Objective
The aim of this study is to assess early homing of placenta-derived stem cells after perinatal intracerebral transplantation in rats.
Study Design
Neonatal Wistar rats (2-4 days old) were anesthetized, and 250,000 human placenta–derived mesenchymal stem cells (MSC) injected into the lateral ventricle or the paraventricular white matter using a stereotactic frame. Donor MSC were detected by immunohistochemistry using an antihuman HLA-ABC antibody.
Results
In all, 84% of the animals survived the transplantation. Donor cells were detected in the brain ventricle 1-2 hours posttransplantation. After 4 hours, donor cells migrated throughout the ventricular system. At 1-4 weeks after transplantation, some cells had migrated into the periventricular white matter.
Conclusion
Human placenta–derived MSC were successfully transplanted into the lateral ventricles of neonatal rats. Donor cells survived, homed, and migrated in the recipient brains. Proliferation and differentiation analysis and functional tests will assess the therapeutic effects of stem cell transplantation.
Preterm delivery is one of the major causes of neonatal morbidity and mortality. Despite enormous efforts, brain injury accounts for a major part of the clinical problems in survivors of premature birth. Approximately 63,000 infants are born with a very low birthweight (<1500 g) in the United States, and this group represents 1-5% of all live births. The enormity of the problems is indicated by occurrence of cognitive, behavioral, attentional, or socialization deficits in 25-50% and of major motor deficits in 5-10% of cases in this group. Several pathologies, such as severe intraventricular hemorrhage, periventricular hemorrhagic infarction, hydrocephalus, or cerebellar disease, occur in premature infants, but cerebral white matter injury, due to the unique maturation process of the brain, seems to be predominant.
Hypoxia-ischemia (HI) and inflammation during preterm intrauterine and extrauterine life frequently coexist and are an acquired form of neuronal injury. Several studies indicate that fetal inflammatory response as a consequence of maternal infection contributes to the extent of prenatal brain damage. Several animal models have been established using lipopolysaccharide application followed by induction of HI damage, but most of them inflict damage on postnatal day 7 (P7) rats, which, in terms of brain development, is rather equivalent to a late human preterm. To understand the effect of such a double-hit insult in very premature infants, animal models with inflammation and HI on postnatal day 2 (P2) rats have to be established. Few studies have been published, and most of them investigate the effect of this double hit to characterize the synergistic effect on perinatal damage, but no data on possible route of treatment are available. Stem cells are a promising source for targeted cell therapy for brain injury. A number of transplantation experiments in animal models of neonatal encephalopathy have been described. Human neural stem cells transplanted intracerebrally into P7 mice after HI injury migrated into the injured hemisphere. Human neural stem cells integrated into the damaged tissue, survived, and were shown to differentiate into neurons and astrocytes. Behavioral sensorimotor tests confirmed functional improvement. Progenitor cells derived from human adult white matter differentiated into oligodendrocytes within demyelinated lesions in the rat. Human neural progenitor cells transplanted into the striatum, hippocampus, and the subventricular zone of the developing rat brain migrated, integrated, and differentiated extensively into the corresponding cell types. Other cell types, such as mesenchymal stem cells (MSC), glial progenitor cells, or human umbilical cord blood (HUCB) cells have been used as grafts. Placental MSC, either derived from the deciduas or the chorionic villi, have been transplanted intravenously in a rat model of experimental stroke and resulted in some improvement of the outcome for the decidua group. In a similar model, intravenous transplantation of placental MSC stimulated the proliferation of stem and progenitor cells in the host and migration of these to the site of injury.
Intraperitoneal (ip) transplantation of HUCB cells in a neonatal rat model of HI brain damage resulted in amelioration of developmental sensorimotor reflexes, a neuroprotective effect in the striatum, and a decrease in microglial activation. In similar experiments, intravenous injection of HUCB cells did not improve spatial memory deficits and volumometric decrease of the hemisphere ipsilateral to the arterial occlusion. Amniotic membrane–derived stem cells transplanted into the telencephalic rat ventricles at embryonic day 15.5 migrated into a number of brain regions and were present into adulthood. No signs of immunorejection or tumor formation were found.
