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First published online September 7, 2006
Stem Cells Vol. 25 No. 1 January 2007, pp. 98 -106
doi:10.1634/stemcells.2006-0055; www.StemCells.com
© 2007 AlphaMed Press

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TISSUE-SPECIFIC STEM CELLS

Potential Treatment of Cerebral Global Ischemia with Oct-4+ Umbilical Cord Matrix Cells

Sachiko Jomuraa, Marc Uya, Kathy Mitchellc, Renee Dallasena, Claudia J. Bodec, Yan Xua,b

aDepartment of Anesthesiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA;
bDepartment of Pharmacology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA;
cDepartment of Pharmacology and Toxicology, University of Kansas, Lawrence, Kansas, USA

Key Words. Cerebral global ischemia • Rat umbilical cord matrix cell • Oct-4 • Extracellular signaling • Neurogenesis • Reperfusion Stem cell therapy

Correspondence: Yan Xu, Ph.D., University of Pittsburgh School of Medicine, Biomedical Science Tower 3, Room 2048, 3501 Fifth Avenue, Pittsburgh, Pennsylvania 15260, USA. Telephone: 412-648-9922; Fax: 412-648-8998; e-mail: xuy{at}anes.upmc.edu

Received on January 26, 2006; accepted for publication on August 29, 2006.

First published online in STEM CELLS EXPRESS  September 7, 2006.

   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 Disclosures
 Acknowledgments
 References
 
Potential therapeutic effects of Oct-4-positive rat umbilical cord matrix (RUCM) cells in treating cerebral global ischemia were evaluated using a reproducible model of cardiac arrest (CA) and resuscitation in rats. Animals were randomly assigned to four groups: A, sham-operated; B, 8-minute CA without pretreatment; C, 8-minute CA pretreated with defined media; and D, 8-minute CA pretreated with Oct-4+ RUCM cells. Pretreatment was done 3 days before CA by 2.5-µl microinjection of defined media or approximately 104 Oct-4+ RUCM cells in left thalamic nucleus, hippocampus, corpus callosum, and cortex. Damage was assessed histologically 7 days after CA and was quantified by the percentage of injured neurons in hippocampal CA1 regions. Little damage (approximately 3%–4%) was found in the sham group, whereas 50%–68% CA1 pyramidal neurons were injured in groups B and C. Pretreatment with Oct-4+ RUCM cells significantly (p < .001) reduced neuronal loss to 25%–32%. Although the transplanted cells were found to have survived in the brain with significant migration, few were found directly in CA1. Therefore, transdifferentiation and fusion with host cells cannot be the predominant mechanisms for the observed protection. The Oct-4+ RUCM cells might repair nonfocal tissue damage by an extracellular signaling mechanism. Treating cerebral global ischemia with umbilical cord matrix cells seems promising and worthy of further investigation.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Conclusion
 Disclosures
 Acknowledgments
 References
 
Unless resuscitation is given immediately, cardiac arrest (CA) invariably leads to debilitating brain damage and death due to cessation of oxygen and glucose supply to the brain tissue. Injury patterns after cerebral global ischemia are characterized by the disseminated neuronal loss of the selectively vulnerable pyramidal neurons in the CA1 and CA3 regions of the hippocampus, medium-sized neurons in the striatum, and Purkinje cells in cerebellum [1–4]. Even with a brief primary insult, the selectively vulnerable neurons die hours to days after the ischemia. For a long time, the secondary derangements were thought to be the direct consequence of excitotoxicity and energy failure. More recent investigations [1–3, 5–8], however, suggest that treatment strategies aimed simply at energy metabolism or excitotoxicity would inevitably fail. Finding alternative strategies to rescue injured neurons from dying and to help uninjured neurons to survive has been the subject of intense investigations in recent years [9–11]. One of the possibilities is to promote neurogenesis from neural stem cells and progenitor cells in the adult brain [12]. It has been suggested that neurogenesis is enhanced by transient brain ischemia [13–17]. Moreover, neurotrophic factors are believed to play an important role in this mechanism. Levels of neurotrophic factors such as brain-derived neurotrophic factor [18–20], fibroblast growth factor-2 [21], and erythropoietin [22] in the brain have been shown to increase after ischemic injury. These neurotrophic factors have also been used for experimental treatment of brain ischemia.

Another way to promote neurogenesis and neuronal protection is stem cell transplantation. Several attempts have been made to use stem cells from different origins [23], including bone marrow stem cells [24–26], neuroepithelical stem cells [27], fetal neural stem cells [28], and umbilical cord blood cells [29, 30], for treatment of focal ischemia. These studies showed the migration and differentiation of stem cells in relation to the functional recovery. Some recent studies even suggest that stem cell contributions to organ repair can arise from a circulating pool of the adult bone marrow stromal stem cells because cells from the donor marrow can be found in the brain, liver, kidney, or lung [31–34]. The potential therapeutic application of stem cell factor to focal ischemia was also investigated [35]. Very few studies to date, however, have focused on the use of stem cells for the treatment of global cerebral ischemia due to the dispersed nature of the damage. The possibility of treating cerebral global ischemia with exogenous stem cells has not yet been fully explored.

