Preclinical assessment of purging with VP-16-213: key role for long- term marrow cultures

An evaluation of the effects of VP-16 on normal human marrow cells and representative lymphoma-leukemia cell lines was performed to assess this agent's applicability to ex vivo marrow purging. Tumoricidal dose curves were defined using malignant lymphoid (SK-DHL2 and Reh) and myeloid (HL-60) cells admixed with a 20-fold excess of irradiated marrow cells to simulate a borderline remission marrow. One-hour treatments yielded ID50 of less than 5 mumol/L of VP-16 for clonogenic units from each cell line; rare-to-zero clonogenic units survived exposure to 50 to 100 mumol/L. CFU-Mix, BFU-E, and CFU-GM were equal in their sensitivity to VP-16 (ID50s25 to 30 mumol/L). Marrows treated with 75 mumol/L were completely depleted of these colony-forming cells but produced CFU-GM in one-stage long-term marrow cultures (LTMCs). This dose had little adverse effect on the proliferative capacity of marrow stromal progenitors, as measured by CFU-F (ID50 271 mumol/L) and by the unperturbed development of adherent layers in LTMCs. Furthermore, these stromal layers were able to support hematopoiesis as well as controls in co-culture experiments with autologous marrow cells (two-stage LTMCs). In conclusion, doses of VP-16 that cleanse marrow of lymphoma-leukemia cells spare hematopoietic and stromal progenitors as demonstrated by LTMCs. These data favor the use of VP-16 in the clinical autotransplant setting.     

Myeloablation with diaziquone: in vitro assessment

The promising antineoplastic agent diaziquone is associated with prolonged aplasia and rare instances of bone marrow necrosis, but only mild extramedullary toxicity. To explore the drug's potential as a myeloablative agent prior to bone marrow transplantation, we compared its effects on hematopoietic versus marrow stromal cells. After short- term (one to six hours) or prolonged (three to seven days) exposure to the drug, marrow was assayed for hematopoietic (CFU-Mix, BFU-E, CFU-GM) and stromal (CFU-F) colony-forming cells and studied in long-term marrow culture (LTMC). One- and three-hour treatments produced little cytotoxicity, even at 5000 ng/mL. After six-hour treatments with this dose, marrow was depleted of CFU-Mix, BFU-E, and CFU-GM, but produced CFU-GM in LTMCs, indicating an ongoing input of CFU-GM from a surviving pre-CFU-Mix population. In contrast, elimination of the latter may be inferred from the absence of CFU-GM in LTMCs exposed for three to seven days to diaziquone at only 150 ng/mL. Under these conditions, CFU-F recovery was 40% and adherent stromal layers in LTMCs were similar to untreated controls regarding rate of development and cellular composition. Our in vitro pre-CFU-Mix-ablative regimen correlates with clinical data that show prolonged but reversible myelosuppression at steady-state diaziquone plasma levels of 101 +/- 10 ng/mL (mean +/- standard error of mean) during 7-day constant infusions. In conclusion: hematopoietic cells are more sensitive than marrow stromal cells to the dose- and highly time-dependent cytotoxicity of diaziquone, a direct drug-induced noxious effect on the marrow microenvironment is an unlikely cause of the isolated episodes of marrow necrosis after the use of diaziquone in vivo, and prolonged infusion of diaziquone represents an attractive means for achieving myeloablation in selected clinical situations.     

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.     

Effects of low dose rate irradiation on human marrow hematopoietic and microenvironmental cells: sparing effect upon survival of stromal and leukemic cells

