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Abstract

Stroke remains still the leading cause of long-term disability worldwide. Although interventions such as early reperfusion, intravenous thrombolysis, and endovascular revascularization have shown neurological benefit in stroke patients, there is still lack of effective treatment enabling regeneration of nervous tissue after cerebral ischemic episodes. Cell therapy is an evolving opportunity for stroke survivors with residual neurological deficits. The purpose of this study was to evaluate safety and potential efficacy of multiple administration of Hospital Exemption—Advanced Therapy Medicinal Product (HE-ATMP) comprising 3 × 10⁷ Wharton’s jelly mesenchymal stem cells (WJMSCs). A study group was composed of six patients—three women and three men. The patients were qualified to the treatment with diagnosis of chronic stroke (2–24 months after cerebral ischemic episode), during 2 years. All the patients undergone repeated rounds of HE-ATMP administration to the CSF (cerebrospinal fluid) via lumbar puncture. The control group consisted of six patients (two women and four men) who experienced stroke, treated at the same time (follow-up period: 24 months) using standard treatment methods, without endovascular treatment. To evaluate the results of the therapy, we used both impairment scales [National Institutes of Health Stroke Score (NIHSS)] and functional outcomes scales [Modified Rankin Scale (MRS) and Barthel Index (BI)]. In four patients, who received at least three repeated rounds of HE-ATMP, we reported neurological improvement and reduction of functional neurodeficiency. The biggest improvement concerned the reduction of speech disorders in two cases; significant improvement in the field of motor skills in three patients and reduction of apraxia and improvement of logical communication skills in two patients were also reported. All the patients became more independent. Significant improvement of the neurological condition using the same scales was registered only in two patients from the control group. We did not report any adverse events in the treated group during follow-up. At 1-year follow-up, we demonstrate safety and beneficial effect of WJMSC transplantation including neurological improvement and reduction of functional neurodeficiency. We are aware that the samples size of this study is relatively small. The treatment regimen needs to be further tested in larger group of patients.

Keywords

stroke regeneration cell therapy WJMSC ATMP

Introduction

Stroke is still the second leading cause of death worldwide after heart diseases¹. Although the effective drug treatment in acute ischemic incidence is currently available recombinant tissue plasminogen activator (rTPA), the restricted time window of application and the severe side effects limit its use¹. Effective treatment of damaged nervous tissue after stroke remains still beyond the capabilities of modern medicine^2,3. Patients after stroke often remain under the care of their relatives and the health care system for the rest of their lives. The problem results in very significant social and economic costs¹. Thus, regenerative medicine and stem cell (SC) therapies are becoming a promising strategy for stroke survivors³.

SCs have unique capabilities that allow them to form complex tissues and organs and maintain their functions during the life of an individual⁴. They can be obtained from many sources, including adult, fetal, and perinatal tissues⁵. Recently, numerous clinical trials are performed using different SC types, including mesenchymal stem cells (MSCs)⁶.

MSCs were first discovered by Fridenstein as adherent cells resembling fibroblasts⁷. Their most important properties are an ability to differentiate into different effector cells, to immunomodulate the immune cells, to secrete growth factors and cytokines⁸, including ones that stimulate neurogenesis-related processes⁹.

Studies carried out in animal models have shown that through these pleiotropic interactions with neurons and immune cells MSCs are able to induce regeneration of damaged nervous tissue¹⁰. Pilot clinical studies using MSCs have also demonstrated their positive potential and safety in patients with neurological^11,12 and other disorders^13,14.

MSCs can be used in autologous and allogeneic setting systems. This is due to the fact that overall MSCs are not easily recognized by the immune system and do not activate it^8,15.

MSCs obtained from Wharton’s jelly (WJMSCs—Wharton’s jelly mesenchymal stem cells) are more often used as allogeneic cells. Studies have shown that these cells have good immunomodulating potential and produce many factors, including neurogenesis-stimulating factors^16,17. Studies using umbilical cord–derived MSCs have shown in animal model systems, as well as in clinical setting, their safety and absence of adverse reactions associated with their administration¹⁸. The use of WJMSCs in the treatment of cardiovascular diseases has shown a positive effect in phase I and II studies^19,20.

