Bone marrow stromal cell lines with lymphopoietic activity express high levels of a pre-B neoplasia-associated molecule

Ceil, Vol. 48, 1009-1021,

March

27, 1987, Copyright

0 1987 by Cell Press

Bone Marrow Stromal Cell Lines with Lymphopoietic Activity Express High Levels of a Pre-B Neoplasia-Associated Molecule Cheryl A. Whitlock, George F. Tidmarsh, Christa Muller-Sieburg,’ and Irving L. Weissman Laboratory of Experimental Oncology Department of Pathology Stanford University School of Medicine Stanford, California 94305

Summary Bone marrow stromal cell lines have been isolated that directly support B lymphopoiesis in vitro. Single B-iineage precursors proliferate and differentiate on certain of these stromal cell lines to establish long-term B-lineage cultures. These lymphopoietic stromai cells produce novel soluble factors that support proliferation of in vitro established pm-B cell populations. Lymphoid populations established on iymphopoietic stromal cell lines lack surface lg-bearing cells, but give rise to surface lg+ cells when transferred to mixed bone marrow feeder layers. Several stromal lines expressed a B-lineage neoplasia marker detected by the monoclonai antibody MAb6C3. Remarkably, only the 6C3Aghi stromal lines supported long-term proliferation of B-lineage cells. We propose that the 6C3 antigen-bearing molecule may play a role in stmmal cell-dependent, pm-B cell proliferation, as well as in neopiastic proliferation of pre-B leukemias. Introduction The outcome of hematopoiesis is a striking diversity of cell types, all apparently derived from a common hematopoietic stem cell (Wu et al., 1966; Abramson et al., 1977). The first evidence that selection of the pathway of differentiation could be controlled by particular microenvironments came from the clonal analysis of hematopoietic precursors initiated by Till and McCulloch (1961). Bone marrow cells injected into lethally irradiated hosts at limiting dilutions were found to give rise to clonal colonies of all nonlymphoid hematopoietic cell types, and the ratio of one colony type to another (for example, myeloid vs. erythroid) depended to a high degree on the organ in which the colony arose (for example, bone marrow or spleen) (Wolf and Trentin, 1968; Trentin, 1970). With the establishment of long-term cultures of bone marrow stromal elements, it became clear that stromal cells not only provided the microenvironment for differentiation and proliferation of stem cells and their maturing progeny, but also that microenvironments could be reconstructed in vitro that selectively supported myeloid/erythroid development (Dexter cultures) (Dexter and Lajtha, 1974) or B lymphocyte de* Current address: Lilly Research Jolla, California 92037.

Laboratories,

3252 Holiday

Ct., La

velopment (Whitlock-Witte cultures) (Whitlock and Witte, 1982). Much effort has focused on assigning roles to the various stromal cell types found in the mixed bone marrow feeder layers (MBMFs), which include macrophages, fibroblasts, adipocytes, epithelial cells, reticulum cells, and endothelial cells (reviewed by Zipori, 1967). A number of mouse and human stromal cell lines have been isolated from Dexter cultures in order to examine the microenvironmental factors and cellular interactions that support stem cell survival, proliferation, and directed differentiation. Myelopoiesis and spleen colony forming cell (CFUs) survival are apparently supported by endothelial adipose cells. The factors important in this support have not been completely discerned-no combination of culture supernatants with known colony stimulating factors has been found that will equal direct contact with the stromal cells. In a previous report, we described the use of two stromal cell lines that we isolated from a Whitlock-Witte B-lineage culture for limiting dilution analysis of bone marrow hematopoietic precursors of pre-B cells (MullerSieburg et al., 1966). We report here characterization of eight stromal cell lines and six subclones isolated from that Whitlock-Witte culture. Direct contact with and, to a lesser extent, supernatants from certain of these cell lines supported proliferation of B-lineage lymphocytes harvested from established Whitlock-Witte cultures and of a cloned, stromal layer-dependent pre-B cell normal bone marrow (NBM) line, NBM clone 3 (Whitlock et al., 1983a). Perhaps the most striking finding is that several of these stromal lines express the pre-B neoplasia-associated surface antigen 6C3Ag, and that the functional property of supporting B lymphopoiesis is correlated with expression of 6C3Ag (Pillemer et al., 1984). This antigen, a 160 kd glycoprotein (gp160K3) carrying the epitope recognized by the monoclonal antibody MAb6C3, is expressed at high levels on the cell surface membranes of most neoplastic pre-B cells, whether they are transformed by a variety of retroviral oncogenes including abl, src, ras, fes, erb 6, and NS-7 (but not myc), or arise via spontaneous or chemical transformation (Pillemer et al., 1984; Morse et al., 1987). This molecule is invariably expressed by fully neoplastic pre-B cells transformed by Abelson virus in vitro or in vivo, but is usually absent from the surface of preneoplastic cells early after A-MuLV infection when tumorigenic potential is low and proliferation in vitro requires stromal cells(Whitlock and Witte, 1981; Whitlocket al., 1983b; Tidmarsh et al., 1985; Tidmarsh et al., 1986). In this study we demonstrate that MAb6C3 precipitated a protein of approximately 135 kd (gp1356c3) from most of our stromal cell lines, and the amount of precipitated protein directly paralleled the ability of each cell line to support pre-B cell proliferation in vitro. A model for the role of this antigen in normal pre-B cell growth and pre-B cell neoplasia is discussed.

Cdl 1010

Figure

1. Phase

Microscopy

of AC-6.21

(A, B) Subconfluent cultures of AC-6.21 examined under the 10x objective of a Nikon phase microscope. Long arrow, large binucleated cell; short arrow, contact between membrane extensions and main cell body; arrowhead, detached cells in the process of dividing.

Results Isolation of Stromal Cell Lines from Bone Marrow Cultures Supporting B Lymphopoiesis A five-month-old Whitlock-Witte BALB/c bone marrow culture that was actively supporting pre-B cell proliferation was chosen as the source for the stromal cell lines. The culture medium was pipetted vigorously to suspend the nonadherent lymphocytes and dislodge a small number of loosely attached, presumably mitotic, adherent stromal cells. The supernatant and cells were transferred to a new tissue culture dish and maintained for 1 month with weekly changing of the culture medium. While the majority of the transferred lymphocytes died within the first week, a few lymphocytes that were associated with adherent cells survived. After 4 weeks of culture, there were several isolated patches of adherent cells varying in size from 5 cells to greater than 50 cells each. Twelve patches of stromal cells that had large numbers of viable lymphocytes associated with them were chosen for passage. Stromal cell patches were isolated by using glass cloning rings, and the stromal cells were detached with collagenase. Nonadherent cells disappeared after the first two

passages in the nine surviving cultures. Subsequently, we used a mixture of collagenase with the neutral protease dispase, which allowed efficient detachment of the stromal cells while preserving their viability. Nine stromal cell lines survived multiple passages with collagenase, and varied in their growth characteristics. Six of the nine primary stromal adherent cell (AC) lines (AM, 5, 6, 9, 10, 11) ceased to divide after a confluent layer of cells was formed. AC-6.21, a subclone selected by plating of AC-6 at 0.3 cells per well, was extremely sensitive to omission of 8-mercaptoethanol from the culture medium. Viability was decreased and cell division ceased. Two cell lines (AC-2 and 3) grew slowly at confluence but continued to divide, such that after 3 or 4 weeks, areas with multiple layers of cells were seen. One cell line, AC-8, appeared to contain two morphologically distinct cell types, one of which had short membrane extensions and continued to divide after confluency, thus forming a loosely adherent cell fraction that was nonlymphoid in morphology. The second morphological type of AC-8 cells was similar to the contact-inhibited cells of AC-6. The six stromal cell lines that showed contact inhibition (AC-4, 5, 6, 9, 10, 11) all had long, branching extensions of the

