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

Jing-wen Niu; Bin Zhang; Hu Chen

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

Jing-wen Niu, Bin Zhang, Hu Chen
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 Publication

5-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

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 2023 Dec 3];1:1-8. Available from: https://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

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

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

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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

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]

References

1.	American Psychiatric Association (2000) Diagnostic and Statistical Manual of Mental Disorders, 4th Edition, Text Revision (DSM-IV-TR).
2.	Council for International Organizations of Medical Sciences (2002) International ethical guidelines for biomedical research involving human subjects. Bull Med Ethics:17-23.
3.	Cui YB, Ma SS, Yao N, Qu RN, Wang XX, Xing Q, Meng N, Yang B, Guan FX (2015) Effects of human umbilical cord mesenchymal stem cell transplantation on expression of aging related gene in brain and liver of Alzheimer's disease mice. Zhengzhou Daxue Xuebao: Yixue Ban 50:25-29.
4.	Cummings JL, Mega M, Gray K, Rosenberg-Thompson S, Carusi DA, Gornbein J (1994) The Neuropsychiatric Inventory comprehensive assessment of psychopathology in dementia. Neurology 44:2308-2308.
5.	Dalous J, Larghero J, Baud O (2012) Transplantation of umbilical cord-derived mesenchymal stem cells as a novel strategy to protect the central nervous system: technical aspects, preclinical studies, and clinical perspectives. Pediatr Res 71:482-490.
6.	Erkinjuntti T, Bowler JV, DeCarli CS, Fazekas F, Inzitari D, O'Brien JT, Pantoni L, Rockwood K, Scheltens P, Wahlund LO, Desmond DW (1999) Imaging of static brain lesions in vascular dementia: implications for clinical trials. Alzheimer Dis Assoc Disord 13 Suppl 3:S81-90.
7.	Fan CG, Zhang QJ, Zhou JR (2011) Therapeutic potentials of mesenchymal stem cells derived from human umbilical cord. Stem Cell Rev 7:195-207.
8.	Galea M, Woodward M (2005) Mini-Mental State Examination (MMSE). Aust J Physiother 51:198.
9.	Hu JW, Zhang H, Xu M, Tang YY, Wang J, Sheng HX, Yang ZL, Zhang B, Chen H (2012) Study on culture of mesenchymal stem cells isolated from whole human umbilical cord and its differentiation into chondrocyte in vitro. Chuangshang Waike Zazhi 14:531-534.
10.	Johnson N, Barion A, Rademaker A, Rehkemper G, Weintraub S (2004) The Activities of Daily Living Questionnaire: A validation study in patients with dementia. Alzheimer Dis Assoc Disord 18:223-230.
11.	Lee JK, Jin HK, Bae JS (2009) Bone marrow-derived mesenchymal stem cells reduce brain amyloid-beta deposition and accelerate the activation of microglia in an acutely induced Alzheimer's disease mouse model. Neurosci Lett 450:136-141.
12.	Liu J, Xue M, Zhu L, Ding L, Yan HM, Wang ZD, Han DM, Guo ZK, Wang HX (2010) Clinical analysis on the treatment of spinocerebeilar ataxia and multiple system atrophy-cerebellar type with umbilical cord mesenchymal stem cells. Zuzhigongcheng yu Chongjian Waike Zazhi 6:257-260.
13.	Ma LH, Cheng X, Xu JP, Luo HM (2012) Effects of hUC-MSCs on learning ability and memory in mice with Alzheimer's disease. Guangdong Yixue 33:1366-1369.
