Embryonic Mouse STO Cell–Derived Xenografts Express Hepatocytic Functions in the Livers of Nonimmunosuppressed Adult Rats
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《干细胞学杂志》
a Department of Pharmacology and
b Center for Molecular Genetics, University of California, San Diego, California;
c Ordway Research Institute and
d New York State Health Department, Albany, New York;
e Marion Bessin Liver Research Center, Albert Einstein College of Medicine, Bronx, New York, USA
Key Words. Mouse STO progenitor cells ? Xenotransplantation ? Nonimmunosuppressed rats
Correspondence: K.S. Koch, and H.L. Leffert, Department of Pharmacology and Center for Molecular Genetics, University of California, San Diego, La Jolla, California, 92093-0636 USA. Telephone: 858-534-2354; Fax: 858-822-4184; e-mail: kkoch@ucsd.edu and hleffert@ucsd.edu
ABSTRACT
Embryonic stem cells undergo self-renewal coupled with differentiation into mature parenchymal cells of mesodermal, ectodermal, and endodermal origin . In the developing embryo, tissue progenitor cells may have less potential than embryonic cells but also have the capacity to give rise to different mature cells types . Such cells are variously unrestricted in their biological potential, and it is these properties that provide and hold so much promise for therapeutic transplantation regimens . However, unless transplantation is accompanied by immunosuppression, engraftment of stem or progenitor cells across strain or species difference fails as a result of expression of major histocompatibility markers by the progeny of the stem cells and graft rejection .
The development of stem or progenitor cell lines with capacities for differentiation and unrestricted transplantation might resolve these immunological problems . Recent transplantation studies with stromal stem cells and progenitor cells of bone marrow and neural origin and attenuated major histocompatibility complex (MHC) expression in these and similar cells suggest this may be possible. In this report, evidence is presented that shows that embryonic mouse STO cell lines behave like liver progenitor cells in nonimmunosuppressed rats. These findings are provocative for their biological implications to liver stem cell biology and gene therapy and also because mouse STO cells have been, and continue to be, widely used as feeder layers to support culture and development of embryoid bodies , embryonic stem cells , and liver progenitor cells .
MATERIALS AND METHODS
Mouse STO Cell Lines Can Be Xenotransplanted into Nonimmunosuppressed DPPIV– Rats
To investigate differentiation potential of a putative rat liver progenitor cell line, 3(8)21-EGFP cells were injected into spontaneously mutated DPPIV– rats . The DPPIV gene is naturally mutated in DPPIV– rats with lack of cell surface expression and enzyme activity. This readily permits identification of transplanted cells with DPPIV enzyme activity, e.g., hepatocytes, which abundantly express DPPIV in bile canalicular domains. Similarly, stem or progenitor cells lacking DPPIV activity at the outset can be identified in the liver of DPPIV– recipients after cell differentiation along appropriate lineages . As previously described, 3(8)21-EGFP cells were clonally derived specifically from cocultures of putative 3(8)21 rat liver progenitor cells and STO cells that had been retrovirally transduced with EGFP and neomycin resistance (neoR) genes . Thus, because 3(8)21-EGFP cells were thought to be derived from the rat liver progenitor cells, they were assumed to be syngeneic with DPPIV– rats and to express properties of adult hepatocytes or biliary cells .
EGFP+ cells were seen inside spleens and livers soon after injection (Fig. 1). Four weeks later, DPPIV+ cells were detected in liver plates, mainly in portal spaces or periportal and midzonal sinusoids and occasionally in perivenous areas (Fig. 2A). Mitotic figures and host inflammatory responses were undetectable. DPPIV+ cells coexpressed glycogen and G-6-Pase and hepatocyte-like conjoint patterns of linear DPPIV and ATPase bile canalicular staining, which varied from 10%–80% of transplanted cells at 1–2 and 4–12 weeks. The numbers of DPPIV+ cells per liver comprised approximately 2.0%, 1.7%, and 2.1% of injected cells at 1, 2, and 4 weeks (p < .05) or approximately 2 to 3 donor cells/104 recipient hepatocytes; a single rat liver contains approximately 8 x 108 hepatocytes . The fraction of DPPIV+ cells in vascular areas declined with time; approximately 30% of them remained in portal or sinusoidal areas at 12 weeks. Virtually all wild-type DPPIV+ hepatocytes and bile canalicular domains were stained red in positive control wild-type liver tissue samples (Fig. 2A, panel i), whereas neither red nor background staining reactions were observed in DPPIV– control liver tissues incubated with substrate (glycyl-L-proline-4-methoxy-2-naphthylamide) plus fast Blue B salt (for color development) in PBS, pH 7.4 (Fig. 2A, panel j). Under similar transplantation conditions, grafts of syngeneic rat hepatocytes survive through 3 months, whereas hepatocytes from outbred rats, hamsters, and human fetuses and cell lines die within 72 hours .
Figure 1. Detection of donor 3(8)21-EGFP cells in the vascular spaces of recipient dipeptidylpeptidase IV–negative rat spleen and liver 1 hour after intrasplenic injection: immunohistochemical staining of EGFP. (A): Spleen (x40). Arrows show collections of stained EGFP+ (dark brown) donor cells in red pulp. No staining was observed in control tissue sections incubated without primary antibody. (B): Spleen (x200). (C, D): Liver (x200). Stained cells are observed in the portal vein. (E, F): Liver (x400). Stained cells are observed in sinusoids (E) and partially in a portal venule and sinusoid (F). Abbreviation: EGFP, enhanced green fluorescent protein.
Figure 2. Xenoengraftment of 3(8)21-EGFP cells in DPPIV– rats. (A): Histochemistry reveals DPPIV+ donor cells (red) in vascular spaces (a, c, e) or parenchyma (b, d, f) of recipient livers 4 weeks after intrasplenic injections. DPPIV+ cells in portal areas: a, x100; b, inset in a (x400). Costaining of DPPIV/glycogen (magenta cytoplasm) (c, d) and DPPIV/G-6-P (brown cytoplasm) (e, f). Donor cells in Pa or sinusoids show less glycogen or G-6-P (c, e) compared with parenchymal donor cells (d, f); the latter show linear DPPIV staining (arrows: b, d, f, h) and conjoint bile canaliculi (g, h) with DPPIV (donor cells, arrow) and ATPase (dark brown, native hepatocytes, arrowheads) activities. Toluidine blue (a–d) or methyl green counterstains (g, h). Positive and negative control liver tissues (x200) are from histochemical staining of wild-type DPPIV+ F344 liver (i) and recipient DPPIV– liver (j). Both tissues were subjected to DPPIV staining with incorporation of the substrate; all wild-type hepatocytes and bile canaliculi are stained (i), whereas there is no background staining in DPPIV– liver (j). (B): Recipient rat livers contain mouse COX1 products. Polymerase chain reaction assays were performed as described in Materials and Methods (lanes 2 through 20). Experimental and control (mock-transplanted and harvested at 3 months) results from separate rats are shown in lanes 5 through 16 and 17 through 19, respectively; results from cultured donor cells and from normal mouse and rat livers are shown in lanes 3 and 4, 2, and 20, respectively. DNA quality and loading were monitored by ?-actin determinations (bottom row: lanes 2–4, 240-bp mouse product; lanes 5–20, 270-bp rat product). Abbreviations: Cv, central vein; DPPIV, dipeptidylpeptidase IV; EGFP, enhanced green fluorescent protein; G-6-P, glucose-6-phosphate; Pa, portal area.
