Intracerebral Transplants of GMP-Grade Human Umbilical Cord-Derived Mesenchymal Stromal Cells Effectively Treat Subacute-Phase Ischemic Stroke in a Rodent Model

In subacute and chronic phases of the stroke, there are no therapeutics available at present to promote functional recovery. Human umbilical cord-derived mesenchymal stromal cells (hUC-MSCs) are one of the candidate cell types for treating subacute-phase stroke. The benefits of cell-based therapy largely depend on the migratory capacity of products administered, as well as their potential for engraftment in targeted tissues and paracrine activities. Timing and delivery modes may also influence the outcomes of stem-cell therapy. Still, the functional recuperative effects of differing hUC-MSC delivery modes, about cell replacement and cell-to-cell paracrine activity levels, have yet to be clarified in subacute phases of stroke.This study was conducted to compare the therapeutic effects of various delivery routes when administering Good Manufacturing Practice (GMP)-grade hUC-MSCs in a rodent model of subacute-phase stroke. Cell aliquots (1 × 106) were given to rats as intravenous (IV) injections or intracerebral (IC) transplants 1 week after middle cerebral artery occlusion (MCAo). Transplanted rats were examined up to 7 weeks later using various behavioral tests and immunohistochemical analyses. Most IC-transplanted cells survived for short periods (i.e., <4 weeks after receipt) and gradually disappeared, whereas IV-injected cells were undetectable in the brain at the same time points (i.e., 3 days, 4 weeks, or 7 weeks after injection). Although short-lived, IC-transplanted cells effectively improved behavioral deficits, serving to reduce infarct volumes and glial scar formation, increase subventricular counts of proliferating neuroblasts, and promote cerebrovascular ingrowth in ischemic penumbra regions. IV injection, however, failed to improve behavioral function or histologic parameters during the same 7-week time frame. These findings overall suggest that IC transplantation is preferable to IV injection for delivery of hUC-MSCs during subacute phases of stroke.

