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 Table of Contents  
STUDY PROTOCOL
Year : 2016  |  Volume : 1  |  Issue : 1  |  Page : 1-8

Safety and efficacy of human umbilical cord-derived mesenchymal stem cells in patients with Alzheimer's disease: study protocol for an open-label self-control trial


Department of Hematopoietic Stem Cell Transplantation, Cell and Gene Therapy Center, the 307th Hospital of Chinese People's Liberation Army, Beijing, China

Date of Web Publication5-Apr-2016

Correspondence Address:
Bin Zhang
Department of Hematopoietic Stem Cell Transplantation, Cell and Gene Therapy Center, the 307th Hospital of Chinese People's Liberation Army, Beijing
China
Hu Chen
Department of Hematopoietic Stem Cell Transplantation, Cell and Gene Therapy Center, the 307th Hospital of Chinese People's Liberation Army, Beijing
China
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Source of Support: None, Conflict of Interest: None


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  Abstract 

Background: Animal model experiments have demonstrated the efficacy and safety of human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) in the treatment of Alzheimer's disease. However, the clinical efficacy of hUC-MSCs in the treatment of Alzheimer's disease in humans remains unclear. This study protocol for a phase I/II clinical trial was designed to investigate the clinical safety (primary outcome) and efficacy (secondary outcome) of hUC-MSCs in the clinical treatment of Alzheimer's disease.
Methods/Design: This open-label, self-control, single-center prospective phase I/II clinical trial will be performed at the Department of Hematopoietic Stem Cell Transplantation, Cell and Gene Therapy Center, the 307 th Hospital of Chinese People's Liberation Army, China. Thirty patients with moderate to severe Alzheimer's disease will be included. The primary outcome is the number of patients who have adverse events at 10 weeks and 1 year after hUM-MSCs therapy. Secondary outcomes include cognitive function, overall clinical status, daily living activities, neuropsychiatric symptoms, blood biochemical Alzheimer's disease markers.
Discussion: The findings from this trial will provide valuable evidence for the use of hUM-MSCs in the clinical treatment of Alzheimer's disease.
Trial registration: ClinicalTrial.gov identifier: NCT01547689; registered on 5 March 2012. This study protocol was approved by the Ethics Committee of the 307 th Hospital of Chinese People's Liberation Army.

Keywords: clinical trial; neurodegenerative disease; Alzheimer′s disease; umbilical cord-derived mesenchymal stem cells; stem cells; cell transplantation; self-control trial


How to cite this article:
Niu Jw, Zhang B, Chen H. Safety and efficacy of human umbilical cord-derived mesenchymal stem cells in patients with Alzheimer's disease: study protocol for an open-label self-control trial. Clin Trials Degener Dis 2016;1:1-8

How to cite this URL:
Niu Jw, Zhang B, Chen H. Safety and efficacy of human umbilical cord-derived mesenchymal stem cells in patients with Alzheimer's disease: study protocol for an open-label self-control trial. Clin Trials Degener Dis [serial online] 2016 [cited 2018 Dec 14];1:1-8. Available from: http://www.clinicaltdd.com/text.asp?2016/1/1/1/179741

Conflicts of interest
None declared.
Author contributions
BZ and HC conceived and designed the trial protocol. JWN wrote the paper. BZ read and revised the paper. All authors approved the final version of this paper.
Plagiarism check
This paper was screened twice using CrossCheck to verify originality before publication.
Peer review
This paper was double-blinded and stringently reviewed by international expert reviewers.



