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Abstract

Stem cell therapy is an emerging therapeutic modality in the treatment of stroke. We assessed the safety and feasibility of the cotransplantation of neural stem/progenitor cells (NSPCs) and mesenchymal stromal cells (MSCs) in patients with ischemic stroke. Eight patients were enrolled in this study. All patients had a hemisphere with infarct lesions located on one side of the territories of the cerebral middle or anterior arteries as revealed with cranial magnetic resonance imaging (MRI). The patients received one of the following two types of treatment: the first treatment involved four intravenous injections of MSCs at 0.5 × 10⁶/kg body weight; the second treatment involved one intravenous injection of MSCs at 0.5 × 10⁶/kg weight followed by three injections of MSCs at 5 × 10⁶/patient and NSPCs at 6 × 10⁶/patient through the cerebellomedullary cistern. The patients' clinical statuses were evaluated with the National Institutes of Health Stroke Scale (NIHSS), the modified Rankin Scale (mRS), and the Barthel index (BI). Six patients were given four cell transplantations. The most common side effect of stem cell transplantation in these six cases was low fever that usually lasted 2–4 days after each therapy. One patient exhibited minor dizziness. All side effects appeared within the first 2–24 h of cell transplantation, and they resolved without special treatment. There was no evidence of neurological deterioration or neurological infection. Most importantly, no tumorigenesis was found at a 2-year follow-up. The neurological functions, disability levels, and daily living abilities of the patients in this study were improved. While these observations support the use of the combination transplantation of NSPCs and MSCs as a safe and feasible method of improving neurological function, further studies that include larger samples, longer follow-ups, and control groups are still needed. This manuscript is published as part of the International Association of Neurorestoratology (IANR) special issue of Cell Transplantation.

Keywords

Neural stem/progenitor cells (NSPCs)Mesenchymal stromal cells (MSCs)Ischemic stroke Cell transplantation

Introduction

Stroke is the second leading single cause of death worldwide (after ischemic heart disease) and accounts for over 5 million deaths per year globally (15). Stroke is also the most common cause of adult disability. Of all strokes, 80% are ischemic. Despite advances in acute care and secondary preventative strategies, stroke remains a major burden on healthcare resources worldwide. Thrombolysis with tissue plasminogen activator is now a well-established treatment for acute ischemic stroke and is associated with significant improvements in outcomes. However, the use of this treatment is limited to a narrow time window of only 4.5 h from the onset of symptoms. Many stroke survivors are left with permanent neurological disabilities. Although rehabilitation therapy is important for the maximization of functional recovery after stroke, once neurological deficits are fixed, there are few options for recovery. It has been suggested that any therapy that leads to even slight recovery might be helpful (28).

Neurorestoratology seeks therapeutic solutions to this problem. Neurorestoratology is the subdiscipline of neuroscience that studies neural regeneration, repair, and replacement of damaged components of the nervous system, neuroplasticity, neuroprotection, and neuromodulatory mechanisms of recovery. The goal of neurorestoratology is to promote recovery of neural function (8,16).

Within neurorestoratology, stem cell therapy is an emerging therapeutic modality for the treatment of stroke. The basis of stem cell therapy stems from the observation that certain parts of the adult brain are capable of regeneration. Neurogenesis in the adult brain has been demonstrated in the dentate nucleus of the hippocampus and the subventricular zone. In a study of patients with ischemic stroke, neurogenesis was demonstrated in the ischemic penumbra, where cells were found to preferentially localize in the vicinity of blood vessels (21).

In contrast to neurodegenerative disorders such as Parkinson's disease, which destroys a relatively homogenous population of neurons, stroke affects multiple neuronal phenotypes. An infarct might involve the thalamus, hippocampus, and striate visual cortex and affect three or more very different neuronal populations. Additionally, oligodendrocytes, astrocytes, and endothelial cells are also affected. Reconstitution of the complex and widespread neuronal– glial–endothelial interrelationships may require that cells for transplant initially remain immature and phenotypically plastic to differentiate into the appropriate neural, glial, or endothelial cell types, depending on the damaged area.

