Megakaryocytic cells in mixed haemopoietic colonies (CFU-GEMM) from the peripheral blood of normal individuals

The cellular composition of mixed haemopoietic colonies CFU-GEMM from the peripheral blood of normal individuals was investigated using both an improved in vitro culture assay and a monoclonal antibody reactive with the earliest megakaryocytic precursor cells. The frequency of CFU-GEMM was found to be 22 +/- 7/ml blood (mean +/- SE; n = 8) and 4.9 +/- 1.8/10(6) mononuclear cells. Double-labelling of single colonies with monoclonal antibodies against megakaryocytic as well as granulocytic cells revealed that only 18% of mixed colonies from the peripheral blood contained megakaryocytic cells as compared to 87% of mixed colonies from the bone marrow, while no difference existed with regard to granulocytic cells. It is concluded that circulating multilineage progenitor cells either require different culture conditions to express their megakaryocytic component or, alternatively, that the circulating pluripotent stem cell pool mainly consists of bipotent rather than multipotent progenitor cells.     

Modified in vitro conditions for cord blood-derived long-term culture-initiating cells

OBJECTIVE: The aim of this study was to compare the in vitro growth of cord blood-derived progenitors with that of bone marrow and peripheral blood. MATERIALS AND METHODS: We analyzed 192 umbilical cord blood (UCB), 35 normal bone marrow (NBM), and 35 granulocyte colony-stimulating factor (G-CSF)-primed normal peripheral blood (NPB) samples. Standard clonogenic assays (colony-forming unit granulocyte-macrophage [CFU-GM], burst-forming unit erythroid [BFU-E], CFU-granulocyte erythroid megakaryocyte macrophage [GEMM]) and standard long-term culture-initiating cell (LTC-IC) assay were performed. LTC-IC frequency also was tested under modified culture conditions. The variables tested were incubation temperature (37 degrees C and 33 degrees C) and supportive stromal cell lines (NIH3T3 and M210-B4). RESULTS: The CFU-GM and CFU-GEMM frequencies of UCB samples were similar to NPB and higher compared to NBM samples (p < 10(-4) and p < 0.007 respectively). On the other hand, the BFU-E frequency was lower in cord blood samples (5.2 +/- 5.6/10(4) MNC) compared to bone marrow (7 +/- 3.8/10(4) MNC; p < 0.005) and peripheral blood (15.2 +/- 11.1/10(4) MNC; p < 10(-4)). All colony types (CFU-GM, BFU-E, CFU-GEMM) generated from cord blood progenitors were larger with respect to the other tissues. The LTC-IC frequency was markedly decreased (8.8 +/- 3.8/10(6) MNC) in cord blood with respect to bone marrow (40.7 +/- 7.4/10(6) MNC; p < 10(-4)) and peripheral blood (28.8 +/- 3.8/10(6) MNC; p < 0.04). However, when culture conditions (temperature, stromal layers) were modified, UCB-LTC-IC frequency significantly increased, while the growth of early progenitors derived from adult tissues (BM and PB) did not show any variation. Whatever culture conditions were used, the proliferative potential of UCB LTC-IC was significantly higher with respect to bone marrow and G-CSF-primed PB (10.6 +/- 7.7 colonies vs. 5.9 +/- 5 vs 3.2 +/- 2.2 colonies; p < 0.02 and p < 0.001 respectively). CONCLUSIONS: Optimal conditions for estimation of the LTC-IC frequency in cord blood samples seem to be different from those usually applied to PB and BM progenitors. Although UCB hemopoietic progenitors have a higher proliferative potential than those from bone marrow and G-CSF-primed peripheral blood, their quantitation depends on the culture conditions, which makes it difficult to establish their exact number. This problem and the fact that a significant proportion of UCB samples grew poorly in culture make it necessary to develop suitable and standardized functional assays to test UCB progenitor content before the transplantation procedure.     

Red cell salvage and reinfusion in pediatric bone marrow donors.

