Contribution of hematopoietic stem cells to skeletal muscle.

Cells from adult bone marrow participate in the regeneration of damaged skeletal myofibers. However, the relationship of these cells with the various hematopoietic and nonhematopoietic cell types found in bone marrow is still unclear. Here we show that the progeny of a single cell can both reconstitute the hematopoietic system and contribute to muscle regeneration. Integration of bone marrow cells into myofibers occurs spontaneously at low frequency and increases with muscle damage. Thus, classically defined single hematopoietic stem cells can give rise to both blood and muscle.

Robust in vivo transduction of nervous system and neural stem cells by early gestational intra amniotic gene transfer using lentiviral vector.

Presently, in vivo methods to efficiently and broadly transduce all major cell types throughout both the central (CNS) and peripheral adult nervous system (PNS) are lacking. In this study, we hypothesized that during early fetal development neural cell populations, including neural stem cells (NSCs), may be accessible for gene transfer via the open neural groove. To test this hypothesis, we injected lentiviral vectors encoding a green fluorescent protein (GFP) marker gene into the murine amniotic cavity at embryonic day 8. This method (i) efficiently and stably transduced the entire nervous system for at least 80% of the lifespan of the mice, (ii) transduced all major neural cell types, and (iii) transduced adult NSCs of the subventricular zone (SVZ) and subgranular zones (SGZs). This simple approach has broad applications for the study of gene function in nervous system development and adult NSCs and may have future clinical applications for treatment of genetic disorders of the nervous system.

Weight gain after lung transplantation.

BACKGROUND: Weight gain is frequently observed after lung transplantation, but the magnitude, predictors and implications of weight gain after lung transplant are unknown. METHODS: This retrospective cohort study included 826 lung transplant recipients randomly selected from 12 international transplant centers. We included adult patients with available weight data at baseline and 1 year post-transplant. We examined demographic and clinical predictors of first year weight gain using a multiple linear regression model (n = 579) with percent weight change as the dependent variable. To study the association between first year weight gain and subsequent survival, we performed a Cox proportional hazards analysis. p < 0.05 was considered statistically significant. RESULTS: The median weight change was 10% (range -32% to 84%). On multi-variate analysis, increasing age and prolonged mechanical ventilation were inversely associated with weight gain; obstructive disease, interstitial disease and increasing ischemic time were positively associated with weight gain. Increasing baseline weight was negatively associated with weight gain in patients with obstructive and interstitial disease. The model accounted for 14% of the variance in weight gain. Patients with weight gain above the median had better subsequent survival (adjusted hazard ratio 0.61, 95% confidence interval 0.41 to 0.90). Infection was a more common cause of death in these patients, whereas malignant deaths were more frequent in patients with below-median weight gain. CONCLUSIONS: Substantial weight gain occurs in the first year after lung transplantation. The predictors of weight gain may be used to target high-risk patients for early intervention. Higher weight gain is associated with better subsequent survival.

Optimizing techniques for tracking transplanted stem cells in vivo.

The potential for bone marrow-derived cells (BMDCs) to contribute to nonhematopoietic tissues has generated considerable debate in recent years. Causes for the controversies include disparities in the techniques used to track engraftment of BMDCs, inappropriate tissue preparation, a lack of appropriate positive and negative controls, and basic misunderstandings about how to properly collect and interpret images from epifluorescent and confocal microscopes. Our laboratory was among the first to use bone marrow transplants from transgenic mice constitutively expressing enhanced green fluorescent protein (GFP) to study the ability of BMDCs to give rise to nonhematopoietic tissue types, a system that is now in widespread use. During our 6 years of experience using GFP, as well as beta-galactosidase and the Y chromosome, to track BMDCs in vivo, we have identified many difficulties and have developed techniques to resolve them. We discuss several of these methods, and, in particular, we describe ratiometric analysis techniques for improving detection of transplanted cells derived from genetically modified bone marrow. Finally, to help resolve reported discrepancies regarding the frequency with which BMDCs contribute to skeletal myofibers, we demonstrate that the pattern of highly autofluorescent myofibers in skeletal muscle is clearly distinct from that of GFP-expressing myofibers and describe how unambiguous conclusions can be drawn from such data.