In the present study, we combined the use of rat umbilical cord matrix (RUCM) cells and a clinically relevant outcome model of CA and resuscitation in rats [1, 2] to investigate the potential therapeutic effects of Oct-4+ RUCM cells in mitigating cerebral global ischemic damage after 8-minute normothermic CA.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Conclusion
 Disclosures
 Acknowledgments
 References
 
Isolation and Culture of RUCM Cells
RUCM cells were isolated from the umbilical cords of female Sprague-Dawley rats at 16-day gestation in accordance with a protocol approved by the Institutional Animal Care and Use Committee (IACUC) at University of Kansas (Lawrence, KS). The procedure is similar to that used for isolating porcine and human UCM cells [36]. Briefly, cords were washed in betadine, rinsed in sterile phosphate-buffered saline (PBS), incubated in hyaluronidase (40 U/ml) (MP Biomedicals, Solon, OH, http://www.mpbio.com) and collagenase type I (0.4 mg/ml) (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com) for 30 minutes at 37°C, and rinsed with sterile PBS. Cords were finely minced, plated in six-well plates, and maintained in defined media (DM), which are composed of Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA, y http://www.invitrogen.com) and MCDB-201 medium (Sigma-Aldrich) supplemented with 1x insulin-transferrin-selenium (Invitrogen), 0.15% lipid-rich bovine serum albumin (Albumax; Invitrogen), 0.1 nM dexamethasone (Sigma-Aldrich), 10 µM ascorbic acid-2-phosphate (Sigma-Aldrich), 1x penicillin/streptomycin (Thermo-Fisher, Suwanee, GA, http://new.fishersci.com), 2% fetal bovine serum (BD Biosciences, San Jose, CA, http://www.bdbiosciences.com), 10 ng/ml recombinant human epidermal growth factor, and 10 ng/ml rat platelet-derived growth factor BB (R&D Systems, Inc., Minneapolis, http://www.rndsystems.com). On day 5, cord remnants were removed, and the attached cells were washed three times with PBS, followed by addition of fresh DM. Cells were passaged by lifting with 0.05% trypsin EDTA. Viable cells were counted with a hemocytometer and trypan blue exclusion and usually replated at an initial density of 30%. Cells were passaged when they reached 80% confluency.

Flow Cytometry
RUCM cells at 1 x 106 cells per milliliter were fixed with methanol at 4°C for 5 minutes and blocked with PBS and 5% bovine serum albumin at 4°C for 1 hour. Cells were incubated with mouse primary antibodies (1 µg/ml) against Oct-4, smooth muscle actin (SMA), or vimentin (Chemicon International, Temecula, CA, http://www.chemicon.com) at 4°C for 1 hour. Cells were then washed three times with PBS and incubated with goat anti-mouse secondary FITC conjugate (1:100; Invitrogen) for 30 minutes at 4°C. Thereafter, cells were washed twice in PBS and analyzed using a FACSCalibur flow cytometer (Beckman Coulter, Fullerton, CA, http://www.beckmancoulter.com). Ten-thousand cells (no gating) were collected and analyzed in the FL1 channel. Control cells were incubated with mouse isotype-specific immunoglobulin G to establish the background signal.

Reverse Transcription-Polymerase Chain Reaction Analyses
RNA was isolated from cultured RUCM cells with RNeasy Quick spin columns (Qiagen Inc., Valencia, CA, http://www1.qiagen.com) and converted to cDNA using random hexamers and SuperScript II reverse transcriptase (Invitrogen). Polymerase chain reaction (PCR) amplification was performed using a Bio-Rad I-Cycler (Bio-Rad, Hercules, CA, http://www.bio-rad.com) for 35 cycles with the following primer pairs: Oct-4, forward 5'-GAAGGATGTGGTCCGAGTGT-3', reverse 5'-GTGAAGTGAGGGCTCCCATA-3' (expected product size of 183 base pair [bp]); vimentin, forward 5'-ATGTCCACCAGGTCCGTG-3', reverse 5'-TTATTCAAGGTCATCGTG-3' (expected product size of 1.4 kbp); and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, as a positive control), forward 5'-ATCTTCCAGGAGCGAGAT-3' and reverse 5'-TGGTCATGAGTCCTTCCACGATA-3' (expected product size of 300 bp). For negative control, PCR was performed in the presence of cDNA but without primers. Products were resolved by 2% agarose gel electrophoresis and visualized by ethidium bromide staining.

Immunofluorescence
RUCM cells from passage 10 were grown to 80% confluency in chamber slides. Cells were fixed with 4% paraformaldehyde for 10 minutes at room temperature, quenched in 100 mM glycine for 5 minutes, permeabilized with 0.2% Triton X-100 for 5 minutes, and blocked in blocking buffer (0.2% Triton X-100, 2% normal goat serum, 0.4% bovine serum albumin in PBS) for 1 hour. Cells were incubated with primary antibody for 1 hour (mouse monoclonal antibodies to Oct-4 and SMA, 1:100; Chemicon). Cells were washed three times with PBS and incubated with secondary antibody (Alexa Fluor 546 donkey anti-mouse, 1:200; Invitrogen) for 1 hour. Nuclear DNA was stained with SYTOX Blue nucleic acid stain (Invitrogen). For negative controls, cells were incubated with the labeled secondary antibodies and SYTOX Blue only. Images were obtained with a 510 Zeiss laser scanning microscope (Carl Zeiss, Jena, Germany, http://www.zeiss.com) under x63 oil-immersion lens.