While these investigations were ongoing, one of us (K.S.K.) observed, from studies of Giemsa-stained chromosome spreads, that cloned 3(8)21-EGFP cells and the parental line of transduced 3(8)21 cells originated not from rat liver progenitor cells but, rather, from the aneuploid -irradiated mouse STO cells that had been used as feeder layers to isolate the so-called rat liver progenitor cell lines (details are given in ). These observations suggested that xenogeneic engraftment of a genetically engineered embryonic ‘fibro-blast’ cell line, derived from E15-E17 mouse embryos , had occurred in nonimmunosuppressed rats. This was confirmed by findings of 354-bp mouse COX1 products in livers of 11 of 12 rats between 1 and 12 weeks (Fig. 2B). In contrast, mouse COX1 was undetectable in untreated and mock-transplanted rats or in rat livers 4 weeks after intrasplenic injection of 107 cell equivalents (~15 μg) of 3(8)21-EGFP DNA (not shown). Assay fidelity and DNA quality were validated by exclusive detection of 258-bp rat COX1 in samples of rat origin and ubiquitous detection of ?-actin.
To augment sensitivity of detection of mouse COX1 in 3(8)21-EGFP engrafted livers, COX1 PCR products were analyzed by Southern blots before and after treatment with PleI, which recognizes specifically 354-bp mouse COX1 products with 80%–90% cutting efficiency . As shown in validation studies (Fig. 3A), PleI cut specifically the mouse COX1 primer-generated mouse liver COX1 PCR product into 189-bp and 165-bp fragments; rat COX1 products generated by rat-specific primers were uncut (top row). COX1-sized PCR products were undetectable using rat or mouse PCR primers in control samples containing mouse or rat liver DNA substrates, respectively (top row), yet DNA quality was intact (bottom row). No hybridization signals were seen on Southern blots of untreated or PleI-treated rat-specific 258-bp COX1 products (middle row). In contrast, specific signals were seen on blots of untreated 354-bp mouse liver products; after PleI treatment, although two digestion products were insufficiently resolved, authentic mouse products were cut into fragments of predicted size (middle row). The positive hybridization signal at the higher position (middle row) likely reflected uncut mouse COX1 products (top row).
Figure 3. Specific detection of mouse COX1 DNA in xenoen-grafted livers from DPPIV–rats. (A): Validation of PleI-specific digestion and Southern blot detection of mouse COX1 PCR products. PCR and restriction digest reactions were performed with normal mouse or rat liver DNA samples using ?-actin, rat, or mouse COX1 primers as described in Materials and Methods. Reaction products are shown in EtBr-stained agarose gels (top and bottom rows) or after hybridization on Southern blots using a labeled mouse COX1-specific 17-mer (middle row). Control and experimental autoradiograms were exposed for 1 and 2.5 days, respectively. (B): Identification of mouse COX1 DNA in recipient rat livers by PleI-coupled Southern blots. Control (mock-transplanted) and experimental livers were obtained from DPPIV– rats 4 weeks after intrasplenic injections of mouse 3(8)21-EGFP or STO cells. PCR products were analyzed on EtBr-stained agarose gels (row one). Undigested (PleI–, row two) or PleI-digested PCR products (PleI+, row three) were hybridized on Southern blots to a labeled mouse COX1-specific 17-mer. Control and experimental autoradiograms were exposed for 1 and 2.5 days, respectively. Internal controls for rat COX1 and ?-actin PCR products on EtBr-stained agarose gels (rows four and five, respectively). Abbreviations: DPPIV, dipeptidylpeptidase IV; EGFP, enhanced green fluorescent protein; EtBr, ethidium bromide; PCR, polymerase chain reaction.
Although PleI-coupled Southern analyses were applied to xenografts of 3(8)21-EGFP cells harvested at 4 weeks (Fig. 3B) and mouse COX1 products were detected by EtBr staining in only one of three rats (top row), PleI-sensitive mouse COX1 hybridization products were detected in all three (rows two and three). In contrast, hybridization signals were undetectable in untreated and mock-transplanted rats or in rat livers 4 weeks after intrasplenic injection of 3(8)21-EGFP DNA (not shown). PleI-sensitive 220-bp hybridization signals arose from differences between normal tissues and donor cell lines, because these signals were generated by donor cells (not shown) but not mouse livers (Figs. 3A, 3B).
To exclude immune privilege that might have been conferred artifactually by -irradiation (used initially to prepare STO feeder layers as described elsewhere ) or by transduced DNA or EGFP and neoR expression associated with transduced 3(8)21-EGFP cells (as mentioned above), untreated parental STO cells were similarly transplanted. DPPIV+ STO cells were detected at 1, 2, and 4 weeks (Fig. 4A) at frequencies of approximately 2% of injected cells. STO cell differentiation into hepatocytes was revealed by colocalized DPPIV and glycogen staining and by integration of donor cells into liver plates as shown by linear and colocalized DPPIV and ATPase canalicular staining. Mouse COX1 products were detected on EtBr-stained gels in two of three rats at 2 weeks (Fig. 4B, top row) and on PleI-coupled Southern blots of undigested samples from 11 of 12 rats between 1 and 12 weeks (Fig. 4B, row two). Mouse COX1 165-bp PleI-digestion products were detected in roughly half of the PleI-treated samples (row three). Incomplete digestion (Fig. 3A) and dilute and low abundance of specific targets are likely to have accounted for failure to detect specific restriction fragments in the remaining samples, because more efficient detection of specific 165-bp fragments in PleI-treated samples was observed on similar PleI-coupled Southern blots in an independent experiment (Fig.3B, lanes six through eight). Mouse COX1 products were undetectable in untreated and mock-transplanted rats or in rat livers 1 month after intrasplenic injection of 107 cell equivalents (~15 μg) of STO cell DNA (not shown).