In subacute and chronic phases of the stroke, there are no therapeutics available at present to promote functional recovery. Human umbilical cord-derived mesenchymal stromal cells (hUC-MSCs) are one of the candidate cell types for treating subacutephase stroke. The benefits of cell-based therapy largely depend on the migratory capacity of products administered, as well as their potential for engraftment in targeted tissues and paracrine activities. Timing and delivery modes may also influence the outcomes of stem-cell therapy. Still, the functional recuperative effects of differing hUC-MSC delivery modes, about cell replacement and cell-to-cell paracrine activity levels, have yet to be clarified in subacute phases of stroke.This study was conducted to compare the therapeutic effects of various delivery routes when administering Good Manufacturing Practice (GMP)-grade hUC-MSCs in a rodent model of subacute-phase stroke. Cell aliquots (1 × 10 6 ) were given to rats as intravenous (IV) injections or intracerebral (IC) transplants 1 week after middle cerebral artery occlusion (MCAo). Transplanted rats were examined up to 7 weeks later using various behavioral tests and immunohistochemical analyses. Most IC-transplanted cells survived for short periods (i.e., <4 weeks after receipt) and gradually disappeared, whereas IV-injected cells were undetectable in the brain at the same time points (i.e., 3 days, 4 weeks, or 7 weeks after injection). Although short-lived, IC-transplanted cells effectively improved behavioral deficits, serving to reduce infarct volumes and glial scar formation, increase subventricular counts of proliferating neuroblasts, and promote cerebrovascular ingrowth in ischemic penumbra regions. IV injection, however, failed to improve behavioral function or histologic parameters during the same 7-week time frame. These findings overall suggest that IC transplantation is preferable to IV injection for delivery of hUC-MSCs during subacute phases of stroke.
Keywords: stroke, intracerebral transplantation, umbilical cord (UC), mesenchyaml stem cells, GMP (good manufacturing process) INTRODUCTION Severe brain damage inflicted by strokes leads to long-term disability or even death, and the need for more effective therapies is dire. Ischemic stroke is triggered through multiple mechanisms, involving a cascade of pathophysiologic events that include excitotoxicity, oxidative stress, apoptosis, and inflammation. Spontaneous neurogenesis, astrocytic activation, and angiogenesis may occur in response (Jauch et al., 2013;Powers et al., 2015). In recent years, experimental and clinical trial data have indicated that cell-based therapies of various sorts, incorporating mesenchymal stromal cells (MSCs), neural stem cells, or induced pluripotent stem cells, may offer real opportunities for cell replacement and endogenous brain repair in patients with strokes .
The therapeutic benefits of cell-based therapies depend on their capacities for cellular migration and the extent to which grafted cells inhabit the targeted tissues (Chrostek et al., 2019). Delivery routes are likewise highly influential in the migration and final destination of transplanted cells, impacting therapeutic efficacy. The best means of stem-cell delivery to promote recovery at sites of ischemic brain damage has thus become a major focus of research. Many investigators have explored the efficacy of various routes, including intravenous (IV; Li et al., 2015), intracerebral (IC; Jin et al., 2005;Denham et al., 2012), intraventricular (Jin et al., 2005), and intrathecal injections by lumbar puncture (Lim et al., 2011). MSCs administered as treatment of stroke typically are delivered by IV injection, which is safe and practical. However, recent clinical trials have shown that IV injection of MSCs produces a modest or little therapeutic effect in patients at subacute phases of stroke (Lee et al., 2010;Hess et al., 2017). Although intraventricular or intranasal transfer are alternate options for stem cell delivery, there have been few reported cases, and standardization of these methods has yet to be optimized (Rodríguez-Frutos et al., 2016). On the other hand, IC delivery may offer an ideal route for stem-cell transplantation to damaged brain areas, despite the rather invasive surgical procedures required. Importantly, the outcomes of IC transplantation seem to surpass those of IV injection in terms of functional recovery (Chrostek et al., 2019).
Another issue for cell therapy is the timing of cell delivery. Because the therapeutic window for thrombolytic therapy is quite narrow in clinical situations, <10% of patients with strokes benefit from thrombolytic therapy (National Institute of Neurological Disorders and Stroke rt-PA Stroke Study Group, 1995). Beyond this time frame, there is otherwise a void in established neuroprotective/neurorestorative treatments that is perhaps best addressed by cell therapy. However, data is scarce on direct comparisons of therapeutic effects by MSC delivery routes in subacute or chronic phases of ischemic stroke.
Herein, we have compared efficacies of IC transplantation and IV injection, using Good Manufacturing Practice (GMP)grade hUC-MSCs in a rat model of subacute stroke. IC transplantation ultimately proved to be the preferred route, culminating in functional recovery. Our data provide critical insights into optimal modes of stem-cell delivery for future clinical applications of MSCs aimed at subacute or chronic phases of stroke.

Generation of GMP-Grade hUC-MSCs
This study was authorized by an Institutional Review Board at CHA Bundang Medical Center, permitting the use of human umbilical cord (hUC) samples (IRB no.: BD2013-004D). The investigational protocol fully adhered to institutional guidelines and regulations. Preparation of hUC-MSCs, including their isolation and expansion, was undertaken within a GMP facility and performed in accord with Good Clinical Practice (GCP) guidelines of the Master Cell Bank (MCB) at CHA Bundang Medical Center, as previously described (Oh et al., 2018). A healthy volunteer at the center granted informed consent, releasing an hUC specimen for this purpose. Processing was completed within 24 h.
After removing the umbilical vessels, we sliced Wharton's jelly into explants of 1-2 cm. The slices were attached and placed on culture plates in 500 ml of α-MEM (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 50 ml of 10% FBS (Thermo Fisher Scientific, Waltham, MA, USA), 25 ng/ml FGF4 (R&D Systems, Minneapolis, MN, USA), and 0.2 U/ml heparin (Sigma-Aldrich, St. Louis, MO, USA) for incubation, changing the medium every 3 days. After 15 days, the cord fragments were discarded. The cells were subsequently passaged using TrypLE Select (Thermo Fisher Scientific, Waltham, MA, USA) and expanded to the point of sub-confluence (80-90%). They were incubated under hypoxic conditions (3% O 2 , 5% CO 2 ) at 37 • C, housing hUC-MSCs of passage 2 in the Master Cell Bank (MCB). We then thawed cells for subculture under the same conditions. At passage 4, the cells were stored in the Working Cell Bank (WCB). Again, cells were thawed and further cultured to passage 7, mixing a collection of 2 × 10 7 cells with 1 ml of Cryostor CS10 (BioLife Solutions, Bothell, WA, USA) in an AT-Closed Vial (Aseptic Technologies, Les Isnes, Belgium). The final product was stored at −35 • C. Only hUC-MSCs in passage 7 served in the present study.
To test the eligibility of the final cell product, karyotyping and viral tests were done. Karyotype analysis confirmed a normal human karyotype. Using reverse transcriptase PCR, the absence of viral pathogens (human immunodeficiency virus-1 and 2, cytomegalovirus, hepatitis B virus, hepatitis C virus, human T lymphocytic virus, Epstein-Barr virus, mycoplasma, and syphilis) in cell pellets was ensured. The hUC-MSCs produced also expressed high levels of expected cell surface markers (CD44, CD73, CD90, and CD105), showing extremely low expression levels of hematopoietic stem-cell markers (CD31, CD34, and CD 45) and HLA-DR.

