Preclinical assessment of thrombin‐preconditioned human Wharton’s jelly‐derived mesenchymal stem cells for neonatal hypoxic‐ischaemic brain injury

Abstract Hypoxic‐ischaemic encephalopathy (HIE) is a type of brain injury affecting approximately 1 million newborn babies per year worldwide, the only treatment for which is therapeutic hypothermia. Thrombin‐preconditioned mesenchymal stem cells (MSCs) exert neuroprotective effects by enriching cargo contents and boosting exosome biogenesis, thus showing promise as a new therapeutic strategy for HIE. This study was conducted to evaluate the tissue distribution and potential toxicity of thrombin‐preconditioned human Wharton's jelly‐derived mesenchymal stem cells (th‐hWJMSCs) in animal models before the initiation of clinical trials. We investigated the biodistribution, tumorigenicity and general toxicity of th‐hWJMSCs. MSCs were administered the maximum feasible dose (1 × 105 cells/10 µL/head) once, or at lower doses into the cerebral ventricle. To support the clinical use of th‐hWJMSCs for treating brain injury, preclinical safety studies were conducted in newborn Sprague‐Dawley rats and BALB/c nude mice. In addition, growth parameters were evaluated to assess the impact of th‐hWJMSCs on the growth of newborn babies. Our results suggest that th‐hWJMSCs are non‐toxic and non‐tumorigenic in rodent models, survive for up to 7 days in the brain and hold potential for HIE therapy.


| INTRODUC TI ON
Hypoxic-ischaemic encephalopathy (HIE) is a type of brain dysfunction occurring in newborns, which is induced by oxygen deprivation and limited blood flow, known as neonatal hypoxia-ischemia. 1,2 HIE is a major intractable developmental brain disease that accounts for 23% of all neonatal deaths and is reported to occur in 1.15 million babies annually worldwide. 3 In the case of brain disease, severe damage from HIE results in 25%-50% of deaths. Further, 65%-75% of babies that survive can suffer permanent neurological disorders such as cerebral palsy, cognitive disorders, convulsions, learning disorders, and hearing and vision impairments. 4,5 Despite severe damage to the brain and associated mortality, there are currently no well-established treatment strategies for HIE, except for therapeutic hypothermia. 6 Therapeutic hypothermia reduces the extent of permanent brain damage by lowering body temperature. 7,8 However, it has been reported that the effects of therapeutic hypothermia are insignificant for severe brain damage in newborns. 9,10 However, improved therapeutic effect was confirmed when a combination treatment of stem cell transplantation and therapeutic hypothermia was used. 11,12 Therefore, the application of human umbilical cord bloodderived stem cells can be considered as an improved therapeutic method for the treatment of HIE.

Mesenchymal stem cells (MSCs) exert their therapeutic mech-
anism of action through paracrine effects that regulate various functions such as cell-to-cell communication and immunomodulation through molecules released from the cells. 13,14 Various methods have been studied to increase the efficacy of MSCs. Previous studies have found that thrombin preconditioning of naive MSCs under certain conditions improves their paracrine property, the main mechanism of action of MSCs, without causing a loss in the biological functions of MSCs such as cell viability. 15 Sung suggested that thrombin preconditioning improves the therapeutic properties of MSC-derived extracellular vesicles and increases their cargo protein levels through the activation of PAR-1 and other pathways. 15 Kim also suggested that thrombin preconditioning significantly improves neuroprotective properties such as antioxidative, anti-apoptotic and anticytotoxic effects in human. Wharton's jelly-derived MSCs attenuated severe brain infarction induced by HIE with enhanced paracrine anti-astroglial, anti-inflammatory and anti-apoptotic effects. 16 However, stem cell therapy products have the risk of tumorigenicity or potential toxicity owing to their inherent property of multipotency, cell culture and other manipulations made during the manufacturing process. Hence, it is essential to ensure safety of stem cell therapy products through preclinical studies including tumorigenicity and general toxicity studies before clinical trials in humans. MSCs are known to have a relatively low risk of toxicity, such as tumorigenicity, compared to pluripotent stem cells, but the possibility of tumour growth because of their stem cell characteristics and external factors should not be excluded. 17 This is because genetic modification caused by various culture conditions, such as long-term culture and excessive growth promotion caused by immoderate growth factors during the manufacturing process can lead to tumour formation. 18 Therefore, there is need for thorough evaluation of the safety of thrombin-preconditioned MSCs obtained from an advanced culture before clinical use.
To assess the in vivo safety, toxicity and biodistribution of thrombin-preconditioned human Wharton's jelly-derived mesenchymal stem cells (th-hWJMSCs) used for neonatal HIE treatment, we performed all preclinical studies as follows: a 6-month tumorigenicity study in immunodeficient nude mice, an in vivo biodistribution study in a disease model using real-time PCR and a subchronic general toxicity study in neonatal Sprague-Dawley (SD) rats. Because it is a stem cell treatment for newborns, the evaluation of developmental behaviour was included in the general toxicity study. In addition, in order to evaluate the possibility of excessive immunosuppression by overdose and changes in the expression patterns of histocompatibility antigens, changes in immune cells in blood were evaluated.
In line with the preclinical study that demonstrated the therapeutic efficacy of hWJMSCs in the neonatal HIE rat pup model, 16

