Walter C. Low

Pluripotency of mesenchymal stem cells derived from adult marrow

We report here that cells co-purifying with mesenchymal stem cells—termed here multipotent adult progenitor cells or MAPCs—differentiate, at the single cell level, not only into mesenchymal cells, but also cells with visceral mesoderm, neuroectoderm and endoderm characteristics in vitro. When injected into an early blastocyst, single MAPCs contribute to most, if not all, somatic cell types. On transplantation into a non-irradiated host, MAPCs engraft and differentiate to the haematopoietic lineage, in addition to the epithelium of liver, lung and gut. Engraftment in the haematopoietic system as well as the gastrointestinal tract is increased when MAPCs are transplanted in a minimally irradiated host. As MAPCs proliferate extensively without obvious senescence or loss of differentiation potential, they may be an ideal cell source for therapy of inherited or degenerative diseases.

Reperfusion injury after focal cerebral ischemia: The role of inflammation and the therapeutic horizon

Recent evidence indicates that thrombolysis may be an effective therapy for the treatment of acute ischemic stroke. However, the reperfusion of ischemic brain comes with a price. In clinical trials, patients treated with thrombolytic therapy have shown a 6% rate of intracerebral hemorrhage, which was balanced against a 30% improvement in functional outcome over controls. Destruction of the microvasculature and extension of the infarct area occur after cerebral reperfusion. We have reviewed the existing data indicating that an inflammatory response occurring after the reestablishment of circulation has a causative role in this reperfusion injury. The recruitment of neutrophils to the area of ischemia, the first step to inflammation, involves the coordinated appearance of multiple proteins. Intercellular adhesion molecule-1 and integrins are adhesion molecules that are up-regulated in endothelial cells and leukocytes. Tumor necrosis factor-alpha, interleukin-1, and platelet-activating factor also participate in leukocyte accumulation and subsequent activation. Therapies that interfere with the functions of these factors have shown promise in reducing reperfusion injury and infarct extension in the experimental setting. They may prove to be useful adjuncts to thrombolytic therapy in the treatment of acute ischemic stroke.

Tauroursodeoxycholic acid, a bile acid, is neuroprotective in a transgenic animal model of Huntington's disease

Huntingtons disease (HD) is an untreatable neurological disorder caused by selective and progressive degeneration of the caudate nucleus and putamen of the basal ganglia. Although the etiology of HD pathology is not fully understood, the observed loss of neuronal cells is thought to occur primarily through apoptosis. Furthermore, there is evidence in HD that cell death is mediated through mitochondrial pathways, and mitochondrial deficits are commonly associated with HD. We have previously reported that treatment with tauroursodeoxycholic acid (TUDCA), a hydrophilic bile acid, prevented neuropathology and associated behavioral deficits in the 3-nitropropionic acid rat model of HD. We therefore examined whether TUDCA would also be neuroprotective in a genetic mouse model of HD. Our results showed that systemically administered TUDCA led to a significant reduction in striatal neuropathology of the R6/2 transgenic HD mouse. Specifically, R6/2 mice began receiving TUDCA at 6 weeks of age and exhibited reduced striatal atrophy, decreased striatal apoptosis, as well as fewer and smaller size ubiquitinated neuronal intranuclear huntingtin inclusions. Moreover, locomotor and sensorimotor deficits were significantly improved in the TUDCA-treated mice. In conclusion, TUDCA is a nontoxic, endogenously produced hydrophilic bile acid that is neuroprotective in a transgenic mouse model of HD and, therefore, may provide a novel and effective treatment in patients with HD.

Highly resolved in vivo 1H NMR spectroscopy of the mouse brain at 9.4 T

An efficient shim system and an optimized localization sequence were used to measure in vivo 1H NMR spectra from cerebral cortex, hippocampus, striatum, and cerebellum of C57BL/6 mice at 9.4 T. The combination of automatic first‐ and second‐order shimming (FASTMAP) with strong custom‐designed second‐order shim coils (shim strength up to 0.04 mT/cm2) was crucial to achieve high spectral resolution (water line width of 11–14 Hz). Requirements for second‐order shim strengths to compensate field inhomogeneities in the mouse brain at 9.4 T were assessed. The achieved spectral quality (resolution, S/N, water suppression, localization performance) allowed reliable quantification of 16 brain metabolites (LCModel analysis) from 5–10‐μL brain volumes. Significant regional differences (up to 2‐fold, P < 0.05) were found for all quantified metabolites but Asp, Glc, and Gln. In contrast, 1H NMR spectra measured from the striatum of C57BL/6, CBA, and CBA/BL6 mice revealed only small (<13%, P < 0.05) interstrain differences in Gln, Glu, Ins, Lac, NAAG, and PE. It is concluded that 1H NMR spectroscopy at 9.4 T can provide precise biochemical information from distinct regions of the mouse brain noninvasively that can be used for monitoring of disease progression and treatment as well as phenotyping in transgenic mice models. Magn Reson Med 52:478–484, 2004.