In Vivo Experimental Groups
The CA and resuscitation procedures were approved by the IACUC at the University of Pittsburgh. Thirty-three male Sprague-Dawley rats (Harlan Sprague Dawley, Inc., Indianapolis, http://www.harlan.com), weighing 234 ± 27 g, were used. Rats were randomized into four groups. In group A (sham-operated, n = 7), rats were subjected to the same surgical and CA and resuscitation procedures as detailed below but were resuscitated immediately after the induction of CA without asphyxia. In groups B (n = 9), C (n = 9), and D (n = 8), rats underwent 8 minutes of CA, followed by rapid resuscitation. Rats in group C and group D were pretreated 3 days prior to CA with an intracranial microinjection of sterilized defined cell culture medium and RUCM cells, respectively. In all groups, the rat body temperature was measured by a rectal temperature probe and controlled to 36.5°C ± 0.5°C throughout the experiment using a heating pad and warm light source.

CA and Resuscitation
Rats were prepared as described previously [1, 2] with a few minor modifications. Under approximately 3% isoflurane anesthesia, rats were quickly intubated orotracheally. After intubation, rats were mechanically ventilated with a 50:50 mixture of air and O2. Anesthesia was maintained with 1.5%–2% isoflurane, and paralysis was produced by pancuronium bromide (2 mg/kg). The arterial blood pH and gases were measured using a Ciba-Corning blood gas analyzer (model 278; Bayer HealthCare, Tarrytown, NY, http://www.bayerdiag.com) or an i-STAT portable clinical analyzer (Abbott Laboratories, Abbott Park, IL, http://www.abbott.com). Ventilation rate (1 ml/100 g of body weight, 40–45 strokes per minute) and positive end-expiratory pressure were carefully adjusted to control the arterial blood gas values in the normal range before CA [1, 3, 8]. Both femoral arteries and the left femoral vein were catheterized. One of the arterial catheters was used for continuous monitoring and recording of arterial blood pressure and heart rate. The other was used for arterial blood sampling and later for retrograde infusion of oxygenated blood during resuscitation. Approximately 15 minutes before CA, ventilation was switched to 100% oxygen and approximately 5 minutes later, oxygenated blood was withdrawn from the same rat. To prevent spontaneous breathing during the asphyxial CA, a booster dose of short-acting muscle relaxant (vecuronium bromide, 1 mg/kg) was injected intravenously 3 minutes before CA. CA was induced by asphyxia (stoppage of mechanical ventilation) combined with an i.v. bolus injection of an ultra-short-acting ß1-blocker, esmolol (6.25 mg). The latter ensures a very tight control of the time from the onset of asphyxia to the electromechanical dissociation leading to circulatory arrest. Isoflurane anesthesia was discontinued during CA. Resuscitation was started 8 minutes after the induction of CA by 100% O2 ventilation along with retrograde infusion of oxygenated blood mixed with the resuscitation mixture containing heparin (5 U/ml), sodium bicarbonate (0.05 mEq/ml), and epinephrine (8 µg/ml) through one of the catheterized femoral arteries into the abdominal and thoracic aorta. Infusion was performed manually to maintain the mean arterial blood pressure approximately 40 mmHg and was stopped at the first sign of restoration of spontaneous circulation (ROSC). The rats were continually ventilated for at least 2 hours with anesthesia reinstated as required. Thereafter, arterial and venous catheters were surgically removed, and the wound was closed. Mechanical ventilation with air was continued until the effects of muscle relaxant subsided and sustained spontaneous breathing was observed. Animals were then extubated and returned to individual cages for postresuscitation evaluation for 7 days.

Stem Cell Transplantation
RUCM cells were obtained and cultured in the same way as described above. To ensure that a clonal population of cells was transplanted, RUCM cells at passage 53 were plated in 96-well plates with cell densities approximately 1 cell per well. After several days of culturing, cells from a single well were slowly expanded and harvested at passages 67, 69, 96, or 97 for transplantation. Chromosome analysis was done at passage 61 and 78 to confirm that cells from these passages have the same composite karyotype. Immunohistostaining and reverse transcription (RT)-PCR were repeated to confirm that these cells remained Oct-4-positive. Sixteen hours before transplantation, cells were labeled with 5 µM green fluorescent carboxyfluorescein diacetate (CFDA) (Invitrogen) for later histology tracking. Once inside the cells, the CFDA dye is converted to anionic CFDA succinimidyl ester (CFDA-SE) by intracellular esterases and couples to amine groups on proteins to achieve long-term intracellular labeling. Thus, after the acetate groups are cleaved off, CFDA-SE dye can be transferred to other (or daughter) cells only through cell division or cell fusion.