Pre-B Lymphopoiesis 1011

on 6C3Ag+

Stromal

Cell Lines

plasma membrane. If cultured at subconfluency after irradiation (2000 rads), they remained viable for 3 or more weeks, did not divide, and their membranes continued to expand until they covered an extensive area of the dish. These six lines also formed adipocytes with large fat globules if maintained without passage for 1 week or more. Adipocytes disappeared each time the cultures were passaged. AC-10 and 11 consistently showed adipocyte formation within 1 or 2 weeks. Adipocytes appeared infrequently in cultures of AC-4, 5, 6, and 9, and required culture for 3 or more weeks. The subclone AC-6.21 did not form adipocytes even after 4 weeks of culture. Morphology of AC-6 and AC-6.21 The morphology of AC-6.21 is shown in Figures 1 and 2. As will be demonstrated below, this clone was the most active, among those isolated, at supporting B lymphopoiesis in vitro. Analysis by phase microscopy revealed that the cells in confluent cultures were cobblestone in appearance, with little overlap at the areas of contact between adjacent cells (not pictured). This stromal line most resembled the endothelial, or endothelial-like, cell lines described by Zipori et al. (1985). Binucleation (Figure 1A) was a common feature of the large cells. Long, thin extensions of the plasma membranes often had expanded and branching termini that came in contact with the main body of adjacent cells (Figure 1B). Electron micrographs of AC-6 (Figure 2A) and AC-6.21 (Figure 28) after release from the culture dish by either collagenase-dispase (AC-8) or EDTA (AC-6.21) showed an extensive amount of plasma membrane that contracted into villous-like structures. Bundles of microfilaments were seen dispersed throughout the cytoplasm (not visible at magnifications shown). The outer membranes contained abundant vesicular structures, some in arrays suggesting pinocytosis and/or endocytosis; these were especially prominent on the AC-6 cells, which were cultured longer (not pictured). The AC-8 cells also contained numerous inclusion bodies, including fat-filled liposomes. Also notable were multiple Golgi stacks and numerous dilated rough endoplasmic reticula filled with proteinaceous material, suggesting that these may be secretory cells. Multiple electron micrographs of all of the stromal lines and clones were examined, and no viral particles or neurosecretory granules have been noted (see Hunt et al., 1987, for stromal cells with neurosecretory-like granules).

Figure

2. Electron

Microscopy

of AC-6 (A) and AC-6.21

(B)

Cells were harvested by collagenase-dispase (AC-6) or 5 mM EDTA (AC-6.21) and cell pellets were processed for electron microscopy as described in Experimental Procedures. (A) Approximately 3792x. (B) Approximately 13,430x.

Some Stromal Cell Lines Support In Vitro Proliferation of B-Lineage Cells The ability of the stromal cell lines to support B lymphocyte proliferation was first tested by culturing nonadherent B-lineage lymphocytes from Whitlock-Witte bone marrow cultures in direct contact with confluent stromal cell layers, and observing lymphocyte viability and increase in number by phase microscopy. From five independent assays, the following patterns of long-term growth support emerged (data not shown): One group (represented by AC4 and 6) consistently supported long-term proliferation of the nonadherent B-lineage populations; a second group (AC-3 and 8) supported proliferation in only some assays; and

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Table 1. Proliferation of Pm-B Stromal Cell Supernatants

TIME Figure 3. Growth Stromal Lines

of

Nonadherent

(hours)

Pre-B

Cells

on

Four

Different

Confluent cultures of AC4, AC-6.21, AC-IO, and AC-II were harvested with dispasecollagenase as described in Experimental Procedures and counted, and equivalent numbers were plated in wells of 96well microtiter plates. At 0 time, nonadherent pre-B cells from established Whitlock-Witte cultures were pooled and plated on the stromal cell layers. Duplicate wells were retained with stromal cells only. At the times indicated, each well received 2.5 nCi 3H-thymidine and was reincubated for 2.5 hr, then harvested, and TCA-precipitable radioactivity assessed. The data plotted represent counts per minute for ten replicates of wells containing AC-6.21 (0) AC-4 (w), AC-11 ( l ), or ACIO (0) with nonadherent pre-B cells minus background incorporation of stromal cells alone and pre-B cells alone.

a third group (AC-10 and 11) did not support growth in long-term cultures. Stromal lines and subclones that were representative of these three groups were chosen for quantitative analysis of their potential for supporting B-cell proliferation. In a preliminary experiment measuring 3H-TdR incorporation into nonadherent lymphocytes pooled from 5week-old Whitlock-Witte cultures, or into the Whitlock-Witte stromaldependent clone NBM 3 (Whitlock et al., 1983a) 2 days after seeding onto the stromal cells, the following order of growth support was observed: AC-a.21 ti MBMF 2 AC-6.4 > AC-8 > AC-3 > AC-6 > AC-4 > AC-10 B AC-l 1 (data not shown). Four stromal lines were studied in greater detail (Figure 3). Clone 6.21 supported cell growth and DNA synthesis best by Whitlock-Witte culture pre-B cells, while AC4 supported these cells less well at early time points, but quite well by 7-11 days of culture. Stromal cell lines AC-10 and AC-11 failed to support these pre-B cells. By phase microscopy, the number of viable cells in the cultures could be seen to parallel the amount of 3HTdR incorporation. Accurate enumeration of viable lymphocytes was not possible because of the tendency of lymphoid cells to migrate beneath the stromal cells. The most dramatic differences in support by the stromal cell lines were seen after 1 to 2 weeks of culture of the pooled nonadherent cells. At this time point, cultures with AC-3, 4, 6, 6.4, and 6.21 contained a nearly confluent layer of lymphocytes, as well as much cellular debris. AC-10 contained rare foci of viable lymphocytes, numbering less than 20 cells each. AC-11 was generally devoid of lymphocytes. AC-8 stromal cells were overgrown at this point; therefore, lymphoid cells were absent or rare. The MBMF