| Test article
The procedure for cell culture was approved by the Institutional Human Wharton's jelly-derived mesenchymal stem cells (hWJMSCs) were isolated and expanded as described previously, after obtaining consent from pregnant mothers. 19 hWJMSCs from a single donor at passages 5-6 were used in this study. The stemness of the hWJMSCs was confirmed using in vitro differentiation assays for osteogenesis, adipogenesis, and chondrogenesis and flow-cytometric analysis of cell surface markers (CD73, CD90, CD105, CD166, CD14, CD11b, HLA-DR (MHCII), CD34, CD45 and CD19), as described previously. 20 After reaching more than 90% confluence, the hWJMSCs were preconditioned with thrombin (2 U/mL; Sigma-Aldrich, Steinheim, Germany) in culture medium (α-MEM; Gibco, Life Technologies) for 3 h as per an optimal preconditioning regimen protocol. 21 The th-hWJMSCs were appropriate in appearance and microbial sterility (48 h

| Dose selection
In the in vivo studies, the highest dose of th-hWJMSCs was 1 × 10 5 cells/10 µL/head, the maximum feasible dose, based on the

Guidance on Nonclinical Safety Studies for the Conduct of Human
Clinical Trials and Marketing Authorization for Pharmaceuticals M3 (R2). 23 In addition, the additional one or two lower doses were selected to define a dose-dependent response on any findings observed at the highest dose in the tumorigenicity and general toxicity studies. The vehicle control group was administered the same culture medium.
In the tumorigenicity test, the positive control material, U87MG cells, was administered in the mouse cerebral ventricle via ICV injection at 3 × 10 4 cells/10 µL/head based on the preliminary test. In the preliminary test, ICV injection was administered to six male mice. It was confirmed that all animals died within 2 months at 3 × 10 4 cells/10 µL/head, and tumours were formed in their brain tissues.

| Animal husbandry and maintenance
The subchronic general toxicity study, in vivo tumorigenicity study and biodistribution studies were performed with rodent species purchased from Orient Co. The animals were housed in a room maintained at a temperature of 23 ± 3°C, relative humidity of 50 ± 10%

| Surgical procedure and ICV injection
The surgical procedure and ICV injection techniques were adapted from Schmidt's method, with minor modifications. 24 Under deep isoflurane anaesthesia, the animals were placed in the stereotaxic frame. After making a midline incision from the frontal cranial bones, th-hWJMSCs were administered using an infusion pump with 31G insulin syringe into the area of AP (Anterior-Posterior)

| Subchronic general toxicity study in neonatal SD rats
The subchronic general toxicity study was conducted under Good Laboratory Practice (GLP) regulations of Organisation for Economic Co-operation and Development (OECD) 25 and US Food and Drug Administration (FDA). 26 The study was designed in accordance with

| In vivo Biodistribution study in disease model
The in vivo biodistribution study was designed based on the

