Presence Of Human Mesenchymal Stem Cells Research Articles

Engraftment of Human MDS Samples in Xenotransplanted Mice Depends on Human Cytokines but Not Mesenchymal Stem Cells (MSC)

Background: Myelodysplastic syndromes (MDS) are a heterogeneous group of diseases with limited treatment options. Development of a patient derived xenotransplantation model for pre-clinical studies is a priority goal in the field. It has been hypothesized that patient derived mesenchymal stem cells and/or human cytokines are necessary to establish MDS engraftment in immunocompromised mice. In this study we evaluated the ability of mesenchymal stem cells (MSC) to facilitate the engraftment process having as a goal to generate a robust patient-derived xenograft (PDX) mouse model for MDS. We also assessed the contribution of human cytokines as expressed in transgenic mice.Methods: MSC from normal donors and MDS patient samples were generated and characterized using standard culture methods. NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) and NOD.Cg-Prkdcscid Il2rgtm1Wjl Tg(CMV-IL3,CSF2,KITLG)1Eav/MloySzJ (NSG-S) mice were used as recipients. Mice were injected with 1 x 106 bone marrow mononuclear cells (MNC) alone or in combination with 5 x 105 MSC. MSC were from normal donors, from same patient as MNC (autologous), from other MDS patients or normal donors (allogeneic). All cells were injected intrafemorally. Mice were assessed for engraftment by bone marrow aspiration at 6-8, 10 weeks and at necropsy at 12-32 weeks (depending on initial assessment). Long-term engrafted samples were further assessed for presence of stem cells by secondary transplantation into NSG-S mice. 1 x 106 human CD45+ cells were passaged without or with 5 x 105 MSC. Secondary recipients were assessed at 12 weeks for engrafment. MDS samples represented both high risk (refractory anemia with excess blasts, RAEB)) and low risk (refractory anemia (RA), refractory cytopenia with myelodysplasia (RCMD), chronic myelomonocytic leukemia (CMML)) disease. The overall degree of engraftment was assessed by bone marrow aspiration and measurement of human CD45+ cells by means of FACS. Lymphoid, myeloid and erythroid engraftment was assessed similarly. 5 x 105 MSCs from normal donors were transduced with GFP (pELNS.CBR-T2A-GFP) and intrafemorally injected into NSG mice and the presence of human MSC was evaluated by in vivo Imaging (Xenogen Spectrum system and Living Image Version 4.3 software.).Results: 12 out of 12 injected MDS samples showed persistent human cells when assessed at 6-8 weeks. For most samples, engraftment at that time-point was low (<2%) and was not consistently influenced by the presence or absence of MSC. On week 10, 4 out of 12 injected MDS patient samples showed increased engraftment and only one showed higher engraftment levels in the mice co-transplanted with MSC. Two of these were from patients with RAEB and CMML and showed both myeloid and erythroid engraftment. Molecular analysis showed consistent engraftment of the malignant clone in 2 out of 2 patient samples tested. Overall, only 3 out of 12 patients showed long-term engraftment (> 12 weeks) and these samples were transferred to secondary animals. Secondary transplanted mice injected with selected hCD45+ cells with or without MSC showed variable engraftment levels on week 10 after injections. One of them reached long-term engraftment (>12 weeks), whereas the second gradually lost engraftment after week 10 and the third is ongoing. MSC tracking showed a gradual loss of MSC after intrafemoral injections to the point that no MSC were detected 3 weeks later. Comparison of engraftment in NSG mice to NSG-S mice (the latter producing human cytokines) showed a slight increase in engraftment at week 6 which continued at later time points.Conclusions: Passive transfer of MDS hematopoietic cells as assessed at 6-8 weeks after intrafemoral injection is highly consistent and not dependent on MSC when total MNC fraction is used. The latter may be explained since MSC are lost within the first three weeks after injections. Direct comparison of NSG vs. NSG-S mice showed that the presence of hGM-CSF, hIL-3 and h-SCF in the NSG-S mice increased the engraftment levels. Successful long-term engraftment of MDS cells in xenotransplanted mice was achieved for one patient sample so far, indicating that MDS initiating cells can be maintained in NSG-S mice. However, establishing long-term engraftment can only be achieved with a subset of patient samples. MDS engraftment is influenced by human cytokines but not with MSC cells which may suggest that MDS does not depend on MSC's for long term growth. DisclosuresNunez-Cruz:Novartis: Research Funding. Milone:Novartis: Patents & Royalties, Research Funding.

