Clinical use of recombinant human hematopoietic growth factors (GM-CSF, IL-3, EPO) in patients with myelodysplastic syndrome.

We have conducted several phase I/II clinical studies in a total of 65 MDS patients utilizing recombinant human hematopoietic growth factors including GM-CSF, IL-3, and EPO. Twenty-seven patients with MDS were treated with either continuous i.v. infusion or single daily s.c. injection of rhGM-CSF at dosages from 15 micrograms/m2 to 1000 micrograms/m2. All of them exhibited white cell responses during the treatment cycles, but no sustained rise in reticulocytes or platelets was recorded. In four of the patients, all with > or = 15% blast cells in the bone marrow, the percentage of circulating blast cells increased during treatment with rhGM-CSF (at dosages of 500 micrograms/m2 and 1000 micrograms/m2, respectively), although no leukemic conversion occurred. Of 9 patients treated so far with rhIL-3 at single daily s.c. dosages of 60 micrograms/m2, all exhibited white cell responses; 8 exhibited significant improved platelet and reticulocyte counts. Nineteen further patients received rhEPO for a period of 14 weeks by s.c. (10,000 U five times weekly) or i.v. bolus administration (150-450 U/kg). None of these patients experienced an increase in white cell and platelet counts. A significant increase of the reticulocyte count was recorded in 3 patients only. Another strategy involves the recruitment of leukemic cells into the cell cycle by hematopoietic growth factors followed by treatment with cycle-specific cytostatic agents. Therefore in 10 patients administration of rhGM-CSF (250 g/m2/day x 14, s.c.) was combined with Ara-C treatment (20 mg/m2/day x 14; s.c.). Initial results of this pilot study available in 5 patients indicated that this approach may control leukemic cell proliferation and may increase number of mature myeloid cells in both bone marrow and peripheral blood. A similar approach utilizing rhIL-3 in conjunction with Ara-C is on-going.     

Effect of liposomal muramyl tripeptide phosphatidylethanolamine and indomethacin on hematopoietic recovery in irradiated mice

The effects of liposomal muramyl tripeptide phosphatidylethanolamine (MTP-PE/MLV, radioprotective immunomodulator; 10 mg/kg) and indomethacin (INDO, inhibitor of prostaglandin production; 2 mg/kg) on post-irradiation recovery of hematopoietic functions in mice were investigated. Two agents with distinct radioprotective mechanisms were administered alone or in combination 24 h and 3 h before exposure to 7 Gy (60)Co radiation. In the post-irradiation period (3-14 days) combined pre-treatment of mice accelerated recovery of bone marrow cellularity, weight of spleen and myelopoietic and erythropoietic activity in both hematopoietic organs, compared to treatment with MTP-PE/MLV or indomethacin alone. In the peripheral blood, improved radioprotective effects of combined drug administration were found in the recovery of reticulocytes and platelet count. No further significant differences in the recovery of leukocyte count were observed in the examined groups until post-irradiation day 14. Within the first 3-6 post-irradiation days, the bone marrow and peripheral blood smears of mice pre-treated with indomethacin alone or its combination with MTP-PE/MLV more frequently featured blast cells and large cells with abundant cytoplasm which could be considered the hematopoietic stem cells.     

EFFECT OF CYCLOPHOSPHAMIDE ON THE HEMATOPOIETIC MICROENVIRONMENTAL FACTORS WHICH INFLUENCE HEMATOPOIETIC STEM CELL PROLIFERATION

Transplanted hematopoietic stem cells (HSC) regenerate more rapidly in the femoral marrow of lethally irradiated hosts pretreated with cyclophosphamide (CY) 4 days prior to X-irradiation than they do in that of uninjected irradiated hosts (control). On the other hand, regeneration of HSC transplanted into irradiated hosts given CY 7 days before X-irradiation is slower than in controls.
The microenvironment in the femoral marrow was studied at various times after giving CY. Four days after injecting CY, the number of colony forming units (CFU), total nucleated hematopoietic cells, and mature myeloid and erythroid cells in the femoral marrow is markedly reduced. Seven days after injecting CY, the number of CFU in the femoral marrow is still reduced, the total nucleated cell count is back to normal, but the number of mature myeloid elements in the marrow are significantly increased. These observations suggest the conclusion that the rate of proliferation of HSC is modulated by the number of mature myeloid cells in the microenvironment.     