Source of Supernatant

3H-Thymidine Incorporationa Pre-B Culture

Media alone MBMFC AC3 AC4 AC6 AC6.4 AC6.21 AC0 AC10 AC11

492 1,812 1,430 903 1,366 1,292 2,679 693 777 759

f f f f f & + f f f

12 102 99 s 134 126 160 142 10 100

Cells

of Cells

in Cultures

Containing

3H-Thymidine lncorporationb of NBM Clone 3 Cells 3,294 10,605 9,082 7,168 9,603 9,678 15,408 6,720 5,532 4,588

f f 2 f f f f f f f

258 842 496 362 305 185 472 370 351 250

a Pooled nonadherent cells from Whitlock-Witte cultures or NBM Clone 3 cells were cultured in the presence or absence (Media) of stromal cell- or MBMF-conditioned media, as described in Experimental Procedures. b Thymidine incorporations of quadruplicate cultures were averaged and standard deviations determined. c MBMF = mixed bone marrow feeder cells.

layer typically contained discrete foci of lymphocytes randomly distributed over the stromal layer, and no cellular debris (because of the presence of phagocytic cells). Culture Supernatants from Stromal Cell Lines Support Proliferation of B-Lineage Cells Supernatants from individual stromal cell cultures were assayed for their ability to support proliferation of pooled nonadherent cell8 from E&week-old bone marrow cultures and of NBM Clone 3 in a 3H-TdR incorporation assay 1 day after placement in culture (Table 1). The patterns of growth support were similar to those seen with the test populations in direct contact with the stromal cells; proliferation in the presence of supernatants was generally about 5-fold lower for the pooled nonadherent cells and 2.5fold lower for NBM clone 3. Supernatants from AC-6.21 and MBMFs were approximately B-fold and 3-fold, respectively, above that in unconditioned media. Supernatants from AC-6.4,6,3,4,8, and 10 stimulated progressively less incorporation, and the AC-1 1 supernatant was only slightly better than unconditioned medium. Limiting Dilution Cloning of Bone Marrow Cells on Stromal Cell Feeder Layers Limiting dilution culture of a mixed cell population allows one to analyze not only the frequency of cells in a population capable of proliferation, but also the presence of cells in the population that can either enhance or inhibit proliferation of the cells of interest. In addition, it provides a method for obtaining clonal populations of cells that are not amenable to agar cloning. We attempted limiting dilution culture of fresh bone marrow in contact with the stromal cell lines for two reasons: first, to determine whether cloned B-lineage cell lines could be established from a single B-lineage precursor under the influence of a single stromal cell type; and second, to test whether clonal proliferation of any other hematopoietic lineage would be supported directly by the stromal cell lines.

Pre-B 1013

Lymphopoiesis

on 6C3Ag+

Table 2. Effect of Hydrocortisone and CFUs Survival by Coculture with AC-6 Stromal Cells

Stromal

Cell Lines

on Lymphopoiesis, Myelopoiesis, of Fresh Bone Marrow

-HC No. ceils per culture Q 3 weeks CFUs per culture r@ 3 weeks Staining 8220 SlQ Thy-l Mac-i 6C5

3x107 80

+HC 56x 290

IO’

Q 4 weeks: X7%8 0% <0.5% c 4%


Bone marrow was harvested from 3week-old BALE/c mice, and 10’ were cultured without removal of adherent cells in each of two T-75 flasks containing confluent layers of AC-6 stromal cells. One culture (- HC) was established and maintained under standard Whitlock-Witte culture conditions. Another (+ HC) had added to the culture medium 1 x 10-s M hydrocortisone acetate. Cultures were fed weekly. Nonadherent cells were harvested after 3 weeks of culture, and 10% were returned to the corresponding flasks. Fresh medium with and without hydrocortisone was added and the flasks incubated for an additional week. Nonadherent cells harvested at 3 weeks were injected into irradiated BALB/c mice for assessment of CFUs (for procedure, see Muller-Sieburg et al., 1966). Nonadherent cells harvested at 4 weeks of culture were immunofluorescently stained for the surface markers shown. a Staining with RA3-6B2 (anti-8220) showed a wide range of staining intensities. The number given (67%) represents a minimum estimate of the number of B220+ cells since only those with staining clearly above background were counted. b At or below the level of background staining. c Unable to assess because of low numbers of cells binding MAC-l and the presence of approximately equal numbers of autofluorescing cells.

As a preliminary experiment, whole bone marrow cell suspensions (without depletion of adherent cells) that were cultured under Whitlock-Witte medium conditions on established AC-6 feeder layers rapidly proliferated with production of large foci. Lymphoid cells predominated in cultures maintained for 4 weeks. Initiation and maintenance of a parallel culture in the presence of 10-S M hydrocortisone suppressed lymphoid cells, but, surprisingly, allowed expansion of an even greater number of myeloid cells (Table 2). Survival of CFUs in both cultures containing stromal cells was much prolonged as compared to our previous data on cultures established without them. (CFUs are undetectable by 1 week in standard Whitlock-Witte cultures [Whitlock et al., 1964; C. A. Whitlock and C. Muller-Sieburg, unpublished data].) The question raised by these observations was whether the AC-6 layer was directly supporting CFUs and myeloid precursors or whether it was enhancing establishment by other accessory cells of an in vitro microenvironment conducive to their survival and/or proliferation. A satisfactory answer to this question could only come from limiting dilution analysis, which physically separated the CFUs and myeloid precursors from potential accessory cells. Bone marrow cells were cultured in multiple experiments at 37 to 9000 cells per well on the B lymphopoiesis-supportive stromal cell lines AC-4 and AC-6. By 1 week, a number of nonadherent cell colonies appeared that exhibited a wide range of colony sizes and morpholo-

Figure 4. Morphology from Limiting Dilution

of the Three Most Common Culture of Fresh Bone Marrow

Colonies Arising on AC-4 or AC-6

Bone marrow cells were harvested from the femurs of 3-week-old BALB/c mice and cultured without depletion of adherent cells at numbers ranging from 37 to 9fIOO cells per well on confluent layers of unirradiated AC-4 or AC-6 (placed into well 24 hr prior to addition of bone marrow cells). Above are pictured Wrights-stained, cytocentrifuged preparations of the three most common types of colonies grown in the absence of added hydrocortisone, which were easily discernible by phase microscopy: (A) large, granular cells, (6) small, lymphoid-like cells, (C) medium-sized myeloid cells. Magnification of all three = 64x.

gies as detected by phase microscopy (see Experimental Procedures). The most frequent colony type consisted of large cells (20-25 urn in diameter) that, after another 7 to 10 days of culture, became granular and vacuolated; these spread out and attached to the stromal cell layer. Wright’s staining of one such colony is shown in Figure 4A.

Cell 1014

Figure

5. Limiting

Dilution

Culture

of Bone

Marrow

on AC-6.21

and AC-11

Bone marrow was harvested from the femurs of three-week-old BALBlc mice, and single-cell suspensions were made in culture medium or without (A and B) 10-s M hydrocortisone acetate. Cells were cultured at 37, 111, 333, 1000, and 3000 cells per well on confluent layers of (A) or AC-11 (B and C). After 1 week, each well was fed with 50 ~1 of the appropriate medium. After 2 weeks, each well was scanned with microscope to detect the presence of colonies of small cells (O), medium-sized cells (0), or large, granular cells (0). In (C), A represents quency of wells containing either small-cell, medium-sized cell, or mixed small cell/large, granular cell colonies.

