Blast cell colony assay for umbilical cord blood and adult bone marrow progenitors

We previously described candidate human blast cell colonies in culture of umbilical cord blood cells. However, their replating efficiencies were low, and we were unable to grow colonies from adult marrow cells. We report here a consistent method of growth and identification of human blast cell colonies that are supported by low serum culture and by delayed addition of medium conditioned by a T lymphoblast cell line, C5MJ. Nonadherent mononuclear cord blood and bone marrow cells were prepared by use of Ficoll-Paque and overnight adherence to plastic. Bone marrow cells were further enriched for progenitors by panning with monoclonal anti-My-10 antibody. Cells were plated in methylcellulose culture containing 2% fetal calf serum and supplemented with bovine serum albumin, lecithin, cholesterol, and transferrin. On day 14 of culture, concentrated C5MJ-conditioned medium was carefully added to each dish. Blast cell colonies consisting of 18 to 100 cells were detected on days 21 to 28. Forty percent to 75% of the blast cell colonies in individual samples yielded secondary colonies upon replating (positive colonies). The replating efficiency of the positive colonies ranged from 3% to 100%. The largest secondary colony contained 7,800 cells. In addition to single-lineage colonies, multilineage colonies revealing two to five lineage combinations were seen. These results suggest that human primitive progenitors are dormant in cell cycle and that they survive in the absence of colony-stimulating factors. Human blast cell colonies may provide a unique population of progenitors for studies of the early process of human hemopoiesis.     

Monoclonal antibody J11d.2 recognizes cell cycle-dormant, primitive hematopoietic progenitors of mice

It was reported that monoclonal antibody (MoAb) J11d.2 reacts with mature blood cells of mice but not with their progenitors. We tested in culture studies whether this antibody could be used for enrichment for primitive marrow progenitors. The majority of colony-forming cells including multipotential progenitors in the marrow cells from 5- fluorouracil (5-FU)-treated mice were J11d.2+, whereas most of the progenitors from normal mice were J11d.2-. In addition, formation of multilineage colonies from J11d.2+ in both 5-FU-treated and normal mice was augmented by interleukin 6. These observations indicated that MoAb J11d.2 recognizes cell cycle-dormant progenitors. We have recently described a simple method that provides 800-fold enrichment for the progenitors in post-5-FU marrow cells using MoAb D7 (anti-Ly-6A/E). When this method was modified to include sorting with MoAb J11d.2, D7+ J11d.2+ cells were 2,250-fold enriched for multipotential progenitors. Micromanipulation and culture of individual D7+ J11d.2+ cells showed that average plating efficiency of the cell population is approximately 70% and that about 30% of the progenitors are lymphohematopoietic in nature. These data demonstrate that J11d.2 is a useful MoAb for the isolation of primitive hematopoietic progenitors of mice.     

Thrombopoietin alone stimulates the early proliferation and survival of human erythroid, myeloid and multipotential progenitors in serum-free culture

We examined the effects of recombinant human thrombopoietin (TPO, c-Mpl ligand) on the proliferation and differentiation of human haemopoietic progenitors other than megakaryocytic progenitors using serum-free cultures. TPO alone supported the generation of not only megakaryocytic (MK) but also blast cell (blast) colonies from cord blood CD34⁺ cells. Delayed addition of a cytokine cocktail (cytokines; interleukin (IL)-3, IL-6, stem cell factor, erythropoietin, granulocyte-macrophage colony-stimulating factor, and TPO) to cultures with TPO alone on day 7 induced various colonies including granulocyte-macrophage (GM) colonies, erythroid bursts (E), granulocyte-erythrocyte-macrophage-megakaryocyte (GEMM) colonies. Replating experiments of blast colonies supported by TPO alone for culture with cytokines revealed that approximately 60% of the blast colonies contained various haemopoietic progenitors. Single cell cultures of clone-sorted CD34⁺ cells indicated that TPO supported the early proliferation and/or survival of both primitive and committed haemopoietic progenitors. In serum-free suspension cultures, TPO alone significantly stimulated the production of progenitors for MK, GM, E and GEMM colonies as well as long-term culture-initiating cells. These effects were completely abrogated by anti-TPO antibody. These results suggest that TPO is an important cytokine in the early proliferation of human primitive as well as committed haemopoietic progenitors, and in the ex vivo manipulation of human haemopoietic progenitors.     

