Multilineage hemopoiesis induced by cloned stromal cells

Long-term hemopoiesis in culture depends upon the presence of an adherent layer composed of a variety of stromal cells. A subtype of endothelial-adipocytes from the bone marrow stroma (clone 14F1.1) was previously shown to induce long-term myelopoiesis and renewal of pluripotent stem cells. One of a series of stromal cell lines and clones from mouse thymus stroma (STAC-1.2) has now been found to support long-term hemopoiesis. These marrow- and thymus-derived stromal cell clones also have lymphopoietic activities: precursor T cells, or pre-B cells accumulated in co-cultures of thymus cells and the stromal clones, as indicated by cell surface markers, T cell receptor and immunoglobulin gene rearrangements. The predominance of a cell type in these cultures depended upon the serum used to supplement the medium. Recombinant interleukin 2 (IL-2) and the 14F1.1 clone synergistically promoted the proliferation of thymocytes, while a thymus hormone, THF-gamma 2, shifted the population to a relatively mature phenotype. It is proposed that one major function of stromal cells, whether from the bone marrow or thymus, is to restrain the maturation flow and preferentially support the accumulation of cells at early differentiation stages.     

Expression of CD28 by bone marrow stromal cells and its involvement in B lymphopoiesis

Young mice lacking CD28 have normal numbers of peripheral B cells; however, abnormalities exist in the humoral immune response that may result from an intrinsic defect in the B cells. The goal of this study was to assess whether CD28 could be involved in the development of B cells. CD28 mRNA was detected preferentially in the fraction of bone marrow enriched for stromal cells. Flow cytometry and RT-PCR analysis demonstrated that CD28 was also expressed by primary-cultured stromal cells that supported B lymphopoiesis. Confocal microscopy revealed that in the presence of B-lineage cells, CD28 was localized at the contact interface between B cell precursors and stromal cells. In addition, CD80 was detected on 2-6% of freshly isolated pro- and pre-B cells, and IL-7 stimulation led to induction of CD86 on 15-20% of pro- and pre-B cells. We also observed that stromal cell-dependent production of B-lineage cells in vitro was greater on stromal cells that lacked CD28. Finally, the frequencies of B-lineage precursors in the marrow from young (4- to 8-wk-old) CD28(-/-) mice were similar to those in wild-type mice; however, older CD28(-/-) mice (15-19 mo old) exhibited a 30% decrease in pro-B cells and a 50% decrease in pre-B cells vs age-matched controls. Our results suggest that CD28 on bone marrow stromal cells participates in stromal-dependent regulation of B-lineage cells in the bone marrow. The localization of CD28 at the stromal cell:B cell precursor interface suggests that molecules important for T cell:B cell interactions in the periphery may also participate in stromal cell:B cell precursor interactions in the bone marrow.     

Early pre-B-cell transformation induced by the v-fms oncogene in long-term mouse bone marrow cultures.

Murine long-term bone marrow cultures that support B-lymphoid-cell development were infected with a helper-free retrovirus containing the v-fms oncogene. Infection of B-lymphoid cultures resulted in the rapid clonal outgrowth of early pre-B cells, which grew to high cell densities on stromal cell feeder layers, expressed v-fms-coded glycoproteins, and underwent immunoglobulin heavy-chain gene rearrangements. Late-passage cultures gave rise to factor-independent variants that proliferated in the absence of feeder layers, developed resistance to hydrocortisone, and became tumorigenic in syngeneic mice. The v-fms oncogene therefore recapitulates known effects of the v-abl and bcr-abl oncogenes on B-lineage cells. The ability of v-fms to induce transformation of early pre-B cells in vitro underscores the capacity of oncogenic mutants of the colony-stimulating factor-1 receptor to function outside the mononuclear phagocyte lineage.     

Systematic analysis of the ability of stromal cell lines derived from different murine adult tissues to support maintenance of hematopoietic stem cells in vitro.

