Umbilical Cord Cell Therapy Improves Spatial Memory in Aging Rats

There is a growing interest in the potential of adult stem cells for implementing regenerative medicine in the brain. We assessed the effect of intracerebroventricular (icv) administration of human umbilical cord perivascular cells (HUCPVCs) on spatial memory of senile (27 mo) female rats, using intact senile counterparts as controls. Approximately one third of the animals were injected in the lateral ventricles with a suspension containing 4.8 X 105 HUCPVC in 8 μl per side. The other third received 4.8 X 105 transgenic HUCPVC overexpressing Insulin-like growth factor-1 (IGF-1) and the last third of the rats received no treatment. Spatial memory performance was evaluated using a modified version of the Barnes maze test. In order to evaluate learning ability as well as spatial memory retention, we assessed the time spent (permanence) by animals in goal sector 1 (GS1) and 3 (GS3) when the escape box was removed. Fluorescence microscopy revealed the prescence of Dil-labeled HUCPVC in coronal sections of treated brains. The HUCPVC were located in close contact with the ependymal cells with only a few labeled cells migrating into the brain parenchyma. After treatment with naïve or IGF-1 transgenic HUCPVC, permanence in GS1 and GS3 increased significantly whereas there were no changes in the intact animals. We conclude that HUCPVC injected icv are effective to improve some components of spatial memory in senile rats. The ready accessibility of HUCPVC constitutes a significant incentive to continue the exploration of their therapeutic potential on neurodegenerative diseases.


Introduction
There is a growing interest in the therapeutic potential of adult mesenchymal stem cells (MSCs) in the brain. Although adult tissues are a frequent source of MSCs, they can also be obtained from birth-associated tissues including placenta, amnion, and umbilical cord with the advantage of their availability, avoiding the need for invasive procedures and eliminating other ethical concerns. A number of MSC types can be obtained from the umbilical cord depending on whether they are isolated from the whole umbilical cord (UC-MSCs), Wharton's Jelly (WJ-MSCs), the umbilical cord blood (CB-MSCs) [1], perivascular areas (human umbilical cord perivascular cells or HUCPVCs) [2], In particular, HUCPVCs are advantageous candidates for cell therapy due to their lower donor variability, faster doubling time, and ready availability [3].
Insulin-like growth factor-1 (IGF-1) is a neuroprotective peptide produced by astroglial cells in response to brain insults. We have previously demonstrated that IGF-1 gene therapy in old rats is neuroprotective in different areas of the brain including the hippocampus [4][5][6]. We therefore reasoned that HUCPVC, genetically modified to express IGF-1 may display a significant restorative effect on spatial memory performance in old rats. In this brief report we describe our results.

Materials and Methods
Cells Human umbilical cord perivascular cells were obtained from healthy donors after written informed consent and protocol was approved by the BInstitutional Evaluation Committee^(CIE) from School of Biomedical Sciences, Austral University (CIE #16-038). The protocol authorizes a number of Hospital researchers to use the human umbilical cords for a number of experimental studies. HUCPVCs were isolated from umbilical cord using a protocol adapted from Sarugaser et al. [2] and were cultured in DMEM low glucose (lg) (Cat# 31600-034; Thermo Fisher Scientific, Buenos Aires, Argentina), as previously described [7].
Cell Transduction HUCPVCs were seeded at 70% of confluence in complete medium. Medium was then removed, and cells were transduced with either an adenoviral vector expressing the cDNA for rat IGF-1 (RAd-IGF-1), or an adenoviral vector (RAd-GFP) expressing the reporter gene encoding humanized green fluorescent protein (GFP) in both cases at a multiplicity of infection (MOI) of 30 in DMEM lg and 2% fetal bovine serum (FBS; Natocor, Inc. Cordoba, Argentina) in half of total volume for 2 h. The RAd-GFP-transduced cells were incubated for 5 min with (1 μg/ml) DAPI (4,6-Diamidino-2-phenylindole, dihydrochlorid; Sigma Chem Co. cat# 10236276001), only.
Subsequently, medium was completed with 10% FBS in DMEM lg. Three days later cells were processed and injected as described below.
Animals Senescent (27 mo.) female Sprague-Dawley (SD) rats were used. The animals were housed in a temperaturecontrolled room (22 ± 2°C) on a 12:12 h light/dark cycle. Food and water were available ad libitum. All experiments with animals were performed in accordance with the Animal Welfare Guidelines of NIH (INIBIOLP's Animal Welfare Assurance No A5647-01) and institutional IACUCapproved protocol P05-02-2015.
Rats were grouped, as follows: Group Intact, consisted of 6 senile rats that received no treatment. Group HUCPVC, consisted of 8 senile rats that were stereotaxically injected with HUCPVC only and Group HUCPVC + IGF-I, consisted of 9 senile rats that were stereotaxically injected with HUCPVC transduced with RAd-IGF-1.
Experimental Design Nine days before HUCPVC injection (experimental day −9) the pre-treatment Barnes test began.
On experimental day 0, experimental rats were icv injected with HUCPVCs as described below. From experimental day 21 to 26 all rats were submitted to the post-treatment Barnes test (Fig. 1a). On experimental day 27 all rats were sacrificed as described below.

