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First published online October 18, 2007
Stem Cells Vol. 26 No. 1 January 2008, pp. 72 -78
doi:10.1634/stemcells.2007-0744; www.StemCells.com
© 2008 AlphaMed Press

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EMBRYONIC STEM CELLS

Embryonic Stem Cells Cultured in Serum-Free Medium Acquire Bovine Apolipoprotein B-100 from Feeder Cell Layers and Serum Replacement Medium

Michiko Hisamatsu-Sakamotoa,b, Norihisa Sakamotoa,b, Amy S. Rosenberga

aDivision of Therapeutic Proteins, Center for Drug Evaluation and Research, U.S. Food and Drug Administration, Bethesda, Maryland, USA;
bDepartment of Gastroenterology and Hepatology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan

Key Words. Apolipoprotein B-100 • Low-density lipoproteins • Fetal calf serum • Conditioned medium • Murine embryonic fibroblasts

Correspondence: Correspondence: Norihisa Sakamoto, M.D., Ph.D., Division of Therapeutic Proteins, Center for Drug Evaluation and Research, U.S. Food and Drug Administration, Bethesda, Maryland 20892, USA. Telephone: 301-827-1790; Fax: 301-480-3256; e-mail: norihisha.sakamoto{at}fda.hhs.gov

Received on September 5, 2007; accepted for publication on October 14, 2007.

First published online in STEM CELLS EXPRESS  October 18, 2007.

   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Previous studies have demonstrated that cell populations that are cultured with heterologous animal products can acquire xenoantigens, potentially limiting their clinical utility because of immune responses. Embryonic stem cells (ESCs) are an attractive source of multiple potential cellular therapies and are typically derived and routinely cultured on murine embryonic fibroblast (MEF) feeder cell layers in commercially available serum replacement (SR) medium or fetal calf serum (FCS)-containing medium. Recently, we found that a strong antibody response was generated in human subjects after the second infusion of therapeutic cells cultured in FCS-containing medium. This response was specific for bovine apolipoprotein B-100 (apoB-100), which is the major protein component of low-density lipoproteins (LDL) and which targets its binding to abundant low-density lipoprotein receptors on the cell surface, from which it is internalized. Here, we have shown that ESCs cultured on MEFs in SR medium acquired bovine apoB-100 from MEFs and from the SR medium as well. Our findings also suggest that bovine LDL are used as critical nutrients for ESC propagation.

Disclosure of potential conflicts of interest is found at the end of this article.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Embryonic stem cells (ESCs) are of increasing interest as a source of cellular therapies for repair or replacement of damaged or destroyed tissues [1, 2]. However, a major concern for all such therapies is the potential for immune-based rejection, due to allogeneic or xenogeneic responses, the latter involving medium components of animal origin [3–5]. Thus, the culture conditions under which ESCs are grown and differentiated are of critical importance [6, 7]. Currently, ESCs are cultured on mouse embryonic fibroblast (MEF) feeder layers, most frequently in commercially available serum replacement (SR) medium to circumvent the need for fetal calf serum and its associated risks of xenogeneic proteins, carbohydrates, and adventitious agents. Indeed, the predominant xenoantigen in fetal calf serum (FCS) is bovine apolipoprotein B-100, to which a strong antibody response was generated in patients after the second infusion of therapeutic cells cultured in FCS-containing media [8]. Apolipoprotein B-100 (apoB-100) is the major protein constituent of low-density lipoproteins (LDL), which bind to cell surface LDL receptors and proteoglycans and are internalized into the cell [9–12]. LDL-delivered cholesterol is vital in maintaining homeostasis of individual cells, being used for membrane synthesis and as a potential source of energy. It is also critical on an organism level for steroid production [12]. Indeed, the presence of LDL in culture media inhibits lipid biosynthesis in the cell, as cells preferentially ingest lipids from the culture medium [13].