Hematopoietic stem cells interact with a complex microenvironment both in vivo and in vitro. In association with this microenvironment, murine stem cells are maintained in vitro for several months. Fibroblast-like stromal cells appear to be important components of the microenvironment, since several laboratories have demonstrated that cloned stromal cell lines support hematopoiesis in vitro. The importance of the tissue of origin of such cell lines remains unknown, since systematic generation of stromal cell lines from adult tissues has never been accomplished. In addition, the capacity of stromal cell lines to support reconstituting stem cell has not been examined. We have previously described an efficient and rapid method for the immortalization of primary bone marrow stromal cell lines (Williams et al., Mol. Cell. Biol. 8:3864-3871, 1988) which can be used to systematically derive cell lines from multiple tissues of the adult mouse. Here we report the immortalization of primary murine lung, kidney, skin, and bone marrow stromal cells using a recombinant retrovirus vector (U19-5) containing the simian virus large T antigen (SV40 LT) and the neophosphotransferase gene. The interaction of these stromal cells with factor-dependent cells Patterson-Mix (FDCP-Mix), colony forming units-spleen (CFU-S), and reconstituting hematopoietic stem cells was studied in order to analyze the ability of such lines to support multipotent stem cells in vitro. These studies revealed that stromal cell lines from these diverse tissues were morphologically and phenotypically similar and that they quantitatively bound CFU-S and FDCP-Mix cells equally well. However, only those cell lines derived from bone marrow-supported maintenance of day 12 CFU-S in vitro. One lung-derived stromal cell line, ULU-3, supported the survival of day 8 CFU-S, but not the more primitive CFU-S12. A bone marrow-derived stromal cell line, U2, supported the survival of long-term reconstituting stem cells for up to 3 weeks in vitro as assayed by reconstitution 1 year post-transplant. These studies suggest that adherence of HSC to stromal cells is necessary but not sufficient for maintenance of these stem cell populations and that bone marrow provides specific signals relating to hematopoietic stem cell survival and proliferation.     

Structure and function of bone marrow environment

Bone marrow and spleen are the major hematopoietic tissue in adult mice. However, little is known about the specific mechanism regulating hematopoiesis within these tissues. Since Dexter et al. first described conditions to maintain bone marrow hematopoiesis, long term bone marrow culture (LTBMC) has been developed in order to analyze the mechanism of the maintenance of proliferation and differentiation of hematopoietic stem cells in vitro. Furthermore, several stromal cell lines which are able to support the growth and differentiation of hematopoietic lineage, has been established from LTBMC. Although it is well known that bone marrow stromal cell lines are able to produce colony stimulating factors, it has been suggested that the stromal cell factors which involve membrane bound moieties must have a key role in the regulation of hematopoiesis. We expect that monoclonal antibodies to the surface of bone marrow stromal cells could detect such a critical stroma-associated protein that bounds the cell surface of the bone marrow stroma.     

Generation of murine stromal cell lines supporting hematopoietic stem cell proliferation by use of recombinant retrovirus vectors encoding simian virus 40 large T antigen.

The bone marrow is a complex microenvironment made up of multiple cell types which appears to play an important role in the maintenance of hematopoietic stem cell self-renewal and proliferation. We used murine long-term marrow cultures and a defective recombinant retrovirus vector containing the simian virus 40 large T antigen to immortalize marrow stromal cells which can support hematopoiesis in vitro for up to 5 weeks. Such cloned cell lines differentially supported stem cells which, when transplanted, allowed survival of lethally irradiated mice, formed hematopoietic spleen colonies in vivo, and stimulated lymphocyte proliferation in vitro. Molecular and functional analyses of these cell lines did not demonstrate the production of any growth factors known to support the proliferation of primitive hematopoietic stem cells. All cell lines examined produced macrophage colony-stimulating factor. The use of immortalizing retrovirus vectors may allow determination of unique cellular proteins important in hematopoietic stem cell proliferation by the systematic comparison of stromal cells derived from a variety of murine tissues.     

Oncogene cooperation in lymphocyte transformation: malignant conversion of E mu-myc transgenic pre-B cells in vitro is enhanced by v-H-ras or v-raf but not v-abl.

