A brain injury is defined as an alteration in brain function or other evidence of brain pathology caused by trauma, including trauma resulting from motor vehicle accidents, falls, hypoxic insults, infections, and other conditions. Survivors—not only in severe cases but also in moderate or mild brain injury cases—experience the significant burdens of physical and neuropsychological disabilities. These disabilities disrupt the lives of patients and their families and result in substantial health care and social costs [1]. Chronic histopathological changes, such as cell death, axonal injury, vascular damage, and inflammation, have long-term persistence in brain injury survivors [2]. Thus, it is important to develop a novel approach to treating chronic brain injury.

The intravenous infusion of mesenchymal stem cells (MSCs) has shown therapeutic efficacy in experimental animal models of neurological diseases and injuries, including cerebral ischemia [3-12], spinal cord injury (SCI) [13-16], chronic epilepsy [17], and peripheral nerve injury [18-20]. The suggested therapeutic mechanisms of MSCs from animal studies include the secretion of neurotrophic factors that can provide neuroprotection [14,17,21], neovascularization [22,23], the restoration of the blood-brain barrier [13,24], the regeneration of axonal injury [13,25], remyelination [13], synaptogenesis [12,25], induced neural plasticity [12,25], and remote effects [15]. These therapeutic mechanisms may have beneficial effects on chronic brain injury as well. We also conducted clinical studies in which the intravenous infusion of autologous MSCs in patients with stroke [26,27] and SCI [28] was performed, and we showed its safety and improvements in neurologic symptoms. Thus, we hypothesize that the intravenous infusion of MSCs may have therapeutic efficacy for patients with chronic brain injury.

The objectives of the proposed clinical trial include evaluating the safety and efficacy of intravenously infused autologous MSCs that are expanded with autosera in patients with chronic brain injury.

The (phase 2) trial will be a single-arm, open-label trial. The outline of the clinical protocol is shown in Figure 1. We will infuse the MSCs at least 180 days after onset. This trial will be carried out at Sapporo Medical University Hospital, Japan. The study protocol was based on advice provided by the Pharmaceuticals and Medical Devices Agency (PMDA) in Japan.

We propose to provide an extensive rehabilitation protocol to all participants prior to MSC infusion in order to exclude the potential effects of rehabilitation alone and evaluate MSC-specific effects. Briefly, all patients with chronic brain injury will receive formal rehabilitation (at least 80 min/weekday for 4 weeks) and continue formal rehabilitation until they show no further improvements in Fugl-Meyer assessment (FMA) scores for the last 2 weeks prior to MSC infusion. Thus, we expect to evaluate the therapeutic effects of MSC infusion in addition to the effects of rehabilitation therapy. We will evaluate FMA scores approximately every week. The clinical data will be collected by at least 1 physical therapist and at least 1 Japanese board-certified neurosurgeon at Sapporo Medical University Hospital. If the patients require more than 2 weeks to reach an FMA score plateau, we will continue formal rehabilitation until they show no further improvements for the next 2 weeks.

For cell preparation, peripheral blood draws from each patient for autoserum and bone marrow collection will be performed. The autologous MSCs will be cultured in autologous sera, and the autologous human MSCs (called STR-01) will be manufactured in a cell processing center at Sapporo Medical University. STR-01 will be prepared under good manufacturing practice conditions by personnel, who have received formal good manufacturing practice training, within a facility with highly controlled temperature, room air, pressure, and environmental conditions and then cryopreserved at −150 °C until its use, as previously described [28]. On the day of infusion, cryopreserved units will be thawed at the bedside in a 37 °C water bath and will be administered with saline to each patient for approximately 60 minutes. The total number of cells in the STR-01 product is based on our previous study (0.5 × 10⁸ to 2 × 10⁸ cells per patient) [27]. Hospital treatment, including rehabilitation for a target of 80 min/weekday, will be performed for 180 (±14) post-MSC infusion days, after which final outcome measures will be evaluated.

