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MINISTRY OF EDUCATION AND TRAINING MINISTRY OF HEATH HANOI MEDICAL UNIVERSITY ====== VU THI THU TRANG APPLICATION OF AUTOLOGOUS BONE MARROW STEM CELLS THERAPY IN CHRONIC OBSTRUCTIVE PULMONARY DISEASE[.]

HANOI MEDICAL UNIVERSITY

======

VU THI THU TRANG

APPLICATION OF AUTOLOGOUS BONE MARROW STEM CELLS THERAPY IN CHRONIC OBSTRUCTIVE PULMONARY DISEASE

The thesis has been completed at

HANOI MEDICAL UNIVERSITY

Supervisors: A Prof Phan Thu Phuong MD PhD

Reviewer 1: A Prof Nguyen Huy Luc MD PhD

Reviewer 2: A Prof Bach Khanh Hoa MD PhD

Reviewer 3: A Prof Nguyen Viet Nhung MD PhD

The thesis will be present in front of board of university examiner and reviewer lever at… on … 2023

This thesis can be found at:

National Library

National Medical Informatics Library

Library of Hanoi Medical University

THESIS

1 Ngô Quý Châu, Phan Thu Phương, Nguyễn Tuấn Tùng, Vũ Văn Trường, Vũ Thị Thu Trang*, Nguyễn Thanh Thủy, Đào Ngọc Phú, Đặng Thành Đô, Nguyễn Đức Nghĩa (2019) “Kết quả bước đầu ghép tế bào gốc tự thân từ tuỷ xương điều trị bệnh phổi tắc nghẽn mạn tính tại Trung tâm Hô hấp, Bệnh viện Bạch Mai” Tạp chí Y học Việt Nam, tập 477 số đặc biệt tháng 4 năm 2019, tr30-38

* Tác giả chịu trách nhiệm chính

2 Vu Thi Thu Trang, Phan Thu Phuong, Nguyen Tuan Tung, Vu Van Truong, Nguyen Huy Binh, Pham Cam Phuong, Nguyen Thanh Thuy, Dao Ngoc Phu, Dang Thanh Do, Nguyen Duc Nghia, Vu Van Giap, Ngo Quy Chau (2020)

“Clinical effects of autologous bone marrow derived stem cell therapy for patients with chronic obstructive pulmonary disease at Bach Mai hospital” Journal Of Medical Research 136 (12) – 2020: p99-109

3 P Thu Phan, T.T Thu Vu*, G Van Vu, T Tuan Nguyen, T Van Vu, P Cam Pham, T Thanh Nguyen, B Huy Nguyen, K Ba Nguyen, Q Ngoc Tran, T Huyen Tran, H Quoc Phan, P Ngoc Dao, N Duc Nguyen, D Thanh Dang, H Thi Chu, K Trong Mai, C Quy Ngo (2021).“Quality and safety of autologous bone marrow derived stem cell separation procedure in chronic obstructive pulmonary disease patients” Journal of functional ventilation and pulmonology, Issue 36 Volume 12, 4/2021, p1-6

* Correspondent author

INTRODUCTION Chronic obstructive pulmonary disease (COPD) is the fourth leading cause of death worldwide Although the disease progresses slowly, is chronic in nature and does not cause death at an early stage, the consequences caused by the disease to the patients, families and society are very severe In the advanced stages of the disease, the patient becomes disabled, severely impaired in health and quality of life, even with treatment Today, the drugs to treat the disease are increasingly optimized, but they do not work to prevent the pathogenesis of COPD The disease continues to progress to severe stages, eventually to death Stem cells with characteristics such as high differentiation ability, immunoregulatory ability, tissue regeneration are expected to bring benefits to patients and improve quality of life In Vietnam as well

as in the world, the number of studies applying stem cells therapy in general and bone marrow (BM) stem cells in particular in the treatment of COPD is still small and the follow-up time is limited In addition, autologous BM stem cell sources have been studied in normal people and in the treatment of some diseases, but there have been

no studies evaluating the characteristics of bone marrow stem cells in patients with COPD Therefore, we conducted a study on the topic "Application of autologous bone marrow stem cells in chronic obstructive pulmonary disease management" with two objectives:

1 Characterization of bone marrow fluid and autologous bone marrow stem cell grafts of patients with COPD

2 Outcomes of application of autologous BM stem cells therapy in COPD management

Necessity of the thesis:

