Transplantation of modified human bone marrow‐derived stromal cells affords therapeutic effects on cerebral ischemia in rats

Abstract Aims SB623 cells are human bone marrow stromal cells transfected with Notch1 intracellular domain. In this study, we examined potential regenerative mechanisms underlying stereotaxic transplantation of SB623 cells in rats with experimental acute ischemic stroke. Methods We prepared control group, empty capsule (EC) group, SB623 cell group (SB623), and encapsulated SB623 cell (eSB623) group. Transient middle cerebral artery occlusion (MCAO) was performed on day 0, and 24 h after MCAO, stroke rats received transplantation into the envisioned ischemic penumbra. Modified neurological severity score (mNSS) was evaluated, and histological evaluations were performed. Results In the mNSS, SB623 and eSB623 groups showed significant improvement compared to the other groups. Histological analysis revealed that the infarction area in SB623 and eSB623 groups was reduced. In the eSB623 group, robust cell viability and neurogenesis were detected in the subventricular zone that increased significantly compared to all other groups. Conclusion SB623 cells with or without encapsulation showed therapeutic effects on ischemic stroke. Encapsulated SB623 cells showed enhanced neurogenesis and increased viability inside the capsules. This study reveals the mechanism of secretory function of transplanted SB623 cells, but not cell–cell interaction as primarily mediating the cells' functional benefits in ischemic stroke.


| INTRODUC TI ON
Ischemic stroke is a leading cause of long-term disability, representing one of the most serious health problems in the world. 1,2 Although there have been various innovative therapies for ischemic stroke, such as rt-PA and mechanical thrombectomy, many patients still suffer from this disorder. 3 Cell transplantation treatment has emerged as an experimental treatment for ischemic stroke. 4,5 In particular, bone marrow stromal cells (BMSCs) transplantation has been demonstrated as safe and effective for experimental ischemic stroke. 6 BMSCs possessed cell regenerative features including their capacity for cell replacement and secretion of neurotrophic factors [7][8][9] with the latter by-stander effects of BMSCs gaining more compelling laboratory evidence than the former mechanism of cell differentiation into neurons. 10 However, the specific mechanism mediating SB623 cells' therapeutic effects still remains uncertain.
SB623 cells are modified human BMSCs transfected transiently with a vector encoding the human Notch1 intracellular domain (NICD). 11,12 Transplantation of rat NICD transfected cells exerted more functional benefits in stroke rats than transplantation of untransfected BMSCs. 13 Interestingly, SB623 cells secrete higher levels of various neurotrophic factors like IL-6, IL-8, FGF1, FGF2, and MCP-1 than those of BMSCs. [14][15][16] The enhanced supply of these trophic factors is considered a key to strong therapeutic effects of SB623, compared to control BMSCs. Intracerebral transplantation of SB623 cells showed promising results in clinical trials, despite a clear understanding of the cells' mechanism of action. [17][18][19] Cognizant of the cell replacement and by-stander effects of BMSCs 7,10 , optimizing the cell delivery of SB623 cells to achieve such regenerative mechanisms stands as an important factor to improve the efficacy/safety of cell transplantation therapy. Various cell delivery routes, such as venous or arterial injection and direct injection to the brain, have been examined. 20, 21 We previously reported that encapsulation of different cell lines promotes functional recovery through the secretion of neurotrophic factors. Our past studies showed encapsulation of BMSCs enhanced their cell regenerative features. 22 Most xenogeneic or allogeneic cells transplanted directly into rodent's brains survive for only a few days or weeks because they are attacked by the host's immunoreaction. [22][23][24][25][26] In contrast, encapsulated cells survive longer because the capsule protects the cells from hosts' immunoreaction, allowing the grafted cells to continue to secrete neurotrophic factors. [22][23][24][25][26] In this study, we tested a two-pronged hypothesis; first, that encapsulation would allow SB623 cells to survive more robustly when directly transplanted into brains of stroke rats; second, with encapsulation preventing cell-to-cell contact between the host and the grafted cells, we would be able to ascribe any therapeutic effects of SB623 cells to the by-stander mechanism, i.e., secretion of trophic factors.

| Animals
Adult male Wistar rats (Charles River Laboratories Japan, Inc., Yokohama, Japan), weighing 280 to 320 g at the beginning of the experiment, were used in this study. They were group-housed with two animals per cage in the temperature and humidity-controlled room, maintained on a 12 h light/dark cycle, with free access to food and water.
The time course of this experiment is shown in Figure 1A.
MCAO was induced according to the intraluminal suture method used in our previous studies. 25,27,28,29 Under general anesthesia (2% sevoflurane in 70% N₂O and 30% O₂), a 4-0 monofilament nylon suture with silicone-coated tip (Xantopren L blue & ACTIVATOR Universal Liquid, Heraeus Kulzer GmbH & Co. KG) was inserted through an arteriotomy of the right external carotid artery into the origin of the right MCA. After MCAO for 90 min, the filament was withdrawn to restore the blood flow and the wound was sutured.

