Timothy R. Brazelton

The Evolving Concept of a Stem Cell: Entity or Function?

We wish to thank A. Banfi, B. Colyear, and N. Gewertz for expert assistance with artwork. We are indebted to M. Baron, J. Botas, G. Cossu, E. Gussoni, Y.N. Jan, T. Palmer, T. Tlsty, B. Wold, and members of our laboratory for in-depth critique of the manuscript. We gratefully acknowledge review of the tissue-specific material by E. Fuchs, M. Kay, P. Khavari, A. Oro, and L. Reid. We apologize in advance to those whose work we were not able to cover due to space constraints. This work was supported by a Lutheran Fellowship and an NIH predoctoral training grant GM07149 to T.R.B. and by NIH grants CA59717, AG09521, HD 18179, and HL65572 to H.M.B.

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-μm-thick, formaldehyde-fixed, paraffin-embedded sections that encompassed up to ≈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.

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.

Molecular mechanisms of action of new xenobiotic immunosuppressive drugs: tacrolimus (FK506), sirolimus (rapamycin), mycophenolate mofetil and leflunomide

Among all the new immunosuppressive molecules being investigated either preclinically or clinically, four stand out: tacrolimus (FK506), sirolimus (rapamycin), mycophenolate mofetil and leflunomide (and its malononitriloamide analogs). Each drug has distinct mechanisms of immunosuppressive action, and in the past year significant advances have been made in our understanding of the actions of these drugs at the molecular and even atomic levels. Data from recent clinical trials demonstrate that these drugs very effectively suppress graft rejection or autoimmune diseases, validating the pivotal role played by each of their distinct molecular targets in the normal functioning of immune cells.

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.

The role of respiratory epithelium in a rat model of obliterative airway disease.

BACKGROUND The etiology and pathogenesis of obliterative bronchiolitis after lung transplantation remain to be fully elucidated. Using a rat model of heterotopically transplanted trachea grafts, we have examined the role airway epithelium plays in obliterative airway disease (OAD). METHODS Rat trachea isografts were denuded of epithelium by protease digestion. Grafts were inoculated either with or without native airway epithelial cells and transplanted into the omentum of recipient animals. RESULTS Airway epithelium removal resulted in OAD in denuded isogeneic trachea grafts. Reseeding of the denuded grafts with epithelial cells significantly reduced airway obliteration from 78% to 22% luminal occlusion. CONCLUSIONS Non-immune-mediated injury will cause OAD, and epithelial cell replacement in denuded isografts can significantly reduce the fibrotic progression of the disease.

Epithelial re-growth is associated with inhibition of obliterative airway disease in orthotopic tracheal allografts in non-immunosuppressed rats.

Background. Because epithelial cells are targets of alloimmune injury leading ultimately to airway obliteration, we tested whether epithelial re-growth could prevent obliterative airway disease (OAD) in orthotopic tracheal allografts. Methods. Brown Norway tracheal segments were orthotopically transplanted into nonimmunosuppressed Lewis rats. Allografts were removed on days 2–10 (n=13), 30 (n=4), and 60 (n=5) for histology, computerized morphometry (obliteration), and immunohistochemical detection of mononuclear cells, smooth muscle &agr;-actin, and tissue phenotype. Normal tracheas, host tracheas, and heterotopically transplanted allografts served as controls. Results. Orthotopic allografts removed on days 2–10 exhibited epithelial damage and re-growth and mononuclear cell infiltration. On days 30 and 60, partially ciliated cuboidal or attenuated epithelium completely covered the lumen. Although mononuclear cells declined, numerous T cells with a high CD4/CD8 ratio were found in the epithelium till day 60. Orthotopic allograft epithelium expressed donor phenotype on day 7, but recipient phenotype on days 30 and 60. Despite subepithelial &agr;-actin positive myofibroblast proliferation, obliteration did not progress from day 7 to 30 and 60 (35, 30, and 33%, respectively). Although more than in normal or host tracheas, the obliteration in orthotopic allografts on days 30 and 60 was significantly less (P <0.001) than in heterotopic allografts. Conclusions. We describe, for the first time, long-term patency of fully histoincompatible orthotopic tracheal allografts in nonimmunosuppressed rats. Despite acute alloimmune injury and induction of myofibroblast proliferation, epithelial re-growth from the host limited the progression of OAD, thus emphasizing the role of epithelium in the control of airway obliteration.

