enitors, in the absence of added cytokines. Under these conditions c-kit ligand, interleukin-7. (IL-7), and Flt3 ligand (Flt3-L) are added to the cultures to ensure ...
5 Differentiation of B Lymphocytes From Hematopoietic Stem Cells Paulo Vieira and Ana Cumano Summary Differentiation of B lymphocytes can be efficiently obtained when multipotent hematopoietic precursors are cocultured with stromal cell lines and soluble growth factors. Stromal cell lines provide yet-undefined signals required for the expansion of the precursor population and/or lineage commitment and soluble mediators. In consequence, the type of the exogenously added interleukins depends on the stromal support used in the assay. In contrast to S17 and OP9 stroma, the fibroblast line NIH3T3 does not support B-cell precursor expansion of CD19+ fetal liver cells; neither does it induce B-lineage differentiation from embryonic multipotent progenitors, in the absence of added cytokines. Under these conditions c-kit ligand, interleukin-7 (IL-7), and Flt3 ligand (Flt3-L) are added to the cultures to ensure optimal B-cell differentiation. Another cytokine, stroma-derived lymphopoietin, can also be used instead of IL-7 in embryonic but not adult hematopoietic precursors.
Key Words Stromal cells; lymphocytes; cytokines.
1. Introduction Lymphocytes are ultimately generated from hematopoietic stem cells (HSCs), which are able to sustain blood cell production throughout life owing to two main properties: self-renewal and multilineage differentiation. Common lymphoid progenitors (CLPs) (1), which are more advanced than HSC in the hematopoietic hierarchy, are also capable of multilineage differentiation: they can give rise to B, T, and NK cells, although they lack erythromyeloid differentiation potential. However, CLPs lack self-renewal capacity and can only originate a wave of mature cell production, in vivo. Both cell types are found in the adult bone marrow (1) where they differentiate in direct contact with the local cytokine-producing stromal compartment. From: Methods in Molecular Biology, vol. 271: B Cell Protocols Edited by: H. Gu and K. Rajewsky © Humana Press Inc., Totowa, NJ
67
68
Vieira and Cumano
HSCs are generated once in the lifetime of mammals—during embryonic development—and colonize the hematopoietic organs where they expand and differentiate. Two populations of hematopoietic precursors coexist and are independently generated in the mouse embryo (2). The first to be detected is generated in the yolk sac (YS) at 7.5 d postcoitum (dpc) and the second in the intraembryonic mesoderm around the aorta rudiment, starting at 9 dpc. These populations differ in the two main aspects of HSCs: The former does not differentiate into lymphocytes and does not support long-term blood production in irradiated hosts. Therefore they are neither multipotent nor self-renewable. The latter population of hematopoietic precursors colonizes adult recipients and contributes for more than 6 mo to all hematopoietic lineages of recipient mice (3). The intraembryonic-derived hematopoietic precursors therefore qualify as HSC. Both embryonic and adult HSCs as well as CLPs can differentiate into multiple hematopoietic lineages in vitro. This development is strictly dependent on the presence in the cultures of soluble growth factors. In contrast to what is observed in vivo, T lymphocytes are conspicuously absent in these cultures, reflecting requirements for differentiation and/or survival that remained, for long, elusive. T cells can, however, be obtained when HSCs or CLPs are assayed in fetal thymic organ cultures (FTOCs) (4), where precursors are allowed to differentiate in a fetal thymic lobe depleted of hematopoietic components. T-cells obtained under these conditions closely resemble thymocytes, and the system is highly efficient in that single cells are capable of generating large numbers of T lymphocytes. We will not discuss here the FTOC cultures that have been extensively described elsewhere. For many years, the attempt to maintain unmanipulated HSCs in vitro for long periods of time has been pursued, and different culture systems were devised, most dependent on the presence of bone marrow-derived stromal cells. So far no culture system allowed self-renewal and expansion of either embryonic or adult HSCs. Multipotent precursors with multilineage long-term reconstituting capacity cannot be maintained in vitro and rapidly differentiate. This constraint creates serious restrictions on the genetic manipulation required for autologous transplantation after gene transfer. The understanding of the requirements for self-renewal of stem cell populations remains therefore a major focus of research in hematopoiesis.
