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* Immunobiology and Cancer Program, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104;
Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73190;
Department of Immunology, University of Toronto, Sunnybrook and Women’s Research Institute, Toronto, Ontario, Canada; and
The Scripps/Florida Research Institute, Jupiter, FL 33458
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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+/– prolymphocytes were the most efficient of adult bone marrow cells in short-term cultures, but the assay does not necessarily reflect cells normally responsible for replenishing the adult thymus. Although marrow-derived progenitors with Ig DH-JH rearrangements acquired T lineage characteristics in this model, that was not the case for more B committed cells with VH-DHJH rearrangement products. |
Introduction |
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However, the range of target cells that can respond to Notch receptor ligation with a lineage choice decision and the extent to which partially committed progenitors can be reprogrammed remain unclear. Indeed, we need to rigorously test whether there are single branch points where progenitors at a discrete stage are irreversibly diverted into a particular pathway. Moreover, it is not known if B lineage inhibition and T lineage induction represent completely separate or related events (7, 8). Controversy remains about the role of Notch in determining the relative production of TCR
vs TCR
lymphocytes and biasing the formation of CD4 and CD8 subsets (9, 10, 11). Additionally, most of the studies to date have used fetal lymphoid progenitors and direct comparisons with their counterparts in adult bone marrow have not been reported.
One of the most unique and important features of hemopoietic stem cells (HSCs)3 is their ability to extensively self-renew while retaining the ability to give rise to multiple specialized types of blood cells. Understanding how this process is regulated may someday facilitate expansion of stem cells in culture, opening possibilities for genetic engineering and other therapeutic applications. In that context, it is interesting that human stem cells were expanded 10-fold in culture by ligation of Notch receptors (12), and stem cell lines were established by introduction of constitutively active Notch 1 (13). Therefore, it is possible that Notch receptor ligation might trap cells in a proliferative mode and block their differentiation. If so, lymphohematopoietic cells might be expanded and maintained for prolonged periods on Delta 1-transduced stromal cells.
We have now used RAG-1/GFP-knockin mice, cell sorting, and coculture with Notch ligand-transduced stromal cells to address these questions. Unlike their fetal counterparts, multiple subsets of adult bone marrow cells were able to generate long-term, Notch ligand, and IL-7-dependent cell lines paused at the CD4–D8–thymocyte (DN)2/3 thymocyte stage. Progression of adult progenitors in the T lineage was blocked in the presence of 5 ng/ml IL-7. These findings provide a basis for further investigation of adult lymphoid progenitors in short- and long-term cultures.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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RAG-1/GFP-knockin mice have been described previously (14). Female B6.PL-Thy1a/CyJ (Thy-1/CD90.1) congenic, and C57BL/6 mice were bought from The Jackson Laboratory. Mating homozygous male RAG-1/GFP-knockin mice with wild-type C57BL/6 strain female mice generated heterozygous RAG-1/GFP embryos, as well as mice that were studied as adults. Mating homozygous male RAG-1/GFP-knockin mice with female B6.PL-Thy1a/CyJ mice yielded heterozygous RAG-1/GFP animals bearing the Thy-1/CD90.1 Ag. Timed pregnancies were determined by designating the day of vaginal plug observation as day 0.5 postcoitus (E0.5). For experiments with adult mice, 5- to 10-wk-old male mice were used. All of the studies were reviewed and approved by an institutional review committee.
Antibodies
FITC-conjugated CD45R/B220 (RA3-6B2), CD3
(145-2C11), CD8(53-6.7), CD43, CD25, and Mac-1 Abs, as well as PE-conjugated CD3(145-2C11), TCR, NK1.1(PK136), CD45R/B220 (RA3/6B2), CD19 (ID3), and IL-7R(SB/199) Abs were all purchased from BD Pharmingen. The same source was used for biotinylated-CD106/VCAM-1, biotinylated-CD19 (clone 1D3), CD90.1 (OX-7) and allophycocyanin-conjugated CD11b/Mac-1 (M1/70), DX-5, c-Kit (2B8), CD44 (1M7), TCR, CD4, and CD45R/B220. Allophycocyanin-Cy-7-CD62L (MEL-14) and PE-Cy5-Sca-1 (D7) were obtained from eBioscience. A PE-Texas red tandem-conjugated streptavidin (PE-TR-streptavidin) was purchased from Caltag Laboratories. Purified rabbit anti-TdT polyclonal Ab and a FITC-conjugated F(ab')2 fraction of goat anti-rabbit IgG Ab were purchased from Supertechs. FITC-conjugated goat anti-mouse IgM Ab was purchased from Zymed Laboratories.