  Background Top


Alzheimer's disease (AD) is a progressive neurodegenerative disease clinically characterized by loss of memory and severe cognitive impairment. The primary pathological features of AD include amyloid-β (Aβ) plaque formation, neuronal loss and the presence of neurofibrillary tangles induced by tau hyperphosphorylation. Aβ deposition in the brain (Lee et al., 2009), in particular in the hippocampus and cerebral cortex, further results in neuronal loss. The majority of AD patients develop inflammatory plaques, neurofibrillary tangles, and neurodegenerative symptoms. At this stage, an effective method to treat patients with AD is to block Aβ deposition, increase cell survival rate and supplement the lost cells (Parekkadan and Milwid, 2010). Stem cell replacement therapy has come into the spotlight. Its mechanism of action is to infuse healthy stem cells into a patient's body which then repair or replace injured cells or tissues. After infusion, healthy stem cells can migrate toward the sites of injury and then differentiate to replace lost or injured neurons, thereby treating nerve injury. Embryonic stem cells, embryonic germ cells, neural stem cells, bone marrow-derived stem cells, mesenchymal stem cells, umbilical cord blood-derived stem cells, amniotic fluid stem cells, skin-derived stem cells and adipose tissue-derived stem cells can be used in stem cell replacement therapy (Shihabuddin and Aubert, 2010; Zhan et al., 2010, 2011).

Human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) have recently become an area of interest in stem cell replacement therapy for neurodegenerative diseases. hUC-MSCs are a kind of stem cells derived from umbilical cord and perivascular tissue. They are considered ideal seed cells to replace bone marrow mesenchymal stem cells because they are abundant in content and easy to harvest, proliferate rapidly, and have stable biological properties and no ethical issues (Shetty et al., 2010; Fan et al., 2011; Taghizadeh et al., 2011; Dalous et al., 2012). hUC-MSCs show high self-renewal ability and multi-directional differentiation potential (Liu et al., 2010). Under certain induction conditions, hUC-MSCs can differentiate into fibroblasts, adipose cells, chondrocytes, myocytes and vascular endothelial cells, neurons and glial cells and they can also express neurotrophic factors, vascular endothelial growth factors, brain-derived neurotrophic factors and glial cell line-derived neurotrophic factors (Wang et al., 2004), which brings new hope to AD treatment.

Recent animal experiments have demonstrated that intracerebroventricular administration of hUC-MSCs can improve learning and memory abilities in mouse models of Alzheimer's disease induced by chemical (D-galactose + aluminum chloride) and physical injuries (Ma et al., 2012). Intravenous administration of hUC-MSCs can greatly improve learning and memory abilities and postpone aging in APP transgenic mouse models of AD, possibly through regulating the expression of senescence-related genes p21, p53, silence information regulator 2 (Sir2) and proliferating cell nuclear antigen (PCNA) (Cui et al., 2015). Studies are needed to further investigate the safety and efficacy of hUC-MSCs transplantation for AD treatment in the clinic.

We designed a prospective self-control, phase I/II clinical trial to validate the safety (primary outcome) and efficacy (secondary outcome) of hUC-MSCs transplantation for AD treatment in the clinic.


  Methods/Design Top


Study design and setting

This is an open-label, self-control, single-center prospective phase I/II clinical trial that will be performed at the Department of Hematopoietic Stem Cell Transplantation, Cell and Gene Therapy Center, the 307 th Hospital of Chinese People's Liberation Army, China. The endpoint of this trial will be completion of final follow-up of the last recruited AD patients. This trial was registered at ClinicalTrial.gov (identifier: NCT01547689).

Patients

Patients with moderate to severe AD who will receive treatment in the Department of Hematopoietic Stem Cell Transplantation, Cell and Gene Therapy Center, the 307 th Hospital of Chinese People's Liberation Army, China will be included in this trial. Under the assistance of a physician in charge, a clinical research associate will examine whether the patients meet the inclusion criteria and ensure that each patient provides signed informed consent prior to entry into trial. A sample size of 30 is required.

The patients who fulfill the criteria detailed below will receive hUC-MSC transplantation.

