Ex Vivo Culture of CD34⁺/Lin⁻/DR⁻ Cells in Stroma-Derived Soluble Factors, Interleukin-3, and Macrophage Inflammatory Protein-1α Maintains Not Only Myeloid But Also Lymphoid Progenitors in a Novel Switch Culture Assay

ALTHOUGH PRIMITIVE progenitors of both myeloid and lymphoid lineage can be found within the CD34 positive bone marrow population,1-4 it is unclear if ex vivo culture to expand stem cells or to induce proliferation for retroviral gene transduction will maintain cells capable of multilineage differentiation, self-renewal, and engraftment. Cultivation of the CD34 positive, lineage negative, HLA-DR negative/dim population (CD34⁺/Lin⁻/DR⁻) in stromal-dependent long-term culture (LTC) results in differentiation into committed myeloid progenitors and maintenance of long-term culture initiating cells (LTC-IC).5,6 Although stromal cells in LTC are a requirement for the maintenance of myeloid progenitors with LTC-IC capacity, direct contact with intact stromal feeders is not a requirement for this process. When progenitors are physically separated from the stroma layer by a microporous membrane (stroma noncontact culture), CD34⁺/Lin⁻/DR⁻ LTC-IC are maintained.7 Moreover, LTC-IC are maintained to a greater extent under stroma noncontact (SNC) conditions than when CD34⁺/Lin⁻/DR⁻ cells were cultured in direct contact with stroma. We have also shown that addition of macrophage inflammatory protein-1α (MIP-1α), a member of the chemokine family, and the growth promoting cytokine interleukin-3 (IL-3) to SNC culture results in a fourfold to sixfold expansion of LTC-IC after 2 weeks.8-10 Without stroma, no combination of known growth promoting and growth inhibitory cytokines could maintain LTC-IC, suggesting that other factors produced by stroma may be essential.

To evaluate lymphoid development, we have developed a modified long-term marrow culture system, which generates natural killer (NK) cells from CD34⁺/Lin⁻/DR⁻ adult marrow cells, the population known to contain LTC-IC.11Purified CD34⁺/Lin⁻/DR⁻cells are plated directly on allogeneic, irradiated primary marrow stromal feeders with medium containing IL-2. When progenitors are plated in the absence of IL-2 or in the presence of hydrocortisone, no NK cell differentiation is seen. However, when CD34⁺/Lin⁻/DR⁻ cells are cultured on allogeneic irradiated stroma with IL-2 and in the absence of hydrocortisone, differentiation of NK cells results. However, progeny from these cultures are unable to reinitiate secondary long-term cultures suggesting that cultures do not maintain NK cell progenitors and lead to terminal NK cell differentiation. The requirement for direct contact with intact stromal layers distinguishes this population from more committed progenitors (CD34⁺/CD7⁺), which do not require direct contact with stroma.12 The importance of progenitor-stroma contact interaction for lymphoid progenitor differentiation has been described for several lymphoid culture systems and is the basis for the murine Whitlock-Witte culture.13 

Primitive myeloid progenitors can be assessed as blast cell colony cells, high proliferative potential–colony-forming cell (HPP-CFC) or LTC-IC. Long-term NK cultures,11,12,14 fetal thymic organ cultures,15 and human modified B-cell assays13,16,17 have been used to assess lymphoid capacity. However, few in vitro assays exist that measure more primitive human cells with both myeloid and lymphoid capacity. One approach is to initiate long-term cultures under myeloid “Dexter” conditions (hydrocortisone containing) followed by a “switch” to conditions favoring lymphoid growth (removal of hydrocortisone) similar to the Whitlock-Witte assay. Such an assay would allow recognition of lymphoid progenitors that are maintained under myeloid conditions and may serve as a system to study conditions of primitive lymphoid progenitor maintenance. In this report, we describe a novel myeloid/NK cell switch culture system.

Bone marrow was obtained from normal donors after informed consent using guidelines approved by the Committee on the Use of Human Subjects in Research at the University of Minnesota. Bone marrow mononuclear cells (BMMNC) were obtained by Ficoll-Hypaque (specific gravity 1.077) (Sigma Diagnostics, St Louis, MO) density gradient centrifugation.