Reconstitution of the functional human hematopoietic microenvironment derived from human mesenchymal stem cells in the murine bone marrow compartment

Hematopoiesis is maintained by specific interactions between both hematopoietic and nonhematopoietic cells. Whereas hematopoietic stem cells (HSCs) have been extensively studied both in vitro and in vivo, little is known about the in vivo characteristics of stem cells of the nonhematopoietic component, known as mesenchymal stem cells (MSCs). Here we have visualized and characterized human MSCs in vivo following intramedullary transplantation of enhanced green fluorescent protein-marked human MSCs (eGFP-MSCs) into the bone marrow (BM) of nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice. Between 4 to 10 weeks after transplantation, eGFP-MSCs that engrafted in murine BM integrated into the hematopoietic microenvironment (HME) of the host mouse. They differentiated into pericytes, myofibroblasts, BM stromal cells, osteocytes in bone, bone-lining osteoblasts, and endothelial cells, which constituted the functional components of the BM HME. The presence of human MSCs in murine BM resulted in an increase in functionally and phenotypically primitive human hematopoietic cells. Human MSC-derived cells that reconstituted the HME appeared to contribute to the maintenance of human hematopoiesis by actively interacting with primitive human hematopoietic cells.

243. Transplantation of Neonatal NOD/SCID/MPSVII Mice with Human Mesenchymal Stem Cells

In this study we assessed the transfer and expression of the gene for beta-glucuronidase (GUSB) into multiple tissues by infusing normal human mesenchymal stem cells (MSC) into unconditioned neonatal NOD/SCID/MPSVII (GUSB null) mice. In this study, pups were transplanted before 3 days of age using the facial vein injection strategy. We hypothesized that neonatal pups would provide a rapidly growing environment for engraftment and expansion of transplanted normal human MSC into different tissues. The Sands laboratory has used the MPSVII mouse for many years to study disease progression, including neural defects, and to study correction of the disease by gene replacement therapies. In order to study cellular therapies using normal human cells, the murine MPSVII mutation was backcrossed for ten generations onto the NOD/SCID strain. Human umbilical cord blood CD34+ cells engraft in the resultant NOD/SCID/GUSB null strain to levels of 86% marrow replacement, similar to the NOD/SCID parent (Hofling 2004). The tissues of the mice can be homogenized and subjected to a GUSB enzyme assay, and plotted against a standard curve composed of normal human and GUSB null mouse cells, to quantitate the percentage of human cells that had been residing in the tissue of interest at the time of harvest. There is also a FACS|[ndash]|based assay to detect levels of the GUSB protein, and to allow sorting of donor-derived cells from any tissue after transplantation. The aspect of the NOD/SCID/GUSB null strain that makes it so particularly well suited for the study of human stem cell therapy is the clear-cut enzymatic stain for normal, GUSB positive human cells transplanted into the mice. On tissue slides prepared from mice that had been transplanted at birth with human mesenchymal stem cells, the histochemical stain for GUSB revealed normal cells in multiple tissues. Almost all tissues surveyed (N=19) demonstrated the presence of human MSC by staining for GUSB. The stain is quite specific, and although the released enzyme can be taken up by neighboring cells, it appears to be in very low abundance or, more likely, in a processed form where it is no longer detectable by the histochemical analysis. Thus, the individual transplanted human cells stand out vividly against the background murine tissues. In summary, the use of the NOD/SCID/MPSVII neonatal injection model has allowed accurate tracking and quantitation of human MSC into multiple tissues of mice injected by facial vein within three days of birth. This study may set the stage for neonatal gene/cell therapy for newborns with lysosomal storage diseases.