2. Materials 2.1. Dissection of Embryo 1. 70% ethanol. 2. Phosphate-buffered saline (PBS) with calcium and magnesium.
Differentiating B Lymphocytes and Stem Cells
69
2.2. Organ Culture In Toto and Analysis of Hematopoietic Potential 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.
13.
14.
24-Well plate (TPP). OptiMem (Invitrogen). Fetal calf serum (FCS from ICN). Penicillin–streptomycin (Invitrogen). β-Mercaptoethanol (Invitrogen). OP9 stromal cells (T. Nakano, S.-I. Nishikawa, Kyoto, Japan). S17 cells (from K. Dorshkind), NIH-3T3. Trypsin–ethylenediaminetetraacetic acid (EDTA) (Invitrogen). 96-Well plates (TPP). Hank’s balanced salt solution (HBSS from Invitrogen). Trypan blue (Invitrogen). Irradiator (X-ray or cesium source). Interleukin-7 (IL-7) supernatant. We use the supernatant of J558 cells transfected with the complementary deoxyribonucleic acid (cDNA) encoding IL-7, a kind gift from F. Melchers (Basel, Switzerland). Cells are grown to confluency and the supernatant is collected. Titration is done in 2E8 cells, a kind gift from P. Kincade (Oklahoma, USA), dependent on IL-7 for growth. The supernatant is serially diluted on the 2E8 cells, and growth is scored 3 d later either by counting the cells or by thymidine incorporation. The highest dilution that gives maximum proliferation is chosen. Murine thymic stroma-derived lymphopoietin (TSLP) can be purchased from R&D. Flt3 ligand (Flt3-L) supernatant: we use the supernatant of the Sp2.0 myeloma transfected with the cDNA encoding Flt3-L and Baf3 cells transfected with the cDNA encoding Flt3, kind gifts from R. Rottapel (Toronto, Canada). Supernatant collection and titration is done as in step 12. A c-kit-ligand (kL) supernatant: we use the supernatant of CHO cells transfected with the cDNA encoding kL, a kind gift from Genetics Institute (Boston, MA). Titration is done as in step 12 on freshly prepared bone marrow mast cells cultured for 1 wk in kL.
2.3. Flow Cytometry Analyses 2.3.1. Monoclonal Antibodies From Pharmingen (Becton-Dickinson) 1. 2. 3. 4. 5. 6.
CD19–fluorescein isothiocyanate (FITC; clone 1D3). Ter119–phycoerythrin (PE). Mac-1– allophycocyanin (APC) (clone M1/70). CD4-PE. CD8-APC. Ly5.1–FITC.
2.3.2. Other Products 1. 1X PBS. 2. Round-bottom 96-well plate (CML). 3. Propidium iodide (PI) (Sigma).
70
Vieira and Cumano
3. Methods 3.1. Culture System 3.1.1. Stromal Cell Lines
Multipotent hematopoietic progenitors do not efficiently give rise to B lymphocytes in suspension culture without the presence of stromal cells, reflecting the requirement for yet-undefined survival and/or proliferation signals. We have successfully used two different mouse stromal lines obtained from the bone marrow. S17 is capable of supporting B lymphocyte, myeloid, and erythroid cell differentiation from single HSCs. More recently we have been using the OP9 stromal cell line, derived from bone marrow of the op/op mouse. This line is therefore deficient in macrophage colony-stimulating factor (M-CSF) production and does not sustain macrophage overgrowth that can impair lymphocyte expansion. In contrast, OP9 produces IL-15, strictly required for NK cell development. Consequently, the development of hematopoietic multipotent cells in OP9 stroma leads to the differentiation of large numbers of B-cell precursors and of NK-cells (see Fig. 1). OP9 stroma is 30% more efficient than S17 stroma in B-cell development from single HSC cultures (5,6). The interaction between committed B-cell precursor and the surrounding stroma does not require a specialized cell type as shown by our finding that a fibroblast line, NIH-3T3, can replace classic stromal lines in these cultures (7). The use of fibroblasts affords a better control of the cytokines in the culture because 3T3 cells produce no factors able to support differentiation of B lineage cells. Indeed committed B-cell precursors from either fetal liver or bone marrow do not proliferate on NIH-3T3 feeder layers in the absence of exogenous growth factors. All cell lines can be amplified and maintained in OptiMem, 15% fetal calf serum (FCS), 1% antibiotics, and 5 × 10–5 M β-mercaptoethanol, at 37°C, 5% CO2. For the preparation of the culture plates coated with stromal cells, cells are trypsinized by adding 4 mL of trypsin–EDTA to the culture flask that has been preemptied of culture medium. Trypsin is allowed to act for 15 min at room temperature. Cells are then collected and washed once in HBBS containing 5% of FCS. The pellet is recovered in 1 mL complete medium (OptiMem, 10% FCS, 1% antibiotics). Stromal cells are numbered, after dilution in trypan blue, and plated at a concentration of 5 × 104 living cells per mL of complete medium for
Fig. 1. (see facing page) Phenotypic analysis of B-cell precursors derived from single multipotent embryonic precursors, in culture with OP9 cells, IL-7, and Flt3-L. Cells were stained with anti-NK1.1 and CD19. Gaited populations are shown in the right panels.