OP9 cocultures
Transduced OP9-MigR1 (OP9-vector) and murine Delta-like 1-transduced OP9 cells (OP9-DL1) stromal cell lines have been described previously (5). They were maintained in modified MEM (-MEM; Invitrogen Life Technologies) supplemented with 20% FCS (HyClone). Flt3-L, stem cell factor, and IL-7 were all purchased from R&D Systems and used at concentrations specified in the text. Coculture of hemopoietic progenitors was modified to allow calculation and comparisons of proliferation and differentiation from stem cells/progenitors (5, 15). Briefly, 1 day before plating of progenitors, 2 x 104 stromal cells were plated into each well of 24-well plates. Progenitors were then added to the stromal cells at different densities to determine the numbers of input cells that gave optimal lymphocyte yields. On a weekly basis, stromal cells and hemopoietic cells were harvested using 0.53 mM EDTA/PBS (pH 7.4). Then, 3,000–10,000 hemopoietic cells together with stromal cells were subcultured onto freshly prepared stromal cells. For weekly monitoring of differentiation, stromal cells were excluded by staining with an Ab to VCAM-1, and dead cells were excluded by staining with 7-aminoactinomycin D. Culture conditions and cell densities were considered acceptable when >95% of the recovered hemopoietic cells were viable.
Cell sorting and flow cytometry
E13.5-15.5 fetal livers were harvested and subjected to cell sorting as described previously (16). Bone marrow cells were harvested and enriched for lineage-negative cells by incubation with a mixture of Abs to lineage markers, CD45RA (supernatant from clone 14.8), Gr-1 (Ly-6G; RB6-8C5), CD11b/Mac-1 (M1/70), CD19 (1D3), and Ter-119, followed by negative selection with the MACS cell separation system (Miltenyi Biotec). These partially lineage-depleted cells were further stained with rat anti-mouse lineage markers (biotin-Gr-1, Mac-1, CD2, CD3, CD8, Ter-119, CD45R, DX-5, and CD19), as well as allophycocyanin-labeled c-Kit and PE-Sca-1. Lineage-positive cells were electronically gated out, and lineage negative fractions were sorted on the basis of RAG-1/GFP, c-Kit, and Sca-1 expression with a MoFlo (DakoCytomation). Similar separations were done to isolate the HSC (Lin–Sca-1+c-KithighCD90.1low), L-selectin-positive progenitors (LSPs, Lin–CD90.1–Sca-1+c-KithighCD62L+), early lymphoid progenitors (ELPs, Lin–Sca-1+c-KithighRAG-1+), and common lymphoid progenitors (CLPs, Lin–Sca-1lowc-KitlowIL-7R+), as described in Fig. 6. These previously described fractions (17, 18, 19, 20) were sorted on a FACSAria (BD Biosciences) after thoroughly lineage depleting bone marrow cells and then staining with biotin-CD90.1 revealed by PE-TR, allophycocyanin-Cy7-CD62L, PE-IL-7R, PE-Cy-5-Sca-1, and allophycocyanin-c-Kit. Postsort evaluation of sorted preparations revealed purities of >97%. All analyses were performed on a FACSCalibur (BD Biosciences).
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Congenic Ly 5.1 mice were lethally irradiated with a single dose of 960 rad 4 h before transplantation. Then, 105 recipient whole bone marrow cells were mixed with 105 sorted Lin–c-Kit+Sca-1+ cultured cells and injected i.v. into recipients. Cells were harvested from bone marrows, thymuses, and spleens 21–28 days after injection and stained to check lineage differentiation. Additional transplanted animals were observed for 2 mo and autopsied to see whether tumors developed.