Figure 4. Xenoengraftment of STO cells in DPPIV– rats. (A): Histochemistry reveals intrahepatic donor cells (x400) that express hepatocytic markers 1 (a, b), 2 (c, d), and 4 weeks (e, f) after intrasplenic injections. DPPIV+ cells (arrows in a–f) are costained for glycogen (arrows in b, d, f) or counterstained with toluidine blue (a, c, e). DPPIV+ staining is seen in perivascular cells (c, e; inset in f); linear DPPIV+ staining (b, d, f) is seen in cells costained for ATPase (arrowheads, insets in d and f). (B): Recipient rat livers contain mouse COX1 products. Polymerase chain reaction assays (rows 1, 4, 5) and Southern blots (rows 2, 3) were performed as described in Materials and Methods (lanes 2–17). Autoradiograms were exposed for 1 day (control samples) or 2.5 days (experimental samples). Abbreviation: DPPIV, dipeptidylpeptidase IV; Pa, portal area.
STO Cells and 3(8)#21-EGFP Cells Are Derived from Swiss Mice
To be certain of the Swiss mouse origin of STO cells and 3(8)#21-EGFP cells that were used for xenotransplantation, MHC H-2K allele typing was performed on both of the donor cell lines by PCR analysis using standard D17Mit28 primers, followed by determination of the microsatellite DNA sequences expected in the PCR products. The results are summarized in Table 1. The size of the C57BL/6J PCR product was approximately 120 bp; the product contained a (CA)24 dinucleotide repeat interrupted by 5 nucleotides between its sixth and seventh repeat. In contrast, the PCR products from Swiss mouse, STO, and 3(8)#21-EGFP cells were smaller but similar in size (~100 bp); all contained shorter, continuous, and essentially identical elements of (CA)17 or (CA)17–18 dinucleotide repeats.
Table 1. Determination of H-2K allele types by PCR and DNA sequencing
Mouse STO Cell Lines Can Be Xenotransplanted into Nonimmunosuppressed DPPIV+ Rats
DPPIV– rats display normal immunity , yet DPPIV is also a lymphocyte surface protein designated CD26 (GenBank #AAH22183), and mutated CD26 might alter T-cell function . Thus, 3(8)21-EGFP or STO xenografts might be tolerated by DPPIV– rats but rejected in wild-type recipients. This possibility was tested by injecting either 3(8)21-EGFP or normal STO donor cells into wild-type DPPIV+ rats. Whereas EtBr-stained mouse COX1 products were undetectable in 3(8)21-EGFP and STO xenografts, Southern blot analyses revealed mouse COX1 products in three of four 3(8)21-EGFP xenografts at 8–9 weeks (two of which yielded PleI-sensitive 165-bp fragments) and in 8 of 12 STO xenografts between 1 and 12 weeks (six of which yielded PleI-sensitive 165-bp fragments). Mouse COX1 products were undetectable in untreated and mock-transplanted rats or in rat livers 1 month after intrasplenic injection of donor cell DNA (not shown).
Hepatocyte-Specific Molecules Are Expressed in Cultured STO Cell Lines
In log-phase cultures at low cell densities, neither hepatocyte nor bile duct markers were observed cytochemically. In contrast, in confluent cultures at high densities (Fig. 5A), clusters of DPPIV+ cells were observed frequently in 3(8)21-EGFP and occasionally in STO cultures; GGT staining was undetectable. 3(8)21-EGFP cells formed interspersed clusters with intenseG-6-Pase activity; STO cells were largely negative, but many showed faint precipitates. 3(8)21-EGFP cells showed intense glycogen staining with dark multilayered areas; STO cells stained relatively weakly.
Figure 5. Mouse STO cell lines express hepatocyte-specific properties in culture and in xenografts.(A):Cytochemicalstains (x400) in cultures of 3(8)21-EGFP (a, c, e, g) or STO cells (b, d, f, h). (a, b): DPPIV+ cell clusters, dark and faint orange brown. (c, d): GGT– cells. (e, f): G-6-Pase+, intense and faint brown. (g, h): Glycogen+, intense and weak magenta. Toluidine blue counter-stains (a–h). (B): Mouse albumin and DPPIV mRNA expression in cultured donor cells and 3(8)21-EGFP xenografts. Reverse transcription–polymerase chain reaction assays (rows 1, 2, and 3) and Southern blots (row 4) were performed as described in Materials and Methods. Autoradiograms were exposed for 1 day (control samples) or 7 days (experimental samples). Cultured STO (lane 3) and 3(8)21-EGFP cells (lane 4). Abbreviations: DPPIV, dipeptidylpeptidase IV; EGFP, enhanced green fluorescent protein; G-6-P, glucose-6-phosphate; GGT, -glutamyl-transpeptidase; ND, not determined.
DPPIV expression was confirmed independently in confluent cultures by nested RT-PCR (Fig. 5B) with primers specific for mouse DPPIV (exons 10–14, first-stage 351-bp product ; exons 10–12, nested 193-bp product ). Both exon products were detected in each culture system; differences in band intensities of first-stage products (3(8)21-EGFP > STO) were consistent with cytochemical findings. Species and mRNA specificities of RT-PCR products were validated by DNA sequencing, which revealed identical mouse DPPIV sequences (exons 10–12) in mouse liver tissue and both culture systems (not shown).
Albumin synthesis was undetectable in donor cell cultures after immunoprecipitation of -methionine-labeled proteins or Western blot analyses (not shown). However, albumin mRNA was detected with equivalent intensity in both culture systems by nested RT-PCR (Fig. 5B) using primers specific for mouse albumin (exons 3 and 4). Species and mRNA specificities of nested 185-bp RT-PCR products were validated by DNA sequencing, which revealed identical mouse albumin sequences (exons 3 and 4) in control mouse liver tissue and both culture systems (not shown). Albumin mRNA expression was donor cell–specific, being undetectable in cultured Swiss mouse NIH3T3 cells (not shown).
Xenografts of STO Cell Lines Express Albumin and DPPIV mRNAs in DPPIV– Rats
Mouse albumin mRNA was detected with increasing frequency in 3(8)21-EGFP xenografts between 1 and 12 weeks (Fig. 5B). Similar results were obtained in a second set of rats injected with 3(8)21-EGFP or STO cells (not shown). In contrast, mouse albumin mRNA was undetectable in untreated and mock-transplanted rats or in recipient livers 1 month after intrasplenic injection of 3(8)21-EGFP DNA (as above). The nested RT-PCR product (Fig. 5B, top row, lane 15) from a 3(8)21-EGFP xenograft at 12 weeks was sequenced, and the albumin sequence obtained from the xenograft was identical to those of control mouse liver and STO and 3(8)21-EGFP cells (not shown).