In this work, we have examined whether use of SR medium for culture of ESCs bypasses the immunogenicity problems associated with FCS. We have shown that ESCs cultured on MEFs in SR medium acquired highly immunogenic bovine apoB-100 from both the MEFs and the SR medium. Our studies also suggest that bovine LDL from MEFs and SR medium is a nutrient vital for cell propagation.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Cell Lines and Culture
The BL6.9 cell line was derived in the Transgenic Mouse Facility of the Johns Hopkins School of Medicine from a C57BL/6 blastocyst by culture on mitotically inactivated (mitomycin C-treated) primary mouse embryonic fibroblasts (Specialty Media, Phillipsburg, NJ, http://www.specialtymedia.com) in high-glucose Dulbecco's modified Eagle's medium (DMEM) supplemented with 15% fetal calf serum (HyClone, Logan, UT, http://www.hyclone.com) or 20% KnockOut SR (Invitrogen, Carlsbad, CA, http://www.invitrogen.com) in KnockOut DMEM (Invitrogen). Both media included minimal essential medium nonessential amino acids (100 µM), glutamine (2 mM), penicillin (100 U/ml), streptomycin (100 µg/ml), 2-mercaptoethanol (5 µM), and leukemia inhibitory factor (1,000 U/ml) (ESGRO; Chemicon, Temecula, CA, http://www.chemicon.com). Sodium pyruvate (1 mM) was added to 15% FCS ES medium. Undifferentiated cultures were characterized by expression of stage-specific embryonic antigen-1 (Chemicon) [14] and confirmed by teratoma generation in C57BL/6 mice. BW5147.3 cell lines were purchased from American Type Culture Collection (American Type Culture Collection, Manassas, VA, http://www.atcc.org) and cultured with DMEM containing 10% fetal calf serum (HyClone) or 10% mouse serum (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com). One day before ESC spread, mitotically inactivated MEFs were thawed and cultured on 0.1% gelatin-coated flasks and plates in high-glucose DMEM supplemented with 10% FCS. For the preparation of the supernatant or conditioned medium (CM) from MEFs, mitotically inactivated MEFs were thawed and cultured on 0.1% gelatin-coated six-well plates in high-glucose DMEM supplemented with 10% FCS. After a minimum of 10 hours, MEFs were washed once with either serum-free DMEM or SR medium and reconstituted with the same medium as used in the wash. MEFs were then cultured for 10–12 hours, and supernatants were collected, filtered, and used.

Antibodies
Fluorescein isothiocyanate (FITC)-conjugated control mouse isotype IgG1, horseradish peroxidase (HRP)-conjugated anti-mouse IgG antibodies, HRP-conjugated anti-rabbit IgG antibodies, and FITC-conjugated streptavidin were purchased from BD Pharmingen (San Diego, http://www.bdbiosciences.com/index_us.shtml). Rabbit anti-human apoB polyclonal antibody was purchased from Cortex Biochem (San Leandro, CA, http://www.cortex-biochem.com). Goat anti-mouse low-density lipoprotein receptor (LDLR) antibody was purchased from R&D Systems Inc. (Minneapolis, http://www.rndsystems.com). Control goat IgG fraction was affinity-purified by protein G from goat serum (Sigma-Aldrich). 3E8.1 monoclonal antibody, specific for bovine apoB-100, was described previously [8].

LDL Releasing Assay
BW5147.3 cells were washed three times and resuspended (2.5 x 107cells per milliliter) in serum-free DMEM. One hundred microliters of the cell suspension was placed in the upper wells of Transwell chambers (5-µm pore size; Corning Life Sciences, Lowell, MA, http://www.corning.com/lifesciences). Serum-free DMEM (650 µl) was placed in the lower chambers, and the Transwell was incubated at 37°C in a moist atmosphere containing 5% CO2. At each time point, the supernatant in the lower chamber was collected. One hundred fifty microliters of each supernatant was used for enzyme-linked immunosorbent assay (ELISA).

ELISA for Bovine LDL in the Medium
One hundred fifty microliters of medium or supernatant was put onto the ELISA plate (Nunc A/S, Roskilde, Denmark, http://www.nuncbrand.com) and incubated at 4°C overnight. After washing with ELISA washing buffer (R&D Systems), plates were blocked with 2% human serum albumin (Sigma-Aldrich) in phosphate-buffered saline (PBS) and then washed before addition of 3E8.1 monoclonal antibody (mAb) and control mouse isotype IgG1 (BD Pharmingen). The plates were washed and then probed with HRP-conjugated anti-mouse IgG1 antibody. The plates were developed using an ELISA development system (R&D Systems) and read at 450 nm with a SpectraMax Plus spectrophotometer (Axon Instruments/Molecular Devices Corp., Sunnyvale, CA, http://www.moleculardevices.com).