These large, granular (LG) cells were present at a frequency of 1:50 to 1:650, depending on the experiment and stromal cell line used for the analysis. Attempts to characterize these large, granular ceils have been hampered by their limited proliferative capacity plus their tendency to autofluoresce and bind most antibodies and fluoresceincoupled reagents nonspecifically. The next most frequent colony type was composed of small (7 to 10 pm) lymphoid (SC) cells resembling the patches of lymphocytes that establish themselves in Whitlock-Witte cultures (Figure 48). The third most frequent colony consisted of medium-sized (approximately 15 pm) myeloid (MC) cells (Figure 4C). In the experiment presented in Figure 5, bone marrow cells were cultured at limiting dilution on AC-6.21 and AC11. Hydrocortisone acetate was added to one set of cultures to enhance myelopoiesis. After 2 weeks of culture, each well was screened by phase microscopy for the presence of colonies of the three morphological categories described above (Figure 5). Wright’s stain of cytospins of nonadherent cells harvested from multiple wells from each group was also examined. The frequencies of lymphoid precursors and LG cell precursors could be easily assessed by this type of analysis. SC colonies on AC-6.21 without hydrocortisone were at a frequency of approximately 1 in 350 (Figure 5A). All wells analyzed by Wright’s stain contained lymphoid cells. On AC-1 1, in the absence of hydrocortisone, the frequency of wells with SC colonies was very low, and these contained granulocytes, not lymphocytes (Figure 58). LG cell colonies were so frequent and so large under these conditions that all colony types were suppressed at 1000 and 3000 cells per well. This confirmed our previous data, which showed AC-11 to be poor at supporting proliferation and/or differentiation of lymphoid precursors. To assess myeloid precursor support by the stromal cells, hydrocortisone was added. Hydrocortisone suppressed LG colony numbers and size (Figure 58 vs. 5C),

with (C) AC-6.21 a phase the fre-

thus allowing the myeloid colonies, which were 50- to lOOfold less frequent, to establish themselves. The frequency of MC colonies on AC-6.21 with hydrocortisone was about 1 in 5600 (data not shown), and on AC-11 was 1 in 1600 (Figure 5C). Because of the low frequency of positive wells, the linearity of the curve obtained by limiting dilution on AC-6.21 could not be assessed. The curve obtained by using AC-11 was nearly linear, suggesting that AC-11 may directly support myeloid precursor development. We conclude that about 1 in 35 to 40 bone marrow cells is capable of proliferating on AC-6.21 or AC-11 (Figure 5). Lymphoid colony formation is supported by AC-6.21 (and by AC-3,4, 6 in experiments not shown). Approximately 1 in 350 bone marrow cells forms a lymphoid colony on AC-6.21, but only a subpopulation of these precursors is capable of longterm proliferation. A low frequency of myeloid precursors (approximately 1 in 1600) capable of forming colonies on AC-11 under limiting dilution conditions is also found. Cloning of B-Lineage Cells from Whitlock-Witte Cultures Using Stromal Cell Line AC-4 In the experiments above we showed the capacity of AC4 (and other stromal cell lines) to support lymphopoiesis in limiting dilution culture of fresh bone marrow suspensions. Whether the stromal cell line AC4 would support proliferation of single cells from established WhitlockWitte cultures was also investigated by using limiting dilution culture. Nonadherent cells from a 5-week-old Whitlock-Witte culture were cultured at 1000, 100, 10, and 0.1 cell per well on AC-4 in 96-well plates after two adherence steps (1 hr each) at 37% to remove any harvested adherent cells. Wells containing colonies of nonadherent cells (which varied in size from 10 cells to 1000 cells) were scored as positives at 1 week and 3 weeks of culture. All wells initiated with 100 or 1000 cells were positive at both 1 and 3 weeks. The frequency of positive cultures at the remaining three cell concentrations is shown in Figure 6. One in 10 cells cultured were capable of proliferation for

Pre-B Lymphopoiesis 1015

on 6C3Ag+

CELLS/WELL

Stromal

Cell Lines

CELLS/WELL

(x10-‘)

Figure herent

6. Limiting Dilution Culture Cells from a Whitlock-Witte

of NonadCulture

Nonadherent cells from five-weekold WhitlockWitte cultures were harvested, then depleted of adherent cells by adherence to plastic for 2 hr at 37%. Nonadherent cells were then cultured at 104, IOs, lOa, IO, and 1 cell per well on AC-4 as described in Experimental Procedures. Wells were screened by phase microscopy for colonies of nonadherent cells after one ( l ) and 3 (0) weeks of culture. (A) Small, lymphoidlike colonies as pictured in Figure 48. (B) Large, granular cell colonies as pictured in Figure 4A.

1 week; 1 in 15, for 3 weeks. The data points all fall on straight lines that pass through the origin of the plot shown in Figure 6. This indicated that cooperation of two or more limiting cell types from the nonadherent population was not required for formation of a colony, and confirmed that the environment created by these stromal cell lines was alone sufficient for supporting growth of single B-lineage cells. lmmunofluorescent staining showed that these cells expressed the 6220 antigen characteristic of B-lineage cells (data not shown). In this limiting dilution analysis, colonies of LG cells were also seen (Figure 6B). The precursor that gave rise to these colonies was at a lower frequency (1 in 690) in the 5week-old bone marrow culture than in fresh bone marrow. The frequency of these precursors was found to decline as age of the bone marrow cultures increased (data not shown). Although the precursors of these large cells were maintained in significant numbers (103-lo4 per culture at 2 to 3 weeks) in the Whitlock-Witte cultures, colonies of LG cells were not seen. These colonies were also not seen upon culture of fresh bone marrow populations on MBMF. Support of LG cell growth was, therefore, a unique property of the stromal cell lines. All of the stromal lines analyzed showed this property. Stromal Cell Lines Support Only Limited Differentiation of B-Cell Precursors The B lymphoid populations expanded from culture of fresh bone marrow subpopulations on AC-4 and AC-6 were analyzed for expression of surface immunoglobulin. Although all subpopulations contained B220-bearing cells, none of those tested expressed surface IgM (Table 3). This suggested that differentiation of pre-B cells to or survival of mature 6 cells was not supported by the stromal cell lines. To analyze this further, nonadherent cells from a Whitlock-Witte bone marrow culture were transferred to AC-3, 4, and 6, then cultured for 3 weeks. Aliquots from each were then transferred back to mixed bone marrow stromal cell layers. After 3 weeks of culture on AC-3, 4, or 6, none of the nonadherent populations contained surface IgM-bearing cells (Table 4). Even after three more weeks of culture, mature B cells did not appear. In contrast, populations transferred from the stromal cell cultures to MBMF were found to contain surface IgM-bearing cells by 1 week in two cases and by 3 weeks in all cases.

