Safety of autologous bone marrow mononuclear cell transplantation in patients with nonacute ischemic stroke

Stroke is the third leading cause of death in developed countries [1] and the leading cause of death in some developing countries [2] including Brazil [3]. Furthermore, stroke remains the leading cause of disability in the world; at least 30% of stroke survivors do not make a complete recovery, and a further 20% require assistance for activities of daily living [4]. Although acute-stroke care facilities such as stroke units and the use of thrombolytics may improve prognosis, there are few clinical trials evaluating effective ways to enhance recovery in patients with residual disability [5]. In recent years, several studies have investigated the potential neuroprotective and restorative role of stem cells from different sources in animal models of brain ischemia [6]. Although the mechanisms of action are still unclear, these studies demonstrated that stem cell administration ameliorates the functional loss observed after ischemia, making stem cell transplantation an attractive approach to restore brain function after stroke in humans.

To date, only a few clinical studies evaluated stem cell transplantation in stroke patients [6]. Some of these studies employed cells derived from a human teratocarcinoma [7,8], in one study porcine cells were used [9] and in all of the studies the patients included were in the chronic phase of the stroke. Cell therapies using bone marrow-derived stem cells have also been reported in stroke patients. For example, Bang and colleagues examined the safety and efficacy of cultured expanded autologous mesenchymal bone marrow-derived cells administered intravenously 4–5 and 7–9 weeks after symptom onset in five stroke patients [10]. More recently, bone marrow mononuclear cells (BMMCs) harvested from the patients were stereotactically implanted into the perilesional area in five patients bearing chronic sequels of stroke [11]. Both studies concluded that bone marrow-derived stem cells are safe in patients with stroke in the subacute or chronic phase.

In our study we have used autologous BMMCs, as they are easily obtained and can be isolated in a short period of time just prior to transplantation, minimizing the chances of contamination and allowing treatment of the patients in the acute or subacute phase of stroke. Furthermore, the mononuclear fraction contains several types of bone marrow cells including stem cells and precursors, which can produce large amounts of cytokines and trophic factors that promote angiogenesis, neuroprotection and neuroregeneration after CNS injury in animal models of neurological diseases [12–18].

An important question to be addressed in stroke studies is related to the time window for treatment. Transplantation of stem cells during the first few days of ischemia presents several obstacles since the patients are very unstable in the acute phase. On the other hand, in the nonacute phase, the formation of scar tissue and the restoration of the blood–brain barrier might adversely affect the migration and homing of the transplanted cells. In order to address these issues, in our study a small percentage of the transplanted cells were labeled with ^99mTechnetium (^99mTc). We were able to demonstrate that the intra-arterially injected BMMCs were able to migrate and locate to the lesion at least in the first 2 h after transplantation [19,20]. However, it remained to be determined whether this method of transplantation of BMMCs was safe and whether these cells were also well tolerated for longer periods of time. The results reported here showed no adverse effects related to the transplanted cells during the procedure and during the follow-up period of 180 days in these patients. We conclude that intra-arterial transplantation of autologous BMMCs seems to be feasible and safe in nonacute stroke patients.

Patients & methods

▪ Subjects

This is an unblinded, uncontrolled Phase I study. The protocol and the consent form were approved by the Institutional Research Ethics Committee and the National Committee of Ethic and Scientific Research of Brazil. Written informed consent was obtained from all subjects. This study is registered at ClinicalTrials.gov (NCT00473057) [101].

Individuals aged 18–75 years of both sexes were eligible for the study if they had the following characteristics:

• ▪ Ischemic stroke in the middle cerebral artery (MCA) territory evidenced by computed tomography or MRI occurring within 90 days;

• ▪ Recanalization of the involved MCA as assessed by transcranial Doppler studies or by magnetic resonance angiography;

• ▪ A score between four and 17 according to the National Institutes of Health Stroke Scale (NIHSS).

