed in a short period of time following stroke onset in patients who meet the clinical criteria (Brzica et al., 2017).Another problem is the phenomenon of potential Drug-Drug Interactions (pDDIs).This happens in the setting of altered impacts of one drug by other concomitantly applied drug(s).The appearance of pDDIs in cases who suffered from acute ischemic stroke was most related to the total number of drugs prescribed, length of hospitalization and older age (Aleksic et al., 2019).Indeed, the brain is physiologically different from other organs.Blood vessels have a strong endothelial layer, and blood-brain barrier has made it difficult for drugs to pass through it.The blood-brain barrier protects the brain from other toxins and infections but is one of the biggest barriers to drug delivery, too (Pardridge, 2001).

Challenges in categorizing stem cells as drugs
Stem cells, as a therapeutic method, represent a paradigm shift in medicine that offers revolutionary potential in the treatment of various diseases and injuries (J.Wang et al., 2024).Stem cells are unique in their ability to self-renew and differentiate into specialized cell types, making them valuable tools for regenerative medicine and tissue engineering (Jin et al., 2023).In the field of drug development and regulation, stem cells present unique challenges and considerations.Unlike traditional small molecule drugs that exert their effects through specific molecular interactions, stem cells are living organisms with complex biological properties (Mansour et al., 2023).Mechanisms of action of stem cells are multifaceted, involving processes such as cell differentiation, paracrine signaling, and immune modulation.In this way, the development and evaluation of stem cell therapies requires a different regulatory framework and approach compared to conventional drugs (George, 2011).
With all these attributes in the definition of stem cells as a drug or a tool, different ideas are raised.A drug is any substance, other than food, that produces a physiological change in the body when taken in various forms, such as inhalation, injection, smoking, consumption, absorption through an adhesive on the skin, or dissolution under the tongue (Jin et al., 2023).In pharmacology, drugs, also known as pharmaceutical drugs, are used to treat, cure, prevent, or diagnose diseases, as well as to promote well-being.According to this definition, drugs must meet certain criteria, including having an indication to treat various diseases and being available as off-the-shelf products (Rubin and Haston, 2011;Wittich et al., 2012).Therefore, according to this definition, stem cell drugs are defined as off-the-shelf products based on stem cells for the treatment, cure, prevention, or diagnosis of diseases, or the promotion of well-being (Van Pham, 2016).
Stem cell drugs, as off-the-shelf products are used in allogeneic stem cell transplantation.There are important differences between allogeneic stem cell transplants and stem cell drugs.The main difference is that stem cell drugs are products, while allogeneic stem cell transplantation is a method of using these drugs.Furthermore, the former is approved as a drug, while the latter is approved as a medical device (Falkenburg et al., 2008).The defining characteristics of stem cell drugs include 1-Derivation from stem cells, 2-Readily available for use, 3-Manufactured in large quantities with consistent quality, 4-Subject to quality control measures by Good Manufacturing Practice guidelines, and 5-Officially approved as drugs.However, the features of allogeneic stem cell therapy encompass 1-The methodologies employed in cell utilization, 2-Utilization either as off-the-shelf products or directly from donors, 3-Variation in quality across batches based on donors and production methods, and 4-Approval as medical devices (Van Pham, 2016).
In recent years, regulatory agencies around the world have developed guidelines and regulations specifically for the development and evaluation of cell-based therapies, including stem cell products (Louria, 1969).The purpose of these regulations is to ensure the safety, efficacy, and quality of stem cell-based products while facilitating their translation from preclinical research to clinical practice.However, navigating the regulatory landscape for stem cell therapies remains complex and requires collaboration among researchers, clinicians, regulatory agencies, and industry stakeholders (Abubakar et al., 2023).The administration of stem cells in various diseases faces contradictions at different levels, including social acceptance, regulatory barriers, and scientific uncertainties.These contradictions highlight the need for balanced dialogue, informed decision-making, and sustainable settings to ensure responsible progress in medical science and technology (Rosen, 2006).

Utilizing stem cell therapy for ischemic stroke treatment
There is a growing interest in using stem cells for the treatment of numerous neurological diseases such as ischemic stroke (Leader et al., 2008).Several cell types are lost during the development of an ischemic stroke and repairment of blood vessels (pericytes, smooth muscle cells, and endothelial cells) as well as oligodendrocytes, neurons, and astrocytes is of paramount importance.The advantage of cell-based treatments and other restorative therapies is their ability to be used for longer periods of time (Bath and Sprigg, 2006).Stem cell-based therapy may be administered within days, weeks, or even months following injury, providing more benefits to patients (Hess and Borlongan, 2008).
Stem cell therapy has been considered in research fields as a promising regenerative therapy and a more promising therapeutic strategy for induced brain damage by various types of strokes.Stem cells, whether induced pluripotent stem cells or endogenous neural stem cells have the potential to replace damaged brain cells.Cell replacement strategies have been evaluated in numerous animal stroke models within decades of research (Caplan, 2007).Indeed, beneficial paracrine effects have been observed using pluripotent stem cells.Reduced cell death, facilitated growth/nutritional support for host cells and enhanced regeneration has been observed in the host brain using this therapeutic strategy (Wei et al., 2017).

