Design

A clinical retrospective study.

Time and setting

The study was accomplished from July 2010 to March 2011 in the Second Affiliated Hospital of Kunming Medical University, China. 

Subjects

Twenty-five patients with spinal cord injury in the late stage (injury time > 6 months) were included as the treatment group, aged 18–48 years.

All cases had complete or incomplete traumatic cervical, thoracic and lumbar injury. Cases with primary spinal cord disease, such as myelitis, infection and tumor, were excluded.

Another 25 patients with spinal cord injury in the late stage (injury time > 6 months), aged over 18 years, who received only traditional rehabilitation therapy and no stem cell therapy were included as the control group. The inclusion and exclusion criteria were the same as treatment group.

Before receiving rehabilitation, all patients underwent spinal surgery in the orthopedics department of different hospitals. All patients had normal blood cell counts and had no tumor or coagulation disorders. There were 5 cases of cervical spinal injury, 11 cases of thoracic spinal cord injury and 9 cases of lumbar spinal cord injury. No patient had shown any neurological improvement before stem cell therapy. All patients received somatosensory evoked potentials tests^[18] and other tests of neurological function before and after the stem cell therapy. We examined the sensation and muscle strength in key points of bilateral limbs, and used the American Spinal Cord Injury Association score to evaluate the sensory and motor function of patients. All patients were followed-up for 12 months after stem cell therapy. The moral principles of this study were in accordance with the Administrative Regulations of Medical Institutions formulated by the State Council of the People’s Republic of China^[33].

Methods

Preparation of human umbilical cord blood stem cells

Umbilical cord blood (100–150 mL) was collected from healthy unrelated donors, after obtaining signed informed consent forms in accordance with the sterile procurement guidelines for cord blood in each hospital^[34]. Mononuclear cells were collected and washed twice in saline. Contaminating erythrocytes were lysed with lysis buffer (Beyotime, Shanghai, China) comprising of injection-grade water. Cell density was adjusted to 2–6 × 10⁶/mL and seeded in DMEM/F12 culture medium with basic fibroblast growth factor and epidermal growth factor (Peprotech, Rocky Hill, NJ, USA) at a concentration of 20 ng/mL. Culture media (DMEM/F12; Gibco, New York, USA) was mixed with 2% v/v B-27 Stem Cell Culture Supplement (Gibco). Cells were cultured at 37°C with saturated humidity and 5% CO₂ by volume. At this stage, all relevant information about the initial culture was entered in the batch information record, including test results for sterility, mycoplasma and endotoxins. Cell growth was regularly monitored and the inspection records were updated accordingly. Cells were harvested for clinical application after 1 week of cultivation with cell quantity ≥ 1 × 10⁷ and viability ≥ 95%.

To ensure the quality of the umbilical cord blood-derived mononuclear cells, a number of parameters were confirmed before use. Raw material control: Tests for communicable diseases (hepatitis B virus, hepatitis C virus, human immunodeficiency virus, alanine transaminase and syphilis) for umbilical cord blood units were performed before any processing began. Testing was performed by a third party laboratory under local government-monitored conditions.

In-process control: Non-qualifying cells were eliminated in accordance with Beike’s cell counting and morphology standards, which include a cell quantity of ≥ 1 × 10⁷ and highly homogeneous cells that have a rounded shape and have detached from the culture flask.

Culture control: Any contaminated cell suspensions or unhealthy cells were eliminated upon discovery. Contamination was determined by the presence of mycoplasma or visible microorganisms by microscopy. Furthermore samples were required to have an endotoxin level ≤ 0.5 EU/mL and be negative of free DNA.

Finished product control: This incorporates a final cell count (≥ 1 × 10⁷), containing 1.0–2.0% CD34⁺ cells as determined by flow cytometry (BD Bioscience Pharmingen Inc., San Diego, CA, USA), cell viability   (≥ 95%) and sterility test.

Transplantation of human umbilical cord blood stem cells

Depending on the patient’s condition, they were admitted to receive stem cell infusion by lumbar puncture and intravenous infusion, which was repeated four or five times. Treatments were separated by      1 week intervals. At the first time of therapy, a 30-mL intravenous infusion of cell suspension was administered through an intravenous catheter over a period of 20–30 minutes. Following this, the next three treatments were administered by lumbar puncture, which was performed in the lateral decubitus position, with the patient prepped and draped in sterile fashion, and the needle placed in the lumbar subarachnoid space. Flow of the cerebrospinal fluid into the syringe needle was evidence of the needle being in the correct place in subarachnoid space. Thus, the stem cells could be injected into the correct place successfully and accordingly exert their effects, which was the criterion of successful stem cell transplantation. 4 mL of cerebrospinal fluid was removed and replaced with    4 mL of cell suspension containing 1–3 × 10⁷ cells. The color and pressure of the cerebrospinal fluid were observed and recorded to determine whether they were normal. During the progress, any abnormal reactions of the patients were observed. Stem cell therapy was implemented by Professor Ao, who was the item director of stem cell treatment for spinal cord injury. 