Cell Proliferation Assay by Flow Cytometry
BW5147.3 cells were extensively washed, resuspended in serum-free DMEM or in supernatants from MEFs at 5 x 104 cells per milliliter in 24-well plates, and incubated under standard conditions. After incubation, all cells were collected, and each well was rinsed three times with PBS. For BL6.9 mouse ESCs, 5 x 104 cells were cultured on MEFs in six-well plates with the indicated concentration of human LDL (Sigma-Aldrich) for 72 hours. The cells were then trypsinized, washed with 2% FCS-containing PBS, and resuspended in PBS containing 10 µg/ml propidium iodide. The final volume was adjusted to 500 µl for BW5147.3 cells and 2 ml for BL6.9 cells. Cells were immediately analyzed on a FACSCalibur (Becton, Dickinson and Company, Mountain View, CA, http://www.bd.com) for 10 seconds on high mode. Data were analyzed using the CellQuest program (Becton Dickinson).

Immunoprecipitation and Western Blot Analysis
Phenylmethylsulfonyl fluoride (100 pg/ml) was added to culture medium or supernatants, and medium was precleared with protein G-Sepharose 4Fast Flow (Amersham Biosciences, Piscataway, NJ, http://www.amersham.com) and then immunoprecipitated by 3E8.1 and isotype-matched control antibody. Proteins were separated by SDS-polyacrylamide gel electrophoresis with 3%–8% Tris-acetate gels (Invitrogen). For Western blotting, immunoprecipitates were transferred (60 V for 4 hours in 25 mM Tris, 192 mM glycine, 10% methanol) to polyvinylidene difluoride membranes (Immobilon-P; Millipore, Billerica, MA, http://www.millipore.com). Membranes were incubated for 1 hour in PBS containing 5% membrane blocking agent (Amersham Biosciences). Proteins were detected by HRP-conjugated anti-mouse IgG antibodies or HRP-conjugated anti-rabbit IgG antibodies and developed with enhanced chemiluminescence Western blotting reagents (Amersham Biosciences) by autoradiography.

Confocal Scanning Laser Microscopy
BW5147.3 cells were washed three times, stained with FITC-conjugated 3E8.1 or isotype-matched control mouse IgG1 (BD Pharmingen) antibody at 5 µg/ml, incubated on ice for 15 minutes, and then washed to remove excess antibody. Cells were then fixed in 2% formaldehyde-containing PBS for 20 minutes at 25°C, washed with PBS, and then preincubated with 0.5% saponin (Sigma-Aldrich)-containing PBS for 10 minutes at room temperature. Cells were then incubated with Alexa Fluor 488-conjugated goat anti-mouse IgG (Invitrogen) at 5 µg/ml and Alexa Fluor 647 phalloidin (Invitrogen) for staining of actin filaments. After two washes with 0.5% saponin-containing PBS, cells were washed with PBS. Confocal images were obtained with the Zeiss LSM 5 PASCAL confocal laser scanning microscope installed on a Zeiss Axioskop2 MOT microscope (Carl Zeiss MicroImaging, Inc., Thornwood, NY, http://www.zeiss.com). To visualize cells, the Zeiss Plan Neofluar 1.3 (x100) lens was used. Images were acquired using the Zeiss LSM 5 PASCAL system and Zeiss LSM 5 PASCAL version 3.2 SP2 software.

Immunofluorescence Microscopy
After several passages in either 15% FCS-containing medium or SR medium, BL6.9 cells were seeded onto MEFs in 24-well plates for 3 days. Cells were gently washed three times with PBS and stained with FITC-conjugated 3E8.1 or isotype-matched control mouse IgG1 (BD Pharmingen) at 5 µg/ml on ice for 15 minutes. Following a wash to remove excess antibodies, cells were incubated with Alexa Fluor 488-conjugated goat anti-mouse IgG (Invitrogen) as secondary antibody to enhance the fluorescence. After being washed three times with PBS, cells were fixed with 2% formaldehyde-containing PBS for 20 minutes at 25°C, washed with PBS, and analyzed. Images were acquired using Axiovert 200 installed on an AxioCam MRm camera with an A-Plan x10, 0.25 objective lens and analyzed using AxioVision (Carl Zeiss MicroImaging, Inc.).