The prevalence of moderate and severe COPD in Vietnam ranks highest in the Asia Pacific region, accounting for 6.7% of the population In the near future, the burden of COPD will still tend to increase, due to continued exposure to risk factors and an aging population Current measures to treat COPD include: stopping exposure

to risk factors, using drugs (bronchodilators, corticosteroids, phosphodiesterase 4 inhibitors ), pulmonary rehabilitation, oxygen support, mechanical ventilation etc However, despite comprehensive and optimized treatment, the disease continues to progress over time Therefore, an urgent need today is to find new treatments with the ambition of stopping or even reversing the course of the disease In recent years

in the world, there have been many studies on the use of stem cells in the treatment

of various diseases including respiratory diseases such as pulmonary fibrosis, COPD However, the number of studies and follow-up time of clinical trials on stem cells therapy are still limited, there are many questions about the safety and mechanism of action of exogenous cells after being introduced into chronic damaged lungs still needs to be answered

New contribution of the thesis:

This is the first study to apply autologous BM stem cell therapy in the treatment

of COPD, conducted in Vietnam The study results provide indicators on the characteristics of bone marrow fluid, nucleated and mononuclear cell composition in the bone marrow fluid of patients with COPD as well as characteristics of autologous bone marrow stem cell graft of patients with COPD The study demonstrates the safety of autologous stem cell therapy from bone marrow for COPD treatment, as well as the initial results of the therapy when treating COPD patients The thesis also provides scientific data as a basis for further studies

Thesis outline:

The thesis consists of 119 pages, covering: introduction (2 pages), overview (39 pages), object and method of research (21 pages), results (27 pages), discussion (28 pages), conclusion (2 pages) It has 48 tables, 7 figures, 2 charts 154 References, including English and Vietnamese versions

CHAPTER 1: OVERVIEW 1.1 Overview of COPD

COPD definition

According to the definition of the Global Initiative on COPD (GOLD), COPD

is a common preventable and treatable disease, characterized by chronic airflow obstruction, often progressive with a progressive response Excessive chronic inflammatory response in the respiratory tract and lung parenchyma to noxious particles or gases

COPD pathogenesis

The pathogenesis of COPD is very complex, in which inflammatory factors play

a central role The hallmark of COPD is frequent, excessive inflammation of the entire airways and lung parenchyma This inflammatory reaction is much more amplified than the usual inflammatory responses of the airways Cells involved in the

inflammatory response release a variety of inflammatory mediators that chemotaxis other inflammatory cells and proinflammatory mediators that amplify inflammation and promote tissue remodeling, change the normal structure and function of the lung Imbalance of Proteinase - Proteinase inhibitors is the most important pathogenesis of the development of pulmonary emphysema and loss of lung elasticity In COPD the balance is tilted towards increased proteolysis or increased proteinase

Oxidative activation not only damages lung tissue but also participates in the imbalance of protease - anti-protease Antioxidants also support inflammation by promoting the activity of genes that produce inflammatory chemical mediators such

COPD diagnosis

Thoroughly explore the history of exposure to risk factors for the disease, perform a clinical examination to find diagnostic-oriented signs such as a history of exposure to risk factors (smoking, dust smoke), history of cough , chronic sputum production or persistent dyspnea, increasing with exertion

Spirometry is the gold standard for diagnosing and assessing the degree of airway obstruction in patients with COPD

Diagnosis is confirmed when the obstructive disorder does not completely recover after the bronchodilator test: the FEV1/FVC index < 70% after the test

1.2 Stem cells and sources of stem cells

Definition

Stem cells are unspecialized cells that can self-renewal and can also differentiate into more mature cells with specialized functions In humans, stem cells have been found in early embryos; in some fetal tissues, umbilical cord and placenta; and in some mature organs Sources of stem cells: embryonic stem cells, fetal stem cells, adult stem cells

Bone marrow stem cells

Bone marrow is the home of hematopoietic stem cells (HSCs), mesenchymal stem cells (MSCs), endothelial stem/progenitor cells (EPCs), and a few other rare types of stem cells In which, HSC and MSC have been studied and applied widely

Bone marrow MSCs

Definition of MSC

embryo, which originates mainly in the mesoderm and makes up the majority of connective tissue cells in the adult body Mesenchymal Stem Cells (MSCs) are multipotent stromal cells that can differentiate into many different cell types of connective tissue including osteoblasts, chondrocytes, muscle cells, and stromal cells fat cells, etc

IL-10 and NO

MSCs have immunosuppressive ability, modulating the function of T cells and

B cells MSC also has the ability to modulate immunity through the effect of reducing the maturation and function of dendritic cells, inhibiting dendritic cells proliferation, differentiation and chemotaxis of B cells in vitro