The effects of different dose rates of in vitro irradiation on the proliferative capacity of marrow stromal, hematopoietic and leukemic colony-forming cells (CFC) are described. Marrow cell suspensions, HL-60 cells and trypsin-dispersed fibroblasts were irradiated at 5 or 45 cGy/min and then assayed for CFC. Irradiation at low (5 cGy/min) compared to high (45 cGy/min) dose rate showed a significant difference in survival of stromal and of HL-60 cells, but not of hematopoietic progenitors: the respective D0 values were 170 and 120 (p = 0.003) for marrow fibroblastic progenitors (CFU-F); 145 and 110 (p = 0.005) for passaged marrow fibroblasts (CFU-F); 170 and 140 (p = 0.045) for HL-60 cells; 85 and 85 for multipotential CFC (CFU-mix); 125 and 120 for erythroid progenitors (BFU-E); and 115 and 120 for granulomonopoietic progenitors (CFU-GM) (p = 0.5 for hematopoietic clonogenic cells). Marrow suspensions did not establish confluent stromal layers in long-term marrow cultures following irradiation with 600 cGy at 45 cGy/min, whereas after 840 cGy at 5 cGy/min confluent stromal layers were obtained. This indicates that low dose rate-sparing effect applies to all stromal cell progenitors. Confluent stromal layers derived from progenitors surviving irradiation sustained hematopoiesis as well as controls when co-cultured with fresh hematopoietic cells. Adherent layers in long-term marrow cultures irradiated after establishment with doses less than or equal to 1500 cGy at 5 or 45 cGy/min also showed normal hematopoietic supportive function when co-cultured with freshly isolated hematopoietic cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Radiobiological properties of the human hematopoietic microenvironment: contrasting sensitivities of proliferative capacity and hematopoietic function to in vitro irradiation

We describe the effects of in vitro irradiation on the proliferative capacity and hematopoietic supportive function of human marrow stromal cells. To assess the effects on the proliferative capacity of stromal progenitors and differentiated fibroblasts, marrow cell suspensions and trypsin-dispersed marrow fibroblasts were treated with a single dose of gamma radiation at 100 rad/min. Fibroblastic progenitors (CFU-F) showed an exponential decrease in colony formation with increasing doses of irradiation, with a Do slightly higher than that of granulomonopoietic progenitors (CFU-GM); Do values for CFU-F and CFU-GM were 130 and 115, respectively. However, although the CFU-F survival curve exhibited a shoulder (n = 1.3), the CFU-GM curve did not (n = 1.0), indicating that only fibroblastic progenitors have the potential to repair irradiation- induced damage. Passaged marrow fibroblast colony-forming cells also showed a shouldered exponential survival curve with a Do of 110 and n value of 1.4. Marrow stromal progenitors giving rise to adherent layers in long-term marrow cultures also demonstrated a highly radiosensitive proliferative capacity. Stromal layers derived from irradiated marrow suspensions failed to establish adherent layers after relatively low doses of irradiation (over 240 rad) in a dose-response manner. To assess any functional damage in stromal progenitors surviving irradiation, stromal layers derived from marrow suspensions irradiated up to 240 rad were cocultured with freshly isolated autologous hematopoietic cells and assayed for their capacity to support prolonged CFU-GM production. Confluent stromal layers derived from irradiated marrow suspensions sustained CFU-GM production as well as controls. To study the effects of irradiation on the hematopoietic supportive capacity of established marrow-derived stromal layers, 4 to 6-week-old adherent layers were irradiated as described and cocultured with autologous marrow cells enriched for colony-forming cells. Stromal layers irradiated up to 1,320 rad sustained prolonged CFU-GM production, indicating that the hematopoietic supportive function remained intact at this dose of irradiation. In conclusion, we demonstrated that the proliferative capacity of human marrow stromal progenitors, as well as that of their differentiated descendants, is quite sensitive to in vitro radiation, while the hematopoietic supportive function of differentiated stromal cells is relatively resistant to the effects of radiation.     

Effects of anti-CD33 blocked ricin immunotoxin on the capacity of CD34+ human marrow cells to establish in vitro hematopoiesis in long-term marrow cultures.