Predictive factors for poor peripheral blood stem cell mobilization and peak CD34(+) cell count to guide pre-emptive or immediate rescue mobilization

Background aimsFailure in mobilization of peripheral blood (PB) stem cells is a frequent reason for not performing hematopoietic stem cell transplantation (HSCT). Early identification of poor mobilizers could avoid repeated attempts at mobilization, with the administration of pre-emptive rescue mobilizationMethodsData from the first mobilization schedule of 397 patients referred consecutively for autologous HSCT between 2000 and 2010 were collected. Poor mobilization was defined as the collection of < 2 × 10⁶ CD34⁺cells/kg body weight (BW).ResultsThe median age was 53 years (range 4–70) and 228 (57%) were males. Diagnoses were multiple myeloma in 133 cases, non-Hodgkin's lymphoma in 114, acute myeloid leukemia or myelodysplastic syndrome in 81, Hodgkin's lymphoma in 42, solid tumors in 17 and acute lymphoblastic leukemia in 10. The mobilization regimen consisted of recombinant human granulocyte–colony-stimulating factor (G-CSF) in 346 patients (87%) and chemotherapy followed by G-CSF (C + G-CSF) in 51 (13%). Poor mobilization occurred in 105 patients (29%), without differences according to mobilization schedule. Diagnosis, previous therapy with purine analogs and three or more previous chemotherapy lines were predictive factors for poor mobilization. A CD34⁺cell count in PB > 13.8/μL was enough to ensure ≥ 2 × 10⁶ CD34⁺cells/kg, with high sensitivity (90%) and specificity (91%).ConclusionsThe prevalence of poor mobilization was high, being associated with disease type, therapy with purine analogs and multiple chemotherapy regimens. The threshold of CD34⁺ cell count in PB identified poor mobilizers, in whom the administration of immediate or pre-emptive plerixafor could be useful to avoid a second mobilization.     

Influence of purified recombinant human macrophage colony stimulating factor in mice

The endotoxin-resistant strain of mouse, C3H/Hej, was assessed for hematological responsiveness to multiple injections of high dosages of purified recombinant human macrophage colony stimulating factor (rhu-M-CSF). Mice were administered the rhu M-CSF i.p. at dosages of 40 micrograms per injection, 2 or 3 times per day for 4 days. This resulted in significant increases in circulating leukocytes compared to control mice given sterile pyrogen-free saline. Assessment of the marrow and spleen of these mice on the 5th day noted a significant reduction in the numbers of marrow hematopoietic progenitor cells, with no change in their cycling rates. In contrast, splenic granulocyte-macrophage and erythroid progenitor cell numbers were markedly increased and the cycling rates of these progenitors plus those of multipotential progenitors were significantly enhanced. Marrow and splenic early myeloid cells (blasts, promyelocytes, and myelocytes) and macrophages were increased, while marrow and splenic PMN were decreased. The results suggest that multiple injections of high dosages of rhu-M-CSF to previously untreated mice for a short period of time has a modest enhancing effect on blood leukocyte levels. This is associated with a shift of hematopoietic cell activity from the marrow to the spleen.     