Mouse DPPIV mRNA exons 10 through 12 were undetectable in 3(8)21-EGFP xenografts using unlabeled (Fig. 5B) or end-labeled (not shown) primers. This was possibly the result of low abundance and dilution, because mouse DPPIV mRNAs were seen on Southern blots with a 193-bp probe that specifically hybridized to nested approximately 200-bp products from mouse liver and donor cells (Fig. 5B, bottom row, lane 2 and lanes 3 and 4, respectively) and one of three xenografts at 12 weeks (Fig. 5B, bottom row, lane 15). The slightly higher position of the DPPIV band in the xenograft is due to the presence of two incompletely resolved bands: a more-intense nonspecific higher-Mr band and a less-intense specific approximately 193-bp lower-Mr band (owing to the dilution of target molecules). This conclusion is suggested by the presence of two bands of similar sizes in the sample of normal mouse liver, in which the specific band is more intense than the nonspecific one (Fig. 5B, lane 2). Notably, the nonspecific bands are absent or very faint in both donor cell samples (Fig. 5B, lanes 3 and 4); this difference may reflect differences between tissues and cultured cell samples. No hybridization signals were observed in control rats before (Fig. 5B) or after intrasplenic injection of 3(8)21-EGFP DNA (not shown).
MHC Plasma Membrane Markers Are Neither Expressed Nor Interferon--Inducible in Cultured Swiss Mouse–Derived STO Cell Lines
Consistent with their mouse origin, neither 3(8)21-EGFP nor STO cells expressed rat MHC class I RT-1Alv1 or RT1A (OX-18) determinants by flow cytometry, whereas low levels of expression were observed on 7777 cells, a well-characterized rat cell line (Fig. 6).
Figure 6. Cultured donor cells do not express rat major histo-compatibility complex class I markers. Cells were plated (2 x 105/10-cm dish per 10 ml medium), and cell suspensions were analyzed by flow cytometry as described in Materials and Methods. Three treatment groups consisted of incubations with PBS (A, D, G, J) or fluorescein isothiocyanate–conjugated RT1Alv1 (B, E, H, K) or OX-18 (C, F, I, L) MAbs. (A–C): Swiss NIH3T3 mouse cells (negative control). (D–F): Rat 7777 cells (positive control). (G–I): Mouse 3(8)21 cells. (J–L): Mouse STO cells. Fluorescence curves of cells incubated without PBS were identical to the curves obtained with isotype MAb controls. Abbreviations: MAb, monoclonal antibody; PBS, phosphate-buffered saline.
However, because donor cell lines reportedly originated from inbred Swiss mice, they should constitutively express mouse MHC molecules of q-haplotype; such expression should be induced or augmented by interferon (IFN)- . Unexpectedly, after flow cytometry, neitherclassIH-2Kq, H-2Dq, and H-2Lq nor class II I-Aq determinants were observed before (Fig. 7) or after (Fig. 8) IFN- treatment in vitro. The isotype control and experimental fluorescence curves produced by 3(8)21-EGFP cells were identical (consistent with no detectable MHC-specific fluorescence); but, compared with STO cells (Figs. 7C, 7H, 7M, 8B, 8E, 8H), the curves were right-shifted and split (Figs. 7D, 7I, 7N, 8J). These artifacts were caused by endogenous green epifluorescence interference with FITC and PE fluorophores and nonspecific green fluorescence in a cohort of dead epifluorescent cells, as seen by analysis of parental 3(8)21 cells, which showed unimodal fluorescence profiles and no MHC determinants (Fig. 7).
Figure 7. Cultured donor cells do not express mouse major histocompatibility complex class I and class II markers. Cells were plated (2 x 105/10-cm dish per 10 ml medium), and cell suspensions were analyzed by flow cytometry as described in Materials and Methods. H-2Kq: top group: shaded, isotype control; unshaded, anti-H-2Kq; H-2Dq/H-2Lq: middle group: unshaded upper panels, isotype control; shaded lower panels, anti-H-2Dq/H-2Lq; I-A: bottom group: unshaded upper panels, isotype control; shaded lower panels, anti-I-A. (A, F, K): q-Haplotype controls: Swiss mouse NIH3T3 (top); DBA/1 splenocytes (middle and bottom). (B, G, L): b-Haplotype control: mouse Hepa 1–6. (C, H, M): STO. (D, I, N): 3(8)21-EGFP. (E, J, O): 3(8)21. Fluorescence curves of cells incubated with or without phosphate-buffered saline were identical to the curves obtained with isotype and secondary monoclonal antibody controls. Abbreviation: EGFP, enhanced green fluorescent protein.
Figure 8. IFN- does not induce mouse major histocompatibility complex class I or class II expression in cultured STO cells and STO cell derivatives. Cells were plated (2 x 105/10-cm dish per 10 ml medium) as described in Materials and Methods. IFN- was purchased from MP Biomedicals. Vehicle (PBS supplemented with 0.1% bovine serum albumin; unshaded upper panels of each group) or IFN- (33 ng/ml; shaded bottom panels of each group) were added 24 hours later. On day 5, attached cells were recovered, and flow cytometry assays were performed as described in Materials and Methods. H-2Kq: top group, anti-H-2Kq MAb; H-2Dq/H-2Lq: middle group, anti-H-2Dq/H-2Lq MAb; I-A: bottom group, anti-I-A MAb. (A, D, G): Positive control: Swiss mouse NIH3T3. (B, E, H): STO. (C, F, I): 3(8)21. (J): 3(8)21-EGFP. Fluorescence curves of cells incubated without PBS were identical to the curves obtained with isotype and secondary MAb controls. Abbreviations: EGFP, enhanced green fluorescent protein; IFN, interferon; MAb, monoclonal antibody; PBS, phosphate-buffered saline.
DISCUSSION
This work was supported by the American Cancer Society, NIH (DK46592, DK41296, DK57619, and ES10337), and University of California, San Diego (UCSD) Academic Senate (RE466-H). We thank P. Castiglioni, D. Young and M. Zanetti (UCSD), and H. Crissman (LANL) for help with flow cytometry and helpful discussion; B. Ju and S. Maeda (UCSD) for NIH3T3 cells and mouse liver; and Bruce Herron (Wadsworth Genomics Institute) and Stephanie Ostrowski for their assistance in the mouse MHC sequencing.
