Rhesus Monkey Bone Marrow Cells Research Articles

Efficient detection and selection of immature rhesus monkey and human CD34+ hematopoietic cells expressing the enhanced green fluorescent protein (EGFP).

The feasibility of using the enhanced green fluorescent protein (EGFP) as a selectable reporter molecule of retroviral-mediated gene transfer in immature rhesus monkey and human CD34+ hematopoietic cells was examined. Retroviral transduction with the MFG-EGFP retroviral vector resulted in readily detectable EGFP expression in 27% of human and 11-35% of rhesus monkey bone marrow cells, and in 17-38% of rhesus monkey peripheral blood cells mobilized with FLT3 ligand (FL) and granulocyte colony-stimulating factor (G-CSF). In addition, we used the human CD34+ KG1A cell line as a model to study viability and growth of successfully transduced cells. Cultures of mock- and EGFP-transduced KG1A cells generated equal viable cell numbers for at least 1 month, indicating the absence of a cytotoxic effect of EGFP expression in these cells. FACS selection on the basis of EGFP and CD34 expression resulted in enriched subsets (> or = 87%) of CD34+ EGFP-negative and CD34+ EGFP-positive KG1A, rhesus monkey and human bone marrow cells, demonstrating the potential of obtaining almost pure populations of transduced immature hematopoietic cells. EGFP expression was also readily demonstrated in erythroid and granulocyte/macrophage colonies derived from the CD34+ EGFP-positive rhesus monkey and human bone marrow cells by either inverted fluorescence microscopy or flow cytometry. Using four-color flow cytometry, EGFP expression could also be demonstrated in viable and phenotypically defined immature subpopulations of the CD34+ cells, ie those expressing little or no HLA-DR (rhesus monkey) or CD38 (human) antigens at the cell surface. These results demonstrate that EGFP is a very useful marker to monitor gene transfer efficiency in phenotypically defined immature rhesus monkey and human hematopoietic cell types and to select for these cells by multicolor flow cytometry prior to transplantation.

Improved Amphotropic Retrovirus‐Mediated Gene Transfer into Hematopoietic Stem Cells

The efficiency of amphotropic retrovirus-mediated gene transfer into human Hematopoietic Stem Cells (HSC) is less than 1%. This has impeded gene therapy for hematopoietic diseases. In this study we demonstrate that populations of mouse and human HSC contain low to undetectable levels of the amphotropic virus receptor mRNA (ampho R mRNA), and are resistant to transduction with amphotropic retroviral vectors. In a subpopulation of mouse HSC expressing 7-fold higher levels of ampho R mRNA, transduction with amphotropic retrovirus vectors was 30-fold higher. We conclude that retrovirus transduction of HSC correlates with ampho R mRNA levels. Our results predict that alternative sources of HSC or retroviruses will be required for human gene therapy of hematopoietic diseases. One alternative source of stem cells is from individuals treated with cytokines. We have previously shown that mice treated with G-CSF and SCF have an immediate increase in peripheral blood HSC immediately after treatment, followed by a 10-fold increase in bone marrow HSC 14 days after treatment. In this report we show that when rhesus monkey bone marrow cells collected 14 days after G-CSF and SCF treatment were transduced with amphotropic retroviruses, gene transfer levels were approximately 10%, which was easily detected by Southern blot analysis. We conclude that the increased gene transfer may be the result of increased expression of the amphotropic retrovirus receptor, increased numbers of cycling HSC or both.

Cryopreserved Primate Bone Marrow Cells Can Be Used for Retroviral-Mediated Gene Transfer

Retroviral-mediated gene transfer into Rhesus monkey bone marrow cells, which were cryopreserved, stored, and then transduced at the time of thawing, was studied for potential application in gene therapy protocols. Albumin density gradient fractionation was used to define subpopulations of cryopreserved cells transduced by a murine retroviral vector. The transfer of the bacterial neomycin phosphotransferase gene into Rhesus monkey bone marrow that was cryopreserved and thawed was found to be preferential in a light-density population enriched for granulocyte-macrophage colony-forming units (CFU-GM). These results are similar to results obtained with freshly harvested bone marrow in which this population is enriched for CD34 antigen-positive cells, as well as for cells that were undergoing cell division. This method may be useful in enriching for transduced precursors in future gene transfer experiments in primates.

Development of a High‐Titer Retrovirus Producer Cell Line and Strategies for Retrovirus‐mediated Gene Transfer into Rhesus Monkey Hematopoietic Stem Cells

Retroviral-mediated gene transfer into pluripotent hematopoietic stem cells has been difficult to achieve in large animal models. We have compared several infection protocols in a murine model system and concluded that bone marrow can be maintained and infected in vitro for 2-6 days. We have also developed an amphotropic producer clone that generates greater than 10(10) recombinant retroviral particles (CFU) per milliliter of culture medium. Autologous rhesus monkey bone marrow cells were co-cultured with either high- (2 x 10(10) CFU/ml) or low- (5 x 10(6) CFU/ml) titer producer clones for 4-6 days and reinfused into sublethally irradiated animals. The proviral genome was detected in blood and bone marrow cells from all three animals reconstituted with cells co-cultured with the high-titer producer cells. In contrast, three animals reconstituted with bone marrow co-cultured with the low-titer producer clone exhibited no evidence of gene transfer.

Development of a high-titer retrovirus producer cell line capable of gene transfer into rhesus monkey hematopoietic stem cells.

Retroviral-mediated gene transfer into primitive hematopoietic cells has been difficult to achieve in large-animal models. We have developed an amphotropic producer clone that generates greater than 10(10) recombinant retroviral particles (colony-forming units) per ml of culture medium. Autologous rhesus monkey bone-marrow cells were cocultured with either high (2 x 10(10) colony-forming units/ml) or low (5 x 10(6) colony-forming units/ml) titer producer clones for 4-6 days and reinfused into sublethally irradiated animals. The proviral genome was detected in blood and bone-marrow cells from all three animals reconstituted with cells cocultured with the high-titer producer cells. In contrast, three animals reconstituted with bone marrow cocultured with the low-titer producer clone exhibited no evidence of gene transfer.

Sedimentation velocity characterisation of the cell cycle of granulocytic progenitor cells in monkey hemopoietic tissue.

AbstractSedimentation velocity separation of Rhesus monkey bone marrow cells has demonstrated a reproducible but heterogeneous size distribution of cells capable of forming granulocytic colonies in agar culture (CFC's). This heterogeneity is shown to be due to the cell cycle status of the progenitor cell population. In vitro exposure of bone marrow cells to lethal doses of tritiated thymidine (H3TdR) either before or after separation restricts the size distribution of CFC's, greatly reducing the proportion of rapidly sedimenting cells. The calculation of the volume distribution of such cells before and after H3TdR exposure indicates that 55% of total CFC's in adult marrow are in G0 or G1 with a volume of 410 μ3, 42% are in S phase and of volume 450–950 μ3, and the remainder are in G2 and mitosis with a volume of between 600–950 μ3. CFC's in mid gestation fetal liver were larger than their adult counterparts and were of homogeneous volume indicative of a single non cycling population with no evidence of an S or G2 component. H3TdR exposure confirmed the non‐cycling status of these fetal progenitor cells.