After intravascular delivery of genetically marked adult mouse bone marrow into lethally irradiated normal adult hosts, donor-derived cells expressing neuronal proteins (neuronal phenotypes) developed in the central nervous system. Flow cytometry revealed a population of donor-derived cells in the brain with characteristics distinct from bone marrow. Confocal microscopy of individual cells showed that hundreds of marrow-derived cells in brain sections expressed gene products typical of neurons (NeuN, 200-kilodalton neurofilament, and class III beta-tubulin) and were able to activate the transcription factor cAMP response element-binding protein (CREB). The generation of neuronal phenotypes in the adult brain 1 to 6 months after an adult bone marrow transplant demonstrates a remarkable plasticity of adult tissues with potential clinical applications.

specific adult stem cell may need to be expanded. Accordingly, adult stem cells may not only act locally in the tissues in which they reside, but may also be recruited out of the circulation and enlisted in regeneration of diverse tissues at distal sites. Taken to an extreme,

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 (sexmatched, control) bone marrow transplants. Cerebella were evaluated in 10-m-thick, formaldehyde-fixed, paraffin-embedded sections that encompassed up to Ϸ50% 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.stem cell ͉ plasticity ͉ cell fusion ͉ cell fate change I n humans, bone marrow has been reported to contribute to human epithelium and liver, but not to the brain (1, 2). Here we investigated whether bone marrow-derived cells could cross the blood-brain barrier and contribute to neurons in the CNS. Previous studies in mice have shown that bone marrow-derived cells can contribute to neuronal cell types in the CNS, including a class of highly specialized neurons in the brain, the Purkinje neurons (3-5).Purkinje neurons are generated only during early brain development. In humans, generation of Purkinje neurons starts at 16 weeks of gestation and is complete by the end of the 23rd week (6). Most of the maturation of the characteristic dendritic trees of human Purkinje neurons is finalized during the first year of life (7). By contrast to other neurons in the adult brain, there is no evidence for the generation of new Purkinje neurons after birth, even in cases of severe Purkinje cell loss caused by trauma or genetic disease (8, 9).The human brain contains Ϸ15 million Purkinje cells, which are among the largest neurons in the CNS (10). A typical Purkinje neuron has Ͼ50-fold the volume of neighboring neurons in the brain, and its complex dendritic extensions receive inputs from as many as one million granule cells. Purkinje cells play vital roles in maintaining balance and regulating movement. A loss o...

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.

The enhanced green fluorescent protein (GFP) reporter has been widely adopted for tracking cell lineage. Here, we compare three transgenic mouse strains in which GFP is considered "ubiquitously expressed," with the GFP transgene under control of the chicken ␤-actin (CBA) or human ubiquitin C (UBC) promoter. We compared the expression of GFP using flow cytometry, direct tissue fluorescence, and immunostaining with multiple commercially available anti-GFP antibodies. Mice of CBA-GFP strain 1Osb have strong but variegated expression of GFP in adult liver, kidney, small intestine, and blood. Mice of CBA-GFP strain Y01 have the highest proportion of GFP-positive peripheral blood cells yet limited GFP expression in liver, intestine, and kidney. UBC-GFP mice express GFP only weakly in solid organs and variably in blood. Direct fluorescent detection of GFP in formalin-fixed, paraffin-embedded tissue sections was the simplest approach, but it was useful only in high-expressing strains and potentially subject to artifact because of tissue autofluorescence. Immunofluorescence using either primary goat or primary rabbit antibodies was much more sensitive and allowed better discrimination of authentic signal from autofluorescence. Immunohistochemical staining was less sensitive than direct fluorescence or immunofluorescence and was subject to false-positive signal in the small intestine. In conclusion, there is considerable variability of expression within and between GFP transgenic strains. None of the tested strains gave truly ubiquitous GFP expression. A detailed analysis of GFP expression in one's tissues of interest must guide the choice of reporter mouse strain when GFP is used as a marker of cell lineage or donor origin.

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. Stem 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.

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.