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Although only 5% of steady-state peripheral blood (PB) CD34+ cells were found to express chemokine receptor CXCR4, 45% of the cells became CXCR4+ after incubation at 37 degrees C for 4 hours. In contrast, there were no remarkable differences between PB CD34+ cells before and after the 37 degrees C incubation in their expression of selectin ligand, VLA-4, and VLA-5 or in their affinity for VCAM-1 or fibronectin. This increase in CXCR4 expression level was inhibited by the addition of brefeldin A, actinomycin D, or cycloheximide. When PB CD34+ cells with CXCR4 expression levels enhanced by a 4-hour preincubation at 37 degrees C or bone marrow (BM) CD34+ cells were exposed overnight to stromal cell-derived factor 1 (SDF-1), the expression levels of CXCR4 were greatly reduced, and when SDF-1 was removed, CXCR4 levels were thereafter up-regulated. The reexpressed CXCR4 was able to elicit integrin-dependent migration of hematopoietic progenitor cells. There was no difference in the severe combined immunodeficient mouse repopulating cell activity between PB CD34+ cells with and cells without a 37 degrees C preincubation.

Cord blood (CB) cells are a useful source of hematopoietic cells for transplantation. The hematopoietic activities of CB cells are different from those of bone marrow and peripheral blood (PB) cells. Platelet recovery is significantly slower after transplantation with CB cells than with cells from other sources. However, the cellular mechanisms underlying these differences have not been elucidated. We compared the surface marker expression profiles of PB and CB hematopoietic cells. We focused on two surface markers of hematopoietic cell immaturity, i.e., CD34 and AC133. In addition to differences in surface marker expression, the PB and CB cells showed nonidentical differentiation pathways from AC133(+)CD34(+) (immature) hematopoietic cells to terminally differentiated cells. The majority of the AC133(+)CD34(+) PB cells initially lost AC133 expression and eventually became AC133(-)CD34(-) cells. In contrast, the AC133(+)CD34(+) CB cells did not go through the intermediate AC133(-)CD34(+) stage and lost both markers simultaneously. Meanwhile, the vast majority of megakaryocyte progenitors were of the AC133(-)CD34(+) phenotype. We conclude that the delayed recovery of platelets after CB transplantation is due to both subpopulation distribution and the process of differentiation from AC133(+)CD34(+) cells.

Although the vast majority of hematopoietic progenitor cells (HPCs) reside within the bone marrow (BM), a small number of HPCs also continuously circulate in the peripheral blood (PB). The examination of the fate of blood-borne HPCs in parabiotic mice, which are surgically conjoined and share a common circulation, recently revealed that steady-state PB HPCs play a physiological role in, at least, the functional re-engraftment of unconditioned BM. To assess the possibility that human HPCs have a similar function, in this study we examined the expression level and affinity of the homing-related molecules, as well as the SCID mouse reconstituting cell (SRC) activity of human PB CD34+ cells, and compared adults with neonates. There was no remarkable difference between adults and neonates in the expression of E- and/or P-selectin ligands by PB CD34+ cells or in these cells' affinity to VCAM-1. In contrast, the expression level of CXCR4 on PB CD34+ cells was much lower in adults than in neonates. Adult cells also showed a much lower SRC activity than neonates. These results suggest that human PB HPCs may contribute to steady-state hematopoiesis in the BM of neonates to some extent, but not so much in adults.

Background: An ex vivo culture system was previously established for stem cell expansion using human marrow stromal cells and serum-free medium. However, the stromal cells were prepared using long-term culture medium containing horse serum and FCS, which may transmit infectious diseases of xenogeneic origin. In this study, therefore, a method was established to prepare stromal cells using an AB serum-based medium. In the case that serum from a transplant recipient or PBPC donor is available, additional infectious diseases would not be transmitted., Study Design and Methods: Cord blood CD34+ cells were cultured with thrombopoietin, stem cell factor, and flt3/flk2 ligand on a monolayer of human marrow primary stromal cells prepared using long-term culture medium or AB serum-based medium. After 2 weeks, clonogenic progenitor activity and SCID mouse-reconstituting cell activity were assayed. mRNA expression of cytokines and Notch ligand by stromal cells was also examined., Results: There were no remarkable differences in expansion-supporting activity and mRNA expression between stromal cells established by the two methods., Conclusion: An ex vivo expansion system completely based on AB serum has been established.

