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Bone marrow is the primary site of hematopoiesis in adult humans. Bone marrow can be cultured in vitro but few simple culture systems fully support hematopoiesis beyond a few months. Human bone marrow analogs are long-term in vitro cultures of marrow stromal and hematopoietic stem cells that can be used to produce cells and products normally harvested from human donors. Bone marrow analog systems should exhibit confluence of the stromal cell populations, persistence of hematopoietic progenitor cells, presence of active regions of hematopoiesis and capacity to produce mature cell types for extended periods of time. Although we are still years away from realizing clinical application of products formed by artificial bone marrow analogs, the process of transitioning this research tool from bench to bedside should be fairly straightforward. The most obvious application of artificial marrow would be for production of autologous hematopoietic CD34+ stem cells as a stem cell therapy for individuals experiencing bone marrow failure due to disease or injury. Another logical application is for ‘blood farming’, a process for large-scale in vitro production of red blood cells, white blood cells or platelets, for transfusion or treatment. Other possibilities include production of nonhematopoietic stem cells such as osteogenic stromal cells, osteoblasts and rare pluripotent stem cells. Bone marrow analogs also have great potential as ex vivo human test systems and could play a critical role in drug discovery, drug development and toxicity testing in the future.

Background Application of gene therapy to induce antigen-specific immune tolerance could be important for transplantation or treatment of autoimmune diseases. Hematopoietic stem cell-based gene therapy has been hampered by relatively weak gene expression in vivo and loss of transduced cells over time. Selective expansion of transduced hematopoietic stem cells has been accomplished by incorporating the dihydrofolate reductase (DHFR) gene into the gene transfer vector. Methods To assess whether this strategy could be applied to transplantation, we constructed a retroviral vector plasmid (KA274) containing the cDNA encoding human leukocyte antigen (HLA)-A2.1 and a tyr22 mutant DHFR and generated vesicular stomatitis virus-G-pseudotyped recombinant retrovirus by transfection into 293GPG cells. Bone marrow cells from C57BL/6 mice were infected with KA274 at a multiplicity of infection of 100, and transplanted into lethally irradiated syngeneic mice. Results After transplantation with transduced bone marrow, the proportion of peripheral blood cells expressing HLA-A2 ranged from 3.2% to 38% and increased 2- to 4.9-fold after selection for DHFR-expressing cells using trimetrexate and nitrobenzylmercaptpurine riboside 5' monophosphate. HLA-A2 expression remained above pretreatment levels throughout the study. Cytotoxic spleen cells from reconstituted mice lysed third-party HLA-B7-expressing targets but were unable to lyse HLA-A2-expressing targets. All KA274 reconstituted C57BL/6 mice accepted skin grafts from HLA-A2.1 transgenic mice for more than 245 days but rejected third-party Balb/c skin grafts in 12 days. Conclusion Long-term transgene expression and immunologic tolerance to retrovirus-encoded HLA-A2, equivalent to that obtained by donor bone marrow transplantation, was accomplished, and selective expansion of transduced bone marrow cells was induced using DHFR as a selectable marker.

Introduction. Bone marrow cells expressing foreign MHC antigens survive poorly after transplantation. Stable mixed hematopoietic chimerism requires reconstitution with a relatively large number of foreign bone marrow cells and intensive depletion of host cells. In addition, when foreign MHC-transduced autologous bone marrow cells are transplanted, prolonged hematopoietic transgene expression requires extensive host conditioning. The competitive disadvantage associated with engraftment of donor cells expressing foreign MHC antigens is thought to result from a defect in engraftment secondary to donor-host incompatibility or immunologic resistance by the host. Methods. We used a limiting-dilution competitive repopulation assay with cells from HLA-A2.1 transgenic mice to determine whether and to what extent foreign MHC antigen expression impairs engraftment in C57BL/6 hosts. Transplants were performed with Hoechst 33342 fluorescence-sorted side population (SP) cells, a subset of bone marrow enriched for stem cells. Results. Transplantation with 250 stem cell-enriched HLA-A2.1-transgenic side population cells successfully competed with nearly 5000 host C57BL/6 side population cells to produce stable long-term mixed chimerism. There was a direct relationship between the number of transplanted donor HLA-A2-expressing cells and the percentage of HLA-A2-expressing cells in the peripheral blood of reconstituted C57BL/6 mice (r2=0.1799, P=0.031). This correlation was maintained in secondary transplant recipients. Conclusions. HLA-A2-expressing hematopoietic cells do not have an engraftment defect when transplanted into C57BL/6 hosts and immunologic resistance did not limit chimerism following lethal irradiation. These results may have relevance to understanding long-term gene expression after hematopoietic stem cell based gene therapy.