Neural stem/progenitor cells (NSPCs) are present during embryonic development and in certain regions of the adult central nervous system (CNS) (32). Mobilizing adult neural stem cells (NSCs) to promote the repair of injured or diseased parts of the CNS is a promising approach (2). NSPCs in the adult CNS are capable of generating new neurons, astrocytes, and oligodendrocytes (38). Most importantly, studies have shown that adult NSCs that are transplanted into the sites of injury can differentiate into vascular cells (endothelial cells and vascular smooth muscle cells) for vasculogenesis (17).

Human embryonic stem cell (ESC)-derived NSCs that are grafted into the ischemic boundary in rats that have been subjected to stroke migrate toward the lesion and improve forelimb performance. Electrophysiological recordings have demonstrated that the grafted cells exhibit functional neuronal properties and receive synaptic input from host neurons. Human ESC-derived NSCs implanted into cortical lesions differentiate partially into neurons and induce some improvement in simple sensorimotor functions. Transplanted human fetal NSCs give rise to neurons that migrate toward ischemic lesions in rodents. Human NSCs isolated from the fetal striatum and cortex generate mature neurons after transplantation into the stroke-damaged rat striatum. Taken together, these findings provide evidence that stem cell-derived neurons can exhibit some level of synaptic integration into the host neural circuitries and suggest that neuronal replacement in the stroke-damaged brain may contribute to the observed behavioral improvements (28).

Mesenchymal stromal cells (MSCs) have currently been isolated from most tissues, including the umbilical cord (UC) and UC blood (UCB). UC and UCB MSCs are more primitive than those isolated from other tissue sources and do not express the major histocompatibility complex (MHC) class II human leukocyte antigen-D-related (HLA-DR) antigens. Studies have shown that UC MSCs are still viable and are not rejected up to 4 months after transplantation as xenografts and thus do not require immune suppression, which suggests that these cells are a favorable cell source for transplantation (45). The UC, including the arteries (UCA), veins (UCV), and Wharton's jelly (UCWJ), is a convenient, efficient source of MSCs that can be expanded easily in vitro for numerous clinical applications, such as treatment of nonhematopoietic diseases, and moreover, in studies of tissue regeneration, immunosuppression, and stroke (10,14,18). UC MSCs have been proven to be efficacious in reducing lesion sizes and enhancing behavioral recovery in animal models of ischemic (40) and traumatic CNS injury (29). Recent findings also suggest that neurons derived from UC MSCs can alleviate movement disorders in parkinsonian animal models (37). Therefore, UC MSCs could be a viable alternative to human ESCs or NSCs for transplantation therapy in the CNS (30). Functional restoration after stroke has been reported using various types of non-NSCs, such as those in UCB and bone marrow-derived hematopoietic stem cells (43). In the clinical setting, adult MSCs could be particularly useful because they are easily obtained, can be harvested from autologous donors, and can be rapidly expanded in vitro (34).

In this study, we assessed the safety and feasibility of the cotransplantation of NPCs and MSCs in patients with ischemic stroke.

Materials and Methods

Consecutive patients attending the neurology in-patient clinic were considered for the study between July 2010 and May 2011. The study was approved by the local institutional review board in accordance with the guidelines of Helsinki, and written informed consent was obtained from each patient or their legal guardian.

Inclusion and Exclusion Criteria

Patients with ischemic stroke were considered eligible if they fulfilled the following criteria: one hemisphere with a recent stroke and relevant lesions within the territories of the middle cerebral artery (MCA) and/or the anterior cerebral artery (ACA), no coma, normal renal and liver function, and normal blood cell counts, blood pressure, heart rate, respiration, and body temperature.

Patients meeting the above criteria were excluded from the study if they had any of the following: comorbidity that was likely to limit survival to less than 3 years (e.g., malignant disease or hepatic or renal failure); a hemisphere damaged by a previous stroke; pregnancy; or HIV positivity. The patients did not pay for their inclusion or treatment in this study.

Standard Stroke Care

Full standard stroke care was given to every patient. Concomitant treatment included fluid supplementation, antiplatelet or anticoagulant medications, antibiotics, antihypertension drugs, fever control, statins, and insulin when medically indicated.