Surgical Procedures
Stereotaxic Injections Rats were anesthetized with ketamine hydrochloride (40 mg/kg; ip) plus xylazine (8 mg/kg; im) and placed in a stereotaxic apparatus. In order to access the lateral ventricles (LV), the tip of a 26G needle fitted to a 50 μl syringe was brought to the following coordinates relative to the bregma: −0.8 mm anteroposterior, 4.1 mm dorsoventral and ± 1.5 mm mediolateral [8]. The animals were injected bilaterally with 8 μl per side of a suspension containing 4.8 X 10 5 HUCPVC.

Spatial Memory Assessment
The modified Barnes maze protocol used in this study was based on a previously reported procedure [9]. It consists of an elevated (108 cm above the floor) black acrylic circular platform, 122 cm in diameter, containing 20 holes around the periphery (Fig. 2 A inset). The holes are uniform in diameter (10 cm) and appearance, but only one hole is connected to a black escape box. The escape box is 38.7 cm long × 12.1 cm wide × 14.2 cm in depth and it is removable. A white cylindrical starting chamber (an opaque, 35 cm diameter and 20 cm high, open-ended chamber) is used to place the rats on the platform. Four proximal visual cues are located in the test room, 50 cm from the platform. The escape hole was numbered as hole 0 for graphical normalized representation purposes, the remaining holes being numbered 1 to 10 clockwise, and − 1 to −9 counterclockwise (Fig. 2a inset). Hole 0 was held in a fixed position, relative to the cues to avoid randomization of the relative position of the escape box. During the tests the platform was rotated daily. A 90-dB white-noise generator and a white-light 500 W bulb provided the escape stimuli from the platform.
We used an abbreviated protocol based on three days of acquisition trials (AT), followed by two probe trials (PT), 1 and 5 days after training (PT1 and PT2), to assess recent spatial memory retention. An AT consists of placing a rat, randomly oriented, in the starting chamber for 30s. The chamber is then raised, the aversive stimuli are switched on and the rat is allowed to freely explore the maze for 120 s. A probe trial is defined as a trial where the escape box has been removed, its purpose being to assess the latency to explore the empty escape hole and the error frequency. After the starting chamber is raised, the rat is given 120 s to explore and the number of explorations per hole is recorded. In order to eliminate olfactive clues from the maze and the boxes, the surfaces are cleaned with 10% ethylic alcohol solution, after each trial. On the day before AT, animals were habituated to the starting chamber and escape box by placing them inside each one for 60 s in the starting chamber and 120 s in the escape box Behavioral performance was recorded using a computerlinked video camera mounted 110 cm over the platform. The video-recorded performances of the subjects were measured using the Kinovea v0.7.6 (http://www.kinovea.org) software. The behavioral parameters assessed were as follows.

