3. Luan An Tom Tat - Eng.pdf

108 INSTITUTE OF CLINICAL MEDICAL AND PHARMACEUTICAL SCIENCES LE CHI VIEN APPLICATION RESEARCH ON AUTOLOGOUS BONE MARROW STEM CELLS IN THE TREATMENT OF MIDDLE CEREBRAL ARTERY INFARCTION Specialism Neu[.]

108 INSTITUTE OF CLINICAL MEDICAL AND

Specialism: Neuroscience Code: 9720158

ABSTRACT OF MEDICAL DOCTORAL THESIS

Ha Noi – 2023

MINISTRY OF DEFENCE MINISTRY OF EDUCATION

AND TRAINING

The thesis has been completed at 108 Institute of Clinical Medical

and Pharmaceutical Sciences

Supervisors:

1 Assoc Prof Lê Huu Song

2 Assoc Prof Nguyen Hoang Ngoc

on .th 2023

National Library:

1 National Informatics Library

2 Library of 108 Institute of Clinical Medical and Pharmaceutical Sciences

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BACKGROUND

About 50 to 70% of patients with middle cerebral artery (MCA) infarction suffer from severe sequelae Most of the current treatments being applied in the subacute period of cerebral infarction have limited effectiveness, therefore, it is necessary to research and apply new treatment therapies Bone marrow stem cells (BMSCs), also known as bone marrow-derived mononuclear cells, had shown clear and consistent experimental evidence in terms of safety and effecacy in treating cerebral infarction when administered via the vascular route (intravenous or intra-arterial infusion) However, there is limited evidence about its efficacy in humans, with several pilot studies reporting a tendency to improve in neurological function, such as Taguchi A (2015) or Bhatia V (2018), but only on limited samples Currently, there are no clinical trials on BMSCs for the treatment of stroke in Vietnam This study was conducted with 2 objectives:

Objectives 1: to evaluate the safety of intravenous autologous bone marrow stem cell therapy and intra-arterial autologous bone marrow stem cell therapy in the treatment of MCA infarction

Objectives 2: to evaluate the efficacy of intravenous autologous bone marrow stem cell therapy and intraarterial autologous bone marrow stem cell therapy in the treatment of MCA infarction

Chapter 1 OVERVIEW

1.1 MIDDLE CEREBRAL ARTERY INFARCTION

1.1.1 Diagnosis of MCA infarction

1.1.1.1 Symptoms and signs

According to Mohr J (2012), symptoms of MCA infarction include hemiplagia and/or numbness; ataxia of contralateral extremities; aphasia as a result of a dominant hemisphere lesion; perceptual deficits

as a result of a non-dominant hemisphere lesion

1.1.1.2 Diagnostic imaging

a Parenchymal imaging: Head CT shows hypodense lesions in

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territory of MCA, identified and evaluated using the ASPECT scale During the first week, the infarcted parenchyma demonstrates high DWI signal and low ADC signal on head MRI

b CT angiography (CTA) or MR angiography (MRA) shows the

occlusion of MCA consistent with cerebral infarct territory

1.1.3.1 Short-term and long-term prognosis

MCA infarction has poor prognosis in the short term with 40-80% mortality and severe sequelae in the long term

1.1.3.2 Prognosis of upper limb and aphasia recovery

The ability to recover upper limb motor function, especially the hands and fingers, is low According to SH Jang (2013), the rate of complete recovery of hand and finger motor function is at 0% The ability to recover language function in patients with aphasia is similar 1.2 BONE MARROW STEM CELLS

BMSCs are adult stem cells including hematopoietic stem cells (HSCs), mesenchymal stem cells (MSCs) and endothelial progenitor cells

1.2.1 Hematopoietic stem cells

1.2.1.1 Surface markers used in the identification of HSCs

CD34 is the most important marker used to identify HSCs

1.2.1.2 Characteristics of HSCs

The main characteristics of HSCs are ability of seft renewal, potential for differentiation, "homing" and apoptosis

1.2.1.3 Plasticity of HSCs

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1.2.2 Mesenchymal stem cells

1.2.2.1 Surface markers used in the identification of MSCs

Confirming MSC identity requires the use of several surface markers The Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy defines mesenchymal stem cells as positive for CD105, CD73, CD90, and negative for CD45, CD34, CD14 or CD11b, CD79ᵅ or CD19, MHC-class II

1.2.2.2 Characteristics of MSCs

The main characteristics of MSCs are (1) supporting hematopoiesis, (2) immunoregulatory properties, (3) migration and homing, (4) potential for differentiation