Table 3. Limited Differentiation Bone Marrow When Cultured Bone Marrowa Subpopulation

of B-Cell on Stromal

Feeder Layer

Precursors Cells

from

B220+

slgM+

%

%

Thy-lo

MBMF AC6

66 71

4
Thy-l”’

MBMF AC3 AC4 AC6

48 60 51 53


LD WellsC on AC4

(18/18) 20-600~

(O/l 8) <1

B220-/MAC-I-10C5-

4

a Bone marrow was harvested from the femurs of 3-week-old BALB/c mice and immunofluorescently stained for the markers indicated as described in Experimental Procedures. Cells were sorted on a FACS II (Becton-Dickinson, Mountain View, CA), and those positively stained with anti-Thy-l and not stained with anti-L3T4, anti-Lyt-2, anti-B220, MAC-I, or 8C5 (anti-granulocyte antibody) were selected (MullerSieberg et al., 1986). After 2 to 3 weeks of culture on MBMF or stromal cells, the nonadherent cells were harvested and analyzed for expression of 6220 and slgM. b Below the level of background staining (second-stage reagent only). c Eighteen wells from a limiting dilution culture on AC-4 were selectively expanded onto AC-4 in 24well plates, then stained for 8220 or slgM. Within the parentheses are the number of populations that contained either B220+ or slgM+ cells. The percentage of B220- cells in each population is given as a range.

Stromal Cell Lines Express a Membrane Glycoprotein Associated with Pre-B Cell Neoplastic Transformation Eight stromal cell lines were examined for expression of the following membrane antigens: H-2Kd, IAd, MAC-l, Thy-l, and the pre-B cell lymphoma-associated antigen 6C3Ag. All cell lines examined expressed class I histocompatibility antigens on their membranes, but none expressed detectable levels of class II antigens or high levels of Thy-i (AC-3 and AC-11 showed faint staining with the anti-Thy-l reagent). Approximately 60% of the AC-6 population bound Jl, an anti-NIP antibody with y2B heavy chains that was used to control for nonspecific antibody binding. The small cells, which comprised 60% of the AC-6 population, also bound the anti-Fc,zb,uI receptor antibody 2.4G2 (Unkeless, 1979). AC-6 also bound the anti-MAC-l antibody 70.1, which has y2B heavy chains, and attempts

Cell 1016

Table 4. slgM Expression by Pm-B from Stromal Cells to MBMF

Cells

0 L

% slg+ Nonadherent Origin AC-3 AC-4 AC-6

% 6C3Ag+ Calls

After Transfer

Cells

2O Culture

0 wk.

1 wk.

3 wk.

AC-3 MBMF AC-4 MBMF AC-6 MBMF

0 0 0 0 0 0

0 0 0 4 0 6

0 5 0 7 0 4

Nonadherent cells from B-week-old Whitlock-Witte bone marrow culture were harvested, pooled, and allowed to adhere to plastic for 2 hr. Approximately 5 x 105 were added onto multiple l-m cultures of AC-3, AC-4, and AC-6, and maintained for 3 weeks with weekly changing of the medium. After 3 weeks, aliquots of each set were transferred onto MBMF in a 24-well plate and immunofluorescently stained for slg (0 wk). Cultures were maintained with weekly feeding for 3 additional weeks. After 1 and 3 weeks of additional cultures, portions of the nonadherent cells were harvested and stained for slg.

20 1

30 I

40 1

50 1

60 fl

70 1

so I

MBYF

90 I

100 I

Intensity cd Staining ++

AC3 1

+++

AC4 I-

+++

AC6 -

++ - +++

AC64

6

+++

AC621 3

+++

AC8

- - ++ ++

AC10 1 AC11

Figure

to block this binding with anti-F, receptor antibody or excess Jl antibody were unsuccessful. AC-& but not AC6.21, was also found to be phagocytic (P. Hunt, personal communication). Phagocytosis by the remaining stromal cell lines has not been analyzed. The staining pattern with MAb6C3 was most striking (Figure 7). Five cell lines (AC-3, 4, 6, 6.4, and 6.21) were 100% positive for the marker. AC-3, 4, 6.4, and 6.21 all stained very brightly, while the parental AC-6 population showed a range of fluorescent intensities from medium to very bright. The appearance of the fluorescence was unusual in that there were intensely stained speckles distributed on a uniformly stained membrane (not pictured). This differs from the ring fluorescence we normally see when staining transformed lymphocytes with this antibody, and may be the result of infolding of the extensive membrane after detachment from the dish. Two of the remaining three stromal cell lines, AC-8 and 11, also showed a range of fluorescence staining intensities, but they differed from AC-6 in that a significant portion of the population was unstained or very faintly stained. Only 3% of AC-10 cells showed detectable staining with MAb6C3, and the staining intensity on those cells was less than AC-6.21. The MBMF layer that permits establishment of bone marrow cultures is highly heterogeneous in morphology. We examined the cells from these feeder layers to determine what frequency of cells, if any, expressed 6C3Ag. Three to five percent of the cells bound MAb6C3 in two separate experiments. MAb6C3 reactive proteins synthesized by the stromal cell lines were analyzed by gel electrophoresis of immunoprecipitates of extracts from metabolically labeled cells. Equivalent TCA-precipitable counts per minute from each cell line were reacted with MAb6C3 or control antibody, as described in Experimental Procedures, in an attempt to quantitate the amount of MAbGC9precipitable protein. The results, shown in Figure 8, parallel the staining results shown in Figure 7. The greatest amounts of 6C3Ag were synthesized in decreasing order by AC-6.21,

10 1

- -. ++

7. 6C3Ag

Expression

of Stromal

Cell Lines

Stromal ceils were harvested from subconfluent cultures as described in Experimental Procedures, and immunofluoreecently stained with MAb6C3. Stained cells were observed by using a fluorescent microscope, and the number of 6C3Ag-bearing cells was assessed, as was the brightness of staining. Absence of staining is depicted as white (-), and the brightest staining is depicted as black (+++). Some cell lines, AC-6, for example, contained a population of 6C3Ag-negative cells as well as a population that showed variable amounts of staining with MAb6C3.

