Stem Cells International

Stem Cells International / 2020 / Article
Special Issue

Current Status and Perspectives of Human Mesenchymal Stem Cell Therapy 2020

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Research Article | Open Access

Volume 2020 |Article ID 8877003 | https://doi.org/10.1155/2020/8877003

Sze-Piaw Chin, Mohd-Yusoff Mohd-Shahrizal, Mohd-Zuhar Liyana, Kong Yong Then, Soon Keng Cheong, "High Dose of Intravenous Allogeneic Umbilical Cord-Derived Mesenchymal Stem Cells (CLV-100) Infusion Displays Better Immunomodulatory Effect among Healthy Volunteers: A Phase 1 Clinical Study", Stem Cells International, vol. 2020, Article ID 8877003, 13 pages, 2020. https://doi.org/10.1155/2020/8877003

High Dose of Intravenous Allogeneic Umbilical Cord-Derived Mesenchymal Stem Cells (CLV-100) Infusion Displays Better Immunomodulatory Effect among Healthy Volunteers: A Phase 1 Clinical Study

Academic Editor: Kar Wey Yong
Received24 Apr 2020
Revised18 Jun 2020
Accepted11 Jul 2020
Published28 Sep 2020

Abstract

Background. Mesenchymal stem cells (MSCs) express growth factors and other cytokines that stimulate repair and control the immune response. MSCs are also immunoprivileged with low risk of rejection. Umbilical cord-derived MSCs (UCMSCs) are particularly attractive as an off-the-shelf allogeneic treatment in emergency medical conditions. We aim to determine the safety and efficacy of intravenous allogeneic infusion of UCMSCs (CLV-100) by Cytopeutics® (Selangor, Malaysia) in healthy volunteers, and to determine the effective dose at which an immunomodulatory effect is observed. Methodology. Umbilical cord samples were collected after delivery of full-term, healthy babies with written consent from both parents. All 3 generations (newborn, parents, and grandparents) were screened for genetic mutations, infections, cancers, and other inherited diseases. Samples were transferred to a certified Good Manufacturing Practice laboratory for processing. Subjects were infused with either low dose (LD, 65 million cells) or high dose (HD, 130 million cells) of CLV-100 and followed up for 6 months. We measured cytokines using ELISA including anti-inflammatory cytokines interleukin 1 receptor antagonist (IL-1RA), interleukin 10 (IL-10), pro-/anti-inflammatory cytokine interleukin 6 (IL-6), and the proinflammatory cytokine tumor necrosis factor-alpha (TNF-α). Results. 11 healthy subjects (LD, ; HD, ; mean age of years) were recruited. All subjects tolerated the CLV-100 infusion well with no adverse reaction throughout the study especially in vital parameters and routine blood tests. At 6 months, the HD group had significantly higher levels of anti-inflammatory markers IL1-RA ( vs. ; ) and IL-10 ( vs. L; ); and lower levels of proinflammatory marker TNF-α ( vs. ; ) compared to LD group. Conclusion. Allogeneic UCMSCs CLV-100 infusion is safe and well-tolerated in low and high doses. Anti-inflammatory effect is observed with a high-dose infusion.

1. Introduction

Mesenchymal stem cells (MSCs) are multipotent fibroblast-like cells that reside in various tissues of the human body. MSCs have the capacity to regenerate and replicate as well as to differentiate into various specialized cells and tissues in the body, including chondrocytes, adipocytes, osteocytes, and neuron-like cells [13]. The self-renewal and multilineage potentials of MSCs in providing new cells for tissue repair by replacing the damaged cells suggest its therapeutic potentials in tissue regeneration [2, 48].

Several studies have reported that the mechanism of MSCs in repairing tissue damage is associated to their immunomodulatory properties rather than its capacity for differentiation [9, 10]. One of MSCs’ vital biological function, the immunomodulation, provides MSCs with the ability to migrate and adhere to any injury or inflammation sites found in the body and thereby interact with various immune cells such as T cells, B cells, natural killer cells, dendritic cells, neutrophils, and macrophages before evoking effective immune responses to ameliorate the intense inflammatory reaction of the injured site via direct cell-cell contact mechanism and/or the release of soluble inducible factors [1113].

