Propagation of Human Embryonic Stem Cells on Human Feeder Cells

Mark Richards and Ariff Bongso Summary

Human embryonic stem (hES) cell lines are usually derived and propagated on inactivated murine embryonic fibroblast (MEF) feeders. The use of MEFs and culture ingredients of animal origin for hES cell support increases the risk of cross-contamination of the hES cells with infectious animal agents from the MEFs and animal-based culture medium. This thus makes such hES cells lines undesirable for clinical application. This chapter describes several protocols used in the propagation of hES cells on human fibroblast feeder cells. Two culture methods, the bulk enzymatic culture protocol and the microdissection "cut and paste" protocol are described. Only certain human fetal and adult fibroblast feeders support hES cell growth. Methods for the characterization of pluripotent undifferentiated hES cells grown on human feeders including cell surface marker staining and real-time polymerase chain reaction are also described.

Key Words: Bulk culture; "cut and paste" protocol; human adult fibroblasts; human embryonic stem cells; human fetal fibroblasts; nonsupportive feeder; supportive feeder.

1. Introduction

Human embryonic stem (hES) cell lines were first derived and serially propagated on inactivated murine embryonic fibroblast (MEF) feeder layers (1,2). The use of MEFs and other components of animal origin in the culture media for hES cell support substantially increases the risk of contaminating these lines with infectious animal agents such as retroviruses and severely limits the potential of these lines for clinical usage as well. All National Institutes of Health registered hES cell lines approved for US federal government research funding have been derived on MEF feeder layers and have also been exposed to xeno-proteins. This makes these hES lines undesirable for clinical application

From: Methods in Molecular Biology, vol. 331: Human Embryonic Stem Cell Protocols Edited by: K. Turksen © Humana Press Inc., Totowa, NJ

although they may suffice for most basic research studies. Therefore, many more new hES cell lines derived in "xeno-free" conditions need to be established for clinical investigation and application.

Several groups have shown it possible to derive and propagate hES cell lines on a variety of human feeder layers (3-7). hES cells supported on human feeder layers form colonies that have angular and straight edges as opposed to hES colonies on MEF feeders, which appear circular. hES cell lines derived and serially propagated on human feeder layers remain pluripotent over extended passage; express Oct-4, Nanog, and telomerase; maintain stable karyotypes; individual cells retain a high nucleus to cytoplasm ratio, test positive for all molecular and cell surface markers associated with the pluripotent phenotype, form teratomas in severe combined immunodeficiency SCID mice; and are capable of multilineage in vitro differentiation (8). Apart from differences in gross colony appearance, all other characteristics of MEF and human feeder supported hES cell lines appear to be similar.

In our laboratory, we have found that some human fibroblast feeders are an excellent substitute for MEFs, and we propagate our hES cell lines exclusively on human feeders. We have also been able to derive and maintain a human ES cell line in "xeno-free" conditions on human fetal muscle fibroblasts in culture media containing human based ingredients for more than 20 serial passages without recourse to any animal-based ingredients (3).

Although many labs prefer using MEFs instead human feeder layers for hES support because of convenience, it is likely that the propagation of hES cells on human feeder layers will gain popularity especially with the push to transfer the science to a clinical setting. The production of a supportive human feeder line immortalized with telomerase is technically possible. If such a line were made widely available, this would certainly lead to the widespread use of human feeders for hES cell support.

2. Materials

1. High-glucose Dulbecco's modified Eagle's medium (DMEM) without sodium pyruvate, without L-glutamine 1X (GIBCO, Carlsbad, CA; cat. no. 11960-044), store at 4°C.

2. L-glutamine 200 mM, 100X liquid (GIBCO; cat. no. 25030-081), aliquot and store at -20°C (see Note 1).

3. Penicillin-streptomycin liquid containing 5000 U of penicillin and 5000 mg of streptomycin/mL (GIBCO; cat. no. 15070-63) aliquot and store at -20°C.

4. MEM nonessential amino acids 10 mM, (100X) (GIBCO; cat. no. 11140-050), wrap in foil to protect from light and store at 4°C.

