In the first of a series of experiments we wanted to determine if UCBMC given intravenously could stimulate the proliferation of the endogenous stem/progenitor cells in the hippocampus. We chose to study proliferation as a slowing of the cell cycle and a decrease in proliferation seems to be most affected with age when compared to the ability of the cells to survive and differentiate into neurons which appears to occur at relatively the same rate in young animals 30. The effect of a single intravenous injection of UCBMC on cell proliferation in the granule cell layer in young (3-months old) or aged (20-months old) F344 rats was determined by analyzing BrdU staining 24 hours after the BrdU injections (48 hours following UCBMC injection). Using the optical fractionator method of design based stereology 31, we found that in the aged animals there was a significant increase (t(9) = 4.256; p < 0.005) in the number of BrdU⁺ cells in the UCBMC group (2504 ± 227.3 n = 5) compared to the animals that received media alone (1549 ± 82.07 n = 6) (Figure 1A). In young animals there was no significant effect of the UCBMC treatment (data not shown). To determine if there might be a prolonged effect on proliferation in the aged F344 rats BrdU injections were given 14 days after the UCBMC treatment. Figure (1E) shows the effect of a single intravenous injection of UCBMC on the number of cells that incorporated BrdU on day 14. Stereological analysis revealed that in the aged UCBMC-treated rats there was a significant increase in the number of BrdU⁺ cells (t(12) = 3.468; p < 0.01) (2357 ± 149.4 n = 6) compared to the media-treated group (1548 ± 176.9 n = 4).

To determine if UCBMC would also stimulate neurogenesis in the aged rats, doublecortin (DCX) immunostaining was examined. Counting the number of DCX⁺ cells in the SGZ/GCL, we found a significant increase (t(16) = 2.188; p < 0.05) in the number of DCX⁺ cells in the aged rats 15 days after a single i.v. injection of the UCBMC (2619 ± 212.6 n = 9) compared to animals that received media alone (1843 ± 283.9 n = 9) (Figure 2A). To confirm the results obtained by DCX, and to determine if there was any change in the ability of the proliferating cells to differentiate following a UCBMC treatment, we injected the animals with BrdU (50 mg/kg) for five days beginning 24 hours after our i.v. treatment. Quantifying the number of BrdU⁺ cells in the SGZ/GCL using the optical fractionator method of design based stereology we found a similar increase in the number of BrdU⁺ cells in the aged UCBMC treated group as was found using the neurogenic marker DCX (Figure 2A–D). In aged rats, there was a significant increase (F(2, 14) = 10.94, p < 0.005) in the number of BrdU⁺ cells generated over a period of five days following a single i.v. injection of UCBMC (2772 ± 263.3 n = 5) compared to the rats that received media alone (1498 ± 206.1 n = 5); as determined by the Tukey's Multiple Comparison Test (p < 0.01). In this experiment, we also included a group that was injected with adult human peripheral blood mononuclear cells (PBMC) as a control for the effect of delivering cells. The PBMC group was determined to have significantly fewer BrdU⁺ cells (1712 ± 171.2 n = 5) than the UCBMC treated group (p < 0.01), but this was not significantly different from the group that received media alone (Figure 2E–H). As with the results of the proliferation study, young rats showed no significant effect of UCBMC treatment (data not shown). To determine if the treatment with UCBMC might alter the phenotype of the newborn cells, we double labeled with the antibodies to Tuj1, NeuN and GFAP. While exhaustive sampling was not conducted, 50 BrdU⁺ cells were analyzed from each rat (4 rats per group) using confocal microscopy for each marker and there did not appear to be any change in phenotype due to the treatment (Figure 2I–J). To confirm that the increase in neurogenesis was from the endogenous stem/progenitor cells, sections were stained for HuNu to look for the presence of the transplanted cells in the brain. Cells positive for the HuNu were found in the blood smears of the rats that were treated with UCBMC, but no HuNu immunoreactive cells were found in the hippocampus of the rats (data not shown).

Using the optical fractionator method of design based stereology, we counted the number of OX-6⁺ cells in the dentate gyrus 15 days after a single UCBMC injection; this was at the same time point that we observed an increase in DCX⁺ cells and BrdU⁺ cells. OX-6 is a marker for MHCII and presumably stains for microglia in an activated, proinflammatory state. In aged rats, we found that 15 days after the UCBMC treatment there was a significant decrease (t(12) = 2.699; p < 0.05) in the total number of activated OX-6⁺ microglia in the UCBMC group (678.7 ± 155.3 n = 7) compared to the media control (1217 ± 128.0 n = 8) (Figure 3A). The decrease in OX-6⁺ microglia negatively correlated with the number of DCX⁺ cells (Spearman r(15) = ^-- 0.6429; p < 0.01) (Figure 3E).