We excluded patients who met any of the following criteria:

• ▪ Difficulty in obtaining vascular access for percutaneous procedure;

• ▪ Ipsilateral carotid stenosis (>50%, by Doppler studies);

• ▪ Neurological worsening (>four points in the NIHSS) before injection due to either edema or intracerebral hemorrhage;

• ▪ Thrombophilias or primary hematological diseases;

• ▪ Neurodegenerative disorders;

• ▪ Previous stroke with modified Rankin Scale (mRS) of more than 2;

• ▪ Intracardiac thrombus;

• ▪ Autoimmune disorders;

• ▪ Sepsis (according to the Society of Critical Care Medicine and the American College of Chest Physicians 1992 criteria);

• ▪ History of neoplasia or other comorbidity that could impact a patient’s short-term survival;

• ▪ Any condition that in the judgment of the investigator would place the patient under undue risk;

• ▪ Bone disorders that could increase the risk of the bone-marrow harvesting procedure;

• ▪ Liver failure renal failure (serum creatinine >2 mg/ml);

• ▪ Hemodynamic or respiratory instability;

• ▪ Lacunar stroke;

• ▪ Pregnancy;

• ▪ Previous participation in other clinical trials.

▪ Bone marrow aspiration, cell separation, injection & labeling

Cells were obtained by marrow aspiration of the iliac crest (∼80 ml) and processed by Ficoll density centrifugation. After washing, counting and viability testing, cells were resuspended in 10 ml of saline solution with 5% autologous serum. Cell isolation and manipulation was performed in a laminar flow cabinet with sterile equipment. Bacteriology and culture were also performed to rule out contamination of the material. Viability of the labeled cells was evaluated by the trypan blue exclusion test, and was estimated to be greater than 93% in all cases. Labeling efficiency (%) was calculated by the activity in the pellet divided by the sum of the radioactivity in the pellet plus supernatant, and was estimated to be greater than 90% in all cases. Approximately 1 ml of this solution was labeled with ^99mTc [19,20]. Labeled cells were added back to the total mononuclear cell suspension (final volume 10 ml) and slowly injected into the territory of the MCA, via the femoral artery, after navigation (Seldinger technique) and under local anaesthesia and conscious sedation. A routine coaxial technique with femoral arterial puncture was used. A 6 Fr guiding catheter (Envoy-Cordis, Miami, FL, USA or Guider Soft tip, Boston Scientific, Target Therapeutics, Fremont, CA, USA) was positioned at the cervical level with continuous flushing with normal saline. Intravenous heparin was used (bolus of 80 units/kg of body weight) as needed to maintain the activated clotting time between two- and three-times the baseline values. A large-inner-diameter microcatheter (SL 1018 Boston Scientific, Target Therapeutics) was navigated to the M1 portion of the MCA and the infusion was performed in approximately 10 min.

▪ Imaging

Whole-body, planar scintigraphies and single-photon-emission computed tomography were performed using a Millennium GE camera (General Electric Medical Systems, Milwaukee, WI, USA), as previously described [20]. Acquisition protocols were performed 2 and 24 h after cell transplantation. Computed tomography images were acquired before cell therapy with a 40-detector row scanner (Brilliance-40, Philips Medical Systems, Surrey, UK). Computed tomography or MRI were performed before and after the procedure during the follow-up.

▪ Neurologic evaluation

All individuals were evaluated at admission, on the day of transplantation, and 1, 3, 7, 30, 60, 90, 120 and 180 days after cell infusion using the NIHSS, the Barthel Index (BI) and the mRS by a single board-certified neurologist (VB). Routine laboratory tests (complete blood count and biochemical examinations: urea, creatinine and electrolytes) were carried out at these points. An electroencephalogram was obtained within 7 days after transplantation.

Results

The main goal of this study was to examine the safety and feasibility of BMMC transplantation in patients with MCA ischemic stroke between 2 and 3 months after stroke onset. The patients included in the study were evaluated using blood tests, NIHSS, mRS and BI before treatment, and 1, 3, 7, 30, 60, 90, 120 and 180 days after the infusion of the BMMCs. A minimum of 1 × 10⁸ and maximum of 5 × 10⁸ BMMCs were injected 2–4 h after bone marrow aspiration. The pharmacological treatment and rehabilitation therapy of each patient was maintained unaltered during the follow-up period.

▪ Baseline characteristics

The main characteristics of the patients included are shown in Table 1. In summary, six male patients (aged 24–65 years) received intra-arterial BMMCs 59–82 days after mild-to-moderate MCA infarcts (NIHSS between four and 13). Three of the patients had received thrombolytic therapy and one of them was submitted to a craniectomy during the acute stage of stroke. Two patients were taking an oral anticoagulant drug to prevent recurrent strokes, and were switched to low-molecular-weight heparin (enoxaparine 1 mg/kg twice daily) 1 week before the procedure.