Human marrow cells that express the CD34 antigen but lack CD33 are able to initiate sustained, multilineage in vitro hematopoiesis in long-term Dexter cultures and are believed to include the primitive stem cells responsible for effecting long-term hematopoietic reconstitution in vivo following marrow transplantation. In studies described in this report we investigated the effects of a novel anti-CD33 immunotoxin on the clonogenic potential of normal human CD34+ marrow cells and on the ability of these cells to initiate hematopoiesis in two-stage Dexter cultures (long-term marrow cultures, LTMC). This immunotoxin (anti-CD33-bR), shown previously to kill both clonogenic myelogenous leukemia cells and normal mature myeloid progenitor cells (granulocyte-macrophage colony-forming units, CFU-GM), consists of an anti-CD33 monoclonal antibody conjugated to purified ricin that has been modified by blocking the carbohydrate binding domains of the ricin B-chain to eliminate nonspecific binding. For our studies, normal CD34+ human marrow cells were isolated from the light-density (less than 1.070 g/ml) cells of aspirated marrow by positive selection with immunomagnetic beads linked to the monoclonal antibody K6.1. These cell isolates were highly enriched with both multipotential and lineage-restricted clonogenic, hematopoietic progenitors (mixed lineage colony-forming units, CFU-Mix; CFU-GM; and erythroid burst-forming units, BFU-E) which constituted greater than or equal to 20% of the cells. Recovery of clonogenic progenitors from these CD34+ cell preparations, following treatment with anti-CD33-bR (10 nM), was reduced by greater than or equal to 85% for CFU-GM and 20%-40% for CFU-Mix and BFU-E. However, the capacity of these cells to initiate hematopoietic LTMC was preserved. Indeed, the production of high proliferative potential (HPP) CFU-GM, BFU-E, and CFU-Mix in cultures seeded with 10(5) anti-CD33-bR-treated CD34+ marrow cells was substantially greater than that observed in LTMC seeded with equivalent numbers of untreated CD34+ cells. Moreover, concentrations of long-term culture initiating cells in CD34+ cell isolates, quantified by a limiting dilution technique, were found to be increased following anti-CD33-bR treatment. These findings support the potential usefulness of anti-CD33-bR for in vitro marrow purging or in vivo treatment to eliminate CD33+ leukemic clones, while sparing normal CD34+/CD33- stem cells that support normal hematopoiesis and hematopoietic reconstitution in vivo.     

Studies in feline long-term marrow culture: hematopoiesis on normal and feline leukemia virus infected stromal cells.

To study the effects of feline leukemia virus (FeLV) on the hematopoietic microenvironment, a two-step feline long-term marrow culture (LTMC) system was developed and characterized. The adherent, stromal layer of these cultures is composed of fibroblastoid cells (50% to 80%), macrophages (10% to 30%), fat cells (10% to 20%), and large, polygonal cells that express muscle actin (1% to 2%). When fresh, enriched marrow mononuclear cells (MMNC) were added to 3-week-old irradiated stromal cultures, nonadherent erythroid progenitors (BFU-E) and granulocyte/macrophage progenitors (CFU-GM) could be detected for up to 5 and 12 weeks, respectively. LTMC stromal layers established from marrow cells from cats viremic with either a nonpathogenic strain of FeLV (FeLV-A/61E) or the anemogenic strain FeLV-C/Sarma were morphologically equivalent to uninfected LTMC stromal layers, although more than 80% of the stromal cells expressed FeLV gag protein. When FeLV-infected stromal cultures were recharged with uninfected MMNC, altered patterns of hematopoiesis were observed, compared with recharged, uninfected stromal cultures. In cultures with infected stroma, fewer nonadherent cells (NAC), nonadherent BFU-E, and nonadherent CFU-GM were detected during the first 4 to 5 weeks after recharge. In contrast, greater numbers of NAC and nonadherent CFU-GM were found from weeks 5 to 12 after recharge. When FeLV-infected stromal cultures were recharged with MMNC from a cat heterozygous for the X-chromosome-linked enzyme glucose-6-phosphate dehydrogenase (G-6-PD), the percentage of nonadherent CFU-GM expressing the domestic type G-6-PD isoenzyme remained stable over time (mean % domestic [%d], 53% +/- 3%), and was equivalent to that of nonadherent CFU-GM maintained in uninfected cultures (mean %d, 56% +/- 3%), indicating that clonal drift or clonal selection was not responsible for the enhanced maintenance of CFU-GM. Furthermore, as only 10% to 20% of recharged hematopoietic cells became infected with FeLV in vitro, it is unlikely that the altered pattern was due to progenitor infection. We hypothesize that the increase in NAC and nonadherent CFU-GM in FeLV-infected cultures resulted from enhanced growth factor production by stromal cells. The two-step LTMC system may facilitate the characterization of stromal-derived factors that affect progenitor cell engraftment and proliferation.     

