[Show abstract][Hide abstract] ABSTRACT: The long-held concept that transplanted bone marrow (BM)-derived cells contribute only to cells of the hematopoietic system was challenged by data from our laboratory showing that a single male BM-derived cell could not only reconstitute the hematopoietic system of an irradiated female recipient, but could also lead to the generation of mature BM-derived epithelial cells in the liver, lung, skin, and gastrointestinal tract. Careful costaining and single-cell analyses have been used to rule out false positive cells due to inadequate detection techniques in microscopy or cell overlay. Since this initial discovery, we have sought to understand the mechanisms underlying the formation of BM-derived epithelial cells, and to evaluate their therapeutic use for gene therapy and/or tissue regeneration. Several reports have shown that donor BM-derived cells, possibly macrophages, can fuse with existing host epithelial cells to form heterokaryons that express both donor and tissue-specific markers. While this is certainly true for murine tyrosinemia models, we have used a Cre-lox system to demonstrate that fusion is not a requirement for the generation of BM-derived epithelial cells and is likely not responsible for the BM-derived epithelial cells generated after standard BM transplantation. In a proof of principal experiment for potential gene therapy applications, we have shown that autologous BM-derived cells transfected with a transgene prior to BM transplantation are able to develop into mature type-II pneumocytes that express the transgene. We also discuss future research directions in the field and the therapeutic potential of BM-derived epithelia, including ongoing work to test whether combined cell and gene therapy can be used therapeutically in preclinical mouse models of human disease.
[Show abstract][Hide abstract] ABSTRACT: Recent findings suggest that bone marrow-derived cells (BMDC) may contribute to tissue maintenance throughout the body. However, it is not yet known whether marrow-derived epithelial cells are capable of undergoing proliferation. Our laboratory has shown that BMDC engraft as keratinocytes in the skin at low levels (</= 1%) in the absence of injury. Here we show that skin damage affects the degree of engraftment of BMDC as keratinocytes and that the keratinocytes are actively cycling. Female mice reconstituted with sex-mismatched BM were wounded by punch biopsy and incision. At the wound site, engraftment of BMDC as epidermal cells increased within 1 day, and continued to increase to approximately 4% by 3 weeks after injury. Using a Cre-lox system, fusion of BMDC with epithelial cells was ruled out. BMDC-derived epithelial cells at the wound edges expressed Ki67, a marker for actively cycling cells, and this proliferation correlated with an increase in the number of donor-derived cells within the wound. Donor-derived cytokeratin 5-expressing cells were rare, suggesting that BMDC do not engraft as epidermal stem cells, and the level of engraftment peaked and then decreased over time, further suggesting that BMDC may assist in early wound healing by engrafting as transit-amplifying cells, which then differentiate into keratinocytes.
American Journal Of Pathology 12/2004; 165(5):1767-72. DOI:10.1016/S0002-9440(10)63431-1 · 4.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Analysis of developmental plasticity of bone marrow-derived cells (BMDCs) is complicated by the possibility of cell-cell fusion. Here we demonstrate that epithelial cells can develop from BMDCs without cell-cell fusion. We use the Cre/lox system together with beta-galactosidase and enhanced green fluorescent protein expression in transgenic mice to identify epithelial cells in the lung, liver, and skin that develop from BMDCs without cell fusion.
[Show abstract][Hide abstract] ABSTRACT: Stem cell plasticity refers to the ability of adult stem cells to acquire mature phenotypes that are different from their tissue of origin. Adult bone marrow cells (BMCs) include two populations of bone marrow stem cells (BMCs): hematopoietic stem cells (HSCs), which give rise to all mature lineages of blood, and mesenchymal stem cells (MSCs), which can differentiate into bone, cartilage, and fat. In this article, we review the literature that lends credibility to the theory that highly plastic BMCs have a role in maintenance and repair of nonhematopoietic tissue. We discuss the possible mechanisms by which this may occur. Also reviewed is the possibility that adult BMCs can change their gene expression profile after fusion with a mature cell, which has brought into question whether this stem cell plasticity is real.
