Characterization of peripheral blood stem cells in mice

Peripheral blood stem cells (PBSCs) were mobilized in mice by treatment with cytosine-arabinoside on day 0, followed by the administration by injection of granulocyte colony-stimulating factor for 4 days. There were remarkable increases in the numbers of cells with lineage-negative (Lin-) c-kit+ markers, cells with colony-forming unit-cell (CFU-C) and colony-forming unit-spleen (CFU-S) activities, and cells with marrow- repopulating ability (MRA) in the extramedullary sites (the spleen, peripheral blood, and liver) on day 5, whereas the number of these immature hematopoietic cells decreased in the bone marrow (BM) on day 5. This finding suggests the mobilization of immature hematopoietic cells from the BM to the extramedullary sites. Three-color flow cytometric analyses showed that CD4 antigen was not expressed on the Lin-Sca-1+ cells in the mobilized PB cells (PBCs), although CD4lo cells were found in those of normal BM cells. Lin-c-kit+ cells in the mobilized PBCs contained more cells with immature phenotypes (Sca-1+, Thy1.2lo, CD71-, and Rh123dull) than in normal BMCs, indicating an alteration of the hierarchical composition of the Lin-c-kit+ cells. The Lin-c-kit+Sca-1+ cells in the mobilized PBCs had similar CFU-C and CFU- S activities to those in normal BMCs. Electron microscopic studies of these cells in the mobilized PBCs showed that only 10% to 20% of these cells had a thin rim of cytoplasm with poorly developed organelles. Allogeneic transplantation [B6 --> C3H] of PBSCs showed long-term reconstituting activity across the major histocompatibility complex barrier 24 weeks after transplantation, although longer observation is necessary.     

Enhanced antileukemic activity of allogeneic peripheral blood progenitor cell transplants following donor treatment with the combination of granulocyte colony-stimulating factor (G-CSF) and stem cell factor (SCF) in a murine transplantation model

Allogeneic peripheral blood progenitor cells (PBPCs) have mostly been mobilized by granulocyte colony-stimulating factor (G-CSF). There is neither clinical nor experimental data available addressing the question if other hematopoietic growth factors or combinations thereof might influence engraftment, graft-versus-host disease (GvHD), and graft-versus-leukemia (GvL) effects after allogeneic peripheral blood progenitor cell transplantation (PBPCT). We used a murine model to investigate these parameters after transplantation of PBPCs mobilized with G-CSF and SCF either alone or in combination. Treatment of splenectomized DBA and Balb/c mice with 250 microg/kg/day G-CSF for 5 days resulted in an increase of CFU-gm from 0 to 53/microl. The highest progenitor cell numbers (147/microl) were observed after treatment with 100 microg/kg/day SCF administered in conjunction with G-SCF. No differences were detected with regard to the number of T cells (CD3+), T cell subsets (CD4+, CD8+), B cells (CD19+) and NK cells (NK1.1+) in PBPC grafts mobilized by G-CSF plus SCF compared to those mobilized with G-CSF alone. The antileukemic activity of syngeneic and MHC-identical allogeneic PBPC grafts was investigated in lethally irradiated Balb/c mice bearing the B-lymphatic leukemia cell line A20. In this model, PBPCs mobilized by G-CSF plus SCF exerted a significantly higher antileukemic activity compared to grafts mobilized by G-CSF alone (94 vs 71% freedom from leukemia at day 100, P<0.05). The antileukemic effect was lowest after BMT (38% freedom from leukemia). Since significant differences in the incidence of lethal GvHD were not observed, improved GVL-activity resulted in superior overall survival. Our data demonstrate that the utilization of specific hematopoietic growth factors not only improve the yield of hematopoietic progenitor cells but can also significantly enhance the immunotherapeutic potential of allografts.     