Tauroursodeoxycholic acid reduces apoptosis and protects against neurological injury after acute hemorrhagic stroke in rats

Tauroursodeoxycholic acid (TUDCA), an endogenous bile acid, modulates cell death by interrupting classic pathways of apoptosis. Intracerebral hemorrhage (ICH) is a devastating acute neurological disorder, without effective treatment, in which a significant loss of neuronal cells is thought to occur by apoptosis. In this study, we evaluated whether TUDCA can reduce brain injury and improve neurological function after ICH in rats. Administration of TUDCA before or up to 6 h after stereotaxic collagenase injection into the striatum reduced lesion volumes at 2 days by as much as 50%. Apoptosis was ≈50% decreased in the area immediately surrounding the hematoma and was associated with a similar inhibition of caspase activity. These changes were also associated with improved neurobehavioral deficits as assessed by rotational asymmetry, limb placement, and stepping ability. Furthermore, TUDCA treatment modulated expression of certain Bcl-2 family members, as well as NF-κB activity. In addition to its protective action at the mitochondrial membrane, TUDCA also activated the Akt-1/protein kinase Bα survival pathway and induced Bad phosphorylation at Ser-136. In conclusion, reduction of brain injury underlies the wide-range neuroprotective effects of TUDCA after ICH. Thus, given its clinical safety, TUDCA may provide a potentially useful treatment in patients with hemorrhagic stroke and perhaps other acute brain injuries associated with cell death by apoptosis.

Thymidine analogs are transferred from prelabeled donor to host cells in the central nervous system after transplantation: a word of caution

Thymidine analogs, including bromodeoxyuridine, chlorodeoxyuridine, iododeoxyuridine, and tritiated thymidine, label dividing cells by incorporating into DNA during S phase of cell division and are widely employed to identify cells transplanted into the central nervous system. However, the potential for transfer of thymidine analogs from grafted cells to dividing host cells has not been thoroughly tested. We here demonstrate that graft‐derived thymidine analogs can become incorporated into host neural precursors and glia. Large numbers of labeled neurons and glia were found 3–12 weeks after transplantation of thymidine analog‐labeled live stem cells, suggesting differentiation of grafted cells. Remarkably, however, similar results were obtained after transplantation of dead cells or labeled fibroblasts. Our findings reveal for the first time that thymidine analog labeling may not be a reliable means of identifying transplanted cells, particularly in highly proliferative environments such as the developing, neurogenic, or injured brain.

Infusion of human umbilical cord blood ameliorates neurologic deficits in rats with hemorrhagic brain injury

Abstract: Umbilical cord blood is a rich source of hematopoietic stem cells. It is routinely used for transplantation to repopulate cells of the immune system. Recent studies, however, have demonstrated that intravenous infusions of umbilical cord blood can ameliorate neurologic deficits associated with ischemic brain injury in rodents. Moreover, the infused cells penetrate into the parenchyma of the brain and adopt phenotypic characteristics typical of neural cells. In the present study we tested the hypothesis that the administration of umbilical cord blood can also diminish neurologic deficits caused by intracerebral hemorrhage (ICH). Intracerebral hemorrhage is a major cause of morbidity and mortality, and at the present time there are no adequate therapies that can minimize the consequences of this cerebrovascular event. ICH was induced in rats by intrastriatal injections of collagenase to cause bleeding in the striatum. Twenty‐four hours after the induction of ICH rats received intravenous saphenous vein infusions of human umbilical cord blood (2.4 × 106 to 3.2 to 106 cells). Animals were evaluated using a battery of tests at day 1 after ICH, but before the administration of umbilical cord blood, and at days 7, and 14 after ICH (days 6 and 13, respectively, after cord blood administration). These tests included a neurological severity test, a stepping test, and an elevated body‐swing test. Animals with umbilical cord blood infusions exhibited significant improvements in (1) the neurologic severity test at 6 and 13 days after cord blood infusion in comparison to saline‐treated animals (P < 0.05); (2) the stepping test at day 6 (P < 0.05); and (3) the elevated body‐swing test at day 13 (P< 0.05). These results demonstrate that the administration of human umbilical cord blood cells can ameliorate neurologic deficits associated with intracerebral hemorrhage.