RT-PCR analysis of gene expression
The mRNAs were isolated from sorted cells using MicroPoly(A) Pure (Ambion) and converted to cDNA with Moloney murine leukemia virus reverse transcriptase (Invitrogen Life Technologies). cDNA amounts were normalized by comparing the PCR products using probes specific for -actin. The PCR was conducted by using combination with ampli-TaqDNA polymerase (Takara) and TaqStart Ab (BD Clontech) at three different cycles to confirm the reaction is in the exponential phase. To compare the relative expression level of relevant genes, serial 5-fold dilutions of cDNA were amplified at appropriate cycles. In the case of no amplification, secondary PCR was performed by using 2 µl for 20-µl reaction volume from first reaction as a template to confirm the lack of transcripts. Anti-Taq Ab was inactivated by heating at 95°C for 7 min before amplification conducted as follows: 15 s at 94°C, 15 s at 57°C, and 30 s at 72°C. Gene-specific primer sequences are listed in Table I.
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Results |
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Primitive cells in the aorta/gonad/mesonephros and fetal liver can efficiently give rise to T lineage lymphocytes (defined as TCR+CD4+CD8+ or TCR+) when cultured on OP9-DL1 in -MEM supplemented with 5 ng/ml IL-7 and 5 ng/ml Flt3-L (T. Yokota et al., submitted for publication), and we assumed the same would be true for adult bone marrow. However, this was not the case, and only very small yields of + T lymphocytes were obtained (Fig. 1). In the example shown, RAG-1/GFPlow progenitors from day 14 fetal liver were directly compared with Lin–c-KitlowRAG-1/GFP+IL-7R+ cells isolated from adult bone marrow and found to generate at least 1000 times more T cells (Fig. 1B).
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It seemed possible that adult bone marrow progenitors of T cells might benefit from extended culture intervals. However, this was not the case, and surface TCR+ cells represented a minor fraction of recovered populations even after 5 wk of coculture (data not shown). Note that stromal cell overgrowth was detrimental to total cell yields, and we subcultured lymphoid cells at weekly intervals to freshly prepared stromal cell layers. Lymphocyte growth could not be sustained when long-term OP9-DL1-cultured cells were transferred to OP9-vector stromal cells or when IL-7 was withdrawn, indicating that survival and/or proliferation required sustained Notch and IL-7 signaling (data not shown). In contrast, OP9-DL1 cocultures with added cytokines continued to thrive for at least 3 mo (Fig. 3A and data not shown). Although cultures could be established from six discrete fractions of bone marrow, the efficiency of growth during the first week was not uniform. The most efficient Lin–c-KitlowRAG-1/GFP+IL-7R+/– prolymphocytes/CLP produced 5000 cells/input progenitor during this interval, while the yield from other populations was 1 log less. From the second week, each cell that was subcultured produced an average of 65 cells, regardless of the starting population. After becoming established, the cultures could be propagated indefinitely, and we estimate that one progenitor produced at least 1016 lymphocytes within 2 mo (Fig. 3A).
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Long-term cultured cells were CD45+Sca-1+c-Kit+CD43+CD44+/–CD25+CD5+ and thus resembled thymocytes at the DN2/DN3 stage (Fig. 3C). Flow cytometry revealed low to high levels of RAG-1/GFP and RAG-1 transcripts were detected by RT-PCR (data not shown). TdT is a valuable marker of lymphopoiesis that is expressed from the ELP stage in bone marrow and by thymocytes. Long-term cultured cells were TdTlow/+ (Fig. 3D). Components of the TCR complex were also evaluated. Small numbers of cells with cytoplasmic CD3 or TCR were detectable by flow cytometry (Fig. 3D). RT-PCR revealed mRNA for pT (data not shown). We conclude that while only prolymphocytes expand rapidly, many other discrete fractions of adult bone marrow progenitors can be extensively propagated in response to Notch receptor-mediated signals and acquire indistinguishable properties. Our subsequent experiments focused on extensive characterization of these long-term cultured cells.