3, 6, 6.4, and 4. AC-8 and 11 showed barely detectable amounts, consistent with the large frequencies of unstained cells in these populations. AC-10 showed no detectable 6C3-precipitable protein, even on longer exposure of the gel (data not shown). In addition, cell surface expression of 6C3Ag by aliquots of the stromal cells used in the experiment shown in Figure 3 were measured by detecting the amount of MAb6C3 bound to stromal cells with lz51-goat anti-rat immunoglobulin antibodies: After subtraction of background counts and averaging of triplicate samples, the level of expression of each line was compared to AC-6.21, the cell line expressing the highest levels of 6C3Ag. AC-4 bound 33% of the amount of MAb6C3 bound to AC-6.21; AC-11, 24%; and AC-lo, 8%. Thus the ability of these lines to support long-term cultures correlates well, but not exactly, with synthesis and cell surface expression of 6C3Ag (Figures 7 and 8, respectively). Discussion The bone marrow contains all of the precursors and stromal elements necessary for the production of B lymphocytes. A method for establishing B lymphopoiesis in vitro using mouse bone marrow cells was described previously (Whitlock and Witte, 1982) and in such cultures, nonadherent B-cell precursors rapidly proliferate and differentiate in close association with a complex mixture of adherent cells. An obvious next step toward understanding the factors involved in supporting B-cell ontogeny was to clone and characterize the stromal cell ele-

Pre-B 1017

Lymphopoiesis

AC3

4

on 6C3Ag+

6

8

10

Stromal

Cell Lines

11

621

64

ABABABABABABABAB

6. Metabolic Labeling and lmmunoprecipitation of a 6C3AgContaining Molecule from Stromal Cells

Figure

Confluent

stromal

cell layers

were

metabolically

labeled

with %S-

methionine, and cell lysates were immunoprecipitated with isotypematched control antibody (A) or MAb6C3 (6) as described in Experimental

Procedures.

Precipitated

proteins

were

separated

on an

6% SDS-polyacrylamide gel and visualized by exposure of the sodium salicylate-treated gel to X-ray film. MAb6C3 precipitated a protein of 135,000 molecular weight from all of the stromal cell lines tested except AC-1 0.

ments from these cultures. We (and, independently, Hunt et al. [Witte et al., 1984; Hunt et al., 1987) have accomplished this beginning with stromal layers from WhitlockWitte cultures that support pre-6 cell growth. We have established several primary clones and, in one case (AC-8), multiple subclones. All but two of the stromal lines support to varying degrees the proliferation of a stromal layer-dependent pre-B cell line, as well as permit the establishment-at either the multicellular or clonal level-of new pre-B cultures from fresh bone marrow cells. These stromal lines also support proliferation of B-lineage-committed cells taken from Whitlock-Witte cultures. These lines support B lymphopoiesis best when allowed to interact directly with the B-cell precursors, but the stromal cells also clearly produce soluble factors that can substitute, at least in part, as a short-term proliferative stimulus for stromaldependent 6 lymphoid lines (Figure 3; Table 1). Whether the relative growth advantage afforded by stromal cells vs. supernatant is the result of high local growth factor concentrations or the need for cell contact is not yet known, and is the subject of ongoing investigation. When these long-term cloned stromal lines were cocultured in bulk with bone marrow in the presence of hydrocortisone, myelopoiesis, and not B lymphopoiesis, was the result. Somewhat surprising was the rapidity with which the myeloid cells proliferated, as was the extended survival of CFUs. CFUs disappear within a few days under standard conditions for establishing Whitlock-Witte cultures. It is unclear whether hydrocortisone is acting at the level of the adherent stromal cell clone, the hematopoietic precursors, or both. Conceivably, hydrocortisone acts merely at the level of the hematopoietic cells, suppressing lymphopoiesis (a known effect of hydrocortisone in the mouse via lymphocytolysis [Galili et al., 1980]), thus allowing myelopoiesis to flourish. If true, this would indicate that some regulation occurs between lymphopoiesis and myelopoiesis. But what if the effect is on the stromal cells? Here one could conceive of at least two circumstances. First, hydrocortisone may induce a change in the gene expression program of the cloned stromal cells and, therefore, the types of molecules or factors produced by them. Second, the effect of hydrocortisone on the microenvironment may be to allow another cell type from the fresh bone marrow inoculum to be established that has the

effect of supporting myelopoiesis. We favor this second hypothesis. A surprising finding was the high frequency of colonies of a novel cell type (the LG cells) that emerged when culturing either fresh bone marrow cells or nonadherent cells from Whitlock-Witte cultures on the stromal cell lines. It was particularly interesting that the stromal line (AC-11) that was poor at supporting preB cell proliferation was better than AC-8.21 at supporting proliferation of this unknown cell type. By limiting dilution analysis, 2% to 3% of mouse bone marrow will form LG cell colonies. Hydrocortisone suppresses the size and number of these colonies, but does not eliminate them. Because of their high frequency in bone marrow, limiting dilution analysis of infrequent cells (such as myeloid precursors) on stromal cells may require prior separation of the bone marrow population into subpopulations that are enriched for the cells of interest and/or lack the LG cell precursors. Interestingly, there was an almost complete absence of these colonies when MBMF stromal layers were used as feeders instead of the stromal cell lines. A potentially useful finding emerged upon characterization of surface lg expression by the lymphocytes grown on the stromal cell lines. In most cases where clonal populations from limiting dilution analysis were analyzed, 6220 was expressed, but not surface immunoglobulin. Previous studies have shown that the differentiation event that separates B220+ slg- and B220+ slg+ stages of ontogeny is rearrangement and expression of K light chain genes (Maki et al., 1980; Alt et al., 1981; Coffman, 1982; Coffman and Weissman, 1983). That surface lg-bearing cells are often absent from lymphoid populations grown on the stromal cells suggests that this step of differentiation may be regulated by an external stimulus that is diluted or not present in the microenvironment created by the stromal cell lines. We need to verify that lack of slg expression is due to a failure of the pre-B cells to differentiate, and not simply to a failure of mature B cells to survive on the stromal cells. If the former is true, then a search will be made to find the additional stromal cell type that will provide the signal for rearrangement and expression of light chain genes. Perhaps the most striking finding in this study is the presence of the B-cell neoplasia-associated antigen SC3Ag on each of those stromal cell lines that support pre-B hematopoiesis. The degree of expression of the 6C3 antigen on our stromal lines appears to correlate with the ability of cell lines to support long-term pre-B cell cultures (Figures 7, 8, 9). lmmunofluorescent staining of the pre-B supportive stromal lines of ALCS and ALC8 isolated by P Hunt, D. Robertson, and 0. Witte (Witte et al., 1984; Hunt et al., 1987) showed specific binding of MAb6C3 (G. Tidmarsh and C. Whitlock, unpublished data). Six bone marrow stromal cell lines established by D. Zipori, 14F.1, MBA1.11419, MBA-2, MBA-V5/7, MBA-19 and MBA-13.2, were negative for MAb6C3 binding. None of four tested supported pre-B cell growth (G. Tidmarsh and D. Zipori, unpublished data). One cell line (30R), established by D. Rennick (DNAX, Palo Alto, CA), does appear to support B lymphopoiesis from fresh bone marrow (personal com-

