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Acute lymphoblastic leukemia cells from 19 children, including 7 who remain in first complete remission (CR1), were engrafted into nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. High-level infiltration of bone marrow, spleen, and liver was observed, with variable infiltration of other organs. The immunophenotypes of xenografts were essentially unaltered compared with the original patient sample. In addition, sequencing of the entire p53 coding region revealed no mutations in 14 of 14 xenografts (10 from patients at diagnosis and 4 at relapse). Cells harvested from the spleens of engrafted mice readily transferred the leukemia to secondary and tertiary recipients. To correlate biologic characteristics of xenografts with clinical and prognostic features of the patients, the rates at which individual leukemia samples engrafted in NOD/SCID mice were analyzed. Differences in biologic correlates were encountered depending on stage of disease: a direct correlation was observed between the rate of engraftment and length of CR1 for samples harvested at relapse (r ‫؍‬ 0.96; P ‫؍‬ .002), but not diagnosis (r ‫؍‬ 0.38; P ‫؍‬ .40). In contrast, the in vivo responses of 6 xenografts to vincristine showed a direct correlation (r ‫؍‬ 0.96; P ‫؍‬ .002) between the length of CR1 and the rate at which the leukemia cell population recovered following vincristine treatment, regardless of whether the xenografts were derived from patients at diagnosis or relapse. This study supports previous findings that the NOD/SCID model of childhood ALL provides an accurate representation of the human disease and indicates that it may be of value to predict relapse and design alternative treatment strategies in a patient-specific manner. (Blood. 2002;99: 4100-4108)

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Allogeneic (allo) hematopoietic stem cell transplantation is an effective therapy for hematological malignancies but it is limited by acute graft-versus-host disease (GVHD). Dendritic cells (DC) play a major role in the allo T cell stimulation causing GVHD. Current immunosuppressive measures to control GVHD target T cells but compromise posttransplant immunity in the patient, particularly to cytomegalovirus (CMV) and residual malignant cells. We showed that treatment of allo mixed lymphocyte cultures with activated human DC-depleting CD83 antibody suppressed alloproliferation but preserved T cell numbers, including those specific for CMV. We also tested CD83 antibody in the human T cell–dependent peripheral blood mononuclear cell transplanted SCID (hu-SCID) mouse model of GVHD. We showed that this model requires human DC and that CD83 antibody treatment prevented GVHD but, unlike conventional immunosuppressants, did not prevent engraftment of human T cells, including cytotoxic T lymphocytes (CTL) responsive to viruses and malignant cells. Immunization of CD83 antibody-treated hu-SCID mice with irradiated human leukemic cell lines induced allo antileukemic CTL effectors in vivo that lysed 51Cr-labeled leukemic target cells in vitro without further stimulation. Antibodies that target activated DC are a promising new therapeutic approach to the control of GVHD.

Cells resembling bone marrow mesenchymal stem cells (MSC) have been isolated from many organs but their functional relationships have not been thoroughly examined. Here we compared the immunophenotype, gene expression, multipotency and immunosuppressive potential of MSC-like colony-forming cells from adult murine bone marrow (bmMSC), kidney (kCFU-F) and heart (cCFU-F), cultured under uniform conditions. All populations showed classic MSC morphology and in vitro mesodermal multipotency. Of the two solid organ-specific CFU-F, only kCFU-F displayed suppression of T-cell alloreactivity in vitro, albeit to a lesser extent than bmMSC. Quantitative immunophenotyping using 81 phycoerythrin-conjugated CD antibodies demonstrated that all populations contained high percentages of cells expressing diagnostic MSC surface markers (Sca1, CD90.2, CD29, CD44), as well as others noted previously on murine MSC (CD24, CD49e, CD51, CD80, CD81, CD105). Illumina microarray expression profiling and bioinformatic analysis indicated a correlation of gene expression of 0.88-0.92 between pairwise comparisons. All populations expressed approximately 66% of genes in the pluripotency network (Plurinet), presumably reflecting their stem-like character. Furthermore, all populations expressed genes involved in immunomodulation, homing and tissue repair, suggesting these as conserved functions for MSC-like cells in solid organs. Despite this molecular congruence, strong biases in gene and protein expression and pathway activity were seen, suggesting organ-specific functions. Hence, tissue-derived MSC may also retain unique properties potentially rendering them more appropriate as cellular therapeutic agents for their organ of origin.