参芪扶正注射液对急性淋巴细胞白血病化疗患者造血和免疫功能的影响

目的:观察参芪扶正注射液对急性淋巴细胞白血病化疗患者造血及免疫功能的影响。方法:收集96例处于初治诱导缓解治疗阶段的急性淋巴细胞白血病患者,并将其随机分为治疗组和对照组。治疗组48例患者在进行常规化疗的同时给予参芪扶正注射液250 m L,1次/天,共28天;对照组48例患者仅接受常规化疗,两组患者均给予相同的支持对症治疗。治疗结束后,观察两组患者的疾病缓解率、治疗前后造血系统、T和B淋巴细胞亚群的变化情况。结果:化疗结束后,治疗组患者的疾病缓解率为89.6%,对照组为83.3%,两组比较无统计学差异(P0.05);化疗14天及化疗结束后1周,治疗组患者的白细胞、红细胞计数和血红蛋白水平明显高于对照组(P0.05);且化疗结束后1周,治疗组患者的CD3+、CD4+及CD4+/CD8+含量均明显高于对照组(P0.05)。而两组患者治疗前后的血小板计数、CD3-CD19+含量比较均不具有统计学差异(P0.05)。结论:参芪扶正注射液辅助治疗不仅能够改善化疗所致的急性淋巴细胞白血病患者的骨髓抑制,而且能够提高其细胞免疫功能,有助于患者化疗后的恢复。     

Circulating CD34+ hematopoietic progenitors in the harvesting peripheral blood stem cells; enhancement by recombinant human granulocyte colony-stimulating factor

The number of circulating progenitor cells increases during the period of hematopoietic recovery following myeloablative therapy. These progenitor cells were used for autologous transplantation in order to reconstitute hematopoiesis. As an indicator of the circulating progenitor cells, the number of granulocyte-macrophage colony forming units (CFU-GM), which is measured by means of a long-term cell culture, has been widely used. Recently, a cell surface marker, CD34, which can easily be measured by means of flowcytometry, was found to represent immature hematopoietic progenitor cells, which are very close to stem cells. Therefore, the relationship between the number of CD34 positive cells (CD34⁺ cells) and the number of CFU-GM in the peripheral blood following chemotherapy was studied in 9 patients selected to undergo autotransplantation. The number of peripheral blood CD34+ cells was found to be significantly correlated with that of CFU-GM (r = 0.81). When four out of 9 patients received recombinant human granulocyte-colony stimulating factor (rG-CSF) administration, a significant increase in the release of peripheral blood CD34⁺ cells as well as peripheral blood CFU-GM was observed (P<0.01). Thus, the measurement of CD34⁺ cells is useful for predicting the number of circulating CFU-GM.     

Continuous perfusion with interleukin 6 (IL-6) enhances production of hematopoietic stem cells (CFU-S)

The in vivo effect of human recombinant IL-6 on hematopoietic stem cells (colony forming units in spleen, CFU-S) was investigated. Normal mice perfused with IL-6 for 7 days showed an increase in the serum level of IL-6 in a dose-dependent manner. This increase was accompanied by a dramatic enhancement (approximately 8-fold) in the number of spleen CFU-S 7 days after starting perfusion, although heat-treated IL-6 did not exhibit any activities. Enhanced CFU-S number returned to normal at 13 days after cessation of perfusion. These results suggest that IL-6 could be valuable for treating various forms of hematopoietic depletion.     