BMMNC were enriched for CD34⁺ cells using an avidin-biotin column as recommended by the manufacturer (Cellpro, Bothel, WA). Resultant cells were stained with phycoerythrin (PE)-conjugated anti-CD34 antibody (HPCA-2) (Becton Dickinson [BD], San Jose, CA) for isolation of CD34 positive cells or CD34-biotin (Cellpro) for multicolor sorting as previously described.18Fluorescein isothiocyanate (FITC)-conjugated antibodies against CD2, CD3, CD4, CD5, CD7, CD8, CD10, and CD19 were used for the lineage (Lin) cocktail (BD). PE-conjugated anti–HLA-DR (DR) was used and streptavidin SA670 (GIBCO BRL, Grand Island, NY) as the third fluorescent color.

CD34⁺/Lin⁻/DR⁻ cells were plated in long-term culture medium (LTC medium—Iscove's modified Dulbecco's medium [IMDM] [GIBCO BRL, Grand Island, NY], 12.5% fetal calf serum [HyClone Laboratories, Logan, UT], 12.5% horse serum [Stem Cell Technologies, Vancouver, BC], 10^-6hydrocortisone [Upjohn, Kalamazoo, MI]) supplemented with 5 ng/mL IL-3 (R&D Systems, Minneapolis, MN) and 100 ng/mL MIP-1α (R&D Systems) in a collagen-coated Transwell (Costar Corp, Cambridge, MA) above a preestablished, allogeneic, irradiated (2,500 cGy) stromal feeder. The Transwell insert (Costar) separates progenitors from stroma by a 0.45-μm filter.8 At day 7, half of the medium was removed from the lower well and supplemented with the same medium supplemented with fresh cytokines. After an additional 7 days, cells were harvested and inoculated directly into NK cultures or sorted into CD34⁺/CD33⁻ and CD34⁺/CD33⁺ populations as previously described.9 

NK cell progenitors were plated in direct contact with preestablished allogeneic irradiated stroma as indicated. NK progenitor maintenance was tested by plating NK cell progenitors in contact with or separated from stroma by a Transwell for 5 weeks and further differentiation in IL-2 alone. The three-step NK cell switch culture incorporates IL-3 and MIP-1α SNC culture progeny to induce NK cell commitment and further differentiation (Fig 1). Cultures were maintained in a humidified atmosphere at 37°C and 5% CO₂ and medium was half changed weekly. Medium was supplemented as indicated with 1,000 U/mL IL-2 (a gift from Amgen, Thousand Oaks, CA), 10 ng/mL stem cell factor (SCF), a gift from Amgen), and 10 ng/mL IL-7 (R&D Systems). In the final culture step, NK medium consisted of 2:1 (vol/vol) Dulbecco's Modified Eagle Medium (DMEM)/Ham's F12 supplemented with 10% heat inactivated human AB serum and 1,000 U/mL IL-2 as described.19 

Progenitors were plated in bulk or adapted to culture in 96-well plates for limiting dilution analysis (22 replicates at four dilutions: 1,200 to 2,400, 400 to 800, 133 to 266, 45 to 90 cells/well).12Wells were determined as positive by addition of 5,000⁵¹Cr-labeled K562 cells (American Tissue Type Collection [ATTC], Rockville, MD) resulting in a specific lysis greater than three standard deviations above spontaneous lysis. The frequency of NK cell progenitors in each population was calculated as the reciprocal of the concentration of cells that resulted in 37% negative wells using Poisson statistics and the weighted mean method.20,21 In some limiting dilution assays (LDA) experiments, 10 ng/mL FLT-3 ligand (a gift from Immunex, Seattle, WA) was added. To simplify the LDA switch culture, all cytokines were added to the NK based medium (after finding no difference between LTC medium and NK-based medium) at culture initiation and cultures were half changed with NK medium containing IL-2 alone for the remaining culture period.