Morphologically the OX-6⁺ cells expressed two main phenotypes (see Figure 3F). Type 1 microglia appear to be in a more quiescent state based on morphology Type 2 microglia were thought to represent a more activated state. The type 1 microglia make up the majority of the OX-6⁺ cells in the dentate gyrus and were found to be significantly decreased (unpaired t(12) = 2.791; p < 0.05) in aged rats following UCBMC treatment (426.6 ± 117.0 n = 7) compared to controls (842.4 ± 94.67 n = 8) (Figure 3G). The type 2 microglia, while representing a smaller percentage of the total OX-6⁺ microglia, were significantly reduced (t(12) = 3.281; p < 0.01) to a greater extent by the UCBMC treatment (53.30 ± 13.27 n = 7) compared to controls (212.9 ± 43.82 n = 8) than the total microglia (Figure 3H). Fifteen days after the UCBMC treatment, there was a 4 fold change in the number of type 2 OX-6⁺ microglia, whereas there was only a 1.8 fold change in the total number of OX-6⁺ microglia. It appears that the highly activated microglia are being reduced to a greater extent by the UCBMC treatment, although all OX-6⁺ microglia are affected.

Cryopreserved human umbilical cord blood mononuclear cells (UCBMCs) were obtained from Saneron CCEL Therapeutics, Inc. (Tampa, FL, USA). Cryopreserved Human peripheral blood cells (PBMC) (mononuclear fraction) were obtained from AllCells, LLC (Emeryville, CA, USA). Just prior to intravenous (i.v.) injection, the UCBMC or PBMC were thawed into media (Hanks' balanced salt solution, HBSS, Gibco) at 37°C, washed, and the number of viable cells was determined using the trypan blue exclusion method 23. Cell viability ranged from 85 to 88%. Cell concentration was adjusted to 10⁶ viable cells/500 μl. Rats were then anesthetized with 3% isofluorane and randomly chosen to receive a single i.v. injection via the penile vein of UCBMC at a dose 10⁶ cells shown most effective in a stroke model 22, 10⁶ PBMC, or media for both the aged and young rats.

All experiments were conducted in accordance with the National Institute of Health Guide and Use of Laboratory Animals, and were approved by the Institutional Animal Care and Use committee of the University of South Florida, College of Medicine. Male Fisher 344 (F344) rats (NIA contract colony, Harlan Sprague Dawley, Indianapolis, IN), were pair-housed in environmentally controlled conditions (12:12 h light:dark cycle at 21 ± 1°C) and provided food and water ad lib. Two age groups of animals young (3 months old) and aged (20 months old) were used in this study. The mean life span of the F344 rats is approximately 29 months with a maximal life span of 36 months 50.

Rats were then divided in three groups. Group 1 received 50 mg/kg of bromodeoxyuridine (5-bromo-2-deoxyuridine, BrdU; Sigma, St. Louis, MO, USA), intraperitoneal (i.p.) twice a day beginning 24 hours the injection of UCBMC, and were sacrificed the subsequent day. Rats in group 2 received BrdU (50 mg/kg, i.p.) twice a day, beginning fourteen days after the administration of UCBMC and were sacrificed on the following day. Rats in Group 3 received BrdU (50 mg/kg, i.p.) for five consecutive days, beginning the day after the administration of UCBMC and were the sacrificed day fifteen.

The rats were anesthetized with pentobarbital (50 mg/kg, i.p.). Blood was collected by cardiac puncture and smears were made of the blood to look for the presence of the transplanted cells. The rats were transcardiac perfusion with phosphate-buffered (PB), followed by 4% paraformaldehyde in PB. The brains were postfixed in 4% paraformaldehyde for 12 h, after which they were transferred into 30% sucrose in phosphate-buffered saline (PBS) for at least 16 h, and stored at 4°C. Exhaustive sagittal sections of the left hemisphere were made, at 40 μm using a Microm cryostat (Richard-Allan Scientific, Kalamazoo Michigan) and stored in cryoprotectant at 4°C.