Mobilization of early hematopoietic progenitor cells with BB-10010: a genetically engineered variant of human macrophage inflammatory protein- 1 alpha

BB-10010 is a genetically engineered variant of human macrophage inflammatory protein-1 alpha with improved solution properties. We show here that it mobilizes stem cells into the peripheral blood. We investigated the mobilizing effects of BB-10010 on the numbers of circulating 8-day spleen colony-forming units (CFU-S8), CFU-S12, and progenitors with marrow repopulating ability (MRA). A single subcutaneous dose of BB-10010 caused a twofold increase in circulating numbers of CFU-S8, CFU-S12, and MRA 30 minutes after dosing. We also investigated the effects of granulocyte colony-stimulating factor (G- CSF) and the combination of G-CSF with BB-10010 on progenitor mobilization. Two days of G-CSF treatment increased circulating CFU-S8, CFU-S12, and MRA progenitors by 25.7-, 19.8-, and 27.7-fold. A single administration of BB-10010 after 2 days of G-CSF treatment increased circulating CFU-S8, CFU-S12, and MRA even further to 38-, 33-, and 100- fold. Splenectomy resulted in increased circulating progenitor numbers but did not change the pattern of mobilization. Two days of treatment with G-CSF then increased circulating CFU-S8, CFU-S12, and MRA by 64-, 69-, and 32-fold. A single BB-10010 administration after G-CSF treatment further increased them to 85-, 117-, and 140-fold, respectively, compared with control. We conclude that BB-10010 causes a rapid increase in the number of circulating hematopoietic progenitors and further enhances the numbers induced by pretreatment with G-CSF. BB- 10010 preferentially mobilized the more primitive progenitors with marrow repopulating activity, releasing four times the number achieved with G-CSF alone. Translated into a clinical setting, this improvement in progenitor cell mobilization may enhance the efficiency of harvest and the quality of grafts for peripheral blood stem cell transplantation.     

Progenitor content of autologous grafts: mobilized bone marrow vs mobilized blood

The progenitor content of autologous peripheral blood progenitor and stem cell collections is a major determinant of prompt hematopoietic recovery following autologous stem cell transplantation. We analyzed unstimulated bone marrow (BM) and peripheral blood (PB) apheresis products in comparison to those collected following G-CSF or GM-CSF stimulation. We quantitated their committed (CFU-GM) and primitive (long-term culture-initiating cells, LTC-IC) progenitors in relation to hematologic recovery in 63 patients undergoing autografting for lymphoid malignancies. G-CSF, but not GM-CSF, substantially enriched the committed progenitor content (2.5-3.6-fold) of both PB and BM grafts. G-CSF also enriched the LTC-IC content of BM and PB compared to control grafts. GM-CSF augmented (11.5-fold) the LTC-IC content of stimulated BM, but not GM-CSF-mobilized PB. Neutrophil recovery was substantially quicker in recipients of BM or PB mobilized with G-CSF or GM-CSF. In contrast, red cell and platelet recovery was accelerated in recipients of GM-CSF-stimulated BM (but not PB) and G-CSF-stimulated PB (but not BM). No direct correlation between progenitor dose and hematopoietic recovery for neutrophils, platelets or red cells was observed. Cytokine stimulation can augment the committed and more primitive multilineage progenitor content of BM and PB grafts, to a differing extent. The uncertain relationship with multilineage myeloid recovery emphasizes the limitations in using clonogenic progenitor analyses to assess the adequacy of an autologous graft prior to transplantation.     

Kinetics of engraftment of CD34(-) and CD34(+) cells from mobilized blood differs from that of CD34(-) and CD34(+) cells from bone marrow