We evaluated the use of a semi-automated processing technique to salvage red blood cells from pediatric bone marrow donors to minimize the risk of severe anemia following bone marrow harvest and ABO incompatibility in the recipient. Sixty healthy, HLA-matched, pediatric donors of bone marrow hematopoietic cells with a median age 8.0 years (2-19) were studied. Thirteen of the donor-recipient pairs were ABO incompatible. There were 60 recipients with a median age of 8.6 years (2 months to 20.8 years). Bone marrow was harvested under general anesthesia, filtered in the operating room and then transferred to the stem cell laboratory for processing. Samples were obtained for cell count, CD34+ quantification, colony assay, viability, and bacteriologic cultures before and after processing. The cells were processed in a semi-automated closed system (Stericel, Terumo) by density gradient separation with Ficoll-Hypaque and then washed. Two aliquots were obtained: one containing the mononuclear cell layer to be infused to the recipient and the other the washed red cells to be infused to the donor. The median volume harvested was 608 +/- 40.42 ml (278-1409), while the final volume infused was 174 +/- 10.75 ml (30.2-380) P < 0.0001, representing a decrease of 72% of the volume infused. The nucleated cell count harvested was 1.6 x 10(10) +/- 0.1 (0.56-3.2), while the count infused was 6.9 x 10(9) +/- 0.1 (0.12-5.4) P < 0.0001. The median mononuclear cell count (MNC) per kg harvested was 0.67 x 10(8) +/- 0.05 (0.18-2.0) vs an infused cell number of 1.3 x 10(8) MNC/kg +/- 0.1 (0.6-33.6) P < 0. 0001. The CD34+ cells harvested were 2.8 x 10(6)/kg +/- 0.1 (0.25-10.2) vs an infused number of 6.0 x 10(6)/kg +/- 0.5 (0.84-31.0) P < 0.0001. The viability before and after processing was 99%. Red cell salvage performed in a semi-automated closed system is safe and reduces the risk of post-bone marrow harvest anemia in pediatric donors, decreases the volume infused into the donor and enriches the mononuclear and CD34+ cell population, without affecting hematopoietic reconstitution.     

Sustained long-term hematologic recovery despite a marked quantitative defect in the stem cell compartment of patients with aplastic anemia after immunosuppressive therapy

Previously, we reported that patients with aplastic anemia (AA) have profoundly decreased numbers of hematopoietic progenitor and stem cells as measured in the long-term culture initiating cell (LTC-IC) assay (Blood 1996;88:1983-1991). We now present results of a long-term prospective study of LTC-IC numbers in peripheral blood (PB) and bone marrow (BM) of patients treated with antithymocyte globulin and cyclosporin A. Numbers of secondary colony forming cells (secondary CFC) in long-term bone marrow culture (LTBMC) were used to quantitate LTC-IC. BM (N = 35) and PB (N = 41) secondary CFC from both untreated severe AA patients and responders to immunosuppressive therapy who were sampled up to 6 years after initial treatment were compared. Normal controls showed 148 +/- 38 (N = 17) and 16 +/- 3 (N= 14) secondary CFC per 10(6) in BM and PB, respectively. In cross-sectional analysis, prior to therapy, AA patients showed 2.6 +/- 1 (mean +/- SD) secondary CFC/10(6) BM MNC; within the first year after initial treatment (N = 14), secondary CFC number rose modestly to 8.2 +/- 2.2/10(6) MNC, and further increased to 15.8 +/- 7 (N = 17) at 2 years and 16.2 +/- 7/10(6) MNC (N = 25) 3 years after treatment. There was no further improvement in the secondary CFC numbers at 4, 5, and > or =6 years (N = 37). Thus, while BM secondary CFC increased about 6-fold at 3 years post-therapy compared to presentation, they remained about only 10% of normal despite hematologic recovery. Similar data were obtained for PB, with approximately 4-fold increase in secondary CFC numbers within 2 years of therapy, to about 15% of normal values. We confirmed these observations in patients studied serially over a period of 4 years: initial secondary CFC were 2.35 +/- 1/10(6) BM MNC and 0.11 +/- 0.1/10(6) PB MNC improving to an average of 6 +/- 1. 2 (BM; N = 12) and 2.4 +/- 1/10(6) MNC (PB; N = 14). In many cases of partial recovery, PB counts improve but do not normalize. When we studied secondary CFC numbers only in patients who achieved complete normalization of PB counts (ANC >1,500/mm(3); platelets >10(5)/mm(3) and absolute reticulocytes >5 x 10(4)/mm(3)), BM secondary CFC were significantly higher than in patients with partial recovery; the PB secondary CFC number was modestly increased but remained below the normal values. Within the group of patients with complete recovery, there was no correlation between the secondary CFC and time after initial treatment. In addition, there also was no correlation between the secondary CFC number at presentation and the quality of hematopoietic recovery. Despite a limited expansion potential of a severely reduced stem cell pool, their numbers are sufficient to provide a long-term supply of mature blood cells. Am. J. Hematol. 65:123-131, 2000. Published 2000 Wiley-Liss, Inc.     

Rapid and automated processing of bone marrow grafts without Ficoll density gradient for transplantation of cryopreserved autologous or ABO-incompatible allogeneic bone marrow.