Considerations in Biodistribution Assessment of Stem Cell Therapy
Product of Korea MFDS 30 and published literature. 31 The HIE disease model in PND 7 SD rats was induced by ligation of the unilateral (right side) carotid artery, followed by exposure to 8% oxygen for 2 h in 35 males and 35 females, including sham controls at the Samsung Medical Center. The animals were evaluated for sufficient brain damage by checking the infarction area using brain diffusion-weighted imaging. After administration of th-hWJMSCs at a dose of 1 × 10 5 cells/10 µL/head into the cerebral ventricle of the HIE disease animals, 5 animals/sex/ timepoint were provided to KIT to collect organ samples on each scheduled sacrifice day (1, 2, 4, 7, 28 and 91 days after administration). The sham controls were sacrificed on day 1, without any administration.
After sacrifice, the human Alu gene expression was measured from the collected organs (the brain, spinal cord, blood, heart, lung, liver, spleen, kidney, mesenteric lymph node, pancreas, epididymis, ovary, testis and uterus) using real-time PCR (Applied Biosystems ViiA™ 7 Real-Time PCR System) from all animals, and the results were analysed using the analysis software (ViiA RUO S/W v 1.2.2).

The validated quantitative conditions for the analysis of the human
Alu gene in SD rat tissue using real-time PCR are shown in Table S1.

| Tumorigenicity study in nude mice
The tumorigenicity study was conducted under GLP regulations of Haematology was conducted prior to necropsy, except in the positive control group. All animals were euthanized using isoflurane at the end of the study period, and macroscopic observations and histopathological examinations of the liver with the gall bladder, adrenal glands, lungs with bronchi, mandibular lymph node, brain (injection site), mesenteric lymph node, heart, spleen, kidneys and stomach were conducted. In addition, immunohistochemistry to assure accurate analysis of tumour origin was performed using a cell-specific marker, anti-human mitochondria antibody (ab92824; Abcam) and anti-mouse cyclophilin A (D2Y4 M; Cell Signaling Technology) for the tumour tissues. 35

| Statistical analysis
Multiple comparison tests for different dose groups were conducted using the means and standard deviations from each group. Variance homogeneity was assessed using the Bartlett's test. Homogeneous data were assessed using one-way analysis of variance (ANOVA), and significant differences between some groups were assessed using Dunnett's test. Heterogeneous data were assessed using the Kruskal-Wallis test, and significant differences between the control and treated groups were assessed using Dunn's rank-sum test. Categorical data were assessed using the χ 2 -test followed by

| Subchronic general toxicity study in neonatal SD rats
No mortality or obvious clinical signs related to the th-hWJMSCs were observed during the study period. In addition, no abnormal changes were observed in body weight and food consumption, clinical pathology, macro/microscopic observation, lymphocyte analysis, and developmental evaluation, including growth pattern, behaviour, learning and memory function, and sexual maturity in the th-hWJMSC-treated groups. The results are presented in Tables 1-3, Tables S2 and S3 and Figure 1.
A significant decrease in absolute basophil count and increase in inorganic phosphorus were observed in females at 1 × 10 5 cells/10 µL/head. In addition, significant increases in absolute and relative (over brain and terminal body weight) weights of the thyroid were observed in females at more than 3 × 10 4 cells/10 µL/head (data not shown). However, histopathological examination did not reveal any associated lesions.
Based on these results, no adverse effect level (NOAEL) of th-hWJMSCs was considered at 1 × 10 5 cells/10 µL/head in newborn SD rats (PND 7) under the condition of a single ICV administration.  Figure 2B). The gene expression in other tissues (blood, heart, lung, liver, spleen, kidney, mesenteric lymph nodes, pancreas, testes, epididymis, ovaries and uterus) was not detected in either sex.