长期和短期限制性束缚对小鼠造血干细胞的不同影响

慢性心理应激会导致机体多种功能紊乱,其中包括免疫力下降。慢性心理应激能够负调节免疫系统,但机制尚未完全阐明。免疫细胞,包括髓系来源细胞,由骨髓造血干细胞(hematopoietic stem cell, HSC)分化而来,在机体免疫中发挥重要作用。本实验采用短期高强度束缚和长期温和束缚慢性心理应激小鼠模型,探讨不同限制性束缚模式对小鼠骨髓HSC和髓系来源细胞的影响。长期温和束缚模型中,对小鼠连续束缚4周,每天束缚2次,每次束缚2 h (9:00到17:00间完成);短期高强度束缚模型中,小鼠连续束缚5天,每天束缚16 h (当日17:00到次日9:00)。束缚完成后,取小鼠骨髓和外周血进行白细胞计数,流式细胞术检测小鼠骨髓HSC (Lin^-CD117^+Sca1^+CD150^+CD48^-)和髓系细胞(CD11b^+Ly6C^+)的比例和绝对数,以及外周血髓系细胞的比例和绝对数,BrdU掺入实验检测小鼠HSC增殖能力。实验结果表明,长期温和应激导致小鼠骨髓HSC的比例和绝对数增加,而短期高强度应激导致小鼠骨髓HSC的绝对数下降,伴有HSC的增殖下降。两种束缚模式都使小鼠骨髓和外周血CD11b^+Ly6C^+细胞的总数增加或呈现增加的趋势。综上所述,长期温和应激和短期高强度应激对小鼠HSC的比例和绝对数影响不同;两种应激模型都可以使CD11b^+Ly6C^+细胞总数增加,HSC增多可能并不是CD11b^+Ly6C^+细胞增多的主要机制。     

Ex vivo expansion and clonality of CD34+ selected cells from bone marrow and cord blood in a serum-free media

Although umbilical cord blood is increasingly being used in allogeneic marrow transplantation, delayed platelet engraftment is often a concern for cord blood transplant recipients. We evaluated the potential of ex vivo expansion and clonality in CD34+ cells separated from a bone marrow source, and cord blood, in a serum-free Media. The CD34+ cells, selected from bone marrow (BM) and umbilical cord blood (CB), were expanded with hematopoietic growth factors. They were then cultured for burst-forming units of erythrocytes (BFU-E), colony-forming units of granulocytes and monocytes (CFU-GM) and colony-forming units of megakaryocytes (CFU-Mk) at days 0, 4, 7, and 14 under the combination of growth factors, with cell counts. The cytokines included the recombinant human megakaryocyte growth and development (100 ng/ml), interleukin-3 (10 ng/ml), stem cell factor (100 ng/ml), flt-3 ligand (50 ng/ml) and interleukin-11 (200 ng/ml). The CB-selected CD34+ cells showed significantly higher total cell expansion than those from the BM at day 7 (3.0 fold increase than BM), day 14 (2.4 fold), and day 17 (2.6 fold). The colony count of the BFU-E/CFU-E per CD34+ cell at day 0 was 0.14 +/- 0.023 in the CB, which was significantly higher than 0.071 +/- 0.015 in the BM. The CB-selected CD34+ cells produced more BFU-E colonies than the BM on culture days 4, 7, and 14. The BFU-E colonies from the CB cells increased markedly on culture days 4 and 7, with a 4-fold increase at day 14. The colony count of the CFU-Mk per CD34+ cell at day 0 was 0.047 +/- 0.011 in the CB-selected CD34+ cells cultures, which was higher than the 0.026 +/- 0.014 in the BM. The CB-selected CD34+ cells produced more CFU-Mk colonies than the BM on culture days 4, 7 and 14. In conclusion, the ex vivo expansion of the CB cells may be very promising in producing total cellular expansion, CFU-Mk and BFU-E compared with BM, especially at day 7. The ex vivo expansion of the CB may have rationale in making an ex vivo culture for 7 to 14 d.     

Safe and efficient methods of autologous hematopoietic stem cell transplantation for biomedical research in cynomolgus monkeys

We have established safe and efficient methods for autologous hematopoietic stem cell (HSC) transplantation in cynomolgus monkeys (Macaca fascicularis) that include regimens of supportive care to ensure survival during hematopoietic reconstitution following otherwise lethal total body irradiation. Eleven young adult cynomolgus monkeys were studied. Bone marrow was aspirated from the ilium and/or tuber ischiae after administration of recombinant human stem cell factor (SCF) and granulocyte colony-stimulating factor (G-CSF). Using the immunomagnetic selection method, CD34+ cells were then isolated (90 to 95% pure) as a fraction containing HSCs. Just prior to transplantation, the animals received myeloablative total body irradiation-500 to 550 cGy daily for two days. The monkeys re-infused with CD34+ cells developed moderate to severe myelosuppression, with some animals requiring intravenous hyperalimentation, antibiotic administration, and blood transfusion. Hematopoiesis was restored in all animals after transplantation. It took 12 days, on average, until the peripheral white blood cell count reached more than 1,000 cells/microl. Up to two years after transplantation, signs of radiation-induced pneumonitis or other radiation-related disorders were not evident at the aforementioned dose of irradiation. This transplantation model will be useful for testing new approaches using HSCs for therapy of many diseases and will offer unique insights into the biology of these cells.     