The growing number of BMTs has increased interest in safe and standardized in vitro bone marrow processing techniques. We describe our experience with a rapid automated method for the isolation of mononuclear cells (MNC) from large volumes of bone marrow using a Fenwal CS-3000 cell separator without employing density gradient materials. Forty bone marrow harvests with a mean volume of 1650 +/- 307 ml were processed. A mean of 75 +/- 34% (50 percentile range 54-94%) of the original MNCs were recovered in a volume of 200 ml with only 4 +/- 2% of the starting red blood cells (RBC). Removal of granulocytes, immature myeloid precursors and platelets proved to be sufficient to permit safe cryopreservation and successful autologous BMT (n = 25). Allogeneic BMT (n = 14, including three major ABO-incompatible) could be performed without additional manipulation. In both groups of patients timely and stable engraftment comparable to historical controls receiving Ficoll gradient processed autologous (n = 17) or unprocessed allogeneic BMT (n = 54) was observed. Moreover, 70 +/- 14% of the RBC could be recovered from the grafts. They were used for autologous RBC support of donors, rendering unnecessary autologous blood pre-donations.     

Comparison of monocyte-dependent T cell inhibitory activity in GM-CSF vs G-CSF mobilized PSC products

This study compares the immune properties of peripheral blood stem cell (PSC) products mobilized with different hematopoietic growth factors (HGFs) as well as apheresis products and peripheral blood leukocytes (PBL) from normal individuals. We found that monocytes in mobilized PSC products appear to inhibit T cell function independent of whether granulocyte colony-stimulating factor (G-CSF) or granulocyte-macrophage colony-stimulating factor (GM-CSF) was used for mobilization. In addition, the GF used to mobilize the stem cell product may be less important to the CD4:CD8 ratio than the extent of prior chemotherapy, as we found an inverse correlation between chemotherapy and the CD4:CD8 ratio. In other observations, all apheresis products, whether mobilized or unmobilized, contained significantly more monocytes compared to normal PBL. The mononuclear cells (MNC) from G-CSF or GM-CSF mobilized PSC products had a similar T cell phytohemagglutinin (PHA) mitogenic response that was significantly lower (P = 0.001 and P = 0.005, respectively) than non-mobilized apheresis products. We also examined the T cell inhibitor (TI) activity of the MNC from the PSC products for allogeneic lymphocyte proliferation and found that PSC products significantly reduced the proliferation of allogeneic PBL to PHA. A significant correlation (P = 0.001, r = 0.517) between the frequency of monocytes and TI activity also was observed.     

In vitro growth of bone marrow-derived multipotent and lineage-restricted hematopoietic progenitor cells in myelodysplastic syndromes

The aim of the present study was to evaluate the incidence of bone marrow-derived multipotent (CFU-GEMM), megakaryocytic (CFU-Mk), erythroid (BFU-E), and granulocyte-macrophage (CFU-GM) progenitor cells in 22 patients with myelodysplastic syndromes (MDS), and to investigate the role of hematopoietic accessory cells (T-lymphocytes and monocytes) as a possible cause of growth derangement. As compared to normal controls (n = 15), growth values in the 22 patients (mean +/- SEM) were significantly reduced for CFU-GEMM (0.4 +/- 0.1 versus 7 +/- 1, P less than 0.0005), CFU-Mk (1.4 +/- 0.5 versus 18 +/- 4, P less than 0.0005), BFU-E (2.2 +/- versus 40 +/- 6, P less than 0.0005), and CFU-GM (19 +/- 5 versus 65 +/- 10, P less than 0.0005). The growth of CFU-GEMM was abnormal at an early stage in the clinical development of MDS, sometimes even when CFU-GM formation was still normal. Colony-formation was unaffected by removal of hematopoietic accessory cells. Although no correlation was found between the incidence of lineage-restricted progenitors and the degree of peripheral cytopenia, derangement of colony growth was more pronounced in patients with worse prognosis. We conclude that: (i) the grossly defective CFU-GEMM growth supports the concept of MDS as clonal disorders of hematopoietic multipotent stem cells; (ii) a progressive impairment of in vitro hematopoiesis occurs in association with the clinical progression of the myelodysplastic syndromes.     

Interferon is a mediator of hematopoietic suppression in aplastic anemia in vitro and possibly in vivo.

We have investigated interferon as a mediator of hematopoietic suppression in bone marrow failure. Interferon production by stimulated peripheral blood mononuclear cells from patients with aplastic anemia was significantly higher than that observed in controls; spontaneous interferon production by these cells was also high for more than half of aplastic anemia patients. Circulating interferon, not detectable in normal individuals, was detected in 10 of 24 patients. Interferon is a potent inhibitor of hematopoietic cell proliferation and, therefore, may be the mediator of suppression in many in vitro models employing patients' cells and sera. The possible pathogenic importance of interferon in aplastic anemia was suggested by an increase in hematopoietic colony formation in vitro after exposure of bone marrow cells to antiinterferon antisera (277 +/- 71% increase for patients compared to 1.6 +/- 1.6% for normal individuals). Interferon levels in the bone marrow sera of aplastic anemia patients were high (mean = 203 international units (IU)/ml, n = 8), even in comparison to circulating levels in the same patients. Normal bone marrow sera also contained measurable interferon but at lower levels (41 IU/ml, n = 16), indicating that interferon may be a normal bone marrow product. High concentration of bone marrow interferon, possibly due to abnormal immunologic activity or a reaction to virus infection of the bone marrow, may mediate hematopoietic suppression in aplastic anemia patients.     