Inclusion criteria

  • 50-85 years of age
  • Either gender
  • Correspondence with the NINCDS-ADRDA Alzheimer's Criteria proposed by the National Institute of Neurologi­cal and Communicative Disorders and Stroke and the Alzheimer's Disease and Related Disorders Association (McKhann et al., 2011)
  • Mini-Mental State Examination (MMSE) (Galea and Wood­ward, 2005) score of 3-20 (including score 3 and score 20)
  • Have previously not received stem cell therapy
  • Informed consent provided, signed by the patients them­selves or their guardians, and approval by the Hospital Ethics Committee


Exclusion criteria

  • Malignant tumors
  • Human immunodeficiency virus (HIV)-positive
  • Unable to receive MRI or CT examination
  • Mental disorders (such as depression, schizophrenia and manic depression)
  • Dementia caused by other causes excluding Alzheimer's disease (such as central nervous system infection, Creutzfeldt-Jacob disease, severe traumatic brain injury, Pick's disease, Huntington disease and Parkinson's disease)
  • Vascular dementia diagnosed according to the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition (American Psychiatric Association (2000)) and Erkinjuntti's vascular dementia imaging criteria (Erkinjuntti et al., 1999)
  • Have severe white matter hyper-intensities (periventricular cap or band) ≥ 10 mm or a deep white matter lesion ≥ 25 mm
  • Have a history of stroke in the most recent 3 months
  • Have severe hepatic and renal function disorder (total bilirubin ≥ 3 mg/dL; serum creatinine ≥ 1.5 mg/dL)
  • Have suspected active pulmonary disease according to chest X-ray film
  • Hemoglobin < 9.5 g/dL (male), < 9 g/dL (female); total white cell count < 3,000/mm3
  • Platelet count < 150,000/mm3; plasma prothrombin time (PT) ≥ 1.5; international normalized ratio (INR) or activated partial thromboplastin time (APTT) ≥ 1.5-fold baseline
  • Highly allergic constitution or have a history of hypersensitiveness
  • Generalized infection or severe local infection
  • Those who have poor compliance or cannot accomplish the clinical trial or those who the researchers consider cannot enter the clinical trial


Withdrawal or replacement criteria

  • Participants can withdraw from the trial at any time for any reason
  • Researchers will determine whether the trial should continue according to the participant's condition
  • If any participants withdraw from the trial, patient recruitment will continue until the required sample size is reached


Cell transplantation

Culture and preparation of stem cells


Isolation, culture and sub-culture of hUC-MSCs: umbilical cords will be harvested from 18-48-year-old healthy pregnant women who will have a normal delivery (with signed informed consent). After removal of umbilical cord vessels, the left tissue will be chopped into small blocks and digested with 0.05% type II collagenase solution at 37°C for 2-3 hours. The suspension will be filtered through a stainless steal mesh, washed twice with PBS, and then cultured in serum free medium in a 5% CO 2 -saturated incubator at 37°C. Culture medium will be refreshed for the first time 48 hours after cell isolation (no cell observation) and then once every 3 or 4 days (cell observation daily). When cells reach near confluency (10-14 days), they will be digested with 0.05% Trypsin and 0.01% EDTA. A single cell suspension will be prepared and sub-cultured at a ratio of 1:2-3. Passage 3 cells will be used for later experiments.

Prior to the clinical trial, cell quality will be ensured: (1) Adherence to plastic in standard culture conditions; (2) phenotype: CD105, CD73, CD90 positive (≥ 95%); CD45, CD34, CD14 or CD11b, CD79a or CD19, HLA-DR negative (≤ 2%); (3) in vitro differentiation: osteoblasts, adipocytes, chondroblasts (demonstrated by staining of in vitro cell culture; (4) by a sterility test (negative result); and (5) by an endotoxin test (negative result).

hUC-MSC transplantation

Dose: 0.5×10 6 UC-MSCs/kg.

Course of treatment: Intravenous injection for 8 times, once every 2 weeks in the first month of each quarter.

Infusion methods: The patients will be asked to lie in a supine position and hUC-MSCs will be administered via the peripheral vein using a transfusion apparatus. In total, 30 mL of hUC-MSC suspension will be controlled to drip completely within 10 minutes and then 10 mL of 0.9% sodium chloride solution will be used to flush the passage. At the end of the first infusion, headache, dizziness, nausea and vomiting, jerks, and fever will be monitored. Once any of these occurs, date of symptom presence, management method and the possible relationship with treatment should be recorded in the details. The management protocol should be reported to the responsible researchers and clinical ethics committee.

Blinding

This trial is open label, so patients and the cell infusion operators will know grouping and intervention methods.