A Rodent Model of Ischemic Stroke
All animal experiments followed guidelines of the Institutional Animal Care and Use Committee (IACUC140012) at CHA University. A model of transient middle cerebral artery occlusion (MCAo) was generated as described by Longa et al. (1989), using adult male Sprague-Dawley rats (Orient Bio, Seoul, South Korea) 270-300 g in weight. Once anesthesia was achieved by injecting ketamine (1%, 30 mg/kg) and xylazine hydrochloride (4 mg/kg) intraperitoneally, a ventral neck incision was made, exposing the right common carotid (CCA), external carotid (ECA), and internal carotid (ICA) arteries. In total, a 22-mm length of 4-0 monofilament nylon suture (PN.403756PK10; LMS Company, Seoul, Korea) was inserted through ECA into ICA lumen to block MCA at its origin for 90 min. This suture was then carefully removed. The rats were maintained at body temperatures of 37 ± 0.5 • C using a heating pad (monitored by the rectal probe) and kept warm during recovery. To select appropriate stroke models, acute neurologic assessments (scored 0-5) of forelimb and hindlimb placement and appraisals of circling behavior took place 24 h after MCAo induction. Only those animals with scores ≤3 qualified for experimentation.

IV Injection or IC Transplantation of hUC-MSCs
One week after MCAo induction (typically considered a subacute phase of stroke), hUC-MSCs were prepared for IV injection or IC transplantation by thawing stored cryovials in a water bath (37 • C) within 1 min. For IV injection, a full cryovial was transferred to a 15-ml conical tube containing saline, gently swirled, and centrifuged at 1,000 rpm for 5 min. The supernatant was then aspirated, resuspending the pellet in saline. Doses of 1 × 10 6 cells in 500 µl of saline (IV injection) or 1 × 10 6 cells in 8 µl of saline (IC transplantation) were administered. Experimental groups were designated as follows: (1) IV saline only (500 µl) infused into tail veins (n = 13); (2) IC saline only (8 µl) delivered to ipsilateral hemispheres of the brain (n = 12); (3) IV hUC-MSCs (as above) infused into tail veins (n = 11); and (4) IC hUC-MSCs (as above) delivered to ipsilateral hemispheres of the brain (n = 11). Coordinates for stereotaxic injections of saline or hUC-MSCs were as follows (Tornero et al., 2013): anteroposterior (AP), +1.0 mm from Bregma; mediolateral (ML), −2.5 mm from midline; and dorsoventral (DV), −4.0/−7.0 mm from the surface. All transplanted rats were immunosuppressed through daily intraperitoneal injections of cyclosporine A (5 mg/kg/day; CKD Pharmaceutical Company, Seoul, Korea) starting 1 day before transplantation and continuing for up to 7 weeks post-transplantation.
Rats receiving IV-injected or IC-implanted hUC-MSCs were later sacrificed at 3 days, 4 weeks, and 7 weeks post-treatment for histologic examinations of cell survival within the brain (n = 5 at each time point per group).