Multifactor stimulation of megakaryocytopoiesis: effects of interleukin 6.

We have previously demonstrated that interleukin 3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), and granulocyte colony-stimulating factor (G-CSF) stimulate various aspects of megakaryocytopoiesis. We have investigated the capacity of interleukin 6 (IL-6) to stimulate megakaryocyte colony formation from both normal Balb/C marrow and light-density marrow extensively depleted of adherent, pre-B, B and T cells. Human recombinant IL-6 (167 ng/ml) stimulated megakaryocyte colony formation from normal marrow (8.6 +/- 1 megakaryocyte colony-forming units [CFU-meg]/10(5) cells) as compared to control (1.5 +/- 4 CFU-meg/10(5) cells) in 16 determinations (p less than 0.01). IL-6 (167 ng/ml) also stimulated CFU-meg formation from depleted marrow (control, 10.8 +/- 4 CFU-meg/10(5) cells versus IL-6, 68 +/- 19 CFU-meg/10(5) cells in 12 determinations, p less than 0.01). IL-6 synergistically augmented IL-3-induced colony formation (139% IL-3 control, 120% calculated IL-3 plus IL-6 control, n = 11, p less than 0.01) in normal marrow and showed an additive effect in depleted marrow (133% IL-3 control, p less than 0.01, 114% of IL-3 plus IL-6, value not significant [NS] at 0.05 level). Studies with recombinant murine IL-6 gave similar results. There was an increasing level of megakaryocyte colony-stimulating activity from G-CSF (16,667 U/ml, 2.47 +/- 0.6 CFU-meg/10(5) cells, n = 17), to IL-6 (167 ng/ml, 8.47 +/- 0.96 CFU-meg/10(5) cells, n = 19), to GM-CSF (52 U/ml, 23 +/- 4 CFU-meg/10(5) cells, n = 14), to IL-3 (167 U/ml, 48 +/- 5 CFU-meg/10(5) cells, n = 20) as compared to media-stimulated marrow (range 1.29-1.86 CFU-meg/10(5) cells). A similar hierarchy was seen with depleted marrow. Combinations of factors (including IL-3, GM-CSF, G-CSF, and IL-6) tested against normal unseparated murine marrow did not further augment CFU-meg numbers over IL-3 plus IL-6 but did increase colony size. These data suggest that IL-6 is an important megakaryocyte regulator, that at least four growth factors interact synergistically or additively to regulate megakaryocytopoiesis, and that combinations of growth factors, possibly in physical association, might be critical in stimulating megakaryocyte stem cells.     

Differential sensitivity of adherent CFU-blast, CFU-mix, BFU-E, and CFU-GM to mafosfamide: implications for adjusted dose purging in autologous bone marrow transplantation.

The availability of an in vitro assay able to detect hematopoietic progenitor cells closely related to those responsible for marrow engraftment following autologous bone marrow transplantation (ABMT) prompted us to establish a procedure aimed at maximally increasing the concentration of the cyclophosphamide derivative mafosfamide used for marrow purging. It, therefore, was the aim of the present study to investigate in a group of patients with acute nonlymphoblastic leukemia (ANLL; n = 19) and acute lymphoblastic leukemia (ALL; n = 19) in complete remission the effect of mafosfamide at the level of adherent blast colony-forming units (blast colony-forming units, CFU-Blast), as well as multipotential (granulocyte erythrocyte macrophage megakaryocyte colony-forming units, CFU-GEMM), erythroid (erythroid burst-forming units, BFU-E), and granulocyte-macrophage (granulocyte-macrophage colony-forming units, CFU-GM) progenitor cells. When nonadherent marrow mononuclear cells (MNCs) were incubated (30 min, 37 degrees C) with increasing doses of mafosfamide (30-120 micrograms/ml), a statistically significant (p less than or equal to 0.0005) dose-dependent suppression of CFU-Blast growth was observed. The mean (+/- 1 standard error of the mean [SEM]) values of 50% inhibition (ID50) of the CFU-Blast growth were not significantly different for ANLL (106 +/- 5) and ALL (107 +/- 5) patients. Analysis of CFU-Blast ID50 distribution demonstrated that ID50 ranged from 100 to 120 micrograms/ml in 17 cases (45%), whereas it ranged from 60 to 100 micrograms/ml in 12 cases and from 120 to 160 micrograms/ml in 9 cases. A statistically significant (p less than or equal to 0.05), dose-dependent suppression of colony growth from multi-potential and lineage-restricted progenitor cells was also observed. However, the value of CFU-Blast ID50 was significantly higher (p less than or equal to 0.05) than CFU-GEMM, BFU-E, and CFU-GM ID50 and ID95 values. In conclusion, our data demonstrate that: 1) the CFU-Blast assay allows to detect on an individual basis the doses of mafosfamide used for marrow purging, and 2) the concentrations of mafosfamide extrapolated by using the CFU-Blast assay are significantly higher than those obtained with the CFU-GM assay. The absence of any detrimental effect on marrow engraftment in vivo supports the safety of the CFU-Blast assay to evaluate the dose of mafosfamide used for marrow purging before ABMT.     