Quantitative and qualitative in vitro analysis of the stem cell potential of hematopoietic cells purified from murine skeletal muscle

The murine skeletal muscle contains hematopoietic stem cells, but this potential has so far not been studied quantitatively or qualitatively in vitro. To quantify the hematopoietic stem cell potential, we have used highly purified SP/CD45^＋ cells in long-term culture initiating cell （LTC-IC） assays. The SP/CD45^＋ cell population purified from murine muscle was found to have significant stem cell activity with an LTC-IC frequency of 1/640. Single-cell-sorted SP/CD45^＋ cells from muscle exhibited robust proliferative activity in vitro at day 16 （380-fold amplification）, especially after culture with OP-9 layers that also support embryonic stem cells. Amplified cell populations originating from single cells exhibited multilineage differentiation ability with evidence of myeloid, lymphoid and NK cell markers. Thus, our results demonstrate that hematopoietic stem cells that can be quantified by LTC-IC assays exist in the murine skeletal muscle and show also for the first time, at the single-cell level, that these cells exhibit multilineage differentiation ability and major proliferative potential.     

Peripheral blood progenitor cell cycle kinetics following priming with pIXY321 in patients treated with the "ICE" regimen.

PURPOSE: Treatment with hematopoietic growth factors increases the percentage of hematopoietic progenitor cells in cell cycle. Following withdrawal of certain growth factors, preclinical data suggest that there is a transient fall in the percentage of progenitor cells in cycle below the baseline, thus providing a window to administer chemotherapy with reduced risk of myelotoxicity. PATIENTS AND METHODS: Patients with histologically confirmed, previously untreated neoplasia, were treated with pIXY321 by subcutaneous injection at a dose of 375 microg/m2 twice daily (total dose 750 microg/m2/day) for seven days (days -8 to -2), followed by a two-day rest (days -1 to 0). Patients received ICE (ifosfamide, carboplatin and etoposide) on days 1 to 3. On day 4, pIXY321 was resumed until hematologic recovery. Peripheral blood was collected on days -8, -2, -1, 1, and cell cycle distribution was determined using flow cytometry. RESULTS: Twenty patients were treated in this study and received a total of 54 cycles. Partial responses were observed in three of 13 patients with non-small cell lung cancer (23 percent) and two of five patients with small cell lung cancer (40 percent). Six of 15 patients had an increased number of cells in S+G2/M on day 1 of ICE following seven days of pIXY321 and two days off (days -1 to 0). The average increase was 63 percent (range 6-253). Seven patients had a decreased number of cells in S+G2/M. The average decrease was 55 percent (range 6.3-78). There were no significant differences among the fifteen patients with regards to the observed toxicity of the chemotherapy. DISCUSSION: pIXY321 in this schedule did not consistently decrease the percentage of cycling progenitor cells in the peripheral blood. Future studies should define whether other growth factors and/or schedules can synchronize progenitor cell cycling and protect the marrow compartment from cycle specific chemotherapy.     