| SB623 cells derivation and preparation
SB623 cells were provided by SanBio, Inc., and prepared in reference to previous reports. SB623 characterization were described previously. 11,16,17,19,30,31 In brief, bone marrow aspirates of healthy young adult human donors were transfected with an expression vector encoding NICD (amino acids 1703-2504), expanded, harvested, cryopreserved and stored in the vapor phase of liquid nitrogen. Frozen vials containing SB623 cells were placed into a 37°C water bath until completely thawed. Once thawed, they were immediately removed, and cells were transferred into a 15 ml conical centrifuge tube containing 10 ml of cold Dulbecco's phosphate-buffered saline (PBS).
The preparations were centrifuged at 1000 rpm (200 × g) in a swinging bucket rotor for 8 min at room temperature (RT) to form a pellet of cells. The supernatant was carefully removed and Dulbecco's modified Eagle's medium (DMEM; Sigma-Aldrich, St. Louis, Missouri, United States), without serum or antibacterial agent, was added to yield a final cell concentration of about 8.0 × 10 4 cells/μl. Cell counting was done to obtain the desired cell count and to check viability.
After cell grafting, the remaining cells were checked and the number of viable cells was verified by trypan blue exclusion. 30,31 The results of cell concentration were in the range of 80,500 ± 25,400 cells/μl, with a viability range of 96.8 ± 2.29%. F I G U R E 1 Experimental design, and evaluation of behavioral and infarction area. Experimental design for this study is shown (A). All rats underwent MCAO on day 0, and transplantation was performed on day 1. Modified neurological severity score was evaluated on day 0, 1, 7, and 15. All rats were euthanized on day 15. BrdU was administered to all rats at a concentration of 50 mg/kg body weight, with four consecutive intraperitoneal injections every 12 h from day 13 to 15. BrdU/Dcx double positive cells in the subventricular zone (SVZ) was evaluated like Figure (B). BrdU/Dcx double positive cells were counted bilaterally in four defined areas (200 × 60 μm) of the bilateral lateral ventricle wall. We counted 16 areas (4 areas × 2 sections × 2 sides) in each rat. Behavioral evaluation was shown (C). SB623 and eSB623 groups showed significantly better mNSS score than control and EC groups at day 15 (n = 7 in control group, n = 9 in EC group, n = 8 in SB623 group, and n = 9 in eSB623 group, the means ± SEM, F (3, 29) = 11.33, *p < 0.05). On day 7, functional improvement was observed in the treatment group, but there was no significant change (F (3, 29) = 2.391; eSB623 vs. control group: p = 0.425, eSB623 vs. EC group: p = 0.190, SB623 vs. control group: p = 0.165, SB623 vs. EC group: p = 0.053). Representative Nissl stains of all groups are shown (D). We calculated the infarction area ratio, defining LT, RT, and RI. LT is the area of the left hemisphere in mm 2 , RT is the area of the right hemisphere in mm 2 and RI is the infarction area in mm 2 . Infarction area ratio = [LT − (RT − RI)] × 100 / LT (%). LT is yellow, RT is blue, and RI is uncolored (E). The results of the infarction area ratio are shown (F). The infarction area ratio on day 15 after MCAO was reduced significantly in SB623 and eSB623 groups compared to control and EC groups (F (3,16) = 17.64, each n = 5, the means ± SEM, *p < 0.05 **p < 0.01). BrdU, 5-bromo-2′-deoxyuridine; Dcx, Doublecortin; EC, empty capsule; eSB623, encapsulated SB623; MCAO, middle cerebral artery occlusion; mNSS, modified neurological severity score.

| Encapsulation
Encapsulation was performed as in our previous studies. [22][23][24][25] Sterile polymeric hollow fibers (15 cm in length) consisting of a semipermeable membrane (Amicon) were cut to 7 mm in length and used as capsules. These fibers were made of polysulfone (molecular cut-off: 100 kDa), and the capsules had an inner diameter of 700 μm. Capsules (7 mm in length) were sealed at the distal end just before cell loading by applying photo-curable cement. SB623 cells were prepared as a single-cell suspension at a density of 8.0 × 10 4 cells/μl resembling the same protocol for SB623 cell transplantation as described above. 5 μl of the cell solution was loaded into the proximal end of the hollow fibers. The access port was sealed with photo-curable cement. The encapsulated SB623 cells were implanted within 1 h of encapsulation.