Cell surface antigens were determined by direct staining of cells with mouse monoclonal antibodies. FITC-or PE-coupled antibodies (BD) were directed at CD2, CD3, CD7, CD8, CD10, CD16, CD19, CD56, and NKB1. FITC- and PE-coupled isotype matched immunoglobulins were used as controls. All analyses were performed with a FACS Star Plus flow cytometer (BD) equipped with a Consort 32 computer (BD). Cytotoxicity assays were performed in triplicate using K562 (ATTC) and the Raji (ATTC) cell lines in a 4-hour Cr⁵¹ release assay.22,23 

Progeny of switch cultures were harvested and total mRNA extracted using RNeasy spin columns according to the manufacturer's recommendations (Qiagen, Santa Clarita, CA). Reverse transcription was performed as previously described in detail.18 Briefly, samples were subjected to 40 cycles of denaturation at 95°C for 20 seconds, annealing at 55°C for 15 seconds, and extension at 72°C for 1 minute in a Perkin Elmer 480 thermal cycler (Foster City, CA). Oligonucleotide primer sequences were: CD3ζ 5′ primer: 5′-CTCTGCCTCCCAGCCTCTTT-3′; CD3ζ 3′ primer: 5′-GCGTCGTAGGTGTCCTTGGT-3′; CD3ε 5′ primer: 5′-AGTTGGCGTT-TGGGGGCAAGATGGTAATGAAGAAA-3′; CD3ε 3′ primer: 5′-CCCAGGAAACAGGG-AGTCGCAGGGGGACTGGAGAG-3′. Amplified products were size separated on 1.5% agarose gels and transferred to Hybond N+ nucleic acid transfer membranes (Amersham, Arlington Heights, IL). Probes were labeled with³²P-deoxyadenosine triphosphate (dATP) using a TdT 3′-end labeling kit (Boehringer Mannheim, Indianapolis, IN) using probe sequences: CD3ζ 5′-ACTGTAGGCCTCCGCCA-3′; CD3ε 5′-TTCT-CACACACTCTTGCCCTCAGG-3′.

Results of experimental points obtained from multiple experiments were reported as mean ± 1 standard error of the mean (SEM). Significance levels were determined by two-sided Student's t-test analysis.

Primitive progenitors can develop along the NK cell lineage by culture in direct contact with allogeneic stromal feeders and IL-2 (termed “NK cell differentiation culture”), but this system does not maintain NK progenitors. Using this model for NK cell development, we evaluated whether NK cell progenitors were present after SNC cultures supplemented with IL-3 and MIP-1α, conditions known to expand myeloid LTC-IC. CD34⁺/Lin⁻/DR⁻cells were cultured in IL-3 and MIP-1α SNC cultures (n = 6). After 14 days under these conditions, no phenotypic NK cells were identified and progeny from these cultures were unable to lyse K562 tumor targets (data not shown). Progeny of IL-3 and MIP-1α SNC cultures were then evaluated for their capacity to initiate NK cell differentiation cultures. NK cells could not be generated under these conditions suggesting that the IL-3 and MIP-1α SNC cultures did not maintain progenitors of the NK cell lineage or alternatively, that other factors were missing to induce primitive cells to develop along the NK cell lineage.

Because early B, T, and NK progenitors require interaction with stroma for survival and differentiation,12,24,25 we introduced an intermediate culture step that promotes direct contact with stromal feeders in an attempt to induce NK progenitor commitment from IL-3 and MIP-1α SNC LTC-IC expansion cultures (Fig1, “NK cell progenitor switch conditions”). The addition of direct contact without additional cytokines was insufficient to induce NK progenitor commitment. IL-2, IL-7, or the combination of IL-2 and IL-7 poorly induced NK cell commitment, as measured after the final culture step with IL-2 alone (Fig 2). Therefore, in addition to IL-2, IL-7, and stromal-derived contact factors, we evaluated the addition of SCF, which has importance in lymphoid commitment and expansion.15,26-29 SCF alone did not induce commitment of previously cultured cells to give rise to NK cell progeny. In contrast, the combination of IL-2, IL-7, and SCF induced NK cell progenitor commitment resulting in significant generation of NK cells after a final switch to conditions favoring NK cell differentiation (Fig 2). NK cells were phenotypically similar to those already described from fresh CD34⁺/Lin⁻/DR⁻ cells (data not shown) and exhibited characteristic function in cytotoxicity assays (Fig 3, left panel).