Synergism of interferon-gamma and stem cell factor on the development of murine hematopoietic progenitors in serum-free culture

We examined the effects of interferon-gamma (IFN-gamma) on the growth of murine hematopoietic progenitors supported by interleukin-3 (IL-3) or stem cell factor (SCF) in a serum-free culture system. IFN-gamma inhibited IL-3-dependent granulocyte-macrophage colony growth by normal bone marrow cells, but increased the number of pure and mixed megakaryocyte colonies by post-5-fluorouracil bone marrow cells. The addition of IFN-gamma to the culture containing SCF resulted in a synergistic action on the development of primitive hematopoietic progenitors as well as on the development of mature populations. Primitive progenitors responding to SCF + IFN-gamma were suggested to be supported by SCF in the early stage of development and require IFN- gamma for subsequent growth. Replating experiments of blast cell colonies and comparison of total colony growth among SCF + IFN-gamma, SCF + IL-3, and SCF + IFN-gamma + IL-3 suggest that multipotential progenitors supported by SCF + IFN-gamma are a part of those reactive to SCF + IL-3. These findings suggest that IFN-gamma has bifunctional activity on murine hematopoiesis.     

Induction of mixed erythroid-megakaryocyte colonies and bipotential blast cell colonies by recombinant human erythropoietin in serum-free culture

The effects of recombinant human erythropoietin (rEp) on murine hematopoietic progenitors were studied using a serum-free culture. A high concentration of rEp stimulated the formation of mixed erythroid-megakaryocyte colonies (EM colonies) and blast cell colonies, as well as erythroid colonies, erythroid bursts, and megakaryocyte colonies from normal mouse bone marrow cells. Direct effects of rEp on EM colony, megakaryocyte colony, and erythroid burst formation were confirmed by depletion of accessory cells such as T cells, B cells, and macrophages from crude bone marrow cells, and inhibition of the colonies by the addition of rabbit anti-rEp antibody to the culture in a dose-dependent fashion. Replating experiments were performed to confirm the differentiating ability of blast cell colonies grown in the presence of rEp. Most of the blast cell colonies yielded not only secondary erythroid colonies but also megakaryocyte colonies in the presence of 2 IU/mL rEp. Some of the blast cell colonies produced secondary EM colonies in the presence of 16 IU/ml rEp of 2 IU/mL rEp plus interleukin-3, although no granulocyte-macrophage colonies were found in the secondary culture. These results suggest that Ep acts not only as a late-acting factor that is specific for erythroid progenitors, but also as a bipotential EM-stimulating factor for murine hematopoietic cells.     

A bispecific antibody enhances cytokine-induced killer-mediated cytolysis of autologous acute myeloid leukemia cells

An anti-CD3 Fab' x anti-CD13 Fab' bispecific antibody (BsAb) was generated. This BsAb reacted with both CD3+ T cells and CD13+ acute myeloid leukemia (AML) cells. We investigated whether cytokine- stimulated peripheral blood mononuclear cells (PBMC) could lyse patient AML cells after addition of the BsAb. When interleukin-2 (IL-2)- stimulated PBMC were assayed for their cytotoxicity against 51Cr- labeled allogeneic and autologous CD13+ AML cells, their activity was markedly enhanced by the addition of the BsAb. PBMC stimulated with IL- 2 plus anti-CD3 monoclonal antibody (MoAb) showed higher proliferative ability and higher cytotoxicity if this was expressed as lytic units per culture. IL-7-stimulated PBMC also exhibited enhanced cytotoxicity against CD13+ AML cells after addition of the BsAb. Ultrastructurally, CD13+ AML cells incubated with IL-2 plus anti-CD3 MoAb-stimulated PBMC and the BsAb showed apoptotic morphologic changes. A colony assay for AML blast progenitors showed that the colony formation of CD13+ AML cells was inhibited by the addition of autologous IL-2 plus anti-CD3 MoAb-stimulated PBMC, and that this inhibition was further enhanced by the addition of the BsAb. A colony assay for normal bone marrow progenitor cells showed that the addition of autologous IL-2 plus anti- CD3 MoAb-stimulated PBMC and the BsAb inhibited the formation of granulocyte-macrophage colonies and mixed-cell colonies. However, the degree of inhibition was smaller than that for the AML blast colonies. Taken together, these findings suggest that this BsAb may be useful for ex vivo purging of CD13+ AML cells in autologous bone marrow transplantation.     