Phase I and II clinical trials using MSCs have shown the positive results in treatment of stroke^21–23. Intravenous injection of MSCs can improve functional results reported in the Barthel Index (BI), the National Institutes of Health Stroke Score (NIHSS), and the Modified Rankin Scale (MRS), without any adverse event^21–23. Mechanisms of action of MSCs can be divided into two levels. Peripheral level involves reducing the inflammation and immunomodulation. The central level involves activation of angiogenesis, neurogenesis, remodeling of astrocytes, axons, and oligodendrocytes²⁴. Interestingly, WJMSCs could be a better source of SCs for regenerative medicine due to their different pattern of secretion and distinct immunomodulatory properties^25,26. Neuroprotection is the fundamental property of WJMSCs action in animal model of stroke²⁷. Wu et al.²⁷ concluded in their study of the rat model of stroke that beneficial function of WJMSCs does not require the survival of the grafted cells, but their secretion products. MSCs can repair injured tissue or replace the lost neurons after stroke. The process is based on modulation of the microenvironment in affected brain toward more regenerative and less inflammatory^28–31.

Although in the past 10 years, there were about 78 of preclinical studies on the use of MSCs for stroke recovery; there are still only eight clinical trials with reliable large study groups³². Three of the clinical trials were conducted with placebo control groups and these investigations were still at the proof-of-concept stage³².

In this report, we show safety and possibly efficacy of WJMSC administration in group of six stroke patients in comparison with the control group.

As part of the therapy for the chronic cerebral stroke, experimental therapy using Hospital Exemption—Advanced Therapy Medicinal Product (HE-ATMP) (3 × 10⁷ WJMSCs) was performed in six patients—three women (aged 42–72 years) and three men (aged 41–62 years). The treatment was composed of repeated rounds of HE-ATMP administration every 3 months to the CSF (cerebrospinal fluid) via lumbar puncture (intrathecally subarachnoid). The treatment protocol for the cases in the experimental group was planned for minimum one to maximum five administrations of ATMP in each case. We performed, in total, 20 applications; five rounds of SC implantations in three cases and three rounds in one case. The patients were qualified to the treatment after 2 months and not longer than 24 months after episode. During the course of the study, six patients observed in Neurological Ambulatory Center, who suffered from a stroke, were selected to the control group.

ATMP Product Manufacturing

The WJMSCs were harvested from the unrelated donor during labor. An isolation, expansion, and final preparation of WJMSCs were done according to current Good Manufacturing Practice (cGMP) rules as HE-ATMP. Umbilical cords were obtained from healthy donors after obtaining informed consent of volunteer donor mothers. Then, tissue was placed into sterile transportation container in 0.9% solution of natrium chloride and transported to the manufacturing facility. A sample of maternal blood was obtained on the day of tissue harvest for performing the following infectious agents test (HIV, HCV, HBV, CMV, toxoplasmosis, syphilis). Umbilical cords were placed in the antibiotic/antimicotic solution (10,000 units/ml of penicillin, 10,000 μg/ml of streptomycin, and 25 μg/ml of Gibco Amphotericin B), followed by the fresh 0.9% natrium chloride solution for dissection of vessels and tissue cutting. Wharton’s jelly explants were placed into earlier prepared culture dishes for cell isolation. Isolated cells were reseeded into culture bottles for MSC expansion. Expanded cells were cryopreserved in cryoprotectant prepared with 10% dimethyl sulfoxide (DMSO) solution in 5% human serum albumin. Then, the product was cooled down in a controlled-rate freezer and placed in vapor phase of liquid nitrogen. Mesenchymal cells were evaluated for post-thawed sterility, prior to cryopreservation, and post-thawed cell number and cell viability, post-thawed immunophenotype, as well as functional test.

Pretreatment and Follow-up Efficacy Assessment

Pretreatment and follow-up neurological examinations were performed according to the both impairment scales (NIHSS) and functional outcomes scales (MRS and BI). The Ashworth scale was used to assess spasticity. Lovett scale (0–5 terms) was used to evaluate muscle strength. We also evaluated changes in patients’ quality of life and their individual evaluation of functional improvement using simple internal and nonstandardized questionnaire.

One of the set goals was to assess the safety of the treatment. The safety criteria of the transplantation procedure in the treated group included infection, fever, pain, headache, increased level of CRP (C-reactive protein), leukocytosis, allergic reaction—shock, and perioperative complications. The safety criteria of therapy also included cancer development, deterioration of the neurological state, appearance of a neuropathic pain, secondary infections, urinary tract infections, or pressure ulcers.

