Introduction

Ischemic stroke, a leading cause of death and disability worldwide, is associated with high mortality, disability, and recurrence rate. Severe ischemic stroke patients usually suffer neurological deficits and severe complications (i.e., hypostatic/aspiration pneumonia, bedsore, deep vein thrombosis, etc.), despite acute therapy that protects them from death. When neurological deficits persist, there is limited treatment to enhance recovery currently. As a result, severe stroke remains a significant unmet clinical need, imposing the urgent hope for novel treatments.

Laboratory studies have shown that cell-based therapy is a potential new treatment approach of regenerating the injured brain beyond the acute phase of ischemic stroke [1, 2]. Various cell types have been used to provide functional and structural benefits after stroke, including embryonic stem cells (ESCs), immortalized pluripotent stem cells (iPSCs), neural stem/progenitor cells (NSCs), and non-neuronal adult stem cells such as mesenchymal stem cells (MSCs) and bone marrow mononuclear cells (MNCs) [3]. MSCs are not ethically controversial and have no risk of tumor formation as the case in ESCs and iPSCs. Among the various stem cells, MSCs have been most commonly used in the clinical trials for patients with stroke.

MSCs, originally isolated from bone marrow, can differentiate into multiple tissues, including the bone, fat, cartilage, neurons, hepatocytes, and cardiocytes. Also, MSCs can be isolated from other tissues, such as the umbilical cord, peripheral blood, adipose tissue, endometrial polyps, and menses blood [4]. MSCs are found to play multiple roles in the treatment of cerebral ischemia. Transplanted MSCs can improve the outcome through differentiating into neurons and astrocytes, increasing cytokines and neurotrophic factors, promoting angiogenesis and cerebral blood circulation, facilitating a proliferation in endogenous neurogenesis, reducing apoptotic cells, and encouraging axonal sprouting, myelin remodeling, and restoration of neural circuits. Moreover, MSCs play an important role in immunomodulatory function [5].

A meta-analysis examined preclinical studies of MSCs in the treatment of ischemic stroke and found that this cellular therapy improves outcome, with very large effect sizes. Effects were robust across species, delivery route, time of administration in relation to stroke, MSC immunogenicity, and MSC dose [6]. These results support the urgent need for further studies of MSC transplantation in the treatment of ischemic stroke in humans. In recent years, several clinical studies have administrated MSCs in stroke through intracerebral injection or intravascular injection [7,8,9,10,11,12,13,14]. These trials varied in terms of the patient characteristics, timing, and dose of cell therapy (Table 1). Moreover, the assessments of functional improvement, adverse effects, and pretreatment screening tests for safety have varied greatly among the studies. None of the studies aimed to determine the efficacy of MSC therapy in patients with stroke. All of the studies aimed to assess the feasibility and safety of stem cell treatments, and most were small series and did not include a control group [15].

Table 1 Clinical trials of MSCs in the treatment of patients with ischemic stroke
Full size table

Intrathecal injection of autologous or allogenic MSCs has been administrated in many kinds of human neurological disorders, such as basilar artery dissection [16], progressive multiple sclerosis [17, 18], spinal cord injury [19, 20], amyotrophic lateral sclerosis [21], spinal muscle atrophy [22], progressive supranuclear palsy [23], cerebral hemorrhage [24], and spinocerebellar ataxia and multiple system atrophy [25]. No serious stem cell-related adverse effect was reported and some patients had functional improvement in the studies above. At present, there is no clinical report on the treatment of cerebral infarction with MSCs through intrathecal injection. Although intracerebral transplantation may allow more directly cell homing, local injection may lead to poor cell distribution in the injured brain, and it is an invasive method that may result in bleeding, seizures, and other complications. So there are many hurdles for getting intracerebral injection into clinical application. Intra-arterial injection, delivering cells to the infarcted brain by intra-carotid injection, may lead to a better distribution of stem cells compared with intracerebral transplantation. However, a potential problem of this approach is that MSCs might be unable to pass the blood-brain barrier [26, 27]. Additionally, several laboratorial and clinical studies have shown that MSCs can adhere to each other and form microemboli after intra-arterial administration (IA), which aggravate the brain damages [28, 29]. Intravenous injection (IV) represents the least invasive method of delivery, but it has the same problem as the case in IA that MSCs might be unable to pass the blood-brain barrier. And injected cells by IV also migrate to perivascular locations in other organs, and there is a potential risk of leading to ectopic growth or elaboration of secreted proteins in other organs [30]. What’s more, cells delivered by IV have to first pass through the lungs before they can be distributed throughout the body. This presents a major problem with what has been termed the pulmonary “first-pass” effect, which results in significant entrapment of active stem cells and the greatly increasing of the treatment dose [31, 32]. Furthermore, intravenous injection of a large dose of stem cells may lead to pulmonary embolism [33, 34]. Intrathecal injection is a safe and feasible approach that infuses stem cells into the subarachnoid space of the patient by lumbar puncture. It allows higher concentrations of stem cells to migrate to the lesion site. Moreover, it is safer than intracerebral injection. So, MSC transplantation via intrathecal injection may be the best routine of stem cell therapy in patients with ischemic stroke.