Preclinical studies on the use of selective antibody-ricin conjugates in autologous bone marrow transplantation

Whole-ricin immunoconjugates were synthesized with the pan-T cell antibodies T101 and 3A1 and assayed in the presence of 0.1 mol/L lactose. Their toxicity for cell lines, peripheral blood T lymphocytes, and normal bone marrow progenitors was compared with that of whole ricin. In the presence of 0.1 mol/L lactose, normal cells and cell lines exhibited the following sensitivities to ricin: 8392 (human malignant B cell line) less than E rosette-positive lymphocytes less than bone marrow progenitors less than 8402 (human T ALL) less than CEM (human T ALL). Ricin sensitivities correlated with ricin binding as determined by immunofluorescence. In the presence of lactose, peripheral blood T cells were resistant to 0.1 nmol/L ricin, but a similar concentration of T101-ricin inhibited normal and malignant T colony formation by greater than 98%. 3A1-ricin was slightly less effective. At a conjugate concentration of 0.1 nmol/L, bone marrow progenitor colony formation was inhibited by 30% or less; T101-positive cells were at least tenfold more sensitive than normal progenitors. When mixtures of 10% CEM cells and marrow cells were incubated with T101-ricin, 95% of CEM colonies were killed, and 96% of marrow granulocyte/ macrophage progenitors survived. Some free ricin was released from immunotoxin-treated cells, producing minimal inhibition of protein synthesis or cell growth. We conclude that (a) normal blood cells and malignant cell lines exhibit varying degrees of ricin sensitivity in the presence of lactose; (b) T101-ricin is at least tenfold more toxic to T lymphocytes than to bone marrow progenitor cells and is effective in mixtures of normal and malignant cells; and (c) treatment of infiltrated marrow with anti-T cell immunotoxins should safely remove target T cells without excessively damaging normal progenitors or producing excessive free ricin. Anti-T cell, whole-ricin immunotoxins merit trials for autologous transplantation.     

Production of T lymphocyte colony-forming units from precursors in human long-term bone marrow cultures

T cell differentiation in human marrow was studied in Dexter type long- term bone marrow cultures. In these cultures, T lymphocyte colony- forming units (TL-CFU), E rosette-forming cells (E+), and T3+, T4+, and T8+ cells (assayed by indirect immunofluorescence) were found to be present for at least 7 weeks. It was investigated whether the existence of T cells in long-term culture resulted from the persistence of inoculated T lymphocytes or from the production by immature progenitors. No significant numbers of E+, T3+, T4+, or T8+ cells were detected in cultures that were established from E+ lymphocyte-depleted bone marrow, indicating little or no production of T lymphocytes from E- negative precursors. On the other hand, bone marrow cells purged of E+ lymphocytes did not contain TL-CFU, but appeared to regain high numbers of TL-CFU during Dexter culture; this suggested that an earlier step in T cell differentiation may take place in this culture system. The generation of TL-CFU in the E-negative long-term marrow cultures only occurred when an adherent stroma layer had been established in the culture flask; it did not require added mitogens or detectable interleukin 2 in the culture medium. TL-CFU in fresh marrow (TL-CFU II) are mature (E+, T3+) T cells and are capable of producing helper (T4+) and suppressor/cytotoxic (T8+) phenotype cells in colonies. The TL-CFU newly formed in E-depleted Dexter cultures (TL-CFU I) are distinct from this population, as they are E-negative and give rise to colonies of the helper type only. T3 cell depletion of the marrow inoculum prior to culture did not prevent the appearance of TL-CFU I in long-term culture; this suggests that TL-CFU I are derived from an E- and T3- precursor (pre-TL-CFU).     

