Resting and activated subsets of mouse multipotent hematopoietic stem cells.

The fluorescent vital dye rhodamine 123 (Rh-123), which preferentially accumulates in mitochondrial membranes, can be used as a probe to indicate mitochondrial and hence cellular activity. In this study, mouse bone marrow hematopoietic stem cells were subdivided into Rh-123lo, Rh-123med, and Rh-123hi populations. The Rh-123lo (resting) population was significantly enriched in cells with a higher proliferative potential compared to the Rh-123hi (activated) population. The resting population exhibited a 20-fold greater ability to differentiate into splenic colony-forming units (CFU-S) relative to the activated population, whereas the activated population contained about 4-fold more day 13 CFU-S on primary transfer relative to the resting population. The two populations produced morphologically distinct splenic colonies; however, the frequency and morphology of in vitro colonies were very similar. Only the resting population provided sufficient stem cells to transfer long-term hematopoietic repopulation to secondary recipient animals after lethal irradiation. On a single cell level, the resting and activated populations exhibited an equivalent ability to differentiate into lymphoid and myeloid progeny. These observations provide further insight into the heterogeneous nature of CFU-S and directly demonstrate that multipotent hematopoietic stem cells are heterogeneous with regard to their clonogenic capacities.     

Effects of caspase inhibitors on hematopoietic engraftment after short-term culture

The induction of apoptosis during cytokine-induced proliferation of hematopoietic stem and progenitor cells (HSPC) may result in the loss of hematopoietic function. We tested the ability of several caspase inhibitors to maintain transplantation potential of mouse HSPC during in vitro culture. HSPC were isolated from mouse bone marrow by cell sorting and cultured in the presence of steel factor (STL) with or without various caspase inhibitors. After incubation, cells were harvested and tested for in vitro colony-forming cell (CFC) potential and transplantation activity in both short- and long-term in vivo assays. HSPC required STL to retain CFC activity during a 24-h culture at 37 degrees C, and none of three caspase inhibitors could substitute for STL in this respect. In transplant assays, a twofold higher frequency of animals showed donor-derived blood cells 12 weeks after competitive transplantation of 50 HSPC cultured for 4 h in the presence of STL plus n-acetyl-Tyr-Val-Ala-Asp-chloromethyl ketone (ac-YVAD) compared with 50 cells cultured in STL alone. To evaluate the effect of ac-YVAD on short-term engraftment, 500 cultured HSPC were transplanted into lethally irradiated mice. Animals transplanted with cells cultured in the presence of ac-YVAD showed a higher survival rate and a faster recovery of platelets and hematocrit compared with animals transplanted with cells cultured in STL alone. We conclude that both the short-term and the long-term engraftment potentials of HSPC cultured in the presence of STL + ac-YVAD were superior to that obtained from cells cultured in STL alone.     

The spleen is a major site of megakaryopoiesis following transplantation of murine hematopoietic stem cells

The stem cell pool can be fractionated by using the mitochondrial dye, rhodamine-123, into Rho(low) hematopoietic stem cells and Rho(high) progenitors. Rho(low) stem cells permanently engraft all lineages, whereas Rho(high) progenitors transiently produce erythrocytes, without substantial platelet or granulocyte production. We hypothesized that the inability of the Rho(high) cells to produce platelets in vivo was due to the fact that these cells preferentially engraft in the spleen and lack marrow engraftment. Initially, we demonstrated that Rho(high) progenitors produced more megakaryocytes in vitro than Rho(low) stem cells did. To study the activity of the Rho(low) and Rho(high) subsets in vivo, we used mice allelic at the hemoglobin and glucose phosphate isomerase loci to track donor-derived erythropoiesis and thrombopoiesis. Rho(low) stem cells contributed to robust and long-term erythroid and platelet engraftment, whereas Rho(high) progenitors contributed only to transient erythroid engraftment and produced very low numbers of platelets in vivo. Donor-derived megakaryopoiesis occurred at higher densities in the spleen than in the bone marrow in animals receiving Rho(low) stem cells and peaked around day 28. Blockade of splenic engraftment using pertussis toxin did not affect the peak of splenic megakaryopoiesis, supporting the hypothesis that these megakaryocytes were derived from progenitors that originated in the bone marrow. These data emphasize that in vitro behavior of hematopoietic progenitor cell subsets does not always predict their behavior following transplantation. This study supports a major role for the spleen in thrombopoiesis following engraftment of transplanted stem cells in irradiated mice.     