MSCs can be isolated from various tissues including bone marrow, peripheral blood, adipose tissue, cord blood, and umbilical cord. Recent studies have shown that MSCs derived from human umbilical cord (UCMSCs) possess several advantages compared to MSCs isolated from other tissues, including high-proliferation and self-renewal capacity and multilineage differentiation capability. Umbilical cord is considered as a medical waste, and the collection of UC-MSCs is noninvasive which eliminates any ethical concern from its collection. [14, 15]. In addition, UCMSCs possess low immunogenicity allowing them to be utilized in allogeneic transplantation without any rejection and thereby providing a new approach for the treatment of autoimmune diseases [16].

Consequently, UCMSCs have been developed as an “off-the-shelf” cell therapy for a variety of diseases especially in autoimmune diseases. Clinical studies in graft-versus-host disease (aGVHD) have demonstrated that UCMSCs dramatically improved the patients’ conditions with no adverse effects and no evidence of cancer recurrence throughout the trial period ([17, 18]). Moreover, UCMSCs treatment in active systemic lupus erythematosus (SLE) resulted in amelioration of the disease activity, serologic changes, and stabilization of proinflammatory cytokines in the patients [19].

The production of UCMSCs cells products from manufacturing methods must be tested for its safety prior to be used as therapeutic agents in cell therapy ([20]). Therefore, this Phase 1 clinical study was conducted to establish a new UCMSCs cell line (CLV-100) by assessing the safety and efficacy of intravenous allogeneic infusion of our manufactured UCMSCs (CLV-100) among healthy volunteers. This study also sought to compare the immunomodulatory effect of different dosage of CLV-100 between high-dose and low-dose infusion in healthy volunteers based on several clinical assessments and measurements of changes in systemic biomarkers. The findings of this study will act as a guideline and benchmark for future CLV-100 clinical research.

2. Materials and Methods

2.1. Study Design

This is an open-label nonrandomized Phase 1 study assessing the safety and efficacy of CLV-100 infusion among 11 healthy subjects recruited at NSCMH Medical Centre in Seremban, Malaysia. The subjects were divided into 2 groups; low-dose group received 65 million cells (equivalent to about 1 million cells per kg body weight) (LD, ), while high dose group received 130 million cells (equivalent to about 2 million cells per kg body weight) (HD, ) of allogeneic infusion of CLV-100. This study was approved by the Medical Research and Ethics Committee (MREC) Ministry of Health Malaysia (NMRR-13-1152-17400) and monitored by independent Data Safety and Monitoring Board (DSMB). All subjects provided written informed consent before participating in the study. The inclusion and exclusion criteria were listed in Table 1.


Key inclusion criteria
(i) Men and women aged 40 years and older
(ii) Subjects with normoglycemia
(iii) Subjects with normotension
(iv) Subjects with normal fasting lipid profile
(v) Subject must understand patient information sheet and signed informed consent form

Key exclusion criteria
(i) Subject who has enrolled in another investigational drug trial or innovative therapeutics product-related trial or has completed the aforesaid within 3 months
(ii) Subject with history of current or past use (within 1 year) of alcohol, smoking, or drug abuse
(iii) Pregnant or nursing women
(iv) Subject with known documented drug allergies
(v) Subject who is required of the following medicines on a regular basis: anti-histamine, steroid, antibiotic, anti-inflammatory, immunosuppressant, and pain killer medications
(vi) Subject who is currently on any hormone replacement or hormone suppressive therapy for any indication
(vii) Subject with any acute or chronic infections or communicable diseases including hepatitis B, hepatitis C, or HIV
(viii) Subject with any active or past history of neoplasia and primary hematological disease
(ix) Subject with any renal impairment indicated by serum creatinine ≥120 μmol or creatinine clearance <60 mL/min
(x) Subject with any cardiovascular disease including documented coronary disease of more than 50% stenosis, angina, myocardial infarction, heart failure, stroke, transient ischemic attack, and/or peripheral artery disease
(xi) Subject with any diabetes mellitus
(xii) Subject with any liver impairment indicated by serum aspartate transaminase and alanine transaminase greater than 1.5 times upper limit normal
(xiii) Subject with any chronic pulmonary or airways disease
(xiv) Subject with any current or past history of mental illness or cognitive impairment.