Marrow purging in autologous bone marrow transplantation for T-lineage acute lymphoblastic leukemia: efficacy of ex vivo treatment with immunotoxins and 4-hydroperoxycyclophosphamide against fresh leukemic marrow progenitor cells

A lymphoblast progenitor cell assay was used to evaluate the antileukemic efficacy of marrow-purging protocols that employed intact ricin immunotoxins (IT) and 4-hydroperoxycyclophosphamide (4-HC) against clonogenic primary T-lineage marrow blasts freshly obtained from 12 T-lineage acute lymphoblastic leukemia (ALL) patients. Residual T-lineage blast colonies were observed after treatment with 1 micrograms/mL T101 (anti-CD5)-Ricin (R) + G3.7 (anti-CD7)-R in eight of 12 cases and after 100 micrograms/mL 4-HC in six of nine cases. By comparison, a combination of IT and 4-HC proved very effective against T-lineage leukemic progenitor cells, and no residual blast colonies were observed in any of the eight cases studied. We conclude that future trials should consider combined treatment protocols such as IT + 4-HC for more effective purging of autologous marrow grafts.     

Evidence for a multipotential stem cell disease in some childhood Philadelphia chromosome-positive acute lymphoblastic leukemia

Children with Philadelphia chromosome (Ph+) acute lymphoblastic leukemia (ALL) have a poorer prognosis than do most pediatric patients with ALL. Because of this poor prognosis and the presence of the Ph chromosome, we have asked whether or not Ph + ALL involves a multipotential stem cell. We cultured hematopoietic progenitors from two children with Ph+ ALL and examined individual BFU-E and CFU-GM colonies for the Ph chromosome. We studied cells from two patients after 18 to 34 months of first complete clinical remission; direct cytogenetic analyses showed 26% and 13% Ph+ metaphases in these patients' marrow cells. BFU-E colonies were obtained from light density marrow cells cultured in methylcellulose supplemented with erythropoietin and CFU-GM colonies from agar or methylcellulose cultures stimulated with leukocyte feeder layers. Fifty-seven G-banded metaphases were recovered from 33 colonies. Ten metaphases from seven colonies were Ph+. Ph+ metaphases were found in three of 12 and three of five BFU-E colonies from the two patients. One of 16 CFU-GM colonies from one patient had the Ph+ chromosome; analyzable metaphases were not obtained from CFU-GM of the other patient. No colonies contained both Ph+ and Ph- cells. These results indicate that Ph+ ALL with persistence of Ph+ cells in remission involves a multipotential stem cell for erythroid and granulocyte/macrophage as well as lymphoid lineages. Multipotential stem cell involvement in the pathogenesis of some childhood Ph+ ALL suggests similarities to Ph+ chronic myelocytic leukemia and may contribute to the poor prognosis of these patients.     

Blast colony-forming cells and precursors of colony-forming cells detectable in long-term marrow culture express the same phenotype (CD33- CD34+).

We have previously shown that CD33- CD34+ human marrow cells are capable of giving rise to colony-forming cells (CFC) in long-term marrow culture (LTMC) but are mainly depleted of progenitors that directly form colonies in semi-solid media. In contrast, the CD33+ CD34+ population contains most of the CFC but not the precursors of these cells detectable in LTMC. The purpose of the present study was to determine if a form of CFC with self-renewal potential, the blast CFC, is contained within the CD33+ CD34+ or CD33- CD34+ population. The results demonstrate that blast CFC segregate within the CD33- population, representing a distinct progenitor population that may be analogous to progenitors for CFC detectable in LTMC.     

Clinical utility of marrow cell tissue cultures in acute leukemia of childhood

To determine the clinical value of tissue culture techniques in the study of acute leukemia, marrows from 50 children (40 acute lymphoblastic [ALL], 10 acute myeloblastic [AML]) were cultured by the use of an AML blast colony assay and the CFU-GEMM assay. In the AML assay, AML marrow gave rise to high numbers of blast colonies within 24 to 48 hr; in sharp contrast, 37 of 40 ALL marrows did not yield colonies in this assay. In the CFU-GEMM assay, AML marrow produced excessive numbers of monocyte-macrophage CFU-C colonies, an obvious background of individual macrophages and clusters, and occasional blast colonies. ALL marrow yielded very low numbers of granulocytic CFU-C colonies, no background of macrophage cells, and no blast colonies. These clear-cut differences in cellular growth kinetics in vitro between ALL and AML marrows should allow confirmation of the type of acute leukemia within 24 to 48 hr. The colony assays may also be valuable in differentiating ALL from AML in difficult diagnoses when conventional approaches are nondiagnostic.     

