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Development of a Xenofree Non Contact Co-culture System, Embryonic Stem Cells

Development of a Xenofree Non Contact Co-culture System, Embryonic Stem Cells

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STEM CELL BIOLOGY
Development of a xeno-free non-contact co- culture systemfor derivation and maintenance of embryonic stem cellsusing a novel human endometrial cell line
Nina Desai
&
Jennifer Ludgin
&
Jeffrey Goldberg
&
Tommaso Falcone
Received: 15 January 2013 /Accepted: 6 March 2013 /Published online: 11 April 2013
#
The Author(s) 2013. This article is published with open access at Springerlink.com
Abstract
 Purpose
Mouse embryonic fibroblast feeder layers (MEF)have conventionally been used to culture and maintain the pluripotency of embryonic stem cells (ESC). This studyexplores the potential of using a novel human endometrialcell line to develop a non-xeno, non-contact co-culture sys-tem for ESC propagation and derivation. Such xeno-freesystems may prove essential for the establishment of clinicalgrade human ESC lines suitable for therapeutic application.
 Methods
A novel line of human endometrial cells were seed-ed in a 6-well dish. Filter inserts containing mouse ESCswere placed on these wells and passaged 2
 – 
3 times per week.Inner cell masses derived from mouse blastocysts were alsocultured on transwells in the presence of the feeder layer. In both cases, staining for SSEA-1, SOX-2, OCT-4 and alkaline phosphatase were used to monitor the retention of stem cells.
 Results
ESCcoloniesretainedtheirstemcellmorphologyandattributesfor over120 days inculture and 44 passages todate.Inner cell mass derived ESC cultures were maintained in a  pluripotent state for 45 days, through 6 passages with reten-tion of all stem cell characteristics. The stem cell coloniesexpressed stem cell specific markers SSEA-1, Sox 2, Oct-4and alkaline phosphatase. Upon removal of the human feeder layer, there was a distinct change in cell morphology withinthe colonies and evidence of ESC differentiation.
Conclusions
Human feeder layers offer a simple path awayfromtheuseofMEFfeedercellsorMEFconditionedmediumforESCculture.Furthermore,indirectco-cultureusingporousmembranes to separate the two cell types can prevent contam-ination of stem cell preparations with feeder cells during passaging.
Keywords
Embryonicstemcells.Feedercells.Inner cellmass.Co-culture.Humanendometrialcellline.Microporousmembrane.Laser 
Introduction
Embryonic stem cells (ESCs) are derived from the inner cellmass (ICM) of mammalian blastocysts. Original studies in theearly 1980
s showed that stem cells derived from the inner cellmass of mouse blastocysts were pluripotent and could prolife-rate continuously in an undifferentiated state [13,22]. The first  derivationofahumanESClinewasreportedbyThomsonetal.[31]. The human embryonic stem cell (hESC) line retained its pluripotency during in vitro propagation and was capable of differentiation into all three germ layers, i.e. endoderm, meso-derm and ectoderm. Derivation of human ESC lines in earlystudies required the use of a mouse embryonic fibroblast (MEF) feeder layer [25,31]. Cytokines, growth factors, and extracellular matrix (ECM) components,as well as a myriadof as yet undefined components in the MEF cell culture milieu,apparently have the ability to sustain the pluripotency of hu-man, as well as monkey and mouse ESC in vitro.TheuniqueabilityofESCstodifferentiateintoadesiredcelllineage with specific signaling makes them an invaluable toolfor research in drug development, cell regeneration and deliv-ery of gene therapies. For such applications, ESC culture withanimal feeder cell layers may be undesirable due to the poten-tialriskofpathogentransmissionandviralinfection[4,26,27]. For the therapeutic potential of ESCs to be effectivelyexploited,itwilllikelybe necessarytodevelop
clinical grade
Capsule
We developed a non-contact co-culture system using a novelhuman endometrial cell line that allowed propagation of embryonic stemcells. This offers a simple path away from the use of mouse embryonicfeeder (MEF) cells for stem cell culture. N. Desai (
*
)
:
J. Ludgin
:
J. Goldberg
:
T. FalconeCleveland Clinic Fertility Center, Department of OB-GYN/ Women
s Health Institute, Suite 220 South Bldg, 26900 Cedar Rd.,Beachwood, OH 44122, USAe-mail: desain@ccf.orgJ Assist Reprod Genet (2013) 30:609
 – 
615DOI 10.1007/s10815-013-9977-1
 
ESC lines that have been cultivated without direct exposure toanimal tissues.Attempts to overcome this problem have led to the culti-vation of hESCs in MEF conditioned media [21,33] or on an extracellular matrix with growth factors [19]. Others havesuggested that hESC encapsulation and 3-D culture in a hydrogel in combination with MEF
– 
conditioned mediummay provide some benefit, allowing self renewal without  passaging [15]. Xeno and feeder-free culture in chemicallydefined medium supplemented with ECMs, high levels of growth factors and signaling molecules is another attractive path towards creating therapeutic grade hESC lines, standard-izing culture conditions and reducing work load. [2,4,5,21, 33
 – 
35].However, suchmedia systems can becomplex,costlyand require diligent attention to prevent undesired differenti-ation of ESC colonies on the periphery of the cultures.An alternate approach has been the use of a variety of different human cell types as feeder layers for co-culture of ESCs. The cells used include fetal fibroblasts [12,28], fore- skin fibroblasts [3,7,16], placental fibroblasts [14], human marrow stromal cells [6] and human endometrial cells [20]. Commercially available human embryonic stem cell linesthemselves may also be useful as feeder cells for propagationof other hESC lines as well as for the derivation of new hESClines from human embryos [32]. Still, any type of direct contact between the hESCs and the feeder cell layer, even if of human origin, carries the potential threat of contaminationwith the feeder cell population, making their use for futuretherapeutic transplantation into patients more difficult.In the present study we describe a non-contact co-culturemodel for ESC propagation using a novel human endome-trial cell line as a potential alternative to MEF feeder cells.We tested this model first for propagation of a commerciallyavailable mouse ESC line and then for establishment of ESCcultures from the inner cell mass of mouse blastocysts.