OBJECTIVE: Mobilized peripheral blood (PB) progenitors are increasingly used in autologous and allogeneic transplantation. However, the short- and long-term engraftment potential of mobilized PB or bone marrow (BM) has not been directly compared. Although several studies showed that BM-derived Lin(-)CD34(-) cells contain hemopoietic progenitors, no studies have addressed whether Lin(-)CD34(-) cells from mobilized PB contain hemopoietic progenitors. Here, we compared the short- and long-term engraftment potential of CD34(+) cells and Lin(-)CD34(-) cells in BM and PB of normal donors who received 5 days of granulocyte colony-stimulating factor (G-CSF). MATERIALS AND METHODS: 35 x 10(3) CD34(+) or Lin(-)CD34(-) cells from G-CSF mobilized BM and PB of normal donors were transplanted in 60-day-old fetal sheep. Animals were evaluated 2 and 6 months after transplantation for human hemopoietic cells. In addition, cells recovered after 2 months from fetal sheep were serially passaged to secondary and tertiary recipients to assess long-term engrafting cells. RESULTS: Mobilized PB CD34(+) cells supported earlier development of human hemopoiesis than BM CD34(+) cells. When serially transferred to secondary and tertiary recipients, earlier exhaustion of human hematopoiesis was seen for PB than BM CD34(+) cells. A similar degree of chimerism was seen for Lin(-)CD34(-) cells from PB or BM in primary recipients. We again observed earlier exhaustion of human hemopoiesis with serial transplantation of PB than BM Lin(-)CD34(-) cells. CONCLUSIONS: Differences exist in the short- and long-term repopulating ability of cells in PB and BM from G-CSF mobilized normal donors, and this is independent of the phenotype. Studies are ongoing to examine if this reflects intrinsic differences in the repopulating potential between progenitors from PB and BM, or a lower frequency of long-term repopulating cells in PB than BM CD34(+) and Lin(-)CD34(-) cells, that may not be apparent if larger numbers of cells are transplanted.     

Improved retroviral gene transfer into murine and Rhesus peripheral blood or bone marrow repopulating cells primed in vivo with stem cell factor and granulocyte colony-stimulating factor.

In previous studies we showed that 5 days of treatment with granulocyte colony-stimulating factor (G-CSF) and stem cell factor (SCF) mobilized murine repopulating cells to the peripheral blood (PB) and that these cells could be efficiently transduced with retroviral vectors. We also found that, 7-14 days after cytokine treatment, the repopulating ability of murine bone marrow (BM) increased 10-fold. In this study we examined the efficiency of gene transfer into cytokine-primed murine BM cells and extended our observations to a nonhuman primate autologous transplantation model. G-CSF/SCF-primed murine BM cells collected 7-14 days after cytokine treatment were equivalent to post-5-fluorouracil BM or G-CSF/SCF-mobilized PB cells as targets for retroviral gene transfer. In nonhuman primates, CD34-enriched PB cells collected after 5 days of G-CSF/SCF treatment and CD34-enriched BM cells collected 14 days later were superior targets for retroviral gene transfer. When a clinically approved supernatant infection protocol with low-titer vector preparations was used, monkeys had up to 5% of circulating cells containing the vector for up to a year after transplantation. This relatively high level of gene transfer was confirmed by Southern blot analysis. Engraftment after transplantation using primed BM cells was more rapid than that using steady-state bone marrow, and the fraction of BM cells saving the most primitive CD34+/CD38- or CD34+/CD38dim phenotype increased 3-fold. We conclude that cytokine priming with G-CSF/SCF may allow collection of increased numbers of primitive cells from both the PB and BM that have improved susceptibility to retroviral transduction, with many potential applications in hematopoietic stem cell-directed gene therapy.     

Single leukapheresis products collected from healthy donors after the administration of granulocyte colony-stimulating factor contain ten-fold higher numbers of long-term reconstituting hematopoietic progenitor cells than conventional bone marrow allografts