The L4F3 antigen is expressed by unipotent and multipotent colony- forming cells but not by their precursors

Antibody L4F3 is a murine monoclonal antibody that recognizes an antigen expressed on in vitro colony-forming cells, including virtually all CFU-GM, CFU-Meg, BFU-E, and CFU-Mix. In the present study we examined whether cells that do not express the L4F3 antigen include precursors of hematopoietic colony-forming cells. Colony-forming cells were depleted from marrow by treatment with L4F3 and complement. The remaining cells generated CFU-GM, BFU-E, and CFU-Mix when cultured in the presence of irradiated adherent cell layers from long-term marrow cultures. Marrow cells not expressing the L4F3 antigen, which were separated by cell-sorting techniques, were depleted of colony-forming cells but nevertheless generated CFU-GM when cultured over irradiated adherent cell layers. These data suggest that there are marrow precursors that do not express the L4F3 antigen and that give rise to colony-forming cells of multiple types. Negative selection techniques should allow the enrichment of these precursors of colony-forming cells, thereby enabling direct studies of these immature stem cells.     

Human multilineage progenitor cell sensitivity to 4-hydroperoxycyclophosphamide

This institution has documented consistent reconstitution of hematopoiesis in patients treated with marrow lethal chemoradiotherapy who are "rescued" by reinfusion of autologous cryopreserved marrow cells incubated with 4-hydroperoxycyclophosphamide (4-HC) for in vitro purging of occult tumor cells. After 4-HC incubation, the reinfusion marrow cells showed marked reduction in committed progenitor cell (BFU-E, CFU-GM) frequency, and often total absence of detectable progenitors, without significant loss of marrow reconstituting ability. Since BFU-E and CFU-GM assays did not predict marrow reconstituting ability after 4-HC incubation, we sought to determine whether multilineage progenitor cells (CFU-GEMM) might be more resistant to 4-HC incubation and therefore a more reliable predictive assay in this setting. We found that BFU-E, CFU-GM, and CFU-GEMM all show similar dose-related sensitivity to in vitro incubation with 4-HC and do not appear representative of the cell(s) responsible for marrow reconstitution.     

Effects of interleukin-6 on fetal hematopoietic progenitors

Effects of interleukin-6 (IL-6) on cycling status and clonogenic maturation of human fetal (cord blood) and adult hematopoietic progenitors were compared. Adult marrow cells were incubated for various lengths of time with various concentrations of IL-6, in a serum- free system, after which tritiated thymidine suicide studies were performed. After incubation of 2 to 5 x 10(6) cells/mL for 4 hours in 5.0 ng IL-6/mL, increased thymidine suicide rates were observed for multipotent progenitors (CFU-Mix), granulocyte-macrophage progenitors (CFU-GM), and erythroid burst-forming units (BFU-E). Similar incubations of fetal cells in IL-6 resulted in similar increases in tritiated thymidine suicide rates. In other studies, IL-6 used alone did not support colony formation from adult progenitors. However, it did support colony formation from fetal CFU-Mix (P less than .05), CFU- GM (P less than .001), and BFU-E (P less than .05). In cultures of adult progenitors, IL-6 acted synergistically with IL-3 to support CFU- Mix colony formation (P less than .001), but synergistic actions on CFU- GM and BFU-E were not seen. In contrast, IL-6 acted synergistically with IL-3 and with GM-CSF to support colony formation by fetal CFU-Mix, CFU-GM, and BFU-E. Thus, IL-6 appears to have a wider spectrum of action on fetal progenitors from cord blood than on adult progenitors; including not only the induction of cycling, but also the support of clonogenic maturation of CFU-Mix, CFU-GM, and BFU-E.     

In vivo infection of marrow stromal fibroblasts by feline leukemia virus.

Marrow stromal fibroblasts (FBs) likely play an important role in the regulation of hematopoiesis within the marrow microenvironment. Infection of these cells by feline leukemia virus (FeLV) might not only contribute to the pathogenesis of FeLV-induced hematologic diseases, but could provide a reservoir for virus in the infected cat. To determine the frequency of FeLV infection among marrow FB precursor cells (fibroblast colony-forming units, CFU-F) of cats viremic with FeLV-C/Sarma and FeLV-A/61E, marrow FBs and FB cell clones were isolated and assayed for expression of FeLV gag protein. From 30% to 86% and 64% to 88% of marrow FB precursors were infected with FeLV-C/Sarma and FeLV-A/61E, respectively. CFU-F from a cat viremic with FeLV-A/61E were not affected by exposure to antibody against FeLV envelope glycoprotein gp70 and heterologous complement, whereas similarly treated hematopoietic progenitors (erythroid colony-forming units, CFU-E; erythroid burst-forming units, BFU-E; and granulocyte-macrophage colony-forming units, CFU-GM) and culture-propagated, FeLV-infected marrow FBs were effectively lysed, suggesting that infected CFU-F within the marrow microenvironment do not express a significant amount of gp70 on their cell membranes. Thus, marrow FB precursor cells appear to be a major target for FeLV in vivo. Furthermore, the low level of gp70 antigen expression on the surface of these cells in vivo may allow them to escape immune surveillance and provide a reservoir of virus during active or latent infection.     