1.3 BMMCS IN THE TREATMENT OF CEREBRAL INFARCTION

1.3.1 Mechanism of intravenous or intra-arterial BMSCs therapy

in cerebral infarction

1.3.1.1 The ability of BMMCs to cross the blood-brain barrier

By labeling BMSCs before infusion and monitoring its presence in brain tissues after infusion, several experimental studies have demonstrated that BMSCs cross the blood-brain barrier to cerebral parenchyma when infused through the vascular system: Bing Yang (2013); Vasconcelos-dos-Santos A (2012) or Zhang H L (2018)

1.3.1.1 BMSC-derived paracrine factors

After being infused into the circulatory system, BMSCs release several crucial paracrine factors, including chemokines, cytokines, growth factors and extracellular vesicles These factors promote the endogenous repair process to regenerate new neurovascular units (NVU) through endogenous angiogenesis, endogenous neurogenesis, anti-inflammation and immune regulation

1.3.1.2 Angiogenesis

There is clear experimental evidence on the angiogenic effect of BMSCs in cerebral infarction (Youshi Fujita, 2010)

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1.3.1.3 Neurogenesis

The neurogenic effect of intravascularly injected BMSCs has been evidenced in experimental studies, including research by Zhang HL (2018) Direct mechanism: Stem cells pass through the blood-brain barrier to the cerebral infarction area and differentiate directly into brain cells Indirect mechanism: stimulating endogenous neurogenesis through paracrine substances released from BMSCs

1.3.1.4 Anti-inflammatory and immunomodulatory effects

1.3.1.5 Differences in mechanism of action between intravenous BMSCs and intra-arterial BMSCs in cerebral infarction

A larger number of stem cells can pass through the blood-brain barrier to the area of cerebral infarction when BMSCs are infused via

the arterial route compared to using the venous route, according to

Bing Yang (2013); Zhang H L (2018) In terms of neurogenic effect, Bing Yang (2013) reported no difference between the two routes, while according to Zhang HL (2018), the arterial route was more effective In terms of the angiogenic effect, there was no difference between two routes according to Bing Yang (2013)

1.3.2 Experimental evidence on the safety and efficacy of BMSCs

in the treatment of cerebral infarction

1.3.2.1 The safety and efficacy

The experimental evidence on the safety and effectiveness of both intravenous BMSCs infusion and intra-arterial BMSCs infusion is quite strong and consistent, the difference between the 2 groups is only

in terms of TBG TX dose level and infusion time, shown by the results

of some meta-analyses, such as Vahidy FS (2016) (IV route); Guzman

R (2018) (IA route) Also according to some meta-analyses, BMSCs infusion via IA route has the advantage of being more effective

1.3.2.2 BMSCs doses in experimental studies

The dose of 1x106 cells/kg is not effective in terms of clinical improvement, the minimum effective dose is 10x106 cells/kg in IV

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BMSCs infusion and 5x106 cells/kg in IA BMSCs infusion The doses

in several studies are listed in Table 1.3 of the full text of the thesis

1.3.3 Clinical evidence on the safety and efficacy of BMSCs in the treatment of cerebral infarction

1.3.3.1 Safety

a IV route: Several meta-analyses have shown that IV BMSCs

infusion was safe A few adverse events were recorded such as fever, mild infection, seizures, recurrent stroke however there was no difference compared to the control group Savitz S (2011) used high dose (10x106 cells/kg) and concluded that the therapy was safe Lee J

S (2010) used dose of 5x107 cells and did not record any cancer-related adverse events after 5 year follow-up

b IA route: Bhatia V (2018) used dose of 1,02 x 108 BMSCs and recorded safety According to Guzman R (2018), no embolic complications were recorded when infusing BMSCs at high doses

1.3.3.2 Efficacy on neurological function

a IV route: Taguchi A (2015) used dose of 3,4×108 cells and concluded a tendency of improved neurological function

b IA route: Several preliminary studies reported that there was a

tendency of improved neurological function in patients having IA BMSCs infusion, while Bhatia, V (2018) reported that the outcome in

IA BMSCs group (10 patients) was better than that in control group (10 patients), shown by the proportion of patients achieving mRS≤2 (80% in IA BMSC group vs 40% in control group) Similarly, Friedrich et al (2021) reported that the pecentage of patients achieving mRS≤2 after 3 months using IA BMSCs infusion was 40% Some studies that published negative results all used doses of <3,1x108 cells: Ghali AA (2016) (1x106 cells); Savitz SI (2019) (3,08x106 cells)