Although transgenic mice bearing a c-myc gene controlled by the immunoglobulin heavy-chain enhancer (E mu) eventually develop B-lymphoid tumors, B-lineage cells from preneoplastic bone marrow express the transgene but do not grow autonomously or produce tumors in mice. To determine whether other oncogenes can cooperate with myc to transform B-lineage cells, we compared the in vitro growth and tumorigenicity of normal and E mu-myc bone marrow cells infected with retroviruses bearing the v-H-ras, v-raf, or v-abl oncogene. The v-H-ras and v-raf viruses both generated a rapid polyclonal expansion of E mu-myc pre-B bone marrow cells in liquid culture and 10- to 100-fold more pre-B lymphoid colonies than normal in soft agar. The infected transgenic cells were autonomous, cloned efficiently in agar, and grew as tumors in nude mice. While many pre-B cells from normal marrow could also be induced to proliferate by the v-raf virus, these cells required a stromal feeder layer, did not clone in agar, and were not malignant. Most normal cells stimulated to grow by v-H-ras also cloned poorly in agar, and only rare cells were tumorigenic. With the v-abl virus, no more cells were transformed from E mu-myc than normal marrow and the proportion of tumorigenic pre-B clones was not elevated. These results suggest that both v-H-ras and v-raf, but apparently not v-abl, collaborate with constitutive myc expression to promote autonomous proliferation and tumorigenicity of pre-B lymphoid cells.     

Activated EGFR Promotes the Survival of B-Lineage Acute Leukemia in the Absence of Stromal Cells

B-lineage acute leukemia (B-ALL) cells often require stromal cell support for optimal proliferation and apoptotic resistance. In addition, stromal cell contact can promote resistance to chemotherapeutic agents. However, the precise biochemical pathways within the leukemic cell that are activated by the bone marrow microenvironment which result promotion of cell proliferation and apoptotic protection are not fully characterized. We have recently reported that simultaneous inhibition of the MEK and PI3K pathways or the MEK and mTOR pathways promote rapid apoptosis of the stromal cell dependent B-lineage ALL cell line BLIN-2 in the presence of stromal cell support. These data indicated that stromal cell induced apoptotic protection is mediated by PI3K/mTOR and MEK in a mechanism(s) that suggests cross-talk or points of convergence. The EGF receptor (EGFR) has been reported to activate both MEK and PI3K. We report herein that use of the EGFR inhibitor, AG1478, inhibits BLIN-2 survival in the presence of stromal cells. FACS analysis revealed that EGFR is expressed on the surface of BLIN-2 cells. The addition of EGF to BLIN-2 cultures in the absence of stromal cells prolongs BLIN-2 survival. Similarly, introduction of a constitutively active form of EGFR, v-ErbB, into BLIN-2 prolongs the survival of BLIN-2 cells in the absence of stromal cell support. These data provide evidence that stimulation of the EGFR pathway is one mechanism by which the bone marrow microenvironment may contribute to the growth and survival of B-cell acute leukemia.     

Normal B cell precursors responsive to recombinant murine IL-7 and inhibition of IL-7 activity by transforming growth factor-beta

The ability of stromal cells in bone marrow to support B lymphopoiesis may be partially mediated by secretion of biologically active factors. The first cytokine with lymphopoietic activity to be molecularly cloned from stromal cells, IL-7, was originally identified by its growth-promoting activity on long term cultured lymphocytes. We now report that murine rIL-7 is a potent proliferative stimulus for B cell progenitors isolated from fresh bone marrow. Proliferation was initially most obvious among large precursor cells which bear the B lineage associated Ag, Ly5/220 and BP1. A majority of these also contained cytoplasmic Ig mu H chains. Extended culture with IL-7 resulted in a predominance of immature c mu- lymphocytes. No effect by IL-7 was observed on the proliferation of mature lymphocytes. It also did not induce maturation in a number of early B lineage cell lines, or promote the formation of LPS-responsive, clonable B cells from precursors. When incorporated into semisolid agar medium, IL-7 specifically and rapidly induced the formation of pre-B cell colonies in a linear fashion with respect to numbers of cells cultured from either purified B cell progenitor preparations or unfractionated bone marrow. In both liquid and agar culture conditions, the IL-7 proliferative activity was inhibitable by two related forms of transforming growth factor (TGF) beta, TGF-beta 1 and TGF-beta 2. Taken together, these results indicate that IL-7 is a stimulus for replication of normal B lineage cells at an early stage of differentiation, and its activity can be modulated by other cytokines. IL-7 also provides a means of studying the progeny of a single B cell progenitor, and of enumerating clonable pre-B cells in the absence of colony formation by other cell types in bone marrow.     