Background: In vitro maintenance and expansion of human hematopoietic stem cells is crucial for many clinical applications, and investigators have been using xenogeneic, especially murine, stromal cells for stem-cell expansion. In addition, many such culture systems utilize FCS-containing medium or serum-free medium that contains human- or animal-derived proteins. However, the possible transmission of infectious diseases has led to a debate about the safety of the delivery of grafts expanded in culture using cells and proteins of allogeneic or xenogeneic origin. Using primary human BM stromal cells, we have established an AB serum-based co-culture system to expand human primitive progenitors and transplantable stem cells., Methods: Cord blood CD34+ cells were cultured on a monolayer of human BM-derived primary stromal cells with thrombopoietin (TPO), stem-cell factor (SCF) and flt3/flk2 ligand (FL) in the presence of either FCS or AB serum. One to three weeks later, cells were examined for total cells, CD34+ cells, CD34+ CD38- cells, and clonogenic progenitors. SCID mouse reconstituting cell (SRC) activity was also studied., Results: Three weeks of culture with TPO, SCF, and FL supported more than a 250-fold expansion of CD34+ cells, CD34+ CD38- cells and CFU-C, regardless of the kind of serum used. SRC assay revealed that transplantable stem cells were moderately expanded as well., Discussion: This ex vivo expansion system should prove valuable in clinical settings in which stromal cells and serum are available from recipients or stem-cell donors.

Objective: In an attempt to maintain and expand human stem cells, many investigators have used xenogeneic, especially murine, stromal cells and fetal calf serum. Because of the possible transmission of infectious diseases, however, the safety of the delivery of grafts expanded in culture using xenogeneic cells and serum has been debated. Using primary human marrow stromal cells, we established a novel serum-free culture system to expand human primitive progenitors and transplantable stem cells., Materials and Methods: Cord blood CD34(+) cells were cultured on a monolayer of human primary marrow stromal cells in the presence of thrombopoietin (TPO), flt3/flk2 ligand (FL), and/or stem cell factor (SCF) under serum-free conditions. After 2 or 4 weeks of culture, cells were examined for clonogenic progenitors and severe combined immunodeficient disorder (SCID) mouse-reconstituting cells (SRC)., Results: In the presence of TPO, FL, and SCF, marrow stromal cells supported more than a 100- and 1,000-fold expansion of CD34(+) cells and colony-forming units in culture after 2 and 4 weeks of incubation, respectively. In addition, cobblestone area-forming cells were expanded more than 18- and 60-fold after 2 and 4 weeks of culture, respectively. Furthermore, SRC assay demonstrated augmented engraftment by cultured cells., Conclusion: This ex vivo expansion system should prove valuable in clinical settings in which stromal cells are available from recipients or stem cell donors.

In vitro maintenance and expansion of human hematopoietic stem cells is crucial for many clinical applications. Thrombopoietin (TPO) and flt3/flk2 ligand (FL) have been suggested to support the proliferation of primitive hematopoietic progenitors and the expansion of transplantable stem cells in culture. In this study, we examined the synergistic effects of the murine stromal cell line MS-5 and a combination of the two cytokines, TPO and FL, on the ex vivo expansion of human cord blood primitive progenitors and transplantable stem cells. A monolayer of MS-5 cells with TPO/FL synergistically supported a more than 600-fold expansion of human cord blood CD34+ cells and CD34+CD38- cells in 2 weeks of culture. Colony-forming unit in culture (CFU-C) and 5-week and 8-week cobblestone area-forming cells (CAFC) were also expanded approximately 300-, 4- and 13-fold, respectively. When MS-5 cells were physically separated from progenitors by a Transwell filter, the synergy was reduced to a quarter of the control, suggesting that direct cell-cell contact between MS-5 cells and progenitors is required for maximum expansion. The severe-combined immunodeficient (scid) mouse-reconstituting cell (SRC) assay demonstrated the slight augmentation of transplantable stem cell activity in culture. These results indicated that MS-5 cells provide a milieu that stimulates the proliferation of primitive progenitors including transplantable stem cells.