5. Defined fetal bovine serum (Hyclone, Logan, UT; cat. no. SH30070-03) aliquot and store at -20°C.

6. Qualified fetal calf serum (GIBCO; cat. no. 10099) aliquot and store at -20°C.

7. Dulbecco's phosphate-buffered saline (PBS) (+) (GIBCO; cat. no. 14040-133), store at 4°C.

8. Dulbecco's PBS, Ca2+, Mg2+ free, PBS(-) (GIBCO; cat. no. 14190-144), store at 4°C.

9. Sterile water, tissue culture grade (GIBCO; cat. no. 15230-162), store at 4°C.

10. Porcine gelatin (Sigma, St. Louis, MO; cat. no. G1890), store at 4°C.

11. Insulin-transferrin-selenium growth supplement (GIBCO; cat. no. 41400-045), store at 4°C.

12. 0.05% Trypsin-EDTA (1X) (GIBCO; cat. no. 25300-062), aliquot and store at -20°C.

13. P-Mercaptoethanol (GIBCO; cat. no. 21985-023), store at 4°C.

14. Dispase (GIBCO; cat. no. 17105-041), store at 4°C.

15. Collagenase IV (GIBCO; cat. no. 17104-019).

16. F12/DMEM, HEPES buffered (GIBCO; cat. no. 11330-057).

17. KnockOut Serum Replacement (GIBCO; cat. no. 10828-028).

18. Basic fibroblast growth factor, human recombinant (GIBCO; cat. no. 13256-029).

19. 1 M HEPES solution (GIBCO; cat. no. 15630-080), store at 4°C.

20. Ethylene glycol (Sigma; cat. no. E9129), store at room temperature.

21. Dimethyl sulfoxide, hybridoma tested, 5 x 10 mL in flamed sealed ampules (Sigma; cat. no. D2650), store at room temperature and protect from light.

22. Mitomycin C, cell culture grade (Sigma; cat. no. M4287), store at 4°C (see Note 2).

23. One-well dish, 60-mm diameter, well area: 2.89 cm2 (BD, Franklin Lakes, NJ; cat. no. 353652).

24. Four-well plate, well area: 1.39 cm2 (BD; cat. no. 353653).

25. Four-well culture slides, well area: 1.7 cm2 (BD; cat. no. 354114).

26. 75 cm2 canted neck, vented tissue culture flask (BD; cat. no. 353136).

27. 175 cm2 canted neck, vented tissue culture flask (BD; cat. no. 353112).

28. 15-mL conical centrifuge tubes, high-clarity polypropylene (BD; cat. no. 352196).

29. 50-mL conical centrifuge tubes, high-clarity polypropylene (BD; cat. no. 352070).

30. 1-mL individually wrapped serological pipet (BD; cat. no. 357522).

31. 5-mL individually wrapped serological pipet (BD; cat. no. 357543).

32. 10-mL individually wrapped serological pipet (BD; cat. no. 357551).

33. 25-mL individually wrapped serological pipet (BD; cat. no. 357525).

34. 500-mL Stericup-GP filter unit (Millipore, Billerica, MA; cat. no. SCGP U05 RE).

35. Sterivex-GP 2000 filling bell filter unit (Millipore; cat. no. SVGP B10 10).

36. 33-mm Millex-GP filter unit (Millipore; cat. no. SLGP 033 RS).

37. Nunc System 100 cryogenic vials with silicon gasket (Nalgene, Rochester, NY; cat. no. 5000-1012).

38. Glass capillaries, 1.0-mm OD (Clark Electromedical Industries, Kent, UK; cat. no. GC100T-15).

39. Sterile 30-G hypodermic needles (BD; cat. no. 511252/511256).

40. Media for hES cells (hES media). hES cells are maintained in cell culture media comprising 80% high-glucose DMEM (v/v), 20% defined fetal bovine serum (v/v), 2 mmol/L L-glutamine, 50 IU/mL penicillin and 50 |ig/mL streptomycin, 1X

nonessential amino acids, 1X insulin-transferrin-selenium G supplement, and 0.1 mmol/l P-mercaptoethanol. To prepare 400 mL media, combine 320 mL DMEM, 80 mL defined FBS, 4 mL L-glutamine, 2 mL penicillin-streptomycin, 4 mL nonessential amino acids, 4 mL ITS-G supplement, and 720 |L P-mercaptoethanol. Filter-sterilize before use. Wrap bottle with aluminium foil to protect from light and store at 4°C for up to 3 wk. The addition of antibiotics to culture media is optional (see Note 3).