Continued, low-level T and NK lineage differentiation from long-term propagated lymphocytes arrested at the DN 2/3 stage
It has been reported that Notch can promote self-renewal of HSCs (21) and the above results show that bone marrow cells could be expanded for months on the Delta 1 ligand. Thus, it seemed possible that long-term propagated cells generated from the various marrow fractions were reprogrammed and assumed stem cell properties. This was addressed with a more extensive RT-PCR and functional analysis of the cultured cells.
Semiquantitative RT-PCR analyses revealed that the GATA-3 and PU.1 and E47 transcription factors were expressed, and the Id-1 repressor of the bHLH family of transcription factors was just detectable. Consistent with the growth selection conditions, the cells expressed Bcl-2, Notch 1, Hes-1, and IL-7R transcripts. Levels of the IL-7R were beneath that detectable by flow cytometry, even though their survival required the continuous presence of this cytokine. In contrast, the c (CD132) cytokine receptor component was easily seen by staining. Although the cells expressed the IL-2R-chain (CD25), the IL-2R (CD122) component was absent (data not shown).
A recent study identified genes whose expression changes sharply as cells progress through early differentiation stages in the thymus (22). Therefore, we evaluated four of these by RT-PCR analysis, comparing long-term cultured lymphocytes to freshly sorted CD4–CD8–CD3–CD19–CD11c–DX-5– thymocytes (Fig. 4). This revealed that cells propagated in Notch-dependent cultures resembled CD44+CD25+ (DN2) and CD44–CD25+ (DN3) cells within a normal thymus. Additional PCR analyses were performed to assess the status of receptor gene rearrangements. As expected, evidence was obtained for rearrangement of all of four TCR gene segments (V3, V6, V8, and V17) that were evaluated (data not shown). Although long-term cultures always contained very small percentages (
3%) of surface TCR+ or NK1.1+ lymphocytes, it seemed possible that these were self-renewing. Therefore, we rigorously depleted these differentiated cells by cell sorting and returned the remaining cells to culture. Although the majority population remained immature Sca-1+c-Kit+ cells 1 wk later, T and NK lineage cells were again detectable (Fig. 5A).
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Adult lymphoid progenitors have unique requirements for T lineage differentiation in culture
Recent studies have demonstrated remarkable differences in cytokine requirements for fetal and adult lymphopoiesis (23, 24, 25). In particular, IL-7 is only essential for formation of T and B cells beyond the early neonatal period (26). Therefore, we considered that T cell progenitors within adult bone marrow might require special conditions. Therefore, cultures were initiated with prolymphocytes and maintained for the first 2 wk in the presence of 5 ng/ml IL-7. They were then subcultured weekly with a range of IL-7 concentrations for an additional 2 wk (Fig. 6A). This revealed that low doses of IL-7 dramatically increased percentages of CD4+CD8+ thymocyte (DP) T lineage cells. However, total yields of TCR+ cells were much less, and slightly fewer TCR+ cells were produced when IL-7 was low during the last 2 wk (data not shown).
Four subsets of adult marrow were then isolated and placed on OP9-DL1 stromal cells with 1 ng/ml IL-7. They were passaged to fresh OP9-DL1 cells just once after 8 days. The Lin–Sca-1lowc-Kitlow IL-7R+ (CLP) fraction generated lymphocytes on day 18, and they emerged in other cultures somewhat later. Impressive percentages of CD4+CD8+ DP cells, and even CD4+ cells were present in all of the cultures when they were evaluated after a total of 24 days (Fig. 6B). Similar results were obtained in parallel cultures that were held with 0.2 ng/ml IL-7. Total yields of DP T lineage cells were highest in cultures initiated with CLP and when the higher concentration of IL-7 was used (Fig. 6C).
The frequency of subculture is another variable that might influence T lineage differentiation, and we sought conditions that might favor T cell production from stem cells. Therefore, Lin–Sca-1+c-KithighThy-1/CD90.1low HSC were isolated from adult bone marrow and placed in OP9-DL1 cocultures for 8 days with 1 ng/ml IL-7. A subset of those cultures was passaged and held for an additional 19 days (Fig. 6D). An additional group was subcultured a second time after 16 days, and all were evaluated at the same time. Although absolute yields of DP cells were not dramatically changed by the additional passage, numbers of DN cells substantially increased.