Statistical Analysis
Data were analyzed with the Sigma Plot v. 11 software (San Jose, CA). Comparisons between groups were made using one-way ANOVA for the probe trials. Data are presented as mean ± SEM. Tukey's post-hoc tests was used where appropriate. Criteria for significant differences were set at 95% probability level.

Time-Course of HUCPVC in the Cerebral Ventricles
Dil-fluorescence in coronal sections of HUCPVC-injected brains revealed that five days after injection, Dil-labeled cells were located in close contact with the ependymal cells (Fig. 1b). The number of labeled cells decreased progressively from Experimental day 5 on. On the day of sacrifice labeled cells were still observable close to the ependymal cells (data not shown).

Cognitive Changes
Hole Exploration The hole exploration frequency of senile rats before and after treatment was assessed and compared in different goal and nongoal sectors. No significant differences were detected (data not shown).

Time Spent in GS 1 and GS 3
The time spent by rats from different experimental groups in GS 1 and GS 3 before and after treatment at PT1 and PT2 was compared. In the intact senile rats there was no significant difference between pre and post-treatment groups in GS 1 (Fig. 2a). In contrast, in the HUCPVC and HUCPVC+IGF-1 groups, the time spent in GS 1 was significantly longer at PT1 and PT2 than at pre-treatment. For GS 3 , in the intact senile rats there was no significant difference between pre-and posttreatment whereas in the HUCPVC and HUCPVC+IGF-1 groups the time spent in GS 3 was significantly longer at PT1, but not PT2, than at pre-treatment (Fig. 2b).