CD34 expression by stromal precursors in normal human adult bone marrow.

Normal bone marrow cells were isolated by fluorescence-activated cell sorting (FACS) on the basis of CD34 antigen expression and then assayed in vitro for colonies of fibroblastic cells (fibroblast colony-forming units [CFU-F]). Greater than 95% of detectable CFU-F were recovered in the CD34+ population, while their numbers were markedly depleted in the CD34- population. Additional experiments showed that the majority of CFU-F exhibited high forward and perpendicular light scatter and low-density CD34 antigen. Growth of sorted cells in medium optimized for long-term marrow culture (LTMC) produced a complex mixture of adherent stromal elements including fibroblasts, adipocytes, smooth muscle cells, and macrophages. Monoclonal antibody STRO-1, which identifies bone marrow stromal cells, reacted with approximately 5% of CD34+ cells, which included all CFU-F and stromal precursors in LTMC. Experiments using soybean agglutinin (SBA) further showed that these stromal elements were restricted to a population of bone marrow cells with the phenotype CD34+/SBA+. These properties of stromal precursors are quite distinct from those of primitive hematopoietic progenitors, showing that although the precursors of the hematopoietic and stromal systems share expression of CD34, they are otherwise phenotypically distinct cell types.     

Recombinant alpha-interferon inhibits colony formation of bone marrow fibroblast progenitor cells (CFU-F).

Alpha-Interferon (IFN-alpha) has been shown to inhibit colony formation of hematopoietic progenitor cells, including colony-forming unit-granulocyte, erythroid, macrophage, megakaryocyte (CFU-GEMM), day 7 colony-forming unit granulocyte-macrophage (CFU-GM), day 14 CFU-GM, burst-forming unit erythroid (BFU-E), pluripotent stem cells (CFU-S), and colony-forming unit megakaryocyte (CFU-MK). The present study was designed to see whether IFN-alpha also has inhibitory effects on bone marrow fibroblast progenitors (CFU-F). We found that IFN-alpha exerted a significant inhibitory effect on both rabbit and human CFU-F formation. Inhibition of human marrow CFU-F formation by alpha interferon was unaffected by removal of 98% of monocytes/macrophages and T lymphocytes from light density marrow cells. This finding suggests that IFN-alpha probably exerts a direct inhibitory effects.     

Autologous bone marrow transplantation in acute myelogenous leukemia: in vitro treatment with myeloid-specific monoclonal antibodies and drugs in combination

We report the results of a preclinical study comparing four different purging protocols using a promyelocytic human cell line HL-60 and myeloid leukemic progenitor cells (colony-forming unit-leukemic [CFU-L]) from acute myelogenous leukemia (AML) patients assayed in semisolid culture. We studied the antileukemic effect of (1) Single-cycle complement-mediated lysis by two different monoclonal antibodies (MoAbs) (M195 [CD33] and F23 [CD13] 40 micrograms/mL), reactive with distinct antigens found on early myeloid cells and monocytes, used alone and in combinations; (2) 4-Hydroperoxycyclophosphamide (4-HC) (80 mumol/L or 100 mumol/L) alone; or (3) combined with VP-16 (5 micrograms/mL) and (4) a cocktail of 1 through 3 as above (combined immunochemotherapy). More than 4 logs of HL-60 tumor cell elimination were observed after 1 hour of incubation with both MoAbs plus 4-HC + VP-16 while the single treatment (immunotherapy or chemotherapy) provided 1.5 and 3.5 logs of colony-forming inhibition, respectively. When the same protocols were tested on cryopreserved leukemic cells from eight patients with AML, we observed a mean value of CFU-L inhibition of 92.3% +/- 2.5% SD, 95.5% +/- 1.4% SD, and 99% +/- 0.8% SD after MoAbs and complement lysis, 4-HC, and 4-HC + VP-16 treatment, respectively. The combined treatment of MoAbs and 4-HC + VP-16 produced more than 3-log reduction of CFU-L colony formation. By comparison, the mean recovery of committed normal bone marrow progenitors after incubation with MoAbs and complement was 12% for CFU-granulocyte-macrophage (CFU-GM), 22.9% for burst-forming unit erythroid (BFU-E), and the recovery following 4-HC + VP-16 treatment was 4.4% for CFU-GM and 5.6% BFU-E. In subsequent experiments, highly purified CD34+ blast cells, enriched by positive selection, and stimulated in liquid culture by cytokines (interleukin-1 [IL-1], IL-3, and combination of both) or MO-conditioned medium (MoCM), demonstrated that immunochemotherapy spares hematopoietic colony-forming cells earlier than day 14 CFU-GM, in vitro.     