Statistical Analysis
Each experiment was repeated at least two times. Data are shown as mean ± SD. The statistical significance of differences was analyzed using Student's t test.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Cells Cultured in FCS Bound Bovine apoB-100 on Cell Surface via LDL Receptor: Kinetics of Shedding from the Cell Surface
3E8.1, a monoclonal antibody to bovine apolipoprotein B-100 (apoB-100), bound to the surfaces of all cell lines grown in FCS-containing media [8] and that such binding was significantly blocked by anti-mouse LDL receptor antibodies and heparin [8]. Moreover, we demonstrated that immune responses to bovine apoB-100 are generated in numerous and diverse patient populations treated with cells cultured in FCS and are correlated with poor engraftment in some cases, suggesting that bovine apoB-100 binding to the cell surface offers a target for immune-mediated attack. Because ESCs require trypsinization, which removes LDLR and other proteoglycans from the cell surface to which apoB-100-containing LDL binds [8], we used a murine T-cell lymphoma line, BW5147.3, which expresses abundant LDLR and does not require trypsinization for initial studies. Indeed confocal microscopic analysis indicated that bovine apoB-100 binds robustly to the cell surface, providing a potential target for antibody-mediated binding (Fig. 1). As previously shown [8], 3E8.1 fails to bind to cells cultured with mouse serum, indicating that 3E8.1 mAb is specific to bovine apoB-100.


Figure 1
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  		Figure 1. Cells cultured in FCS bound bovine apolipoprotein (apo) B-100 on their cell surface via low-density lipoprotein receptor. Cell surface staining with fluorescein isothiocyanate-conjugated 3E8.1 showed the abundant cell surface bovine apoB on BW5147.3 cells cultured in FCS-containing medium but not in mouse serum medium. Abbreviations: cont., control; FCS, fetal calf serum.

 
However, a key issue for therapeutic use of cell populations cultured in FCS, to which bovine apoB-100 has bound, is how rapidly bovine apoB-100 is lost from the surface on transfer of cells into a bovine apoB-100-free medium, such that the target antigen is no longer expressed. To address the kinetics of loss of cell surface bovine apoB-100 into serum-free medium, BW5147.3 cells were vigorously washed and resuspended in serum-free DMEM and then placed in the upper chamber of a Transwell system. At successive time points, the supernatant in the lower chamber was collected and analyzed for bovine apoB-100. Bovine apoB was shed over time in a linear fashion, suggesting that a considerable amount of bovine apoB-100 resident on the cell surface or from internal stores was shed and populated serum-free medium (Fig. 2). This is consistent with previous data in which we showed that a significant loss of bovine apoB from the surface of cells requires several hours following placement in media totally devoid of FCS [8].


Figure 2
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  		Figure 2. Bovine apolipoprotein B-100 was shed from cell surface and released into serum-free medium. BW5147.3 cells were washed three times with sufficient nonserum Dulbecco's modified Eagle's medium (DMEM), resuspended in nonserum DMEM, and then placed in the upper chamber of the Transwell. At each time point, the supernatant in the lower chamber was collected, and enzyme-linked immunosorbent assay was performed with 3E8.1 (solid line) and isotype-matched cont. antibody (dotted line). Data are reported ± SD. Abbreviations: cont., control; min., minutes; O.D., optical density.

 
Sources of Bovine apoB-Containing LDL in Cultured Cell Populations
A second key issue regards the sources from which cultured therapeutic cell populations may acquire bovine apoB-100. We hypothesized that acquisition of bovine apoB-100 not only could come from medium sources, but also could be shed from the surface or released from internal stores of MEFs. To assess whether bovine apoB-100 was shed from MEFs, we thoroughly washed and cultured MEFs in serum-free DMEM and analyzed the supernatants over time by ELISA (Fig. 3A) and Western blotting (Fig. 3B). Such supernatants clearly contained bovine apoB-100, indicating that MEFs are a source from which ESCs and other therapeutic cell populations could acquire this immunogen. Western blotting using anti-human apoB polyclonal antibody, which cross-reacts with bovine apoB [8] confirmed this finding (data not shown).