Steroid modulation of naturally occurring diurnal variation in circulating pluripotential haematopoietic cells (CFU-GEMM)

The number of committed granulocyte-monocyte precursors (CFU-GM) in circulation has been shown to increase following steroid administration in humans. To see if steroids could be used to improve the collection of peripheral blood stem cells for haematopoietic reconstitution, their effect on circulating CFU-GEMM was investigated. Six healthy young adults, three men and three women, were given 60 mg of prednisone orally at 8 a.m. on day 1. Blood CFU-GEMM and committed precursors (CFU-GM and BFU-E) were cultured in methylcellulose. Samples were taken at 4 p.m. the day before and at 8 a.m. on the day of steroid administration, and at 4, 8, 24 and, in a few cases, 48 h after steroid. Pre-treatment CFU-GEMM levels (per unit volume of blood) at 4 p.m. was 180 +/- 23% (mean +/- SEM) of that at 8 a.m., showing a significant (P less than 0.025) diurnal variation. 8 h after steroids there was a fall in CFU-GEMM to 28 +/- 4.5% of the 8 a.m. presteroid level. 24 h following steroid administration, CFU-GEMM rose significantly (P less than 0.05) to 188 +/- 33% of 8 a.m. baseline values; CFU-GM and BFU-E changes generally paralleled those noted for CFU-GEMM. These results suggest that blood levels of CFU-GEMM exhibit a significant diurnal variation. Oral prednisone given 24 h in advance of collection increases the 8 a.m. value to that found at 4 p.m. The steroid effect may be due to a resetting of the diurnal control mechanism. Use of this information may be important in collection of circulating haematopoietic stem cells for use in bone marrow reconstitution in man.     

Interleukin-8 induces rapid mobilization of hematopoietic stem cells with radioprotective capacity and long-term myelolymphoid repopulating ability

Interleukin-8 (IL-8) belongs to a family of chemoattractant cytokines involved in chemotaxis and activation of neutrophils. As in vivo administration of IL-8 induces granulocytosis and the release of immature white blood cells into the circulation, we assessed a possible mobilizing effect of IL-8 on myeloid progenitor cells. IL-8 was administered at intraperitoneal doses ranging from 0.1 to 100 micrograms per mouse to female Balb/C mice (aged 8 to 12 weeks; weight, 20 to 25 g). Animals were killed at time intervals ranging from 1 to 240 minutes after IL-8 administration, and blood, bone marrow, and spleen cells were harvested. Injection of 30 micrograms IL-8 resulted in an increment from 25 +/- 9 to 418 +/- 299 granulocyte-macrophage colony-forming units (CFU-GM) per milliliter blood at 15 minutes after a single intraperitoneal injection. Sixty minutes after the injection of IL-8, the numbers of circulating CFU-GM per milliliter blood had almost returned to pretreatment values (82 +/- 39 CFU-GM per milliliter). A dose of 100 micrograms IL-8 per animal did not result in a further increment in the number of circulating CFU-GM. Transplantation of 5 x 10(5) blood-derived mononuclear cells (MNC) obtained at 30 minutes after IL-8 injection (30 micrograms) resulted in 69% survival of lethally irradiated (8.5 Gy) recipients at 60 days versus 22% for animals transplanted with an equal number of nonprimed blood-derived MNC. Transplantation of 1.5 x 10(6) MNC obtained from IL- 8-treated donors resulted in 100% survival. Six months after transplantation, female recipients of MNC derived from IL-8-treated male donors were killed, and chimerism was determined in bone marrow, spleen, and thymus using a Y chromosome-specific probe and fluorescent in situ hybridization (FISH). The majority of bone marrow, spleen, and thymus cells (83% +/- 25%, 89% +/- 5%, and 64 +/- 28%, respectively) consisted of Y chromosome-positive cells, showing that the IL-8- mobilized cells had myelolymphoid repopulating ability. We conclude that IL-8 is a cytokine that induces rapid mobilization of progenitor cells and pluripotent stem cells that are able to rescue lethally irradiated mice and that are able to completely and permanently repopulate host hematopoietic tissues.     