Mature T cells are part of adherent cells in human long-term bone marrow cultures

We determined the proportion of lymphocytes in nonadherent and adherent fractions of human bone marrow cells cultured by a Dexter-type continuous marrow culture method. T-lymphocytes, B-lymphocytes, and common alloantigen (CALLA)-positive cells (cells with receptors for CALLA) were sequentially enumerated using commercially available appropriate monoclonal antibodies. Nonadherent cells from weeks 2-5 of culture contained relatively fixed proportions of OKT3-positive (4%-10.4%) and CALLA-positive (5%-6.6%) cells. The adherent cells during the culture period between weeks 6 and 18 contained an average of 34% OKT3-positive cells with a range from 6% to 68.5%, despite a high hydrocortisone concentration of 5 X 10(-5) M added to the growth medium. The T cells retrieved from the adherent layers and resuspended in steroid-free medium responded to PHA stimulation and to mixed leukocyte culture in the same manner as did freshly drawn peripheral blood leukocytes. These results indicate that adherent cell populations include mature T-lymphocytes in human long-term bone marrow cultures. In view of well-documented interactions of nonlymphoid hematopoietic progenitors with T-lymphocytes in the clonogenic culture system, it can be speculated that the adherent T cells also may play a role in proliferation and differentiation of granulocyte-macrophage and erythroid progenitors in this long-term culture system.     

Synergism between interleukin-6 and interleukin-3 in supporting proliferation of human hematopoietic stem cells: comparison with interleukin-1 alpha

Currently available evidence suggests that in the steady state, the majority of hematopoietic stem cells are dormant in cell cycle and reside in the so-called G0 period. Studies in our laboratory indicated that once a stem cell leaves G0, its subsequent proliferation requires the presence of interleukin-3 (IL-3). Recently it was reported that interleukin-1 (IL-1) may stimulate stem cells to become sensitive to IL- 3. In a separate study, we observed that interleukin-6 (IL-6, also known as B cell stimulatory factor-2/interferon beta 2) possesses synergism with IL-3, shortening the G0 period of murine hematopoietic stem cells. We report here that human IL-6 and IL-3 act synergistically in support of the proliferation of progenitors for human blast cell colonies and that IL-1 alpha reveals no synergism with IL-3 when tested against purified human marrow progenitors. Panned My-10+ human marrow cells were plated in culture and on day 14 of incubation, either IL-3, IL-6, IL-1 alpha or a combination of these factors was added to the cultures. Blast cell colony formation was analyzed daily between days 18 and 32 of culture. IL-6 or IL-1 alpha alone failed to support blast cell colony formation. In the presence of IL-3 alone, blast cell colonies continued to emerge between days 21 and 27. When a combination of IL-3 and IL-6 was added, blast cell colonies developed earlier than in cultures with IL-3 alone and twice as many blast cell colonies were identified. IL-1 alpha failed to augment IL-3-dependent blast cell colony formation. Replating studies of the individual blast cell colonies revealed various types of single as well as multilineage colonies. These observations suggest that IL-6 shortens the G0 period of human hematopoietic stem cells and that the reported synergistic activities of IL-1 on primitive hematopoietic cells may be indirect.     

A simple method for culturing myeloma cells from human bone marrow aspirates and peripheral blood in vitro