Embryonic stem cells
Embryonic stem cells (ESCs), derived from the inner cell mass of the embryo before implantation, have unlimited self-renewal ability and the potential to differentiate into almost any cell type.ESCs could be differentiated into neural lineages using specific in vitro culture conditions (Bain et al., 1995).ESCs are identified as an optimal source of cell transplantation in order to treat neurological disorders.ESCs-derived cells which express cell surface markers of endothelial cells, astrocytes, neurons, and oligodendrocytes are observed in the lesion following transplantation of mouse ESCs into the cortex of mice suffering from ischemic stroke with subsequent functional recovery and structural improvement (Wei et al., 2005).Intrastriatal transplantation of ESCs-derived neuron-like cells or mouse ESCs was associated with improved dopaminergic function and consequently behavioral impairment in rates with focal ischemia with middle cerebral artery occlusion (MCAO) (Yanagisawa et al., 2006;Ye et al., 1998).Improved sensory and motor function of mice with MCAO have been observed following intracerebral transplantation of mouse ESCs which subsequently diminished infarct size.
Teratoma formation and malignant transformation are considered as disadvantages of ESC application.Limited resources, ethical issues, and high likelihood of malignant transformation are factors that limit the widespread ESC usage.There are very limited investigations on the applicability of ESCs in the treatment of stroke (Reubinoff et al., 2000).Malignant transformation of in vivo injected ESCs could be prevented using transplantation of differentiated cells derived from ESCs.Neural derivatives of ESCs are potential cells for the treatment of stroke.Numerous investigations have evaluated the impacts of ESC-derived neural stem/progenitor cells (NSPC) in the treatment of animal models of stroke (Daadi et al., 2008).Improved behavioral deficits, enhanced differentiation into neuronal cells, and diminished infarct area were observed following cell transplantation, regardless of the applied source of transplanted cells, the type of animal stroke model, and the kind of injection route.Meanwhile, numerous investigations have demonstrated the risk of teratoma formation using transplanted ESC-derived neurons (Sonntag et al., 2007).Culture conditions may reduce the risk of tumorigenesis of neural cells derived from transplanted ESCs.For example, an expandable and homogeneous population of NSCs called SD56 was isolated from ESCs using a medium supplemented with epidermal growth factor.After their implantation in the brain of ischemic rats, NSCs migrated to the parenchyma of the adult brain injured by ischemia.Besides, independent use of the stroke-impaired forelimb was improved two months following transplantation (Daadi et al., 2008).A higher density of cerebral blood vessels is parallel with a lower probability of stroke with a later occurrence.Any treatment which facilitates angiogenesis has a fundamental role in improving the performance of stroke patients.Intra-arterially transplanted human ESCs-derived endothelial and mural cells were associated with significantly increased vascular density and cerebral blood vessels in the ischemic striatum, diminished apoptosis and infarct volume, and facilitated neurological recovery in mice models of transient MCAO (Oyamada et al., 2008).

Inducible pluripotent stem cells
Inducible Pluripotent Stem Cells (iPSCs) are primarily induced from mouse adult fibroblasts or embryonic cells following transfection of four factors Sox2, Oct3/4, Klf4 and c-Myc (Takahashi and Yamanaka, 2006).These cells show the morphological and developmental characteristics of ESCs and express ESCs marker genes.In vivo subcutaneous transplantation of iPSCs into mice led to the development of tumors that contain diverse tissues derived from all three germinal layers (Tat et al., 2010).The advantage of iPSCs is the ability to proliferate and multipotent differentiation.Unlike ESCs, there is no ethical problem with using iPSCs.Chen et al. evaluated the therapeutic impacts of subdural transplantation of iPSCs combined with fibrin glue in mice with MCAO-induced ischemic stroke (S.J. Chen et al., 2010).Subdural transplantation of iPSCs has been demonstrated to diminish the total infarct volume.Diminished inflammatory responses in ischemic brain could be attributed to the protective and beneficial impacts of iPSCs.Other researchers have demonstrated migration of transplanted iPSCs derived from adult human fibroblasts into the damaged brain area with significant improvement of sensorimotor function in a rat model of MCAO (M.Jiang et al., 2011).However, behavioral improvement was not observed in one study using transplantation of iPSCs into mice brains with transient MCAO (Kawai et al., 2010).Differentiation of iPSCs into neurons and neuroblasts for ischemic stroke was observed representing a promising therapeutic strategy to supply sufficient neurons.The tumorigenic property is one of the concerns of iPSCs.Indeed, iPSCs have been demonstrated to form teratoma following transplantation into mouse ischemic brain (Yamashita et al., 2011).If tumorigenesis is properly controlled, iPSCs are potent candidates for the treatment of ischemic stroke.