Cytokine-mobilized peripheral blood progenitor cells (PBPCs) have been used successfully for hematopoietic reconstitution following allogeneic transplantation. The ease of harvest, the faster engraftment and the high yield of CD34+ cells have made this source of hematopoietic progenitor cells (HPCs) an attractive alternative to bone marrow (BM). In the present study we compared the engraftment potential of conventional BM allografts and single leukapheresis products (LPs) collected from healthy donors following the administration of granulocyte colony-stimulating factor (G-CSF). For this, lineage-committed and primitive HPCs were assessed by flow cytometry and by colony- and cobblestone area-forming cell (CFC, CAFC) assays. Mean numbers of CD34+ cells in LPs (n = 11) were similar to that of BM grafts (n = 12) (278+/-57 vs 227+/-34 x 10(6) CD34+ cells). The frequencies of CFCs, week 5 CAFCs and week 8 CAFCs were 1.6-, 8.4- and 10.3-fold higher in the CD34+ compartment of mobilized blood than that of marrow, resulting in significantly higher yields of clonogenic HPCs in LPs when compared to BM grafts. We conclude that G-CSF preferentially mobilizes clonogenic progenitors capable of short- and, in particular, longterm reconstitution, and that the engraftment potential of single LPs is superior to that of BM allografts. Hence, the use of PBPCs may be favorable for protocols that include graft manipulations with expected cell loss (eg T cell depletion, CD34+ selection). PBPCs may also be advantageous for gene therapy trials due to their high numbers of potential target cells (eg CAFCs).     

Developmental fate of hematopoietic stem cells: the study of individual hematopoietic clones at the level of antigen-responsive B lymphocytes

We have shown previously that hematopoiesis in mice reconstituted with retrovirally marked hematopoietic stem cells (HSCs) is provided by multiple, mainly short-lived clones, as measured by retroviral insertion site analysis of individual spleen colony-forming unit (CFU-S)-derived colonies. However, the CFU-S is the relatively early progenitor and the contribution of each CFU-S in the steady-state hematopoiesis is uncertain. Here, we have studied the fate of individual mature B cells, as well as CFU-S, representing the progeny of retrovirally transduced marrow-repopulating cells (MRC). B-cells-generated hybridomas and CFU-S-derived colonies were used to determine the clonal composition of hematolymphopoiesis at the single-cell level. Bone marrow (BM) cells and splenocytes (approximately 1/3-1/2 of spleen at a time) from mice reconstituted with retrovirally marked syngeneic BM cells were repeatedly collected at 3, 10, and 16 months post-transplant. The percentage of retrovirally marked CFU-S and B-cell-produced hybridomas was about 50% at 3 months and decreased to 10-15% at 10 months after reconstitution in spite of stable degree of chimerism. The clonal origin of BM-derived CFU-S and spleen-derived B-cell hybridomas was detected by Southern blot analysis. Overall, DNA obtained from 159 retrovirally marked spleen colonies, 287 hybridomas and 43 BM samples were studied. Multiple simultaneously functioning clones of MRC-derived B cells were observed. The same individual clones among hybridomas and CFU-S were identified in three out of 11 mice. Thus, hematopoiesis is generated by multiple hematopoietic clones some of which can simultaneously contribute to both mature lymphoid cells and myeloid progenitors. These data establish that the stem cell compartment functions by continuously producing progeny, which fully but transiently repopulate all lineages.     

Hematologic effects of flt3 ligand in vivo in mice

We have investigated the effects of in vivo treatment with flt3 ligand (FL) on murine hematopoiesis, including mobilization of progenitors into the peripheral blood (PB). Mice were injected once daily with 10 micrograms recombinant human FL for 15 days. On days 3, 5, 8, 10, 15, and 22, mice were killed and analyzed for the number of leukocytes and colony-forming units (CFU) in bone marrow (BM), spleen, and PB. Splenic and PB cellularity increased with time in FL-treated mice. In the spleen, there was an increase in B cells, myeloid cells, and nucleated erythroid cells; in the PB, there was an increase in lymphocytes, granulocytes, and monocytic cells. The maximal number of CFU in the BM was observed after 3 days of FL treatment, giving 3.7- and 7.3-fold increases in CFU-granulocyte-macrophage (CFU-GM) and CFU-granulocyte, erythrocyte, monocyte, megakaryocyte (CFU-GEMM), respectively, compared with mouse serum albumin (MSA)-treated controls. After 8 days of FL treatment, there was a maximal 123- and 108-fold increase in splenic CFU-GM and CFU-GEMM, respectively. The maximal number CFU-GM and CFU- GEMM were seen in PB on day 10, with 537- and 585-fold increases, respectively. Burst-forming units-erythroid (BFU-E) increased in the same time frame as those of CFU-GM and CFU-GEMM in BM, spleen, and PB, although the magnitude was not as great. Primitive day-13 CFU-spleen (CFU-S) and phenotypically defined stem cells were also mobilized into the PB of FL-treated mice with similar kinetics and magnitude to that of CFU-GM and CFU-GEMM. We conclude from these studies that FL, when administered as a single agent, is a potent mobilizer of hematopoietic progenitors into the PB.     