Treatment

The cells used in this study were NSPCs and MSCs that were provided by the Hongtianji Neuroscience Institute (Beijing, China). MSCs were taken from UC. NSPCs were taken from the subependymal zone of the fetal brain (unknown gender). The detailed method of obtaining MSCs and NSPCs is provided in Chen et al. (11). The patients were divided into two treatment groups: the first group was given four intravenous infusions of MSCs (0.5 × 10⁶/kg weight in 250 ml of saline). The second group was given an intravenous infusion of MSCs (0.5 × 10⁶/kg weight in 250 ml of saline) followed by three treatments with MSCs (5 × 10⁶/patient) and NSPCs (6 × 10⁶/patient) in 10 ml of saline that was injected through the cerebellomedullary cistern under local subdermal anesthesia with 2% lidocaine (Tianjin Pharmaceutical Group, China). The interval between each cell transplantation treatment was 1 week. The treatment of the first group was intended for use with patients who could not endure the cistern puncture operation.

Baseline Assessment

The baseline assessment included clinical and laboratory tests. The patient characteristics and outcomes are shown in Table 1.

Table 1.

Participant Characteristics

Case Number	1	2	3	4	5	6
Age (year)/sex	85/F	81/F	66/F	45/M	57/M	3/M
Territory and side	MCA/left	MCA/right	MCA/right	ACA/right	MCA/right	MCA/left
Previous stroke	N	N	N	N	N	N
Diabetes	N	N	N	N	Y	N
Dyslipidemia	N	Y	N	N	N	N
Hypertension	Y	Y	N	Y	N	N
Therapy time from onset	2 months	3 months	1 week	2 weeks	10 months	2 years
Antiplatelet/anticoagulant	Clopidogrel	Clopidogrel	Clopidogrel	Aspirin	Clopidogrel	N
Type of therapy received	1	1	2	2	2	2
NIHSS at baseline	16	15	6	6	3	3
BI at baseline	0	10	65	25	80	65
mRS at baseline	5	5	3	5	3	3
Side effect	Fever	Fever and dizziness	–	–	Fever	Fever
Total time(s) of side effect	2	2	–	–	2	1

MCA, middle cerebral artery; ACA, anterior cerebral artery. Therapy 1: Four intravenous infusions of mesenchymal stromal cells (MSCs; 0.5 × 10⁶/kg weight in 250 ml of saline). Therapy 2: One intravenous infusion of MSCs (0.5 × 10⁶/kg weight in 250 ml of saline) followed by three injections of MSCs (5 × 10⁶/patient) and NSPCs (6 × 10⁶/patient in 10 ml of saline) into the cerebellomedullary cistern under local subdermal anesthesia with 2% lidocaine. The interval between each cell transplantation was 1 week. NIHSS, National Institutes of Health Stroke Scale (lower scores indicate better outcomes, and higher scores indicate worse outcomes); BI, Barthel index (scores range from 0 to 100, higher score indicate better outcomes, and lower scores indicate the worse outcomes); mRS, modified Rankin scale (scores range from 0 to 5, lower scores represents better outcomes, and higher scores represent worse outcomes).

Clinical Parameters

Patient's neurological deficit(s) were assessed using the National Institutes of Health Stroke Scale (NIHSS) (22), the Barthel index (BI) (41), and the modified Rankin Scale (mRS) (39). The NIHSS, BI, and mRS assessments were repeated every day for 4 weeks and at 3 months, 6 months, 1 year, and 2 years after cell transplantation. For NIHSS, lower scores indicate better outcomes, and higher scores indicate worse outcomes; for BI, scores range from 0 to 100, and higher scores indicate better outcomes, and lower scores indicate the worse outcomes; for mRS, scores range from 0 to 5, and lower scores represents better outcomes, and higher scores represent worse outcomes.