We have previously shown that CD34⁺/CD33⁻cells present in 14-day IL-3 and MIP-1α SNC cultures are highly enriched for LTC-IC, with a cloning frequency up to 30%.9We next assessed whether lymphoid cells are present after IL-3 and MIP-1α SNC cultures. After 14-day IL-3 and MIP-1α SNC culture, CD34⁺/Lin⁻/DR⁻ progeny did not express any marker of lymphoid progenitors (CD2, CD7, CD10)12,30 or mature lymphocytes (CD3, CD8, CD10, CD19, or CD56). When CD34⁺/CD33⁻ and CD34⁺/CD33⁺ cells from CD34⁺/Lin⁻/DR-initiated IL-3 and MIP-1α SNC cultures were purified by flow cytometry and plated directly into NK cell differentiation cultures, no NK cells resulted (n = 6). In contrast, culture under NK cell progenitor switch conditions followed by conditions supporting NK cell differentiation resulted in significant NK cell generation. CD34⁺/CD33⁻ cells gave rise to significantly more NK cells than CD34⁺/CD33⁺cells (154 ± 64-fold expansion v 18 ± 9-fold expansion, n = 9; P = .044). The number of NK cells generated by CD34⁺/CD33⁻ cells was similar to that reported by us for fresh CD34⁺/Lin⁻/DR⁻cells.11 These cells exhibited cytolytic function in cytotoxicity assays similar to that derived from CD34 positive marrow populations (Fig 3, right panel). The phenotype of these 9-week cultured NK cells was similar to the phenotype of NK cells derived from fresh CD34⁺/Lin⁻/DR⁻cells. Although NK cells expressed other lymphoid markers (CD2, CD7, CD8), switch cultures do not give rise to mature CD3⁺ T cells or CD19⁺ B cells (Table1). To estimate progenitor frequency, CD34⁺/CD33⁻ cells selected after IL-3 and MIP-1α SNC cultures were then plated in a modified limiting dilution assay incorporating NK cell progenitor switch conditions. Similar to the cloning frequency observed with fresh CD34⁺/Lin⁻/DR⁻cells,12 the cloning frequency of resorted CD34⁺/CD33⁻ cells was approximately 0.1% (n = 3).

The addition of stromal ligands, IL-2, IL-7, and SCF was sufficient to induce NK cell progenitor commitment from CD34⁺/CD33⁻ cells cultured for 14 days in IL-3 and MIP-1α SNC cultures, but it was still uncertain how these conditions contributed to NK cell generation. Further experiments were performed to address these questions. NK cell progenitors were defined as cells with the capacity to give rise to NK cells when switched to IL-2–containing NK medium under NK cell differentiation conditions. To fully test NK cell progenitor survival conditions, culture duration was lengthened to 5 weeks. Freshly sorted CD34⁺/Lin⁻/DR⁻progenitors were plated in LTC medium with or without additional cytokines in direct contact with stroma or separated from stroma by a Transwell membrane. After the 5-week culture, medium was changed to conditions that support terminal NK cell differentiation (direct contact, human serum, IL-2). NK cell progenitors did not survive in LTC medium alone whether progenitors were in contact with or separated from stroma during the initial culture period. Addition of IL-7 alone failed to increase NK cell progenitor survival (data not shown). However, IL-2 and especially the combination of IL-2 and IL-7 increased NK cell progenitor survival, but only if CD34⁺/Lin⁻/DR⁻ cells were in contact with stroma for the 5-week culture period (Fig 4).