Autologous MSCs have some disadvantages compared with allogenic MSCs although they are the best safe cells. First of all, the culture of MSCs usually takes 4–7 weeks in view of cell amount, and the cell therapy may fail because the number of cells is insufficient, while it is easy to get enough allogenic MSCs within 2 weeks following admission, which has been isolated from healthy donors. Second, patients with cerebral infarction usually take antiplatelet or anticoagulant drugs, so the extraction of MSCs from bone marrow may lead to local bleeding. Third, patients with cerebral infarction are mostly elderly, the proliferation and differentiation capacity of stem cells is greatly decreased, and it is difficult to obtain sufficient stem cells. Fourth, MSCs will not pose any immunological problems because MSCs express low levels of HLA class I major histocompatibility complex (MHC) molecules and no class II MHC or costimulatory molecules [35]. So, allogeneic MSC transplantation is more suitable for the treatment of cerebral infarction patients, especially elderly patients.

In addition, mechanisms of MSC therapy in patients with ischemic stroke are unknown, and the researches on the mechanisms are limited to animal experiments [5, 6]. Moreover, whether there is a difference between human and animal important immune systems is still under controversy [36, 37]. Hence, it is necessary to explore the therapeutic mechanism of MSC transplantation in clinical trials.

The aim of the study is to determine the safety, efficacy, and therapeutic mechanisms of allogenic intrathecal MSC therapy in severe ischemic stroke (phase II).

Materials and Methods

Design

This is a prospective, randomized, controlled, observer-blinded phase II trial. The study will be held in the neurology department of the Sun Yat-sen Memorial Hospital, Sun Yat-sen University, China. The included subjects (n = 118) will be randomly divided into experimental (n = 59) and control (n = 59) groups according to a random number table, which is generated by computer. Patients with ischemic stroke at the subacute phase (30 to 90 days following onset) will receive four intrathecal infusions of allogenic BM-MSCs (1 × 106 cells/kg body weight) once a week. The primary objective will be assessed in 90 days after treatment. After four infusions, follow-up evaluations will be performed in 7, 30, 90, 180, and 360 days. So this trial includes ten visits, namely from V1 to V10. Details of patient follow-up are summarized in Table 2 and Fig. 1.

Table 2 Flowchart of a patient follow-up
Full size table
Fig. 1
figure 1

A schematic flow chart of the clinical trial

Full size image

Patient Population—Inclusion and Exclusion

The ischemic stroke included in this trial is based on the diagnosed standards of guidelines from the American Heart Association/American Stroke Association [38]. The detailed inclusion and exclusion criteria are shown in Table 3.

Table 3 Inclusion and exclusion criteria
Full size table

Randomization

The statistics office of Sun Yat-sen University will use a computer to generate a randomized sequence table for subjects. The subjects will be randomly divided into the experimental (n = 59) and control (n = 59) groups at a ratio of 1:1. To ensure the allocation concealment, the third party will place paper strips with black characters on a gray background into non-transparent envelopes which will be sealed with adhesive tape. Care providers and patients will not be masked; however, the outcome assessors will be masked to treatment allocation.

Interventions

Preparation of Allogenic BM-MSCs

All procedures were approved by the Ethics Committee and are accomplished at the Center for Biotherapy, Sun Yat-sen Memorial Hospital, Sun Yat-sen University (Guangzhou, China). All healthy donors were informed of the scientific contributions, possible risks and complications, and the corresponding prevention and treatment measures for bone marrow aspirations and signed the informed consent form. The protocols for isolation, expansion, passaging, and storing of BM-MSCs were performed as described by our previous works [39, 40]. After identifying MSC immunophenotype markers by flow cytometry, passages three to five will be used for the clinical trial.

Intervention Dose and Route

Intrathecal injection of allogenic BM-MSCs will be administrated in patients with ischemic stroke. The allogenic MSCs (1 × 106 cells/kg body weight) in 10 ml normal saline are slowly injected over approximately 10 min after the mixture with 2 mg (0.4 ml) dexamethasone and 0.6 ml normal saline (to prevent aseptic chemical meningitis) is injected. After the infusion of the MSCs, 2 ml of normal saline is injected to flush the syringe and spread the MSCs.

Clinical Outcomes

The primary outcome and secondary outcomes have been summarized in Table 4.

Table 4 Clinical outcomes of the trial
Full size table

Sample Size Estimates

Sample size is calculated for superiority hypothesis on the percentage of effective treatment. The effective treatment is defined as modified Rankin Scale (mRS) ≤ 3 in 90 days after cell transplantation, while mRS > 3 means an effective treatment. According to the two previous trials, sample size is calculated [8, 41] with a standard formula [42] to yield a sample size of 53 per group. To allow approximately 10% of patients to be excluded from the population, a total of 118 subjects (59 per arm) should be randomized to provide a study power of 90% with an alpha risk of 5%.

Statistical Analyses

Statistical analysis will follow the intention-to-treat (ITT) principle. The primary effect parameter is defined as the relative risk for improvement on the mRS and will be compared between the MSC treatment group and the control group using ordinal logistic regression [43]. The analysis will be repeated after adjustment for sex, age, basic scale scores, time since onset, previous stroke, hypertension, diabetes mellitus, hyperlipemia, atrial fibrillation, and history of smoke and alcoholic intemperance using multivariable regression analysis. For the analysis of the secondary outcomes, Student’s t test, Mann–Whitney tests, chi-square test, analysis of variance, and multivariable linear and logistic regression models will be used, where appropriate. All of the statistical tests will adopt a two-tailed test and P values < 0.05 are considered statistically significant.

Summary

This paper summarizes the methodology for a prospective, randomized, controlled, observer-blinded phase II trial. We believe that our study will provide a high level of evidence and a better understanding of allogenic MSC therapy via intrathecal injections in patients with severe ischemic stroke.