Behavioral Tests
Effects of administered hUC-MSCs were assessed by experimenters blinded to treatment status, performing stipulated behavioral tests [rotarod, stepping, and modified neurological severity score (mNSS)] weekly after MCAo for up to 8 weeks. In the rotarod test (Jeong et al., 2003), all rats are placed on a rotating rod set to progressively accelerate from 4 to 40 rpm in 120 s Times at which animals fell during rotation were recorded as the average of three trials.
For the stepping test (Olsson et al., 1995), rats were stationed on a tabletop in a forelimb stance at nearly 90 • bodily orientation. Once they seemed relaxed, they were nudged forward along the tabletop, counting the number of forepaw placements when moved slowly in forehand and backhand directions across a distance of 90 cm over 5 s The right and left steps were separately counted. This test was always performed by the same operator, and the rats were familiarized with the experimenter's grip before testing.

Tissue Preparation
For immunohistochemical analysis, we sacrificed rats at 8 weeks post-MCAo, perfusing the hearts with saline and 4% paraformaldehyde (PFA). The brains were extracted and post-fixed overnight in 4% PFA at 4 • C, then incubated in (E) Behavioral recovery rates for three behavioral tests. The recovery rate is defined as the percentage of the final score (Week 8) to the baseline score (Day 0). Cell transplantation ("TP") performed 1 week after MCAo induction. All data expressed as mean ± SEM values. *p < 0.05; **p < 0.001 (IV saline group vs. IC hUC-MSC group); # p < 0.05; ## p < 0.001 (IC saline group vs. IC hUC-MSC group); $ p < 0.05; $$ p < 0.001 (IV hUC-MSC group vs. IC hUC-MSC group) by mixed ANOVA.

Determination of Infarct Volume
Eight coronal cryosections serially cut at 16 µm were stained with Cresyl violet in each group (n = 8 each). Ischemic and total hemispheric areas in each section were traced and measured, summing values of eight serial sections in the coronal plane for each brain. Based on intact contralateral hemispheres, infarct sizes were gauged as follows (Oh et al., 2015): estimated infarct size (%) = [1 − (area of remaining ipsilateral hemisphere/area of intact contralateral hemisphere)] × 100. Areas of interest were measured via ImageJ software (National Institutes of Health, Bethesda, MD, USA), summing values from eight serial coronal sections in each brain.

Statistical Analysis
All computations relied on standard software (Enterprise v4.1; SAS Software, Cary, NC, USA), powered by the CHA University mainframe computer. Histologic parameters and infarct measurements were assessed by the Mann-Whitney U test, invoking a two-way analysis of variance (mixed-ANOVA) for behavioral data. Results were expressed as mean ± SEM (standard error of mean) values, setting significance at p < 0.05.

Intracerebral Transplants of hUC-MSCs Ameliorate Neurologic Deficits in MCAo Rats
Effective IV doses of MSCs have ranged from 5 × 10 5 to 3 × 10 6 in rat models of stroke previously reported ; and we have observed functional recovery in an acute model of stroke by infusing 1 × 10 6 hUC-MSCs intravenously (Oh et al., 2018). Consequently, doses of 1 × 10 6 cells were stipulated for the current experiment, assessing behavioral outcomes of IV injection and IC transplantation 8 weeks after MCAo induction.
Results of rotarod testing indicated significant improvement by Week 8 (vs. Week 1) in IC-treated rats, compared with IV-treated animals or those in either control group (IV or IC saline recipients; Figure 1B). Degrees of improvement gradually increased during the 7 weeks post-transplantation. In the stepping test, IC-treated rats also showed significant recovery between 3 and 7 weeks post-treatment, compared with IV-treated or control animals ( Figure 1C); and mNSS assessments paralleled rotarod test results, the IC-treated rats demonstrating significant recovery between Week 1 and Week 7 weeks post-treatment, compared with IV-treated animals or those of either control group ( Figure 1D). Collectively, final behavioral scores after IC transplantation of hUC-MSCs reflected significant improvement from baseline levels in all three testing categories, relative to scores achieved after IV-injection of hUC-MSCs or saline-only IV or IC delivery ( Figure 1E).
Compared with controls, IV injections of hUC-MSCs yielded no functional improvement whatsoever by any behavioral measure 7 weeks after treatment. Hence, IC transplantation seemed preferable to IV injection for delivery of hUC-MSCs in our rodent model of subacute-phase ischemic stroke.