The collection, preservation and function of peripheral blood hematopoietic cells in dogs.

Semicontinuous flow centrifugation (SFC) was employed in canines to obtain adequate numbers of cells for autologous marrow repopulation following supralethal cyclophosphamide administration (100 mg per kg). Four cycles using a 225 ml bowl and 30 ml per minute flow rate were carried out for procurement. Collections averaged 8.4 +/- 0.4 x 10(9) (n = 30) leukocytes. Mononuclear cells (MNC) comprised 75 +/- 2% of the population and granulocyte colony-forming units (CFU-C) totaled 1.9 +/- 0.09 x 10(5) colonies per collection. Eighty-six percent of mononuclear cells were "T" cells in the 30 to 90 second fraction compared to 49% at 150 to 120 seconds. CFU-C fractionation revealed a peak at 30-210 seconds and a second peak at 120-210 seconds. Following programed freezing and rapid thawing 77.7 +/- 11.4% of CFU-C were recovered. Using a dose of 1 x10(9) MNC per kg for reinfusion, marrow repopulation and clinical recovery occurred in four out of four dogs. It was concluded that 1) SFC was effective for obtaining adequate numbers of peripheral blood stem cells for autologous marrow repopulation. 2) A rapid thawing and direct transfusion technique appears satisfactory for administration. 3) Some separation of "T", "B" and CFU-C peripheral blood components is possible by centrifugation.     

Growth characteristics and expansion of human umbilical cord blood and estimation of its potential for transplantation in adults.

We estimated whether single collections of cord blood contained sufficient cells for hematopoietic engraftment of adults by evaluating numbers of cord blood and adult bone marrow myeloid progenitor cells (MPCs) as detected in vitro with steel factor (SLF) and hematopoietic colony-stimulating factors (CSFs). SLF plus granulocyte-macrophage (GM)-CSF detected 8- to 11-fold more cord blood GM progenitors [colony-forming units (CFU)-GM] than cells stimulated with GM-CSF or 5637 conditioned medium (CM), growth factors previously used to estimate cord blood CFU-GM numbers. SLF plus erythropoietin (Epo) plus interleukin 3 (IL-3) enhanced detection of cord blood multipotential (CFU-GEMM) progenitors 15-fold compared to stimulation with Epo plus IL-3. Under the same conditions, bone marrow CFU-GM and CFU-GEMM were only enhanced in detection 2- to 4- and 6- to 8-fold. Increased detection of cord blood CFU-GEMM correlated directly with decreased detection of cord blood erythroid burst-forming units (BFU-E). In contrast, adult bone marrow CFU-GEMM and BFU-E numbers were both enhanced by SLF plus Epo plus IL-3. This suggests that most cord blood BFU-E may actually be CFU-GEMM. Cord blood collections (n = 17) contained numbers of MPCs (especially CFU-GM) similar to the number found in nine autologous bone marrow collections. To assess additional sources of MPCs, the peripheral blood of 1-day-old infants was assessed. However, average concentrations of MPCs circulating in these infants were only 30-46% that in their cord blood. Expansion of cord blood MPCs was also evaluated. Incubation of cord blood cells for 7 days with SLF resulted in 7.9-, 2.2-, and 2.7-fold increases in numbers of CFU-GM, BFU-E, and CFU-GEMM compared to starting numbers; addition of a CSF with SLF resulted in even greater expansion of MPCs. The results suggest that cord blood contains a larger number of early profile MPCs than previously recognized and that there are probably sufficient numbers of cells in a single cord blood collection to engraft an adult. Although the expansion data must be considered with caution, as human marrow repopulating cells cannot be assessed directly, in vitro expansion of cord blood stem and progenitor cells may be feasible for clinical transplantation.     

Detection of myeloid precursors (granulocyte/macrophage colony forming units) in the bone marrow adjacent to rheumatoid arthritis joints.

Various cytokines were recently found to be involved in the pathogenesis of rheumatoid arthritis (RA) and particularly, cytokines with hematopoietic activity have been detected in synovial tissues. We counted the number of myeloid precursors in terms of granulocyte/macrophage colony forming units (CFU-GM) and the number of stromal cell progenitors in terms of fibroblast colony forming units (CFU-F) in the tibial bone marrow adjacent to the joints affected by RA (n = 21), osteoarthritis (OA) (n = 10), and trauma (n = 2) using the colony formation unit assay. We also quantitated the amounts of interleukin 1 beta (IL-1 beta), IL-6, and granulocyte/macrophage colony stimulating factor (GM-CSF) in the culture supernatant of synovial tissue explants of these patients by enzyme linked immunosorbent assay (ELISA). The mean number (+/- SEM) of CFU-GM in patients with RA (7.4 +/- 4.9) was greater than that in patients with OA (0.5 +/- 0.2), while CFU-GM was not detected in trauma patients. The number of CFU-GM in the tibial bone marrow of patients with RA correlated well with the amount of IL-1 beta (r = 0.64, p < 0.01), but not with GM-CSF or with IL-6 from synovial tissues. These findings suggest that active bone marrow is present adjacent to the affected joints in patients with RA and that hematopoietic activity is influenced by IL-1 beta produced in nearby synovial tissues.     