▪ Imaging

The patients included in this study were in the nonacute phase of stroke and the blood–brain barrier was likely closed by this time. To assess whether the injected cells were still able to reach the lesioned region we investigated the distribution of BMMCs labeled with ^99mTc 2 and 24 h after transplantation. Whole-body scans obtained 2 h after cell transplantation showed the presence of ^99mTc-labeled cells in the brains of all patients. The activity of the radioisotope in the brain was 0.6–5.1% of the activity in the whole body as described previously [20]. A representative image illustrating the whole-body biodistribution of BMMCs is shown in Figure 1.

At 24 h, cell homing could only be visualized in the brains of two patients, while in all patients uptake was seen in the liver, lungs, spleen, kidneys and bladder [20]. The absence of labeled cells in the brain of the remaining patients 24 h after the transplant could be due to the decay of the radioactivity compound below the levels of detection and/or to the decrease in the number of cells at the lesion site.

▪ Safety of the procedure

Serial clinical, laboratory and radiographic evaluations showed no deaths, stroke recurrence or cell-related adverse events during the procedure or during the follow-up period of 180 days. These results confirm the excellent tolerance to the procedure.

The electroencephalogram performed after the transplantation showed polymorphic slow activity in the ischemic area and only one patient exhibited spike-wave activity without clinical manifestation. Two patients suffered generalized seizures after the end of follow-up (around 200 days after the BMMC infusion); one was successfully treated with phenytoin and the other was treated with a combination of oxcarbazepine and lamotrigine.

▪ Neurological evaluation

Table 1 shows a summary of the values of the NIHSS, BI and mRS before and after transplantation of BMMCs.

Only one patient (patient three) was independent in daily activities at BMMC infusion day (BI: 95, mRS: 1). No signs of worsening in the neurological condition were observed immediately after the procedure or during the follow-up period. In fact, at the 180-day follow-up evaluation, all patients had improved their scores in comparison with the values before transplantation. For example, the NIHSS scores improved (range -1 to -8 points) during follow-up in all patients (Table 1 & Figure 2).

Discussion

In the present series of patients, BMMC transplantation proved to be safe and feasible in six patients with ischemic stroke in the MCA territory within 90 days after onset. There were no complications or unexpected outcomes related to the procedure. All patients improved their neurological scores (NIHSS, BI and mRS) at the end of follow-up.

A small percentage of the injected cells were labeled with ^99mTc, which allowed us to demonstrate that BMMCs were capable of migration and homing to the lesion site. ^99mTc has a half-life of 6 h, which is a significant advantage over ¹⁸F-fluorodeoxyglycose, which has a half-life of 110 min. ¹¹¹Indium-oxine (¹¹¹In-oxine) has a half-life of 2.81 days and allows monitoring for approximately 96 h, whilst ^99mTc permits tracking for 24–48 h; however, ¹¹¹In-oxine has shortcomings including suboptimal photon energies, low-resolution images and the 18–24-h period between injection and imaging that is generally necessary [21,22]. Moreover, studies have suggested that ¹¹¹In-oxine may have deleterious effects on different cells, including hematopoietic progenitor cells [23] and mesenchymal stem cells [24]. ^99mTc allows imaging for 24–48 h and results in higher image resolution and a lower radiation burden to the patient [21,22].

Because of this window for cell tracking, it was not possible to assess exactly how long the cells remained in the brain. This has been an unsolved issue in the few clinical trials performed so far and even in animal models, and new methods of cell labeling have to be developed in order to demonstrate the presence of transplanted cells for longer periods of time and/or cell fate. In spite of that, we were able to demonstrate that BMMCs could be used as therapy in the nonacute phase of the stroke since they were able to migrate to the ischemic region even after restoration of the blood–brain barrier.

The injection of large amounts (up to 5 × 10⁸) of bone marrow-derived cells intra-arterially and the observation that some of these cells may be able to cross the blood–brain barrier could raise many safety concerns that have to be addressed in clinical trials. We have used the maximal amount of cells that could be obtained with local anesthesia and mild sedation. The use of general anesthesia, which could allow us to increase the volume of bone marrow aspirate, may also potentially cause several adverse events. Moreover, the amount of cells used was comparable to other clinical trials using BMMCs for myocardial diseases [25–31].

