We report that prospectively isolated, human CNS stem cells grown as neurospheres (hCNS-SCns) survive, migrate, and express differentiation markers for neurons and oligodendrocytes after longterm engraftment in spinal cord-injured NOD-scid mice. hCNS-SCns engraftment was associated with locomotor recovery, an observation that was abolished by selective ablation of engrafted cells by diphtheria toxin. Remyelination by hCNS-SCns was found in both the spinal cord injury NOD-scid model and myelin-deficient shiverer mice. Moreover, electron microscopic evidence consistent with synapse formation between hCNS-SCns and mouse host neurons was observed. Glial fibrillary acidic protein-positive astrocytic differentiation was rare, and hCNS-SCns did not appear to contribute to the scar. These data suggest that hCNS-SCns may possess therapeutic potential for CNS injury and disease.behavioral assessment ͉ differentiation ͉ stem cell transplantation R ecent studies have used a variety of immortalized, engineered, or isolated rodent-derived precursor͞stem cells transplanted into rodent models of spinal cord injury. Many of these studies focused on cell survival and did not address differentiation, functional recovery, or the causal relationship between successful engraftment and observed behavioral improvements. When differentiation was investigated, embryonic and adult neural stem cells were reported to principally assume glial fibrillary acidic protein (GFAP)-positive astrocytic phenotypes after grafting into nonneurogenic regions of uninjured adult CNS (1, 2) or injured spinal cord (3-5). Furthermore, although in vitro predifferentiation paradigms designed to generate neural lineage restricted precursors successfully generated ␤-tubulin III (Tuj-1)-positive neuronal phenotypes either in vitro or after transplantation into uninjured spinal cord, this commitment was overridden by environmental cues in the injured spinal cord (6).Transplants of human brain-derived stem cells or human spinal cord tissue into injured rat spinal cord have been described (7-9). Moreover, several human cell transplantation paradigms recently have been reported to promote locomotor recovery: human umbilical cell infusion in a rat spinal cord injury model, although only within 3 weeks or less postgrafting (10); neurons differentiated in vitro under retinoic acid from human embryonal teratocarcinoma cells and transplanted into a rat spinal cord injury model (11); human ES cells differentiated in vitro to oligoprogenitors and transplanted into a rat spinal cord injury model (12); and human neural stem͞progenitor cells transplanted into a monkey spinal cord injury model (13). In general, these studies lack some or all of the following: definitive identification of transplanted cells, longterm survival and engraftment data, evidence of differentiation, and͞or direct evidence of functional integration of human cells in the injured spinal cord. The current study addresses three previously unexplored issues in stem cell transplantation research for spinal co...

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Of the eight nuclear genes in the plant multi‐gene family which encodes the small subunit (rbcS) of Petunia (Mitchell) ribulose bisphosphate carboxylase, one rbcS gene accounts for 47% of the total rbcS gene expression in petunia leaf tissue. Expression of each of five other rbcS genes is detected at levels between 2 and 23% of the total rbcS expression in leaf tissue, while expression of the remaining two rbcS genes is not detected. There is considerable variation (500‐fold) in the levels of total rbcS mRNA in six organs of petunia (leaves, sepals, petals, stems, roots and stigmas/anthers). One gene, SSU301, showed the highest levels of steady‐state mRNA in each of the organs examined. We discuss the differences in the steady‐state mRNA levels of the individual rbcS genes in relation to their gene structure, nucleotide sequence and genomic linkage.

Direct isolation of human central nervous system stem cells (CNS‐SC) based on cell surface markers yields a highly purified stem cell population that can extensively expand in vitro and exhibit multilineage differentiation potential both in vitro and in vivo. The CNS‐SC were isolated from fetal brain tissue using the cell surface markers CD133+, CD34–, CD45–, and CD24–/lo (CD133+ cells). Fluorescence‐activated cell sorted (FACS) CD133+ cells continue to expand exponentially as neurospheres while retaining multipotential differentiation capacity for >10 passages. CD133–, CD34–, and CD45– sorted cells (∼95% of total fetal brain tissue) fail to initiate neurospheres. Neurosphere cells transplanted into neonatal immunodeficient NOD‐SCID mice proliferated, migrated, and differentiated in a site‐specific manner. However, it has been difficult to evaluate human cell engraftment, because many of the available monoclonal antibodies against neural cells (β‐tubulin III and glial fibrillary acidic protein) are not species specific. To trace the progeny of human cells after transplantation, CD133+‐derived neurosphere cells were transduced with lentiviral vectors containing enhanced green fluorescent protein (eGFP) expressed downstream of the phosphoglycerate kinase promoter. After transduction, GFP+ cells were enriched by FACS, expanded, and transplanted into the lateral ventricular space of neonatal immunodeficient NOD‐SCID brain. The progeny of transplanted cells were detected by either GFP fluorescence or antibody against GFP. GFP+ cells were present in the subventricular zone‐rostral migrating stream, olfactory bulb, and hippocampus as well as nonneurogenic sites, such as cerebellum, cerebral cortex, and striatum. Antibody against GFP revealed that some of the cells displayed differentiating dendrites and processes with neurons or glia cells. Thus, marking human CNS‐SC with reporter genes introduced by lentiviral vectors is a useful tool with which to characterize migration and differentiation of human cells in this mouse transplantation model. © 2002 Wiley‐Liss, Inc.