To investigate the clinical applicability of prophylaxis of post-transplant graft-versus-host disease by UV-B irradiation of stem cell preparations, the UV-B sensitivities of human lymphocytes and primitive hematopoietic progenitors were compared. The mononuclear cell fractions (MNC) derived from human cord blood and granulocyte-colony-stimulating factor-mobilized peripheral blood were used. After UV-B irradiation, lymphocyte proliferation ability, hematopoietic colony-forming cells, and apoptotic cells were analyzed. At a dose of 33 J/m(2), significant differences were observed in the residual percentages of hematopoietic progenitors and lymphocyte functions [ANOVA, F (5,46) = 19.4; P <.0001], and the difference between CFU-C (85.2% + 24.0%; n = 8) and MLR (12.7% + 12. 6%; n = 10) was significant (P <.0001). There were no significant differences in the residual percentages of CFU-C, HPP-CFC, and LTC-IC. Percentages of annexin V-positive cells in the total MNC and the CD34(+) cell population in MNC after UV-B irradiation were 69.8% and 18.7%, respectively. In conclusion, there was a range of UV-B doses over which T lymphocytes were inactivated but hematopoietic progenitors, including HPP-CFC and LTC-IC, were preserved.

To study the cytokine regulation of early stages of human B-lymphopoiesis, we developed a stroma-free two-step culture system. Single human cord blood CD34+CD38- cells were individually cultured by micromanipulation with interleukin (IL)-3, stem cell factor (SCF), fIt3 ligand (FL), IL-6 and granulocyte colony-stimulating factor (G-CSF). About 10% of the cells formed primary colonies, which were individually tested for myeloid and B-lymphoid potentials by reculturing aliquots of the primary colony cells into secondary myeloid and B-lymphoid cultures. One third of the primary colonies proved capable of differentiation into CD19+IgM+ cells, as well as into myeloid lineage cells. RT-PCR analyses revealed that some cells in the primary culture had already matured to express B cell-specific transcripts. Thus, the combination of IL-3, SCF, FL, IL-6 and G-CSF supported the differentiation of CD34+CD38- lymphohematopoietic progenitors toward B cell lineage in addition to myeloid lineages. Screening of cytokines to identify the minimum requirement of cytokines in the primary culture revealed that IL-3 and SCF were essential and that the addition of FL, and to a lesser extent IL-6 or G-CSF, to the combination of IL3 and SCF remarkably enhanced the primary colony formation and the generation of CD19+ cells in the secondary B-lymphoid culture.

Earlier, we described a stromal cell-free two-step clonal culture system in which murine primitive lymphohematopoietic progenitors produce myeloid and B-lymphoid lineage cells. In the same culture T-cell potential of the progenitors was maintained. We now report that, in addition to myeloid and B-lymphoid cells, putative T-cell progenitors are also produced in culture. Lineage-negative (Lin-) Ly-6A/E+ c-kit+ bone marrow cells from 5-fluorouracil-treated mice were cultured in methylcellulose in the presence of SF (Steel factor), interleukin (IL)-11, and IL-7, and the resulting primary colonies were picked and pooled. When injected into severe combined immune deficiency (scid) mice, the pooled cells reconstituted the T-cell compartment of the scid mice earlier than freshly prepared primitive marrow cells. This reconstitution activity of the pooled primary colony cells was enriched in the Ly-6A/E+ and FcgammaRII/III-/low cell fractions. Reverse transcriptase-polymerase chain reaction (RT-PCR) and DNA-PCR analyses showed that some of the primary colony cells are differentiated sufficiently to express messenger RNA (mRNA) of T-cell receptor (TCR) beta-chain and pre-TCR alpha (pTalpha) and, although not frequently, to perform Dbeta-Jbeta rearrangement of the TCR gene. Micromanipulation studies confirmed the clonal origin of myeloid lineage cells and the cells positive for the T-cell-specific transcripts and D-J rearrangement of TCR beta-chain. These results suggested that, in the presence of SF, IL-11, and IL-7, primitive lymphohematopoietic progenitors differentiate toward T-cell lineage in addition to myeloid and B-cell lineages.