Cdl 1018

munication), and this cell line contained a minor population (l%-2%) of cells that stained brightly with MAb6C3 (C. Whitlock, unpublished data). The remainder were weakly stained (
ports growth of a subline of NFSGO (isolated by J. Ihle, Frederick Cancer Center, MD) that was derived by D. Rennick (DNAX Corp., Palo Alto, CA) and that will grow in either IL-3 or in medium containing granulocyte-colonystimulating factor (G-CSF) (C. Whitlock and D. Rennick, unpublished data). Another subline derived by D. Rennick, DA-3.15 (from DA-3, isolated by J. Ihle), will grow in either IL-3 or in medium containing granulocyte/macrophage-colony-stimulating factor (GM-CSF), but failed to grow in direct contact with AC-6.21. An IL-3/lL4-dependent mast cell line, MC 9 (Nabel, 1981), also failed to grow on AC-6.21. Thus the stromal line AC-6.21 supports the proliferation of pre-B lineage cells and their differentiation from hematopoietic progenitors in the absence of known growth factors. In a companion paper, Hunt et al. (1987) have isolated similar cloned stromal lines (ALC) supporting pre-B and myeloid cell proliferation, and these lines also produce novel growth factors. In the future it shall be important to analyze the cell surface and secreted polypeptides produced by our cloned stromal cells under conditions favoring preB lymphopoiesis, and to identify analogous cells and molecules in humans. Experimental

Procedures

Bone

Cultures

Marrow

Bone marrow cultures that support B lymphopoiesis were established and maintained by the protocol of Whitlock and Witte (Whitlock et al., 1984; Whitlock and Witte, 1986). Nonadherent lymphocytes were harvested from these cultures by gentle pipetting of the medium to dislodge the lymphocytes that loosely adhere to the stromal cell layer. To deplete any stromal cells that might contaminate the harvested lymphocytes, the suspended cells with the conditioned media were transferred to a new tissue culture dish and incubated for 2 hr. The lymphocytes were then suspended by gentle swirling or pipetting of the medium. Mixed bone marrow feeder layers were prepared as described elsewhere (Whitlock et al., 1984; Whitlock and Witte, 1987).

Cell Lines Stromal cell lines were isolated from mixed bone marrow stromal cell layers as described in the text. All lines except AC-8 and, to a lesser extent, AC-3 grew to confluency and ceased dividing. Confluent stromal cell layers were maintained for up to 3 to 4 weeks without passage by changing of the tissue culture medium every 5 to 7 days. To passage, the stromal cell layers were washed three times with serumfree medium, then overlayed with 2.5 ml (T-25 flasks) to 7.5 ml (T-75 flasks) of 0.5 mglml collagenase-dispase (Soehringer-Mannheim, Indianapolis, IN) in serum-free medium. The cultures were allowed to incubate 15 to 30 min at 37%; then the enzyme-containing medium was aspirated, and medium with serum was added. The stromal cells were suspended by pipetting with a Pasteur pipet, then cultured directly at one-fifth to one-fiftieth the original cell concentration. In general, confluent stromal layers subcultured at 1:lO reached confluency again after 5 to 7 days, Subclones of AC-6 were obtained by limiting dilution culture from 30 to 0.3 cells per well. The limiting dilution plot was a straight line through the origin with a frequency of colony-forming cells of one in 1.1 plated. Six clones from the 0.3 cell/well plate were chosen for expansion. Two of these, AC-6.4 and AC-6.21, were chosen for complete analysis. NBM Clone 3 is a stromal layer-dependent, pre-B cell clone that was isolated by limiting dilution culture of nonadherent cells from an established Whitlock-Witte bone marrow culture onto MBMF in 96-well plates. Characterization of this cell line has been published elsewhere (Whitlock et al., 1983a).

lmmunofluorescent

Staining

For surface staining of stromal cells, cultures were passaged to 50% confluency 1 day prior to staining with collagenase-dispase. To remove

yre:

Lymphopoiesis

on 6C3Ag+

Stromal

Cell Lines

the cells for staining, the adherent ceils were washed three times with PBS lacking calcium and magnesium, then were overlayed with the same PBS containing 5 mM EDTA. After approximately 5 to 10 min at room temperature, the adherent cells could be seen (by phase microscopy) to detach from the plate. Medium with serum was then added to the culture vessel, and the detached ceils were harvested by pipetting with a Pasteur pipet. One to 2 x 105 stromal cells were stained by a standard technique described elsewhere (Muller-Sieburg et al., 1988). Nonadherent cells were harvested after gentle suspension with a Pasteur pipet. One to 10 x 10s nonadherent cells were stained by using the same immunofluorescent techniques. All stainings were done in the presence of 0.02% sodium azide, and nonviable cells were excluded from the analysis by using propidium iodide to stain the nuclei of dead cells (Parks et al., 1983). First-stage reagents used included MAb6C3 (Pillemer et al., 1964); R7D4, anti-mouse idiotype with the same heavy chain isotype, y2a, as MAb6C3; 70.1 anti-mouse MAC1 (Springer et al., 1979); 8C5, marker on mature mouse granulocytes (R. Coffman, personal communication), generously donated by R. Coffman (DNAX Research Institute, Palo Alto, CA); Jl, rat anti-NIP with the same heavy chain isotype, y2b, as MAC-l and 8C5, donated by R. Coffman; 30-H.12, anti-mouse Thy-l (Marshak-Rothstein et al., 1979); NIP-conjugated MKDG, anti-mouse 1-A”. donated by R. Coffman; RA36B2, anti-mouse 8220 (Coffman, 1982); affinity-purified polyvalent rabbit anti-mouse p heavy chains (Whitlock and Watson, 1979); 1185, rat anti-mouse u heavy chain (McGrath et al., 1980); and FITCconjugated rabbit anti-mouse lg. Second-stage reagents included FITC-conjugated Avidin (Vector Labs, Burlingame, CA): FITCconjugated sheep anti-rabbit lg (Pasteur Institute, Paris, France) absorbed with mouse spleen cells; FlTCconjugated goat anti-rat lg (Pelfreeze, Rogers, AK); and FITC-conjugated J3, rat anti-NIP monoclonal antibody, donated by R. Coffman. Staining was assessed with a Zeiss fluorescence microscope (Microscopes West, Inc., Sunnyvale, CA). Metabolic Labeling and SDS-PAGE Confluent adherent cell layers in T-25 flasks were washed with Hanks Balanced salt solution (GIBCO, Lawrence, MA), then incubated for 21 hr in 5 ml RPM1 medium (JR Scientific, Woodland, CA) containing 100 nCi 35S-methionine (Amersham, Arlington Heights, IL). The medium was then removed and the adherent cells were lysed with 1 ml of phosphate lysis buffer (PLB) (Witte et al., 1978). Each lysate was precleared with 50 pl of 1.2 mg/ml goat anti-rat lg (Pelfreeze, Rogers, AK) and 100 )II of 10% fixed Staphylococcus aureus particles (The Enzyme Center, Malden, MA) on ice for 1 hour. The lysates were microfuged for 15 min at 4OC, and 5 ul of each was diluted in PBS + 5% FCS before precipitation with 10% trichloracetic acid (TCA). Lysate volumes were adjusted with PLB to equivalent TCA-precipitable counts per ml; the 0.5 ml samples of each were removed. One hundred microliters of Mab n-11 (antiTEPC-15 idiotype [Pillemer and Weissman, 19791) culture supernatant were added to one sample as control, and 100 ul of MAb6C3 culture supernatant was added to the other. The lysates were placed at 4OC overnight, then 80 ug of goat anti-rat lg was added to each tube and allowed to bind on ice for 1 hr. lmmunoprecipitates were collected onto 50 ul of 10% Staphylococcus aureus particles, which were washed four times with phosphate lysis buffer, then resuspended in 80 ul gel sample buffer containing 8-mercaptoethanol (Witte et al., 1978). Immunoprecipitates were dissociated by heating in a boiling water bath for 4 min and then the Staphylococcus aureus particles were removed by microfuge centrifugation at room temperature. Each sample was separated on an 8% SDS-polyacrylamide gel as previously described (Witte et al., 1978). 35S-labeled immunoprecipitated proteins were visualized by soaking the gel for 0.5 hr in 1 M sodium salicylate, then exposing Kodak XAR-5 film at -70°C for various times, Limiting Dilution Analysis One day prior to limiting dilution culture, the stromal cells were harvested by using collagenase-dispase, then cultured at 2 to 4 x 10s per well in 100 ~1 medium in 98-well plates. Medium used was RPMI1840 containing 10% FCS (Irvine Scientific #302066, Irvine, CA), 50 units per ml penicillin and streptomycin, 2 mM glutamine, 5 x 10-s M 5-mercaptoethanol, and 1 mM sodium pyruvate. The following day, the cells to be analyzed were diluted in the above medium, and 100 ul of each dilution was cultured in 96 wells. The plates were wrapped loosely in Saran wrap to prevent dehydration, and then incubated at