Rats were anesthetized with isoflurane and placed on a stereotactic apparatus for precise intracranial microinjection. Using predetermined coordinates based on the Paxinos atlas [37], the CFDA-labeled RUCM cells were injected into the following four sites in the left hemisphere: dorsal thalamic nucleus (DTN), dorsal hippocampus (H), corpus callosum (CC), and dorsal cortex (Fig. 1A). Because injuries after global ischemia are disseminated, these sites are selected based either on their vulnerability to ischemia or on their ability to allow cell migration. Approximately 4 x 104 cells in 10 µl (2.5 µl at each site) were transplanted at an infusion rate of 0.1 µl/minute using a programmable infusion pump (model UMC4; World Precision Instruments, Inc., Sarasota, FL, http://www.wpiinc.com) and a Mity Flexfil-microsyringe (model 500,818; World precision Instruments, Inc.) with a 200-µm outer diameter flexi-tip titanium needle. After transplantation, the needle was left in the brain for an additional 15 minutes before removal. As a negative control of the transplantation procedure, rats in group C received microinjections of the same volume of DM at exactly the same four coordinates. Three days after the cell transplantation or DM injection, the CA procedure was performed.


Figure 1
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  		Figure 1. Anatomic references. (A): The microinjection sites, as marked by the gray dots, for rat umbilical cord matrix cell transplantations. The stereotactic coordinates are 1 mm left and 2.1 mm posterior to the bregma and 1.5 mm (cortex), 2.6 mm (corpus callosum), 3.5 mm (dorsal hippocampal region), and 5.0 mm (dorsal thalamic nucleus) from the top of the brain. (B): Cresyl violet staining of a rat brain section at the dorsal hippocampus level showing the predetermined areas (circles) in the CA1 regions where neuronal counting was performed. Abbreviations: CC, corpus callosum; DG, dentate gyrus; DV3, dorsal third ventricle; LV, lateral ventricle.

 
Outcome Evaluation
Rats were observed for 7 days after CA and resuscitation. The functional recovery was evaluated using the neurological deficit scores (NDS), which have a value ranging from 0 for brain death to 500 for neurologically normal, as detailed previously [1, 38]. After final NDS evaluation, rats were anesthetized with isoflurane and perfused with buffered 10% formalin phosphate. The brain was extracted from the skull and stored in buffered 10% formalin for 48 hours. The brain section containing the dorsal hippocampal region was embedded in paraffin and sliced into 6-µm-thick coronal sections. Alternating sections were stained with cresyl violet to visualize neuronal damages and deparaffinized to evaluate the survival, engraftment, and migration of the transplanted RUCM cells by the fluorescence microscopy of CFDA. The dorsal hippocampus of coronal sections was photographed. To quantify the histology damages, normal and damaged neurons were counted using Adobe Photoshop software (Adobe Systems Incorporated, San Jose, CA, http://www.adobe.com) in four predetermined regions (two in each hemisphere) in the CA1 of the coronal hippocampal sections between 3.3 and 3.6 mm posterior to the bregma (Fig. 1B). Each circled region in the hippocampus had, on average, 89 ± 21 neurons. Every region was counted by at least two investigators who were blinded of the treatment groups to minimize bias in judgment. The histology damage was quantified as the percentage of damaged neurons against the total neuronal counts in the same region.

Three-Dimensional Rendering of Cell Migration
To better visualize the fate of transplanted RUCM cells, three-dimensional (3D) reconstruction of cell migration was rendered using a total of 114 consecutive coronal sections (thickness 6 µm) from the brain of a typical cell-transplanted rat, killed 7 days after CA and 10 days after transplantation. The sections were serially prepared using a microtome and were digitally imaged using a Leica DMR fluorescence microscope (Leica, Heerbrugg, Switzerland, http://www.leica.com) to visualize the CFDA dye in the RUCM stem cells. The images were imported into the Reconstruct software [39] (http://www.synapses.bu.edu) and aligned using the ventricles and other physical structures as reference points. The height and width scale of the sections was determined by the Measure program in Leica software module and was used as a parameter to scale the images in Reconstruct. Each fluorescent stem cell in the brain sections was contoured using Reconstruct. The Paxinos rat brain atlas [37] was used as a reference for creating anatomical structural groups, including the lateral and third ventricles, the hippocampus, and the corpus callosum, to show the locations of the transplanted cells and their migration. The 3D surface reconstructions generated by Reconstruct were exported to 3D Studio MAX (Autodesk, Inc., San Rafael, CA, http://usa.autodesk.com) for final rendering.

Data Analysis
Statistical analysis was performed using the Origin software (OriginLab Corporation, Northampton, MA, http://www.originlab.com) and GraphPad PRISM (GraphPad Software, Inc., San Diego, http://www.graphpad.com). One-way analysis of variance was used to compare the physiological parameters (Table 1 and the Bonferroni multiple-comparison test was used to determine the differences among groups. A p value of <.05 was considered statistically significant. All data are reported as mean ± SD except for neuronal counting, which is presented as mean ± SE.