We describe a two-step clonal culture assay system for human lymphohematopoietic progenitors present in umbilical cord blood which are capable of differentiation along both myeloid and B-lymphoid lineages. Human cord blood CD34+ cells were plated in methylcellulose in the presence of stem cell factor (SCF), granulocyte-colony stimulating factor (G-CSF), interleukin (IL)-7, and the murine stroma cell line, MS-5. The growing primary colonies were individually examined for their potentials to differentiate along both myeloid and B-lymphoid lineages by reculturing aliquots of the primary colonies in methylcellulose culture containing IL-3, G-CSF and erythropoietin (Epo), and on a monolayer of MS-5 in the presence of SCF and G-CSF. Approximately 10-15% of the primary colonies generated various combinations of myeloid cells and CD19+ sIgM+ cells. Subsequent studies using micromanipulated single CD34+ cells unequivocally demonstrated the clonal origin of the lymphohematopoietic progenitors. This culture system should prove valuable for elucidation of the mechanisms regulating early stages of human lymphohematopoiesis.

The receptors for interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), and IL-5 share a common signaling subunit betac. However, in the mouse, there is an additional IL-3 signaling protein, betaIL-3, which is specific for IL-3. We have previously reported that IL-3 abrogates the lymphoid potentials of murine lymphohematopoietic progenitors and the reconstituting ability of hematopoietic stem cells. We used bone marrow cells from betac- and betaIL-3-knock-out mice to examine the relative contributions of the receptor proteins to the negative regulation by IL-3. First, we tested the effects of IL-3 on lymphohematopoietic progenitors by using lineage-negative (Lin-) marrow cells of 5-fluorouracil (5-FU)-treated mice in the two-step methylcellulose culture we reported previously. Addition of IL-3 to the combination of steel factor (SF, c-kit ligand) and IL-11 abrogated the B-lymphoid potential of the marrow cells of both types of knock-out mice as well as wild-type mice. Next, we investigated the effects of IL-3 on in vitro expansion of the hematopoietic stem cells. We cultured Lin-Sca-1-positive, c-kit-positive marrow cells from 5-FU-treated mice in suspension in the presence of SF and IL-11 with or without IL-3 for 7 days and tested the reconstituting ability of the cultured cells by transplanting the cells into lethally irradiated Ly-5 congenic mice together with "compromised" marrow cells. Presence of IL-3 in culture abrogated the reconstituting ability of the cells from both types of knock-out mice and the wild-type mice. In contrast, addition of GM-CSF to the suspension culture abrogated neither B-cell potential nor reconstituting abilities of the cultured cells of wild-type mice. These observations may have implications in the choice of cytokines for use in in vitro expansion of human hematopoietic stem cells and progenitors., (Copyright 1998 by The American Society of Hematology.)