Rapid mobilization of murine hematopoietic stem cells with enhanced engraftment properties and evaluation of hematopoietic progenitor cell mobilization in rhesus monkeys by a single injection of SB-251353, a specific truncated form of the human CXC chemokine GRObeta

SB-251353 is an N-terminal truncated form of the human CXC chemokine GRObeta. Recombinant SB-251353 was profiled in murine and rhesus monkey peripheral blood stem cell mobilization and transplantation models. SB-251353 rapidly and transiently mobilized hematopoietic stem cells and neutrophils into the peripheral blood after a single subcutaneous injection. Transplantation of equivalent numbers of hematopoietic stem cells mobilized by SB-251353 into lethally irradiated mice resulted in faster neutrophil and platelet recovery than stem cells mobilized by granulocyte colony-stimulating factor (G-CSF). A single injection of SB-251353 in combination with 4 days of G-CSF administration resulted in augmented stem and progenitor cell mobilization 5-fold greater than G-CSF alone. Augmented stem cell mobilization could also be demonstrated in mice when a single injection of SB-251353 was administered with only one-day treatment with G-CSF. In addition, SB-251353, when used as a single agent or in combination with G-CSF, mobilized long-term repopulating stem cells capable of hematopoietic reconstitution of lethally irradiated mice. In rhesus monkeys, a single injection of SB-251353 induced rapid increases in peripheral blood hematopoietic progenitor cells at a 50-fold lower dose than in mice, which indicates a shift in potency. These studies provide evidence that the use of SB-251353 alone or in combination with G-CSF mobilizes hematopoietic stem cells with long-term repopulating ability. In addition, this treatment may (1) reduce the number of apheresis sessions and/or amount of G-CSF required to collect adequate numbers of hematopoietic stem cells for successful peripheral blood cell transplantation and (2) improve hematopoietic recovery after transplantation.     

Inhibition of day-12 spleen colony-forming units by a monoclonal antibody to the murine macrophage/monocyte colony-stimulating factor receptor

Primitive hematopoietic stem cells differentiate into committed progenitors that are thought to selectively express hematopoietic growth factor receptor(s), thereby acquiring hematopoietic growth factor responsiveness. To assess whether hematopoietic stem cells express hematopoietic growth factor receptors, the progenitor activity of bone marrow (BM) fractions, isolated by expression of receptors for macrophage/monocyte colony-stimulating factor (M-CSF), were examined. Recovery of day-12 spleen colony-forming units (CFU-S) is diminished in both M-CSF receptor-positive (M-CSFR+) and M-CSFR- fractions, indicating antibody inhibition of day-12 CFU-S. Incubation of BM cells with antibody without fractionation inhibits 50% to 60% of day-12 CFU- S. This inhibition is specific (control antibodies have no effect) and reversible by removal of bound antibody at low pH. Incubating BM cells with control or antireceptor antibody does not affect day-8 CFU-S, which are predominantly erythroid. Treating sublethally irradiated mice with antibody inhibits endogenous day-12 CFU-S. These results indicate that some early progenitors express M-CSFRs, and blocking M-CSFRs inhibits the ability of these progenitors to form colonies, possibly because of inactivation caused by prolonged receptor blockade.     