Figure 3
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  		Figure 3. Bovine apolipoprotein B-100 existed in Sup. from murine embryonic fibroblast (MEFs) and functioned as a nutrient. (A): Enzyme-linked immunosorbent assay analysis of Sup. from MEFs cultured in serum-free Dulbecco's modified Eagle's medium (DMEM) was performed with 3E8.1 (filled column) and isotype-matched cont. antibody (open columns). Serum-free DMEM was also used as negative cont. (B): Sup. from MEFs was immunoprecipitated by 3E8.1 or isotype-matched cont., and Western blotting was performed with 3E8.1 antibody. (C): BW5147.3 cells (5 x 104) were cultured in nonserum DMEM or Sup. from MEFs. Cell count analysis was performed by FACS on day 1 (open columns), day 2 (gray filled columns), and day 3 (black filled columns). (D): BW5147.3 cells (5 x 104) were cultured in Sup. from MEFs with blocking of anti-mLDLR antibody (black filled columns) or cont. antibody (open columns). Cell count analysis was performed after a 72-hour culture by FACS. (E): BW5147.3 cells (5 x 104) were cultured in nonserum DMEM with the indicated conc. of LDL. Cell count analysis was performed by FACS on day 1 (open columns), day 2 (gray filled columns), and day 3 (black filled columns). Data are reported ± SD. Statistical differences among groups were assessed with Student's t test. *, p < .001; **, p < .01. Abbreviations: 3, immunoprecipitation with 3E8.1 monoclonal antibody; C, immunoprecipitation using isotype-matched control antibody; conc., concentration; cont., control; FACS, fluorescence-activated cell sorting; IP, immunoprecipitation; LDL, low-density lipoprotein; mLDLR, mouse low-density lipoprotein receptor; NS, no-serum Dulbecco's modified Eagle's medium; O.D., optical density; PBS, phosphate-buffered saline; sec., seconds; Sup., supernatant.

 
Next, we examined whether the supernatants from MEFs, cultured in serum-free DMEM, were sufficient to support cell growth. BW5147.3 cells were cultured in serum-free DMEM or in culture supernatants of MEFs. BW5147.3 cells cultured in MEF supernatant proliferated. In contrast, cells cultured in serum-free DMEM not only failed to proliferate but died (Fig. 3C). To investigate whether LDL within the MEF supernatants provided significant nutritional support for cell proliferation, we blocked LDL uptake through LDLR by coculture with anti-mLDLR antibodies. Although LDLR antibodies blocked LDL binding [8] and suppressed cell proliferation by only ~26% (Fig. 3D), this nonetheless had a significant impact on cellular proliferation, suggesting that LDLR-mediated uptake may be more efficient than other pathways of LDL internalization, including the cell surface proteoglycan pathway [15, 16]. To confirm the ability of LDL to support cell growth, human LDL was added to cells cultured in serum-free DMEM. BW5147.3 cells proliferated in an LDL dose-dependent manner, suggesting that one of the major nutrients for cell growth is LDL (Fig. 3E). Taken together, these results indicate that LDL shed from feeder layer cells are a major nutritional source supporting cell growth in serum-free medium.

Bovine apoB-100 Is Present in SR Medium, and Its Content Is Increased in MEF-Conditioned Medium
We next examined whether the ability of SR medium and MEF-conditioned medium (MEFCM) to support growth of ESCs in the absence of FCS was attributable to the presence of bovine LDL. Bovine LDL, as detected by bovine apoB-100, was present in SR medium and in MEFCM at a greater level (Fig. 4A, 4B). This was confirmed by Western blot analysis using anti-human apoB polyclonal antibody (data not shown). As SR medium is not recommended in the plating of feeder cells [17], which require culture in FCS-containing medium, it is highly possible that the success of SR medium in supporting growth of ESCs depends on release of LDL from MEFs previously cultured in FCS. That MEFs provide a sustained source of bovine LDL was supported by the observation that even after the fourth wash, bovine apoB-100 was still released from MEFs into the medium (Fig. 4C). Addition of supplemental human LDL to ESCs cultured on MEFs in SR medium only minimally bolstered cell growth, suggesting that the lipid content present in SR medium and released from MEFs is sufficient to optimally support ESC growth (Fig. 4D).


Figure 4
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  		Figure 4. Bovine apolipoprotein B-100 is present in SR medium and increased in its CM. (A): Enzyme-linked immunosorbent assay (ELISA) analysis of SR medium and CM were performed with 3E8.1 (filled columns) and isotype-matched cont. antibody (open columns). (B): SR medium and CM were immunoprecipitated by 3E8.1 or isotype-matched cont., and Western blotting was performed with 3E8.1 antibody. (C): SR medium, CM after first wash, and CM after fourth wash were analyzed by ELISA with 3E8.1 (filled columns) and isotype-matched cont. antibody (open columns). (D): BL6.9 mouse ESCs cultured in SR medium with the indicated conc. of LDL. Cell count analysis was performed after a 72-hour culture by FACS (filled columns). Data are reported ± SD. Statistical differences among groups were assessed with Student's t test. *, p < .01; **, p < .05. Abbreviations: 3, immunoprecipitation with 3E8.1 monoclonal antibody; C, immunoprecipitation using isotype-matched control antibody; CM, conditioned medium; conc., concentration; cont., control; FACS, fluorescence-activated cell sorting; IP, immunoprecipitation; LDL, low-density lipoprotein; O.D., optical density; PBS, phosphate-buffered saline; sec., seconds; SR, serum replacement.