We identified the cell cycle status of CD34(+) cells of steady-state bone marrow (BM) and peripheral blood (PB) obtained from healthy volunteers, and those of apherasis PB samples collected from healthy donors who had been administered granulocyte colony-stimulating factor (G-CSF). More than 10% of CD34(+) cells in BM were in S+G2/M phase. In contrast, regardless of whether G-CSF treatment was performed, less than 2% of CD34(+) cells in PB were cycling. BM CD34(+) cells showed greater VLA-4 expression and adherence to stromal cells than PB CD34(+) cells. In addition, when cycling and dormant BM CD34(+) cells were analyzed separately, the cells in S+G2/M phase expressed more VLA-4 and adhered to the stromal cell monolayer more efficiently than the cells in G0/G1 phase. Furthermore, this adhesion of CD34(+) cells to the stromal cell layer was almost completely inhibited by anti-VLA-4 antibody. Taken together, these results suggest that CD34(+) progenitors in G0/G1 phase of the cell cycle differ from those in S+G2/M phase in adhesiveness mediated by VLA-4 in the hematopoietic microenvironment., (Copyright 1998 by The American Society of Hematology.)

We established a co-culture system with a monolayer of the murine bone marrow (BM) stroma cell line, MS-5, in which human cord blood CD34+ cells differentiated to CD19+ cells. The addition of stem cell factor (SCF) and granulocyte colony-stimulating factor (G-CSF) highly enhanced the production of CD19+ cells. The expansion of the cell numbers was over 10(3)-fold. Furthermore, a significant proportion (<45%) of the cells expressed surface IgM (sIgM) after 5 weeks of co-culture. CD34+CD19- cells also showed a similar development of CD19+ cells and CD19+sigM+ cells. Filter separation of MS-5 cells and CD34+ cells did not inhibit the growth of CD19+ cells. However, when further purified CD34+CD19-CD13- CD33- cells were cultured in the presence of MS-5 cells with or without a separation filter, CD19+ cells did not appear in the non-contact setting. This result suggested that the highly purified CD34+CD19-CD13-CD33- progenitors require the cell-cell contact for the development of CD19+ cells, whereas other CD34+ fractions contain progenitors that do not require the contact. This co-culture system should be useful for the study of early human B-lymphopoiesis.

We have established a clonal cell culture system that supports the proliferation of committed natural killer (NK) cell progenitors of mice to investigate the pathway and cytokine regulation of NK cell development. Day 14 fetal thymocytes cultured in methylcellulose with interleukin-7 (IL-7), IL-15, and steel factor (SF) formed diffuse colonies that could not be classified to known colony types. Single-cell origin of the colonies was established by micromanipulation of the colony-forming cells. Cells in the colonies are very blastic, showing no cytoplasmic differentiation, and express Ly5, Thy-1, and CD25 but not myeloid, B, mature T, or NK cell markers. The cells lack T, B, and myeloid potentials but can differentiate to mature NK cells in fetal thymus organ culture, suggesting that the colonies consist of NK committed progenitors. Examination of the minimal cytokine requirement for the NK colony formation showed that IL-7 and SF are indispensable for the formation of immature NK cell colonies. Both IL-2 and IL-15 increased the frequency of colonies. In contrast to IL-2, IL-7, and IL-15, IL-4 strongly inhibited the formation of the colonies. This quantitative clonal culture will provide a useful means to examine the mechanism of NK cell development.

In an effort to establish the specificity of the thrombopoietin (TPO) effects on murine multipotential progenitors, we tested the effects of soluble TPO receptor (sTPOR; sMpl) on multilineage colony formation that was supported by a combination of TPO and steel factor (SF). Surprisingly, sTPOR did not suppress colony formation from primitive progenitors. This led to the discovery that sTPOR synergizes with SF or Flt3/Flk2 ligand (FL) to support the formation of various types of hematopoietic colonies including multilineage colonies. The colonies supported by the combination of sTPOR and SF were capable of expressing both myeloid and B-lymphoid potentials. Studies using micromanipulation and serum-free culture showed that the effects of sTPOR and SF on the primitive progenitors are direct, not mediated by contaminating stromal cells, and not dependent on factors present in the serum. TPOR belongs to the cytokine receptor group that includes granulocyte colony-stimulating factor receptor (G-CSFR) and erythropoietin receptor (EPOR). Therefore, we tested the effects of sG-CSFR and sEPOR on primitive progenitors. sG-CSFR, but not sEPOR, was able to synergize with SF or FL in supporting the proliferation of primitive progenitors. The direct effects of the soluble receptors appear to be mediated through interactions with their respective membrane-bound receptors expressed on the primitive hematopoietic progenitors.