Neural stem cells
Neural Stem Cells (NSCs) are present in the subventricular zone of the adult brain (Gage, 2000).After the onset of ischemic stroke, endogenous NSCs can proliferate and migrate to the damaged area and promote tissue repair (Yamashita et al., 2006).Endogenous NSCs contribute to post-ischemic brain repair.However, insufficient amount of endogenous NSCs in some instances results in improper replenishment of lost neurons, and a small number of NSCs differentiate into neurons (Arvidsson et al., 2002).Transplantation of NSCs can increase neurogenesis and is considered a promising therapeutic approach for ischemic stroke (Andres et al., 2011).Neuroprotection is induced with delay by intravenous NSC transplantation 3 days following ischemic stroke through suppression of inflammation and subsequent glial scar formation indicating the potential of NSCs for extension of the therapeutic window in the setting of ischemic stroke (Bacigaluppi et al., 2008).NSCs modified with Akt-1 or VEGF also improved neuronal function following ischemic stroke through enhancement of angiogenesis and neuronal survival (H.J. Lee et al., 2007).Therefore, NSCs are recognized as an effective candidate for the treatment of ischemic stroke.

Mesenchymal stem cells
The therapeutic capabilities of mesenchymal stem cells (MSCs) has been extensively investigated in the ischemic brain (Eckert et al., 2013).So far, it was not clear how transplanted cells contribute to improved functioning after ischemic stroke.Due to their limited neural differentiation capacity, MSCs have been found to exert their beneficial impacts principally through immunomodulatory and paracrine mechanisms rather than cell replacement (X.Liu et al., 2014).In vitro studies have demonstrated that the MSC environment significantly increased neurite outgrowth of dorsal root ganglion (Neuhuber et al., 2005).MSCs co-cultured with glutamate-exposed neurons significantly ameliorated neuronal damage induced by glutamate by releasing soluble neuroprotective factors (Hokari et al., 2008).Studies have shown that cultured mesenchymal stem cells can release a variety of chemokines.Cooperation of these chemokines with MSCs is essential for participation in the healing of ischemic brain tissue damage (Ringe et al., 2007).Mesenchymal stem cells can release anti-inflammatory substances such as interleukins (ILs) to regulate the function of the immune system after ischemic stroke, and play their anti-inflammatory role through the PL3K/AKT pathway by inhibiting pyroptosis (Fig. 1) (Walkowski et al., 2022;Zhou et al., 2019).In vivo investigations have demonstrated that MSC injection into mice following ischemic stroke was associated with reduced destruction of the blood-brain barrier and improved neurobehavioral recovery through inhibition of inflammation and induced angiogenesis and neurogenesis (J.Chen et al., 2003).In vivo transplantation of genetically engineered MSCs overexpressing IL-10 could significantly enhance mitophagy and autophagy with reduced markers of neuroinflammation and cell death compared with mere MSCs.
In stroke rat models, MSC transplantation was associated with enhanced axonal plasticity and interhemispheric and intracortical connections (Z.Liu et al., 2010).A number of investigations have recently demonstrated enhanced neurotrophic therapeutic impacts of MSCs by gene modification.For instance, intravenous injection of human MSCs modified with brain-derived neurotrophic factor (BDNF) and/or a combination of human MSCs with vascular endothelial growth factor and angiopoietin-1 into ischemic mice showed better therapeutic effects than yielded unmodified MSCs (Nomura et al., 2005).

Stem-cell-based gene therapy
In the last few years, a lot of experimental, preclinical and clinical data have been published showing the possibility of transferring new genetic information with relatively high efficiency in diverse cells or tissues such as differentiated cells and hematopoietic progenitor cells.Addition of normal gene to the cells with endogenous gene deletion, mutation, or alteration has been demonstrated experimentally to reverse phenotype and in some cases restore functional defect (Bagnis and Mannoni, 1997).Stem cell-based gene therapy is considered as a potential novel therapeutic strategy for the treatment of ischemic stroke in the future.Stem cells secrete different neurotrophic factors by themselves.Transplanted gene-modified stem cells overexpress various neurotrophic factors including noggin, BDNF, VEGF, HGF, GDNF, EPO, and NGF, and have been associated with significant improvement in Fig. 1.Therapeutic potential of mesenchymal stem cells in the ischemic brain.Cultured mesenchymal stem cells can release a variety of chemokines.These chemokines collaborate with mesenchymal stem cells to participate in improvement of damage or tissue repair of the ischemic brain.These cells can also release antiinflammatory substances such as interleukins to regulate the operation of post-stroke immune system and play their anti-inflammatory role by inhibiting pyroptosis through the PL3K/AKT path.