Case Presentation

HE-ATMP Application Procedures and Patients’ Evaluation

A 72-year-old woman was qualified to the experimental therapy using HE-ATMP cells after stroke localized in both hemispheres of cerebellum (Fig. 1). The patient’s risk factor for stroke was arterial hypertension. Therapy was started 18 months after the stroke. Before the treatment, the patient was examined for a very discreet spastic right-sided paresis, with Babinski’s symptom on the same side and excessive reflexes. The most serious disorders concerned the cognitive sphere, especially speech disorders—dysprosody. In addition, the patients complained of severe ataxia of the trunk, right lower limb and imbalances while walking, resulting in difficulty in independent, efficient movement. The patient underwent five rounds of administration every 3 months with intensive courses of neurorehabilitation beginning every second week after each transplant. The patient improved after the first round of HE-ATMP administration. The second and third administration helped to minimalize the neurological deficits, especially reduction of ataxia of the trunk, which was observed after second round of SCs. After the fourth and fifth series of SCs, we recorded the stabilization in neurological condition. The biggest improvement concerned the reduction of speech disorders; this became more pronounced and faster. The patient also reported significant improvement in the field of motor skills: the quality of gait improved, became more independent, balance disorders withdrew (Table 1).

Figure 1.

Cerebellar stroke in acute-phase T2W—scan with hypertensive areas in cerebellum and cerebellar pedunculus, showing areas of ischemia.

Table 1.

Neurological Condition Before Treatment and Improvement in Neurological Examination After the Experimental Treatment in Study Group.

Case	1						2						3				4						5		6
Sex (F—female, M—male)	F						M						M				M						F		F
Age (years)	72						41						55				62						68		42
Time from illness to treatment (months)	18						2						24				12						24		24
Diagnosis	Cerebellar stroke						Stroke in the area of the right occipital region						Right hemisphere stroke				Hemisphere infarction						Left hemisphere stroke		Right hemisphere stroke
Treatment	Before treatment	WJMSC 1	WJMSC 2	WJMSC 3	WJMSC 4	WJMSC 5	Before treatment	WJMSC 1	WJMSC 2	WJMSC 3	WJMSC 4	WJMSC 5	Before treatment	WJMSC 1	WJMSC 2	WJMSC 3	Before treatment	WJMSC 1	WJMSC 2	WJMSC 3	WJMSC 4	WJMSC 5	Before treatment	WJMSC 1	Before treatment	WJMSC 1
NIHSS	7	6	5	4	4	4	1	1	1	1	1	1	8	8	8	8	9	9	9	9	9	9	15	15	3	3
MRS	1	1	1	1	1	1	1	1	1	1	1	1	3	3	3	3	3	3	3	3	3	3	4	4	1	1
Barthel scale	100	100	100	100	100	100	100	100	100	100	100	100	95	90	90	90	80	80	80	80	80	80	25	25	95	95
Epilepsy	No	No	No	No	No	No	No	No	No	No	No	No	No	No	No	No	No	No	No	No	No	No	Yes	Yes	No	No
Conscious	Normal	Normal	Normal	Normal	Normal	Normal	Normal	Normal	Normal	Normal	Normal	Normal	Slightly limited	Slightly limited	Slightly limited	Slightly limited	Normal	Normal	Normal	Normal	Normal	Normal	Limited	Limited	Normal	Normal
Swallowing reflex	Normal	Normal	Normal	Normal	Normal	Normal	Normal	Normal	Normal	Normal	Normal	Normal	Choking	Choking	Choking	Choking	Normal	Normal	Normal	Normal	Normal	Normal	Trouble swallowing	Trouble swallowing	Normal	Normal
Breathing	Normal	Normal	Normal	Normal	Normal	Normal	Normal	Normal	Normal	Normal	Normal	Normal	Normal	Normal	Normal	Normal	Normal	Normal	Normal	Normal	Normal	Normal	Normal	Normal	Normal	Normal
Emotional dysfunction	Depression	Depression	Depression	Normal	Normal	Normal	Normal	Normal	Normal	Normal	Normal	Normal	Depression	Depression	Depression	Depression	Normal	Normal	Normal	Normal	Normal	Normal	Post-ischemic psychosis	Post-ischemic psychosis	Normal	Normal
Memory dysfunction,	None	None	None	None	None	None	None	None	None	None	None	None	None	None	None	None	None	None	None	None	None	None	Fresh memory	Fresh memory	None	None
Speech dysfunction	Dysarthria, dysprosody	Mild dysarthria, dysprosody	Significant reduction of dysarthria and dysprosody	Significant reduction of dysarthria and dysprosody	Significant reduction of dysarthria and dysprosody	Significant reduction of dysarthria and dysprosody	None	None	None	None	None	None	None afatic pragnozia	Better understanding, logical communication	Better understanding, logical communication	Better understanding, logical communication	None	None	None	None	None	None	Mixed aphasia	Mixed aphasia	None	None
Cranial nerves dysfunction	Right VII	Right VII	Right VII	Right VII	Right VII	Right VII	None	None	None	None	None	None	None	None	None	None	Left VII	Left VII	Left VII	Left VII	Left VII	Left VII	Right VII	Right VII	Left VII	Left VII
Lovett scale kd P	4	4	4	4	4	4	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5	1	1	5	5
Lovett scale kd L	5	5	5	5	5	5	5	5	5	5	5	5	3	3	3	3	3	3	3	3	3	3	5	5	4	4
Lovett scale kg P	4	4	4	4	4	4	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5	1	1	5	5
Lovett scale kd L	5	5	5	5	5	5	5	5	5	5	5	5	3	3	3	3	2	2	3	3	3	3	5	5	3	3
Oppenheim reflex	No	No	No	No	No	No	No	No	No	No	No	No	No	No	No	No	No	No	No	No	No	No	0	0	0	0
Babinski reflex	Yes right	Yes right	Yes right	Yes right	Yes right	Yes right	No	No	No	No	No	No	No	No	No	No	Yes left	Yes left	Yes left	Yes left	Yes left	Yes left	Yes left	Yes left	Both sides	Both sides
Independent in everyday living	No	No	No	No	No	No	Yes	Yes	Yes	Yes	Yes	Yes	No	No	No	No	No	No	No	No	No	No	No	No	No	No
Walking	Imbalanced	Imbalanced	Independent walk	Independent walk	Independent walk	Independent walk	Independent walk	Independent walk	Independent walk	Independent walk	Independent walk	Independent walk	Dependent gait	Dependent gait	Improvement in independent walk	Improvement in independent walk	Dependent gait	Dependent gait	Improvement in independent walk	Improvement in independent walk	Improvement in independent walk	Improvement in independent walk	Wheelchair	Wheelchair	Dependent gait	Dependent gait
Ataxia	Right lower limb, trunk	Right lower limb, trunk	Reduced in trunk	Reduced in trunk	Reduced in trunk	Reduced in trunk	No	No	No	No	No	No	No	No	No	No	No	No	No	No	No	No	No	No	No	No