Techniques for hUC-MSCs culture, isolation and transplantation

The precise details of hUC-MSC culture, isolation and transplantation are shown in [Figure 1].
Figure 1: A scheme of culture, isolation and transplantation of human umbilical cord-derived mesenchymal stem cells (hUM-MSCs).

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Patient recruitment, treatment and evaluation post-treatment

For precise information, please see [Figure 2].
Figure 2: A flow chart of patient recruitment, treatment, and evaluation post-treatment.
ADAS-Cog: Alzheimer's Disease Assessment Scale-cognitive subscale; MMSE: Mini-Mental State Examination; CIBIC: Clinician Interview Based Impression of Change; CIBIC-plus: Clinician Interview Based Impression of Change-Plus; ADL: Activity of Daily Living Scale; NPI: Neuropsychiatric Inventory.


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Adverse events

Mesenchymal stem cells themselves have no limitation of major histocompatibility complex and cannot cause rejection reaction. Because of the specificity of medical science and individual difference, allergic reactions may appear during and after cell infusion, including fever, urticaria, hypotension and shortness of breath and even allergic shock as well as some other unexpected circumstances.

Primary outcomes (safety evaluation):

(1) Number of participants who have adverse events within 10 weeks after the last infusion of hUM-MSCs.

(2) Number of participants who have adverse events within 1 year of follow up.

Secondary outcomes (efficacy evaluation):

Evaluation of improvement of Alzheimer's disease 10 weeks after the last infusion of hUM-MSCs.

The outcome measures are shown in [Table 1].
Table 1: Evaluation indices and time schedule of outcome measures


Click here to view


Statistical analysis

Numerical data such as the incidence of adverse events, will be compared using a chi-square test or Fisher's exact test between data collected prior to cell infusion and data collected at different time points after cell infusion. Measurement data, such as blood routine, hepatic function, renal function, cerebrospinal fluid Aβ 42 , total tau protein and phosphorylated tau level, peripheral blood Th1/Th2 level, serum level of carrier protein for thyroxine, ADAS-Cog score, CIBIC score, CIBIC-plus score, MMSE score, ADL score, NPI score will be compared using paired t-test or Wilcoxon signed rank test. A level of P < 0.05 is considered statistically significant. All statistical data will be processed by professional statisticians using SPSS 15.0 software.



Ethics statement

The trial will be performed in accordance with World Medical Association's Declaration of Helsinki. The trial protocol is in accordance with relevant international principles for medical research involving human participants (Council for International Organizations of Medical Sciences, 2002). A written approval regarding the protocol procedure, informed consent, patient information and any advertisement materials will be obtained from the Medical Ethics Committee of the Affiliated Hospital of Academy of Military Medical Sciences of China. If necessary, the study team will acquire written approval from the Medical Ethics Committee of the Affiliated Hospital of Academy of Military Medical Sciences of China regarding revision of the items that influence trial conduction, patient interest or safety, including trial design, sample size, trial procedure or any intervention method.


  Discussion Top


Our team has performed a series of studies on hUC-MSCs: in vitro experiments as the culture, preservation, and differentiation of hUC-MSCs; proliferation of hematologic malignant tumor cells when co-cultured with hUC-MSCs; as well as the effects of MSCs on immunological responses of SD rats after skin radiation injury ([Table 2] and [Table 3]). In terms of AD, the results from C57 mice with acute AD have confirmed the efficacy and safety of hUC-MSCs in animal experiments. This study protocol for phase I/II clinical trials was designed to investigate the efficacy and safety of hUC-MSCs for AD treatment in the clinic. Outcomes from this study will add reliable clinical data to clinical AD treatment using the potential hUC-MSC replacement therapy.
Table 2: Preliminary experiments involving hUC-MSCs transplantation


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Table 3: Preliminary experiments regarding culture and preservation of hUC-MSCs


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Trial status

Recruitment of participants at the time of submission.[29]

 
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    Figures

  [Figure 1], [Figure 2]
 
 
    Tables

  [Table 1], [Table 2], [Table 3]



 

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