Intracerebral Transplants of hUC-MSCs Reduce Infarct Volumes
To determine how IC-transplanted hUC-MSCs impact neuronal injury after MCAo, we evaluated changes in infarct volumes by Cresyl violet staining of samples taken 7 weeks after treatment (Figure 2A). Mean infarct volume was significantly less in IC-treated rats (38.6 ± 4.5%) than in IV-treated animals (51.9 ± 4.3%) or in saline-only controls (IV, 50.6 ± 2.5%; IC, 54.2 ± 3.2%; Figure 2B). IV-treated rats did not differ from the two saline-only control groups in terms of infarct volume. These results suggest that a significant reduction in infarct volume is

Survival of IC-Transplanted vs. IV-Injected hUC-MSCs Within Brain Tissue
We evaluated the survival of IV-injected and IC-implanted hUC-MSCs (Figure 2C), examining the hNu marker in three coronal sections of brain tissue (AP: +1.0, 0, −1.0 from the Bregma) obtained at 3 days, 4 weeks, and 7 weeks after treatment (n = 5 per group at each time point; Figure 2C). In rats given IC transplants of hUC-MSCs, striatal hNu + cells were present 3 days after treatment (Figure 2D), although their numbers declined by 4 weeks, becoming undetectable at 7 weeks ( Figure 2E). In rats given IV injections of hUC-MSCs, hNu + cells were undetectable within in any regions of the brain at any time point (Figure 2E). This implies that IV-injected hUC-MSCs do not promote functional recovery, failing to migrate into the ischemic brain, whereas IC-transplanted hUC-MSCs (however shortlived) likely confer functional recovery through bystander effects of engrafted cells.

Transplants of hUC-MSCs Promote Endogenous Neurogenesis
We evaluated the potential impact of hUC-MSC delivery mode on endogenous neurogenesis, staining for BrdU, and DCX in ipsilateral SVZs (Figures 6A,B). Compared with saline-only IV and IC control groups, numbers of DCX + neuroblasts were significantly greater in IC-treated rats ( Figure 6C) but proved similar in IV and IC treatment groups. BrdU + cells proliferating within SVZs were also significantly increased in IC-treated rats, relative to IV-treated animals and saline-only control groups ( Figure 6D); and those with dual markers (DCX + /BrdU + cells) were proportionately more numerous in IC-treated rats (20.4 ± 2.9%), exceeding totals in IV-treated animals (11.2 ± 0.7%) or in either control group (IV, 6.3 ± 1.4%; IC, 7.8 ± 1.8%; Figure 6E).
To validate this finding, we stained for both PSA-NCAM and PCNA (Figures 6F,G). PSA-NCAM + neuroblasts were significantly more numerous in IC-treated rats, compared with saline-only IV and IC groups (Figure 6H), again showing proportionate similarity to IV-treated animals. Proliferating PCNA + cells in IC-treated rats were proportionately more numerous than in the other three groups (Figure 6I). Neuroblasts with dual markers (PSA-NCAM + /PCNA + cells) were also more frequent in IC-treated rats (10.8 ± 2.8%), compared with IV-treated animals (2.7 ± 0.8%) or saline-only controls (IV, 3.4 ± 1.4%; IC, 3.2 ± 0.6%; Figure 6J). Proportions of DCX + /BrdU + cells and PSA-NCAM + /PCNA + cells in IV-treated animals and saline-only control groups did not differ. Overall, our findings provide compelling evidence that IC-transplanted hUC-MSCs promote endogenous neurogenesis within the SVZs of stroke-damaged rat brains.