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  		Table 1. Parameters of cardiac arrest and resuscitation

 

   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Conclusion
 Disclosures
 Acknowledgments
 References
 
Characterization of RUCM Stem Cells
The typical morphology of RUCM cells in culture at passage 10 is shown in a bright-field micrograph in Figure 2A. The cell morphology and growth rate remained essentially the same at later passages. The nuclear localization of the embryonic transcription factor, Oct-4 (red), was demonstrated by its colocalization with SYTOX nuclear stain (blue) (Fig. 2B). The cytoplasmic localization of the SMA filaments (red) is shown in Figure 2C. The negative control sample with the secondary antibody only shows no immunoreactivity (Fig. 2D). Flow cytometry results obtained from one of four different isolations of RUCM cells are shown in Figure 2E. Oct-4 was expressed in nearly 90% of the total number of cells counted as determined by flow cytometry with an average of 87% ± 5% (±SD, n = 4) for the four different isolations of RUCM cells. SMA and vimentin were expressed by an average of 85% ± 11% (n = 4) and 80% ± 11% (n = 4) for the four different isolations of RUCM cells, respectively. Expression of vimentin and Oct-4 was determined by RT-PCR with GAPDH as a positive control. The negative control was done where PCR was performed in the presence of cDNA but no primers (Fig. 2F). PCR products of the expected sizes for Oct-4 and vimentin were detected.


Figure 2
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  		Figure 2. Characterization of rat umbilical cord matrix (RUCM) cells. Cells were imaged by (A) bright-field microscopy or immunostained for (B) Oct-4 and (C) smooth muscle actin (SMA) (red). Confocal images were overlaid with SYTOX Blue nucleic acid stain (blue). (D): Negative control with secondary antibody overlaid only with SYTOX Blue shows no immunoreactivity. (E): Flow cytometric analysis of RUCM cells. Each panel represents a single antibody assay. Black lines represent control cells with mouse immunoglobulin G alone plus fluorescein isothiocyanate-labeled secondary antibody. Black-filled lines represent primary mouse antibodies for vimentin, Oct-4, or SMA. (F): Reverse transcription-polymerase chain reaction of mRNA isolated from one representative RUCM cell line demonstrating the expression of the myofibroblast marker vimentin (Vim, 1.4 kilobase pair), the stem cell marker Oct-4 (183 base pair [bp]), and the housekeeping gene GAPDH (300 bp). The negative control (Neg) demonstrates the absence of product in a reaction with no added primers. Abbreviations: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MW, molecular weight.

 
Stem Cell Treatment of Global Cerebral Ischemia
CA and resuscitation were highly reproducible in all experimental groups subjected to 8-minute CA. Four out of 33 animals (one each in groups A and B, two in group C) died of unidentified causes before the end of the planned recovery period. None of the animals in group D with RUCM cell transplantation died unexpectedly. Table 1 summarizes the important parameters of CA and resuscitation. In groups B (untreated CA), C (CA pretreated with defined medium), and D (CA pretreated with RUCM cells), all rats showed the same arterial blood pressure changes as reported previously [1], and the arterial blood pH and gases were within the normal physiological range before CA (pH 7.43 ± 0.03, pCO2 36.1 ± 3.1, SpO2 99.7 ± 0.1). The esmolol injection induced a rapid onset of CA. The time from esmolol bolus injection to CA was 0.37 ± 0.10 minutes. No significant differences were detected among groups. There were no differences in the resuscitation time among groups B (untreated CA), C (CA pretreated with defined medium), and D (CA pretreated with RUCM cells). The time from the initiation of the resuscitation effort to ROSC in these three groups was 0.68 ± 0.14 minutes (0.70 ± 0.19 minutes for the four-group average and 0.77 ± 0.33 minutes for group A alone). In groups B, C, and D, 8 minutes elapsed between the esmolol injection and the onset of resuscitation. The actual duration of CA (from electromechanical dissociation to ROSC) was 8.29 ± 0.16 minutes, and there were no significant differences among the three groups. Rats in sham group (group A) were injected with esmolol and resuscitated immediately by infusion of oxygenated blood and resuscitation mixture. The transient "no-flow" time for rats in group A was 0.99 ± 0.43 minutes. A larger standard deviation of no-flow time in group A compared with other groups was likely due to the varying responses to the possible drug interaction between esmolol and epinephrine when the short-action esmolol was still effective in the sham group while the resuscitation mixture was infused.