All immunohistochemical staining was conducted on free-floating sections for every sixth section for the entire hippocampus beginning with a random start and including sections before and after the hippocampus to ensure that the entire structure was sampled; with one exception, in the aged animals from group 3 a one in three series was stained to allow for sampling of an adequate number of BrdU⁺ cells. For BrdU staining, sections were pretreated with 50% formamide/2× SSC (0.3 M NaCl, 0.03 M sodium citrate) at 65°C for 2 hours, rinsed in 2× SSC, incubated in 2 N HCL for 30 minutes at 37°C, washed with borate buffer (pH 8.5), then PBS. This was followed by quenching endogenous peroxidase activity in 0.3% H₂O₂ solution in 30% methanol; then one hour in blocking solution (0.1 M PBS supplemented with 3% normal horse serum and 0.25% Triton X-100: PBS-TS); followed by incubation overnight with mouse-anti-rat-BrdU (1:100; Roche) in PBS-TS. The following day the sections were washed and then incubated for one hour in a biotinylated secondary antibody (1:200; Vector Laboratories, Burlingame, CA) in PBS-TS; then washed before one hour incubation in avidin-biotin substrate (ABC kit, Vector Laboratories, Burlingame, CA); and then washed before 10 minutes incubation in DAB solution (Vector Laboratories, Burlingame, CA). Sections were then mounted onto glass slides and coverslipped with mounting medium.

Doublecortin (DCX) is a marker of migrating neurons that is expressed for approximately three weeks after the cell is born and has been shown to be a reliable indicator of neurogenesis 5152. For DCX immunohistochemistry a polyclonal goat antibody raised against human DCX (1:200; SC-8066, Santa Cruz biotechnology, Santa Cruz, CA, USA) was used following a similar protocol to BrdU except the antigen retrieval steps were omitted and Goat serum (Vector Laboratories, Burlingame, CA) was used instead of horse serum. For OX-6 immunohistochemistry a monoclonal antibody directed against the rat major histocompatibility II (MHCII) (RT1B, Becton, Dickinson Pharmingen, San Diego, CA, USA) was used at a concentration of 1:750 in place of the other primary antibodies all other steps were the same.

Tissues were pretreated with 2 N HCL for 2 hours at room temperature, washed, and incubated in blocking solution (0.1 M PBS containing 10% goat serum and 0.3% Triton X-100) for 1 hour at room temperature. Tissues were then incubated in rat anti-BrdU (1:400; Accurate Chemical, Westbury, NY) and additional primary antibodies [anti-GFAP (1:500; Dako, Carpinteria, CA), mouse anti-NeuN (1:100; Chemicon, Temecula, CA), mouse anti-TUJ1 (1:800; Convance, Berkeley, CA)], overnight at 4°C. Tissues were then rinsed 3 times in PBS and the appropriate secondary antibody conjugated to an Alexafluor probe (Molecular Probes, Eugene, OR) was applied for 2 hour. Following 6 washes in PBS, tissues were mounted on slides and coverslipped using Vectashield (Vector Labs, Burlingame, CA).

To detect for the presence of the transplanted cells, blood smears and tissue sections were stained with a mouse monoclonal antibody that recognizes Human Nuclei antigen (HuNu) (MAB 1281; 1:50; Chemicon, Temecula, CA), and does not react with rat nuclei. Prior to incubation overnight at 4°C in the HuNu antibody, the samples were washed in PBS and incubated in blocking solution (0.1 M PBS containing 10% goat serum and 0.3% Triton X-100) for 1 hour at room temperature. The HuNu antibody was visualized by secondary antibody conjugated to an Alexafluor probe (Molecular Probes, Eugene, OR).

To determine cell numbers the optical fractionator method of unbiased stereological cell counting techniques 31 was used with a Nikon Eclipse 600 microscope and quantified using Stereo Investigator software (MicroBrightField, Colchester, VT). For the proliferation study, because of the low number of BrdU⁺ cell in the aged animals the virtual grid and counting frame were both 125 μm × 125 μm in order to count all the cells that were present in the section. For all other counts sampling was optimized to count at least 200 cells per animal with error coefficients less than 0.07. Outlines of the anatomical structures were done using a 10×/0.45 objective and cell quantification was conducted using a 60×/1.40 objective. OX-6⁺ cells were counted in the entire dentate gyrus including the subgranular zone (SGZ: defined as a two cell diameter band on both sides of the granular cell layer (GCL)). All other cell counts were done in the SGZ/GCL. The phenotype of the BrdU⁺ cells were analyzed using an inverted Zeiss LSM 510 confocal microscope with a 40×/1.30 NA oil immersion objective. Argon and HeNe laser lines in conjunction with 488 and 555 band pass filters were applied to excite the samples using line switching to minimize crosstalk between fluorochromes. Images and Z-stacks were produced with dual photomultiplier detectors and the LSM 5 version 3,2,0,115 software suite, and optical Z stacks where created at 2 μm intervals throughout the 40 μm of the sections with a guard region of 2 μm excluded from top and bottom of the Z stack. The Z stacks were rotated in all planes to verify double labeling.

Data are presented as mean cell number ± SEM. Statistical analysis was performed using an unpaired, two-side t-test, or a one-way ANOVA followed by a Tukeys post-hoc test. p < 0.05 was considered to be significant.