Discussion
Adult stem cells constitute a promising therapeutic tool for the treatment of human neurodegenerative processes. Thus, a number of irreversible neurodegenerative diseases, such as Alzheimer's Disease (AD), amyotrophic lateral sclerosis (ALS) and Parkinson's disease (PD), stem cell therapy constitutes a promising new avenue of research. Although a number of pre-clinical studies have been launched, quite a few clinical studies have been performed at present. Evidence suggests that autologous bone-marrow derived mesenchymal stem cells are safe and effective when transplanted unilaterally in PD patients. Interestingly, no adverse effects of these stem cells were detected, a fact that paved the way for future studies [10]. Human umbilical cord blood derived MSC were transplanted in AD patients at two different doses and endpoint analysis was measured by ADAS-cog scoring, PET imaging, and the measurement of Aβ and tau levels in CSF [11]. In China, a research group recruited AD patients in phase I/II trial with thirty probable AD participants, using a similar study design as the one outlined above, where patients are intravenously administered with 20 X10 [6] human UCB-MSCs [12]. Medipost Co Ltd. started a double blinded, placebo controlled, phase I/IIa trial. Patients with mild to moderate AD will be subjected to repeated intraventricular administrations of UCB-MSCs and will be evaluated 24 weeks after the first transplantation [13]. Concerning animal studies, in 2015 we demonstrated that lifelong intravenous (iv) injection of hMSCs begun in a rat at 6 months of age, markedly prolonged lifespan (44 months versus the typical 36 months of standard laboratory rats) [14]. Our findings are in line with a previous study where lifelong iv injection of amniotic membrane-derived hMSCs (AM-MSCs) or adipose tissue-derived MSC (AD-MSC) to 10-month-old male F344 had a similar life-extending effect. The AM-MSC and AD-MSC improved cognitive and physical functions of naturally aging rats, extending life span by 23 and 31%, respectively [15]. This and other [16] evidence prompted us to undertake the present study. We reasoned that icv administration of HUCPVC to aged rats could be able to restore their deficient cognitive performance.
The time-course results reveal a slowly declining number of HUCPVC in the cerebroventricular space until the end of the experiment. A rather modest number of Dil(+) cells were observed in the brain parenchyma at any time. The initial assumption concerning the mechanisms by which MSCs Fig. 2 Effect of MSCs treatment in old rats on GS 1 and GS 3 sector permanence-Sector permanence (expressed as the time spent in the corresponding sector) in the Senile (intact) group showed no significant changes 27 days after the initial tests (equivalent to pre versus post treatment period in the other groups) in either GS 1 or GS 3 . In contrast, in both, the HUCPVC and the HUCPVC+IGF-1 experimental groups posttreatment sector permanence increased significantly for PT1 and PT2. In all cases, comparisons were made versus the corresponding pre treatment counterparts. Inset-Barnes maze platform showing GS1 and GS3. *, P < 0.05: **, P < 0.01, **, P < 0.001, N = 6, 8 and 9 for Senile control, HUCPVC and HUCPVC+IGF-1, respectively in both PT1 and PT2 improve central nervous system (CNS) injury, was that they migrate to the injured tissues and transdifferentiate to replace injured neural cells [17,18]. However, subsequent studies showed that transplanted MSCs may exert their therapeutic effect without evidence of engraftment [19,20], thus indicating that their regenerative and differentiating abilities do not play a role in limiting tissue destruction or enhancing tissue repair. Instead, it seems that inflammatory signals, induce MSCs to secrete a variety of bioactive molecules, such as anti-inflammatory molecules and trophic factors able to modulate the host microenvironment [21], which seems to be the main mechanism responsible for their therapeutic effects when they are administered icv or iv [22]. Therefore, the putative neurogenic factors released by our HUCPVC into the cerebroventricular space should have reached hippocampal cells thus inducing a moderate improvement in function.
In laboratory rodents, spatial memory has been extensively evaluated using the Morris Water Maze (MWM). However, a potential disadvantage of the MWM in age-related studies is that it requires a substantial degree of physical fitness. Thus, we chose the less physically demanding Barnes maze for evaluating spatial memory in aging rats. In both tests, rats learn to use spatial cues to guide them to a hidden platform or tunnel. While, the MWM involves immersion in water, a stimulus that induces considerable corticosterone secretion and corticotropin release [23], in the Barnes maze rodents are placed on an circular platform for them to walk in search of the escape hole.
The MWM probe trial, which specifically measures the spatial component of target-finding, has no counterpart in the Barnes maze. However, in more recent times, removal of the escape box was used to measure spatial memory extinction [24]. By removing the escape box at the end of the training period the PT was introduced in our variant of the test, which enabled us to assess memory-dependent variables as well as recent (PT1) and longer-term (PT2) retention of spatial memory, whose levels are proportional to the inverse of spatial memory extinction. In this study we found that the permanence in GS 1 and GS 3 during PT1 and PT2 is a sensitive parameter to assess the effect of HUCPVC-treatment on spatial memory. Hole exploration frequency was less sensitive to the treatement.
Concerning the effect of HUCPVC on the permanence in GS1 and GS3, our findings are in line with documented evidence of cognitive improvement in rodent models of neurodegenerative diseases. Thus, it has been reported that in an AD mouse model, intra-hippocampal administration of human umbilical cord blood-derived mesenchymal stem cells (hUC-MSCs) significantly rescued cognitive performance as determined by the MWM test [25]. In another study, adult human MSCs were administered (10 5 cells/bilaterally) into the lateral ventricle of Flinders sensitive line (FSL) rats, an animal model for depression. When 12 days later, animals were assessed by the forced swim test and the dominant-submissive relationship (DSR) paradigm, results revealed that MSCstransplanted FSL rats had a clear improvement in their behavioral performance in both tests [26].
It has been previously shown that transgenic mouse bone marrow-derived mesenchymal stromal cells overexpressing IGF-1 seem to possess an anti-fibrogenic effect on experimental hepatic fibrosis induced in mice by chronic thioacetamide application or bile duct ligation [27]. Our results suggest that overexpression of IGF-1 in our HUCPVC does not enhance the restorative activity of HUCPCV per se on the permanence performance of old rats in GS 1 and GS 3 . It could be speculated that at hippocampal level, the restorative action of HUCPVCderived soluble factors exert a maximal restorative action on neurons and/or glia, thus preventing further improvement by transgenic IGF-1 action.