Effects of granulocyte–colony-stimulating factor on progenitor cell mobilization and heart perfusion and function in normal mice

Background aimsMobilization of stem cells and progenitor cells from the bone marrow (BM) into the peripheral blood (PB) by granulocyte–colony-stimulating factor (G-CSF) is being investigated for cardiac regeneration in ischemic heart disease. However, hematopoietic (HPC), mesenchymal (MPC) and endothelial (EPC) progenitor mobilization have not been optimized and the effect of G-CSF on myocardial perfusion and cardiac function in a normal heart has never been studied.MethodsNormal mice were injected daily for 1–10 days with subcutaneous recombinant human G-CSF. PB and BM were evaluated for HPC and EPC by flow cytometry and HPC and MPC by hematopoietic (CFU-GM) and mesenchymal (CFU-F) colony assays. Echocardiography, microSPECT imaging, cardiac catheterization and immunohistochemistry were performed in mice treated for 10 days.ResultsHPC and CFU-GM in PB peaked after 2 days, CFU-F after 4 days and EPC after 3 days. Thereafter, while HPC temporally decreased before showing a second peak, EPC remained detectable only at low levels. In BM, hematopoietic stem cells (HSC) and CFU-GM did not increase much overall but peaked twice on days 2 and 7. EPC (peak on day 7) production increased in the BM, but CFU-F formation declined considerably after day 2. G-CSF enhanced myocardial perfusion and vascularization but impaired hemodynamic performance of the heart through apparently increased ventricular wall rigidity.ConclusionsG-CSF induces the mobilization of HPC, EPC and CFU-F progenitors in PB according to very different patterns, and has a significant impact on perfusion and function of the normal heart.     

In vitro expansion of long-term repopulating hematopoietic stem cells in the presence of immobilized Jagged-1 and early acting cytokines

There is an increasing body of evidence that suggests that genes involved in cell fate decisions and pattern formation during development also play a key role in the continuous cell fate decisions made by adult tissue stem cells. Here we show that prolonged in vitro culture (14 days) of murine bone marrow lineage negative cells in medium supplemented with three early acting cytokines (stem cell factor, Flk-2/Flt-3 ligand, thrombopoietin) and with immobilized Notch ligand, Jagged-1, resulted in robust expansion of serially transplantable hematopoietic stem cells with long-term repopulating ability. We found that the absolute number of marrow cells was increased approximately 8 to 14-fold in all cultures containing recombinant growth factors. However, the frequency of high quality stem cells was markedly reduced at the same time, except in cultures containing growth factors and Jagged-1-coated Sepharose-4B beads. The absolute number of hematopoietic cells with long-term repopulating ability was increased approximately 10 to 20-fold in the presence of multivalent Notch ligand. These results support a role for combinatorial effects by Notch and cytokine-induced signaling pathways in regulating hematopoietic stem cell fate and to a potential role for Notch ligand in increasing cell numbers in clinical stem cell transplantation.     

Effect of pyridoxal phosphate on gamma-aminobutyric acid metabolism in different sections of the brain in irradiated animals]

A study was made of the effect of X-rays (4,5 Gy) and pyridoxal phosphate (3 mg/kg, v/v) on the activity of pyridoxal enzymes of GABA metabolism (e.g. glutamate decarboxylase, E.C. 4.1.1.15) and aminobutyrate aminotransferase (GABA-T, E.C. 2.6.1.19), as well as on GABA and glutamate content of the hemisphere cortex, brain stem and cerebellum of rabbits 6 and 10 days following irradiation and injection of a coenzyme. The height of the radiation sickness in rabbits was characterized by the manifest changes in glutamate decarboxylase and GABA-T activity, as well as in GABA and glutamate content of various brain parts differing in the structural and functional functions. The administration of pyridoxal phosphate produced pronounced activation of glutamate decarboxylase, particularly 6 days after irradiation and administration of the co-enzyme, and, to a lesser extent, influenced GABA-T function. Pyridoxal phosphate favored maintaining the GABA level above the control level in the hemisphere cortex and brain stem 6 and 10 days after exposure. The injection of pyridoxal phosphate did not normalize the glutamate content of the brain parts 6 days after exposure, but favored the normalization of GABA-T activity on day 10.     