Use of long-term human marrow cultures to demonstrate progenitor cell precursors in marrow treated with 4-hydroperoxycyclophosphamide

The continued retrieval of progenitor cells (CFU-GEMM, BFU-E, CFU-E, CFU-GM) from human long-term marrow cultures (LTMC) is not uncommonly used as evidence that proliferation and differentiation are occurring in more primitive hematopoietic stem cells (HSC) in these cultures. Alternatively, the continued presence of progenitors in LTMC could be the result of survival and/or limited self-renewal of progenitor cells present when the culture was initiated, and such progenitors would have little relevance to the parent HSC. The following studies were designed to determine the relative contributions of precursors of progenitor cells to the total progenitor cells present in LTMC using a two-stage regeneration model. The adherent layer in LTMC was established over 3 weeks, irradiated (875 rad) to permanently eliminate resident hematopoietic cells, and recharged with autologous cryo-preserved marrow that was either treated or not treated (control) with 4-hydroperoxycyclophosphamide (4-HC, 100 micrograms/ml for 30 min). The 4-HC-treated marrow contained no progenitor cells, yet based on clinical autologous bone marrow transplant experience, has intact HSC. Within 1-3 weeks, progenitor cells reappeared in the irradiated LTMC recharged with 4-HC-treated marrow, and were preferentially located in the adherent layer. By 2-6 weeks, the number of progenitor in the adherent layer of LTMC recharged with 4-HC marrow was equivalent to control LTMC. The progenitors regenerating in the irradiated LTMC recharged with 4-HC-treated marrow appear to originate from precursors of progenitor cells, perhaps HSC. We propose this model may be useful in elucidating cellular and molecular correlates of progenitor cell regeneration from precursors.     

Isobutyramide, an orally bioavailable butyrate analogue, stimulates fetal globin gene expression in vitro and in vivo

Summary. Clozapine, a novel antipsychotic drug that is particularly effective in treatment-resistant schizophrenia, causes severe agranulocytosis of unknown aetiology in approximately 0·8% of U.S. patients. We evaluated potential toxic mechanisms of drug-induced agranulocytosis. Clozapine, the two major metabolites N-desmethylclozapine and N-oxide clozapine, and five other clozapine derivatives were screened for toxicity to normal haemopoietic precursors. For all compounds except N-des-methylclozapine, toxicity to CFU-GM, BFU-E and CFU-GEMM occurred at concentrations at least 10 times the normal serum levels reported in unaffected patients. In contrast, the LD₅₀ for N-desmethylclozapine was 2·5 μg/ml for CFU-GM, 3·2 μg/ml for BFU-E, and 2·4 μg/ml for CFU-GEMM, only 3–6 times the normal serum concentration. Bone marrow from patients with acute clozapine-induced agranulocytosis was not more sensitive to clozapine or N-desmethylclozapine than bone marrow from normal donors. These studies suggest that N-desmethylclozapine, the major metabolite of clozapine, is itself toxic or is further metabolized to an unstable compound which is toxic to haemopoietic precursors of both myeloid and erythroid lineages.     

Factors affecting volume reduction and red blood cell depletion of bone marrow on the COBE Spectra cell separator before haematopoietic stem cell transplantation

The COBE Spectra is used to volume/red blood cell (RBC) deplete BM before transplantation or cryopreservation. We have audited our results to identify the effect of transit time, refrigerated storage, age and cellular composition on mononuclear cells (MNC) and CD34+ cell recoveries, volume/RBC depletion and neutrophil engraftment. In total, 88 consecutive collections from autologous (n = 25) and allogeneic (n = 63) donors were included. The mean collection volume was 1250 +/- 398 ml with RBC content of 341 +/- 113 ml. The MNC and CD34+ cell recoveries were 83.3 +/- 18.5 and 88.1 +/- 18.9%, respectively, volume depletion was 88.2+/-4.4% and RBC depletion 98.3 +/- 1.8%. Neutrophil engraftment was achieved in 20.1 +/- 6.4 days. Factors affecting MNC and CD34+ cell recoveries were transit time (P = 0.0060), overall age (P < 0.0210) and MNC/CD34+ cell concentrations (P < 0.0313). The presence of crenated RBC also reduced CD34+ cell recovery (P = 0.0028). Refrigerated storage did not adversely affect cell recovery (P > 0.8161) or neutrophil engraftment (P = 0.8959). This study demonstrates that time in transit, overall age, MNC and CD34+ cell concentrations and RBC condition were important factors affecting processing. RBCs show artefacts soon after collection at ambient temperatures and these may interfere with the separation and collection of MNC/CD34+ cells. Refrigeration at 4-6 degrees C during transit and storage may reduce formation of RBC artefacts and maximize MNC and CD34+ cell recoveries.     

