Treatment of acute ischemic stroke by minimally manipulated umbilical cord-derived mesenchymal stem cells transplantation:A case report

Hyunjun Ahn,Sang Yeon Lee,Won Ju Jung,Kye-Ho Lee

Hyunjun Ahn,Sang Yeon Lee,Kye-Ho Lee,bio Beauty&Health Company(bBHC)-Stem Cell Treatment &Research Institute(STRI),Seoul 04420,South Korea

Won Ju Jung,97.7 Beauty&Health(B&H)Clinics,Seoul 04420,South Korea

Abstract BACKGROUND Stroke is one of the major causes of disability and death worldwide.Some treatments for stroke exist,but existing treatment methods have limitations such as difficulty in the regeneration of damaged neuronal cells of the brain.Recently,mesenchymal stem cells(MSCs)have been studied as a therapeutic alternative for stroke,and various preclinical and case studies have been reported.CASE SUMMARY A 55-year-old man suffered an acute stroke,causing paralysis in the left upper and lower limbs.He intravenously transplanted the minimally manipulated human umbilical cord-derived MSCs(MM-UC-MSCs)twice with an 8-d interval.At 65 wk after transplantation,the patient returned to his previous occupation as a veterinarian with no adverse reactions.CONCLUSION MM-UC-MSCs transplantation potentially treats patients who suffer from acute ischemic stroke.

Key Words:Acute ischemic stroke;Behavioral disorder;Umbilical cord-derived mesenchymal stem cells;Allogenic;Cell therapy;Minimal manipulation;Case report

INTRODUCTION

Stroke is a major cause of disability and death worldwide[1-3].Acute ischemic stroke is treated by injecting thrombolytic medication into the peripheral vein[4-7].Meanwhile,hemorrhagic stroke requires treatment involving the removal of the thrombus and intracerebral blood using open surgery or minimally invasive surgery[8-10].However,since these methods do not directly regenerate the damaged brain,and side effects can occur,they are not a complete treatment for stroke[10-12].In this respect,treatment using stem cells has received much attention as a novel strategy for treating stroke[13].Notably,mesenchymal stem cells(MSCs)can differentiate into neuronal cells and secrete cytokines and growth factors with neuroprotective effects[14-17].Numerous studies are ongoing regarding the use of MSCs in the treatment of stroke.Previous studies have shown the efficacy of autologous MSCs in treating patients with ischemic stroke,thereby verifying the potential of stem cell therapy using MSCs for ischemic stroke[18-21].The safety of MSC transplantation has been well established based on the results of numerous studies and clinical tests[21,22].

Umbilical cord-derived MSCs(UC-MSCs)rarely cause immune rejection despite being allogeneic cells[23-26].This means transplantation can occur without immunosuppressants.Moreover,among all tissues from which MSCs can be harvested,the largest number of these cells can be obtained from the UC;this effectively eliminates the need for cultured MSCs[27,28].Uncultured UC-MSCs are known to have the most similar characteristics to MSCs in the body[29].Uncultured UC-MSCs are also safer due to the absence of cellular abnormal caused by aging and replication of DNA fromin vitromanipulation[29-32].Furthermore,the absence of cellular aging allows a high level of cellular activity,and as the cells are small,a greater number of cells pass through the blood-brain barrier(BBB)after intravenous injection[33-35].Based on this evidence and the verified safety of MSC transplantation,we applied intravenous transplantation of minimally manipulated UC-MSCs(MM-UC-MSCs)as treatment of a patient with acute ischemic stroke.To date,there have been no other reported cases in which this novel method has been applied to treat stroke.

CASE PRESENTATION

Chief complaints

On March 13,2018,a 55-year-old man,suffering from stroke,visited our clinic.He has paralysis in the left upper and lower limbs.