There was no specific ischemic damage in group A (sham group); neurons stained with crestyl violet show clear round nuclei and cell bodies in dark purple (Fig. 3A). Only 3.8% ± 0.5% and 3.2% ± 0.5% of the hippocampal neurons were damaged in the left and right CA1 regions, respectively. The same amount of "apparent damage" is also seen in animals without having CA surgical manipulations. Seven days after resuscitation, six out of seven animals in group A recovered fully with normal NDS (Table 1). Group B (untreated CA group) had typical ischemic changes in the CA1 neurons, including nuclear pyknosis (the condensation of chromatin), vacuolization (formation of large membrane-bound vacuoles), and karyorhexis (the fragmentation of the nucleus) (Fig. 3B, 3C), and 50.1% ± 6.0% and 51.3% ± 6.2% of the pyramidal neurons in the left and right CA1 regions were damaged, respectively. Despite severe histological damage, the rats that survived in group B showed normal NDS 7 days after resuscitation (Table 1). It is a characteristic finding that behavioral recovery from CA as measured by the NDS is often an all-or-none phenomenon: animals either die within days or appear neurologically normal [3]. Hence, histology outcome as measured by the neuronal loss in the CA1 region is a more quantifiable measure of the damage. CA1 neurons in group C (CA pretreated with DM) had slightly more severe damage than in group B, with 67.9% ± 5.5% and 62.4% ± 5.8% of neurons injured in the left and right CA1 regions, respectively (Fig. 4). The difference between group B (untreated CA) and group C (CA pretreated with DM) is significant only on the ipsilateral (injection) side (p = .04) and not significant on the contralateral side (p = .20). Four animals in group C were nonresponding to tail clamping 7 days after resuscitation (NDS 495 ± 5.3). The percentage of damaged hippocampus neurons in group D (CA pretreated with RUCM cells) was significantly reduced (Fig. 4, p < .001), being only 31.9% ± 2.2% and 24.9% ± 2.6% in the left and right CA1 regions, respectively. Seven days after resuscitation, one of the seven animals in group D was nonresponsive to pain stimulation in the tail.


Figure 3
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  		Figure 3. Representative cresyl violet-stained sections of the CA1 region of hippocampus from rats in groups A (sham-operated), B (untreated cardiac arrest), C (cardiac arrest pretreated with defined medium), and D (cardiac arrest pretreated with Oct-4+ rat umbilical cord matrix cells). Typical global ischemic damages, including vacuolization (arrow in frame B), nuclear pyknosis (arrow in frame C), and karyorhexis (arrowhead), are found in groups B, C, and D, but not in group A. The number of injured neurons is significantly reduced in group D compared with groups B and C. Scale bars = 20 µm.

 


Figure 4
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  		Figure 4. The histological damage, quantified by the percentage of injured neurons counted in the predetermined areas in the CA1 regions of the hippocampus. Results from one-way analysis of variance with Bonferroni multiple comparisons are marked: # and ¶ indicate significant difference (p < .001) between groups of the same side; {ddagger} and * indicate significant difference (p < .05) between groups of the same side and within the same group of the opposite sides, respectively. Abbreviations: LT, left; RT, right.

 
The percentages of the damaged pyramidal neurons in the left and right CA1 regions of the dorsal hippocampus in the four experimental groups are summarized in Figure 4. For groups A (sham group), B (untreated CA), and C (CA pretreated with DM), the injuries are symmetrical, and there is no significant difference between left and right hemispheres. In group D (CA pretreated with RUCM cells), although both sides had significantly less injury compared with groups B (untreated) and C (pretreated with DM) (p < .001), the side with microinjection had slightly more damage than the contralateral side (p = .0496).

The transplanted RUCM cells were identified by the loaded green CFDA dye under a fluorescent microscope. It is evident that the transplanted RUCM cells have survived after the microinjection (Fig. 5). A significant amount of RUCM cells have migrated away from the injection sites. Figure 6 shows the 3D rendering of the migration of CFDA-labeled RUCM cells from the injection sites. Unlike in focal cerebral ischemia, the neuronal damage in global cerebral ischemia is not localized. There seems no clear direction for RUCM cell migration after ischemia, and migration patterns vary from rat to rat. In some rats, a majority of the cells injected into the cortex migrated toward the CC, and those injected directly into the CC migrated the furthest medially toward the contralateral side. In other rats, cells injected in the dorsal hippocampus showed migrations in the medial-lateral and rostral-caudal directions. Cells transplanted in DTN had shorter migration distances. Only a few RUCM cells were found directly in the CA1 regions of the transplantation side, and no CFDA-labeled RUCM cells were found on the contralateral side.


Figure 5
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  		Figure 5. Representative fluorescence images of carboxyfluorescein diacetate-dyed rat umbilical cord matrix cells transplanted 3 days prior to cardiac arrest into the rat brains. Brains were sectioned 7 days after the cardiac arrest and resuscitation. The transplanted cells were found to have survived and migrated away from the injection sites in (A) cortex, (B) corpus callosum, (C) dorsal hippocampus, and (D) dorsal thalamic nucleus (x100 magnification). The areas within the white rectangles in (B) and (C) are further magnified at x400 to show details of the transplanted cells in (E) corpus callosum and (F) dorsal hippocampus, respectively. A section from a medium-injected rat is provided in (G), showing the level of background autofluorescence for comparison.