 
Mouse ESCs Express LDL Receptor on Their Surface and Bind Bovine apoB-100 Acquired from the Medium
Next, we investigated whether bovine LDL originating from MEFs and SR medium bound to cocultured ESCs. First, we documented the significant expression of LDLR on the ESC surface (Fig. 5A). Next, we found that bovine apoB-100 from MEFs and SR culture medium bound substantially to the ESC surface, although in diminished amounts compared with cells cultured in FCS (Fig. 5B). Taken together, our results indicate that the potent immunogen bovine apoB-100 is present not only in FCS but also in SR medium, is released from feeder cells previously cultured in FCS, and is acquired by cocultured ESCs, potentially targeting them for immune-mediated elimination.


Figure 5
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  		Figure 5. Low-density lipoprotein receptor was expressed on mouse ESCs (mESCs), and bovine apolipoprotein B-100 in the medium bound on their cell surface. (A): stage-specific embryonic antigen-1-positive BL6.9 mESCs were stained with anti-mLDLR polyclonal antibodies (solid line) with fluorescein isothiocyanate (FITC)-conjugated streptavidin. FITC-conjugated streptavidin alone was used as a negative cont. (dotted line). (B): BL6.9 mESCs cultured on murine embryonic fibroblasts in SR medium or FCS-containing ES medium were stained with FITC-conjugated 3E8.1 or isotype-matched cont. monoclonal antibody and observed by fluorescent microscopy. Alexa Fluor 488-conjugated goat anti-mouse IgG was used as secondary antibody to enhance the fluorescence. Abbreviations: cont., control; ES, embryonic stem; FCS, fetal calf serum; mLDLR, mouse low-density lipoprotein receptor; SR, serum replacement.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosure of Potential...
 Acknowledgments
 References
 
There has been intense interest in developing optimal culture conditions for stem cells and other cellular populations intended for therapeutic use [6, 7]. Numerous reports have detailed the problems associated with culture media that include animal-derived materials, principally, transmission of infectious agents and elicitation of immune responses to immunogens. Two principal immunogens have been identified in therapeutic cell populations cultured in fetal bovine serum, bovine apolipoprotein B-100, which elicited a strong antibody response in patients infused with cultured cells on more than one occasion [8], and the carbohydrate moiety Neu5Gc, to which preformed antibody exists in humans [18, 19]. The potential to sabotage cellular engraftment via immune responses to such moieties has been shown and has been suggested [8, 18]. The response to bovine apoB appeared to be boosted by successive exposure to cell transplants or vaccines. At minimum, two exposures were required, both in human and mouse, and boosts from subsequent cellular infusions would be expected to sustain a high titer of antibody capable of binding of newly infused cells, causing their destruction [8].

ESCs of both human and murine origin have typically been cultured on MEFs as feeder layers in SR medium or FCS-containing medium [2]. Recognition of the problems associated with FCS and MEFs has brought forth novel reagents: SR medium, considered a significant advance over FCS, and human feeder cells to replace MEFs [20–22]. Furthermore, feeder cell-free culture systems have been devised using Matrigel or extracellular matrix materials, including laminin or fibronectin, with CM [23] or with SR medium [24]. These methods, however, proved to be inferior to traditional MEF cultures, with lower growth rates and cloning efficiency and a higher differentiation rate [24].