History of present illness

The patient had symptoms of temporary weakness on February 17 and 18,2018.He woke up in the morning and suffered an acute stroke,causing paralysis in the left upper and lower limbs.The patient was diagnosed with an Rt striatocapsular infarct at the ER of a university hospital(Figure 1A).He was discharged from the hospital on March 2,2018,after receiving only an aspirin prescription because he had normal brain blood vessels based on a brain computed tomography(CT)scan,even though his condition worsened during hospitalization(Figure 1).On the day of discharge,he was admitted to a hospital specializing in rehabilitation to receive long-term rehabilitation treatment.And then he visited our clinic to receive stem cell therapy on March 13,2018.

Figure 1 Brain computed tomography images of patient before minimally manipulated human umbilical cord-derived mesenchymal stem cells transplantation.These are brain computed tomography images of patient at 02/19/2018(A)and 02/28/2018(B).The white ovals indicate the lesion site.

History of past illness

The patient had no history of specific illnesses.

Personal and family history

The patient had a free personal and family history.

Physical examination

The patient was paralyzed in the left upper and lower limbs and was unable to walk because of an impaired sense of balance.In addition,he had speech impairment due to facial paralysis(Video 1).

Laboratory examinations

The laboratory examination showed that the values of WBC(6.5 × 109/L),RBC(4.58 ×1012/L),Hemoglobin(136 g/L),Platelet(258 × 109/L),MCV(87.1 × 10-15L),MCH(29.7× 10-12g),MCHC(34.1%),and Neutrophil(64.9%)were in the normal range.However,the values of AST(69 U/L)and ALT(46 U/L)were higher than normal.It is known that high values of AST and ALT are associated with stroke[36].

The degree of stroke was evaluated based on the National Institute of Health Stroke Scale(NIHSS)at the time of his visit.The score was 16.

Imaging examinations

Immediately after the pathogenesis,the T2/FLAIR-axial and T1-axial images from the patient showed an RT striatocapsular infarct in the right striatum of the brain.The size of the lesion was 3 cm × 2 cm(Figure 1).

FINAL DIAGNOSIS

The patient was diagnosed with acute ischemic stroke based on the history of present illness,blood test,and the brain CT images.The grade of stroke was assessed the moderate to severe stroke based on the NIHSS score.

TREATMENT

UC procurement

UC was donated by the Obstetrics and Gynecology Department at Lynn Woman’s Hospital.The agreement of UC donation was obtained from the mother.To confirm the safety of the donated UC,we performed a total of seven blood and urine tests from the mother,including for hepatitis B surface antigen,hepatitis B surface antibody,hepatitis C antigen,hepatitis C antibody,human immunodeficiency virus,syphilis rapid plasma reagin,and human T-cell lymphotropic virus type I and II antibody.

Isolation of MM-UC-MSCs

The donated UC were 25 cm in length,and 4.0 × 108MSCs were obtained from UC.The process of isolating cells from the UC is as follows[37,38].The amnion and three blood vessels of the UC were removed.Next,the UC tissue was cut and ground using operating scissors and disposable tissue grinder.Then,the enzyme mixture of collagenase and hyaluronidase treated to the ground tissue and placed in a 37 °C,50 mL/L CO2incubator for 0.5-1.0 h.After that,the solutions contained the isolated cells were filtered through a 100 μm cell strainer and centrifuged to obtain cells.The cells were immediately frozen and stored at -197 °C.

Evaluation of quality and purity of isolated MM-UC-MSCs

We confirmed the isolated cells expressed the MSC markers such as CD73,CD90,and CD105 using a CyFlow?Cube 6(Sysmex)and FCS Express 5 software(Figure 2).We confirmed the isolated cells expressed MSC markers CD73(79.59%),CD90(99.82%),and CD105(97.97%).We determined that the isolated cells were of the minimally manipulated umbilical cord mesenchymal stem cells(MM-UC-MSCs)with same quality as our previous results because the MM-UC-MSCs isolated by the same method uniformly expressed CD73(70%-80%),CD90(90%-100%),and CD105(90%-100%).The isolated cells were evaluated using a sterility test,mycoplasma test,endotoxin test,and testing for adventitious agents of biological products,according to regulations from the Ministry of Food and Drug Safety in the Republic of Korea(data not shown).