 


Figure 6
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  		Figure 6. Three-dimensional reconstruction of the migration of the transplanted carboxyfluorescein diacetate (CFDA)-labeled rat umbilical cord matrix stem cells in the brain of a rat 10 days after the cell transplantation and 7 days after cardiac arrest and resuscitation. Images show (A) the front view in the rostral-to-caudal direction, (B) top view in the dorsal-to-ventral direction, and (C) left-side view in the lateral-to-medial direction. Images (D), (E), and (F) depict the zoom-in views of the regions enclosed in the white boxes in (A), (B), and (C), respectively. Anatomic references are color-coded: magenta, outline of the brain; cyan, corpus callosum; purple, hippocampus; and red, ventricles. The green spheres mark the injection sites, and the yellow spheres are outlines of the CFDA-labeled rat umbilical cord matrix stem cells.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Conclusion
 Disclosures
 Acknowledgments
 References
 
One of the characteristics of brain damage caused by cerebral global ischemia after CA is the disseminated neuronal loss, particularly in the selectively vulnerable CA1, CA3, and dentate gyrus regions of hippocampus. Often the dying or dead neurons are found side-by-side to "good" neurons that appear histologically normal. The underlying molecular and cellular events determining the fate of each neuron after the same ischemic insult are not fully understood.

Using a highly reproducible outcome model of CA and resuscitation, we demonstrated the potential therapeutic effects of transplanted UCM cells on mitigating neuronal loss after severe global ischemia. We found that pretreatment with RUCM cells 3 days before CA can significantly reduce, but not eliminate, brain damage characterized by the pyramidal neuron loss in the CA1 region of the hippocampus. As shown in Figures 3 and 4, the group pretreated with RUCM cells (group D) had significantly less CA1 damage than the untreated group (group B). Possible artifacts unrelated to the direct therapeutic effects of RUCM cells include immune response to microinjection and brain preconditioning to ischemia due to microinjection procedures. However, others [40] have shown that injection of porcine UCM cells into rat brain do not elicit immune response. In this study, we further ruled out any possible preconditioning artifacts by adding group C (pretreated with defined medium), in which the exact same pretreatment procedures were performed as in group D except for receiving RUCM cells. Group C showed no improvement in histology outcome due to preconditioning alone. In fact, microinjection with sterilized DM followed by CA 3 days later had the tendency to slightly worsen the outcome compared with untreated group (group B), and the difference between groups B (untreated) and C (pretreated with DM only) was statistically significant on the injection side. Thus, it can be concluded that the observed improvement in histological outcome in group D was a direct consequence of RUCM cell treatment.

The general assumption about stem cells being beneficial in treating a stroke is their pluripotency. In embryonic stem cells, pluripotency has been linked to the expression of Oct-4, a Pit-Oct-Unc transcription factor [41]. Oct-4 is expressed almost exclusively in embryonic stem cells [42]. In the mature animal, Oct-4 expression was thought to be restricted to the germline. However, Oct-4 expression was recently observed in a population of bone marrow stromal cells after serum deprivation [43] and porcine UCM cells [44]. Oct-4 expression has also been found in amniotic fluid cells [45] and in tumors [46, 47]. Although pluripotency and functionality in these latter cases are yet to be established, these findings nevertheless suggest that Oct-4 might play a role in determining the fate of other types of stem cells.

The Oct-4+ cells used in this study were derived from the RUCM. These cells have the potential to differentiate into other tissue types. For example, it was demonstrated that human UCM cells had the capacity to differentiate into a neuronal phenotype in vitro [36] and that porcine UCM cells not only could survive after transplantation into the rat brain, but also appeared to differentiate into neurons [40]. We showed here that RUCM cells are myofibroblast-like stem cells similar to those isolated from porcine and human umbilical cord. They express markers similar to those found in the adult bone marrow stromal stem cell (SMA and vimentin) and embryonic stem cells (Oct-4). These findings suggest that the UCM is an abundant, easily obtainable source of primitive Oct-4+ stem cells that might be intermediate between embryonic and adult stem cells. Unlike embryonic stem cells, UCM cells do not form teratomas (K. Mitchell, unpublished observations) nor do they elicit a detectable immune response [40, 48]. The UCM cells might in fact have an immune-suppressive effect as has been observed for mesenchymal stem cells [49]. Mesenchymal stem cells from cord blood, while having many of the same characteristics as the UCM cells, are less abundant and might be less primitive than those found in UCM. Because of these properties, UCM cells might be a better alternative to embryonic, bone marrow stromal, or umbilical cord blood cells for cell-based therapies.

We chose a pretreatment strategy in this study to evaluate the possible mode of action of stem cells in preventing neuronal damage after an intrinsically disseminated insult. Our rationale was that with pretreatment, the transplanted stem cells could be activated by the acute exposure to ischemia. If pretreatment can significantly improve the histological outcome after a controlled global ischemia, which is known to lead to nonfocal damages, then neither of the two popular hypotheses about stem cell protection (namely, stem cell differentiation into neurons [transdifferentiation] and stem cell fusion with host cells) would be sufficient to explain the protective effects. Other mechanisms should be considered and explored.