WJMSC: Wharton’s jelly mesenchymal stem cell; NIHSS: National Institutes of Health Stroke Score; MRS: Modified Rankin Scale.

The youngest of the patients, 41-year-old man, suffered from a stroke in the area of the right occipital region. The only risk factor for the patient’s ischemic episode was stressful lifestyle. The patient had left-sided hemopiaxia; no other signs of focal Central Nervous System (CNS) damage were found. The therapy started very early in the second month after the episode. After the first stage of treatment, there was an improvement in the man’s condition. In ophthalmology examination, the range of 20% of vision improvement was observed. The second administration of the HE-ATMP brought further improvement—another 10% of vision improvement. The third and fourth administrations resulted in next improvements—ophthalmological examination revealed 50% view improvement after third round of SCs and 65% after fourth round. There was no further improvement after fifth round of WJMSC.

A 55-year-old patient, after a right hemisphere stroke, with a deep left hemiparesis and non-aphatic speech disorders of the nature of speech apraxia, was qualified for therapy within 2 years of the vascular episode. The standard treatment has not improved the patient’s condition. Three repeated rounds of SCs were used in the treatment. The first two administrations were performed at intervals of 3 months, and the third one after a 6-month break. The patient reported improvement, especially in the sphere of speech, better understanding, reduction of apraxia, and improvement of logical communication skills, and positively assessed the first two stages of therapy. The last administration did not bring any measurable benefits in the cognitive or motor sphere. The patient decided to discontinue the therapy (Table 1).

A 62-year-old patient with a history of modifiable risk factors for CNS vascular diseases (hypercholesterolemia, atherosclerosis, nicotinism, chronic stress) was qualified for WJMSC therapy 1 year after the right hemisphere infarction (Fig. 2A, B). The patient found dominant motor disorders resulting from left-sided hemiparesis with predominance in the upper limb (2/5 according to Lovett scale) on admission. The man underwent five rounds of WJMSCs. After each administration, the patient declared an improvement in his neurological condition in the motor sphere and the superficial sensation in the area of paretic limb. The paresis was reduced especially in the left lower limb, distally what was observed after second implantation of SC. The patient also reported the improvement in reducing unpleasant paresthesia involving the left limb (Table 1). The treatment improved patient’s quality of life in his own opinion. He becomes more independent in his everyday life. The control computed tomography (CT) scan did not reveal morphological changes of the brain tissue after the whole SC treatment.