Figure 2 Mesenchymal stem cell marker expression in minimally manipulated umbilical cord-derived mesenchymal stem cells.The expression marker tested was A:CD73(79.59%);B:CD90(99.82%);C:CD105(97.97%).

Preparation of injection solution

Frozen UC-MSCs(6 × 107cells)were thawed and washed with 1xPhosphate-Buffered Saline(PBS)three times.The cells collected through centrifugation were resuspended in 20 mL of 0.9% normal saline solution and then divided evenly into two 10 mL syringes(HWAJIN,Cheonan-si,Chungcheongnam-do,South Korea).

Treatment of patient

In one treatment,the 10 mL injection solution containing MM-UC-MSCs were injected the 100 mL of 0.9% sodium chloride injection,USP.Then,the 110 mL of mixture were intravenously transplanted for 1-1.5 h to the patient.Immediately after the transplant was over,the same process was repeated once more.The MM-UC-MSCs transplantation was performed twice with an 8-d interval.

Combination of stem cell therapy and rehabilitation exercise

The patient was treated with stem cell therapy and rehabilitation for 4 mo.

OUTCOME AND FOLLOW-UP

The changes in the behavior of the patient were recorded by videography.One month after the first transplantation,the patient recovered from the left upper limb and facial paralysis(Video 2).The patient was able to lift his left arm up to chest level,and recovery of his left arm and hand muscles allowed the patient to control the brakes of his wheelchair.After 8 wk,the patient recovered from left leg paralysis and could walk with an orthosis(Video 3).After 15 wk,the patient showed recovery of the left lower limb muscles and could walk without an orthosis(Video 4).After 36 wk,the patient could remain the arm in the initial position for the full 10 s.Also,the left leg drifted to an intermediate position prior to the end of the full 5 s,but at no point touches the bed for support(Video 5 and Video 6).After 60 wk,recovery from leftsided paralysis,restoration of the respective muscular function,and sense of balance allowed the patient to climb up and down the stairs without an orthosis(Video 7).Moreover,his left arm no longer suffered from tremors,enabling the patient to perform sophisticated tasks(Video 8).After 65 wk,the patient,previously a veterinarian,could return to work,as the patient had recovered to the point that they could maintain a standing position for a long time as required in surgery.The patient was admitted from March 2 to July 7,2018,to the hospital specializing in rehabilitation and received rehabilitation treatment.After discharge from the rehabilitation hospital,the patient continued to perform rehabilitation exercises by himself(Video 9).

The grade of stroke was evaluated using the NIHSS at 0,5,15,24,36,and 60 wk after the first stem cell transplantation.The score was 16 at week 0(before transplantation),which gradually decreased to 0 by 60 wk(Figure 3).

The patient took a brain CT images to confirm the size of the lesion about 30 mo after stem cells transplantation.The size of the lesion reduced to 0.6 cm × 0.3 cm(Figure 4).

Figure 3 National Institute of Health Stroke Scale score of patient.The patient’s National Institute of Health Stroke Scale score gradually decreased for 60 wk after the first treatment.NIHSS:National Institute of Health Stroke Scale.

Figure 4 Brain computed tomography images of patient after minimally manipulated human umbilical cord-derived mesenchymal stem cells transplantation.These are brain computed tomography images of patient at 30 mo after first transplantation.The lesion size decreased from 3 cm × 2 cm to 0.6 cm × 0.3 cm.The white ovals indicate the lesion site.LPH:Left posterior head;LAF:Left anterior frontal.

Report of side effects

The patient reported depressive symptoms following the stroke.However,no side effects,dysfunction,or other symptoms were reported following stem cell therapy.Depressive symptoms disappeared approximately 4 mo after the first transplantation.During the treatment and follow-up period,no adverse reactions related to mobility were observed or reported by the patient.