2.2. Establishing UCMSCs Culture

Umbilical cord samples were collected after delivery of full-term, healthy babies with written consent from both parents. All 3 generations (newborn, parents, and grandparents) were screened for genetic mutations, infections, cancers, and other inherited diseases before the samples were transferred to the laboratory for processing. All cell processing was done in a certified Good Manufacturing Practice (GMP) laboratory in accordance with Malaysia Guidelines for Stem Cell Research and Therapy as published previously [4, 5]. Isolation and culturing have been established and reported previously [21]. The high-quality umbilical cord was digested, and MSCs were isolated based on adherence to flask’s surface. The cells then were expanded in proprietary growth medium kept in 37°C, 5% CO2, and 95% air incubator. After three days, nonadherent cells were discarded and replaced with new growth medium until it reached confluence. Then, the MSCs were cultured in new flasks until the required cell number was achieved. The first few early passages of the cells were cryopreserved and served as a seed for future use. For this study, cells were thawed and expanded from the seed up until Passage 6. Throughout the process, UCMSCs were tested for quality control purposes including immunophenotyping, differentiation assays, as well as to confirm the absence of bacterial, fungal, and mycoplasma contamination.

2.3. Infusion of UCMSCs, Monitoring, and Follow-Up

On the day of CLV-100 infusion, the eligible subjects registered to a medical centre as outpatients. The subjects underwent a routine physical examination, and their vital signs were measured to ensure they were fit and suitable for CLV-100 infusion. Once the subjects were confirmed fit for CLV-100 infusion, a cannula was placed in the subjects’ vein. Before CLV-100 infusion, 200 mL of normal saline was infused into subjects for 0.5-1 hours. While waiting for normal saline infusion, 65 million CLV-100 for LD group and 130 million cells for HD group were thawed, washed, and resuspended in 200 mL of normal saline before being infused intravenously to the subjects for 1 hour. Upon completion, 50 mL of normal saline solution was infused to keep the vein open. All standard precautions for intravenous procedure were observed according to routine and standard practice at the medical centre. The subjects were monitored for vital sign and adverse event (AE) (if any) every 15 minutes during infusion and later on an hourly basis for a minimum of 6 hours in the medical centre. We followed the Good Clinical Practice (GCP) guidelines of the International Council for Harmonization (ICH) in defining our AE. The classifications of AE in this study include any untoward medical occurrence in the study subject administered with CLV-100 which may or may not related to the Investigational Product (IP). The monitored AE included but not limited to fever, headache, injection site swelling, or pain. The subjects were discharged if there were no other complications observed after the monitoring period.

Subjects were required to do the 4 times of follow-up (2, 30, 90, and 180 days) postinfusion. During all follow-up, 20 mL blood was withdrawn from the subjects for blood analysis. Subjects were required to immediately inform the medical centre if there is any AE or severe adverse event (SAE).

2.4. Outcome Measures

Baseline data were collected from each subject prior to CLV-100 infusion and information regarding subject particulars; demographic data and medical history were properly recorded. Several clinical assessments were performed at baseline (before CLV-100 infusion) and during postinfusion follow-ups at 2, 30, 90, and 180 days, including routine blood tests, hypersensitivity tests (specifically white cell count, subfraction, and immunoglobulin E), vital signs, lung function tests specifically in the ratio of forced expiratory volume in 1 second to forced vital capacity (FEV1/FVC) via spirometry, renal function tests, liver function tests, full blood count, level of the proinflammatory and the anti-inflammatory markers such as high-sensitivity C-reactive protein (hs-CRP), and albumin globulin ratio (A/G), respectively, as well as cytokines, to examine any changes in the results in each of the subjects.