Laboratory Parameters

Clinical chemistry monitoring, including tests for blood counts, C-reactive protein (CRP), electrolytes, liver enzymes, renal function, lipids, and coagulation parameters, was performed before cell transplantation and at 3 months, 6 months, and 1 year after cell transplantations. Blood counts were tested by Sysmex 4000i, (Kobe, Japan). CRP was tested by Quik Read 101 from Orion Diagnositica Oy (Espoo, Finland). Electrolytes, liver enzymes, renal function, and lipids were tested by C501 automatic biochemical analyzer (Roche, Mannheim, Germany). Coagulation parameters were tested by ACL top 500 from Werfen Group (Tokyo, Japan). Significant toxicity was assumed if liver enzymes increased to 50% of baseline, and the numbers of platelets and/or red blood cells decreased to below 50% of baseline (9).

Neuroradiological Investigations

Magnetic resonance imaging (MRI) (Siemens Novus1.5T, Munich, Germany) was performed before cell transplantation and at 3 months after cell transplantation. The following MR sequences were used: T1-weighted (TR450.0, TE 10.0), T2-weighted (TR 5520.0, TE 103.0), fluid-attenuated inversion recovery (FLAIR) (TR7040.0, TE126.0), and diffusion-weighted MRI (DWI) (TR3600.0, TE99.0, b value 1000).

Statistical Methods

Descriptive analyses were used. Data are presented as the mean ± standard error of the mean (SEM), and all analyses were performed using SPSS software (version 16.0; IBM, Armonk, NY, USA).

Results

Eight cases were enrolled. All patients presented with unilateral strokes. Infarctions in the territories of the MCA or ACA were confirmed by cranial MRI. Six cases received standard cell transplantation treatments (four treatments). Two cases withdrew their consent to participate in the study after the first or second transplantation. Both of these patients were diagnosed with acute cerebral infarction, and their muscle tones recovered quickly to normal or nearly normal just prior to the first or second therapy.

The characteristics of the remaining six patients are shown in Table 1. Of these six patients (aged 3–85 years, 56.17 ± 30.00 years, three females and three males), cell transplantations were carried out in the acute stage (<1 week from onset) for one patient, in the subacute stage (1 week to 1 month from onset) for three patients, and during stroke sequelae for two patients (0.5–2 years from onset). Two patients (81 and 85 years old) received four intravenous infusions of MSCs because they refused the intrathecal injections. Four patients completed one intravenous infusion of MSCs and three cotransplantations of MSCs and NSPCs through the cerebellomedullary cistern. One of the two patients (case number 1) who received four intravenous infusions of MSCs was bedridden until death 10 months after the cell transplantations. Her cause of death was diagnosed as recurrent massive cerebral infarction. This patient refused an autopsy.

The most common adverse event after stem cell transplantation that was observed in these six cases was low fever (<38.5°C), which usually began after 2–24 h after cell transplantation and lasted 2–4 days. The incidence of fever was approximately 32% in this study. A single patient exhibited slight and temporary dizziness after one transplantation. Low fever and slight dizziness, both of which disappeared without special treatment, were the only adverse events observed in this trial. Adverse events did not occur in all patients. As shown in Table 1, no adverse events were reported by two patients. The patients experienced their adverse event(s) only after the first or second transplantation(s), and no patients experienced any adverse events after the third or fourth transplantations.

All tested laboratory parameters remained stable throughout the 90 days of observation with the exception of instant neutrophil granulocyte counts, which increased slightly in the patients with fever. No significant enlargements of infarct volumes were observed on serial MRIs after cell transplantation. Most importantly, no tumors were found over the 2-year follow-ups of the five patients who survived 2 years.

Among the five patients who took antiplatelet treatments, three received cerebellomedullary cistern operations (Table 1). No hemorrhagic cerebral spinal fluid or ecchymoma was found. No anticoagulant therapy was included in this study. However, intravenous infusion, but not cerebellomedullary cistern injection, should be considered due to the potential bleeding caused by the anticoagulant therapy.

No patient experienced neurological deterioration in this study. Indeed, the patients exhibited different degrees of clinical improvement. The NIHSS scores (Fig. 1) indicated that the patients' neurological deficits improved. Five cases exhibited decreases in NIHSS scores of more than two points (i.e., they exhibited clinical improvement) 3 months after the completion of the cell transplantations. The patients' daily living abilities improved, and the patients' disability levels were reduced as indicated by the BI and mRS scores, respectively (see Fig. 1 for details).