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b Center for Molecular Genetics, University of California, San Diego, California;
c Ordway Research Institute and
d New York State Health Department, Albany, New York;
e Marion Bessin Liver Research Center, Albert Einstein College of Medicine, Bronx, New York, USA
Key Words. Mouse STO progenitor cells ? Xenotransplantation ? Nonimmunosuppressed rats
Correspondence: K.S. Koch, and H.L. Leffert, Department of Pharmacology and Center for Molecular Genetics, University of California, San Diego, La Jolla, California, 92093-0636 USA. Telephone: 858-534-2354; Fax: 858-822-4184; e-mail: kkoch@ucsd.edu and hleffert@ucsd.edu
ABSTRACT
Embryonic stem cells undergo self-renewal coupled with differentiation into mature parenchymal cells of mesodermal, ectodermal, and endodermal origin . In the developing embryo, tissue progenitor cells may have less potential than embryonic cells but also have the capacity to give rise to different mature cells types . Such cells are variously unrestricted in their biological potential, and it is these properties that provide and hold so much promise for therapeutic transplantation regimens . However, unless transplantation is accompanied by immunosuppression, engraftment of stem or progenitor cells across strain or species difference fails as a result of expression of major histocompatibility markers by the progeny of the stem cells and graft rejection .
The development of stem or progenitor cell lines with capacities for differentiation and unrestricted transplantation might resolve these immunological problems . Recent transplantation studies with stromal stem cells and progenitor cells of bone marrow and neural origin and attenuated major histocompatibility complex (MHC) expression in these and similar cells suggest this may be possible. In this report, evidence is presented that shows that embryonic mouse STO cell lines behave like liver progenitor cells in nonimmunosuppressed rats. These findings are provocative for their biological implications to liver stem cell biology and gene therapy and also because mouse STO cells have been, and continue to be, widely used as feeder layers to support culture and development of embryoid bodies , embryonic stem cells , and liver progenitor cells .
MATERIALS AND METHODS
Mouse STO Cell Lines Can Be Xenotransplanted into Nonimmunosuppressed DPPIV– Rats
To investigate differentiation potential of a putative rat liver progenitor cell line, 3(8)21-EGFP cells were injected into spontaneously mutated DPPIV– rats . The DPPIV gene is naturally mutated in DPPIV– rats with lack of cell surface expression and enzyme activity. This readily permits identification of transplanted cells with DPPIV enzyme activity, e.g., hepatocytes, which abundantly express DPPIV in bile canalicular domains. Similarly, stem or progenitor cells lacking DPPIV activity at the outset can be identified in the liver of DPPIV– recipients after cell differentiation along appropriate lineages . As previously described, 3(8)21-EGFP cells were clonally derived specifically from cocultures of putative 3(8)21 rat liver progenitor cells and STO cells that had been retrovirally transduced with EGFP and neomycin resistance (neoR) genes . Thus, because 3(8)21-EGFP cells were thought to be derived from the rat liver progenitor cells, they were assumed to be syngeneic with DPPIV– rats and to express properties of adult hepatocytes or biliary cells .
EGFP+ cells were seen inside spleens and livers soon after injection (Fig. 1). Four weeks later, DPPIV+ cells were detected in liver plates, mainly in portal spaces or periportal and midzonal sinusoids and occasionally in perivenous areas (Fig. 2A). Mitotic figures and host inflammatory responses were undetectable. DPPIV+ cells coexpressed glycogen and G-6-Pase and hepatocyte-like conjoint patterns of linear DPPIV and ATPase bile canalicular staining, which varied from 10%–80% of transplanted cells at 1–2 and 4–12 weeks. The numbers of DPPIV+ cells per liver comprised approximately 2.0%, 1.7%, and 2.1% of injected cells at 1, 2, and 4 weeks (p < .05) or approximately 2 to 3 donor cells/104 recipient hepatocytes; a single rat liver contains approximately 8 x 108 hepatocytes . The fraction of DPPIV+ cells in vascular areas declined with time; approximately 30% of them remained in portal or sinusoidal areas at 12 weeks. Virtually all wild-type DPPIV+ hepatocytes and bile canalicular domains were stained red in positive control wild-type liver tissue samples (Fig. 2A, panel i), whereas neither red nor background staining reactions were observed in DPPIV– control liver tissues incubated with substrate (glycyl-L-proline-4-methoxy-2-naphthylamide) plus fast Blue B salt (for color development) in PBS, pH 7.4 (Fig. 2A, panel j). Under similar transplantation conditions, grafts of syngeneic rat hepatocytes survive through 3 months, whereas hepatocytes from outbred rats, hamsters, and human fetuses and cell lines die within 72 hours .
Figure 1. Detection of donor 3(8)21-EGFP cells in the vascular spaces of recipient dipeptidylpeptidase IV–negative rat spleen and liver 1 hour after intrasplenic injection: immunohistochemical staining of EGFP. (A): Spleen (x40). Arrows show collections of stained EGFP+ (dark brown) donor cells in red pulp. No staining was observed in control tissue sections incubated without primary antibody. (B): Spleen (x200). (C, D): Liver (x200). Stained cells are observed in the portal vein. (E, F): Liver (x400). Stained cells are observed in sinusoids (E) and partially in a portal venule and sinusoid (F). Abbreviation: EGFP, enhanced green fluorescent protein.
Figure 2. Xenoengraftment of 3(8)21-EGFP cells in DPPIV– rats. (A): Histochemistry reveals DPPIV+ donor cells (red) in vascular spaces (a, c, e) or parenchyma (b, d, f) of recipient livers 4 weeks after intrasplenic injections. DPPIV+ cells in portal areas: a, x100; b, inset in a (x400). Costaining of DPPIV/glycogen (magenta cytoplasm) (c, d) and DPPIV/G-6-P (brown cytoplasm) (e, f). Donor cells in Pa or sinusoids show less glycogen or G-6-P (c, e) compared with parenchymal donor cells (d, f); the latter show linear DPPIV staining (arrows: b, d, f, h) and conjoint bile canaliculi (g, h) with DPPIV (donor cells, arrow) and ATPase (dark brown, native hepatocytes, arrowheads) activities. Toluidine blue (a–d) or methyl green counterstains (g, h). Positive and negative control liver tissues (x200) are from histochemical staining of wild-type DPPIV+ F344 liver (i) and recipient DPPIV– liver (j). Both tissues were subjected to DPPIV staining with incorporation of the substrate; all wild-type hepatocytes and bile canaliculi are stained (i), whereas there is no background staining in DPPIV– liver (j). (B): Recipient rat livers contain mouse COX1 products. Polymerase chain reaction assays were performed as described in Materials and Methods (lanes 2 through 20). Experimental and control (mock-transplanted and harvested at 3 months) results from separate rats are shown in lanes 5 through 16 and 17 through 19, respectively; results from cultured donor cells and from normal mouse and rat livers are shown in lanes 3 and 4, 2, and 20, respectively. DNA quality and loading were monitored by ?-actin determinations (bottom row: lanes 2–4, 240-bp mouse product; lanes 5–20, 270-bp rat product). Abbreviations: Cv, central vein; DPPIV, dipeptidylpeptidase IV; EGFP, enhanced green fluorescent protein; G-6-P, glucose-6-phosphate; Pa, portal area.