Indeed, careful analysis of the engraftment and migration of the transplanted cells suggests that RUCM cell transdifferentiation and fusion might not be the predominant mechanisms for the observed protection. Although significant cell migration within hippocampus was observed in some of the animals (Fig. 6), relatively few RUCM cells were found directly in the CA1 pyramidal cell lining on the transplantation side, and no fluorescent cells were detectable on the contralateral side. Thus, even if RUCM cell transdifferentiation into neurons or RUCM cell fusion with neuronal cells does occur during the reperfusion and recovery period, neither mechanism can account for the significant protection seen in group D (pretreated with RUCM cells). Hence, our results seem to suggest the possibility of a third novel mechanism of stem cell repair—one that elicits one or multiple synergistic extracellular signaling pathways. This possibility is strongly supported by the recent studies in which i.v. injection of human umbilical cord blood (HUCB) cells into rats was shown to reduce brain injury during a 1-hour middle cerebral artery occlusion [50, 51]. These focal ischemia studies unequivocally demonstrated that cell entry into the central nervous system is not absolutely required for the neuroprotection by the peripherally injected HUCB cells. As the authors of these studies concluded, the secretion of the "therapeutic molecules" (including the neurotrophic factors) and the nonimmune anti-inflammatory effects are the two necessary components of the observed HUCB cell neuroprotection.

In our case, it can be speculated that the presence of Oct-4+ RUCM cells during ischemia activates and accelerates the proliferation and recruitment of the endogenous neuronal stem cells, including re-entry of quiescent stem cells, into the rescue effort. Other possibilities include the creation of an extracellular milieu that enhances and restores the intrinsic ability of the brain tissue in self-repair [52]. For example, it is possible that the very presence of the transplanted stem cells serves as the first responder to the stress signals from ischemia, priming the activation and reintegration of the Notch and Wnt signaling [53] to renew the adult brain tissue to re-enter a youthful state [54, 55] in the recovery stage. After the initial increase in the proliferation of endogenous neuronal progenitor cells, asymmetric antagonization of Notch signal can lead to rapid differentiation of one of the two daughter cells into vascular lineage for angiogenesis or neuronal lineage for neurogenesis. Another possibility is that, like the cord blood cells, the transplanted RUCM cells can suppress the inflammatory response after ischemia [56], thereby helping injured neurons to recover and promoting the viable neurons to remain alive. The transplanted-cell-host-cell communication as the primary stem cell repair mechanism is further suggested in our study by the improved outcome in the contralateral side where no transplanted RUCM cells were found—a strong indication that retrograde signaling from long-distance connections might also play an important role in determining the fate of neurons after ischemia and reperfusion injuries.

Although the most desirable intervention for cerebral ischemia is post-treatment, preventative therapy by pretreatment to avoid brain damage due to circulatory arrest is also clinically relevant. For example, patients receiving an implantable automatic internal cardiac defibrillator usually undergo two tests of total cerebral ischemia. Also, in pediatric cardiac surgery for repairing complex congenital cardiac malformations [57], in certain adult cardiac surgeries involving the aortic arch [58, 59], and in adult neurosurgery for giant intracranial aneurysms [60, 61], a controlled total circulatory arrest to create a bloodless operative field is often essential. At present, the only commonly used preventative measure in these surgical cases is deep hypothermia, which is not without devastating complications. Thus, devising novel pretreatment strategies aimed at alleviating acute and delayed neurological morbidities is highly beneficial to the future development of innovative medical procedures. Most importantly, the potential future clinical applications of stem cell therapy require a better understanding of the protection mechanisms, for which pretreatment clearly has the advantage over post-treatment in many cases, as discussed above.

Finally, it is worth mentioning that, although we have attempted to use clonal cells for transplantation, it is difficult to conclude that the endothelial cells from the umbilical cord tissue are completely depleted after multiple passages. However, endothelial and vascular cells do not express Oct-4 nor would they proliferate as long in culture as the UCM cells. There is a remote possibility that a very small fraction (<1%) of the injected cells might be endothelial precursors, which might have persisted in culture over multiple passages. Based on the percentage of cells that are positive for SMA and Oct-4 (markers that would not be expressed by endothelial precursors), the very small percentage of endothelial precursors, if any, would be unlikely to contribute substantially to the overall effect observed.


   CONCLUSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
Disclosures
 Acknowledgments
 References
 
RUCM cell transplantation indirectly reduces the percentage of damaged hippocampal neurons after CA. Although more studies will be needed to ascertain the protection mechanism, the results of the present study indicate that Oct-4+ UCM cell treatment of brain injury from global ischemia, particularly through cell signaling pathways, is a distinct possibility and warrants further investigation. A better understanding of the extracellular signaling molecules that are secreted by UCM cells in different environments (e.g., hypoxia) can also help identify potential targets for the development of novel drugs that—when given after CA—can potentially trigger the same healing process that the UCM cells appear to have initiated.


   DISCLOSURES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 Disclosures
Acknowledgments
 References
 
The authors indicate no potential conflicts of interest.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 Disclosures
 Acknowledgments
References
 
We thank Katayoun Ghajarnia for participating in histology tissue preparation and neuronal counting and Deanna Nachreiner for help with histology analysis and 3D rendering of stem cell migration. This work was supported by a grant from the National Institutes of Health (R01NS/HL036124 to Y.X.) and was presented at the November 12–16, 2005 Society for Neuroscience annual meeting.


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Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 Disclosures
 Acknowledgments
 References
 

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