Quantitative and cell-cycle differences in progenitor cells mobilized by recombinant human interleukin-7 and recombinant human granulocyte colony-stimulating factor

Administration of recombinant human interleukin-7 (rhIL-7) to mice increases the exportation of myeloid progenitors (colony-forming unit [CFU]-c and CFU-granulocyte erythroid megakaryocyte macrophage [CFU- GEMM]) from the bone marrow (BM) to peripheral organs, including blood, and also increases the number of primitive progenitor and stem cells in the peripheral blood (PB). We now report that combined treatment of mice with rhIL-7 and recombinant human granulocyte-colony stimulating factor (rhG-CSF) stimulates a twofold to 10-fold increase in the total number of PB CFU-c, and a twofold to fivefold increase in the total number of PB CFU-spleen at day 8 (CFU-S8) over the increase stimulated by rhIL-7 or rhG-CSF alone. In addition, the quality of mobilized cells with trilineage, long-term marrow-repopulating activity is maintained or increased in mice treated with rhIL-7 and rhG-CSF compared with rhIL- 7 or rhG-CSF alone. These differences in mobilizing efficiency suggest qualitative differences in the mechanisms by which rhIL-7 and rhG-CSF mobilize progenitor cells, in fact, the functional status of progenitor cells mobilized by rhIL-7 differs from that of cells mobilized by rhG- CSF in that the incidence of actively cycling (S-phase) progenitors obtained from the PB is about 20-fold higher for rhIL-7-treated mice than for mice treated with rhG-CSF. These results suggest the use of rhIL-7-mobilized progenitor/stem cells for gene-modification and tracking studies, and highlight different functions and rates of repopulation after reconstitution with PB leukocytes obtained from mice treated with rhIL-7 versus rhG-CSF.     

Use of limiting-dilution type long-term marrow cultures in frequency analysis of marrow-repopulating and spleen colony-forming hematopoietic stem cells in the mouse.

We have developed an in vitro clonal assay of murine hematopoietic precursor cells that form spleen colonies (CFU-S day 12) or produce in vitro clonable progenitors in the marrow (MRA cells) of lethally irradiated mice. The assay is essentially a long-term bone marrow culture in microtiter wells containing marrow-derived stromal "feeders" depleted for hematopoietic activity by irradiation. To test the validity of the assay as a quantitative in vitro stem cell assay, a series of unsorted and physically sorted bone marrow cells were simultaneously assayed in vivo and overlaid on the feeders in a range of concentrations, while frequencies of cells forming hematopoietic clones (cobblestone area forming cells, CAFC) were calculated by means of Poisson statistics. Linear regression analysis of the data showed high correlations between the frequency of CFU-S day 12 and CAFC day 10, and between MRA cells and CAFC day 28. A majority of MRA activity and CAFC day 28 was separable from CFU-S day 12 and CAFC day 10. This correlation study validates the CAFC system as a clonal assay facilitation both the quantitative assessment of a series of subsets in the hematopoietic stem cell hierarchy and the study of single long-term repopulating cells in vitro.     

A comparative study of the phenotype and proliferative capacity of peripheral blood (PB) CD34+ cells mobilized by four different protocols and those of steady-phase PB and bone marrow CD34+ cells