While these investigations were ongoing, one of us (K.S.K.) observed, from studies of Giemsa-stained chromosome spreads, that cloned 3(8)21-EGFP cells and the parental line of transduced 3(8)21 cells originated not from rat liver progenitor cells but, rather, from the aneuploid -irradiated mouse STO cells that had been used as feeder layers to isolate the so-called rat liver progenitor cell lines (details are given in ). These observations suggested that xenogeneic engraftment of a genetically engineered embryonic ‘fibro-blast’ cell line, derived from E15-E17 mouse embryos , had occurred in nonimmunosuppressed rats. This was confirmed by findings of 354-bp mouse COX1 products in livers of 11 of 12 rats between 1 and 12 weeks (Fig. 2B). In contrast, mouse COX1 was undetectable in untreated and mock-transplanted rats or in rat livers 4 weeks after intrasplenic injection of 107 cell equivalents (~15 μg) of 3(8)21-EGFP DNA (not shown). Assay fidelity and DNA quality were validated by exclusive detection of 258-bp rat COX1 in samples of rat origin and ubiquitous detection of ?-actin.
To augment sensitivity of detection of mouse COX1 in 3(8)21-EGFP engrafted livers, COX1 PCR products were analyzed by Southern blots before and after treatment with PleI, which recognizes specifically 354-bp mouse COX1 products with 80%–90% cutting efficiency . As shown in validation studies (Fig. 3A), PleI cut specifically the mouse COX1 primer-generated mouse liver COX1 PCR product into 189-bp and 165-bp fragments; rat COX1 products generated by rat-specific primers were uncut (top row). COX1-sized PCR products were undetectable using rat or mouse PCR primers in control samples containing mouse or rat liver DNA substrates, respectively (top row), yet DNA quality was intact (bottom row). No hybridization signals were seen on Southern blots of untreated or PleI-treated rat-specific 258-bp COX1 products (middle row). In contrast, specific signals were seen on blots of untreated 354-bp mouse liver products; after PleI treatment, although two digestion products were insufficiently resolved, authentic mouse products were cut into fragments of predicted size (middle row). The positive hybridization signal at the higher position (middle row) likely reflected uncut mouse COX1 products (top row).
Figure 3. Specific detection of mouse COX1 DNA in xenoen-grafted livers from DPPIV–rats. (A): Validation of PleI-specific digestion and Southern blot detection of mouse COX1 PCR products. PCR and restriction digest reactions were performed with normal mouse or rat liver DNA samples using ?-actin, rat, or mouse COX1 primers as described in Materials and Methods. Reaction products are shown in EtBr-stained agarose gels (top and bottom rows) or after hybridization on Southern blots using a labeled mouse COX1-specific 17-mer (middle row). Control and experimental autoradiograms were exposed for 1 and 2.5 days, respectively. (B): Identification of mouse COX1 DNA in recipient rat livers by PleI-coupled Southern blots. Control (mock-transplanted) and experimental livers were obtained from DPPIV– rats 4 weeks after intrasplenic injections of mouse 3(8)21-EGFP or STO cells. PCR products were analyzed on EtBr-stained agarose gels (row one). Undigested (PleI–, row two) or PleI-digested PCR products (PleI+, row three) were hybridized on Southern blots to a labeled mouse COX1-specific 17-mer. Control and experimental autoradiograms were exposed for 1 and 2.5 days, respectively. Internal controls for rat COX1 and ?-actin PCR products on EtBr-stained agarose gels (rows four and five, respectively). Abbreviations: DPPIV, dipeptidylpeptidase IV; EGFP, enhanced green fluorescent protein; EtBr, ethidium bromide; PCR, polymerase chain reaction.
Although PleI-coupled Southern analyses were applied to xenografts of 3(8)21-EGFP cells harvested at 4 weeks (Fig. 3B) and mouse COX1 products were detected by EtBr staining in only one of three rats (top row), PleI-sensitive mouse COX1 hybridization products were detected in all three (rows two and three). In contrast, hybridization signals were undetectable in untreated and mock-transplanted rats or in rat livers 4 weeks after intrasplenic injection of 3(8)21-EGFP DNA (not shown). PleI-sensitive 220-bp hybridization signals arose from differences between normal tissues and donor cell lines, because these signals were generated by donor cells (not shown) but not mouse livers (Figs. 3A, 3B).
To exclude immune privilege that might have been conferred artifactually by -irradiation (used initially to prepare STO feeder layers as described elsewhere ) or by transduced DNA or EGFP and neoR expression associated with transduced 3(8)21-EGFP cells (as mentioned above), untreated parental STO cells were similarly transplanted. DPPIV+ STO cells were detected at 1, 2, and 4 weeks (Fig. 4A) at frequencies of approximately 2% of injected cells. STO cell differentiation into hepatocytes was revealed by colocalized DPPIV and glycogen staining and by integration of donor cells into liver plates as shown by linear and colocalized DPPIV and ATPase canalicular staining. Mouse COX1 products were detected on EtBr-stained gels in two of three rats at 2 weeks (Fig. 4B, top row) and on PleI-coupled Southern blots of undigested samples from 11 of 12 rats between 1 and 12 weeks (Fig. 4B, row two). Mouse COX1 165-bp PleI-digestion products were detected in roughly half of the PleI-treated samples (row three). Incomplete digestion (Fig. 3A) and dilute and low abundance of specific targets are likely to have accounted for failure to detect specific restriction fragments in the remaining samples, because more efficient detection of specific 165-bp fragments in PleI-treated samples was observed on similar PleI-coupled Southern blots in an independent experiment (Fig.3B, lanes six through eight). Mouse COX1 products were undetectable in untreated and mock-transplanted rats or in rat livers 1 month after intrasplenic injection of 107 cell equivalents (~15 μg) of STO cell DNA (not shown).