Figure 1.

Clinical assessments after cell transplantations. The NIHSS scores indicated that the patients' neurological deficits improved. The patients' daily living abilities improved, and the patients' disability levels were reduced as indicated by the BI and mRS scores, respectively. Data are presented as means ± SEM. Results at 1 and 2 years are from five patients because one patient died 10 months after cell transplantation.

Two cases exhibited clinical improvements after the first transplantation, two cases exhibited improvements after the second transplantation, and one case exhibited improvements after the fourth transplantation. Clinical improvements occurred, on average, 19.81 days after the first cell transplantation. The clinical improvements were stable at the 2-year follow-up.

Compared to the patients who received four intravenous infusions of MSCs, the patients who received NSPC and MSC cotransplantations into the cerebellomedullary cistern exhibited superior improvements as indicated by the NIHSS, BI, and mRS scores after cell therapy. However, it should be noted that the patients who received four intravenous infusions had lower NIHSS scores at the baseline assessment, as well as being essentially a different gender (two females vs. one female and three males).

Compared to the patients who received cell transplantations during the stroke sequelae, the patients who received transplantation during the acute or subacute stages exhibited greater improvements in NIHSS scores. However, this may have been due to the natural process of the recovery of neurological function.

Discussion

The mechanisms of action of stem cell transplantation include the formation of new neuronal circuitry, reduced apoptosis, reduced inflammation, the promotion of angiogenesis, the promotion of neurogenesis, and the promotion of other endogenous repair processes (1,27). Multiple mechanisms are involved in the stroke recovery process. There is a sizeable body of laboratory and clinical data that supports the safety and efficacy of both NSPCs and MSCs (7), and so we hypothesized that cotransplantation of these two types of stem cells would be more beneficial to the functional recovery of stoke patients. Although the small number of patients with 2-year follow-ups limits the definitiveness of this study, this study should help to assuage the fear of any major safety concerns and provide assurance that further studies should be conducted.

There are a wide variety of sources of exogenous NSCs, and much research is being performed regarding the transplantation of embryonic and fetal-derived NSPCs in experimental stroke models. However, the use of such embryonic or fetal tissue is limited by ethico-legal implications. Hence, the plentiful supply of UC MSCs will be a substantial advantage for the wide use of cell therapy. Our study suggests that the combination of NSPCs and MSCs delivered through intravenous and intrathecal routes is safe and feasible in stroke patients.

Fever was the most common adverse event observed in our study, and this finding has been confirmed by others (42). A meta-analysis indicated that there is a significant association between MSC administration and the development of fever (25). The fever caused by stem cell administration was transient, and no special treatment was required. The mechanisms responsible for this fever are not clear but may be related to the acute inflammatory reactions of a subset of patients to particular preparations of stem cells.

Human ES cells can give rise to an unlimited number of NSCs but are also associated with a risk of tumor formation (36). Human fetal NSCs are less tumorigenic than ES cells. However, malignant transformation is more than a theoretical risk (especially for NSPCs) (44). The first glioneuronal neoplasm was reported 4 years after human fetal NSC transplantation in a patient with ataxia telangiectasia (3), though the impurity of the transplanted cells means that it is not entirely certain that the NSCs themselves caused the tumors (4,20). Concerns related to the tumorigenicity of MSCs were raised by preclinical studies that demonstrated increased tumor burden in vivo (13). Studies have suggested that the probabilities of malignant transformation and tumor formation due to MSCs are low (35), and a pooled analysis found no association between MSCs and tumor formation. Malignancy occurred only in studies involving participants with ongoing or previous malignancies. In a 5-year follow-up study of patients with ischemic stroke who received MSC transplantations, significant side effects were not observed (26). This finding is in line with our results; no tumor formation was found in our 2-year follow-up. We suggest that safety concerns should not be an obstacle that slows further progress in stem cell research. In addition to standard stem cell production protocols and quality control (5), the engineering of stem cells that express suicide genes that can be switched on in case of adverse effects and the use of cell sorting to eliminate tumor-forming cells may be viable methods for improving safety in the future.