A double layer agar technique has been developed to grow myeloma colonies (MY-CFUc) from human bone marrow aspirates and peripheral blood. Heavily irradiated HL60 cells (5 x 10(5)/plate) are added to an agar underlay in growth medium containing 0.5% agar. Mononuclear cells from the test bone marrow or blood are overlayered in either 0.2 ml HL60-conditioned medium (HL60-CM) or in 0.5 ml growth medium containing 0.23% agar, and the cultures are incubated at 37 degrees C in an atmosphere of 5% CO2, 10% O2 and 85% N2. Colonies (greater than 50 cells) form between 2 and 3 weeks. Using this method 60/68 samples of bone marrow and 7/12 samples of blood from 54 patients have produced colonies in soft agar and in liquid on an agar underlay. The cells which form these colonies are of two distinct sizes, the larger cells being plasmacytoid and the smaller lymphoid. The two cell types are usually, but not always, present in separate colonies. Both plasmacytoid and lymphoid cells carry the isotype of the respective patient's myeloma protein and the plasma cell marker (HAN PC1). This technique has enabled us to culture myeloma cells from patients with as few as 2% plasma cells in the bone marrow but it does not permit the growth of normal B, T or granulocyte-macrophage colonies (GM-CFUc). The drug sensitivity of myeloma cells (MY-CFUc) compared with normal haemopoietic cells (GM-CFUc) can be measured using dose-response curves in individual patients. Furthermore, this method can detect resistant subpopulations within a given myeloma sample.     

Separation of human blast progenitors from granulocytic, erythroid, megakaryocytic, and mixed colony-forming cells by "panning" on cultured marrow-derived stromal layers

Primitive myeloid progenitor cells will adhere to stromal feeder layers of human bone-marrow-derived adherent cells grown in the presence of methylprednisolone (MP+ layers). These progenitors form colonies of blast cells on the MP+ stromal layers, but not on stromal layers grown in the absence of MP (MP- layers). The present study was designed to determine whether this failure of colony formation was caused by inability of the progenitors to adhere to the MP- layers or inability to proliferate in their presence. We also compared the capacities of the blast progenitors to adhere to MP+ and MP- stromal cells with those of mixed (GEMM-CFC), erythroid (BFU-E), megakaryocytic (Mk-CFC), and granulocyte-macrophage (GM-CFC) colony-forming cells. Incubation of bone marrow mononuclear cells with MP+ stromal layers removed 90% of the blast progenitors, but did not remove the majority of the GEMM-CFC, BFU-E, Mk-CFC, and GM-CFC; incubation of bone marrow mononuclear cells with MP- stromal layers did not remove the blast progenitors or the GEMM-CFC, BFU-E, Mk-CFC, and GM-CFC. Thus, the blast progenitors adhere to MP+ stromal feeder layers, but not to MP- stromal layers. In this respect they differ from the other more mature colony-forming cells that do not show any marked tendency to adhere to either type of stromal layer.     

Direct and synergistic effects of interleukin 11 on murine hemopoiesis in culture.

We have examined the effects of a stromal cell-derived cytokine designated interleukin 11 (IL-11) on the proliferation of murine hemopoietic progenitors in methylcellulose culture. COS cell-conditioned medium containing IL-11 supported formation of granulocyte/macrophage colonies and a small number of multilineage colonies including blast cell colonies in cultures of marrow cells from normal mice. When tested with marrow cells harvested 2 days after injection of 5-fluorouracil at 150 mg/kg, IL-11 enhanced interleukin 3-dependent colony formation, whereas IL-11 alone supported only scant colony formation. Serial observations (mapping studies) of cultures of post-5-fluorouracil spleen cells indicated that the mechanism of the synergistic effect of IL-11 is to shorten the dormant period of stem cells, an effect very similar to that of interleukin 6. When pooled blast cells were plated into medium containing IL-11 and erythropoietin, only macrophage colonies were observed. Thus, IL-11 can directly support the proliferation of committed macrophage progenitors and, and like interleukin 6 and granulocyte colony-stimulating factor, act synergistically with interleukin 3 to shorten the Go period of early progenitors.     

Negative regulation of early T lymphopoiesis by interleukin-3 and interleukin-1 alpha

We recently developed a two-step clonal cell culture system for murine lymphohematopoietic progenitors that are capable of producing myeloid and B-lymphoid progenies and characterized their cytokine requirements. We subsequently observed that addition of interleukin-3 (IL-3) or IL-1 alpha to permissive cytokine combinations in primary culture abrogates the B-lymphoid potential but not the myeloid potential of the lymphohematopoietic progenitors. We now describe a similar negative regulation of the T-cell potential of the lymphohematopoietic progenitors. Lin- Ly-6A/E+ marrow cells from 5-fluorouracil-treated mice were plated individually by micromanipulation in methylcellulose culture with steel factor (SF) and IL-11 for 8 days. The resulting colonies were tested for myeloid potential by reculturing part of each colony in secondary myeloid suspension culture. Remainders of individual primary colonies were injected intravenously into scid mice for determination of T- and B-lymphoid potentials. Approximately 10% of the progenitors that differentiated along myeloid lineages in culture reconstituted T- and B-cell compartments in scid mice. However, when scid mice were injected with colonies pooled from cultures containing steel factor, IL-11, and either IL-3 or IL-1 alpha, there was no reconstitution of thymocytes or spleen T cells. These results suggest negative regulatory roles for IL-3 and IL-1 alpha in the early stages of T lymphopoiesis.     