Genetic approach and phenotype-based complementation screening for identification of stroma cell-derived proteins involved in cell proliferation.

The functional capacities of stromal cell lines to support stem cell activity are heterogeneous and the mechanism of how they support bone marrow cultures remains unclear. Recently, we reported a strategy of functional analysis in which a genetic approach is combined with phenotype-based complementation screening to search for a novel secreted growth factor from mouse bone marrow stroma called ShIF that supported proliferation of bone marrow cells. To investigate the role of stromal cells in hemopoiesis, we extended this strategy to search for stroma-derived proteins that induce cell proliferation by establishing stroma-dependent Ba/F3 mutants of three stroma cell lines from two mouse tissues. Seven stroma-dependent Ba/F3 mutants were used as responder cells to identify cDNAs from stroma cell lines whose products supported proliferation not only to the mutant cells but also to hemopoietic progenitor cells in vitro.     

A stromal cell line from myeloid long-term bone marrow cultures can support myelopoiesis and B lymphopoiesis

The production of B lymphocytes and myeloid cells occurs in the bone marrow in association with a supporting population of stromal cells. To determine whether these processes are dependent upon the same or different populations of stromal cells, stromal cell lines were generated from the adherent layer of a Dexter type long-term bone marrow culture. These cultures support myeloid cells and their precursors, a B cell precursor, and the adherent layer cells with support B cell differentiation under appropriate conditions. Two of the lines examined, S10 and S17, express class I histocompatibility antigens but not other hemopoietic cell surface determinants such as Thy-1, Lyt-1, Ig, Ia, Mac-1, or BP-1. Both lines could support myelopoiesis under Dexter conditions upon seeding with nylon wool-passed bone marrow. The nylon wool passage depletes stromal cells capable of forming adherent layers in vitro but retains hemopoietic precursors. The number of cells and colony-forming units-granulocytes/macrophages in the nonadherent cell population recovered 3 wk post-seeding had increased 19-fold and 10-fold, respectively, in the reseeded cultures of S10 and S17. After 3 wk of growth in Dexter conditions, the reseeded cultures were transferred to conditions optimal for B cell differentiation described by Whitlock and Witte. After 4 wk of growth, hemopoietic cells were consistently recovered from S17 cultures but not those of S10. A proportion of these cells from S17 cultures expressed the 14.8 antigen and were surface IgM positive. Surviving hemopoietic cells present in cultures of S10 were primarily macrophages. These findings indicate that S17 but not S10 can support both myelopoiesis and B lymphopoiesis and suggest that one stromal cell population has the capacity to form a hemopoietic microenvironment for both lineages.     

Thymus-derived stromal cell lines

We derived stromal cell lines from mouse thymus using methods previously established for bone marrow stroma. Two main morphologically distinct groups of cell strains emerged: epithelioid and mixed fibroblast-macrophage. Transmission electron microscopy revealed frequent junctional-complex formations between adjacent cells, a feature that characterized almost all of the thymus stromal lines, but was confined to only one of the five distinct subtypes of cell lines from bone marrow. In contrast to marrow stromal cells, the thymus-derived cell lines were all negative with fat-detecting reagents, had low acid phosphatase and no basic phosphatase activities and were unable to support the in vitro proliferation of myeloid progenitor cells (CFU-gm). Leukemia cell inhibitory activity (LCIA) was detected in one of the thymus stromal cell lines. The differences observed between cell lines derived from the stroma of the thymus and those from bone marrow may relate to the functional specificities of these organs.     

Transformation of murine bone marrow cells with combined v-raf-v-myc oncogenes yields clonally related mature B cells and macrophages.

Murine bone marrow cells infected with replication-defective retroviruses containing v-raf alone or v-myc alone yielded transformed pre-B cell lines, while a retroviral construct containing both v-raf and v-myc oncogenes produced clonally related populations of mature B cells and mature macrophages. The genealogy of these transformants demonstrates that mature myeloid cells were derived from cells with apparent B-lineage commitment and functional immunoglobulin rearrangements. This system should facilitate studies of developmental relationships in hematopoietic differentiation and analysis of lineage determination.     

Human bone marrow stromal cell lines from myeloma and rheumatoid arthritis that can support murine pre-B cell growth.