In comparison to stereotactic implantation, which allows for the precise targeting of defined numbers of cells to desired sites, intravenous and intrathecal approaches have the advantage of readily allowing the repeated administrations of cells. The results of this study suggest that combinations of intrathecal and intravascular therapies are feasible and safe. However, the operation for intrathecal injection will necessitate the exclusion of some patients from this type of cell therapy due to their advanced age.

Regardless of the route of transplantation that is selected, NPCs have the capacity to migrate long distances along chemoattractive gradients toward the sites of brain injury. Indeed, it has been shown that transplanted stem/progenitor cells are able to follow, via the blood stream or circulation of the cerebrospinal fluid, gradients of proinflammatory cytokines and chemokines that are released at the site of brain lesions (19). While promoting interaction between the transplanted NPCs and the activated endothelial/ependymal cells around the inflamed CNS tissues, this chemoattractive gradient leads to selective and specific targeting of transplanted cells to the inflamed CNS areas (6). A recent study showed that NSCs can migrate into ischemic regions, survive, and differentiate into astrocytes and neurons and have the potential to improve neurological function after cerebral ischemia in a rat model (12).

The appropriate time for transplantation after a stroke is unknown. In this study, the patients who received transplantations in the acute or subacute stages tended to exhibit superior neurological improvement compared to the patients who received transplantation during stroke sequelae. This finding is, in fact, the final result of beneficial and harmful environmental contradictions in stroke patients. In the acute setting, the release of excitotoxic neurotransmitters, free radicals, and proinflammatory mediators might threaten new tissue that is introduced into the peri-infarct region. Furthermore, cells may be dying by apoptosis in the penumbra for several weeks after stroke. Inflammation leading to microglial activation may inhibit endogenous neurogenesis and may thereby suppress the growth and survival of transplanted cells (33).

In contrast, during the acute stage, local repair processes, including the release of neurotrophic factors from the intrinsic milieu and the host environment, are active and may facilitate implant growth, survival, differentiation, and/or integration. The ischemic environment also promotes the generation of new neurons in the periventricular regions and in the cerebral cortex (23). However, clinical trials have demonstrated improvements in ischemic stroke patients after transplantation in both the acute and sequelae stages (24).

Our stroke patients exhibited clinical improvements 19.81 days after cell transplantations. These functional improvements were not accompanied by any visible anatomical changes. This finding suggests that stem cells do not operate through a unidirectional mechanism (e.g., the generation of neurons) but rather operate as cellular mediators (31).

It is notable to point out the different parameters included in this study, such as different cell dose, different combined treatment, different delivery route, and also the different starting clinical status. However, we provide evidence for a possible trend after cotransplantation in stroke patients, and this provides us with the confidence to use a bigger sample size in a randomized, double-blinded study in the future.

Conclusion

While our observations have indicated that the transplantation of combinations of NSPCs and MSCs is safe and feasible and may improve neurological function (at least over the follow-up period we investigated), further studies that included larger samples, longer follow-up periods, and control groups are needed. Further studies should also assess intravascular cell therapy as a potential adjunct of thrombolytic or mechanical thrombectomy in ischemic stroke to ensure that patients received the best possible treatment.

Footnotes

Acknowledgments

With great respect and thankfulness, we acknowledge Dr. Haitao Xi and Dr. Feng Zhang from Hongtianji Neuroscience Institute, Beijing, China, for their help in designing and revising the protocol of this study. The authors declare no conflicts of interest.

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A Two-Year Follow-Up Study of Cotransplantation with Neural Stem/Progenitor Cells and Mesenchymal Stromal Cells in Ischemic Stroke Patients

Abstract

Keywords

Introduction

Materials and Methods

Inclusion and Exclusion Criteria

Standard Stroke Care

Treatment

Baseline Assessment

Clinical Parameters

Laboratory Parameters

Neuroradiological Investigations

Statistical Methods

Results

Discussion

Conclusion

Footnotes

Acknowledgments

References