37°C and in 7% CO*. Cultures were fed by adding 50 ul fresh medium after 1 week, and removing 100 ul of medium and adding back 100 ul after 2 weeks. Wells containing bursts of nonadherent cells were scored with a phase microscope. Morphological Assessment of Cell vpes in Limiting Dilution Analyses All wells were assessed initially for morphology by phase microscopy, and scored as SC+ (small cells), MC+ (medium cells), or LG+ (large, granular cells). In the absence of hydrocortisone, most SC+ cells were lymphoid, and most MC+ cells myeloid. However, in the presence of hydrocortisone, all of the wells scored as SC+ did not contain lymphocytes that were easily recognizable by Wright’s stain; this was worrisome. This suggested that phase microscopy was limited in its usefulness for discerning lymphoid vs. myeloid vs. other lineage colonies. Under conditions where lymphoid colony growth was suppressed (e.g., with hydrocortisone) or poorly supported (e.g., with AC-11) this was particularly true. None of the SC+ wells from AC-6.21 or AC-11 with hydrocortisone contained lymphocytes. The presence of small cells correlated instead with the presence of well-differentiated granulocytes with segmented nuclei. The greatest limitation of phase microscopy was assessment of myeloid colony markers. Wright’s stains showed myeloid cells to be contained in colonies with a very wide range of morphologies, as detected by phase microscopy. The presence of MC correlates best with the presence of immature precursors with large, undifferentiated nuclei, dark blue cytoplasm, and sometimes orange or dark blue cytoplasmic granules. In many cases the whole spectrum of granulocyte ontogeny was represented. In a few cases, wells contained predominantly macrophage-like cells with folded nuclei, and highly vacuolated cytoplasms. Wells containing SC that were not lymphocytes generally contained well-differentiated granulocytes with segmented nuclei. In the presence of hydrocortisone, a mixed SClLG cell colony was quite frequent (data not shown), and these too contained predominantly late forms of granulocytes with banded or segmented nuclei; precursors with dark blue cytoplasm were usually absent or rare. Thymidine Incorporation Nonadherent cells pooled from 3- to 6-week-old Whitlock-Witte cultures and NBM Clone 3 cells were depleted of adherent cells as described above, then cultured at 2 x 10“ and 1 x IO4 cells per well, respectively, in 96-well plates. For analysis of stromal cell support of proliferation in direct contact with the pre-B cells, the stromal cells were irradiated 24 hr prior to addition of the pre-B cells with 1000 rads of cesium irradiation, then cultured at 4 x IO3 cells per well in RPMI1640 medium containing heat-inactivated 5% FCS. MBMF layers were prepared by culturing 2 x lo5 fresh bone marrow cells per well 2 weeks prior to the assay. Nonadherent cells were suspended and removed from the MBMF layers twice in the first week of culture, and only a few residual nonadherent cells were seen by phase microscopy at the time of the assay. For analysis of supernatant support, conditioned medium was pooled from 12 l-ml cultures of each stromal cell line and MBMF. After sterile filtration, each supernatant was assayed at a final concentration of 50%. After 1 (supernatant) or 2 (direct contact) days of culture with the pre-B cell populations, 0.5 t&i of 3H-TdR (ICN, Cleveland, OH) was added to each well. Cells were harvested after 5 hr, and thymidine incorporation was assessed. Electron Microscopy Monolayers of AC-6 or AC-a.21 were removed from the tissue culture dish with collagenase-dispase or EDlA. respectively, as described above. Suspended cells were washed once, then resuspended in serum-free RPMI-1640 medium. An equal volume of 3% phosphatebuffered glutaraldehyde solution was added, and the cell suspension was immediately mixed. The cells were then pelleted by centrifugation at 800 rpm in an IEC CRU-5000 centrifuge. The medium was aspirated, and the pellet was overlayed with 1 ml of the undiluted fixative and stored at 4OC until ready for processing. The pellets were rinsed with phosphate buffer, then treated for 1 hr with 1% Verona&buffered 0~04. After dehydration in ethanol, the pellets were embedded in maraglas, and ultrathin sections were cut and stained with uranyl and lead salts. Examination of sections was with an Elmiskop 101 electron microscope.

Cell 1020

Acknowledgments

mitted pre-pre-B cell and a clonogenic cell. Cell 44, 653-662.

We gratefully acknowledge the technical assistance of L. Hu, L. Jerabek, I. Daehne, and R. Ueda, and the secretarial assistance of J. Mason and M. Billingham. We are grateful to K. Bensch for electron microscopic analysis of the stromal cell lines. We also wish to thank W. Paul for testing supernatants from AC-6.21 for the presence of BSF1; D. Rennick for providing cell lines and assays for testing IL-3 IL-l, G-CSF, and GM-CSF production by AC-6.21; P Hunt, 0. Witte, D. Zipori, and D. Rennick for providing their stromal cell lines for immunofluorescent staining with MAb6C3; R. Adkins, S. Heimfeld, and G. Griffiths for reviewing the manuscript; and D. Rennick, D. Zipori, P Sherwood, and R. Adkins for intellectual input into this project. This research was supported by Grant Al 18162 from the U.S. Public Health Service, and by a grant to I. Weissman from the Weingart Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 16 U.S.C. Section 1734 solely to indicate this fact. Received

October

1, 1986; revised

January

15, 1987.

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Stromal

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