71
72
Vieira and Cumano
both S17 and OP9, or 105 per mL in the case of NIH-3T3. 50 μL of stromal cell suspension are dispensed per well of flat-bottom 96-well plates or 500 μL of 24-well plates. Stromal cells are left overnight at 37°C. Plates seeded with stromal cells are prepared 1 d before the culture starts (8). 3.1.2. Culture Conditions for B-Cell Differentiation
Before plating hematopoietic cells in culture, stromal-cell-coated plates are irradiated to arrest cell proliferation. A 10-Gy dose of irradiation is necessary. Hematopoietic precursors are suspended at the required concentrations in complete medium; 50 μL of this suspension is then deposited in each well onto the stromal cell layer. Each well now contains 100 μL (stromal cells + progenitors). Finally, 100 μL of complete medium supplemented with growth factors is added to a final volume of 200 μL. Although IL-7 is an important requirement for B-cell development in vitro, Flt3-L and kL were shown to potentiate B lineage cell recovery. Stromal cells lines, like fresh stroma, secrete cytokines important for hematopoietic development. However, individual cell lines are heterogeneous in cytokine production reflecting either diversity in the stromal bone marrow compartment or the consequence of epigenetic modifications during the establishment of the line. S17 stromal cells produce undetectable levels of IL-7 when expanded in vitro, although they are able to support the expansion and differentiation of precursor B cells, in the absence of added factors, albeit at low frequency. In contrast OP9 cells do not produce M-CSF and Flt3-L, but express kL and IL-7, though in limited amounts. As mentioned in Subheading 3.1.1., NIH-3T3 cells alone are unable to support the expansion of committed B-cell precursors (7). We presently supplement all cultures with Flt3-L, IL-7, and kL to provide an appropriate environment for proliferation and differentiation. Complete medium supplemented with growth factors (each of those twice concentrated) is prepared and 100 μL added to each well. Under those conditions, multipotent hematopoietic precursors can differentiate toward lymphoid, myeloid, and erythroid lineages (if the erythroid lineage differentiation is to be studied, erythropoietin should be added to the cytokine cocktail at a concentration of 8 U/mL). It is noteworthy that OP9 stromal cells are less efficient in sustaining myeloid cell development than S17. The culture lasts 12–14 d, at 37°C, 5% CO2. During the culture period, half of the medium is removed on d 5 and replaced by 100–150 μL of fresh medium in each well (carefully remove the medium with a multichannel preferentially in the surface, so as not to take any developing hematopoietic cells) supplemented with Flt3-L and IL-7 only. An excess of kL favors the development of mast cells that can become predominant in the culture. By the end of the culture period, the well content can be analyzed by flow cytometry. Recently, TSLP, has been
Differentiating B Lymphocytes and Stem Cells
73
Fig. 2. Phase-contrast view of a clone of B-lineage cells developed from single micromanipulated multipotent precursors from embryonic origin.