In this study, we have extended our previous findings by demonstrating that the potent immunogen bovine apolipoprotein B-100 is present not only in FCS but also in SR medium and further originates from feeder cells previously cultured in FCS. Given that commercially available SR medium consists largely of a lipid-rich bovine album fraction [25], it is not surprising that bovine apoB-100 is present in significant amounts. It is also not surprising that feeder cells have abundant bovine apoB-100 given that they are uniformly cultured in FCS-containing medium [26]. Previously, we showed that cells bind copious amounts of bovine apoB-containing LDL on their surface via LDL and other receptors and that these are internalized and stored. We have shown here that bovine apoB-100 is present in SR medium and is also released into the medium from MEFs and that ESCs cultured in these conditions bind bovine apoB-100 and may use it as an energy source for cell propagation. Since less than 0.5% lipoprotein in the medium is enough to fully saturate cell surface receptors with bovine apoB-100 [8], it is highly likely that the amount of bovine apoB-100 present in SR medium is enough by itself to foster the growth of ESCs and that lipoproteins released from MEFs to CM might further bolster cell propagation. Given the potent growth-enhancing effect of bovine LDL, it was surprising that bovine apoB-100 was not identified among the 136 unique proteins in CM involved in cell growth and differentiation, extracellular matrix formation, and remodeling [27]. However, the gel method used to identify these proteins had a 200-kDa cutoff and thus would have excluded bovine apoB-100 with its MW of 520 kDa.

An additional mechanism by which bovine LDL present in the medium may affect the proliferation and differentiation of ESCs regards their binding to heparan sulfate proteoglycans (HS-PGs) expressed on the cell surface, which function not only as receptors for LDL but also as receptors for basic fibroblast growth factors (bFGFs) [28] and bone morphogenetic proteins (BMPs) [29], factors known to exert profound activities on ESC growth and differentiation. As HS-PGs modulate the stability and activity of bFGFs, in formation of their stimulatory binding complexes [28], the binding of LDL may displace bFGFs and stimulate differentiation. An antagonistic relationship also exists for BMPs, which inhibit the cell proliferative effects of LDL [30].

We showed that supernatants from MEFs, cultured in serum-free DMEM, were sufficient to support cell growth and that deprivation of LDL induced cell death in BW cells. Although increasing caspase activity [31] and slowing in the rate of DNA synthesis [32] with serum withdrawal promote apoptotic death of ESCs, unlike other cells, ESCs lack the capacity to undergo G0 exit and fail to accumulate in G1 phase [32]. Thus, although serum deprivation promotes apoptosis, it does not change the cell cycle profile of ESCs [31, 32]. At any rate, it is clear that endogenous cholesterol synthesis cannot keep pace with ESC propagation, in LDL-deprived circumstances.

Delivery of cholesterol in serum-free medium has been a challenging issue, and some strategies have been developed. Animal-derived lipoprotein fractions have been used as replacement for serum in some serum-free media. To reduce the immunogenicity of apoB-100 in LDL, synthetic LDL coupled with an amphiphathic apoB peptide could be one solution [33]. LDL could be replaced by cholesterol complexed with serum albumin [34]. Moreover, plant-derived or synthetic cholesterol combined with β-cyclodextrin added to cultivation medium could also be an effective method for delivering cholesterol to cells in serum-free medium [35–37].

To eliminate problems with animal-derived materials entirely, human feeder cells have recently been cultured with human serum-containing medium, which was completely free of xenoproteins [20, 38]. Furthermore, fully defined serum-free and feeder-independent human ESC culture medium, composed of recombinant or purified sources of human materials (TeSR1), was developed and its components disclosed [39]. Although this medium was unable to support the sustained proliferation of an undifferentiated human ESC line [40], additional testing of a broader range of human ESCs is clearly warranted. While a purely human origin component culture system is an obvious solution for clinical applications of human ESC or their differentiated progeny, the amounts of human AB sera that would be needed from this rarest of blood donor groups would be prohibitive with respect to the need, and infectious agent transmission would require and extensive testing. Moreover, apolipoprotein B has protein polymorphisms that are immunogenic, as they generate alloantibodies in human populations [41, 42]. Thus, autologous serum depleted of anti-Neu5Gc antibodies may be the most practical and safe means for culturing ESCs or other therapeutic cell populations prior to transplantation. An additional potential solution would be genetic manipulation of source bovines to knock out bovine apoB-100 and replace it with its human counterpart, as well as eliminating Neu5Gc, even as galactose-alpha1, 3-galactose ({alpha}1, 3Gal) carbohydrates have been knocked out of swine developed for human organ transplantation [43].


   DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
Acknowledgments
 References
 
The authors indicate no potential conflicts of interest.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
References
 
We thank Drs. Eda Bloom and Brenton McCright for critical reading of the manuscript. M.H.-S. was the principal investigator in this study; N.S. provided guidance for the study and was the primary author; A.S.R. provided the overall project and manuscript guidance. All authors provided guidance for this study and contributed to the writing of the manuscript.


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 

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