Collection of hematopoietic CD34 stem cells in rhesus macaques using Spectra Optia.

BACKGROUND: Nonhuman primates, particularly rhesus macaques, are ideal preclinical large animal models to investigate organ tolerance induction protocols using donor hematopoietic stem cells (HSCs) to induce chimerism. Their relatively small size poses some challenges for the safe and effective collection of peripheral blood HSCs through apheresis procedures. We describe our experiences using the Spectra Optia apheresis unit to successfully obtain HSCs from mobilized peripheral blood of rhesus macaques. METHOD: Mobilization of peripheral blood HSCs was induced using granulocyte stimulating factor (G-CSF) and Mozobil. The Spectra Optia unit was used in 18 apheresis procedures in 13 animals (4.9-10 kg). Animal health was carefully monitored during and after the procedure. Changes in peripheral blood cells before, during and after procedure were determined by complete blood count and flow cytometry. RESULTS: The automatic settings of the Spectra Optia unit were applied successfully to the procedures on the rhesus macaque. All animals tolerated the procedure well with no mortality. Mobilization of HSCs were most consistently achieved using 50 µg/kg of G-CSF for 5 days and a single dose of Mozobil on the 5th day, followed by collection of cells 3 h after Mozobil injection. The final apheresis product contained an average of 23 billion total nucleated cells with 47% granulocytes, 3,871 million total CD3 cells and 77 million CD34 cells which resulted in an average of 10 million CD34+ cells/kg of donor weight. CONCLUSION: Apheresis of peripheral blood mobilized HSCs in rhesus macaques using Spectra Optia is a safe and effective procedure.

Use of CD9 expression to enrich for porcine hematopoietic progenitors.

OBJECTIVE: The aim of this study was to develop novel markers for enrichment of hematopoietic progenitors from bone marrow of swine. MATERIALS AND METHODS: We previously showed that pig bone marrow contains a "side population" (SP) of Hoechst dye-effluxing cells that resembles the hematopoietic stem cell (HSC)-containing murine SP and therefore represents a putative pig stem cell population. We screened a panel of monoclonal antibodies for those that allowed positive or negative enrichment of porcine SP cells and tested one of these for enrichment of hematopoietic progenitors in short-term and long-term in vitro assays. We then screened an expression library to clone the gene whose product is recognized by this antibody. RESULTS: Among a panel of 35 monoclonal lines screened, we found three that were useful for positive enrichment of SP cells and seven for negative enrichment. The 4-6 monoclonal line, allowing around 10-fold negative enrichment of SP cells, recognized the product of the porcine CD9 gene. Hematopoietic progenitors measured by short-term colony-forming unit and long-term cobblestone area-forming cell assays were around 10-fold enriched in the CD9(negative/low) fraction and were significantly depleted in the CD9(high) fraction. CONCLUSIONS: The antibody against the porcine CD9 gene product may be of use for enrichment of porcine hematopoietic stem cells. This approach to identify novel markers for enrichment of hematopoietic progenitors may be applicable to other mammalian species.

Peripheral blood progenitor cell mobilization and leukapheresis in pigs.

BACKGROUND AND PURPOSE: The pig is being investigated as an organ donor for humans. Induction of immunologic tolerance to pig tissues in primates would overcome the major immunologic barriers to xenotransplantation. A proven method of inducing tolerance to allografts is by the induction of mixed hematopoietic chimerism by bone marrow transplantation. We are therefore investigating induction of mixed hematopoietic chimerism in the pig-to-baboon model. METHODS: To obtain large numbers of pig hematopoietic cells, leukapheresis was used to collect blood cell products in miniature swine (n = 5) after progenitor cell mobilization by use of a course of hematopoietic growth factors (cytokines), consisting of porcine interleukin 3, porcine stem cell factor, and human granulocyte colony-stimulating factor. RESULTS: Cytokine therapy and leukapheresis were well tolerated. Cytokine therapy increased the total white blood cell count and allowed large numbers of leukocytes (60 x 10(10)) to be obtained by apheresis, of which approximately 0.1% were granulocyte-erythrocyte-monocyte-megakaryocyte colony-forming units (CFU-GEMMs), which are considered to be representative of hematopoietic progenitors with multi-lineage potential. CONCLUSIONS: The combination of cytokine therapy and leukapheresis enables hematopoietic progenitor cells to be obtained safely from miniature swine.