Unlike fetal progenitors, which produced large numbers of DP cells in 2 wk, adult progenitors required more time, and some subculture was required to maintain optimal yields. Although adult progenitors progressed to the DN2/3 stage and proliferated indefinitely in high IL-7 concentrations, further differentiation was favored by much lower concentrations of this cytokine and a minimal number of passages. In addition, long-term Notch-dependent cell cultures could be easily established from adult but not fetal cells.
Long-term OP9-DL1 cocultures expand marrow progenitors before VHDHJH rearrangement
There are at least two possible explanations why long-term cultures initiated with a wide variety of bone marrow cells are indistinguishable. Rare cells with uniform properties might have been selected for expansion, or alternatively, progenitors dedicated to alternate fates could have been redirected toward the T lineage. Therefore, we prepared cultures with three types of relatively differentiated CD45R/B220+ marrow cells. We know from other studies that several types of stromal cells are suppressive for committed B lineage progenitors (T. Yokota et al., submitted for publication) and that was the case in these experiments. That is, in contrast to prolymphocytes, the three CD45R/B220+ fractions generated very small numbers of cells in 1-wk OP9-DL1 cocultures (data not shown). It is also important to note that even when cultures were initiated with CD19+ cells, numbers of CD19+ cells present in OP9-DL1 cultures 1 wk later were only a fraction of those present in OP9 stromal cell cocultures. However, these could be expanded with three subsequent passages, and sufficient cells were harvested at 4 wk for analysis. At that time, no CD19+ cells were present, and the lymphocytes resembled DN2/DN3 stage thymocytes as described above. That is, the cultures contained 3–8% TCR+, 2–15% NK+, and 75–90% CD44+/–CD25+ cells. We reasoned that CD45R/B220+CD19+CD43+IgM– and CD45R/B220+CD19+CD43–IgM– fractions would be highly enriched for B lineage cells, and genomic PCR analyses confirmed that at least some in the starting populations had undergone VH to DHJH recombination (Fig. 7). However, DHJH, but not VH-DHJH rearrangement, products were found in long-term cultured lymphocytes. We conclude that long-term OP9-DL1 cocultures can suppress expansion of cells that would normally become B cells and provide a very powerful selective pressure to cells not fully committed to the B lymphocyte lineage.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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+ lymphocytes from adult progenitors. This information extends previous indications that fetal and adult progenitors differ in differentiation requirements. A prolymphocyte fraction, including cells designated CLP, grew most efficiently during the first week on OP9-DL1, but established cultures initiated with a wide range of marrow subsets were indistinguishable. This resulted at least in part from the selective expansion of rare progenitors that were not fully committed to the B lymphocyte lineage.
Adult marrow progenitors were dramatically different from their fetal counterparts in several respects. Two Lin–c-Kitlow RAG-1/GFP+IL-7R+/– prolymphocyte fractions of bone marrow expanded as efficiently as fetal liver in 1-wk OP9-DL1 cocultures (Fig. 3A and data not shown). However, the yield of TCR+ lymphocytes 1 wk later was at least 1000-fold less than that obtained from cultures initiated at the same time from fetal liver progenitors (Fig. 1). Although cocultures initiated with fetal cells usually lasted no longer than 4 wk, we found that adult marrow progenitors could be serially transferred to fresh stromal cells an indefinite number of times. These adult-derived cells retained dependence on IL-7 and Notch receptor ligation while acquiring homogeneous characteristics. Indeed, cultures initiated with six distinctly different subsets of bone marrow were remarkably similar by the second passage and subsequently expanded at approximately the same rate. Other fetal/adult differences in T lymphopoiesis have been reported previously (27, 28). In addition, a very recent study indicates that IL-7 blocks adult but not fetal thymocyte differentiation (29, 30).