| Cell transplantation
On day 1, cell transplantation was performed at 24 h after MCAO as in our previous studies. 22,27,29 All rats received anesthesia with 0.3 mg/kg of medetomidine, 4.0 mg/kg of midazolam, and 5.0 mg/kg of butorphanol by intraperitoneal injection and placed in a stereotaxic instrument (Narishige, Japan). They underwent a midline head skin incision and a small hole drilled into their skull. SB623 cells (4.0 × 10 5 cells/5 μl), encapsulated SB623 cells, or an empty capsule was unilaterally implanted into the right striatum. One capsule was implanted for each rat. They were transplanted in accordance with the following stereotactic coordinates 22,24 : 1.0 mm anterior to the bregma, 3.0 mm lateral to the sagittal suture, and 5.0 mm ventral to the surface of the brain for targeting the right striatum. The tooth bar was set at 0.0 mm in all procedures.

| Histological analysis
Histological analysis was performed in all groups using randomly selected animals (n = 5 in each group). Nissl stain was performed to evaluate the infarction area. 27

| Evaluation of the viability of encapsulated SB623 cells and the SB623 cells in the rats' brains
We evaluated the viability of encapsulated SB623 cells and SB623 cells transplanted into the brains of rats using two additional cohorts of animals. Transplantation of SB623 cells or encapsulated SB623 cells was performed in 15 and 12 rats, respectively. SB623 cells or encapsulated SB623 cells was transplanted using the same procedures as described above. Randomly selected rats from each group were euthanized on day 3, 7, and 14 for cell viability analyses. Anti-STEM101/DAPI staining was performed as previously described. 39 All rats were euthanized, and then the brains were post-fixed and cut into a coronal section at 30 μm thickness as described above. were mounted on glass slides and stained for hematoxylin and eosin. 23,24,40,42 Viable cells with nuclei were counted in five sections per capsule. Viable cells were calculated in each capsule, and the averages on day 0, 7, 14 and 28 were used for statistical analyses. In both groups, cell counting was also done to obtain the desired cell count and to check viability as described above immediately prior to transplantation. Both data were statistically analyzed with Mann-Whitney U test.

| Statistical analyses
We analyzed data using IBM SPSS Statistics version 20.0 (IBM) and presented as the means ± standard error (SEM). All data were statistically evaluated using the Kolmogorov-Smirnov test to confirm for normal distribution. Because all data were not rejected the null hypothesis of normal distribution, we analyzed these results using one-way analysis of variance (ANOVA) followed by Tukey's test.
Statistical significance was preset at a p-value <0.05.

| SB623 and eSB623 treatments ameliorate stroke-induced behavioral deficits
Modified neurological severity score was performed on day 0, 1, 7, and 15. Most of the rats of treatment groups showed recovery from hemiparalysis on day 15. ANOVA revealed significant treatment effects of SB623 and eSB623 groups in mNSS ( Figure 1C). SB623 and eSB623 groups showed significantly better mNSS score than control and EC groups at day 15 (F (3, 29) = 11.33, control group: 6.14 ± 0.46, EC group: 6.0 ± 0.53, SB623 group: 2.89 ± 0.48, eSB623 group: 3.56 ± 0.47; eSB623 vs control and EC groups: p < 0.05, SB623 vs. control and EC groups: p < 0.05). On day 7, a trend of functional improvement was observed in the treatment group, but SB623 and eSB623 groups did not reach statistically significant improvement in mNSS score compared to control and EC groups (F (3,29)

| SB623 and eSB623 treatments reduce cerebral infarction
Nissl stain was performed on day15 after MCAO. In the treatment groups, Nissl stain showed reduction of infarction area compared to control and EC group ( Figure 1D,E). ANOVA ( Figure 1F) showed significant treatment effects on cerebral infarction as evidenced by significant reductions in Nissl-stained infarcted areas in SB623 and eSB623 groups compared to control and EC groups (F (

| Significant correlations exist among mNSS score, infarction area ratio, and neurogenesis in the SVZ
Correlational analyses using Pearson correlation coefficient revealed a close interplay among mNSS score, infarction area ratio, and neurogenesis in the SVZ. First, a significant positive correlation between mNSS score and infarction area ratio was observed (n = 20, r 2 : 0.5218, p < 0.05, Figure 3A). Second, a significant negative correlation was also detected between mNSS score and neurogenesis in the SVZ (n = 20, r 2 : −0.5300, p < 0.05, Figure 3B).