Clinical trials in the use of stem cells for the treatment of ischemic stroke
Stem cell-based therapies for the treatment of stroke opened promising horizons due to their ability to address patients' unmet requirements, neuroprotective and regenerative benefits (Mahla, 2016).The advantages of stem cell therapies for the treatment of stroke include neuroregeneration, reducing stroke-induced neuronal loss and neuroprotection, and functioning in the acute phase of stroke in order to restrict the spread of injury (F.Wang et al., 2018).Furthermore, although stroke is a vascular disease, a significant immune response is also present which correlates with the healing process.Certain populations of stem cells have the capacity for immune modulation, promising neuroprotective and neurodegenerative effects, and enhancement of therapeutic effects while reducing inflammatory damages (Caplan and Correa, 2011).Some clinical trials related to the treatment of stroke by stem cells are summarized in Table 1.

Challenges of ischemic stroke treatment with stem cell therapy
Many hurdles must be overcome before successful application of stem cells in the clinic including the strategy of tracking transplanted cells and limited life.In vivo cell tracking is applicable through positron emission tomography (PET), single-photon emission computed tomography (SPECT/CT), bioluminescence imaging (BLI) using GFP protein, and magnetic resonance imaging (MRI) (Zheng et al., 2017).Moshayedi et al. demonstrated enhanced survival of transplanted cells using hyaluronic acid (HA) hydrogels for at least 6 weeks and tracked them in vivo using MRI (Moshayedi et al., 2016).Besides, there are several ethical concerns regarding clinical application of stem cells, mainly with ESCs and NSCs.Application of iPSCs might avoid this problem.In addition, production of autologous iPSCs is possible, but relatively expensive and takes several months to prepare the cells for transplantation.In addition, the clinical usage of stem cells might raise several safety concerns.For example, allograft transplantation might result in immune rejection.Potentially, stem cells could differentiate into undesirable tissues which enhance tumor growth and its metastasis by increasing the production of neo-vessels and altering of tumor microenvironment (Patel et al., 2010).Erdö et al. showed increased tumorigenesis by contamination of undifferentiated Embryonic Stem Cells (Erdö et al., 2003).Amariglio et al. reported the development of brain tumors four years following the initial treatment in cases with ataxia-telangiectasia who were treated with intraspinal and intracerebellar injection of donor-derived neural stem cells (Amariglio et al., 2009).More clinical research is necessary to discover the optimal routes of transplantation, such as the appropriate time, route and dose of injection.

Conclusion and prospective
After the damage induced by ischemic stroke, the application of methods that improve the tissue conditions and reduce the destructive effects is required.Since the general process of ischemic damage is very complicated and the pattern of pathophysiological changes is not the same for all brain cells, a proper understanding of the cellular and molecular alterations caused by cerebral ischemia is necessary to create strategies for the treatment of cerebral stroke.Due to the surgical hazards and the challenges of using chemical drugs for the treatment of stroke, they cannot be considered as safe approach for the treatment of ischemic stroke.On the other hand, stem cells are an attractive candidate for the treatment of ischemic stroke.The beneficial impacts of stem cells include synaptogenesis, neuroprotection, angiogenesis, inflammation, immune responses, and others.However, before clinical application, many important issues including optimal cell sources, dosage, timing, and monitoring of events and side effects must be managed.A better understanding of the mechanisms of action of stem cells in the treating ischemic stroke helps to solve the above problems.
Considering the rapid progress of gene therapy and the widespread use of stem cells in this field, stem cells along with gene therapy in future experiments and clinical programs can play an important role in the treatment of cerebral ischemia.Despite the lingering uncertainties surrounding potential side effects, ongoing research suggests a promising role for stem cell therapy in treating these patients.However, further evidence is required to fully establish its effectiveness and safety.

Ethics statement
Not applicable.

Funding
Non.  CRediT authorship contribution statement Sahar Yaqubi: prepared figures, contributed to the drafting of the text, All authors reviewed the manuscript.Mohammad Karimian: contributed to the conception and design of the study, prepared figures, contributed to the drafting of the text.

Declaration of competing interest
There is no conflict of interest.

Table 1
Clinical Trials in the treatment of stroke using stem cells.