We recently reported that interleukin-3 (IL-3) inhibits B lymphoid lineage expression in methylcellulose culture in a dose-dependent manner. We subsequently used flow cytometric analysis of individual colonies in timed exposure to IL-3 to define more precisely the negative regulatory role of IL-3 in early B lymphopoiesis. When lymphohematopoietic progenitors isolated from 5-fluorouracil (5-FU)-treated mice were cultured in the presence of Steel factor (SF), IL-11, IL-7, and erythropoietin (Epo), B lymphopoiesis appeared to proceed through three stages: lymphohematopoietic proliferation, commitment, and early B lymphoid proliferation. When IL-3 was added to the culture for a 48-hour interval from days 4 to 6 of culture, IL-3 slightly enhanced the formation of pre-B cell colonies. These data appeared to contradict our previous observations that continued exposure to IL-3 from days 0 to 4 or longer severely inhibits B lymphoid potential of the cultured cells. A more frequently timed kinetic observation revealed that in the presence of IL-3 the peak of lymphohematopoietic progenitors was 48 hours earlier but less than one-tenth the number of lymphohematopoietic progenitors in cultures without IL-3. When added to cultures for 48 hours beyond day 6 of culture, IL-3 abrogated the B cell potential of the cultured cells. However, IL-3 failed to negatively modulate B lymphoid progenitors when added on day 14 of culture or later. These observations indicate that IL-3 is a potent negative modulator of the early B lymphopoiesis. IL-3 appears to hasten but suppress the proliferation and commitment of lymphohematopoietic progenitors to B cell lineage. It may also inhibit the proliferation of the progenitors immediately after commitment to B cell lineage.

We have previously described a two-step methylcellulose culture system in which individual primitive progenitors from 5-fluorouracil (5-FU)-treated mice were shown to have both myeloid and B lymphoid differentiation capacity. Highly enriched Lin-Sca+FU2d BM cells were cultured in methylcellulose in the presence of Steel factor (SF), interleukin-7 (IL-7), and pokeweed mitogen stimulated spleen cell conditioned medium (PWM-SCM). Primary mixed myeloid colonies were replated after 8-11 days into secondary cultures containing SF and IL-7, which supported the generation of B220+sIgM- pre-B cell colonies. A number of growth factors, including IL-6, IL-11, granulocyte colony-stimulating factor (G-CSF), and IL-12 were shown to be capable of substituting for PWM-SCM to support the B lymphoid potential of primary colonies. B lymphoid potential was not supported, however, in SF + IL-3 or in SF + IL-3 plus any single growth factor (IL-1 to -12, granulocyte-macrophage colony-stimulating factor [GM-CSF], G-CSF, erythropoietin [Epo], leukemia inhibitory factor [LIF], tumor necrosis factor-alpha [TNF-alpha], transforming growth factor-beta [TGF-beta], gamma interferon [IFN-gamma], or insulin-like growth factor-1 [IGF-1]), but was supported in SF + IL-3 + 5% PWM-SCM. Experiments were designed to identify the factor or factors in PWM-SCM that reverse the inhibitory effects of IL-3 on B lymphoid potential. By substituting various cytokine combinations for PWM-SCM, we determined that combinations of IL-4 + IL-6 or IL-4 + IL-11, but not IL-4 alone, can substitute for PWM-SCM to reverse the inhibitory effect of IL-3 on B lymphoid potential. Neutralizing antibodies to IL-4 completely eliminated the activity in PWM-SCM, but antibodies to IL-6 only partially inhibited the activity. IL-11 was not detected in PWM-SCM, and the activity co-purified with IL-4, but not with IL-6. Thus, IL-4 plus IL-6, IL-11, or one or more unidentified growth factors in PWM-SCM can reverse the inhibitory effects of IL-3 on early B lymphocyte development in culture.