Figure 4. Xenoengraftment of STO cells in DPPIV– rats. (A): Histochemistry reveals intrahepatic donor cells (x400) that express hepatocytic markers 1 (a, b), 2 (c, d), and 4 weeks (e, f) after intrasplenic injections. DPPIV+ cells (arrows in a–f) are costained for glycogen (arrows in b, d, f) or counterstained with toluidine blue (a, c, e). DPPIV+ staining is seen in perivascular cells (c, e; inset in f); linear DPPIV+ staining (b, d, f) is seen in cells costained for ATPase (arrowheads, insets in d and f). (B): Recipient rat livers contain mouse COX1 products. Polymerase chain reaction assays (rows 1, 4, 5) and Southern blots (rows 2, 3) were performed as described in Materials and Methods (lanes 2–17). Autoradiograms were exposed for 1 day (control samples) or 2.5 days (experimental samples). Abbreviation: DPPIV, dipeptidylpeptidase IV; Pa, portal area.
STO Cells and 3(8)#21-EGFP Cells Are Derived from Swiss Mice
To be certain of the Swiss mouse origin of STO cells and 3(8)#21-EGFP cells that were used for xenotransplantation, MHC H-2K allele typing was performed on both of the donor cell lines by PCR analysis using standard D17Mit28 primers, followed by determination of the microsatellite DNA sequences expected in the PCR products. The results are summarized in Table 1. The size of the C57BL/6J PCR product was approximately 120 bp; the product contained a (CA)24 dinucleotide repeat interrupted by 5 nucleotides between its sixth and seventh repeat. In contrast, the PCR products from Swiss mouse, STO, and 3(8)#21-EGFP cells were smaller but similar in size (~100 bp); all contained shorter, continuous, and essentially identical elements of (CA)17 or (CA)17–18 dinucleotide repeats.
Table 1. Determination of H-2K allele types by PCR and DNA sequencing
Mouse STO Cell Lines Can Be Xenotransplanted into Nonimmunosuppressed DPPIV+ Rats
DPPIV– rats display normal immunity , yet DPPIV is also a lymphocyte surface protein designated CD26 (GenBank #AAH22183), and mutated CD26 might alter T-cell function . Thus, 3(8)21-EGFP or STO xenografts might be tolerated by DPPIV– rats but rejected in wild-type recipients. This possibility was tested by injecting either 3(8)21-EGFP or normal STO donor cells into wild-type DPPIV+ rats. Whereas EtBr-stained mouse COX1 products were undetectable in 3(8)21-EGFP and STO xenografts, Southern blot analyses revealed mouse COX1 products in three of four 3(8)21-EGFP xenografts at 8–9 weeks (two of which yielded PleI-sensitive 165-bp fragments) and in 8 of 12 STO xenografts between 1 and 12 weeks (six of which yielded PleI-sensitive 165-bp fragments). Mouse COX1 products were undetectable in untreated and mock-transplanted rats or in rat livers 1 month after intrasplenic injection of donor cell DNA (not shown).
Hepatocyte-Specific Molecules Are Expressed in Cultured STO Cell Lines
In log-phase cultures at low cell densities, neither hepatocyte nor bile duct markers were observed cytochemically. In contrast, in confluent cultures at high densities (Fig. 5A), clusters of DPPIV+ cells were observed frequently in 3(8)21-EGFP and occasionally in STO cultures; GGT staining was undetectable. 3(8)21-EGFP cells formed interspersed clusters with intenseG-6-Pase activity; STO cells were largely negative, but many showed faint precipitates. 3(8)21-EGFP cells showed intense glycogen staining with dark multilayered areas; STO cells stained relatively weakly.
Figure 5. Mouse STO cell lines express hepatocyte-specific properties in culture and in xenografts.(A):Cytochemicalstains (x400) in cultures of 3(8)21-EGFP (a, c, e, g) or STO cells (b, d, f, h). (a, b): DPPIV+ cell clusters, dark and faint orange brown. (c, d): GGT– cells. (e, f): G-6-Pase+, intense and faint brown. (g, h): Glycogen+, intense and weak magenta. Toluidine blue counter-stains (a–h). (B): Mouse albumin and DPPIV mRNA expression in cultured donor cells and 3(8)21-EGFP xenografts. Reverse transcription–polymerase chain reaction assays (rows 1, 2, and 3) and Southern blots (row 4) were performed as described in Materials and Methods. Autoradiograms were exposed for 1 day (control samples) or 7 days (experimental samples). Cultured STO (lane 3) and 3(8)21-EGFP cells (lane 4). Abbreviations: DPPIV, dipeptidylpeptidase IV; EGFP, enhanced green fluorescent protein; G-6-P, glucose-6-phosphate; GGT, -glutamyl-transpeptidase; ND, not determined.
DPPIV expression was confirmed independently in confluent cultures by nested RT-PCR (Fig. 5B) with primers specific for mouse DPPIV (exons 10–14, first-stage 351-bp product ; exons 10–12, nested 193-bp product ). Both exon products were detected in each culture system; differences in band intensities of first-stage products (3(8)21-EGFP > STO) were consistent with cytochemical findings. Species and mRNA specificities of RT-PCR products were validated by DNA sequencing, which revealed identical mouse DPPIV sequences (exons 10–12) in mouse liver tissue and both culture systems (not shown).
Albumin synthesis was undetectable in donor cell cultures after immunoprecipitation of -methionine-labeled proteins or Western blot analyses (not shown). However, albumin mRNA was detected with equivalent intensity in both culture systems by nested RT-PCR (Fig. 5B) using primers specific for mouse albumin (exons 3 and 4). Species and mRNA specificities of nested 185-bp RT-PCR products were validated by DNA sequencing, which revealed identical mouse albumin sequences (exons 3 and 4) in control mouse liver tissue and both culture systems (not shown). Albumin mRNA expression was donor cell–specific, being undetectable in cultured Swiss mouse NIH3T3 cells (not shown).
Xenografts of STO Cell Lines Express Albumin and DPPIV mRNAs in DPPIV– Rats
Mouse albumin mRNA was detected with increasing frequency in 3(8)21-EGFP xenografts between 1 and 12 weeks (Fig. 5B). Similar results were obtained in a second set of rats injected with 3(8)21-EGFP or STO cells (not shown). In contrast, mouse albumin mRNA was undetectable in untreated and mock-transplanted rats or in recipient livers 1 month after intrasplenic injection of 3(8)21-EGFP DNA (as above). The nested RT-PCR product (Fig. 5B, top row, lane 15) from a 3(8)21-EGFP xenograft at 12 weeks was sequenced, and the albumin sequence obtained from the xenograft was identical to those of control mouse liver and STO and 3(8)21-EGFP cells (not shown).