Materials and methods
Human endometrial cell lineThe human endometrial cell line described in this investiga-tion was initially isolated from benign proliferative endome-trium. This cell line has since undergone more than 45 passages, retaining its epithelial cell morphology and charac-teristics [10,11]. The endometrial cell line was maintained in modified
α 
Minimum Essential Medium supplemented with5 % heat inactivated fetal bovine serum (Life Technologies;Grand Island, NY). Culture flasks were incubated in a humid-ified chamber at 37 °C with 6 % CO
2
. The cell line was passaged every 4
 – 
5 days.Transwell permeable supports (Corning Incorporated,Corning, NY) with 12 mm polyester (PET) membranes(pore size 0.4
μ 
M) were used for co-culture in 6-well plates.The porous membrane filters are permeable and transparent,allowing the sharing of media and secretions while creatinga physical separation between the two cell types. Humanendometrial cells were seeded into the dish at a concentra-tion of either 15,000 or 30,000 cells per well. The endome-trial feeder layers were utilized for ESC co-culture whenthey were 40
 – 
50 % confluent. The membrane insert withESC colonies was transferred to a fresh endometrial feeder when the initial feeder layer reached~75 % confluence.Mouse embryonic stem cell lineThe culture medium for all stem cell work was ESC-SureDMEM (Applied Stem Cell; Menlo Park, CA) with 20 %ESC-Sure fetal bovine serum (FBS; Life Technologies;Grand Island, NY) supplemented with 10 ng/ml mouseleukemia inhibitory factor (LIF; Stem Cell Technologies;BC, Canada), 2.0 mML-glutamine, 0.1 mM non-essentialamino acids, 1.0 mM sodium pyruvate, 0.1 mM ßmercaptoethanol, 100units/mL penicillin and 100
μ 
g/mlstreptomycin (all from Invitrogen; Carlsbad, CA).A commercially available mouse ESC line ASE-9005 was purchased (Applied Stem Cell; Sunnyvale, CA). Embryonicstem cells were seeded on the porous membrane of theTranswell insert at a concentration of 2
 – 
3000 cells/well. Amedia half 
– 
change was performed every 48 h. Cultures weremonitored daily and photographed. ESC cultures were pas-saged when colony diameter reached 100-120
μ 
m. Coloniesweretrypsinizedandnewwellswereseeded.InsertswithESCcoloniesweremovedtowellswithnewfeederlayerswhentheendometrial monolayers approached 75 % confluence.Mouse blastocyst derived stem cellsOne cell mouse zygotes from a B6D2F1 x B6C3F1 crosswere purchased for stem cell isolation (Charles River Lab-oratories; Wilmington, MA). Embryos were thawed andcultured in Global medium (Life Global; Guilford, CT)supplemented with 10 % Serum Protein Supplement (SPS;Trumbull, CT) for 6 days. On day 6, the zona pellucida wasremoved from blastocysts using mechanical pipetting alongwith laser opening of the zonae. Embryos were placed onthe Transwell inserts and co-cultured in stem cell mediumwith the human endometrial feeder layer. ICM outgrowthsformed 1
 – 
2 days after plating were microsurgically excised.The ICM clumps were placed in a 20
μ 
l drop of 0.5 %trypsin/EDTA solution for 3 min, after which an equalvolume of FBS was added to inactivate the enzyme. TheICM clumps were then disaggregated using a drawn glassmicropipette. The dispersed ICM cells were then seeded intoa new Transwell insert and placed on a fresh endometrialfeeder layer. Five days after the seeding of ICM cells, dome-
610 J Assist Reprod Genet (2013) 30:609
 – 
615
 
like colonies started to appear. Individual ESC-like colonieswere manually selected and once again disaggregated withtrypsin-EDTA. This process was repeated for further expan-sion of the ICM derived stem cells. However, it was not necessary to manually select colonies after the second pas-sage. Instead, the entire filter was trypsinized. Any colonieslifting off were collected, disaggregated and placed in newTranswell inserts. Colonies were passaged after reachingdiameters of 100
 – 
120
μ 
m. At each passage the insert withstem cells was placed on a fresh feeder of endometrial cells.Characterization of embryonic stem cellsThe mouse ASE-9005 ESC line and the ICM-derivedstem cell cultures were tested for pluripotency using commercially available kit for mouse ESC characteriza-tion (Applied Stem Cell; Menlo Park, CA). We testedfor four markers of undifferentiated mouse stem cells,namely expression of SSEA-1, Sox-2, and Oct4, as wellas alkaline phosphatase activity.Staining was performed as per manufacturer 