| Tumorigenicity study in nude mice
No abnormal changes were observed in clinical signs, body weight or haematology in the th-hWJMSC-treated groups. No tumours were observed in the brain at the site of administration.
One female mouse at a dose of 1 × 10 5 cells/10 µL/head was found dead on day 106, and enlargement of the spleen and lymph nodes was noted on macroscopic observation. Histopathological examination confirmed abnormal lesions as malignant lymphoma.
Additional immunohistochemistry staining for the lymphoma tissues was performed with a human-specific anti-mitochondria antibody, which was confirmed to be negative ( Figure 3). Conversely, a positive reaction was observed for a procedure involving staining with a mouse-specific anti-cyclophilin A antibody (Figure 4).  gov Identifier NCT02256618).
We have been conducting preclinical research on stem cell therapeutics with improved efficacy by preconditioning naïve MSCs with thrombin to overcome the shortcomings of conventional stem cell treatment. The thrombin-preconditioned MSCs were found to have a significantly higher wound healing effect in an in vivo skin wound model than conventional naïve MSCs. 21 In the efficacy study of th-hWJMSCs using a newborn rodent model with severe brain damage, the combination of hypothermia therapy and stem cell therapy induced a decrease in brain cell death and inflammation, along with a reduction in brain damage area and improvement of sensorimotor function. 16 These results suggest that stem cells maximize their paracrine potency because of thrombin preconditioning, thereby enhancing their therapeutic efficacy. 16 We conducted this preclinical study to evaluate the potential toxicity of th-hWJMSCs, which may be caused by the increased paracrine property after thrombin preconditioning, and to assess their tissue distribution to support human clinical trials. In the in vivo tumorigenicity study, immunodeficient BALB/c nude mice were selected as experimental animals in order to minimize immune system rejection against th-hWJMSCs.
However, because neonatal rats have an immature immune system that does not require immunosuppression, they were used for in vivo biodistribution and general toxicity studies without any immunosuppressive agent for the administration of th-hWJMSCs. 38,39 MSCs exert immunomodulatory effects on the naïve T cells, helper T cells and cytotoxic T cells. 40 To analyse changes in immune cells, the percentage (%) and CD4:CD8 T-cell ratio for each lymphocyte subset were generated using flow cytometry, which is conventionally used for the evaluation of immune cell phenotypes in rodent tests. 41,42 On day 50 after administration, a statistically significant increase in the number of cytotoxic T cells was observed only in males at 1 × 10 4 cells/10 µL/head. However, it was not considered to be related to th-hWJMSCs because no dose dependence was observed, and it was within the range of cytotoxic T-cell number in normal rats (14%). 43 In females, no significant changes were observed in

TA B L E 3
Comparison of lymphocyte phenotyping analysis among the four groups in subchronic general toxicity study ratio = 2-4). 44 In addition, when compared to the control group, no statistically significant change was observed in either sex.
In the in vivo biodistribution study performed in the HIE disease model of male and female newborns, human Alu derived from th-hWJMSCs was measured, as this approach is useful for detecting human cells among rodent cells. 45 After administration, the animals were evaluated for the presence and location of the transplanted cells at different time points. It is necessary to examine the potential toxicity and efficacy in relation to the distribution of cells at specific time points. 46 When th-hWJMSCs were administered once intracerebroventricularly to HIE diseaseinduced newborn rats, they were distributed to the brain from 4 to 7 days and disappeared thereafter. In tissues other than that of the brain, the th-hWJMSCs were thought to be distributed in small amounts in only the spinal cord for up to 1 day. Therefore, the later distribution profile showing that th-hWJMSCs were present only As a result of a 26-week tumorigenicity study in male and female nude mice after a single ICV administration, the tumour-generating capacity of th-hWJMSCs was not confirmed. Malignant lymphoma was observed in one female among 26 males and females (approximately 3.8%). However, no positive reaction was observed for lymphoma as a result of immunohistochemical staining using a human cell-specific anti-mitochondrial antibody ( Figure 3). Conversely, a positive reaction was observed after staining with mouse tissuespecific anti-cyclophilin A antibody ( Figure 4). Therefore, lymphoma is not considered a tumour caused by the administration of th-hWJMSCs. In addition, on the microscopic examination after haematoxylin and eosin staining, the findings were histomorphologically similar to those of mouse lymphoma and were considered to be spontaneous changes when considering the incidence of lymphoma in mice. 47   The use of disease models requires consideration of the risk-benefit assessment for inherent variability, limited historical data, difficulty in animal care and technical issues with the physiological and anatomical constraints of the model. 51 In conclusion, the results of the preclinical safety studies indicated that th-hWJMSCs were not oncogenic, and no abnormal changes or findings related to the thrombin-preconditioned MSCs were observed.
The th-hWJMSCs were detected in the brain for up to 4 and 7 days in male and female mice, respectively, and in the spinal cord up to the first day for female mice. Therefore, the NOAEL was considered at 1 × 10 5 cells/10 µL/head under the conditions of this study. This study is expected to provide critical information regarding the clinical use of th-hWJMSCs and thrombin-preconditioned cell therapies.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.