Before receiving traditional rehabilitation, such as strength exercise and electrical stimulation, all patients received spinal fixation surgery by hospital orthopedic departments. Then, the patients in the treatment and control groups received their corresponding treatments between July 2010 and March 2011 in the Second Affiliated Hospital of Kunming Medical University, China. To evaluate the effect of stem cell therapy, the patients were informed to return to the hospital for functional evaluation at different time points during follow-up assessments.

Observation during 12-month follow-up after stem cell therapy

From the first day after infusion, abnormal reactions, such as fever, headache or lumbago, were recorded and the patients received rehabilitation training at an early stage if they had no abnormal reactions. The safety was evaluated by patient complication rates. The neurological functions were evaluated by American Spinal Cord Injury Association score^[34], which evaluates the strength of 10 symmetrical muscle groups and different sensory levels in the body, and scores them as follows: A: complete injury; B: sensory function remains, but the motor function is lost; C: sensory and motor function remains, but more than half of the muscle groups have a  strength < 3 below the injury level; D: sensory and motor function remains, but more than half of muscle groups have a strength > 3 below the injury level; E: normal function.

Other standards that were evaluated included autonomic nerve function (sweating), Ashworth scales, and somatosensory evoked potential values in limbs at different time points before and after treatment^[18]. Sweating tests were performed using dry iodine and amylum. Dry iodine and dry amylum were placed on the skin of the patients’ toe; if the patient had normal sweating function, the iodine and amylum would become wet and the amylum would change color from white to blue. The somatosensory evoked potential tests were detected using an electromyogram instrument (NTS-2000; Nuo Cheng, Shanghai, China). The results of this study were conducted and evaluated by the same doctor. The positive effects of stem cell therapy were defined by improvement of American Spinal Cord Injury Association score (from A to E), improvement of autonomic nerve function (revival of sweating function), decreased spasm (Ashworth score from 5 to 0) and revival of neurological transmit function, which was indicated by a reduced response time(s) of lower limb(s) under stimulation in both paraplegic and quadriplegic patients after treatment. 

Statistical analysis

Statistical analysis was performed using SPSS 17.0 statistical software (SPSS, Chicago, IL, USA). Rates of complication and effectiveness were calculated and expressed as percentage. Statistical data were expressed as mean ± SD, and intergroup comparisons were applied for the mean estimates, Student’s t-test (the data was in accordance with normal distribution) was applied to two non-related parametric samples (independent or non-paired). A level of 5% was set as significant.

1 The safety of human umbilical cord blood stem cell transplantation in the treatment of traumatic spinal cord injury (injury time > 6 months) was observed.
2 The restoration of neurological function was explored at 12 months after stem cell transplantation in patients with traumatic spinal cord injury. In addition, a neuroelectrophysiological monitoring system, the somatosensory evoked potential test, was performed to determine the nerve conduction functions of patients, thus reflecting the conduction of the spinal cord.
1 实验观察了创伤性脊髓损伤时间大于6个月的患者，经人脐血干细胞移植治疗后的安全性。
2 实验观察慢性脊髓损伤细胞移植后12个月患者各项神经功能恢复情况，还采取了特有的神经电生理检测手段--体感诱发电位检测患者神经传导功能，更为精确、客观地反映脊髓的传导功能。

目前，关于干细胞治疗脊髓损伤临床疗效的研究结果都是对于急性脊髓损伤的近期疗效（损伤时间小于3个月），对于后期慢性脊髓损伤患者（损伤时间大于3个月）的远期疗效鲜有报道。中国干细胞治疗脊髓损伤的临床研究报主要集中于观察患者神经功能的恢复。文章除了观察慢性脊髓损伤（损伤时间大于6个月）患者远期的各项神经功能恢复情况外，还采取特有的神经电生理检测手段--体感诱发电位检测患者神经传导功能，更为精确、客观地反应了脊髓的传导功能。有力地证明了该治疗方法的有效性。具有显著的独创性和新颖性。