| Encapsulation prolongs cell survival of SB623 cells transplanted into the stroke brain
The viability of SB623 cells was evaluated on day 3, 7, 14, and the viability of encapsulated SB623 cells was evaluated on day 0, 7, 14, 28.

| Encapsulated SB623 cells showed noninferiority in therapeutic effects comparable to SB623 cells transplanted directly into the stroke brain
This study initially compared the therapeutic effects of direct intracranial transplantation versus encapsulated cell transplantation of SB623 cells. Both treatment groups similarly improved behavioral score and reduced infarction area ratio compared to control and EC groups, but there was no significant difference between the two treatment groups. Encapsulated SB623 cells significantly enhanced neurogenesis in the SVZ compared to control and EC groups. Furthermore, encapsulated SB623 cells survived longer than SB623 cells in the rats' brains. Because the encapsulation prevented host cell-grafted cell interaction and the formation of newly developed neural networks

| Encapsulated SB623 cells survived longer than directly transplanted SB623 cells
Preclinical and clinical studies on transplantation of SB623 cells have demonstrated therapeutic effects in various neurological diseases like stroke, Parkinson's disease, and traumatic brain injury. 13,17,19,30,41 The underlying mechanisms of these therapeutic effects are partially uncertain but implicate multi-pronged regenerative mechanisms. Specifically, SB623 cells secrete factors that protect cells from hypoxic injury, 14 support damaged cells, 15,16,43 promote angiogenesis, 15,16 exert anti-inflammatory effects, 16

| Effects of xenogeneic transplantation
In this study, the human SB623 cell donor and the rat transplant recipient catered to a xenogeneic transplantation approach. The host's immune response poses as a major problem in any xenogeneic cell transplantation procedure. However, there is no detectable significant difference in the therapeutic outcomes between allogeneic intracranial transplantation of rat NICD-transfected BMSCs and xenogeneic transplantation of human SB623 cells in chronic rat stroke model. 41 Furthermore, SB623 cells display immunosuppressive potency comparable to untransfected human BMSCs. In addition, intracranial transplantation of human BMSCs into the ischemic stroke rats even without immunosuppression reduces infarct area and promotes neurogenesis in the SVZ. 46

| Neurotrophic factors secretion of encapsulated SB623 cells
The current study showed good viability of the encapsulated SB623 cells even up to day 14 post-transplantation. Although we did not analyze here growth factor secretion, previous reports showed that secretion of MCP-1, FGF-1, and FGF-2 was enhanced in SB623 cells compared to non-transfected human BMSCs. 14-16 MCP-1 induces migration of neuroblasts from the SVZ to infarct region in rodents, 48 while FGF-1 and FGF-2 play an important role in the growth of neural cells. 14 Similar robust and extended secretion of neurotrophic factors, such as MCP-1, FGF-1, FGF-2, likely complemented the encapsulation of SB623 cells in this study.

| Study limitations
The observation that encapsulated SB623 cells promoted neurogenesis and increased grafted cell viability but did not enhance functional recovery compared to unencapsulated SB623 cells might have been masked by the limited study period. In our previous study, we monitored the therapeutic effects of encapsulated cells for half a year. 45 Here, we examined encapsulated cells for only 2 weeks posttransplantation. A long-term survival period post-transplantation period may reveal improved functional outcomes and histological proof of tissue regeneration with mature neurons by encapsulated SB623 cell. In addition, as noted above, we did not evaluate the secretion of growth factors between encapsulated and unencapsulated SB623 cells. The identification of distinct neurotrophic factors secreted by SB623 may reveal specific regenerative pathways that mediate the cells' therapeutic effects. Recently, sex differences in brain blood vessels, metabolism, and stroke outcomes have been discussed in clinical settings and basic research. 49,50 In our study, only male rats | 1983 were used because of the limited number of animals for animal protection and the uniform results after MCAO. In the future, we need to keep sex differences in mind to perform stroke research.

| CON CLUS ION
We demonstrated SB623 cells with or without encapsulation exerted therapeutic effects on the acute ischemic stroke rats. Encapsulated

ACK N OWLED G M ENTS
The authors acknowledge SanBio members for discussion and critical review of the manuscript. This research was supported by grantsin-aid for Scientific Research of Japan (09156274 and 24592129) and SanBio Inc. We thank Masako Arao and Yoshie Ukai for their technical assistance.

CO N FLI C T O F I NTE R E S T
The authors declare that there is no conflict of interest other than below. Cesar Borlongan is an Editorial Board member of CNS Neuroscience and Therapeutics and a co-author of this article. To minimize bias, they were excluded from all editorial decision-making related to the acceptance of this article for publication.

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.