In order to elucidate the pathologic significance of the bone marrow (BM) microenvironment in multiple myeloma (MM) and rheumatoid arthritis (RA), we established patient- or healthy donor (HD)-derived BM stromal cell lines by transfecting the plasmid for expression of SV40 large T Ag and examined their ability to support the stromal cell-dependent growth of a pre-B cell line, DW34. The means of recovered cell numbers of DW34 co-cultured with MM- and RA-derived BM stromal cell lines ranged from 6- to 10-fold more than those with HD-derived ones. Their enhanced ability to support DW34 cell growth was not caused by cytokines, including IL-6, IL-7, and c-kit ligand, although exogenous IL-7 could augment the growth-supporting ability. DW34 cell growth on the stromal cell lines was abolished by inhibiting cell-to-cell interaction with a membrane filter. FACS analysis revealed that the stromal cell lines did not express LFA-1 alpha, beta, NCAM, or ELAM-1. Both patient and HD BM stromal cell lines variably expressed ICAM-1, VCAM-1, and CD44. However, surface expression levels of these molecules did not correlate with the ability of the stromal cell lines to support DW34 cell growth. Taken together, these results suggested that BM microenvironment might play important roles in the pathogenesis of MM and RA.     

Detection and characterization of a B cell stimulatory factor (BSF-TC) derived from a bone marrow stromal cell line

Stromal cell lines derived from murine bone marrow support the growth of immature pre-B cells and produce cytokines that affect the growth and differentiation of other hematopoietic precursors. Conditioned medium (CM) from one such line (TC-1) stimulated marked proliferation of B cells previously activated by anti-Ig (anti-Ig blasts). Proliferation of anti-Ig blasts was not induced by purified cytokines known to be produced by TC-1 (CSF-1, GM-CSF, or G-CSF) or by IL-1, IL-2, IL-3, IL-4, IL-5, or IL-6. Furthermore, IL-2, IL-4, and IL-5, alone or in combination, failed to support proliferation or differentiation of anti-Ig blasts. TC-1 CM enhanced proliferation of B cells that were co-cultured with LPS, anti-Ig, or dextran sulfate; co-stimulation with anti-Ig was unaffected by the presence of monoclonal anti-IL-4. Proliferation of low, but not high, density B cells isolated from spleen was directly stimulated by TC-1 CM. These results suggest that bone marrow stromal cells produce a novel B cell stimulatory factor (BSF-TC) that induces proliferation of activated B cells.     

Adipogenesis in a myeloid supporting bone marrow stromal cell line.

The bone marrow stroma contains pre-adipocyte cells which are part of the hemopoietic microenvironment. Cloned stromal cell lines differ both in their ability to support myeloid and lymphoid development and in their ability to undergo adipocyte differentiation in vitro. These processes have been examined in the +/+2.4 murine stromal cell line and compared to other stromal and pre-adipocyte cell lines. In long-term cultures, the +/+2.4 stromal cells support myeloid cell growth, consistent with their expression of macrophage-colony stimulating factor mRNA. However, despite the presence of mRNA for the lymphoid supportive cytokines interleukins 6 and 7, +/+2.4 cells failed to support stromal cell dependent B lineage lymphoid cells in vitro, suggesting that these stromal cells exhibit only a myelopoietic support function. The +/+2.4 cells differentiate into adipocytes spontaneously when cultured in 10% fetal bovine serum. The process of adipogenesis can be accelerated by a number of agonists based on morphologic and gene marker criteria. Following induction with hydrocortisone, methylisobutylxanthine, indomethacin, and insulin in combination, a time dependent increase in the steady state mRNA and enzyme activity levels of the following adipocyte specific genes was observed: adipocyte P2, adipsin, CAAT/enhancer binding protein, and lipoprotein lipase. In contrast, adipogenesis was accompanied by a slight decrease in the signal intensity of the macrophage-colony stimulating factor mRNA level, similar to that which has been reported in other bone marrow stromal cell lines. These data demonstrate that although the lympho-hematopoietic support function of pre-adipocyte bone marrow stromal cell lines is heterogeneous, they share a common mechanism of adipogenesis.     

Functional characterization of two stromal cell lines that support B lymphopoiesis.