The results of our study showed that no adverse events were observed up to 180 days after transplantation that could be related to the procedure or to the presence of the transplanted BMMCs in the brain. Similar results have also been reported by our group in patients transplanted in the acute phase of the stroke (up to 10 days after ictus) [Friedrich M et al., Unpublished Data].

In our series, two patients suffered seizures approximately 200 days after the cell infusion that were controlled pharmacologically. These patients are under extended follow-up. Late seizures (at least 2 weeks after the stroke) [32,33] occur in 3–67% of the patients, varying considerably among series [32–35], and although several risk factors for early or late seizures have been identified, there is no clear predictor of poststroke epilepsy [36]. Based on these observations and on the small sample of our study we cannot at present either rule out or conclude that the incidence (two out of six) of seizures is explained by chance or attributed to the cell therapy.

In animal models of ischemia, stem cells from different sources have been used with promising results, and different mechanisms of action have been suggested to explain these functional benefits [37–39]. For example, it was argued that the introduction of neural stem cells into areas of cell loss may result in repair and restoration of circuitry [40] through neuroprotective [41,42] and immunomodulatory effects [43,44], as well as potentially leading to some cell replacement [45,46]. Alternatively, when bone marrow-derived stem cells (mesenchymal or mononuclear cells) are employed, the functional benefits observed are probably due to the release of cytokines and/or trophic factors, which may have an immunomodulatory effect and/or contribute to a reduction in apoptosis in the penumbra area in earlier periods after ischemia [13,14] . In addition, it has been suggested that cell-based therapies amplify the endogenous processes of brain repair and plasticity, including neurogenesis, angiogenesis and synaptogenesis, which could explain the benefits observed in experimental models of stroke, even when cells are administered at later times [18,47–49].

Cell therapies using bone marrow-derived stem cells (mesenchymal cells or the mononuclear cell fraction) have been well documented in several diseases, including myocardial infarction and limb ischemia [25–30,50–52], and may also represent new and important strategies for the treatment of stroke.

At present, only a few clinical studies involving cellular therapies in patients with acute and chronic stroke have been published and some of them involved therapy with autologous bone marrow-derived stem cells [7–10,53–55]. Bang and coworkers investigated intravenous infusion of autologous mesenchymal bone marrow cells, expanded in vitro, in patients with acute cerebral infarcts [10]. The procedure was safe and feasible; the group receiving mesenchymal cells (n = 5) had improved BI and mRS scores after the infusion 3 and 6 months after the onset of symptoms. However, a shortcoming of the study is cell preparation; although feasible, it is time consuming, potentially dangerous and expensive. Two other studies carried out during the acute phase of stroke (3–10 days postictus) evaluated the safety of intra-arterial BMMC transplantation in patients with MCA territory infarcts in two different Brazilian institutions [54,55]. Preliminary reports showed that the procedure is safe and feasible [54,55].

Although the transplantation of bone marrow-derived stem cells in stroke patients seems to be safe and feasible, many questions remain unanswered, including the dose of cells, administration route, timing of the infusion (acute vs nonacute vs chronic stage of stroke), the territory of stroke (cortical vs subcortical territories, anterior vs posterior circulation) and the mechanisms of action of bone marrow-derived stem cells in the setting of stroke. In our study, the rationale for selecting the cell dose was based on the results of the preliminary safety studies described previously [53–55] and the reason for selecting nonacute patients was based on results of some experimental models of cell therapy in stroke [56–58]. However, a therapeutic window should be determined as a function of therapeutic dose, and the repeated-dose regimen could optimize recovery benefit [59]. An additional shortcoming of our study is the absence of a control group and this is particularly relevant in studies involving diseases with spontaneous recovery. For this reason, any conclusions regarding the efficacy of the therapy should be postponed until a Phase II clinical trial with a control group included has been conducted.

Conclusion

Transplantation of BMMCs is feasible and appears to be safe in patients with nonacute ischemic stroke. Further randomized clinical trials are necessary to establish the efficacy and long-term safety of this procedure.