Human adult bone marrow-derived somatic cell therapy results in functional recovery and axonal plasticity following stroke in the rat.

Stroke is the leading cause of adult disability in the United States. To date there is no satisfactory treatment for stroke once neuronal damage has occurred. Human adult bone marrow-derived somatic cells (hABM-SC) represent a homogenous population of CD49c/CD90 co-positive, non-hematopoietic cells that have been shown to secrete therapeutically relevant trophic factors and to support axonal growth in a rodent model of spinal cord injury. Here we demonstrate that treatment with hABM-SC after ischemic stroke in adult rats results in recovery of forelimb function on a skilled motor test, and that this recovery is positively correlated with increased axonal outgrowth of the intact, uninjured corticorubral tract. While the complete mechanism of repair is still unclear, we conclude that enhancement of structural neuroplasticity from uninjured brain areas is one mechanism by which hABM-SC treatment after stroke leads to functional recovery.

Skeletal abnormalities and ultrastructural changes of cartilage in transgenic mice expressing a collagen II gene (COL2A1) with a Cys for Arg-alpha1-519 substitution.

OBJECTIVE: To examine the mechanism by which the Arg-->Cys 519 mutation causes the clinical phenotype employing transgenic mice that express the mutated human COL2A1. METHODS: A DNA construct under the control of a COL2A1 specific promoter was prepared from genomic DNA isolated from fibroblasts from the proband with primary generalized osteoarthritis (OA) associated with a mild chondrodysplasia. Transgenic mice were obtained by injection of the constructs into pro-nuclei of fertilized eggs from the FVB/N inbred mouse strain. Transgenic mice harboring two alleles of the mutated human COL2A1 were examined for morphological abnormalities and for alterations of their skeletal development. Ultrastructural examination was performed to identify changes in the organization and density of collagen II fibrils in articular cartilage of the transgenic mice. RESULTS: Transgenic mice harboring two alleles of the mutated human collagen gene were smaller than their normal littermates, had a cleft palate, and disorganized growth plate. Electron microscopy of articular cartilage showed a decreased density of collagen II fibrils and revealed chondrocytes with dilated Golgi cysternae. CONCLUSIONS: Expression of a COL2A1 with an Arg-->Cys 519 substitution in transgenic mice causes retardation of skeletal development and ultrastructural alterations in articular cartilage with a profound reduction of the density of the collagen II fibrils in the tissue. These alterations may be responsible for the phenotype of precocious generalized OA and chondrodysplasia displayed by patients harboring this COL2A1 mutation.

MicroSAGE analysis of 2,353 expressed genes in a single cell-derived colony of undifferentiated human mesenchymal stem cells reveals mRNAs of multiple cell lineages.

Mesenchymal stem cells (MSCs) isolated from the bone marrow of adult organisms are capable of differentiating into adipocytes, chondrocytes, myoblasts, osteoblasts, and hematopoiesis-supporting stroma. We recently demonstrated that MSCs also adopt glial cell fates when transplanted into the developing central nervous system and hence can produce tissue elements derived from a separate embryonic layer. Despite these remarkable properties, it has been difficult to establish specific criteria to characterize MSCs. Using a modified protocol for micro-serial analysis of gene expression, we cataloged 2,353 unique genes expressed by a single cell-derived colony of undifferentiated human MSCs. This analysis revealed that the MSC colony simultaneously expressed transcripts characteristic of various mesenchymal cell lineages including chondrocytes, myoblasts, osteoblasts, and hematopoiesis-supporting stroma. Therefore, the profile of expressed transcripts reflects the developmental potential of the cells. Additionally, the MSC colony expressed mRNAs characteristic of endothelial, epithelial and neuronal cell lineages, a combination that provides a unique molecular signature for the cells. Other expressed transcripts included various products involved in wound repair as well as several neurotrophic factors. A total of 268 novel transcripts were also identified, one of which is the most abundantly expressed mRNA in MSCs. This study represents the first extensive gene expression analysis of MSCs and as such reveals new insight into the biology, ontogeny, and in vivo function of the cells.

Transgenic mice with inactive alleles for procollagen N-proteinase (ADAMTS-2) develop fragile skin and male sterility.

Transgenic mice were prepared with inactive alleles for procollagen N-proteinase (ADAMTS-2; where ADAMTS stands for a disintegrin and metalloproteinase with thrombospondin repeats). Homozygous mice were grossly normal at birth, but after 1-2 months they developed thin skin that tore after gentle handling. Although the gene was inactivated, a large fraction of the N-propeptides of type I procollagen in skin and the N-propeptides of type II procollagen in cartilage were cleaved. Therefore the results suggested the tissues contained one or more additional enzymes that slowly process the proteins. Electron microscopy did not reveal any defects in the morphology of collagen fibrils in newborn mice. However, in two-month-old mice, the collagen fibrils in skin were seen as bizarre curls in cross-section and the mean diameters of the fibrils were approx. half of the controls. Although a portion of the N-propeptides of type II procollagen in cartilage were not cleaved, no defects in the morphology of the fibrils were seen by electron microscopy or by polarized-light microscopy. Female homozygous mice were fertile, but male mice were sterile with a marked decrease in testicular sperm. Therefore the results indicated that ADAMTS-2 plays an essential role in the maturation of spermatogonia.