characterized as another B-cell growth factor. Although less well studied it apparently targets more mature subsets of B-cell precursors than IL-7 (see Subheading 3.1.3.). 3.1.3. B-Cell Development In Vitro
B-lineage precursors expand extensively and if obtained from embryonic tissues can give rise to large clones (up to 107 cells) of small, round lymphocytes (see Fig. 2). If the starting population consists of HSCs, the first CD19+ cells appear around d 10, whereas CLPs give rise to CD19+ cells within 5 d of culture. The detection of CD19+ cells is followed by differentiation to IgM+ cells that rapidly die thereafter. During this period, cells can respond to lipopolysaccharide (LPS). After an additional 8–10 d, IgM is detectable in the culture supernatant by enzyme-linked immunosorbent assay (ELISA). Mature B cells do not develop easily in vitro, and most keep an immature phenotype IgM+IgD– (see Chapter 7). Cultures of BM-derived B-cell precursors can be maintained for long periods of time by the expansion of CD19+IgM-CD43+ cells. This phenomenon was well characterized in cultures derived from AGM HSCs (9).
74
Vieira and Cumano
Fig. 3. Phenotypic analysis of BM-derived B-cell precursors, after 5 d in culture with NIH-3T3 in the presence of exogenous IL-7 (left panel) or TSLP (right panel). The cells were stained for surface IgM and analyzed in a FACS calibur. FSC, forward light scatter.
The earliest subsets of B lineage cells to respond in vitro to IL-7 are BM fraction A/B. IL-7 is, however, not the only characterized growth factor for B-cell precursors. As previously mentioned, TSLP has been shown to promote the expansion of B-cell precursors from mouse BM. The target population is a later subset than fraction A/B (10) because TSLP cultures do not last as long as IL-7 cultures, and the phenotypic analysis of the cells shows a predominance of small IgM+ cells compared to cultures in IL-7 (Fig. 3). This result, which has been interpreted as evidence that TSLP is a differentiation rather than a growth factor, instead indicates that TSLP promotes expansion only of a more mature BM fraction, leading consequently to the absence of proliferating pro-B cells in the culture (9). When FL precursors are used, however, IL-7 and TSLP have nearly indistinguishable activities. Both factors support proliferation of FL pro-B cells, and in this case, TSLP does not lead to the predominance of small B cells in the culture (7). We have analyzed cultures starting with single micromanipulated embryonic HSCs that lasted for up to 40 d in the presence of OP9 and exogenous kL, Flt3L, and IL-7. Although CD19 expression was first detected after 10 d of culture, IgM+ cells could only be detected between days 19 and 30 (9). As cultures further developed, IgM+ cells could no longer be detected. At this point a phenotypic analysis reveals an increasing fraction of pro-B cells that express
Differentiating B Lymphocytes and Stem Cells
75
Table 1 Cultures of AGM Cells Spleen
Day 10
Day 21
5.25
0.5
0.7
1.25
0.5
0.3
Productive/nonproductive VDJ rearrangements Ratio of VDJ/DJ rearrangements
B cells developed in vitro have an increased ration of productive/nonproductive VDJ rearrangements and a decreased ratio of VDJ/DJ rearrangements. (For experimental details see ref. 9.)