Preliminary in vivo evaluation of the protein transduction domain-modified ATTEMPTS approach in enhancing asparaginase therapy

Asparaginase (ASNase) is an enzyme drug presently approved for the induction of remission in the treatment of patients with acute lymphoblastic leukemia (ALL). The cytotoxic effect of ASNase is derived from its ability to deplete asparagine, an essential amino acid required by certain types of leukemia cells for protein synthesis and survival. Despite its efficacy in enhancing disease remission rate and prolonging complete remission duration in ALL patients, ASNase therapy is nevertheless confounded by a number of serious toxic effects, particularly to organs associated with high protein production (e.g., liver, pancreas), due to the systemic depletion of asparagine. Presented herein is a modified version of our previously established ATTEMPTS protein delivery system that carries the potential to permit a tumor specific, intracellular delivery of ASNase, thereby allowing for a significant reduction of ASNase-induced systemic toxicity. In a previous paper, we already demonstrated the in vitro feasibility of this heparin/protamine-regulated, TAT-mediated system in delivering ASNase directly into ASNase-sensitive murine lymphoma cells. In this article, we further validated the in vivo applicability of this system in animals harboring ASNase-encapsulated L5178Y lymphoma cells. Preliminary results showed that animals inoculated with L5178Y cells containing TAT-ASNase exhibited an extended survival rate of approximately 13% over those harboring L5178Y cells without the encapsulation of ASNase. Furthermore, the TAT-ASNase-treated mice also displayed a significantly improved hematological and liver histological status than the control groups. These findings bring promise to the use of the modified ATTEMPTS delivery system in achieving enhanced ASNase therapy.     

Mouse strain variability in the expression of the hematopoietic stem cell antigen Ly-6A/E by bone marrow cells

The cell surface molecule Ly-6A/E provides a convenient marker for primitive stem cells in the hematopoietic tissues of both fetal and adult mice. However, previous studies have shown that Ly-6A/E expression by lymphocytes is variable depending on the haplotype of the Ly-6 locus. Therefore, strain-specific variation in Ly-6A/E expression by bone marrow (BM) cells was investigated. The results show that Ly-6a mice have, on average, 50% of the number of BM cells expressing Ly-6A/E relative to that for Ly-6b mice. Furthermore, among the 5% of BM cells that do not express antigens characteristic of mature T, B, myeloid, or erythroid lineages, which include the primitive hematopoietic stem cell compartment, Ly-6a mice have, on average, more than fivefold fewer Ly- 6A/E+ cells relative to that for Ly-6b mice. Isolation of Ly-6A/E- and Ly-6A/E+ cells from mice of both haplotypes showed that, whereas 99% of the marrow repopulating activity (MRA) of C57BL/Ka (Ly-6b) mice could be recovered in the Ly-6A/E+ fraction, only about 25% of the MRA of BALB/c (Ly-6a) was recoverable in the same population. On a per-cell basis, the Ly-6A/E+ cells that were isolated from BALB/c mice were essentially equivalent in MRA to those isolated from C57BL/Ka mice. Thus, whereas a large percentage of the hematopoietic stem cells of Ly- 6a mice do not express the Ly-6A/E molecule, the antigen may be used to isolate a subset of stem cells from these mice. These results show that hematopoietic stem cell phenotype can vary between mouse strains and imply that caution should be exercised in the identification of human stem cell antigens such as CD34, because a similar variability may occur between individual humans. To further explore the influence of Ly- 6 haplotype on Ly-6A/E expression by specific cell subsets, lymph-node lymphocytes from a panel of mouse strains were analyzed by multiparameter flow cytometry for correlated expression of Ly-6A/E, CD4, and CD8. All Ly-6a strains examined had less than 20% Ly-6A/E+ cells, and those cells were predominantly CD8+ T lymphocytes. In contrast, the Ly-6b strains had greater than 30% Ly-6A/E+ cells, and those cells included CD4+, CD8+, and B lymphocytes.     