Transplants of hUC-MSCs Promote Angiogenesis
Effects of hUC-MSC delivery modes on angiogenesis (i.e., new blood vessel formation), closely linked to neurogenesis, were assessed by measuring numbers of microcapillary vessels (<30 µm in diameter). RECA-1 immunostaining in peri-infarct border zones (Figures 7A,B) indicated significantly more microcapillary vessels in IC-treated rats (133.4 ± 6.8), compared with IV-treated animals (114.9 ± 3.3) or saline-only control groups (IV, 103.1 ± 6.0; IC, 103.6 ± 6.5; Figure 7C). The latter three groups did not differ in this regard. Dual staining of PECAM1 and BrdU was then pursued to measure actively proliferating peri-infarct blood vessels. Such cells were only detected in peri-infarct boundary zones of IC-treated rats ( Figure 7D), attesting to the likelihood that IC-transplanted hUC-MSCs promote post-stroke angiogenesis in peri-infarct border zones.

DISCUSSION
To maximize the efficacy of transplanted hUC-MSCs, it is important to determine the optimal route and timing of administration, especially in dynamically changing environments of ischemic brain damage. The mode of stem-cell delivery is one issue critical for ensuring therapeutic efficacy in related clinical applications (Lindvall and Kokaia, 2011). Functional neural networks and cell-to-cell communication between grafted cells and the host brain are thus important prerequisites to achieving long-term stem-cell effects in this context.
Because the manner of MSC delivery largely determines cell survival and migratory capacity, we compared the therapeutic efficacy of two delivery modes (IV injection vs. IC transplantation) in a rodent model of subacute-phase stroke. Ultimately, we found that IC transplants of hUC-MSCs significantly improved functional outcomes, whereas IV injection fell short in most aspects. Behavioral recovery was readily observed, starting as soon as 1 week after delivery; and histologic improvements were durable, still observable at 7 weeks. Although our data and that of others (Roh et al., 2008;Li et al., 2016) underscore the short-lived nature of MSCs, the effects of cells that remain viable initially appear to persist, presumably through paracrine reverberations and cell-to-cell exchanges with host brain in terms of brain repair (Rodríguez-Frutos et al., 2016).
To date, IV injection of human MSCs (as opposed to IC transplants) has been favored in clinic settings, owing to the relative safety of this less invasive approach. However, (green) and BrdU (red) in the rat after IC transplantation of hUC-MSCs. Note: No PECAM1 + /BrdU + vessels were found in the other three study groups. DAPI (blue) counterstain. Scale bar = 50 µm. Data expressed as mean ± SEM values. *p < 0.05. the functional recovery achieved by IC transplantation of hUC-MSCs in subacute phases of stroke is lacking after IV injection, as our study shows. This failure reflects an inability to migrate and engraft within brain parenchyma after IV administration, documented in previous publications. Nearly 90% of IV-injected stem cells become trapped in the lungs, liver, or spleen, limiting their effects in treating brain injury (Walczak et al., 2008;Li et al., 2010;Pendharkar et al., 2010). Indeed, migration of circulating MSCs into brain parenchyma is perhaps more difficult in subacute (vs. acute) phases of injury, given the permanent damage to the blood-brain barrier and a dearth of chemotactic signals from the host brain. Another reason for migratory lapse may be the relatively low doses of injected cells that we used. Higher doses of MSCs (3 × 10 6 cells) have produced histologic and behavioral improvements in one previous study (Lin et al., 2011). However, embolic risks (i.e., pulmonary embolism) are increased as doses rise, potentially aggravating hematologic and vascular instability in the aftermath of a stroke. Our observations suggest that IC transplantation is more effective than IV injection as a therapeutic strategy, requiring lower cell doses for therapeutic efficacy and preventing embolism at subacutephases of stroke.