DISCUSSION

We described stem cell therapy using allogeneic MM-UC-MSCs in a patient following an acute ischemic stroke.Through many studies,the role of MSCs in the regeneration of damaged brain is already well known.In the brain,MSCs mainly use secreted factors to promote endogenous neuronal cell growth,reduce apoptosis,and regulate inflammation[39].MSCs transplanted into the damaged brain area promote functional recovery by secreting nutrient factors that induce survival and regeneration of host neurons.Transplantation of MSCs is also known to significantly increase the proliferation of endogenous neural stem cells[40].In addition,MSCs affect blood vessel cells in the brain.MSCs are known to promote angiogenesis and regenerate damaged brain microvessels[41,42].These results provide evidence that MSCs may have a positive role in the treatment of acute ischemic stroke.

For easier and safer transplantation of MSCs,we chose to administer MSCs intravenously rather than surgically implant them in the lesion area.As MSCs exhibit homing effects,we predicted that they would migrate to the lesion area even after intravenous transplantation[43,44].Previous studies have also demonstrated that MSCs can migrate to the brain through the BBB and that smaller MSCs increase the percentage of cells migrating to the brain through the BBB[33-35].The size of a single MSC increases as aging occurs during culture[31,32].Thus,we decided to transplant uncultured MSCs to increase their therapeutic effects.Autologous MSCs were not suitable for our strategy,as fewer cells can be collected from the bone marrow,blood,or adipose tissue.Therefore,we chose UC as the source of MSCs,as it allows the harvesting of an adequate number of cells for transplantation without culture[28].Although UC-MSCs are allogeneic cells,immune rejection is rare,as MSCs can regulate immune responses[27,43].Also,MSCs are used as an immunosuppressant by itself[45,46].Based on this previous study,no immunosuppressants were used throughout the treatment.Following the two transplantations of MM-UC-MSCs,the patient was monitored for 30 mo.The brain CT imaging results indicated that the transplanted MM-UC-MSCs migrated to the striatum of the brain to restore tissue at the lesion site.This supports our hypothesis that stroke may be treated through intravenous transplantation of MM-UC-MSCs,and that MSCs can reliably migrate to the lesion area through the BBB.

Stroke scale assessment based on the NIHSS should be interpreted with care,as it is a subjective assessment.60 wk after the first transplantation,the patient showed gradually decreasing test scores,indicating restoration of motor ability.We assessed the mobility of the patient on the stroke scale and recorded the changes using video clips.The patient's mobility was shown to have improved not only to the level of simple walking and limb movements,but also to the level where he could perform sophisticated tasks and stand for a long period of time,allowing the patient to return to his previous occupation as a veterinarian.

During treatment and follow-up monitoring,no adverse reactions related to mobility were observed or reported by the patient.In addition,the patient exhibited no symptoms such as fever,chills,or nausea that frequently occurred in patients who received MSC transplantation.The patient was administered an antidepressant due to the depressive symptoms caused by early stroke,but no other drugs were used,such as those for immunosuppressants,blood pressure,or blood circulation.

Here,we report the transplantation of MM-UC-MSCs in a patient with stroke.The reported clinical and imaging data are the results of 30 mo of monitoring following transplantation.Based on this case,we are optimistic that intravenous transplantation of MM-UC-MSCs is a safe and effective treatment following a stroke.However,further studies should be conducted to confirm the safety and effectiveness of this treatment before adopting this approach to treat a greater number of stroke patients.In addition,we think that the patient’s constant rehabilitation exercise had a positive effect on the treatment.Therefore,there is a need for studies to understand the correlation between stem cell therapy and rehabilitation exercise in stroke patients.

CONCLUSION

The therapeutic effect of MM-UC-MSCs on acute ischemic stroke is very high as shown in the patient presented here.Recurrence and side effects did not occur during the treatment and follow-up duration of at 30 mo.Based on these results,we expect that MM-UC-MSC transplantation will be an alternative for the treatment of acute ischemic stroke.However,it is necessary to conduct more studies with a greater number of patients.