CD19 and CD43, and the cells do not respond to LPS. B-cell precursors that were expanded for more than 4 wk in culture show a ratio of D–JH vs VH–D–JH rearrangements fivefold higher than in IgM– bone marrow cells, and most VH–D–JH rearrangements are out of frame (see Table 1). Taken together these results indicate the accumulation of cells that cannot progress in differentiation to a pre-B cell and later B cell stages in these cultures. These are apparently blocked in the pro-B-cell stage either by downregulation of the proteins required for immunoglobulin loci rearrangement or by the survival of cells that underwent nonproductive rearrangements. These cells respond well to IL-7, and both would account for the results described above. They can eventually grow indefinitely, and after a crisis period they can be cloned and generate IL-7-dependent cell lines. 3.2. Phenotypic Analysis of Hematopoietic Progeny 3.2.1. Flow Cytometric Analysis 1. After a 2-wk culture, cells from each well are analyzed by flow cytometry for the presence of lineage-specific surface markers: CD19 for lymphoid B cells, Mac-1 for myeloid cells, and Ter119 for erythrocytes. 2. Transfer cells from each culture well into a round-bottom 96-well plate. Spin down the plate 2–3 min at 1300 rpm, 4°C. 3. Excess of medium in the wells is flicked off by inverting the plate vigorously onto a paper towel. 4. Staining mix is prepared in 1X PBS, 3% FCS with 10 mM sodium azide (FACS medium). We use CD19–FITC and NK1.1–PE, at the proper dilutions, previously estimated. Resuspend each cell pellet in 25 μL of staining mix. Incubate 15–20 min at 4°C, in the dark. 5. By the end of the incubation period wash the cells in FACS medium, by adding 200 μL per well, then spin 2–3 min at 1300 rpm, 4°C. During the spin, FACS
76
Vieira and Cumano medium with PI is prepared (0.5 μg/mL PI). PI is a DNA intercalator, entering passively in the nucleus of dead cells. Interestingly, PI gives equal fluorescence in the flow cytometer on filters detecting PE and Cy5–PE conjugates. Therefore it allows the elimination of dead cells that appear in the diagonal of the plot of both parameters (Fig. 1). After washing stained cells, resuspend the pellets in FACS medium–PI and analyze the cells. Use the SSC parameter in linear mode so that you can easily distinguish between mononuclear cells (low SSC) and granulocytes (high SSC).
Acknowledgments This work was supported by the Ligue Contre le Cancer to the Lymphocyte Development Unit as an associated laboratory. References 1. 1 Kondo, M., Weissman, I. L., and Akashi, K. (1997). Identification of clonogenic common lymphoid progenitors in the mouse bone marrow. Cell 91, 661–672. 2. 2 Cumano, A., Dieterlen-Lievre, F., and Godin, I. (1996) Lymphoid potential probed before circulation in mouse, is restricted to caudal intraembryonic splanchnopleura. Cell 86, 907–916. 3. 3 Cumano, A., Ferraz, J. C., Klaine, M., DiSanto, J. P., and Godin, I. (2001) Intraembryonic but not yolk sac hematopoietic precursors, isolated before circulation, provide long-term multilineage reconstitution. Immunity 15, 477–485. 4. 4 Jenkinson, E. J., Anderson, G., and Owen, J. J. (1992) Studies on T cell maturation on defined thymic stromal cell populations. J. Exp. Med. 176, 845–853. 5. 5 Nakano, T., Kodama, H., and Honjo, T. (1994) Generation of lymphohematopoietic cells from embryonic stem cells in culture. Science 265, 1098–1101. 6. 6 Cho, S. K., Webber, T. D., Carlyle, J. R., Nakano, T., Lewis, S. M., and ZunigaPflucker, J. C. (1999) Functional characterization of B lymphocytes generated in vitro from embryonic stem cells. Proc. Natl. Acad. Sci. USA 96, 9797–9802. 7. Voßhenrich, C. A. J., Cumano, A., Müller, W., Di Santo, J. P., and Vieira, P. (2003) Thymic stroma-derived lymphopoietin distinguishes fetal from adult B cell development. Nat. Immunol. 4, 773–779. 8. Godin, I., Dieterlen-Lievre, F., and Cumano, A. (1995) Emergence of multipotent hemopoietic cells in the yolk sac and paraaortic splanchnopleura in mouse embryos, beginning at 8.5 d postcoitus. Proc. Natl. Acad. Sci. USA 92, 773–777. 9. Nourrit, F., Doyen, N., Kourilsky, P., Rougeon, F., and Cumano, A. (1998) Extensive junctional diversity of Ig heavy chain rearrangements generated in the progeny of single fetal multipotent hematopoietic cells in the absence of selection. J. Immunol. 160, 4254–4261. 10. Hardy, R. R., Carmack, C. E., Shinton, S. A., Kemp, J. D., and Hayakawa, K. (1991). Resolution and characterization of pro-B and pre-pro-B-cell stages in normal mouse bone marrow. J. Exp. Med. 173, 1213–1225.