Because of their known myelopoietic activities, both interleukin (IL)-3 and IL-1 are often used in combination with other cytokines for in vitro (ex vivo) expansion of stem cells. We have investigated the effects of IL-3 and IL-1 on in vitro expansion of murine hematopoietic stem cells with long-term engraftment capabilities, using a highly purified progenitor population. Lineage-negative, Ly-6A/E+, c-kit+ bone marrow cells from male mice were cultured in suspension in the presence of stem cell factor, IL-6, IL-11, and erythropoietin with or without IL-3 or IL-1. Kinetic studies revealed an exponential increase in total nucleated cells and about 10-fold enhancement of nucleated cells by IL-3 during the initial 10 days. Addition of IL-3 hastened the development but significantly suppressed the peak production of colony-forming cells. Addition of IL-1 also significantly suppressed the numbers of colony-forming cells. The reconstituting ability of the cultured cells was tested by transplanting the expanded male cells into lethally irradiated female mice. The cells expanded from enriched cells in the absence of IL-3 and IL-1 revealed engraftment at 2, 4, 5, and 6 months, whereas addition of IL-3 or IL-1 to the cultures significantly reduced the reconstituting ability. The results suggest that these cytokines may have a modulatory role on the self-renewal of stem cells and further indicate that the use of IL-3 and IL-1 for in vitro expansion of human stem cells needs to be cautiously evaluated.

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.

We have identified and characterized the lymphohematopoietic progenitors in the bone marrow of normal mice using a single-step methylcellulose culture assay. Lineage-negative Ly-6A/E (Sca-1)+ progenitors isolated from normal mice were plated in methylcellulose culture containing steel factor (SF), interleukin-7 (IL-7), erythropoietin (Ep), and IL-11. After 16 to 17 days of culture, pre-B-cell-containing multilineage myeloid colonies can be microscopically identified; however, flow-cytometric analysis of individual colonies for B220-positive cells proved superior to in situ microscopic identification of lymphomyeloid colonies. Approximately 10% (6/66) of the mixed colonies without a conspicuous B-cell component had B220-positive cells. The single cell origin of the lymphomyeloid colonies was confirmed by micromanipulation. Although the combination of SF, IL-7, and Ep was sufficient to support formation of lymphomyeloid colonies, addition of IL-11, granulocyte colony-stimulating factor or IL-12 to the combination of SF, IL-7, and Ep increased the number of lymphomyeloid colonies. IL-1 alpha and IL-3 independently inhibited the expression of the B-lymphoid lineage when added to the combination of SF, IL-7, Ep, and IL-11. Approximately four times more lymphohematopoietic progenitors are present in normal mice than in mice treated with 5-fluorouracil.

We have examined the effects of the murine ligand (FL) for the flt3/flk2 tyrosine kinase receptor on the proliferation of murine lymphohematopoietic progenitors as well as committed myeloid and B-cell progenitors. In the presence of erythropoietin, FL alone supported scant colony formation from enriched marrow cells of normal mice. However, when it was combined with interleukin-3 (IL-3), steel factor (SF), or IL-11, FL significantly enhanced colony formation. When tested on enriched marrow cells from 5-fluorouracil (5-FU)-treated mice, FL neither enhanced IL-3-dependent colony formation nor synergized with SF in support of colony formation. However, FL synergized with IL-6, IL-11, or granulocyte-colony stimulating factor (G-CSF) in support of formation of various types of colonies, including multilineage colonies. Approximately 30% of these colonies yielded pre-B-cell colonies when replated in secondary cultures containing SF and IL-7, indicating that 2-cytokine combinations, including FL and IL-6, IL-11, or G-CSF can support the proliferation of primitive lymphohematopoietic progenitors. FL, by itself and in synergy with IL-7 or SF, supported the proliferation of B-cell progenitors. These results show that FL has a wide range of activities in early hematopoiesis and B lymphopoiesis.