We report here the first attempt to produce and use whole acellular (AC) lung as a matrix to support development of engineered lung tissue from murine embryonic stem cells (mESCs). We compared the influence of AC lung, Gelfoam, Matrigel, and a collagen I hydrogel matrix on the mESC attachment, differentiation, and subsequent formation of complex tissue. We found that AC lung allowed for better retention of cells with more differentiation of mESCs into epithelial and endothelial lineages. In constructs produced on whole AC lung, we saw indications of organization of differentiating ESC into three-dimensional structures reminiscent of complex tissues. We also saw expression of thyroid transcription factor-1, an immature lung epithelial cell marker; pro-surfactant protein C, a type II pneumocyte marker; PECAM-1/CD31, an endothelial cell marker; cytokeratin 18; alpha-actin, a smooth muscle marker; CD140a or platelet-derived growth factor receptor-alpha; and Clara cell protein 10. There was also evidence of site-specific differentiation in the trachea with the formation of sheets of cytokeratin-positive cells and Clara cell protein 10-expressing Clara cells. Our findings support the utility of AC lung as a matrix for engineering lung tissue and highlight the critical role played by matrix or scaffold-associated cues in guiding ESC differentiation toward lung-specific lineages.

The RUNX transcription factors (RUNX1, RUNX2, and RUNX3) play essential roles in hematopoiesis and skeletal development. Consistent with these roles in differentiation and cell cycle, the activity of both RUNX1 and RUNX3 is perturbed in cancer. To determine a role for the RUNX factors in prostate biology, we investigated the expression of RUNX factors in prostate epithelial cell lines and normal prostate tissue. RUNX1, RUNX2, and RUNX3 were expressed in both normal prostate tissue and an immortalized, non-transformed cell line. We found that prostate cancer-derived cell lines expressed RUNX1 and RUNX2, but not RUNX3. Next, we sought to identify prostate-specific genes whose expression could be regulated by RUNX proteins. Four consensus RUNX sites are located within the prostate-specific antigen (PSA) regulatory region. Chromatin immunoprecipitation (ChIP) analysis showed that RUNX1 is specifically bound to the PSA regulatory region in LNCaP cells. RUNX1 and RUNX2 activated the PSA regulatory region alone or cooperatively with prostate-derived ETS factor (PDEF) and RUNX1 physically associated with PDEF. Taken together, our results suggest that RUNX factors participate in prostate epithelial cell function and cooperate with an Ets transcription factor to regulate PSA gene expression.

Background Successful transduction of hematopoietic stem cells is essential if gene therapy is to be used clinically to induce immunologic tolerance. Methods Hoechst 33342 staining was used to isolate a population of bone marrow cells enriched for stem cells, termed side population (SP) cells. Murine bone marrow SP cells were transduced with HLA-A2.1-expressing VSV-G-pseudotyped lentivirus or retrovirus vectors under identical conditions. Results After transduction without prestimulating cytokines, which minimizes cell cycling and helps maintain stem cell pluripotency, the HLA-A2.1 gene was found in the DNA of 56% of CFU-GM colonies derived from lentivirus-transduced SP cells, but in only 4% of colonies derived from retrovirus-transduced SP cells. Lentivirus and retrovirus transduction including cytokine prestimulation produced the same degree of integration as that following lentivirus-transduction of non-prestimulated cells. Transplantation of 5,000 lentivirus-transduced SP cells into lethally irradiated mice resulted in long-term expression of the HLA-A2.1 transgene in peripheral blood progeny of bone marrow SP cells and prolonged skin graft survival across this class I MHC barrier until the time of animal sacrifice. Conclusions Recombinant lentivirus, but not retrovirus vectors, effectively transduced SP cells that were not prestimulated with cytokines and lentivirus-transduced SP cells successfully repopulated lethally irradiated C57BL/6 mice, animals where there is no selective advantage to repopulation with transduced cells. Transplantation of a relatively small number of transduced SP cells led to prolonged transgene mRNA expression and antigen-specific survival of grafts expressing the foreign MHC transgene.