High-frequency cell surface expression of a foreign protein in murine hematopoietic stem cells using a new retroviral vector

A retroviral vector (pSFF) derived from murine Friend spleen focus forming virus was used to transduce murine hematopoietic stem cells and express a cell surface marker protein, mutated murine prion protein, in vitro and in vivo after transplantation. To enhance retroviral vector integration in bone marrow cells, mice were treated with 5-fluorouracil (5-FU) to increase stem cell mitotic activity, which peaked on day 8 post-5-FU. The infectivity titer of the vector, pSFF-mPrP-3F4, was determined by a novel assay in which antigen-positive foci of infected cells were detected after replication and spread of the vector in cultures of mixed packaging cell lines. Infection of Sca-1+/Lineageneg- low cells with pSFF-mPrP-3F4 resulted in marker protein expression in 40% of the progeny cells after 7 days of culture. Transplantation of marrow cells or sorted Sca-1+/Lineageneg-low cells transduced with vector resulted in 3F4-positive mPrP expression in 11% to 37% of donor- derived peripheral blood leukocytes at 2 weeks. Though the percentage of 3F4-positive blood cells gradually declined, at 28 weeks 23% of recipient mice still maintained expression of the marker gene. Expression was observed in lymphoid, myeloid, and erythroid lineages and was detected in Sca-1+/Lineageneg-low marrow cells. The multilineage, high-frequency expression observed suggests that pSFF may be useful in gene therapy directed at hematopoietic stem cells and their differentiated progeny.     

Distinct roles of IL-7 and stem cell factor in the OP9-DL1 T-cell differentiation culture system

OBJECTIVE: The OP9-DL1 culture system is an in vitro model for T-cell development in which activation of the Notch pathway by Delta-like 1 promotes differentiation of mature T cells from progenitors. The roles of specific cytokines in this culture system have not been well defined, and controversy regarding the role of IL-7 has recently emerged. We examined the roles played by IL-7, Flt3 ligand, and stem cell factor (SCF) in differentiation of adult bone marrow cells in the OP9-DL1 culture system. METHODS: Hematopoietic progenitor cells isolated from mouse bone marrow were cultured with OP9 or OP9-DL1 stromal cells and evaluated for T and B lymphocyte differentiation using immunofluorescent staining. RESULTS: IL-7 provided both survival/proliferation and differentiation signals in a dose-dependent manner. T-cell development from the CD4/CD8 double-negative (DN) stage to the CD4/CD8 double-positive (DP) stage required IL-7 provided by the stromal cells, while differentiation from the DP to the CD8 single-positive (SP) stage required addition of exogenous IL-7. SCF favored the proliferation of DN lymphoid progenitors and inhibited differentiation to the DP stage in a dose-dependent manner. Conversely, blocking the function of SCF expressed endogenously by OP9-DL1 cells inhibited proliferation of lymphoid progenitors and accelerated T-lineage differentiation. Flt3 ligand promoted proliferation without affecting differentiation. CONCLUSION: These results validate the OP9-DL1 model for the analysis of T-cell development from bone marrow-derived progenitor cells, and demonstrate specific roles of SCF, IL-7, and Flt3L in promoting efficient T-lineage differentiation.     

Expression of Fc gamma RIII on HeLa 229 cells: possible effect on in vitro neutralization of Chlamydia trachomatis.

The neutralizing activities of a murine immunoglobulin G3 (IgG3) monoclonal antibody specific for the major outer membrane protein of Chlamydia trachomatis and its monovalent Fab fragments were studied by using Syrian hamster kidney (HaK) cells and human epithelial (HeLa 229) cells. The intact IgG3 antibody was neutralizing for HaK cells but was nonneutralizing for HeLa cells. In contrast, monovalent Fab antibody fragments neutralized chlamydial infectivity for both HaK and HeLa cells. Immunofluorescence analysis of HeLa 229 cells with a panel of monoclonal antibodies specific to human Fc gamma receptors revealed the expression of cell surface Fc gamma RIII. We propose that Fc gamma RIII may obscure the chlamydia-neutralizing activity of certain IgG isotypes by facilitating the Fc gamma R-mediated entry of chlamydiae into HeLa 229 cells. These findings may help explain the inconsistencies that are commonly observed in results when HeLa 229 cells are used in chlamydia neutralization assays.