Bone marrow stromal cells have well documented effects on the production of B lymphocytes, but whether or not stromal cell signals are involved in the pre-B to B cell transition is unclear. The potential of two stromal cell lines, S10 and S17, in this process was examined. Initial experiments, using a short term liquid culture, indicated that S10 and S17 stroma efficiently supported the generation of clonable B cells (B lymphocyte CFU) from their immediate precursors in fresh bone marrow. The contribution of macrophages and other accessory cells in those experiments was minimized through use of a colony assay system that permits the direct effects of stromal cell signals on single B cell progenitors to be evaluated. The results indicated that soluble mediators from the S10 and S17 lines could support colony formation from fresh or cultured surface Ig- bone marrow cells. Colonies supported by S17 stroma appeared on day 15 and contained cells that expressed the B220 Ag; surface IgM expression was never observed. S10 supported colonies appeared on day 7 and routinely included surface IgM+ cells. Individual colonies were capable of undergoing additional growth when picked and replated directly onto the different stroma. Those colonies replated onto S10 stroma generated surface IgM expressing cells in up to 60% of experiments, but colonies transferred onto the S17 cell line included B cells only 10% of the time. These data demonstrate that stromal cells alone can provide the signals necessary for generating a surface IgM+ B cell from precursors but that not all stromal cell lines are equally efficient at doing so.     

Bone marrow stromal cell regulation of B-lymphopoiesis. I. The role of macrophages, IL-1, and IL-4 in pre-B cell maturation

Our experiments have addressed regulation of B lymphocyte formation by bone marrow stromal cells. Stromal cells appear to produce a regulatory factor that acts at the pre-B cell stage to induce the expression of Ig L chains and surface Ig. Bone marrow stromal cell conditioned medium was found to contain this factor and the active component was partially purified by HPLC. This stromal cell-derived factor had a m.w. between 16,000 and 20,000, was specifically neutralized by anti-IL-4 mAb, 11B11, and enhanced the proliferation of anti-mu-stimulated B cells. We also found that rIL-4 induced B cell formation in culture. In our studies, IL-1 had no direct effect on pre-B cell maturation, however, IL-1 was found to stimulate the production of IL-4 by both heterogeneous bone marrow stromal cells and a cloned stromal cell line, SCL-160. These effects of IL-1 on factor production by stromal cells were duplicated by the addition of bone marrow-derived macrophages to SCL-160 cells. We conclude that stromal cell-derived IL-4 is a physiologic stimulator for B cell generation. In addition, macrophages appear to play a role in B cell formation by regulating the production of IL-4 by stromal cells via the secretion of IL-1.     

Xyloside effects on in vitro hematopoiesis: functional and biochemical studies

Xyloside supplementation of long-term bone marrow cultures (LTBMCs) has been reported to result in greatly enhanced proliferation of hematopoietic stem cells. This was presumed to be the result of xyloside-mediated perturbation of proteoglycan synthesis by marrow-derived stromal cells. To investigate this phenomenon, we first studied the effects of xyloside supplementation on proteoglycan synthesis by D2XRadII bone marrow stromal cells, which support hematopoietic stem cell proliferation in vitro. D2XRadII cells were precursor labelled with 35S-sulfate, and proteoglycans separated by ion exchange chromatography, isopyknic CsCl gradient centrifugation, and gel filtration HPLC. Xyloside-supplemented cultures showed an approximately fourfold increase in total 35S incorporation, mainly as free chondroitin-dermatan sulfate (CS/DS) glycosaminoglycan chains in the culture media. Both xyloside supplemented and nonsupplemented cultures synthesized DS1, DS2, and DS3 CS/DS proteoglycans as previously described. In contrast to previous reports, xyloside was found to inhibit hematopoietic cell growth in LTBMC. Inhibitory effects were observed both in cocultures of IL-3-dependent hematopoietic cell lines with supportive stromal cell lines and in primary murine LTBMCs. Xyloside was found to have a marked inhibitory effect on the growth of murine hematopoietic stem cells and IL-3-dependent hematopoietic cell lines in clonal assay systems and in suspension cultures. In contrast, dialyzed concentrated conditioned media from LTBMCs had no such inhibitory effects. These findings suggest that xyloside-mediated inhibition of hematopoietic cell growth in LTBMC resulted from a direct effect of xyloside on proteoglycan synthesis by hematopoietic cells.