Producing BM stem cells graft protocol

Collection of BM stem cells

BM stem cells extraction: manually or automatically

Stem cells preservation

Fresh stem cell grafts

After collection, stem cells are stored at 4°C for a maximum of 72 hours and then infuse to the patient This method is usually applied to facilities that do not have sufficient conditions to be able to store stem cells for a long time

Cryopreserved stem cell grafts

After being collected, if the storage period is longer than 3 days, it is frozenized and stored at -80oC or lower The stem cells grafts were stored for a long time at sub-zero temperatures (-196oC), in liquid nitrogen

Thawing and infusion of stem cells to the patient

Evaluation and quality control of stem cell grafts

Nucleated cells count

Count the percentage of viable cells by Trypan Blue staining method

Cluster culture

Identification of live CD34+ and MSC cells by flow cytometry

Determine the state of infection

1.3 Application of autologous BM stem cells in COPD management

1.3.1 Pre-clinical trials

In general, the studies showing the effectiveness of the treatment of COPD in experimental animals are mainly attributed to the chemical mediators secreted by MSCs These substances reduce the inflammatory response, modulate the immune response, improve epithelial and endothelial cell permeability, and promote tissue damage repair In addition, some pneumocytes are regenerated, which can also contribute to amelioration of the disease These preclinical studies demonstrate the therapeutic potential of MSCs with COPD in humans

1.3.2 Clinical trials applying BM stem cells in COPD

Figure 0.1 Clinical trials applying BM stem cells in COPD

Trial Phase Stem cells Number Results Weiss và cs 2 Allogeneic BM

MSC vs placebo

62 COPD patients,

No toxicity or serious events or deaths

Trial Phase Stem cells Number Results

1x108cells/1 infusion Once a month in 4 months

moderate and severe

There was no difference in lung function or quality of life compared with the control group

Decrease in CRP 1 month after infusion Ribeiro-Paes

và cs

1 Autologous BM

MSC 1x108

cells/kg, single infusion

4 severe COPD patients

No side effects Little improvement in respiratory function, little improvement in health status and quality of life

Stolk và cs 1 Autologous BM

1-2x106cells/kg Twice, 1 week apart

7 COPD patients, severe and much severe

No side effects Alveolar membrane increases CD31 cell expression

1.3.3 Clinical trials applying stem cells in COPD in Vietnam

In 2015, Le Thi Bich Phuong et al conducted a pilot study, without a control group at Van Hanh Hospital (Ho Chi Minh City), Military Hospital 103, National Lung Hospital, initially evaluate the role of mesenchymal stem cells from allogeneic umbilical cord in the treatment of COPD in 20 patients Umbilical cord-derived mesenchymal stem cells (UC-MSCs) were collected from umbilical cord samples from donors at birth Purified UC-MSCs were obtained through multiple stages and qualified for a one-time intravenous infusion to study patients Study results showed that no infusion-related toxicity, death, or serious adverse events occurred that were considered related to the use of UC-MSC Patients receiving UC-MSC infusion showed a significant reduction in mMRC, CAT scores and exacerbations The mMRC value dropped sharply from 1 before treatment to 0 after 1, 3 and 6 months

of treatment Similarly, CAT scores also decreased significantly from 10.05 before treatment to 6.5 at 1 month after treatment, 4.0 at 3 months after treatment and 2.0 at

6 months after treatment COPD exacerbations significantly decreased from 2 before treatment to 0 at 6 months after treatment However, FEV1, CRP and 6-minute walking test were not significantly improved after treatment (1, 3 and 6 months)

compared with before treatment Some other medical facilities also initially conducted TBG treatment for a number of diseases, including COPD However, to date, no other authors have published research results on TBG therapy for COPD

CHAPTER 2 METHODOLOGY 2.1 Subjectives

60 moderate and severe COPD patients at Respiratory Center, Bach Mai hospital

2.1.1 Inclusions criteria

The patient must meet all of the following criteria:

- Male and female patients, between the ages of 40 and 80

- Patient was diagnosed with COPD according to GOLD 2016

- Patient was diagnosed with COPD after 40 years of age

Patients with any of the following criteria were not included in the study:

- Having other lung diseases (asthma, active tuberculosis, restrictive lung disease, idiopathic pulmonary fibrosis, lung cancer, pneumococcal disease )

- Weight < 40 kg

- Have a bacterial or viral infection

- Had an exacerbation of COPD requiring hospitalization within the previous 4 weeks