The pelB and pelE genes from Erwinia chrysanthemi EC16, which encode different pectate lyase enzymes, were sequenced and expressed at a high level in Escherichia coli. The genes possessed little similarity to each other in 5' signal regions, signal peptide sequences, coding sequences, or 3' noncoding regions. Both genes contained their own promoters as well as sequences 3' to the coding regions with considerable secondary structure which may function as rho-independent transcriptional termination signals. High-level expression plasmids were constructed with both genes, which led to 20% or more of E. coli cellular protein. The pectate lyases were secreted efficiently to the periplasm and, to ra lesser extent, the culture medium. The mature proteins in E. coli periplasmic fractions were obtained in milligram amounts and high purity with a single-column affinity purification method. E. coli cells which produced high amounts of the pelE protein macerated potato tuber tissue as efficiently as E. chrysanthemi EC16 cells but cells producing high amounts of the pelB protein were less effective. Thus, the pelE gene product is an important pathogenicity factor which solely enables E. coli to cause a soft-rot disease on potato tuber tissue under laboratory conditions. We cloned genes coding for two different pectate lyase (EC 4.2.2.2) enzymes from the phytopathogenic bacterium Erwinia chrysanthemi EC16 (13) and observed their expression in Escherichia coli. Pectate lyases have previously been shown to account largely or entirely for the maceration or soft rotting of plant tissue caused by Erwinia spp. (4). Confirming this, E. coli cells containing the cloned pectate lyase genes macerated plant tissue, albeit less efficiently than E. chrysanthemi (13). Several groups subsequently cloned similar genes from other strains of E. chrysanthemi (5,14,28,34) and the related bacterium Erwinia carotovora (18,29, 40). The genes that we cloned did not cross-hybridize (13), but coded for enzymes with similar physical properties (molecular weights of ca. 40,000 and isoelectric points of 8.8 and 9.8) which both catalyzed the random eliminative cleavage of sodium polypectate. These enzymes were efficiently secreted to the periplasm and, to a lesser extent, the culture medium of E. coli. For reasons discussed below, the cloned DNA fragments appear to contain pelB and pelE, described by others, and the mature proteins which they encode are PLb and PLe, respectively. Plasmids containing our pel genes were named pPL in the previous paper (13), but this designation was found to be already entered in the Plasmid Reference Center (17). Accordingly, our pel gene plasmid constructs have been renamed pPEL, a designation that we have registered in the Plasmid Reference Center (17).The cloned pelB and pelE genes were both regulated by catabolite repression in E. coli (13) but were not induced by sodium polypectate, as occurs in E. chrysanthemi (4). Due to their pathogenic importance in diseases caused by Erwinia spp. and to their regulation propertie...

We have analyzed the polyadenylation sites for the small subunit of ribulose bisphosphate carboxylase and chlorophyll a/b binding protein genes of Petunia (Mitchell) and the bronze gene of Zea mays. Sequence analysis of multiple cDNA clones revealed that polyadenylation of the transcripts occurred at either 2 or 3 sites for all three groups of genes. In the examples where 3 polyadenylation sites were detected, the middle site was the one predominantly used. Putative polyadenylation signals preceding the poly A tails diverged significantly from the animal consensus sequence AATAAA. In all the genes examined the first A residue in the poly A tail of the cDNA clones corresponded to an A residue in the homologous genomic sequence.

Shiverer-immunodeficient (Shi-id) mice demonstrate defective myelination in the central nervous system (CNS) and significant ataxia by 2 to 3 weeks of life. Expanded, banked human neural stem cells (HuCNS-SCs) were transplanted into three sites in the brains of neonatal or juvenile Shi-id mice, which were asymptomatic or showed advanced hypomyelination, respectively. In both groups of mice, HuCNS-SCs engrafted and underwent preferential differentiation into oligodendrocytes. These oligodendrocytes generated compact myelin with normalized nodal organization, ultrastructure, and axon conduction velocities. Myelination was equivalent in neonatal and juvenile mice by quantitative histopathology and high-field ex vivo magnetic resonance imaging, which, through fractional anisotropy, revealed CNS myelination 5 to 7 weeks after HuCNS-SC transplantation. Transplanted HuCNS-SCs generated functional myelin in the CNS, even in animals with severe symptomatic hypomyelination, suggesting that this strategy may be useful for treating dysmyelinating diseases.

Infantile neuronal ceroid lipofuscinosis (INCL) is a fatal neurodegenerative disease caused by a deficiency in the lysosomal enzyme palmitoyl protein thioesterase-1 (PPT1). Ppt1 knockout mice display hallmarks of INCL and mimic the human pathology: accumulation of lipofuscin, degeneration of CNS neurons, and a shortened life span. Purified non-genetically modified human CNS stem cells, grown as neurospheres (hCNS-SCns), were transplanted into the brains of immunodeficient Ppt1(-/)(-) mice where they engrafted robustly, migrated extensively, and produced sufficient levels of PPT1 to alter host neuropathology. Grafted mice displayed reduced autofluorescent lipofuscin, significant neuroprotection of host hippocampal and cortical neurons, and delayed loss of motor coordination. Early intervention with cellular transplants of hCNS-SCns into the brains of INCL patients may supply a continuous and long-lasting source of the missing PPT1 and provide some therapeutic benefit through protection of endogenous neurons. These data provide the experimental basis for human clinical trials with these banked hCNS-SCns.