[Show abstract][Hide abstract] ABSTRACT: For approximately 5% of autologous transplant recipients and a higher proportion of allogeneic transplant recipients, low level and delayed platelet engraftment is an ongoing problem. Mesenchymal stem cells (MSC), which can be derived from bone marrow as well as other organs, are capable of differentiation into multiple cell types and also support hematopoiesis in vitro. Because cotransplantation of marrow-derived stromal cells has been shown to enhance engraftment of human hematopoietic stem cells, we hypothesized that cotransplantation of MSC could enhance platelet and myeloid cell development.
We tested this hypothesis by transplantation of CD34-selected mobilized human peripheral blood stem cells (PBSC) into sublethally irradiated NOD/SCID mice with or without culture-expanded human MSC and evaluated human myeloid, lymphoid, and megakaryocytic engraftment with flow cytometry and in vitro cultures.
We find that MSC cotransplantation enhances human cell engraftment when a limiting dose (<1 x 10(6)) of CD34 cells is administered. This enhancement is characterized by a shift in the differentiation of human cells from predominantly B lymphocytes to predominantly CD13(+), CD14(+), and CD33(+) myeloid cells with a corresponding increase in myeloid CFU in the marrow. Megakaryocytopoiesis is enhanced by MSC cotransplantation as assessed by an increase in both marrow CFU-MK and circulating human platelets. In contrast, MSC do not affect the percentage of human bone marrow cells that expresses CD34(+).
Cotransplantation of human mesenchymal stem cells with CD34(+)-selected hematopoietic stem cells enhances myelopoiesis and megakaryocytopoiesis.
[Show abstract][Hide abstract] ABSTRACT: Gene therapy application to pulmonary airways and alveolar spaces holds tremendous promise for the treatment of lung diseases. However, safe and effective long-term gene expression using viral and nonviral vectors has not yet been achieved. Adenoviral vectors, with a natural affinity for airway epithelia, have been partially effective, but are inflammatory and induce only transient gene expression. We investigate the novel approach of using retrovirally transduced multipotent bone marrow-derived stem cells (BMSC) to deliver gene therapy to lung epithelium. We have shown previously that up to 20% of lung epithelial cells can be derived from marrow following BMSC transplantation. Here, irradiated female mice were transplanted with male marrow that had been transduced with retrovirus encoding eGFP. Transgene expressing lung epithelial cells were present in all recipients analyzed at 2, 5, or 11 mo after transplant (n = 10), demonstrating that highly plastic BMSC can be stably transduced in vitro and retain their ability to differentiate into lung epithelium while maintaining long-term transgene expression.
American Journal of Respiratory Cell and Molecular Biology 01/2003; 27(6):645-51. DOI:10.1165/rcmb.2002-0056RC · 4.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To better understand the process by which pneumocytes can be derived from bone marrow cells, we investigated the in vivo kinetics of such engraftment following lethal irradiation.
A cohort of lethally irradiated B6D2F1 female mice received whole bone marrow transplants (BMT) from age-matched male donors and were sacrificed at days 1, 3, 5, and 7 and months 2, 4, and 6 post-BMT (n = 3 for each time point). Additionally, 2 female mice who had received 200 male fluorescence-activated cell sorter (FACS)-sorted CD34(+)lin(-) cells were sacrificed 8 months post-BMT.
Lethal irradiation caused histologic evidence of pneumonitis including alveolar breakdown and hemorrhage beginning at day 3. To identify male-derived pneumocytes, simultaneous fluorescence in situ hybridization (FISH) for Y-chromosome and surfactant B messenger RNA was performed on lung tissue. Y(+) type II pneumocytes were engrafted as early as day 5 posttransplant, and eventually from 2 to 14% of the pneumocytes were donor derived in individual mice. Co-staining for epithelial-specific cytokeratins demonstrated that by 2 months, marrow-derived pneumocytes could comprise entire alveoli, suggesting that type I cells derived from type II pneumocytes.
We conclude that alveolar lining cells derive from bone marrow cells immediately after acute injury. Also, the CD34(+)lin(-) subpopulation is capable of such pulmonary engraftment.
[Show abstract][Hide abstract] ABSTRACT: Objective
To better understand the process by which pneumocytes can be derived from bone marrow cells, we investigated the in vivo kinetics of such engraftment following lethal irradiation.