The density and complexity of Notch ligands, APC, and cytokines found in the normal thymus is unlikely to be fully replicated in this coculture model. Despite that, progression of T lymphopoiesis and production of TCR+ cells from fetal progenitors are surprisingly robust. All three of our labs routinely obtain lower cell yields and slower expansion from adult marrow progenitors, but only one routinely observes efficient T cell production from that source (6). We show here that low cytokine concentrations and a minimal number of passages favor lineage progression from adult cells. Subsequent analysis may reveal important technical variables such as serum batches, cytokine source, passage number of stromal cells, and mouse strains may represent other important technical variables.
There is growing evidence that many discrete cell types in bone marrow have the potential to become T lymphocytes, and it is difficult to discern which ones normally replenish the thymus (31, 32). The most primitive Lin–Sca-1+c-Kithigh fraction of bone marrow contains stem cells, LSPs and ELPs, with the potential for sustained T cell production (20, 33). Moreover, cells with these general properties are detectable within the bloodstream and resemble canonical prothymocytes in some respects (7, 34). On the other hand, cells with at least short-term T lymphopoietic potential can be found within Lin–c-Kitlow and even CD45R/B220+ fractions, and it is possible they contribute under some circumstances (18, 20, 35). Although direct comparisons under physiological conditions could be informative, it seems likely that the T vs B fate decision is not as abrupt as generally believed. There are likely to be two factors that contribute to this misconception. One is that multiple types of progenitors can be diverted into the T cell lineage under the conditions found in OP9-DL1 cocultures (present data and Ref.7), although this is not recapitulated under normal physiologic conditions (7). The other is that Notch signaling can, to some extent, redirect the lineage fate of cells that would normally not become T cells.
In any event, the Lin–c-KitlowRAG-1/GFP+IL-7R+/– category of bone marrow gave the most impressive short-term expansion in OP9-DL1 cocultures. Although this includes cells originally described as CLPs, there are reasons to believe they are not the most efficient for replenishing the adult thymus (20, 32). It is also important to note that at least two stromal cell lines can actually suppress the activity of fully committed B lineage progenitors (16). We do not understand the basis for this inhibition but could speculate that there is constitutive low level expression of Notch ligands. Any functional assessment of differentiation potential is subject to bias, and caution is advised when interpreting culture results in terms of in vivo relevance.
Long-term propagated adult lymphocytes closely resembled primitive DN2/DN3 stage thymocytes with respect to expression of four genes (22). Although largely arrested at that stage, the cultures continued to contain small numbers of cytoplasmic TCR+ cells and slowly generated NK and T cells. In addition, they successfully competed with normal bone marrow and progressed to CD4+ and CD8+ lymphocytes in the thymuses of irradiated recipients. Furthermore, we found evidence of polyclonal rearrangement of four TCR genes (data not shown). It is curious that fetal progenitors, such as those freshly isolated from the thymus (5, 7), efficiently complete maturation in the OP9-DL1 culture system, while adult bone marrow progenitors transiently arrest at the DN2/DN3 stage when 5 ng/ml IL-7 is added. When the purified Lin–c-Kit+Sca-1+ cells from long-term cultures were transplanted with equal number of total recipient bone marrow cells, only 0.3% of thymic cells were derived from the cultured cells. This indicates that thymic homing and/or T cell differentiation of bone marrow progenitors cultured on OP9-DL1 may decrease over time. It has been shown previously that T lineage progression from fetal progenitors in a fetal thymic organ culture system is inhibited by high cytokine concentrations (36). However, in the OP9-DL1 coculture system, this is only the case for adult progenitors (this report and Ref.30). These findings again highlight fetal/adult differences and indicate that the cultures do not optimally provide all environmental cues required by adult marrow-derived progenitors. Further study might implicate other Notch ligands (37) or additional components of the normal adult thymus.
The bone marrow, rather than the thymus, is likely to be the source of NK cells during adult life (38). Indeed, the Lin–c-KitlowFlk-2+ prolymphocyte fraction of marrow is the best source of NK progenitors, and before acquisition of the CD122 receptor for IL-15, many of them still retain B lineage potential (39, 40). We did not intentionally promote development of NK cells by addition of IL-15 in the present study. However, small numbers of DX-5+NK1.1+ NK lineage cells were produced in all of our OP9-DL1 cultures, regardless of whether they were initiated with fetal liver or adult bone marrow. These emerged during the first week on OP9-vector control stromal cells and from the second week in OP9-DL1 cocultures (data not shown). Other than this apparent difference in kinetics, we saw no strong evidence that the NK lineage choice was either positively or negatively influenced by Notch receptor ligation. A recent analysis of fetal thymocytes demonstrated that residual NK lineage potential is normally down-regulated at the DN2 stage and suppressed by Notch receptor ligation (8).