Obliterative airway disease after heterotopic tracheal xenotransplantation : Pathogenesis and prevention using new immunosuppressive agents

BACKGROUND The purpose of this study was to investigate whether obliterative bronchiolitis might occur after xenogenic pulmonary transplantation. A model for obliterative airway disease (OAD) after tracheal allograft transplantation in the rat undergoes tracheal obliteration with histologic features characteristic of obliterative bronchiolitis in human lung transplant recipients. Using this model, the pathogenesis of OAD and its prevention with immunosuppressive drugs was studied in rat recipients of hamster tracheal grafts. METHODS Tracheae from 30 hamsters (xenografts) or 23 Brown-Norway rats (allografts) were implanted and wrapped in the greater omentum of untreated Lewis rats. The grafts were removed on day 1, 3, 7, 14, 21, or 28 after transplantation and stained with hematoxylin and eosin and Massons trichrome and by immunohistochemistry and immunofluorescence (IFL) techniques. In addition, 25 recipients were treated with cyclosporine (CsA, 10 mg/kg p.o.), leflunomide (LFM, 20 mg/kg p.o.), or rapamycin (RPM, 6 mg/kg i.p.) for 14 or 21 days (5 animals per treatment group). Visual and morphometric analyses were used to evaluate the extent of airway obliteration, luminal coverage by respiratory or flattened cuboidal epithelium, and extent and density of peritracheal cellular inflammation. RESULTS In all xenografts, a neutrophilic infiltration of the mucosa and submucosa was observed from day 1 until day 14 and was associated with complete loss of tracheal epithelium by day 14. A marked peritracheal mononuclear cellular infiltrate mixed with plasma cells and eosinophils was seen on days 7 and 14. Both the extent of peritracheal inflammation and the density of the mononuclear cell infiltrate were significantly increased in xenograft tracheae when compared with the allografts. Tracheal obliteration began on day 14 and reached a maximum of 43% on day 21 with evidence of intraluminal fibrosis. In contrast to IFL of allografts, IFL of xenografts demonstrated marked deposition of rat immunoglobulin in the peritracheal tissue on days 7 and 14. The effects of treatment with immunosuppressive drugs on tracheal graft narrowing and protection of respiratory epithelium were as follows: After 14 days of treatment, the percentage of tracheal graft narrowing was 12%, 23%, and 19% in the no treatment, CsA, and LFM groups, respectively; the percentage of respiratory epithelium at 14 days was 0%, 21%, and 95%. After 21 days of treatment, the percentage of tracheal graft narrowing was 43%, 49%, 12%, and 5% for the no treatment, CsA, LFM, and RPM groups, respectively; the percentage of respiratory epithelium at 21 days was 0%, 39%, 86%, and 0%. Using computerized morphometry, the extent and densities of the peritracheal cellular infiltrates were significantly reduced in LFM- and CsA-treated groups when compared with untreated xenograft controls. LFM and RPM, but not CsA, significantly reduced the degree of luminal obliteration compared with no treatment (P<0.05). LFM and, to a lesser extent, CsA were able to prevent the loss of normal respiratory epithelium. Analysis by IFL revealed a marked decrease in rat immunoglobulin deposition in xenografts from LFM- and RPM-treated groups compared with xenografts from CsA-treated or untreated rats. CONCLUSIONS (1) OAD occurs not only after tracheal allotransplantation but also after xenotransplantation. (2) Subepithelial infiltration of neutrophils and the appearance of plasma cells and eosinophils in the peritracheal infiltrates distinguished the histology of rejected xenografts from allografts. (3) Antibody deposition was detected by IFL only in xenografts. (4) Treatment with LFM or RPM significantly decreased the severity of luminal obliteration. Importantly, LFM also prevented the loss of respiratory epithelium.

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.