Figure 2.

(A) Hypodense area in the right occipital region of the brain showing ischemia. (B) The same area after contrast administration; cavum septi pellucidi is also seen.

There were no measurable improvements in two patients of the study group.

A 68-year-old woman suffered from hyperlipidemia and impaired glucose tolerance, after left hemisphere infarction with right hemiparesis and mixed aphasia, with diagnosed symptomatic epilepsy, was qualified for experimental therapy 2 years after stroke. Conservative treatment did not bring any significant improvement in the patient’s condition. The patient was under the constant care in the Care and Treatment Institution; she was completely dependent, leading mainly an armchair-bed lifestyle. In addition, the patient suffered from behavioral and emotional disorders, episodes of productive psychosis, and aggressive behavior. Only one HE-ATMP cell administration was performed in this case. We did not observe an improvement in her neurological state and the patient gave up further treatment (Table 1).

A 42-year-old man, with right hemisphere stroke, left-sided hemiparesis, predominantly spastic upper limb, underwent experimental treatment after less than 2 years. The HE-ATMP administration was applied only once. The therapy did not reveal any improvement in the patient’s condition, which prompted the patient to give up further attempts of experimental treatment (Table 1).

The treated group was compared with six patients enrolled in the control group during the study period.

The first patient was a 50-year-old man who suffered from a stroke in the left cerebellar hemisphere. The patient was treated conservatively in the acute stroke phase and intensively rehabilitated 3 weeks after the stroke. In this case, disorders in balance and eye movement predominated. In 24 months after the stroke, the man’s quality of walk and balance had slightly improved. It was not possible to improve the eye movement disorders; massive nystagmus persisted.

The second male patient, aged 66, treated conservatively was diagnosed with the stroke of the right cerebellar hemisphere, resulting in massive ataxia of the trunk and upper limbs with imbalance while walking. In terms of speech, the patient was diagnosed with cerebellar speech and dysprosody. Functional speech therapy was implemented to the patient 4 weeks after the onset of the disease. Despite a year of intensive rehabilitation, the disorders remained at the same level (Table 2). The control magnetic resonance imaging (MRI) examination, performed 1 year after the vascular episode, showed the malacia region in the right cerebellar hemisphere (Fig. 3).

Table 2.

Neurological Condition Before Treatment and Improvement in Neurological Examination After the Standard Treatment in the Control Group.

Case

Sex (F—female, M—male)

Age (years)

Time from illness to neurorehabilitation (weeks)

Diagnosis

Cerebellar stroke in left hemisphere

Cerebellar stroke in right hemisphere

Stroke in the area of the right occipital region

Right hemisphere stroke

Treatment

Conservative

Time from illness

2 days

24 months

2 days

24 months

7 days

18 months

1 day

20 months

1 day

24 months

1 day

18 months

NIHSS

MRS

Barthel scale

100

Epilepsy

Yes

yes

Conscious

Keenly responsive

Normal

Keenly responsive

Swallowing reflex

Normal

Breathing

Normal

Emotional dysfunction

Depression

None

Depression

None

Depression/alcohol psychosis

Depression

Memory dysfunction

None

Intellectual deterioration

None

Intellectual deterioration

Speech dysfunction

Without

Cerebellar dysarthria

None

None afatic pragnozia

Cranial nerves dysfunction

None

Left VII

Lovett scale kd P

Lovett scale kd L

Lovett scale kg P

Lovett scale kd L

Oppenheim reflex

Babinski reflex

Yes left site

Yes left

Independent in everyday living

Slight

Yes

Walking

Unstable gait

Independent walk

Dependent gait

Wheelchair

Ataxia

Upper limb ataxia

Trunk ataxia

NIHSS: National Institutes of Health Stroke Score; MRS: Modified Rankin Scale.

Figure 3.

Stoke in the right hemisphere in the periventricular region in Diffusion – weighted imaging (DWI) sequences.

The control group included a patient with a stroke of the right occipital region. A 54-year-old female patient was treated for familial hypercholesterolemia and arterial hypertension. A woman was a smoker for 30 years. In the first days after a stroke, the patient was treated in general medicine ward, due to the increased blood pressure. Directly after the stroke, there was no targeted treatment for the stroke because the diagnosis was delayed. The anticoagulant treatment was introduced in the department of neurology for 1 week after the first neurological symptoms. The patient was diagnosed with left-sided hemianopia. Despite the treatment used, no significant improvement in the patient’s vision was found within 18 months of follow-up from the onset of the disease (Table 2).