s instruc-tions with slight modifications to accommodate ESC growthon membrane filter inserts. Membrane inserts with ESCcultures were carefully rinsed with phosphate buffered sa-line (PBS) before staining, taking care not to dislodge thecolonies. The ESC colonies were then fixed on the mem- brane by adding 4 drops of fixing solution. After one hour at room temperature, fixative was removed with three 5 minrinses with PBS. Approximately 4 drops of permeablizingsolution were placed on top of each membrane for 30 min at room temperature, followed by another PBS rinse. Blockingsolution was applied to each membrane for one hour beforeapplication of primary antibodies. Aliquots (35-40ul) of SSEA-1, SOX-2 and Oct-4 were placed on separate glassslides. Membranes with ESC colonies were carefully cut out of the Transwell inserts and placed colony side down on thedifferent slides with primary antibody. Slides were enclosedin a humidified chamber and incubated overnight at 4 °C.The next morning, the membranes were rinsed before ap- plication of the appropriate secondary antibody for one hour at room temperature. This last incubation and all subsequent steps were performed in the dark. The specificity of eachantibody was verified with concurrently run negative con-trols. Upon completion of labeling with secondary anti- bodies, the membranes were once again rinsed. A DNAstaining solution was then applied for 8
 – 
10 min. Mem- branes were placed colony side up on slides with mountingsolution and cover slipped. Confocal laser microscopy withoptical sectioning was used to examine immunoflourescent staining for stem cell markers.Alkaline phosphatase in ESC cultures was detected usingthe rapid sensitive alkaline phosphatase test provided in thestem cell characterization kit. Alkaline phosphatase test solution (80
 – 
100
μ 
l) was placed directly on the ESC colo-nies growing on the membrane insert. After 2 h of stainingat room temperature, the membrane was mounted on a glassslide and cover slipped. Alkaline phosphatase activity incolonies was visualized using light microscopy.
Results
In this investigation we explored the possibility of using a novel human endometrial cell line as a feeder layer for ESCculture. We elected to use the Costar Transwell dishes indeveloping this co-culture model to prevent direct contact  between the human feeder layer and the mouse stem cells.This prevented contamination of our stem cell cultures withthe feeder cells. Testing was initially done by following thegrowth of an established stem cell line, ASE 9005, with theendometrial feeder. We later determined that this humanfeeder was also able to support growth of stem cells derivedfrom the inner cell mass of mouse blastocysts.The ASE-9005 mESC line adapted quite nicely to growthon the membrane suspended above the endometrial cellfeeder (Fig.1a-c). ESC colonies proliferated rapidly, neces-sitating at least two passages per week. With time in culture,the growth rate accelerated, requiring the reduction of thenumber of cells seeded onto the insert. Seeding with 2
 – 
3000cells appeared to be optimal. Colony morphology was con-sistent through passages. Stem cells formed a tight amor- phous mass of cells with a distinct border and paralleled themorphology typically observed in our laboratory with ESCgrown directly on a MEF feeder layer (Fig.1d). Cells hadthe high nucleus/cytoplasm ratio associated with pluripotent cells. We often noted signs of differentiation if colonies onthe insert were allowed to grow much past a diameter of 120
μ 
M. Cells started to spread, flatten and there was a lossof border integrity. Cells on the edge of the larger coloniestended to round up and cytoplasmic membranes of individ-ual cells became more clearly evident.This culture model facilitated extended passaging andexpansion of ESC colonies, without feeder cell contamina-tion. Colonies could be selected and removed from the porous membrane insert with a glass micropipette. Withcarefully controlled passaging, ESC colonies have retainedtheir stem cell morphology and attributes for over 120 daysin culture and 44 passages to date. The stem cell coloniescultured in our non-contact co-culture model expressed stemcell specific markers SSEA-1, Sox 2, Oct-4 and alkaline phosphatase (Fig.2). Light microscopic scanning of themembranes revealed that over 80 % of the colonies werestrongly positive for alkaline phosphatase activity. We wereable to successfully cryopreserve these stem cells at various passages. Upon thawing and re-plating in fresh co-culturewells, approximately 70 % of cells were able to attach to the
J Assist Reprod Genet (2013) 30:609
 – 
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