Stem Cell Preparation & Analysis

Director: Diane Krause
Associate Director: Bernard Forget

Services: Assistance in performing murine bone marrow transplantation studies

The YCEMH will provide training and advice regarding murine bone marrow transplantation (BMT) to members of the YCEMH. While the YCEMH can perform BMT as a service, it is expected that most investigators will come to learn about experimental strategies using BMT, and how to perform these assays in their independent laboratories. Specifically, the Cell Preparation and Analysis Core will provide training and expertise in mouse into mouse BMT (both syngeneic and allogeneic) and in human to mouse (xenogeneic) BMT into immunodeficient mice.

Bone marrow transplants in mice

Protocols for transplantation of mouse BM or human cells into mice are similar except for a) the dose of irradiation used, and b) the methods used to assess engraftment.

For murine bone marrow transplantation, one usually uses high (marrow-lethal) dose irradiation (800-1200 centigray using a cesium irradiator), and engraftment can be assessed using some distinguishing feature of the donor cell population. For example, one can use CD45.1 vs. CD45.2 mouse strains and use FACS analysis for the 2 CD45 isoforms to determine engraftment. Alternatively, one can use a donor mouse that expresses a detectable transgene such as GFP so that direct FACS analysis can be used to detect donor-derived cells. In the marrow cell plasticity field, it is also helpful to use male donors and female recipients so that the Y chromosome, which should be present in all donor derived cells (as opposed to transgene expression which can be silenced), can be used as a donor cell marker. Murine BMT can be syngeneic or allogeneic (MHC mismatched). For syngeneic transplantation, the donor derived cells can be administered with minimal manipulation. In contrast, for prevention of graft vs. host disease in allogeneic BMT, the donor marrow cells must be depleted of T lymphocytes prior to transplantation. This T cell depletion can be achieved by specific immunomagnetic depletion of T cells (or subsets thereof) or by lineage depletion using FACS, both of which are performed routinely in the Cell Processing Core.

Xenogeneic Transplantation

There is no perfect assay for human hematopoietic stem cells (HSC). The best assays available are in vivo xenotransplantation assays in which human cells are transplanted into immunodeficient mice. These mice are irradiated with a sublethal dose (usally 250-350 cGy), which makes them more receptive to engraftment and hematopoietic differentiation of human cells. The initial host used was the severe combined immunodeficient (SCID) mouse that lacked both humoral and cell-mediated immunity. From this animal model the SCID-repopulating cell (SRC) was defined. Later the non-obese diabetic/severe combined immunodeficient NOD/SCID (NOD/LtSz-Prkdc scid/ Prkdc scid) mouse was used as a recipient. This strain, which has reduced natural-killer cell activity in addition to the absence of adaptive immunity, is a better host than scid mice for HSC, and does not require exogenous cytokine support. Until recently, this was the most extensively used experimental model for human hematopoiesis in vivo. However, the human engraftment level, although better, was variable between individual mice, hampering the reproducibility of results. In addition, NOD/SCID/ B2m null mice have been developed. This strain has nearly complete absence of NK-cell activity and humoral and cell-mediated immunity, as well as lack of hemolytic complement activity and reduced ability to secrete IL-1 in response to LPS. Thus it is an excellent host for xenogeneic cells. The reports using this xenogeneic model to study human hematopoiesis show that the NOD/SCID/ B2m null host allows biologically distinct subpopulations of HSCs to engraft, that were not previously detected in the NOD/SCID mouse model.

Depending on the human cell population to be assayed, cells will be counted, aliquoted and resuspended in a final volume of 200 ul Iscoves modified medium (IMDM) containing 5% fetal bovine serum. The transplantation of the human cells will be performed via tail vein injection. Thereafter the mice will be maintained in a HEPA-filtered isolator cages in an ultra-clean barrier room and received trimethoprim/sulfamethoxazole for the duration of the experiment. Human cell engraftment will be assayed by FACS analysis for human CD45 vs. murine CD45 expressing cells on peripheral blood samples until time of sacrifice when engraftment will be assessed also be FACS of the BM and by human specific CFU assays. Personnel in our Core have expertise regarding the different immunodeficient mouse strains that are available and, if requested, will advise YCEMH Members regarding which strains are optimal for different purposes.

Issues in BMT for IACUC applications including post-transplant mouse care

Our Core will maintain an approved IACUC protocol in order to perform training and quality control for xenogeneic, syngeneic and allogeneic transplantation. In addition, all animal experiments of individual YCEMH investigators will need to be approved by the Yale University School of Medicine (Institutional) Animal Care and Use Committee.

For investigators who are proposing to perform BMT on mice in their laboratories for the first time, application for approval from the Animal Care and Use Committee can be a daunting task. The applications require a great amount of detail regarding study design, and also how the mice will be taken care of when they are thrombocytopenic and immunosuppressed post-transplantation. For these investigators, our Core will provide training, and advice. The Core will provide information on how to design and describe the experiments to be performed by providing general IACUC training in BMT, and by meeting one-on-one with individual investigators upon request.

Specific concerns to be addressed include how to house the mice before and after transplantation (i.e. sterile food and water and filtered cages), whether to provide antibiotics, what to do if a mouse appears sickly, when to expect engraftment, how best to test for engraftment, what to do if engraftment appears to be delayed, what to do if graft versus host disease develops, and how many animals should be included in each group.

Training

Training in the overall approach to murine BMT, study design including which donor/recipient pairs are optimal, which subpopulations of cells to transplant at what numbers, IV injections, and post-transplant care of the animals are provided by our Core. A 2 hour workshop will be provided at no charge to members of the Yale community at large twice a year. There will be 3 parts to the workshop. First, the Core Director will provide a 50 minute overview of mouse into mouse and xenogeneic BMT approaches and how they are best used. She will also provide an overview/update of the different transgenic mice available for tracking donor derived cells in which the transgene of interest is expressed ubiquitously (optimally or suboptimally) or in a cell-type specific manner, and she will include some of the issues that that must be addressed in applications for approval of animal protocols in which BMT is proposed. Second, the research associate and research assistant will present a 30 minute description of the specific services provided by the Core and how they have been used to facilitate research in the laboratories of Yale investigators, and third, there will be a 30 minute question and answer period.

Prior to initiating this training in BMT, users are required to first undergo training in the use of the Mark I cesium-137 irradiator, which is provided upon request by the Department of Therapeutic Radiology under the auspices of the Yale Cancer Center, and they are strongly urged to take a workshop in basic mouse handling skills provided by the Yale Animal Resources Center. Issues and techniques that will be taught in our Core include selection of murine mouse models, the specific numbers and types of cells to transplant, IV and (tail vein) injection techniques.

Costs

Members of the YCEMH will be charged a minimal hourly fee ($30) for training in techniques and approaches to BMT using mice.