Peripheral blood (PB) CD34+ cells from four commonly used mobilization protocols were studied to compare their phenotype and proliferative capacity with steady-state PB or bone marrow (BM) CD34+ cells. Mobilized PB CD34+ cells were collected during hematopoietic recovery after myelosuppressive chemotherapy with or without granulocyte- macrophage colony-stimulating factor (GM-CSF) or granulocyte colony- stimulating factor (G-CSF) or during G-CSF administration alone. The expression of activation and lineage-associated markers and c-kit gene product were studied by flow cytometry. Proliferative capacity was measured by generation of nascent myeloid progenitor cells (granulocyte- macrophage colony-stimulating factor; CFU-GM) and nucleated cells in a stroma-free liquid culture stimulated by a combination of six hematopoietic growth factors (interleukin-1 (IL-1), IL-3, IL-6, GM-CSF, G-CSF, and stem cell factor). G-CSF-mobilized CD34+ cells have the highest percentage of CD38- cells (P < .0081), but otherwise, CD34+ cells from different mobilization protocols were similar to one another in their phenotype and proliferative capacity. The spectrum of primitive and mature myeloid progenitors in mobilized PB CD34+ cells was similar to their steady-state counterparts, but the percentages of CD34+ cells expressing CD10 or CD19 were lower (P < .0028). Although steady-state PB and chemotherapy-mobilized CD34+ cells generated fewer CFU-GM at day 21 than G-CSF-mobilized and steady-state BM CD34+ cells (P < .0449), the generation of nucleated cells and CFU-GM were otherwise comparable. The presence of increased or comparable numbers of hematopoietic progenitors within PB collections with equivalent proliferative capacity to BM CD34+ cells is not unexpected given the rapid and complete hematopoietic reconstitution observed with mobilized PB. However, all four types of mobilized PB CD34+ cells are different from steady-state BM CD34+ cells in that they express less c-kit (P < .0002) and CD71 (P < .04) and retain less rhodamine 123 (P < .0001). These observations are novel and suggest that different mobilization protocols may act via similar pathways involving the down-regulation of c-kit and may be independent of cell-cycle status.     

Very small embryonic-like stem cells: characterization, developmental origin, and biological significance

Bone marrow (BM) was, for many years, primarily envisioned as the "home organ" of hematopoietic stem cells (HSC). Augmenting evidence demonstrates, however, that BM, in addition to HSC, also contains a heterogeneous population of non-HSC. Recently, our group identified in BM and other adult tissues a population of very small embryonic-like stem cells (VSELs), which express several markers characteristic for pluripotent stem cells that are characteristic for epiblast/germ line-derived stem cells. Thus, we hypothesize that VSELs are a population of epiblast-derived cells that are deposited during early gastrulation in developing tissues/organs and play an important role in turnover of tissue-specific/committed stem cells. In this context, VSELs deposited in BM can give rise to long-term repopulating HSC. VSELs could be also mobilized into peripheral blood (PB), and the number of these cells circulating in PB increases during stress and tissue/organ injuries. Finally, we envision that in pathological situations VSELs are involved in development of some malignancies (e.g., teratomas, germinal tumors).     

Cyclophosphamide/granulocyte colony-stimulating factor causes selective mobilization of bone marrow hematopoietic stem cells into the blood after M phase of the cell cycle

Cytokine-mobilized peripheral blood hematopoietic stem cells (MPB HSC) are widely used for transplantation in the treatment of malignancies, but the mechanism of HSC mobilization is unclear. Although many HSC in bone marrow (BM) cycle rapidly and expand their numbers in response to cytoreductive agents, such as cyclophosphamide (CY), and cytokines, such as granulocyte colony-stimulating factor (G-CSF), MPB HSC are almost all in the G(0) or G(1) phase of the cell cycle. This has raised the question of whether a subset of noncycling BM HSC is selectively released, or whether cycling BM HSC are mobilized after M phase, but before the next S phase of the cell cycle. To distinguish between these possibilities, mice were treated with one dose of CY followed by daily doses of G-CSF, and dividing cells were marked by administration of bromodeoxyuridine (BrdU) during the interval that BM HSC are expanding. After CY and 4 days of G-CSF, 98.5% of the 2n DNA content long-term repopulating MPB (LT)-HSC stained positively for BrdU, and therefore derived from cells that divided during the treatment interval. Next, LT-HSC from mice previously treated with a single dose of CY, which kills cycling cells, and 3 daily doses of G-CSF, were nearly all killed by a second dose of CY, suggesting that CY/G-CSF causes virtually all LT-HSC to cycle. Analysis of cyclin D2 messenger RNA (mRNA) expression and total RNA content of MPB HSC suggests that these cells are mostly in G(1) phase. After CY/G-CSF treatment, virtually all BM LT-HSC enter the cell cycle; some of these HSC then migrate into the blood, specifically after M phase, and are rapidly recruited to particular hematopoietic organs.     

