Study design
This was an open-label, single-center, phase I clinical trial performed at Samsung Medical Center, Seoul, Republic of Korea. First, participants underwent Ommaya reservoir implantation into the right lateral ventricle under local anesthesia, and then 4 weeks later, the participants received their first injection of MSCs followed by an additional two repeated hUCB-MSC injections at 4-week intervals (Fig. 1A, B). The participants received sequential hUCB-MSC (NEUROSTEM®) injections in a dose-escalating manner (Fig. 1). The rationale for the dose of injected hUCB-MSCs is provided in supplementary data. The first two participants (LD_01 and LD_02) received injections of a low dose (1.0 × 107 cells/2 mL) of hUCB-MSCs into the right lateral ventricular space via an Ommaya reservoir. Four weeks after the first injection, the dose-limiting toxicity (DLT) was evaluated. DLT was assessed according to the National Cancer Institute - Common Toxicity Criteria for Adverse Event (NCI-CTCAE) classification which encompassed grade 3 or higher toxicities [22]. After confirming the absence of DLT, the first two participants (LD_01 and LD_02) received their second hUCB-MSC injections, and the next two participants (LD_03 and HD_01) were subsequently enrolled to receive injections of a low (1.0 × 107 cells/2 mL) and high (3.0 × 107 cells/2 mL) dose of MSCs, respectively. During the entire study period, DLT was evaluated 4 weeks after each injection for each participant. We decided to continue repeated injections or enroll the next participants only when the absence of DLT was confirmed.
This trial was registered at ClinicalTrial.gov for 12 weeks of follow-up (Identifier: NCT02054208) and for 36 months of extended observation (Identifier: NCT03172117). We obtained written informed consent from every patient and legally authorized representatives in cases of impaired capacity. This study was approved by the Institutional Review Board of Samsung Medical Center and Ministry of Food and Drug Safety (MFDS).
Participants
We enrolled nine AD dementia patients at Samsung Medical Center from March 2014 to June 2017. Eligible patients were 50 to 85 years of age, fulfilled the criteria for probable AD dementia according to the National Institute on Aging-Alzheimer’s Association workgroup (NIA-AA) [23], and had a Mini-Mental State Examination (MMSE) score of 18–26 at screening. Eligible patients were further screened with amyloid positron emission tomography (PET), [18F] fluoro-2-deoxy-d-glucose (FDG) PET, and magnetic resonance imaging (MRI) and were enrolled when they satisfied the following criteria: (1) had significant amyloid burden assessed by 11C-Pittsburgh compound B (PiB) PET or 18F-Florbetaben PET and (2) had neuronal degeneration assessed by atrophy on structural brain MRI or hypometabolism on FDG PET.
Patients with one or more of the following conditions were excluded: (1) neurodegenerative diseases other than AD dementia; (2) severe white matter hyperintensities on fluid-attenuated inversion recovery (FLAIR) MR images at baseline, which were defined as a cap or band (periventricular white matter hyperintensities) ≥ 10 mm, and deep white matter lesions (deep white matter hyperintensities) ≥ 25 mm as modified from the Fazekas ischemia criteria [24]; (3) major psychiatric disorders; (4) history of stroke within 3 months of enrollment; (4) hepatic, renal, hematologic, or active pulmonary disorders; (5) current or previous history of malignancy; and (6) difficult to perform Ommaya reservoir insertion. Patients were required to have sustained a stable dose of acetylcholinesterase inhibitors or memantine for at least 60 days prior to enrollment and the same dose of medication was continued throughout the study.
Preparation of hUCB-MSCs
To be used for clinical purposes, hUCB-MSC manufacture, cell quality control, and quality assurance were performed in compliance with the Korea Good Manufacturing Practices requirements by the MFDS. hUCB tissue was obtained after receiving written informed consent from normal women in their full-term pregnancy. hUCB-MSCs were grown in α-Minimum Essential Medium (α-MEM, Gibco/Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Gibco/Life Technologies). Cells were cryopreserved at − 150 °C or lower by using 10% dimethyl sulfoxide.
To prepare for intracerebroventricular injection, frozen hUCB-MSCs were first thawed, seeded, and cultured. The cells were harvested 5 days after seeding, repeatedly washed to remove impurities such as fetal bovine serum and trypsin, and re-suspended in an appropriate amount of phenol red-free α-MEM. Therefore, fetal bovine serum was not present in the final drug product, NEUROSTEM®. hUCB-MSCs were tested for viability, phenotype, and presence of endotoxins, bacteria, and mycoplasma. After testing, 50 million cells were re-suspended in 1 mL of phenol red-free α-MEM, and a total volume of 2 mL was used to inject MSCs into a single patient of the low dose group. For the high dose group, from the adjusted final concentration of 150 million cells per 1 mL of phenol red-free MEM-α, a total volume of 2 mL was prepared for administration into each individual patient. hUCB-MSCs for the low dose group were stored at 4–20 °C and had a shelf life of about 60 h from the time of manufacture, and hUCB-MSCs for the high dose group were stored at 4–12 °C and had a shelf life of about 48 h. Based on flow cytometric analyses, the expressions of surface antigens of the cells were consistently positive for CD73, CD90, CD105, and CD166 but negative for CD45, CD14, and HLA-DR. The final products were delivered to the patient’s physician on the day of injection.