There were three patients qualified to the control group with right hemisphere stroke—one woman and two men. A 72-year-old female patient and a 76-year-old male patient had a history of cardiological diseases (valvular heart disease). In both cases, the treatment was carried out in the first hours after the symptoms appeared. Only conservative treatment was administered in both cases—in the female case because the coronary angiography performed 5 days before the episode and in the male case because of active oncological disease. On admission, a neurological examination revealed moderate left hemiparesis (3/5 according to Lovett scale) in both cases. The patients were intensively rehabilitated within 3 months after the episode. There was no reduction of the hemiplegia achieved in those two cases, which significantly influenced the patients’ quality of life (Table 2).

The last patient qualified to the control group was a 44-year-old man with a right hemisphere stroke, which occurred at work. The patient was burdened with numerous modifiable risk factors for CNS vascular disease (hypercholesterolemia, nicotinism, alcoholism) and non-modifiable factors (metabolic syndrome with genetic background). Due to the late diagnosis of stroke (24 hours from the onset of symptoms), the patient was treated conservatively despite his young age. The patient found dominant motor disorders resulting from left-sided hemiparesis with predominance in the upper limb (2/5 according to Lovett scale) on admission and slight non-aphatic speech disorders of the nature of speech apraxia. After 3 weeks of hospitalization, the patient began intensive neurorehabilitation, which took 5 months. At the time of 24 months of follow-up, the patient declared an improvement in his neurological condition in the sphere of superficial sensation in the area of paretic limb without speech or motor improvement (Table 2).

Safety and Feasibility

The early adverse events observed after the HE-ATMP administration include infection, pain, seizure, headache, fever, nausea, vomiting, allergic reaction—shock, drowsiness, fatigue. All of the patients reported increased temperature after each administration of HE-ATMP. This side reaction can be a result of lumbar puncture procedure itself, the disruption of dura continuity, and the administration of a high-protein preparation into the subarachnoid space and was treated with antipyretic (paracetamol, ibuprofen) drugs with good result. One of the treated patients reported transient headache after each implantation, accompanied with the pain in the puncture site. All those symptoms resolved after administration of analgesics. None of the patients from the group reported any late complications (Table 3).

Table 3.

Safety Criteria: Early and Late Treatment Complications and Side Effects in the Study Group.

Complications
	Case 1	Case 2	Case 3	Case 4	Case 5	Case 6
Early
Infection	−−	−	−	−	−	−
Fever	+	+	+	+	+	+
Pain	−	−	+	+	−	−
Seizure	−	−	−	−	−
Headache	−	−	−	−	−	−
Increased level of C-reactive protein (CRP)	−	−	−	−	−	−
Nausea/vomiting	−	−	−	−	−	−
Fatigue	−	−	−	−	−	−
Drowsiness	−	−	−	−	−	−
Allergic reaction—shock	−	−	−	−	−	−
Late
Secondary infection	−	−	−	−	−	−
Deterioration of neurological status	−	−	−	−	−	−
Intracerebral hemorrhage	−	−	−	−	−	−
Carcinogenesis	−	−	−	−	−	−

Quality of Life

During control examination, the patients were asked to fill in the form evaluating patients’ satisfaction and quality of life questionnaire. The questionnaire evaluating the quality of life of our patients was an internal questionnaire prepared specifically for the needs of the hospital. It contains 10 standardized questions to assess the patient’s independence and self-service ability, which can be implemented into the quality of the patient’s functioning. The main goal of our study was to reduce long-term effects and complications of stroke, and to reduce the neurological deficits in long-term follow-up, which was supported by intense neurorehabilitation. Four patients from our group revealed improvement in their neurological condition after repeated rounds of HE-ATMP and confirmed subjective improvement in quality of life. None of the patients from the control group declaimed improvement in his life quality after standard treatment.

Discussion

The therapeutic window for treatment of acute stroke using rTPA is limited—4–5 hours and 6 hours for endovascular treatment and can be executed only by professionals in comprehensive stroke center^23,33,34.

In our trial, only patients who did not receive any other invasive endovascular treatment (including rTPA) were subjected to treatment. However, in other studies, patients receiving rTPA were included because of the increased use of endovascular thrombectomy²³.