Different routes of application, such as ip, intraventricular, intracerebral, intravenous, or intracardiac, have been reported. Intracerebral route seems to be promising, due to exclusion of peripheral immunoreactions affecting the outcome of an experiment. A possible paracrine effect was also demonstrated in a recent study. Intravenously injected HUCB cells reduced HI-induced deficits in motor asymmetry and motor coordination. Treated animals displayed increased levels of nerve growth factor, glial cell-derived neurotrophic factor, and brain-derived neurotrophic factor in their brains after the transplant, suggesting a paracrine effect. Since only few cells were found in the brain after the treatment, the entry of neutrophic factors from the circulation into the brain parenchyma seems possible. Since HUCB cells produce several neutrophic factors and cytokines, and grafting of the transplant is often lacking, it is possible that the paracrine effect results in neuroprotection and antiinflammation.
There are several well-established models of HI in newborn animals. Unilateral or bilateral carotid artery occlusion, eventually followed by reduced oxygen tension, umbilical cord occlusion, or uterine or fetoplacental ischemia results in HI injury of the fetuses. Studies with stem cell grafts in HI brain injury models were exclusively done with rats age ≥P7, which corresponds to human neonates born at term regarding the brain development. Since the majority of cases of neonatal encephalopathy are found in infants of very low birthweight and include both HI and inflammation, we conducted a study to investigate neuroprotective effects of MSC on P2 Wistar rats.
The aim of the study was to develop a method of intracranial application of placenta-derived MSC into the lateral ventricle or into the paraventricular white matter zone using a stereotactic frame to prove that the transplantation of placental stem cells is possible and leads to their survival and homing in neonatal brains.
Materials and Methods
Isolation and expansion of MSC from placental tissue
MSC were isolated and expanded from stromal layer of third-trimester (34-39 weeks of gestation) chorion, as described earlier and from the umbilical cord connective tissue (Wharton jelly). Freshly collected umbilical cords were briefly washed in ethanol 70% and in phosphate-buffered saline (PBS) containing antibiotics/antimycotics (AA) (Invitrogen, Carlsbad, CA). The vessels were flushed with PBS using a syringe and removed with forceps. The Wharton jelly was dissected into small pieces using scalpels. The tissue was incubated for 3 hours in collagenase type 2 (Worthington Biochemical Corp, Lakewood, NJ) (270 U/mL, 25 mL in PBS-AA) at 37°C. The suspension was diluted with the same volume of PBS, and filtered (100-μm pore size) to remove tissue fragments. The filtered cell suspension was further diluted in 8× volume PBS and centrifuged for 10 minutes at 1500 rpm. The resulting cell pellet was resuspended in complete culture medium (Dulbecco’s Modified Eagle’s Medium/F12, fetal calf serum 10%, AA 1×, glutamax 1× [Invitrogen]), and expanded at 37°C, 5% carbon dioxide. To confirm the stem cell character, isolated cells were analyzed by flow cytometry for the cell surface expression of MSC markers (CD73, CD90, CD105) and absence of hematopoietic and major histocompatibility complex markers (CD34, CD45, CD14, HLA-DR), as described earlier.
Placentas and umbilical cords were collected after caesarean sections of healthy donors after obtaining written informed consent. Tissue sampling and experiments were approved by the local institutional review board (Ethics Committee of the Canton of Berne, Switzerland).