1.4 STEM CELL RESEARCH FOR THE TREATMENT OF NEUROLOGICAL DISEASES IN VIETNAM

Prior to the conduct of this study, we have not recorded any

a Inclusion criteria include: (1) patients between 20–75 years old; (2)

ipsilateral MCA infarct confirmed by head MRI; (3) onset of stroke between 7 and < 40 days; (4) NIHSS at day 7 from stroke onset ≥ 7 (at

day 7 after stroke onset); (5) written informed consent

b Patients with at least ONE the following conditions were excluded:

(1) hemorrhagic stroke or symptomatic hemorrhagic transformation; (2) lacunar infarction; (3) previous history of stroke with mRS ≥2; (4) administered IV or mechanical thrombectory after stroke; (5) history

or current diseases of blood forming organs; (6) history or current cancer; (7) acute or chronic heart failure with NYHA Class III or higher; (8) acute or chronic renal failure with decreased renal function; (9) acute or chronic liver failure with decreased liver function; (10) any other severe comorbidity; (11) pregnancy or breast feeding; (12) contraindication for MRI; (13) current participation in any clinical trial in the last three months

All patients received standard medical care and were admitted to a stroke rehabilitation center according to current guideline but patients

in the IV-BMSC group, in addition, received BMSCs infusion as protocol

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Convenience sampling method was used 94 patients with MCA infarction who fulfilled the inclusion and exclusion criteria were allocated into IV group (n=32), IA group (n=31) and control group (n=31) All patients received standard medical care and were admitted

to a stroke rehabilitation center according to current guideline but patients in the IV-BMSC group, in addition, received IV BMSCs infusion and patients in the IA-BMSC group, in addition, received IA BMSCs infusion as protocol

2.3 LOCATION AND TIME OF RESEARCH

Research was conducted at the Stroke Department of 108 Military Central Hospital, from July 2018 to April 2022

2.4 RESEARCH PROCESS STEPS

2.4.1 Patient recruitment, standard medical treatment, group allocation and clinical and head MRI evaluation before autologous stem cell infusion (T0 timepoint)

2.4.1.1 Screening and selecting patients for research

2.4.1.2 Allocation

2.4.1.3 Standard medical treatment

2.4.1.4 Clinical and head MRI evaluation at T0 timepoint

2.4.2 Bone marrow aspiration, cell separation, preservation and quality assessment of BMSCs

240 mL of autologous bone marrow was collected from each patient in IV group and IA group, this technique was performed within

24 hours up to the expected time of BMSCs infusion

2.4.3 Autologous BMSCs infusion

2.4.4 Follow up

a Monitoring adverse events: longitudinal follow-up of 12 months

after ischemic troke

b Follow ups: follow-ups were performed at 6 months and 12 months

after ischemic stroke

2.4.5 Data analysis and conclusion

2.5.1.3 Preventive treatment of recurrent stroke

- Underlying diseases and risk factor control

- Using preventive drugs depending on ischemic stroke types: + Using antiplatelet drugs for ischemic stroke caused by atherosclerosis:

• Aspirin: 81 mg to 300 mg per day or/and Clopidogrel 75 mg per day depending on each specific patient

• If the patient has previously taken aspirin 81 mg/day, the dose could be increased to 325 mg or switched to clopidogrel 75 mg/day + Anticoagulant for thromboembolic ischemic stroke

2.5.2 Procedure for isolating and preserving BMSCs

The process has been approved by 108 Military Central Hospital, including a series of 05 techniques: (1) Bone marrow testing technique (2) Bone marrow havesting technique (3) Technique for isolation and mass formation of BMSCs by density gradient centrifugation method (4) BMSCs preservation technique (5)

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Technique for assessing the quality of BMSCs

2.5.2.1 Bone marrow test technique

A bone marow test was conducted to evaluate the patient's bone marrow function before taking a large amount of bone marrow 2mL

of peripheral blood and 1mL of bone marrow was tested on each patient (anticoagulated with EDTA) Blood cell indices are determined using the XN2000 machine (Sysmex - Japan) The number

of nucleated cells in bone marrow was determined using the XN2000 automatic hematology machine (Sysmex - Japan) The proportion and morphology of cell lines were evaluated on Giemsa-stained specimens

of blood and marrow

2.5.2.2 Bone marrow harvesting procedure

By using Hernigou technique, a total 240 mL of autologous bone marrow was collected, with 60 mL of antifreeze solution in falcol added The collection was performed under local anesthesia in operation room, through a puncture and repeated aspirations at the posterior iliac crest regions