14.	McKhann GM, Knopman DS, Chertkow H, Hyman BT, Jack CR Jr, Kawas CH, Klunk WE, Koroshetz WJ, Manly JJ, Mayeux R, Mohs RC, Morris JC, Rossor MN, Scheltens P, Carrillo MC, Thies B, Weintraub S, Phelps CH (2011) The diagnosis of dementia due to Alzheimer's disease: recommendations from the National Institute on Aging-Alzheimer's Association workgroups on diagnostic guidelines for Alzheimer's disease. Alzheimers Dement 7:263-269.
15.	Parekkadan B, Milwid JM (2010) Mesenchymal stem cells as therapeutics.Annu Rev Biomed Eng 12:87-117.
16.	Reisberg B, Ferris SH (2004) Clinician's interview-based impression of change-plus. Guide to Assessment Scales in Dementia.
17.	Schneider LS, Olin JT, Doody RS, Clark CM, Morris JC, Reisberg B, Schmitt FA, Grundman M, Thomas RG, Ferris SH (1997) Validity and reliability of the Alzheimer's Disease Cooperative Study-Clinical Global Impression of Change. The Alzheimer's Disease Cooperative Study. Alzheimer Dis Assoc Disord 11 Suppl 2:S22-32.
18.	Shetty P, Cooper K, Viswanathan C (2010) Comparison of proliferative and multilineage differentiation potentials of cord matrix, cord blood, and bone marrow mesenchymal stem cells. Asian J Transfus Sci 4:14-24.
19.	Shi R, Xu M, Su YF, Zhang B, Chen H (2012) Notch pathway gene expression after co-culture of umbilical cord mesenchymal stem cells and hematopoietic stem cells. Xibao yu Fenzi Mianyixue Zazhi 28:793-796.
20.	Shihabuddin LS, Aubert I (2010) Stem cell transplantation for neurometabolic and neurodegenerative diseases. Neuropharmacology 58:845-854.
21.	Su ZY, Yang ZL, Tang YY, Hu JW, Sheng HX, Xu M, Zhang B, Chen H (2014) Human umbilical cord mesenchymal stem cells for repair of combined radiation-wound skin injury and tumorigenicity in vitro. Zhongguo Zuzhi Gongcheng Yanjiu 18:5993-5997.
22.	Taghizadeh RR, Cetrulo KJ, Cetrulo CL (2011) Wharton's Jelly stem cells: future clinical applications. Placenta 32 Suppl 4:S311-315.
23.	Wang HS, Hung SC, Peng ST, Huang CC, Wei HM, Guo YJ, Fu YS, Lai MC, Chen CC (2004) Mesenchymal stem cells in the Wharton's jelly of the human umbilical cord. Stem Cells 22:1330-1337.
24.	Wang J, Xu M, Sheng HX, Kong WX, Hu JW, Liao L, Tang YY, Ning HM, Zhang B, Chen H (2012a) Karyotype stability of human umbilical cord mesenchymal stem cells during culture in vitro. Junshi Yixue 36:599-602.
25.	Wang J, Kong WX, Xu M, Sheng HX, Hu JW, Liao L, Tang YY, Ning HM, Zhang B, Chen H (2012b) Effect of cryopreservation on biological features of human umbilical cord mesenchymal stem cells. Junyi Jinxiu Xueyuan Xuebao 33:1152-1155.
26.	Yu CC,Tang YY, Sheng HX, Liu G, Hu ZY, Zhou WX, Li GJ, Zhang B, Chen H (2012) Immune response after transplantation of human umbilical cord-derived mesenchymal stem cells in a mouse model of acute Alzheimer's disease. Zhongguo Zuzhi Gongcheng Yanjiu 16:3476-3481.
27.	Zhan Y, Ma DH, Zhang Y (2011) Effects of cotransplantated Schwann cells and neural stem cells in a rat model of Alzheimer's disease. Neura Regen Res 6:245-251.
28.	Zhan Y, Ma DH, Zhang Y, Chang M, Hu L (2010) Protective effects of transplanted neural stem cells on the brain of Alzheimer's disease rats. Neural Regen Res 5:825-832.
29.	Zhang H, Zhang BX, Cheng M, Tao YL, Hu JW, Xu M, Chen H (2012) Isolation and characterization of mesenchymal stem cells derived from whole human umbilical cord by applying a direct explant technique. Jichu Yixue yu Linchuang 32:71-76.