Expression of MHC I and NK ligands on human CD133+ glioma cells: possible targets of immunotherapy

Mounting evidence suggests that gliomas are comprised of differentiated tumor cells and brain tumor stem cells (BTSCs). BTSCs account for a fraction of total tumor cells, yet are apparently the sole cells capable of tumor initiation and tumor renewal. BTSCs have been identified as the CD133-positive fraction of human glioma, whereas their CD133-negative daughter cells have limited proliferative ability and are not tumorogenic. It is well established that the bulk tumor mass escapes immune surveillance by multiple mechanisms, yet little is known about the immunogenicity of the CD133-positive fraction of the tumor mass. We investigated the immunogenicity of CD133-positive cells in two human astrocytoma and two glioblastoma multiforme samples. Flow cytometry analyses revealed that the majority of CD133-positive cells do not express detectable MHC I or natural killer (NK) cell activating ligands, which may render them resistant to adaptive and innate immune surveillance. Incubating CD133-positive cells in interferon gamma (INF-γ) significantly increased the percentage of CD133-positive cells that expressed MHC I and NK cell ligands. Furthermore, pretreatment of CD133-positive cells with INF-γ rendered them sensitive to NK cell-mediated lysis in vitro. There were no consistent differences in immunogenicity between the CD133-positive and CD133-negative cells in these experiments. We conclude that CD133-posistive and CD133-negative glioma cells may be similarly resistant to immune surveillance, but that INF-γ may partially restore their immunogenicity and potentiate their lysis by NK cells.

Neuroprotection by a Bile Acid in an Acute Stroke Model in the Rat

Tauroursodeoxycholic acid (TUDCA), a hydrophilic bile acid, is a strong modulator of apoptosis in both hepatic and nonhepatic cells, and appears to function by inhibiting mitochondrial membrane perturbation. Excitotoxicity, metabolic compromise, and oxidative stress are major determinants of cell death after brain ischemia-reperfusion injury. However, some neurons undergo delayed cell death that is characteristic of apoptosis. Therefore, the authors examined whether TUDCA could reduce the injury associated with acute stroke in a well-characterized model of transient focal cerebral ischemia. Their model of middle cerebral artery occlusion resulted in marked cell death with prominent terminal deoxynucleotidyl transferase-mediated 2′-deoxyuridine 5′-triphosphate-biotin nick end labeling (TUNEL) within the ischemic penumbra, mitochondrial swelling, and caspase activation. Tauroursodeoxycholic acid administered 1 hour after ischemia resulted in significantly increased bile acid levels in the brain, improved neurologic function, and an approximately 50% reduction in infarct size 2 and 7 days after reperfusion. In addition, TUDCA significantly reduced the number of TUNEL-positive brain cells, mitochondrial swelling, and partially inhibited caspase-3 processing and substrate cleavage. These findings suggest that the mechanism for in vivo neuroprotection by TUDCA is, in part, mediated by inhibition of mitochondrial perturbation and subsequent caspase activation leading to apoptotic cell death. Thus, TUDCA, a clinically safe molecule, may be useful in the treatment of stroke and possibly other apoptosis-associated acute and chronic injuries to the brain.

Neural differentiation and incorporation of bone marrow-derived multipotent adult progenitor cells after single cell transplantation into blastocyst stage mouse embryos.

Previously we reported the characterization of multipotent adult progenitor cells (MAPCs) isolated from the bone marrow of rodents. In that study, single murine MAPCs derived from ROSA-26, β-galactosidase (β-Gal)-positive transgenic mice were injected into E3.5 C57/Bl6 mouse blastocysts. The resultant chimeric blastocysts were then implanted into pseudopregnant females and were allowed to develop naturally through birth and into adulthood. Chimeric mice were sacrificed 6 to 20 weeks after birth, and were processed for histological analysis. β-Galactosidase activity was identified in all organs and tissues examined, and tissuespecific differentiation and engraftment was confirmed by colabeling with antibodies that recognize β-Gal and tissue-specific markers. In the present study we have examined neural engraftment derived from the clonal expansion of a single MAPC during rodent development, and characterized the neural phenotype of MAPCs in the resultant chimeric animals. Donor cell-derived β-Gal activity was evident throughout the brain. Double and triple immunofluorescent labeling studies revealed MAPC-derived neurons (NeuN/β-Gal) and astrocytes (GFAP/β-Gal) in the cortex, striatum, medial septal nucleus, hippocampus, cerebellum, substantia nigra, and thalamus. More specifically, donor-derived neurons contributed to each of the cellular layers of the cortex; the pyramidal and granule cell layers, as well as the hilus, of the hippocampus; Purkinje and granule cell layers in the cerebellum; and GABAergic cells in the caudate and putamen. This study haracterizes the potential for MAPCs to differentiate into specific neuronal and glial phenotypes, and to integrate normally during development, after implantation into blastocysts, and provides additional evidence that MAPCs exhibit properties similar to embryonic stem cells.