In our study, HE-ATMP treatment was performed multiple times to obtain the best effects in the aspect of long-term consequences of stroke and it was safe and well tolerated. The decision on the route of administration (intrathecally via lumbar puncture which means the cells were getting into the subarachnoid space) and dose was based on our previous experience with MSCs and clinical human trial^35–38. Levy et al. used three cohorts enrolled sequentially to the study with dose escalation. The cells were implanted intravenously via metered-dose syringe pump with dose based on body weight³⁹. Kalladka et al.⁴⁰ used increasing doses of CTX0E03 hNSCs: 2, 5, 10, or 20 million. All the cells were administered directly into the brain tissue during surgical procedure using stereotactic frame³⁷. Depending on volume (a maximum of 100 μl) of the implanted cells, the different number of craniotomy tract were used⁴⁰. The invasive technique was also used in Steinberg phase 1/2a trial to treat the patients with chronic stroke. The modified bone marrow–derived MSCs (SB623 cells) were implanted into 18 patients divided into three dose-escalation study groups using MRI stereotactic technique⁴⁰.

We did not observe any adverse side effects of the treatment during 1-year follow-up, and the whole treatment was well tolerated. The adverse events evaluated after the administration of MSCs include infection, pain, seizure, headache, fever, nausea, vomiting, drowsiness, and fatigue^23,32. In the first clinical trial with autologous MSCs, performed in 2005 in South Korea, 1-year evaluation showed no adverse effects⁴¹. However, other studies reported three most common side effects—headache (21.3%), fever (7.8%), and seizures (2.2%)^32,42,43. Our scheme of treatment, the same dose, multiple implantations, route of SC administration were already validated in other studies and seem to be the most safe and effective^35–37. In the largest clinical trial analyzing intravenous autologous MSC administration in ischemic stroke, during 5 years of follow-up, no significant side effects were also observed⁴⁴. The serious side effects observed in the Kalladka’s trial appeared in four of eleven due to the invasive procedure of implantation⁴⁰. One of the treated patients developed cancer disease (malignant melanoma); however it was not related to the therapy. The seizure episodes also were reported in the third cohort of the study group⁴⁰. Although we did not administer the cells into the brain tissue, we also used an invasive procedure that allowed SCs to reach the damaged tissue area. However, in our study, no seizure episodes were registered.

We used standard and widespread scales to assess neurological condition and follow-up: impairment scales (NIHSS) and functional outcomes scales (MRS and BI). The Ashworth scale was used to assess spasticity, and the Lovett scale (0–5 terms) was used to evaluate muscle strength.

We performed the neurological examination every 90 days after HE-ATMP administration and the update of patients’ condition was also performed every 30 days after each procedure by telephone contact. The neurological improvement appeared in four cases from the group of six. In four patients with neurological improvement, the best results were seen after second and third SC implantation. In the largest human trial, Lee et al.⁴⁴ showed possible beneficial effects of autologous MSC transplantations in stroke cases comparing with the control group. The same observation was made in clinical studies in the treatment of drug-resistant epilepsy or Spinal Cord Injury (SCI)^35,37.

Cell-based therapies is a promising alternative or addition to standard stroke treatment⁴³. The SCs used in MASTERS study were multipotent adult progenitor cells (MAPCs) frozen in PlasmaLyte-A, DMSO, and human serum albumin. Such treatment should be considered in the acute phase of stroke because of the specific cells’ properties²². An alternative source of cells to treat stroke in the chronic phase can immortalize hNSCs used in Kalladka et al.’s trial³⁷. The human Neural Stem Cells (hNSCs) administered directly into the brain tissue lead to better survival of the cells than other delivery routes and help to control the cell dose⁴⁵. Although the MASTERS study confirmed mainly the immune regulation function of the cells, the Kalladka study showed the efficacy of the cells in neurological improvement^23,40. We used WJMSCs based on the earlier investigations showing that WJMSCs in comparison with Adult Stem Cells (ASCs) express a wide spectrum of factors with pro-regenerative capabilities related to cell–cell adhesion, neurotrophic effects, and immune modulation²⁶. The most important mechanism of WJMSCs cell activity in the aspect of treating the chronic and subacute nervous system damage appears to be the anti-inflammatory effect associated with expression of anti-inflammatory molecules such as CD200, PD-L1, and non-classical Human Leucoyte Antigens (HLAs) with adhesion molecules such as ICAM1 and VCAM1²⁶. WJMSCs, bone marrow mesenchymal stem cells (BMMSCs), and MAPCs have the ability to reduce T-cell proliferation, which has a direct impact on inflammation environment²⁶. It is known that expression of IDO1 in WJMSC is involved in suppression of T-cell proliferation⁴⁶. WJMSCs have also higher than BMMSC ability to secrete Brain – derived Neurotrophic Factor (BDNF) which is a well-described neurite outgrowth inducer^26,47. WJMSC expression of neurotrophic and angiogenic growth factors and cytokines was responsible for better outcome in animal models of ischemic stroke^48,49.