We next addressed whether NK switch conditions could induce NK progenitor development from a more undifferentiated cell. Fresh CD34⁺/Lin⁻/DR⁻ cells were plated directly in LDA under NK cell differentiation conditions with IL-2 containing medium alone or modified to incorporate contact-mediated NK cell progenitor switch conditions with LTC medium supplemented with IL-2, IL-7, and SCF (n = 3). NK cell progenitor cloning frequency was similar between the two conditions and in no case did LDA's incorporating NK cell progenitor switch conditions result in a higher cloning frequency suggesting that these conditions did not increase commitment of clonogenic NK cell progenitors. An alternative explanation for the increased generation of NK cells in the switch culture is that the NK cell progenitor switch conditions actually expand NK cell progenitors. This was tested by determining the NK cell cloning frequency in the CD34⁺/Lin⁻/DR⁻ starting population (day 0 LDA) and comparing this with the cloning frequency of CD34⁺/Lin⁻/DR⁻ cells cultured for 14 days in a bulk well under NK cell progenitor switch conditions before plating into LDA (n = 4 for all conditions). The day 0 cloning frequency of fresh CD34⁺/Lin⁻/DR⁻ cells was 0.04% ± 0.01%. Bulk culture of the progeny of CD34⁺/Lin⁻/DR⁻ cells under NK cell progenitor switch conditions followed by LDA (plated according to the initial number inoculated at day 0) resulted a 28 ± 4.6-fold increase in cloning frequency compared with the day 0 LDA (Fig 5). The importance of hydrocortisone in NK cell progenitor expansion (LTC medium + IL2, IL-7, SCF) was also tested in LDA. Significantly less NK cell progenitor expansion was observed when hydrocortisone only was eliminated from the culture resulting in only a 5 ± 1.2-fold increase in cloning frequency compared with the day 0 LDA (Fig 5).

In experiments described above, CD34⁺/CD33⁻ cells derived from IL-3 and MIP-1α SNC cultures give rise to NK cells in a switch culture assay using IL2, IL7, and SCF. However, we could not show that these switch conditions could increase the frequency of progenitors to develop along the NK cell lineage. In addition, the approximately 0.1% cloning frequency for cultured CD34⁺/CD33⁻ cells was lower than expected. Several possibilities may explain this low cloning frequency. Either the IL-3 and MIP-1α SNC cultured CD34⁺/CD33⁻ cells poorly support lymphoid progenitor survival or alternatively, the switch culture readout with IL-2, IL-7, and SCF was still inadequate to efficiently induce NK commitment. FLT-3 ligand has been shown to be an important primitive acting cytokine and FLT-3 receptors are selectively found on human CD34 positive cells.31,32 Therefore, we added FLT-3 ligand to our previously tested cytokines (IL-2, IL-7, and SCF) and plated 14 day IL-3 and MIP-1α SNC cultured CD34⁺/CD33⁻ cells in limiting dilution. With the addition of FLT-3 ligand to the switch culture assay, the cloning frequency of NK cells increased 10-fold to 1.2% ± 0.4% (n = 4) compared with cultures where FLT-3 ligand was not present. These data further support the notion that IL-3, MIP-1α, and stromal-derived factors maintain cells capable of lymphoid differentiation and addition of FLT-3 ligand to the switch culture assay induces progenitors to differentiate along the NK cell lineage.

NK cells from FLT-3 ligand switch cultures were CD56⁺/CD3⁻, coexpressed other lymphoid markers (CD2, CD7, CD8), and were able to lyse K562 and Raji targets (data not shown). CD3ζ and CD3ε transcripts were consistently detected in progeny of switch cultures (n = 6), lymphoid genes that are expressed by IL-2–stimulated mature NK cells.33 In addition, a small percentage of NK cells (0% to 8%, n = 6) derived from switch cultures also expressed the killer inhibitory receptor (KIR), NKB1, one of a family of lymphocyte receptors found on NK cells, which recognize class I major histocompatibility complex (MHC).34 Taken together, these data support the notion that cells derived from switch cultures are of lymphoid origin.

Stem cell self-renewal, proliferation, and myeloid versus lymphoid commitment involves complex interactions with the microenvironment through soluble factors and in direct contact with stromal ligands. There has been great interest in exploiting these techniques to cultivate human stem cells for transplantation. Until now, there was no evidence that multilineage progenitors are maintained after ex vivo culture, which also maintain LTC-IC. We developed methods to determine if primitive cells with lymphoid potential are maintained in long-term culture. Sequential modifications of the myeloid long-term culture were used to assess NK cell progenitors as a measure of lymphoid capacity.