- Current or recent quit smoking within 6 months

- Are breastfeeding, pregnant or planning to become pregnant

- Unstable or life-threatening cardiovascular disease:

o Heart failure with left ventricular ejection fraction < 40%

o Myocardial infarction or unstable chest pain in the past 6 months

o Heart valve disease, cardiomyopathy, congenital heart disease

o Severe arrhythmia

- Unstable liver disease such as cirrhosis, hepatic encephalopathy syndrome, coagulopathy, hypoalbuminemia, progressive hepatitis B, C

- Renal failure (creatinine > 2 mg/dl (176.8 µmol/l))

- Uncontrolled diabetes mellitus

- Other clinically significant unstable pathologies/abnormalities Significance was defined as any medical condition that, in the opinion of the investigators, would place subjects at risk of unsafe participation in a study, or compromise the efficacy and safety of the analysis only if the condition worsened during the study

- Have a history of or are being diagnosed with cancer

- Use of a TNF inhibitor within the previous 3 months

- Use of immunosuppressive drugs (azathioprine, methotrexate, .) within at least 8 weeks before

- Allergy to anesthetic drugs, anesthesia is not tolerated when testing stimulation

- Patients with any disease, assess survival time less than 6 months

- The patient is not able to perform the necessary tests and assessments during the study

- Patient is participating in another study

+ Respiratory Center, Bach Mai hospital

+ Nuclear Medicine and Oncology Center, Bach Mai Hospital

+ Hematology and Blood Transfusion Center, Bach Mai Hospital

+ Department of Anesthesiology of Bach Mai Hospital

+ Department of Intensive Care, Bach Mai Hospital

2.2.2 Sample collections

- Simple randomized sampling

- Sample size:

+ Group 1: 30 patients receving autologous BM stem cell infusions

+ Group 2 Placebo group: 30 patients

All COPD patients in the study were maintained on basic treatment

- Sample collection:

+ Patients with COPD who wish to participate in the study are screened according

to the procedure The hospital's expert panel consults with patients to decide if they are eligible to participate in a stem cells treatment study

- The approved patients are divided into 2 groups by lottery method:

+ Prepare the prescribed numbers for 2 groups:

stem cell therapy group (hereinafter referred to as intervention group): No 02

Step 2: Explain to patient and family

Step 3: Perform paraclinical surveys to screen patients participating in the study Step 4: Pre-intervention assessment

Step 5: Patients are randomly assigned to 2 treatment groups and control groups Maintain background treatment of COPD according to GOLD guidelines

Step 6: Intervention techniques

- The patient had BM fluid collected at the operating room of the Anesthesia Department, Bach Mai hospital by aspiration of TX fluid at the posterior upper iliac spines under spinal anesthesia The bone marrow fluid is processed to create stem cell graft using the SEPAX 2 system at the Hematology and Blood Transfusion Center

- The patient continues to be closely monitored the whole condition, the BM suction site to detect and handle possible complications such as bleeding, hematoma

at the bone marrow aspiration site, infection at the aspiration site bone marrow and waiting for a stem cell transplant

- The obtained stem cells graft is sampled for quality assessment tests, the rest will be divided into 2 parts: 1 part is used for the 1st infusion, the rest is stored in liquid nitrogen The preserved portion will be thawed and given to the patient at 6 months after the first infusion

- Autologous stem cell graft from bone marrow was evaluated at the time of the 1st and 2nd infusion

+ Determine the number of cells by automatic counter

+ Determine the percentage of viable cells by Trypan Blue

+ Determine the percentage of CD34 + cells and the surface marker panel of MSCs (CD90+, CD105+, CD73+) flow cytometry technique

+ Culture of bacteria, fungi for each stem cell graft

The criteria for autologous BM stem cell production:

+ Viable cells bef

+ The number of nucleated cells is at least 300 x 106 cells

+ Proportion of stem cells with markers (CD90+, CD73+, CD105+) and stem cells with CD34+ immune phenotype

+ Culture of bacteria and fungi with stem cells to determine sterility

- Perform the first autologous BM stem cell infusion after extraction for the patient After 6 months, the patient is examined and tested as well as evaluated as in step 3 and step 4 If the patient meets the criteria of the indication and has no contraindications, they will receive a second infusion of stem cells with thawed stem cells after storage in liquid nitrogen

- Patients are closely monitored during stem cells infusion and immediately after TBG infusion (1st and 2nd time)

Long term follow up

- Patients in both groups were maintained on the basic treatment regimen of COPD according to GOLD guidelines to maintain the stability of the disease and drugs being treated for other medical conditions not in the exclusion criteria such as: : hypertension, diabetes, The total follow-up time of patients in both groups was 12 months after the first stem cell infusion or from the time of participating in the study with patients in the control group, followed up once a month