Synergistic effects of pegylated recombinant human megakaryocyte growth and development factor and granulocyte colony-stimulating factor on mobilization of hematopoietic progenitor and stem cells with long-term repopulating ability into peripheral blood in mice

We investigated the effects of pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) on peripheral blood progenitor cell (PBPC) mobilization and the combined effect of PEG-rHuMGDF plus recombinant human granulocyte colony-stimulating factor (rhG-CSF) in C57BL/6 mice. Treatment of mice with PEG-rHuMGDF increased the numbers of day 8 and day 12 spleen colony-forming units (CFU-S), and pre-CFU-S in the PB. Ten days administration of PEG-rHuMGDF could mobilize higher numbers of days 8 and day 12 CFU-S than 5 days administration. An optimal dose of PEG-rHuMGDF mobilized a higher number of committed progenitor cells (day 8 CFU-S) and a lower number of immature progenitor cells (pre-CFU-S) into PB than rhG-CSF. The combined administration of optimal or suboptimal doses of PEG-rHuMGDF and rhG-CSF induced synergistic effects on mobilization of CFU-S and pre-CFU-S into PB compared to either factor alone. Four months after sex-mismatched PBPC transplantation, long-term donor-derived engraftment was observed in all recipients that had been transplanted with PBPCs mobilized by rhG-CSF and/or PEG-rHuMGDF, as determined by Y-chromosome polymerase chain reaction (PCR) analysis. Our data suggest that cytokine-induced pathways for PBPC mobilization may be different between PEG-rHuMGDF and rhG-CSF and indicate that PEG-rHuMGDF alone or the combination with rhG-CSF may be useful in effective PBPC mobilization.     

Mobilized peripheral blood stem cells provide rapid reconstitution but impaired long-term engraftment in a mouse model

In this study, we use competitive repopulation to compare the quality and frequency of stem cells isolated from mobilized blood with stem cells isolated from bone marrow (BM) in a mouse model. Lin(-)Sca-1(+)c-Kit(+) (LSK) cells were harvested from control BM and peripheral blood of mice following granulocyte colony-stimulating factor (G-CSF) administration. LSK cells were used because of their resemblance to human CD34(+) cells. We confirmed that transplantation of phenotypically defined mobilized peripheral blood (MPB) stem cells results in rapid recovery of blood counts. However, in vitro results indicated that LSK cells purified from MPB had lower cobblestone area-forming cell day 35 activity compared to BM. Additionally, evaluation of chimerism after co-transplantation of LSK cells purified from blood and BM revealed that MPB stem cells contained 25-fold less repopulation potential compared to BM stem cells. Competitive repopulating unit frequency analysis showed that freshly isolated MPB LSK cells have 8.8-fold fewer cells with long-term repopulating ability compared to BM LSK cells. Secondary transplantation showed no further decline in contribution of hematopoiesis relative to BM. We conclude that the reduced frequency of stem cells within the LSK population of MPB, rather than poorer quality, causes the reduced repopulation potential.Bone Marrow Transplantation (2007) 39, 401-409. doi:10.1038/sj.bmt.1705601; published online 12 February 2007.     

Implantation of bone marrow beneath the kidney capsule results in transfer not only of functional stroma but also of hematopoietic repopulating cells

Renal ossicles are ossified structures developed after the implantation of a bone marrow (BM) plug beneath the kidney capsule. The authors have investigated the origin of the hematopoietic cells in murine renal ossicles by conducting sex-mismatched implants into Ly-5 congenic mice. BM plugs from transgenic mice provided additional genotypic tracers. Flow cytometry analyses on nonadherent cells from long-term cultures established with ossicles excised at 17 to 40 weeks postimplantation evidenced the presence of 5% to 70% of donor-derived myeloid cells. The genetic analysis of the day 12 colony-forming unit (CFU-S(12)) population in ossicles excised at 10 to 40 weeks postimplantation revealed that 16% to 93% of the colonies were of donor origin. Moreover, we describe for the first time the presence of long-term repopulating cells of donor origin in ossicles excised at 10 to 19 weeks postimplantation.