Different distribution of decay-accelerating factor on hematopoietic progenitors from normal individuals and patients with paroxysmal nocturnal hemoglobinuria

Deficiency of decay-accelerating factor (DAF) occurs in blood cells in paroxysmal nocturnal hemoglobinuria (PNH), characterized by an unusual susceptibility to hemolysis by complement activation. This study examined DAF expression on hematopoietic progenitors from normal individuals and PNH patients using a fluorescence-activated cell sorter (FACS) with monoclonal antibodies to DAF. Nonphagocytic mononuclear marrow cells expressing different density distributions of DAF were sorted into DAF-, DAF+/-, DAF+, and DAF++ fractions. The cells from each fraction were cultured in methylcellulose and assayed for CFU-E, BFU-E, CFU-GM, and CFU-Mix. The percentages of distribution of DAF- negative normal progenitors increased in the order of CFU-E, CFU-GM, BFU-E, and CFU-Mix, whereas those of DAF-positive cells inversely decreased in this order. These results indicate that DAF expression may accompany differentiation from CFU-Mix to CFU-E. On the other hand, most progenitors in PNH patients had little, if any, expression of DAF on their cell surfaces. These findings were supported by another approach using a complement-dependent cytotoxicity method with the anti- DAF monoclonal antibodies. Abnormal expression of DAF was found on the progenitors in the bone marrow as well as on mature cells circulating in the blood in PNH.     

Identification of stromal cell precursors in human bone marrow by a novel monoclonal antibody, STRO-1

Murine IgM monoclonal antibody STRO-1 identifies a cell surface antigen expressed by stromal elements in human bone marrow (BM). STRO-1 binds to approximately 10% of BM mononuclear cells, greater than 95% of which are nucleated erythroid precursors, but does not react with committed progenitor cells (colony-forming unit granulocyte-macrophage [CFU-GM], erythroid bursts [BFU-E], and mixed colonies [CFU-Mix]). Fibroblast colony-forming cells (CFU-F) are present exclusively in the STRO-1+ population. Dual-color cell sorting using STRO-1 in combination with antibody to glycophorin A yields a population approximately 100-fold enriched in CFU-F in the STRO-1+/glycophorin A+ population. When plated under long-term BM culture (LTBMC) conditions, STRO-1+ cells generate adherent cell layers containing multiple stromal cell types, including adipocytes, smooth muscle cells, and fibroblastic elements. STRO-1+ cells isolated from LTBMC at later times retain the capacity to generate adherent layers with a cellular composition identical to that of the parent cultures. The STRO-1-selected adherent layers are able to support the generation of clonogenic cells and mature hematopoietic cells from a population of CD34+ cells highly enriched in so-called long-term culture-initiating cells. We conclude that antibody STRO-1 binds to BM stromal elements with the capacity to transfer the hematopoietic microenvironment in vitro.     

Inhibition of in vitro hematopoiesis by hepatitis A virus

Inoculation of human bone marrow with hepatitis A virus (HAV) resulted in a dose- and duration-of-incubation-dependent suppression of hematopoietic progenitor (CFU-GM, BFU-E, CFU-Mix) growth in vitro. Monocytic progenitors appeared to be least affected. While HAV inactivation by heat or beta-propiolactone and neutralization by specific antibodies completely abrogated hematopoietic inhibition, depletion of adherent bone marrow cells, and enrichment of progenitors did not alter the pattern of suppression, which also seemed to be independent of HuIFN-alpha, -beta, -gamma, and TNF. These findings support the concept that direct infection of progenitor cells by HAV may be responsible for hematologic changes commonly seen during early phases of infectious hepatitis and possibly for some cases of bone marrow failure.     