CD34⁺/Lin⁻/DR⁻ cells were cultured in stroma noncontact culture with MIP-1α and IL-3, conditions known to maintain myeloid LTC-IC. These conditions do not promote differentiation of CD34 positive cells into phenotypically identifiable CD2, CD7, or CD10 lymphoid progenitors or mature NK cells. However, a switch to contact with stromal ligands, IL-2, IL-7, and SCF was able to generate NK cells. The NK cell progenitor capacity was mainly found in the CD34⁺/CD33⁻population, which has also been shown to be enriched for LTC-IC. In contrast to the high cloning frequency of LTC-IC within this population (up to 30%), the NK cell progenitor cloning frequency was significantly less. This raises the possibility that despite the ability to initiate myeloid long-term cultures at high frequency, the number of CD34⁺/CD33⁻ progenitors, which also maintain the capacity to generate lymphoid cells, is significantly lower. This is in agreement with studies by Lemieux et al35who developed a murine long-term culture switch assay in which murine marrow is grown in limiting dilutions for 4 weeks under myeloid conditions. Subsequently, a switch to lymphoid conditions and evaluation of B-cell progeny identified progenitors capable of both myeloid and lymphoid growth. The cloning frequency of cells with both myeloid and lymphoid capacity was approximately 10-fold to 15-fold lower than that of cells capable of initiating myeloid long-term cultures alone. The lower frequency of NK cell progenitors in our system, even after addition of FLT-3 ligand, which increased NK progenitor cloning frequency by 10-fold, may be the result of a low number of primitive cells capable of lymphoid differentiation. Alternatively, in vitro conditions may still be inadequate to induce differentiation of primitive cells toward the NK cell lineage.

Glucocorticoids have been shown to suppress mature lymphocytes including NK cells36 and are absent from Whitlock-Witte cultures. Therefore, we did not expect to find that hydrocortisone was important to induce NK cell progenitor expansion as demonstrated in our LDAs. How hydrocortisone effects expansion of NK progenitors is unknown, but may be indirectly related to effects on stroma.37 Glucocorticoids increase SCF in normal marrow fibroblasts.38 However, this alone may not account for our findings given the excess exogenous SCF added to cultures. It is possible that the membrane bound form of SCF is increased and required for expanding NK cell progenitors. Glucocorticoids may also affect expression of other cytokines such as leukemia inhibitory factor produced by marrow endothelial cells.39 Finally, glucocorticoids alter the sulfation pattern of glycosaminoglycans in the extracellular matrix, which may affect progenitor and cytokine binding.40 

The importance of direct contact with stroma for myeloid and lymphoid differentiation seems to be divergent. LTC-IC do not require contact with stroma for maintenance, although soluble factors produced by stroma, such as glycosaminoglycans, are important.41Further work from our group suggests that direct contact with stroma through β1 integrins inhibits myeloid progenitor proliferation suggesting that contact with stroma may function as a negative regulator of myelopoiesis.42 In contrast, for B-cell progenitors or NK progenitors, physical separation from stroma results in substantially decreased proliferation and differentiation of progenitors.12,24 The observation that CD34⁺/CD33⁻ cells obtained after 2 weeks of cytokine supplemented stroma noncontact culture, which would presumable select against lymphoid progenitors, can give rise to NK cells when conditions are switched to facilitate direct contact with stroma supports the notion that contact with stroma is not required for survival of NK progenitors, but contact is required in the differentiation process. However, without further data, the precise mechanism of progenitor differentiation (recruitment, maintenance, and proliferation) in cytokine supplemented, stromal based switch cultures cannot not be determined.

In summary, we developed a switch culture assay that allows differentiation of CD34⁺/Lin⁻/DR⁻ cells cultured in “myeloid” IL-3, and MIP-1α SNC cultures to differentiate into NK cells. Therefore, ex vivo culture in stromal soluble factors, IL-3 and MIP-1α, can maintain cells capable of both myeloid and lymphoid differentiation. To demonstrate whether myeloid and lymphoid progenitors are derived from a single CD34⁺/CD33⁻ cell or from committed myeloid and lymphoid progenitors will require use of single cell deposition assays or retroviral marking.

The authors thank Brad Anderson for his help with flow cytometry and Jeanne Lund for her excellent technical help in PCR assays.