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M. Krevvata et al.
they are thought to arise from a single transformed hematopoietic cell.2-4 Both AML and MDS are genetically heterogeneous making functional characterization of pri- mary human cells essential for studies of disease patho- genesis. However, primary cells from neither of these dis- eases survive well in vitro, making the use of xenotrans- plantation models essential for the study of primary cells. With the rapid growth in the number of immunocompro- mised mouse strains modified to express human proteins, studies using mice as bioreactors for human cells to test specific in vitro observations have become feasible. However, little work has previously been done studying how the recipient mouse affects the biology of the human disease cells. Here we compare the effect of use of NSG versus NSG-S mice on the relative engraftment and growth of human AML and MDS samples.
Collective studies for over three decades have described the contributions of the bone marrow microen- vironment to normal hematopoiesis. Since the descrip- tion of the bone marrow niche by Schofield,5 the regula- tion of normal hematopoietic stem cell homeostasis by mechanisms involving non-hematopoietic cells has been extensively investigated. It is now well understood that normal stem cell self-renewal is tightly regulated, in part, by cell-extrinsic mechanisms.6-10 Taichman and Emerson have shown that cytokines produced by osteoblasts pro- mote proliferation of hematopoietic cells in culture11 whereas increases in osteoblast numbers in a mouse model with constitutively active osteoblast-specific parathyroid hormone resulted in a simultaneous increase of hematopoietic stem cells.12 As with normal hematopoiesis, several hematopoietic malignancies per- sist by maintaining a pool of malignant stem cells that may be partly protected by components of the microen- vironment.13,14 Conversely, leukemic stem cells induce alterations in hematopoietic regulatory functions to gain growth advantage over normal hematopoietic stem cells.15,16 Schepers et al. have shown that leukemic myeloid cells secrete high levels of pro-inflammatory cytokines, creating a paracrine feedback loop that drives myeloid differentiation. At the same time, myeloid cells stimulate mesenchymal stem cells (MSC) to overproduce function- ally altered osteoblastic cells with compromised ability to maintain normal hematopoietic stem cells.17 Thus, cytokine production by the bone marrow microenviron- ment may modify the phenotype of malignant blood dis- eases.
We previously demonstrated that the NSG (NOD-Scid- IL-2Rgcnull) mouse is a robust recipient for human AML xenotransplantation samples, allowing a better under- standing and characterization of AML biology, especially in the context of drug therapy studies.18 However, we observed that a significant proportion of primary AML specimens showed low (0.1 to 1% human blasts in mouse bone marrow) or no (<0.1%) engraftment in NSG mice, suggesting the need for improved xenograft mod- els.18 Transgenic expression of human stem cell factor, granulocyte-macrophage colony-stimulating factor and interleukin-3 in NSG-S mice has been reported to enhance engraftment of primary AML samples, although only a few AML patients were compared between strains.19 These studies did not allow for a rigorous deter- mination of the percentage of patients’ samples that engraft (an assessment for stem cell effects) or the bulk of disease burden (reflecting growth after engraftment).
A recent study attempting to develop a patient-derived xenotransplantation model for human MDS suggested that patient-derived MSC combined with the use of NSG-S immunodeficient mice, could enhance engraft- ment levels.20 Indeed, the use of NSG-S mice appeared to improve engraftment levels and also maintained the malignant clone, but these studies were largely done with accompanying injection of MSC so the critical variables for engraftment of MDS samples were largely undeter- mined.
In this report, we compare the engraftment levels in the two above-mentioned immunodeficient mouse strains as well as the influence of MSC on relative engraftment of human AML and MDS in primary patients’ samples. We describe a comprehensive paired analysis of engraftment of primary AML samples in NSG and NSG-S mice. Consistent with previous studies, the use of the NSG-S strain increased both the percentage of AML samples that engrafted and the level of engraftment. In contrast, MDS engraftment was consistently low and was not influenced significantly by the use of either mouse strain or co-injec- tion of MSC. However, human MSC did not engraft long- term suggesting that a human microenvironment was not established. These results demonstrate that human AML cells respond positively to the three human cytokines as shown by xenografts, while these cytokines appear to be insufficient to enhance the engraftment and expansion of MDS cells. Xenotransplantation models that better mimic the human microenvironment may be necessary to estab- lish robust MDS xenograft models.
Methods
Myelodysplastic syndrome and acute myeloid leukemia specimens
Peripheral blood, leukapheresis product, or bone marrow from AML patients were collected at the Hospital of the University of Pennsylvania after informed consent. French- American-British or World Health Organization classification and cytogenetics were determined at the Hospital of the University of Pennsylvania. For MDS samples, only bone mar- row samples were used and were obtained either from the same source or from Roswell Park Cancer Institute.
Mice
Mice were used in accordance with a protocol reviewed and approved by the Institutional Animal Care and Use Committee of the University of Pennsylvania. NSG or NSG-S mice were ini- tially purchased from Jackson Laboratories (Bar Harbor, ME, USA) and produced at the University of Pennsylvania Stem Cell and Xenograft Core. Male and female mice 6-8 weeks of age were either sublethally irradiated (250 cGy) or chemically condi- tioned by intraperitoneal injection of busulfan (30 mg/kg, Otsuka America Pharmaceutical Inc.) 24 h prior to cell injections. T-cell depleted AML cells (5-10x106 per mouse) were transplant- ed via tail vein injection into mice.21 Mice were euthanized no later than 16 weeks after AML injection and marrow from femo- ra and tibiae, splenocytes and peripheral blood were harvested. Human AML engraftment was assessed by flow cytometry and defined as the percentage of human CD45+CD33+ cells in total live mononuclear cells.22-24
For MDS samples, intrafemoral injections with 1x106 human bone mononuclear cells alone or in combination with 5x105 ex vivo-expanded MSC were performed. For MDS-engrafted mice,
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