Notch is thought to influence a variety of events in hematopoiesis, including the self-renewal of stem cells (12, 13, 21, 41). However, recombinant forms of Delta 1 have been shown to suppress myelopoiesis (42, 43). We consistently observed a reduction in production of myeloid cells in OP9-DL1 cocultures, regardless of whether fetal or adult hemopoietic cells were used. It is unclear whether strong Notch signals killed myeloid progenitors, held them in an undifferentiated, cytokine unresponsive state, or directed them to alternative fates.
It is critical to know if long-term cultured marrow cells were similar because of reprogramming, or alternatively, if rare cells contaminating all of the sorted fractions were selected over time. This is a difficult determination when the starting populations to be compared are all very primitive. Therefore, we started some cultures with highly enriched (98% by postsort analysis) and relatively late CD45R/B220+CD19+CD43–sIgM– (Hardy Fraction E) cells. Although there is almost no proliferative activity in the starting population (44) and we saw no expansion during the first week of coculture, long-term lines were established within 3 wk of serial passage. Remarkably, there was no evidence for Ig VH to DHJH rearrangements and no cytoplasmic µ H chain staining in the expanded lymphocytes, even though small pre-B cells made up almost all of the starting population. We interpret these results as showing that the OP9-DL1 coculture system has tremendous potential for selection and expansion of rare, undifferentiated lymphocytes that might represent contaminants in sorted populations. Although cells can progress substantially in the B lineage and remain Notch ligation sensitive, late-stage progenitors with productively rearranged Ig genes are not reprogrammed to adopt a T cell fate. We demonstrate that an impressive range of different progenitors in adult marrow could be propagated indefinitely in OP9-DL1 cocultures. Regardless of starting population, the cells assumed properties of DN2/DN3 stage thymocytes and retained some potential for terminal T lineage differentiation. Although a degree of lineage reprogramming may occur in this culture model, it can preferentially expand rare, relatively undifferentiated cells. The findings are compatible with a model where Notch regulates multiple events and differentiation options become gradually restricted (45).
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Acknowledgments |
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Disclosures |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Footnotes |
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1 This work was supported by National Institutes of Health Grants AI20069, AI58162, AI33940, and AI53739 and National Center for Research Resources Grant P20 RR15577. J.C.Z.-P. is supported by an Investigator Award from the Canadian Institutes of Health Research. 
2 Address correspondence and reprint requests to Dr. Paul W. Kincade, Immunobiology and Cancer Program, Oklahoma Medical Research Foundation, 825 N.E. 13th Street, Oklahoma City, OK 73104. E-mail address: kincade{at}omrf.ouhsc.edu
3 Abbreviations used in this paper: HSC, hemopoietic stem cell; CLP, common lymphoid progenitor; DN, CD4–D8– thymocyte; DP, CD4+CD8+ thymocyte; ELP, early lymphoid progenitor; LSP, L-selectin-positive progenitor; OP9-DL1, murine Delta-like 1-transduced OP9 cell.
Received for publication May 18, 2005. Accepted for publication July 25, 2005.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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T cell fate in fetal thymus organ culture. Blood 102:2444.-2451. -chain in fetal and adult thymocyte development revealed by analysis of interleukin-7 receptor -deficient mice. Eur. J. Immunol. 28:1859.-1866. [Medline]
cells in fetal thymus organ culture. J. Immunol. 150:2706.-2716. [Abstract]
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) was harvested and stained on day 27. The parallel group (
) was maintained in the same way but passaged one additional time on day 24. Numbers of total cells, CD4–CD8– (DN) and DP cells are shown.
, corresponds to OP9-DL1 cultures. The values are means of duplicate cultures ± SD in this representative of three similar experiments.