The primary endpoint of this study was to evaluate the presence or absence of allergic reaction to the infusion, sepsis, organ failure (clinically apparent or subclinical), hospitalization, cancers, and death, as well as any changes in clinical, functional parameters and blood tests during the 6 months follow-up period.

2.5. Detection of Cytokines and Growth Factors with ELISA

Serum of every subject during day 0, day 2, day 7, day 30, and day 180 postinfusion was collected and kept frozen at -80°C to allow batch analysis at the end of the study. The anti-inflammatory cytokines including interleukin-10 (IL-10), interleukin-1-receptor antagonist (IL-1RA), and prostaglandin E2 (PGE2); proinflammatory cytokines such as interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α); as well as growth factors including transforming growth factor-beta (TGF-β), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF) were detected and measured with enzyme-linked immunosorbent assay (ELISA) kits (R&D System, USA) in duplicates according to manufacturer’s instructions.

2.6. Statistical Analysis

Data analysis was performed by IBM SPSS Statistic v23.0 software (SPSS, Inc., Armonk, NY, USA). Missing data on the primary efficacy variable had their data imputed by the method of Last Observation Carried Forward (LOCF), and the data were presented as . The safety of intravenous infusion of CLV-100 towards the subjects was assessed via descriptive statistic analysis. Differences in side effects and blood test between the group of low dose and high dose were calculated using Fisher’s exact test (for categorical data) and Mann–Whitney test (for numerical data). As for the efficacy analysis, Mc Nemar test (for categorical data) and Wilcoxon signed rank test (for numerical data) were used to assess the difference (if any) between pre- and post-CLV-100 infusion. It was considered statistically significant when the value of .

3. Results

3.1. Baseline Assessments

Throughout the study period, 11 healthy volunteers (male: 4, female: 7) were screened and recruited between May 2017 and January 2018 into the study. As shown in Table 2, the mean age of the subjects during baseline was years old. About million cells per kg and million cells per kg were infused into LD subjects and HD subjects, respectively. There were no significant differences were observed in all clinical routine parameters between the two groups except HD subjects have higher but normal white blood cells (WBC) count ( vs. ; ) and alpha-fetoprotein (AFP) level ( vs. ; ) compared to LD subjects. We have further looked into individual WBC and AFP parameters. All subjects (either LD or HD) were within the normal range ( for WBC and less than 10 ng/mL for AFP). Further on that, from 30 to 180 days (to the end of the study) postinfusion follow-up, no statistical significant differences were observed in WBC and AFP parameters.


ParameterNormal rangeTotal ()Low dose ()High dose () valuea

Age0.36
Body weight0.15
Male4220.82
Female7340.82
Routine blood tests
 WBC (×109/L)4.0-11.00.03
 Hemoglobin (g/dL)11.5-16.50.20
 HCT (%)35-470.41
 MCV (fl)76-960.52
 Platelet (×109/L)150-4000.72
 Creatinine (μmol/L)44-970.36
 ESR (mm/hr)0-200.86
 AST (IU/L)0-400.27
 ALT (IU/L)0-530.52
 Albumin (g/L)30-500.58
 Globulin (g/L)20-500.46
 A/G ratio1.2-2.50.40
 Total cholesterol (mmol/L)<5.20.71
 HbA1c (%)3.0-6.00.07
 FBS (mmol/L)3.9-5.60.93
 Insulin (mU/L)3.0-25.01.00
 IGF-1(ng/mL)87-2380.88
 DHEAS (μmol/L)1.0-11.70.10
 Estradiol (pg/mL)50-1000.83
 Progesterone (ng/mL)0.57-6.110.82
 Testosterone (ng/mL)2.41-8.27-b-b
 hs-CRP (mg/L)<4.70.58
 IgE (IU/mL)<158.00.48
 Total PSA (ng/mL)0.0-4.00.12
 CA125 (U/mL)<35.00.08
 CA15.3 (U/mL)<28.00.72
 CEA (ng/mL)<5.01.00
 CA19.9 (U/mL)<37.00.36
 AFP (ng/mL)<15.00.04
Vital signs
 SBP (mmHg)<1290.14
 DBP (mmHg)<800.71
 Heart rate (beats/min)60-1000.27
Lung function tests
 FEV1 (L)2.5-4.50.36
 FVC (L)2.5-4.50.86
 FEV1/FVC (%)>750.20
Biomarkers
 IL-6 (pg/mL)0.36
 IL-10 (pg/mL)0.30
 PGE2 (pg/mL)0.44
 IL1-RA (pg/mL)0.80
 TNF-α (pg/mL)0.12
 TGF-β (ng/mL)0.80
 VEGF (pg/mL)1.00
 HGF (pg/mL)0.20