Immune interferon enhances functional properties of human granulocytes: role of Fc receptors and effect of lymphotoxin, tumor necrosis factor, and granulocyte-macrophage colony-stimulating factor

We report here a comparative study of the effects of several cytokines known to affect myeloid cell differentiation on functional properties of human mature granulocytes. We show that recombinant interferon-gamma (rIFN-gamma), recombinant granulocyte/macrophage-colony stimulating factor (rGM-CSF), recombinant tumor necrosis factor (rTNF), and lymphotoxin (LT) purified to homogeneity are potent stimulators of polymorphonuclear cells (PMN) activity. All cytokines enhance antibody-dependent cell-mediated cytotoxicity (Ab-CMC) mediated by human PMN; however, rGM-CSF, rTNF, and LT have an immediate and short-lived effect on the PMN, whereas the activation by rIFN-gamma requires several hours of induction but can be observed up to 24 to 48 hr of culture. Only the effect of rIFN-gamma is in part dependent on induction of a high-affinity FcR for monomeric IgG on PMN, as suggested by two-color sorting analysis, and on mechanisms that result in prolonged survival of PMN in a functionally active state to mediate oxidative burst, phagocytosis, and bactericidal activity. Greater enhancement of Ab-CMC is obtained by using rIFN-gamma in combination with the other cytokines. Our data indicate that cytokines previously defined on the basis of their cytotoxic effects mediate a wide spectrum of activities on mature myeloid cells and provide evidence for their possible role in vivo, alone or in combination with rIFN-gamma, in modulating functional activities of cells responsible for non-adaptive systems of defense.     

Human granulocyte colony stimulating factor: effects on human long-term bone marrow cultures

Human granulocyte colony stimulating factor (G-CSF) was previously shown to support the survival and proliferation of early myeloid progenitors (pre-CFU) that are capable of generating more mature CFU-GM progenitor cells. To evaluate the scope of action of G-CSF in the hierarchy of hematopoietic stem cells, we studied the effects of recombinant G-CSF (rhG-CSF) on long-term cultures of normal human bone marrow cells (LTBMC). We found that rhG-CSF predominantly influenced initial cell proliferation and expansion of CFU-GM progenitor cells in LTBMC before establishment of a confluent adherent layer. In rhG-CSF-treated LTBMC, the stromal cell layer was associated with a higher proliferative capacity and progenitor cell content as compared to control cultures. This effect was pronounced early after layer confluence and was gradually lost with culture time. rhG-CSF did not alter the duration of the productive phase of LTBMC, suggesting that it may not be active on the hematopoietic stem cells responsible for LTBMC propagation. Alternatively, stromal cells may exert tight regulatory control over progenitor cells, even in the presence of rhG-CSF.     

Comprehensive analysis of myeloid lineage conversion using mice expressing an inducible form of C/EBP alpha

CCAAT/enhancer-binding protein (C/EBP) alpha is a critical regulator for early myeloid differentiation. Although C/EBPalpha has been shown to convert B cells into myeloid lineage, precise roles of C/EBPalpha in various hematopoietic progenitors and stem cells still remain obscure. To examine the consequence of C/EBPalpha activation in various progenitors and to address the underlying mechanism of lineage conversion in detail, we established transgenic mice expressing a conditional form of C/EBPalpha. Using these mice, we show that megakaryocyte/erythroid progenitors (MEPs) and common lymphoid progenitors (CLPs) could be redirected to functional macrophages in vitro by a short-term activation of C/EBPalpha, and the conversion occurred clonally through biphenotypic intermediate cells. Moreover, in vivo activation of C/EBPalpha in mice led to the increase of mature granulocytes and myeloid progenitors with a concomitant decrease of hematopoietic stem cells and nonmyeloid progenitors. Our study reveals that C/EBPalpha can activate the latent myeloid differentiation program of MEP and CLP and shows that its global activation affects multilineage homeostasis in vivo.