Potential use of stem cells from bone marrow to repair the extracellular matrix and the central nervous system.

A subset of stem-like cells from bone marrow that are referred to as marrow stromal cells (MSCs) have been shown to be capable of differentiating into osteoblasts, chondrocytes, adipocytes, myocytes, astrocytes and perhaps neurons. Recently, conditions have been developed where human MSCs can be expanded almost without limit in culture without apparently losing their multipotentiality for differentiation. The cells appear to be potentially useful for the repair of extracellular matrix and the central nervous system.

Donor variation in the growth properties and osteogenic potential of human marrow stromal cells.

Human marrow stromal cells (MSCs) were isolated from posterior illiac crest marrow aspirates obtained from 17 healthy donors, ages 19-45 years, with no apparent physical disability. First passage hMSCs exhibited growth rates in vitro that varied up to 12-fold between donors. No correlation between growth rate and the age or gender of the donor was evident (P

Marrow stromal cells migrate throughout forebrain and cerebellum, and they differentiate into astrocytes after injection into neonatal mouse brains.

Stem cells are a valuable resource for treating disease, but limited access to stem cells from tissues such as brain restricts their utility. Here, we injected marrow stromal cells (MSCs) into the lateral ventricle of neonatal mice and asked whether these multipotential mesenchymal progenitors from bone marrow can adopt neural cell fates when exposed to the brain microenvironment. By 12 days postinjection, MSCs migrated throughout the forebrain and cerebellum without disruption to the host brain architecture. Some MSCs within the striatum and the molecular layer of the hippocampus expressed glial fibrillary acidic protein and, therefore, differentiated into mature astrocytes. MSCs also populated neuron rich regions including the Islands of Calleja, the olfactory bulb, and the internal granular layer of the cerebellum. A large number of MSCs also were found within the external granular layer of the cerebellum. In addition, neurofilament positive donor cells were found within the reticular formation of the brain stem, suggesting that MSCs also may have differentiated into neurons. Therefore, MSCs are capable of producing differentiated progeny of a different dermal origin after implantation into neonatal mouse brains. These results suggest that MSCs are potentially useful as vectors for treating a variety of central nervous system disorders.

Enhanced in situ detection of beta-glucuronidase activity in murine tissue.

We outline here a protocol for high-resolution in situ localization of beta-glucuronidase in murine tissues processed in glycol methacrylate (GMA). Murine tissues were first stained with 5-bromo-4-chloro-3-indolyl-beta-d-glucuronic acid (x-gluc), followed by histological processing in GMA. Retention of the blue indigo reaction product after overnight incubations in x-gluc allowed high-resolution localization of beta-glucuronidase activity by brightfield microscopy. When illuminated under darkfield, the x-gluc signal was enhanced, permitting detection even in cells with low-level enzyme activity. This technique offers for the first time a more sensitive enzyme histochemical method of detecting beta-glucuronidase activity in animal tissues and also the opportunity to examine expression at high magni-fication.

Plastic adherent stromal cells from the bone marrow of commonly used strains of inbred mice: variations in yield, growth, and differentiation.

Bone marrow stroma contains a unique cell population, referred to as marrow stromal cells (MSCs), capable of differentiating along multiple mesenchymal cell lineages. A standard liquid culture system has been developed to isolate MSCs from whole marrow by their adherence to plastic wherein the cells grow as clonal populations derived from a single precursor termed the colony-forming-unit fibroblast (CFU-F). Using this liquid culture system, we demonstrate that the relative abundance of MSCs in the bone marrow of five commonly used inbred strains of mice varies as much as 10-fold, and that the cells also exhibit markedly disparate levels of alkaline phosphatase expression, an early marker of osteoblast differentiation. For each strain examined, the method of isolating MSCs by plastic adherence yields a heterogeneous cell population. These plastic adherent cells also exhibit widely varying growth kinetics between the different strains. Importantly, of three inbred strains commonly used to prepare transgenic mice that we examined, only cells derived from FVB/N marrow readily expand in culture. Further analysis of cultures derived from FVB/N marrow showed that most plastic adherent cells express CD11b and CD45, epitopes of lymphohematopoietic cells. The later consists of both pre-B-cell progenitors, granulocytic and monocytic precursors, and macrophages. However, a subpopulation of the MSCs appear to represent bona fide mesenchymal progenitors, as cells can be induced to differentiate into osteoblasts and adipocytes after exposure to dexamethasone and into myoblasts after exposure to amphotericin B. Our results point to significant strain differences in the properties of MSCs and indicate that standard methods cannot be applied to murine bone marrow to isolate relatively pure populations of MSCs.

A 1,064 bp fragment from the promoter region of the Col11a2 gene drives lacZ expression not only in cartilage but also in osteoblasts adjacent to regions undergoing both endochondral and intramembranous ossification in mouse embryos.