Granulocytic and stromal progenitors in the bone marrow of patients with primary myelofibrosis

Quantitative and qualitative changes in granulocyte-macrophage (CFU-GM) and fibroblast colony-forming cells (CFU-F) were studied in 7 patients with primary myelofibrosis (MF). Marrow cells were collected from bone biopsy specimens after treatment with collagenase. The number of CFU-GM correlated with the amount of haemopoietic tissue noted in the bone marrow histology and ranged between 0-400/mg of bone. CFU-F were increased in 2 patients with moderate fibrosis. Circulating CFU-GM were increased in all patients studied (169-3749/ml of blood). There was no significant correlation between the number of CFU-GM in the bone marrow and that in the blood. Cytochemical studies showed a high incidence in eosinophil progenitors in the bone marrow and especially in the blood of patients with MF. These data suggest a functional abnormality of myeloid progenitors in this disease.     

An analysis of the multilineage production of human hematopoietic progenitors in long-term bone marrow culture: evidence that reactive oxygen intermediates derived from mature phagocytic cells have a role in limiting progenitor cell self-renewal

To better understand the limited hematopoietic life span of human marrow "Dexter" cultures, we developed a miniaturized, two-stage culture system with which in vitro production of hematopoietic progenitors could be reproducibly detected and quantified. Light- density, gradient-separated human marrow cells were inoculated into Leighton slide tubes, and adherent ("stromal") cell layers were allowed to develop on the removable coverslips within these tubes during an initial 4 weeks of culture. Once stromal cell layers were established, cultures were irradiated (800 cGy) to eliminate all residual hematopoietic progenitors. The cultures were then recharged with autologous, cryopreserved marrow cells (enriched for BFU-E and CFU-GM) to reconstitute stem cell populations and to initiate in vitro hematopoiesis. Most progenitor cells added to irradiated cultures were no longer detectable by clonal assays within one to four days after recharge. Nonetheless, stable populations of adherent BFU-E and CFU-GM became established in these cultures within 24 to 48 hours, and when the total numbers of progenitors (adherent and nonadherent) were measured at weekly intervals thereafter, it was evident that both BFU-E and CFU-GM were generated in vitro. However, progenitor cell production declined as neutrophils and macrophages accumulated in the cultures. Moreover, with this accumulation of mature myeloid cells, increasing levels of O2- and H2O2 could be detected in the cultures, and it was found that the addition of oxidant scavengers (catalase and mannitol) to culture media enhanced the weekly expansions of progenitor cell numbers that could be measured. These findings support the conclusion that reactive O2 intermediates generated by mature myeloid cells have a role in limiting the duration and extent of hematopoietic progenitor cell self-renewal in long-term "Dexter" cultures of human marrow.     

Autologous transplantation of canine long-term marrow culture cells genetically marked by retroviral vectors.

Retroviral infection of bone marrow cells in long-term marrow cultures (LTMCs) offers several theoretical advantages over other methods for gene transfer into hematopoietic stem cells. To investigate the feasibility of this approach in a large animal model system, we subjected LTMCs from nine dogs to multiple infections with retrovirus containing the neomycin phosphotransferase gene (neo) during 21 days of culture. Feeder layers, cocultivation, polycations, and selection were not used. The in vitro gene transfer efficiency was 70% as determined by polymerase chain reaction amplification of neo sequences in colony-forming unit granulocyte-macrophage (CFU-GM) obtained from day-21 LTMCs. Day-21 LTMC cells were infused into autologous recipients with (four dogs) and without (three dogs) marrow-ablative conditioning. At 3 months posttransplant, up to 10% of marrow cells contained the neo gene. This percentage declined to 0.1% to 1% at 10 to 21 months posttransplant. Neo was also detected in individual CFU-GM, burst-forming unit-erythroid (BFU-E), and CFU-Mix progenitors derived from marrow up to 21 months postinfusion and in cultures of peripheral blood-derived T cells up to 19 months postinfusion. There was no difference in the percentage of neo-marked cells present when dogs that received marrow ablative conditioning were compared with dogs receiving no conditioning. Detection of neo-marked marrow cells almost 2 years after autologous transplantation in a large mammalian species shows that retroviral infection of marrow cells in LTMCs is a potentially nontoxic and efficient protocol for gene transfer. Further, our results suggest that marrow conditioning and in vivo selection pressure to retain transplanted cells may not be absolute requirements for the retention of genetically marked cells in vivo.