Hematopoietic stem cell (HSC) transplant is a well established curative therapy for some hematological malignancies. However, achieving adequate supply of HSC from some donor tissues can limit both its application and ultimate efficacy. The theory that this limitation could be overcome by expanding the HSC population before transplantation has motivated numerous laboratories to develop ex vivo expansion processes. Pioneering work in this field utilized stromal cells as support cells in cocultures with HSC to mimic the HSC niche. We hypothesized that through translation of this classic coculture system to a three-dimensional (3D) structure we could better replicate the niche environment and in turn enhance HSC expansion. Herein we describe a novel high-throughput 3D coculture system where murine-derived HSC can be cocultured with mesenchymal stem/stromal cells (MSC) in 3D microaggregates--which we term "micromarrows." Micromarrows were formed using surface modified microwells and their ability to support HSC expansion was compared to classic two-dimensional (2D) cocultures. While both 2D and 3D systems provide only a modest total cell expansion in the minimally supplemented medium, the micromarrow system supported the expansion of approximately twice as many HSC candidates as the 2D controls. Histology revealed that at day 7, the majority of bound hematopoietic cells reside in the outer layers of the aggregate. Quantitative polymerase chain reaction demonstrates that MSC maintained in 3D aggregates express significantly higher levels of key hematopoietic niche factors relative to their 2D equivalents. Thus, we propose that the micromarrow platform represents a promising first step toward a high-throughput HSC 3D coculture system that may enable in vitro HSC niche recapitulation and subsequent extensive in vitro HSC self-renewal.

A tissue stem cell should exhibit long-term self-renewal, clonogenicity and a capacity to differentiate into the tissue of origin. Such a postnatal renal stem cell has not been formally identified. The metanephric mesenchyme (MM) of the developing kidney gives rise to both the renal interstitium and the nephrons and is regarded as the progenitor population of the developing kidney. However, isolated MM does not self renew and requires immortalization for survival in culture. Here we report the isolation and sustained culture of long-term repopulating, clonal progenitors from the embryonic kidney as free floating nephrospheres. Such cells displayed clonal self renewal for in excess of twenty passages when cultured with bFGF and thrombin, showed broad mesodermal multipotentiality, but retained expression of key renal transcription factors (Wt1, Sall1, Eya1, Six1, Six2, Osr1 and Hoxa11). While these cells did display limited capacity to contribute to developing embryonic kidney explants, nephrospheres did not display in vitro renal epithelial capacity. Nephrospheres could be cultured from both Sall1(+) and Sall1(-) fractions of embryonic kidney, suggesting that they were derived from the MM as a whole and not specifically the MM-derived cap mesenchyme committed to nephron formation. This embryonic renal stem cell population was not able to be isolated from postnatal kidney confirming that while the embryonic MM represents a mulitpotent stem cell population, this does not persist after birth.

The online version of this article has a Supplementary Appendix. BackgroundMultipotent mesenchymal stromal cells suppress T-cell function in vitro, a property that has underpinned their use in treating clinical steroid-refractory graft-versus-host disease after allogeneic hematopoietic stem cell transplantation. However the potential of mesenchymal stromal cells to resolve graft-versus-host disease is confounded by a paucity of pre-clinical data delineating their immunomodulatory effects in vivo. Design and MethodsWe examined the influence of timing and dose of donor-derived mesenchymal stromal cells on the kinetics of graft-versus-host disease in two murine models of graft-versus-host disease (major ]) using clinically relevant conditioning regimens. We also examined the effect of mesenchymal stromal cell infusion on bone marrow and spleen cellular composition and cytokine secretion in transplant recipients. ResultsDespite T-cell suppression in vitro, mesenchymal stromal cells delayed but did not prevent graftversus-host disease in the major histocompatibility complex-mismatched model. In the sibling transplant model, however, 30% of mesenchymal stromal cell-treated mice did not develop graft-versus-host disease. The timing of administration and dose of the mesenchymal stromal cells influenced their effectiveness in attenuating graft-versus-host disease, such that a low dose of mesenchymal stromal cells administered early was more effective than a high dose of mesenchymal stromal cells given late. Compared to control-treated mice, mesenchymal stromal cell-treated mice had significant reductions in serum and splenic interferon-g, an important mediator of graft-versus-host disease. ConclusionsMesenchymal stromal cells appear to delay death from graft-versus-host disease by transiently altering the inflammatory milieu and reducing levels of interferon-g. Our data suggest that both the timing of infusion and the dose of mesenchymal stromal cells likely influence these cells' effectiveness in attenuating graft-versus-host disease.Key words: stem cell transplantation, graft-versus-host disease, mesenchymal stromal cells, IFNg.Citation: Christensen ME, Turner BE, Sinfield LL, Kollar K, Cullup H, Waterhouse NJ, Hart DNJ, Atkinson K, and Rice AM. Mesenchymal stromal cells transiently alter the inflammatory milieu posttransplant to delay graft-versus-host disease. Haematologica 2010;95(12):2102-2110. doi:10.3324/haematol.2010 This is an open-access paper. © F e r r a t a S t o r t i F o u n d a t i o n Mesenchymal stromal cells transiently alter the inflammatory milieu post-transplant to delay graft-versus-host disease