Immunologic selection of hemopoietic precursor cells utilizing antibody- mediated plate binding ("panning")

We utilized the property of antibody adherence to plastic to separate and obtain enriched fractions of human myeloid (CFU-GM), erythroid (BFU- E) and pluripotent (CFU-GEMM) hemopoietic precursor cells. Nonadherent buoyant human marrow cells coated with mouse anti-human HLA-DR monoclonal antibody (Mc ab), an anti-pan T lymphocyte Mc ab (Leu 1/17F12) or a granulocyte--monocyte-specific Mc ab (MCS2) were incubated on polystyrene Petri plates coated with affinity purified goat anti-mouse immunoglobulin G (IgG). Cells bound to the coated plates and nonbound cells were separately recovered ("panned") by differential elution. Analysis of the nonadherent buoyant marrow cells demonstrated 12% to possess HLA-DR, 6% T, 40% MCS2 antigens on their surface by indirect immunofluorescence (IMF). After panning, 15% +/- 8%, 14% +/- 4% and 8% +/- 6% cells were plate-bound by their respective antibodies, demonstrating differing binding efficiencies. A substantial degree of purity of the recovered cell fractions was shown for bound 74% +/- 6% and 75% +/- 5% IMF positive cells) and nonbound cells (3% +/- 1% and 0.1% +/- 0.8% positive cells) coated with anti-HLA-DR or anti-T Mc ab respectively, with lesser purity for MCS2 panned cells. Seventy- three percent to 126% CFU recovery was noted, with a sevenfold enrichment of the HLA-DR bound cells for CFU-GM and CFU-GEMM, and 3.5- fold enrichment for BFU-E. Sequential panning, obtaining T nonbound-DR bound-surface immunoglobulin nonbound fractions, resulted in tenfold CFU-GM enrichment (107/10(4) cells, approximately equal to 1/100). Anti- MCS2 antibody was ineffective for panning, but use of this antibody in fluorescence-activated cell sorting (FACS) indicated the absence of the MCS2 antigen on the vast majority of CFU-GM. This study describes a relatively rapid and inexpensive method for obtaining enriched antigenically defined hemopoietic precursors in high yield. These techniques should prove useful for more clearly evaluating cellular and humoral interactions with hemopoietic precursor cells.     

Hematopoietic progenitor cells from allogeneic bone marrow transplant donors circulate in the very early post-transplant period

Despite the therapeutic efficacy of allogeneic bone marrow transplantation (allo-BMT), circulating hematopoietic progenitor cells after bone marrow transplantation have not been well characterized. In the present study, we focused on these 'post-transplant circulating progenitor cells (PTCPC)' which may be on their way to bone marrow. We analyzed the number of myeloid progenitor cells (CFU-GM) per 10 ml of peripheral blood (PB) on days 0 (just before transplantation), 1 (8-15 h after the completion of transplantation), 2, 3, 5, 7, 10, 14, 17, 21, 28 and 35 after allo-BMT in five transplant patients using a standard methylcellulose assay. In addition, high proliferative potential colony-forming cells (HPP-CFC) of the harvested donor bone marrow (BM) and day 1 PB of recipients were assayed in five patients. The origin of HPP-CFC from day 1 PB was analyzed by polymerase chain reaction of a DNA region containing a variable number of tandem repeats. The replating potential of these HPP-CFC was evaluated by a secondary colony assay. The proportion of CD38negative cells among CD34+ cells in the harvested BM and day 1 PB was evaluated by two-color flow cytometric analysis. The number of CFU-GM on day 1 ranged from 6 to 73/10 ml PB, and became undetectable on day 5. The reappearance of PTCPC was observed on day 14, along with hematopoietic recovery. The proportion of HPP-CFC among myeloid colonies from day 1 PB was significantly higher than that from harvested BM (44.3+/-10.4% vs 11.3+/-2.1%, respectively, n=5, P=0.0030). These HPP-CFC from day 1 PB were confirmed to be of donor origin. More than 90% of these HPP-CFC had replating potential. Two-color flow cytometric analysis revealed that the proportion of CD34+CD38negative cells was significantly higher in day 1 PB than in the harvested BM (61.0+/-16.5% vs 9.3+/-3.5%, respectively, n=7, P=0.0002). These observations suggest that both primitive and committed transplanted myeloid progenitor cells may circulate in the very early period following allo-BMT.