Blood erythroid progenitors (CFU-E and BFU-E) in acute lymphoblastic leukemias

Summary Circulating erythroid progenitors from 14 patients with acute lymphoblastic leukemia (ALL) and from 8 healthy subjects were studied in culture to determine the frequence and size of CFU-E- and BFU-E-derived colonies. Cells were cultured in a plasma clot system, and hemoglobinized colonies identified by diaminobenzidine reaction. The numbers of CFU-E and BFU-E per milliliter of peripheral blood were greatly increased in 10 patients when compared to controls. In 13 patients, the size distribution of BFU-E-derived colonies, analyzed by counting the number of subunits in each colony, was also found to differ significantly from controls, with a large excess of small colonies and a low percentage or a total lack of large colonies. This abnormal BFU-E size distribution was partially corrected, in the 5 patients tested, by the addition to the culture medium of 10% phytohemaglutinin-leukocyte-conditioned medium (PHA-LCM). Bone marrow crowding out of the normal progenitors, as well as disturbances in the cellular interactions involved in their normal development, most likely explain these results and these factors could be implicated in the frequent pancytopenia of ALL.     

Generation of osteoclasts from isolated hematopoietic progenitor cells

A variety of studies have shown that osteoclasts originate from bone marrow, but their exact progenitors and differentiation pathway remain unclear. The treatment of mice with a high dose of 5-fluorouracil (5-FU) results in an enrichment for primitive hematopoietic progenitors; using this procedure, we prepared a new class of murine hematopoietic colonies that had very high secondary plating efficiencies in vitro. When spleen cells from mice pretreated in vivo with 5-FU were cultured in the presence of methylcellulose medium containing recombinant interleukin-3 (rIL-3), small colonies consisting of blast cells with little sign of differentiation developed on day 7 of culture. We lifted these blast colonies, pooled them, and replated them as secondary methylcellulose cultures in the presence of rIL-3 and erythropoietin. Approximately 60% of the cells formed colonies comprising various combinations of neutrophils, macrophages, eosinophils, mast cells, megakaryocytes, and erythroblasts. We replated such blast cells into microtiter wells and cultured them in the presence of rIL-3 (100 U/mL) or recombinant granulocyte-macrophage colony stimulating factor (GM-CSF) (100 U/mL) plus 1.25(OH)2D3 (10(-7) mol/L). Multinucleated cells appeared from day 14 of culture and approximately 100 giant cells per well were scored on day 21 of culture. Parathyroid hormone (1 U/mL) also induced the multinucleated cell formation. May-Grunwald-Giemsa staining revealed the large cells containing many nuclei in their cytoplasm, which is characteristic of bone-resorbing cells or osteoclasts. These cells showed a tartrate-resistant acid phosphatase (TRAP) activity. Calcitonin caused a striking shape change in these cells and suppressed the formation of multinucleated cells. Moreover, electron microscopy shows that these cells were able to resorb fetal calvariae. In the presence of r granulocyte-colony stimulating factor, r macrophage-colony stimulating factor, or r interleukin-6 plus 1.25(OH)2D3, formation of TRAP-positive multinucleated cells was lower compared with the support of rIL-3 or rGM-CSF. Mature macrophages collected from colonies did not form the multinucleated cells as described above, even in the presence of rIL-3 and 1.25(OH)2D3. Moreover, to exclude the possibility that osteoclasts generated from non-blast cells, we performed a cloning experiment from one isolated blast cell and demonstrated that single cells differentiate into osteoclasts or macrophages in the presence of rIL-3 with or without 1.25(OH)2D3. This system will provide a useful model for further analysis of osteoclast formation in vitro.