2.5.2.3 Technique for isolation and mass formation of BMSCs by density gradient centrifugation method

The mononuclear cells were isolated by density gradient using a ficoll density gradient centrifugation procedure, 1.077 g/cm3 the remaining mononuclear cells are suspended in buffer (or 0.9% saline solution) to create a final volume of 30 mL

2.5.2.4 BMSCs preservation

- BMSCs were preserved aseptically, at a temperature of 2-8 degrees Celsius, being used within 72 hours from bone marrow collecting time

2.5.2.5 Quality assessment of the final BMSCs product

a Total nucleated cell count

- Total nucleated cells and the bone marrow mononuclear cells were counted using an automated hematology analyzer XN 2000

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b Determination of CD34+ HSCs by flow cytometry method

- Total CD34+ cells, CD34+ cell percentage, viability were tested

on the flow cytometry analyzer FACS – Calibur (BD- USA), using the CD34 Enumeration biological kit Using the BD™ Stem Cell Enumeration Kit from Becton – Dickinson - USA

c MSCs count determination

- The number of mesenchymal stem cells was determined on the Facs Calibur machine with the BD Human Mesenchymal Stem Cell Analysis Kit

- Fibroblast cluster culture (CFU-F): with MesenCult® basal and MesenCult® Stimulatory supplement culture media (STEMCELL Technologies, Inc – Canada)

d Bacteria, fungi, and mycoplasma cultures

Performed on the BacT/Alert 3D60 automated system

e Determination of endotoxin concentration: Performed on Endosafe

system - PTS V7 The normal concentration limit was < 0.5 EU/mL

2.5.3 Intravenous (IV) autologous BMSCs infusion protocol

2.5.3.1 Preparation

Procedure was performed at the emergency room of the Stroke Department by 01 doctor and 01 nurse Facilities: Emergency room; monitor; Tools related to peripheral IV line placement technique BMSCs: stored at the Department of Hematology – 108 Military Central Hospital and only taken out within 1 hour before infusion, and mixed into 200 mL 0.9% NaCl immediately before infusion

2.5.3.2 Execution

a Check medical records and patient status before stem cell infusion

b Performing the infusion:

Peripheral IV line placement technique was performed by nurse

IV BMSCs infusion procedure was perfomed by doctor:

Step 1: Pre-infuse about 150 ml of 0.9% NaCl solution at a rate of 40-60 drops/minute

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Bước 2: Infuse 200 ml 0.9% NaCl solution containing BMSCs intravenously at a rate of 40-60 drops/minute

Bước 3: Infuse the remaining 350ml of 0.9% NaCL solution

2.5.4 Intraarterial (IA) autologous BMSCs infusion protocol

Using a digital subtraction angiography, guide the distal point of diagnostic catheter to the proximal segment of ipsilateral internal carotid artery Through the microcatheter, a total of 30 mL of the BMSC solution was slowly injected

2.5.4.1 Preparation

a Technique was performed at the Intervention Department, using

DSA system for cerebral angiography, and post transfusion monitoring was carried out at the Stroke Department

b 30 mL of autologous BMSCs in falcol was stored at the

Department of Hematology – 108 Military Central Hospital, only taken out within 60 minutes before infusion

Step 4: Remove catheter and compression bandage

2.6 RESEARCH CONTENT

2.6.1 Variables used in research and evaluation methods

2.6.1.1 Variables used in the study at T0 timepoint

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a MCA infarction diagnosis:

- Clinical diagnosis: According to Mohr J (2012), MCA infarction presents with hemiplagia and/or numbness; ataxia of contralateral extremities; aphasia as a result of a dominant hemisphere lesion; perceptual deficits as a result of a non-dominant hemisphere lesion

- Diagnostic imaging:

a Parenchymal imaging: Head CT shows hypodense lesions in territory of MCA, identified and evaluated using the ASPECT scale During the first week, the infarcted parenchyma demonstrates high DWI signal and low ADC signal on head MRI

b CT angiography (CTA) or MR angiography (MRA) shows the occlusion of MCA consistent with cerebral infarct territory

b Clinical characteristics: weakness side, aphasia

c Hematological and biochemical test

e Head MRI 3.0: being performed at T0 and T6 timepoint Infarct

volume: V = AxBxC/2 (A, B, C are the width, length and height of the infarct area, respectively, being determined on DWI image The changed infarct volume = infarct volume/T0 – infarct volume/T6

2.6.1.2 Evaluation of safety result and efficacy result

a Evaluation of safety outcomes

- Short-term adverse events: including events that occured from the time of bone marrow aspiration and stem cell transfusion until discharged timepoint

- Long-term adverse events: Adverse events were recorded from the time of hospital discharge to 12 months after cerebral infarction