We isolated a 1,064 bp promoter fragment that extended from the 3'-end of the adjacent gene for retinoic X receptor-beta to beyond the most clearly defined start site of the mouse Col11a2 gene. The fragment was then joined to a beta-galactosidase gene and used to prepare transgenic mice. Three independent lines of transgenic mice were generated. The reporter beta-galactosidase gene was expressed in essentially all cartilaginous tissues in 15.5-day-old mouse embryos. In addition, the construct was expressed in osteoprogenitors within developing periosteum and in osteoblasts within mineralized bone. This pattern of expression was evident during both endochondral and intramembranous bone formation. Therefore, the results suggest that 1,064 bp promoter fragment can drive tissue-specific expression of the Col11a2 gene.

Localization of transglutaminase in hippocampal neurons: implications for Alzheimer's disease.

The purpose of this investigation was to identify and localize tissue transglutaminase (TGase) within neurons from the hippocampi of normal aged individuals and of those with confirmed Alzheimer's disease (AD). This enzyme may be a factor in the molecular mechanisms of neurodegeneration and formation of insoluble macromolecular complexes found in the neurons of normal aged and AD brain tissue. An antibody made to the extracellular TGase, coagulation factor XIIIa, was found to be specific for purified intracellular guinea pig liver tissue TGase. The specificity for liver tissue TGase has enabled us to identify tissue TGase(s) within rat hippocampal neurons and within neurons from normal aged and AD hippocampal tissues. Degenerating neurons from the AD hippocampus, compared to neurons from the normal aged hippocampus, exhibited increased immunoreactivity for TGase and demonstrated co-labeling for PHF1 and anti-TGase. Our results suggest that TGase may be associated with the neurofibrillary degeneration observed in AD, thereby implicating TGase as a potential factor in the pathogenesis of Alzheimer's disease.

Sequence variation in the src gene product affects metastasis formation: the central, but not exclusive, role of the tumor immune response.

Sequence variation in the src gene product could, in principle, influence metastasis formation through either of 2 effects: an alteration in tumor antigenicity or a non-immune-mediated change in one or more src-associated functions. Our present results establish that both mechanisms underlie the difference in relative levels of metastasis formation induced by the v-src vs. the c-src(527) oncogene. A point that emerges from this analysis is the segregation, within a chicken line genotypically uniform at the major histocompatibility (B) complex (MHC), of a phenotype defined by strong resistance to secondary v-src-induced tumor challenge. The pattern of segregation is consonant with the possibility that a gene unlinked to the MHC governs immune response levels to v-src-encoded tumor antigen.

Endogenous c-src as a determinant of the tumorigenicity of src oncogenes.

We have compared the tumorigenicity of two src oncogenes, v-src and c-src(527), whose respective protein products pp60v-src and pp60c-src(527) show a different spectrum of amino acid substitutions vis-à-vis the c-src protooncogene-encoded product pp60c-src. Whereas the extent of primary tumor growth induced by c-src(527) was quite similar in the two chicken lines tested, the extent of v-src-induced tumor growth showed a marked line dependence. As examined with a line of chickens that shows immune-mediated regression of v-src-induced tumors, a weaker tumor immunity, as correlated with a greater level of primary tumor growth, resulted from inoculation of c-src(527) DNA than of v-src DNA. These observations indicated that the v-src-specific amino acid substitutions define a major tumor antigenicity. That a separate src-associated antigenicity is also targetable by the tumor immune response followed from the finding that the level of protective immunity against the growth of c-src(527) DNA-induced tumors was augmented under conditions of the prior regression of v-src DNA-induced tumors. As this latter antigenicity may include one or more c-src(527)-encoded peptides that are equivalent to c-src-encoded self peptides, these observations suggest that a host tolerance to pp60c-src can be broken so as to permit a tumor immune response based on recognition of self peptides of pp60c-src(527).

Major histocompatibility (B) complex control of the formation of v-src-induced metastases.

Previous observations have shown that the major histocompatibility (B) complex is a determinant of the growth of v-src-induced primary tumors. In the present study, we have observed with two chicken lines congenic for B complex alleles that the control of v-src-mediated oncogenesis by the B complex extends to metastasis formation. In addition, our results show that the differences in metastasis frequencies between these two lines are correlated with the relative strengths of their respective tumor immune responses.

Tumor cells induced by the v-src oncogene are heterogeneous for expression of markers of mesenchyme differentiation.

The observation that v-src-induced tumors contain tumor cells of differing morphology, notably fibroblastoid or polygonal, raised the question as to whether the tumor cells are also heterogeneous with respect to expression of markers of cellular differentiation. Of the markers tested here, consistent reactivity for tumor tissue was noted only for antibody probes reactive to muscle actin (HHF35, alpha sm-1) or to procollagen type I (SP1. D8); for any given tumor, whether induced by v-src DNA or by Rous sarcoma virus, each of these markers was found only in a subpopulation of tumor cells. The observation of marker heterogeneity in the one v-src DNA-induced tumor examined here that typed as monoclonal suggests that v-src-induced transformation is consonant with a degree of plasticity in the phenotypes of the clonal progeny of a single transformant.