Ommaya insertion and intracerebroventricular injection of hUCB-MSCs
Thin-sliced brain CT scans of each patient were acquired for intraoperative navigation before surgery. Under local anesthesia, burr holes (approximately 15 mm) were made in the patient’s right Kocher’s point. An electromagnetic navigation system was used to guide the pathway to the lateral ventricle to avoid possible misplacement of the catheter and intra-parenchymal hemorrhage. The catheter was inserted about 6 cm from the dura opening and was connected to the reservoir chamber (Integra, NJ, USA). To assure that the catheter was securely inserted inside the lateral ventricle, cerebrospinal fluid (CSF) filling following pumping of the reservoir was checked, and intraoperative CT scans were also taken.
The average time interval between screening and Ommaya insertion was 13.7.0 ± 7.3 days. Four weeks following Ommaya reservoir implantation, the participants were admitted to the hospital and received baseline evaluation in the morning, and in the afternoon, a neurologist injected hUCB-MSCs into the intracerebroventricular space via the Ommaya reservoir (low dose 1.0 × 107 cells/2 mL or high dose 3.0 × 107 cells/2 mL). The participants were hospitalized for 2 days and were observed for signs of acute adverse events.
Safety assessments
Our primary goal was to assess the safety and DLT of two ascending doses of hUCB-MSCs in a total of three repeated injections. Safety was evaluated at each visit by examining the vital signs and weight, physical and neurologic examination, electrocardiogram, and clinical laboratory testing (hematologic and serum chemical testing). DLT was evaluated 4 weeks after each hUCB-MSC injection. Adverse events were assessed at each visit according to NCI-CTCAE [22]. In the extended observation study, adverse events were assessed at 18, 24, and 36 months after the end of the phase I trial. C&R Research Inc. served as external monitors of the study.
To detect immunologic reactions between the implanted hUCB-MSCs and the recipient, a mixed lymphocyte reaction was assessed at baseline and at 12 weeks after the first hUCB-MSC injection.
In order to monitor the complications (e.g., parenchymal or intraventricular hemorrhage) related to the Ommaya reservoir implantation, patients underwent brain CT scans at 24 h and 4 weeks after insertion of the Ommaya reservoir.
Clinical and laboratory assessments
To measure the clinical changes, patients underwent 11-item AD Assessment Scale-Cognitive Subscale (ADAS-Cog 11) testing (score range 0–70, lower score indicates better performance) [25], Seoul Instrumental Activities of Daily Living (S-IADL) (score range 0–45, lower score indicates better performance) [26], MMSE (score range 0–30, higher score indicates better performance), Clinician’s Interview-Based Impression of Change Plus Caregiver Input (CIBIC-Plus) [27], and Caregiver-Administered Neuropsychiatric Inventory (CGA-NPI) (sum of frequency × severity of 12 behavioral domains, score range 0–144, higher score indicates worse behavior) [28] at baseline, 4 weeks, 8 weeks, and 12 weeks after the first hUCB-MSC injection. In the extended observation study, the same assessment was done at 12 and 24 months after the end of the phase I trial.
We evaluated the changes in CSF white blood cell (WBC) count, AD biomarkers (CSF Aβ, total-tau, and phosphorylated-tau), and hUCB-MSC–related markers (Galactine-3, sICAM-1, progranulin, GDF-15, and Decorin) at baseline, 1 day, and 4 weeks after the 1st hUCB-MSC injection, 1 day and 4 weeks after the 2nd hUCB-MSC injection, and 1 day and 4 weeks after the 3rd hUCB-MSC injection. CSF was collected via the Ommaya reservoir.
To evaluate the structural changes, patients underwent brain MRI at baseline and 12 weeks after the first hUCB-MSC injection and at 12 and 24 months after the end of the phase I trial. To measure the changes in parenchymal Aβ deposition, patients underwent PiB-PET or florbetaben PET at baseline and 12 weeks after the first hUCB-MSC injection. Changes in standardized uptake value ratio (SUVR) were assessed.
Statistical analysis
Due to the small number of participants, absence of sham-surgery-control, and the fact that it was an open-labeled study design, we did not perform statistical analysis to compare the outcome measures of both the low- and high-dose hUCB-MSC recipients.