Intracerebral injection of the cell graft
To set up transplantation techniques into the neonatal rat brain, we performed stem cell injections into the brain of noninjured, 2- to 4- (mean 2.56) day-old rat pups (n = 44). Animals were anesthetized with ketamine (40 mg/kg body weight [BW], ip) and medetomidine (0.4 mg/kg BW, ip). The BW of the neonates was in the range of 4.8–10.8 g (median, 8.5 g). Proper dosage of the anesthetics is critical in neonates. Anesthetized animals were fixed on a block heated to 36°C in a small animal stereotaxic frame (Kopf Instruments, Tujunga, CA) equipped with soft tissue zygoma ear cups and a neonatal rat adaptor. Proper coordinates for lateral ventricle injection were adapted from a rat brain atlas by comparison of size and shape between the atlas and coronal sections of age-matched freshly euthanized neonatal rats. Bregma and lambda were adjusted to the same horizontal plane. Coordinates determined in the pilot experiments for P2 were 0.18 mm posterior from the bregma, 1.2 mm from the midline, and 1.8 mm below dura. Injections were made using a laboratory animal studies injector (Hamilton Co., Bonaduz, Switzerland) with a 32-gauge needle. A volume of 5 μL was injected over approximately 6 minutes into the left lateral ventricle. To inject at the slowest possible rate, 36 steps of 0.1 or 0.2 μL of suspension were injected, with a delay of 10 seconds between each step. The needle was left in place after injection for 2 minutes and then withdrawn slowly. A total number of 250,000 either human umbilical cord Wharton jelly– (n = 19) or chorion– (n = 252) derived MSC were injected. In all, 200 μL of PBS (prewarmed to 37°C, subcutaneously) and ceftriaxone (75 mg/kg BW, ip) were applied directly after termination of the transplantation, and atipamezole (2 mg/kg BW, subcutaneously) was applied 60 minutes after the induction of anesthesia. The animals were euthanized after 3-4 hours, or 1, 2, or 4 weeks, respectively. Approval for all animal experimentation was obtained from the Veterinary Office of the Canton of Berne, Switzerland.
Analysis of homing and engraftment
Brains were fixed in formaldehyde solution (4%; Merck, Whitehouse Station, NJ) for 2-4 hours at room temperature (RT) followed by 4°C for a total time of 24-48 hours. Fixed brains were embedded in paraffin, sectioned into 7-μm coronal slices, deparaffinized, and stained with hematoxylin-eosin or cresyl violet/Nissl (0.1% in distilled water; Merck). After deparaffinization of the slides, the target was retrieved in citrate buffer (10 mmol/L) in a microwave oven for 15 minutes, slides were washed in PBS 0.1% and Tween 20, and slides were blocked (goat serum 10%, bovine serum albumin 1% in PBS). Human MSC were detected with a mouse antihuman HLA class I ABC antibody (Abcam EMR8-5; Abcam, Cambridge, UK) and the DakoCytomation EnVision+ System-HRP (DAKO, Glostrup, Denmark). The slides were washed in PBS 0.1% and Tween 20 (2× 5 minutes) and incubated with the endogenous peroxidase block solution for 15 minutes, RT. Peroxidase-labeled polymer was applied to the slides for 30 minutes at RT, followed by 3 washes in PBS (5 minutes each) and the addition of 3,3′-diaminobenzidine in chromogen solution in buffer substrate for 10-30 minutes, according to the manufacturer’s instructions. Slides were rinsed in H 2 O and counterstained with cresyl violet (0.1% in H 2 O), dehydrated in a series of ethanol baths (95%, 100%) and xylene, and mounted with Eukitt (Sigma-Aldrich, St. Louis, MO). Alternatively, HLA-ABC expression was detected using a fluorescent setup: the peroxidase blocking was followed by incubation with a fluorescein isothiocyanate-labeled secondary antibody (goat antimouse IgG, 1:100; Sigma-Aldrich) for 1 hour at RT. After 3 washes (5 minutes each) in PBS, the brain slices were counterstained for cell nuclei with DAPI (0.01 mg/mL in PBS, 10 minutes, RT; Fluka, Sigma-Aldrich), washed again, and mounted with Mowiol solution (Mowiol 4-88 8.33% wt/vol, Calbiochem, Merck; polyvinyl alcohol 0.83% wt/vol, Sigma-Aldrich; glycerin 25% vol/vol, Sigma-Aldrich; H 2 O 25% vol/vol; 12 mL Tris-HCL pH 8.0 25 mmol/L).
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