Significant differences among skeletal muscles in the incorporation of bone marrow-derived cells.

While numerous reports indicate that adult bone marrow-derived cells can contribute to nonhematopoietic tissues in vivo in adult mice, the generally low frequency of these events has made it difficult to study the molecular and cellular pathways involved. Here, we show a 1000-fold range in the frequency with which diverse skeletal muscles incorporate adult bone marrow-derived cells in adult mice. Most striking was the finding of one specific muscle, the panniculus carnosus, in which up to 5% of myofibers incorporated bone marrow-derived cells over a 16- month period in the absence of experimentally induced selective pressure. These results suggest that muscles differ physiologically, establishing the panniculus carnosus as an assay for identifying the key regulators, such as trophic, homing, and differentiation factors, as well as the relevant cells within the bone marrow that are capable of circulating throughout the periphery and contributing to adult, nonhematopoietic tissues, such as skeletal muscle. Finally, the 5% incorporation of adult stem cells into skeletal muscle is the highest reported to date in the absence of experimentally induced selective pressure and is at a level that may be consistent with improving the function of defective muscle tissue.

Contribution of transplanted bone marrow cells to Purkinje neurons in human adult brains.

We show here that cells within human adult bone marrow can contribute to cells in the adult human brain. Cerebellar tissues from female patients with hematologic malignancies, who had received chemotherapy, radiation, and a bone marrow transplant, were analyzed. Brain samples were obtained at autopsy from female patients who received male (sex-mismatched) or female (sex-matched, control) bone marrow transplants. Cerebella were evaluated in 10-microm-thick, formaldehyde-fixed, paraffin-embedded sections that encompassed up to approximately 50% of a human Purkinje nucleus. A total of 5,860 Purkinje cells from sex-mismatched females and 3,202 Purkinje cells from sex-matched females were screened for Y chromosomes by epifluorescence. Confocal laser scanning microscopy allowed definitive identification of the sex chromosomes within the morphologically distinct Purkinje cells. In the brains of females who received male bone marrow, four Purkinje neurons were found that contained an X and a Y chromosome and two other Purkinje neurons contained more than a diploid number of sex chromosomes. No Y chromosomes were detected in the brains of sex-matched controls. The total frequency of male bone marrow contribution to female Purkinje cells approximated 0.1%. This study demonstrates that although during human development Purkinje neurons are no longer generated after birth, cells within the bone marrow can contribute to these CNS neurons even in adulthood. The underlying mechanism may be caused either by generation de novo of Purkinje neurons from bone marrow-derived cells or by fusion of marrow-derived cells with existing recipient Purkinje neurons.

Localized arteriole formation directly adjacent to the site of VEGF-induced angiogenesis in muscle.

We have shown previously that implantation of myoblasts constitutively expressing the VEGF-A gene into nonischemic mouse skeletal muscle leads to overgrowth of capillary-like blood vessels and hemangioma formation. These aberrant effects occurred directly at the implantation site. We show here that these regions result from angiogenic capillary growth and involve a change in capillary growth pattern and that smooth muscle-coated vessels similar to arterioles form directly adjacent to the implantation site. Myoblasts genetically engineered to produce VEGF were implanted into mouse leg muscles. Implantation sites were surrounded by a zone of dense capillary-sized vessels, around which was a second zone of muscle containing larger, smooth-muscle-covered vessels but few capillaries, and an outer zone of muscle exhibiting normal capillary density. The lack of capillaries in the middle region suggests that the preexisting capillaries adjacent to the implantation site underwent enlargement and/or fusion and recruited a smooth muscle coat. Capillaries at the implantation site were frequently wrapped around VEGF-producing muscle fibers and were continuous with the circulation and were not observed to include bone-marrow-derived endothelial cells. In contrast with the distant arteriogenesis resulting from VEGF delivery described in previous studies, we report here that highly localized arterioles also form adjacent to the site of delivery.