CD43 is expressed by many types of mature blood cells including T-lymphocytes, activated B-lymphocytes, neutrophils, monocytes, and platelets but not non-hemopoietic cells. Defective CD43 is seen in patients with Wiscott-Aldrich syndrome. We have investigated CD43 expression by hemopoietic progenitors of normal and 5-fluorouracil(5-FU)-treated mice. Bone marrow cells were enriched for progenitors by a combination of density centrifugation and negative immunomagnetic selection using lineage-specific monoclonal antibodies. They were then separated based on expression of Ly-6A/E and CD43. Approximately 50% of the Ly-6A/E+ cells of both normal and 5-FU-treated mice were CD43neg-low, while the rest of the cells were CD43bright. The majority of the colony-forming cells including lymphohemopoietic progenitors that gave rise to both myeloid and B-lymphoid lineages were present in the Ly-6A/E+CD43bright cell population. Therefore, Ly-6A/E+CD43bright cells were approximately 2-times enriched for the progenitors relative to Ly-6A/E+ cells. Anti-CD43 antibody is useful in isolation of primitive murine hemopoietic progenitors.

We have investigated the effects of interleukin (IL)-12 (natural killer cell stimulatory factor/cytotoxic lymphocyte maturation factor) on the proliferation of murine myeloid and lymphohematopoietic progenitors in methylcellulose culture. In the presence of erythropoietin (Ep), IL-12 alone failed to support colony formation by mononuclear and enriched marrow cells of normal mice. Steel factor (SF) alone supported primarily formation of granulocyte/macrophage (GM) colony formation. However, the combination of the two cytokines yielded a significant number of multilineage colonies. When tested on marrow cells from 5-fluorouracil (5-FU)-treated mice, the combination of IL-12 and SF, but not the single factors, was effective in support of formation of various types of colonies. Approximately 25% of these colonies yielded pre-B-cell colonies when replated in secondary culture containing SF and IL-7, indicating that IL-12 can interact with SF in supporting the development of primitive lymphohematopoietic progenitors. These results demonstrate that IL-12, a cytokine believed to be involved in the development of cell-mediated immune responses, has a wider range of activity, including committed myeloid and multipotent lymphohematopoietic progenitors.

We have used a two-step clonal culture system to unequivocally demonstrate that individual primitive lymphohemopoietic progenitor cells have the capacity for differentiation along either the myeloid or the B-lymphoid lineage. Highly enriched murine marrow cells were plated individually in culture by micromanipulation in the presence of pokeweed mitogen-stimulated spleen cell conditioned medium, erythropoietin, steel factor (SF), and interleukin (IL) 7. Forty-five percent of the single cells formed primary colonies expressing multiple hemopoietic lineages. When aliquots from individual colonies were replated in secondary methyl cellulose culture containing SF and IL-7, 41% of the primary colonies gave rise to lymphocyte colonies. Cells of the lymphocyte colonies were blast-like and B220+, sIg-, Mac-1-, Gr-1-, Ly-1-, L3T4-, Ly-2-, and CD3-. Thirty to 70% of the cells were Thy-1+. mu-chain mRNA was detected in most of the cells by in situ hybridization with an antisense RNA probe. When lymphocyte colonies derived from a single cell were pooled and individually injected into scid mice, donor-type IgM was measurable in the serum of mice and spleens contained donor-type B cells. We then carried out initial screening of growth factors to identify growth factors that might replace pokeweed mitogen-stimulated spleen cell conditioned medium in the primary culture. Combinations of two factors that included SF plus IL-6, IL-11, or granulocyte colony-stimulating factor were all effective in the primary culture in the maintenance of the B-lymphoid potential. Interestingly, IL-3 could neither replace nor act synergistically with SF to support the lymphoid potential of the primary cultures. Our observations demonstrate that many primitive progenitors previously believed to be myeloid-committed also possess B-lymphoid potential. This culture system should prove valuable for elucidation of the mechanisms regulating early stages of lymphohemopoiesis.