We will recruit 10 people (minimum: 6; maximum: 20) with a modified Rankin Scale (mRS) grade of 3 to 5. The focus of our trial is to establish safety and potential efficacy. However, it should be noted that if the proportion of cases in which the mRS grade improves by 1 point or more exceeds 10%, the trial will provide significant clinical benefit. To detect a difference by using a Z-test with continuity correction, a minimum of 6 people is required. Thus, the target sample size of people with an mRS grade of 3 to 5 was determined to be 10 (minimum: 6; maximum: 10), under the consideration of dropout.

This study protocol was approved by the institutional review board (IRB) at Sapporo Medical University Hospital (approval number: 29-15). Changes to the protocol will require IRB and Japanese PMDA approval. The trial will be conducted in accordance with the ethical principles of the Declaration of Helsinki, the Japanese Pharmaceutical Affairs Law, and good clinical practice. Trial investigators will obtain written informed consent from each participant before study inclusion. If the participants do not have the ability to write, written informed consent will be obtained from a legal representative alone. Participants will be free to withdraw from the study at any time.

The results from our study will be presented in peer-reviewed publications and meetings without identifying data. Written consent for publication will be obtained from all participants in the study.

Since MSCs will be collected from the participants (autologous MSCs) in our study, we must start the MSC cultures after primary registration in order to initiate the collection of peripheral blood and bone marrow when the protocol therapy starts. Afterward, we must confirm that the MSC product passed the shipping standards before infusion. Thus, we must perform a secondary registration of the participants. Therefore, case registration will require 2 steps in the trial; we will register the participants before blood and bone marrow collections (first registration) and before the infusion of MSCs (second registration).

For the first registration, the inclusion criteria will be as follows: (1) a brain injury oth.er than stroke diagnosed via magnetic resonance imaging (MRI), computed tomography, 3D computed tomography angiography (3D-CTA), or angiography (including a suspected brain injury other than stroke); (2) a classification of grade 3 to 5 on the mRS; (3) patients aged 20 to 80 years; (4) patients whose rehabilitation can be performed for at least 80 minutes per weekday; and (5) written informed consent, which will be obtained as much as possible from the participants. If a participant does not have ability to write, written informed consent will be obtained from a legal representative alone.

For the first registration, the exclusion criteria will be as follows:

For the second registration, the inclusion criteria will be as follows: (1) ≥180 days after the onset of a brain injury other than stroke; (2) patients who, after rehabilitation for at least 80 minutes per weekday or as much as possible over the past 1 month or more, showed no improvement in FMA score over the last 2 weeks (definition of improvement in FMA score: improvement by 1 point or more, as shown by the total FMA score); (3) a classification of grade 3 to 5 on the mRS; and (4) patients who are ready for the infusion of STR-01 that satisfies the specifications of the acceptance criteria.

For the second registration, the exclusion criteria will be as follows:

Outcome measures will be performed by more than 2 Japanese board-certified neurosurgeons at Sapporo Medical University Hospital.

The plan of analysis for study data will be performed by a biomedical statistician (YMI), and statistical analyses will be performed by using JMP 11.1 for Windows (SAS Institute Inc). The details are described in our statistical analysis plan and are described briefly in the following subsections.

The participants of the trial will follow standard clinical procedures during the study; thus, there will be no specific posttrial care. As the participants are the patients within Japan’s National Health Insurance system, postcare will be provided through the National Health Insurance schemes, if necessary. Clinical research insurance for studies will also be covered.

All investigators will have access to the trial data.

All participant trial data will be entered into the electronic data capture system hosted at the Translational Research Center for Medical Innovation (Kobe, Japan). The data collected will be deidentified by using unique study code numbers. To maintain the privacy of the participants, any reports of individual data will only consist of clinical data without any names, addresses, or identifying information. This complies with the university’s IRB guidelines. All patient-related information and data that are generated will be maintained on a secure server. Data monitoring will comply with the university’s policies, its guidelines, and the data management plan that was approved for the study. Data will be audited at an appropriate period by the EPS Corporation (Tokyo, Japan). At the completion of the study, the results will be submitted for publication in a peer-reviewed journal and presented at national and international conferences.