aThe Mann–Whitney test. bData too low to be computed. Significant value at . Abbreviation: WBC: white blood cells; MCV: mean corpuscular volume; HCT: hematocrit; A/G: albumin/globulin; ESR: erythrocyte sedimentation rate; AST: aspartate aminotransferase; ALT: alanine transaminase; HbA1c: hemoglobin A1c; FBS: fasting blood sugar; IGF-1: insulin growth factor-1; DHEAS: dehydropiandrosterone sulphate; SBP: systolic blood pressure; DBP: diastolic blood pressure; FEV1: forced expiratory volume in one second; FVC: forced vital capacity; hs-CRP: high-sensitivity C-reactive protein; IgE: immunoglobulin E; IL-6: interleukin 6; IL-10: interleukin 10; PGE2: prostaglandin E2; IL1-RA: interleukin 1 receptor antagonist; TNF-α: tumor necrosis factor-alpha.
3.2. Tolerability, Hypersensitivity and Adverse Reactions

Similar clinical assessments were examined throughout the 6-months follow-up among all recruited subjects in both groups to assess the safety of allogeneic CLV-100 infusion as shown in Table 3 and Table 4. All subjects tolerated the CLV-100 infusion well. There were no significant different changes in vital signs variables before, during, and after the CLV-100 in both groups. There was a small but significant increase in hemoglobin ( vs. ; ) and MCV ( vs. ; ) at 6 months in HD group.


ParametersBaseline day 0Follow-up period valuea
Day 2Day 30Day 90Day 180

Routine blood tests
 WBC (×109/L)0.51
 Hemoglobin (g/dL)0.74
 Creatinine (umol/L)0.97
 HCT (%)0.59
 MCV (fl)0.72
 Platelet (×109/L)0.96
 ESR (mm/hr)0.69
 AST (IU/L)0.34
 ALT (IU/L)0.92
 Albumin (g/L)0.07
 Globulin (g/L)0.96
 A/G ratio0.06
 Total cholesterol (mmol/L)0.59
 HbA1c (%)0.78
 FBS (mmol/L)0.64
 Insulin (mU/L)0.68
 IGF-1 (ng/mL)0.62
 DHEAS (μmol/L)0.46
 Estradiol (pg/mL)0.90
 Progesterone (ng/mL)0.52
 Testosterone (ng/mL)-b-b-b-b-b
 hs-CRP (mg/L)0.03
 IgE (IU/mL)0.14
 Total PSA (ng/mL)0.90
 CA125 (U/mL)0.90
 CA15.3 (U/mL)0.82
 CEA (ng/mL)0.23
 CA19.9 (U/mL)0.52
 AFP (ng/mL)0.14
Vital signs
 SBP (mmHg)0.89
 DBP (mmHg)0.21
 Heart rate (beats/min)0.40
Lung function tests
 FEV1 (L)0.13
 FVC (L)0.82
 FEV1/FVC (%)0.25
Biomarkers
 IL-6 (pg/mL)0.56
 IL-10 (pg/mL)0.21
 PGE2 (pg/mL)0.76
 IL1-RA (pg/mL)0.22
 TNF-α (pg/mL)0.06
 TGF-β (ng/mL)0.54
 VEGF (pg/mL)0.27
 HGF (pg/mL)0.13

aThe Friedman test. bData too low to be computed. Significant value at .

ParametersBaseline day 0Follow-up period valuea
Day 2Day 30Day 90Day 180

Routine blood tests
 WBC (×109/L)0.69
 Hemoglobin (g/dL)