As we have demonstrated, IC-transplanted hUC-MSCs promote functional recovery through multiple mechanisms. They not only reduce pro-inflammatory responses and glial scarring but also spur SVZ neurogenesis and peri-infarct angiogenesis. These effects served in concert to confer neuroprotection from cerebral ischemia and impart subsequent functional recovery in stroke-damaged rat brains. In conditions of cerebral ischemia, inflammatory responses initiated by resident microglia and blood-borne macrophages persist for several weeks after ischemic insults (Jin et al., 2010). During the early stages of ischemic strokes (1-14 days), there is a proinflammatory shift in immune cells of the central nervous system, creating a prominent proinflammatory phenotype in rodent models of transient cerebral ischemia (Hu et al., 2012). A shift from pro-inflammatory to anti-inflammatory phenotype is activated during subacute phases of the stroke to dampen aberrant inflammation and commence healing of wounded brain tissues (Iadecola and Anrather, 2011). We found that IC transplantation of hUC-MSCs increased the proportion of anti-inflammatory cells (CD206 + -ED1 + cells), relative to proinflammatory counterparts (iNOS + -ED1 + cells), thus reducing neuroinflammation and encouraging the processes of healing. IC-transplanted hUC-MSCs not only repair damaged brain tissue, chiefly through immunomodulation, but they also secrete trophic factors to recruit endogenous stem/precursor cells. These newly recruited cells may facilitate disease recovery and activate endogenous brain tissues by enhancing angiogenesis, neurogenesis, and synaptogenesis (Chopp and Li, 2002;Hsieh et al., 2010Hsieh et al., , 2013Lin et al., 2011;Ernst et al., 2014;Frausin et al., 2015). As already reported, hUC-MSCs may simulate perivascular cells, secreting a multiplicity of proangiogenic cytokines (Choi et al., 2013) to promote angiogenesis in MCAo animal models (Toyama et al., 2009;Horie et al., 2011;Hsieh et al., 2013;Frausin et al., 2015).
Proliferating endothelial cells and new vessels continue to grow in the penumbra for at least 3 weeks after ischemic events (Hayashi et al., 2003). Concurrently, peri-infarct angiogenesis provides an instructive cue for SVZ neurogenesis through the molecular interplay between endothelial cells and neuroblasts (Ohab et al., 2006;Goldman and Chen, 2011). Human MSCs enhance SVZ neurogenesis by orchestrating a broad spectrum of neurotrophic factors (i.e., BDNF and GDNF; Ranganath et al., 2012). This proliferation of neural progenitors within the SVZ is accelerated during the first 2 weeks of the injury and lasts for several months (Arvidsson et al., 2002). Although we did not evaluate the ultimate fates of proliferating SVZ neuroblasts driven by IC transplants of hUC-MSCs, they are known to migrate into injured brain tissue to form mature neurons, thus restoring neural networks and leading to functional recovery (Arvidsson et al., 2002). Based on our reported findings, it is likely that endogenous brain repairs triggered by IC-transplanted hUC-MSCs begin early after treatment and yet are sustained for long periods.
In summary, present findings indicate that IC transplantation of MSCs is a more efficient means of delivering stem cells to target brain regions, reaping diverse functional benefits. Functional outcomes after cell-based therapy are routedependent (Hao et al., 2014), requiring certain amounts of grafted cells to significantly attenuate functional deficits of ischemic injury. IC transplantation is particularly advantageous once a stroke has progressed to the subacute or chronic stage, whereas IV injection offers little therapeutic benefit.
To bolster the survival of IC-transplanted MSCs in patients with strokes, the addition of trophic factors expressed by MSCs has been suggested (Steinberg et al., 2016). In future studies, biochemical and molecular analyses may also help elucidate the molecular mechanisms involved. However, we believe that the various behavioral and histologic improvements we have documented confirm the therapeutic utility of hUC-MSC transplantation in the endogenous repair of strokedamaged brains.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.

ETHICS STATEMENT
The animal studies were reviewed and approved by the CHA University Institutional Animal Care and Use Committee (IACUC140012). The human studies were approved by Institutional Review Board from CHA Bundang Medical Center for the use of human umbilical cord (hUC) samples (IRB no.: BD2013-004D).

AUTHOR CONTRIBUTIONS
J-EN: collection and assembly of data, data analysis, interpretation, and manuscript writing. S-HO and I-HP: data analysis and interpretation, and manuscript writing. JS: conception and design, financial support, administrative support, manuscript writing, and final approval of the manuscript. All authors contributed to the article and approved the submitted version.

FUNDING
This study was supported by the National Research Foundation of Korea (NRF-2017M3A9B4061407), and the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health and Welfare (HI16C1559), South Korea. The authors declare that this study received funding from iPS Bio, Inc. The funder had the following involvement with the study: study design, data analysis and interpretation, and manuscript writing.