Summary

In our study, we used WJMSC-based HE-ATMP product to treat patients with chronic stroke. The most important observation from our study is safety of this treatment regimen. We did not report adverse events during 1-year follow-up except fever. In addition, we also observed some improvement in neurological function in study group in comparison with control. However, we are aware of the fact that our study is too small to reach any definite conclusion about the effectiveness of the treatment.

Supplemental Material

sj-docx-1-cll-10.1177_09636897231195145 – Supplemental material for Comparative Analysis of the Results of Stroke Treatment With Multiple Administrations of Wharton’s Jelly Mesenchymal Stem Cells–Derived HE-ATMP and Standard Conservative Treatment: Case Series Study

Supplemental material, sj-docx-1-cll-10.1177_09636897231195145 for Comparative Analysis of the Results of Stroke Treatment With Multiple Administrations of Wharton’s Jelly Mesenchymal Stem Cells–Derived HE-ATMP and Standard Conservative Treatment: Case Series Study by Olga Milczarek, Jakub Swadźba, Patrycja Swadźba, Anna Starowicz-Filip, Roger M. Krzyżewski, Stanisław Kwiatkowski and Marcin Majka in Cell Transplantation

Supplemental Material

sj-docx-2-cll-10.1177_09636897231195145 – Supplemental material for Comparative Analysis of the Results of Stroke Treatment With Multiple Administrations of Wharton’s Jelly Mesenchymal Stem Cells–Derived HE-ATMP and Standard Conservative Treatment: Case Series Study

Supplemental material, sj-docx-2-cll-10.1177_09636897231195145 for Comparative Analysis of the Results of Stroke Treatment With Multiple Administrations of Wharton’s Jelly Mesenchymal Stem Cells–Derived HE-ATMP and Standard Conservative Treatment: Case Series Study by Olga Milczarek, Jakub Swadźba, Patrycja Swadźba, Anna Starowicz-Filip, Roger M. Krzyżewski, Stanisław Kwiatkowski and Marcin Majka in Cell Transplantation

Supplemental Material

sj-docx-3-cll-10.1177_09636897231195145 – Supplemental material for Comparative Analysis of the Results of Stroke Treatment With Multiple Administrations of Wharton’s Jelly Mesenchymal Stem Cells–Derived HE-ATMP and Standard Conservative Treatment: Case Series Study

Supplemental material, sj-docx-3-cll-10.1177_09636897231195145 for Comparative Analysis of the Results of Stroke Treatment With Multiple Administrations of Wharton’s Jelly Mesenchymal Stem Cells–Derived HE-ATMP and Standard Conservative Treatment: Case Series Study by Olga Milczarek, Jakub Swadźba, Patrycja Swadźba, Anna Starowicz-Filip, Roger M. Krzyżewski, Stanisław Kwiatkowski and Marcin Majka in Cell Transplantation

Footnotes

Author Contributions

OM: idea, project implementation, supervision, writing publications. RK, JS: text editing. MM, SK, JS: supervising the manuscript and the course of the research and checking the text. OM, RK: taking care of neurological parts of our research and qualifications of the patients according to their neurological status. ASF: neuropsychological tests and examination. PS, MM: supervising the manuscript and the course of the research. All authors contributed to the article and approved the submitted version.

Availability of Data and Material

All data and source materials are owned by the authors of the publication and are fully available on request.

Ethical Approval

This study was carried out in RegenMed Hospital, Krakow, Poland, and gained the approval of the Bioethical Committee of Andrzej Frycz Modrzewski Krakow University, Poland. Approval number: KBKA/38/O/2017 and KBKA/66/O/2019.

Statement of Human and Animal Rights

All procedures in this study were conducted in accordance with the Bioethical Committee of Andrzej Frycz Modrzewski Krakow University, Poland. Approval number: KBKA/38/O/2017 and KBKA/66/O/2019.

Statement of Informed Consent

Written informed consent was obtained from the patient(s) for their anonymized information to be published in this article.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

ORCID iDs

Olga Milczarek

Patrycja Swadźba

Supplemental Material

Supplemental material for this article is available online.

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