Mouse DPPIV mRNA exons 10 through 12 were undetectable in 3(8)21-EGFP xenografts using unlabeled (Fig. 5B) or end-labeled (not shown) primers. This was possibly the result of low abundance and dilution, because mouse DPPIV mRNAs were seen on Southern blots with a 193-bp probe that specifically hybridized to nested approximately 200-bp products from mouse liver and donor cells (Fig. 5B, bottom row, lane 2 and lanes 3 and 4, respectively) and one of three xenografts at 12 weeks (Fig. 5B, bottom row, lane 15). The slightly higher position of the DPPIV band in the xenograft is due to the presence of two incompletely resolved bands: a more-intense nonspecific higher-Mr band and a less-intense specific approximately 193-bp lower-Mr band (owing to the dilution of target molecules). This conclusion is suggested by the presence of two bands of similar sizes in the sample of normal mouse liver, in which the specific band is more intense than the nonspecific one (Fig. 5B, lane 2). Notably, the nonspecific bands are absent or very faint in both donor cell samples (Fig. 5B, lanes 3 and 4); this difference may reflect differences between tissues and cultured cell samples. No hybridization signals were observed in control rats before (Fig. 5B) or after intrasplenic injection of 3(8)21-EGFP DNA (not shown).
MHC Plasma Membrane Markers Are Neither Expressed Nor Interferon--Inducible in Cultured Swiss Mouse–Derived STO Cell Lines
Consistent with their mouse origin, neither 3(8)21-EGFP nor STO cells expressed rat MHC class I RT-1Alv1 or RT1A (OX-18) determinants by flow cytometry, whereas low levels of expression were observed on 7777 cells, a well-characterized rat cell line (Fig. 6).
Figure 6. Cultured donor cells do not express rat major histo-compatibility complex class I markers. Cells were plated (2 x 105/10-cm dish per 10 ml medium), and cell suspensions were analyzed by flow cytometry as described in Materials and Methods. Three treatment groups consisted of incubations with PBS (A, D, G, J) or fluorescein isothiocyanate–conjugated RT1Alv1 (B, E, H, K) or OX-18 (C, F, I, L) MAbs. (A–C): Swiss NIH3T3 mouse cells (negative control). (D–F): Rat 7777 cells (positive control). (G–I): Mouse 3(8)21 cells. (J–L): Mouse STO cells. Fluorescence curves of cells incubated without PBS were identical to the curves obtained with isotype MAb controls. Abbreviations: MAb, monoclonal antibody; PBS, phosphate-buffered saline.
However, because donor cell lines reportedly originated from inbred Swiss mice, they should constitutively express mouse MHC molecules of q-haplotype; such expression should be induced or augmented by interferon (IFN)- . Unexpectedly, after flow cytometry, neitherclassIH-2Kq, H-2Dq, and H-2Lq nor class II I-Aq determinants were observed before (Fig. 7) or after (Fig. 8) IFN- treatment in vitro. The isotype control and experimental fluorescence curves produced by 3(8)21-EGFP cells were identical (consistent with no detectable MHC-specific fluorescence); but, compared with STO cells (Figs. 7C, 7H, 7M, 8B, 8E, 8H), the curves were right-shifted and split (Figs. 7D, 7I, 7N, 8J). These artifacts were caused by endogenous green epifluorescence interference with FITC and PE fluorophores and nonspecific green fluorescence in a cohort of dead epifluorescent cells, as seen by analysis of parental 3(8)21 cells, which showed unimodal fluorescence profiles and no MHC determinants (Fig. 7).
Figure 7. Cultured donor cells do not express mouse major histocompatibility complex class I and class II markers. Cells were plated (2 x 105/10-cm dish per 10 ml medium), and cell suspensions were analyzed by flow cytometry as described in Materials and Methods. H-2Kq: top group: shaded, isotype control; unshaded, anti-H-2Kq; H-2Dq/H-2Lq: middle group: unshaded upper panels, isotype control; shaded lower panels, anti-H-2Dq/H-2Lq; I-A: bottom group: unshaded upper panels, isotype control; shaded lower panels, anti-I-A. (A, F, K): q-Haplotype controls: Swiss mouse NIH3T3 (top); DBA/1 splenocytes (middle and bottom). (B, G, L): b-Haplotype control: mouse Hepa 1–6. (C, H, M): STO. (D, I, N): 3(8)21-EGFP. (E, J, O): 3(8)21. Fluorescence curves of cells incubated with or without phosphate-buffered saline were identical to the curves obtained with isotype and secondary monoclonal antibody controls. Abbreviation: EGFP, enhanced green fluorescent protein.
Figure 8. IFN- does not induce mouse major histocompatibility complex class I or class II expression in cultured STO cells and STO cell derivatives. Cells were plated (2 x 105/10-cm dish per 10 ml medium) as described in Materials and Methods. IFN- was purchased from MP Biomedicals. Vehicle (PBS supplemented with 0.1% bovine serum albumin; unshaded upper panels of each group) or IFN- (33 ng/ml; shaded bottom panels of each group) were added 24 hours later. On day 5, attached cells were recovered, and flow cytometry assays were performed as described in Materials and Methods. H-2Kq: top group, anti-H-2Kq MAb; H-2Dq/H-2Lq: middle group, anti-H-2Dq/H-2Lq MAb; I-A: bottom group, anti-I-A MAb. (A, D, G): Positive control: Swiss mouse NIH3T3. (B, E, H): STO. (C, F, I): 3(8)21. (J): 3(8)21-EGFP. Fluorescence curves of cells incubated without PBS were identical to the curves obtained with isotype and secondary MAb controls. Abbreviations: EGFP, enhanced green fluorescent protein; IFN, interferon; MAb, monoclonal antibody; PBS, phosphate-buffered saline.
DISCUSSION
This work was supported by the American Cancer Society, NIH (DK46592, DK41296, DK57619, and ES10337), and University of California, San Diego (UCSD) Academic Senate (RE466-H). We thank P. Castiglioni, D. Young and M. Zanetti (UCSD), and H. Crissman (LANL) for help with flow cytometry and helpful discussion; B. Ju and S. Maeda (UCSD) for NIH3T3 cells and mouse liver; and Bruce Herron (Wadsworth Genomics Institute) and Stephanie Ostrowski for their assistance in the mouse MHC sequencing.
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