41. Media for human feeder expansion (human fibroblast maintenance medium). Human fibroblast feeders are maintained in culture media comprising 90% high-glucose DMEM (v/v), 10% qualified fetal bovine serum (v/v), 2 mmol/L L-glutamine, 50 IU/mL penicillin, and 50 |g/mL streptomycin. To prepare 400 mL media: add together 360 mL DMEM, 40 mL qualified FBS, 4 mL L-glutamine, and 2 mL penicillin-streptomycin. Filter-sterilize media before use and store at 4°C for up to 3 wk. The addition of antibiotics is optional (see Note 3).

42. Mitomycin C solution (see Note 4). Use a 26-G sterile needle to vent the 2 mg mitomycin C ampoule. With an 18-G needle affixed to a 1-mL syringe, add 1 mL sterile PBS (-) to the ampoule, swirl ampoule gently until all powder is dissolved. Remove mitomycin C solution with the syringe and aliquot into sterile microcentrifuge tubes, wrap each tube with aluminium foil to protect from light, and immediately store at 4°C for up to 2 wk (see Note 5).

43. Gelatin solutions (see Note 6). To prepare 1% (w/v) gelatin stock solution: weigh 0.4 g of gelatin powder into a 50-mL conical tube and add 30 mL sterile tissue culture grade water, warm at 37°C for 1-2 h with intermittent pipetting to aid dissolution. After the gelatin has dissolved, top up to the 40-mL mark with sterile water. The 1% stock gelatin solution can be either autoclaved or sterile filtered with a Sterivex-GP 2000 filter bell before use. Stock solutions should be stored at -20°C and can be kept for 6 mo. To prepare 200 mL of 0.1% gelatin working solution: thaw and dilute 20 mL of 1% gelatin stock solution in 180 mL tissue culture-grade water. Aliquots of working solution can be stored at 4°C for up to 2 wk.

44. Dispase solution: dissolve 0.1 g of Dispase in 10 mL of hES media to give a final working concentration of 10 mg/mL of Dispase solution. Filter, sterilize, then warm to 37°C before use.

45. The sources of the monoclonal antibodies for the detection of the cell surface markers are as follows: SSEA-3 (MC-631), SSEA-4 (MC-813-70), Development Studies Hybridoma Bank (Iowa City, IA); Tra-1-60 (MAB-4360) and Tra-1-81 (MAB-4381) Chemicon; Oct-4 (SC-5279) Santa Cruz.

46. The sources of the secondary antibodies for the detection of the primary antibodies are as follows: rabbit anti-mouse immunoglobulin secondary antibody conjugated to fluorescein isothiocyanate (Sigma F2883) and anti-goat immunoglobulin secondary antibody conjugated to fluorescein isothiocyanate (Sigma F7367).

47. Vector Red Alkaline Phosphatase Substrate Kit I, SK-5100 (Vector Labs, Inc., Burlingame, CA; cat. no. SK-5100).

48. KaryoMAX Colcemid solution, liquid (10 |g/mL) in PBS (Invitrogen; cat. no. 15212-012).

49. TRIzol reagent (Invitrogen; cat. no. 15596-026).

50. Ambion DNA-free reagent (Ambion; cat no. 1906).

51. SuperScript III first-strand synthesis system for reverse transcription polymerase chain reaction (RT-PCR) (Invitrogen; cat. no. 18680051).

52. TaqMan probes were purchased from Applied BioSystems (ABI) Assay on Demand and Assay by Design service.

3. Methods

3.1. General Note on hES Culture Methodology

The most critical factor that is essential for success in culturing hES cells is the complete attention to detail in all preparative steps and in cell handling. Good aseptic technique is essential and some prior tissue culture experience will be helpful. The extensive daily observation of cells is important and may be particularly mandatory in the first few weeks of setting up stock cultures. In addition, the use of commercial high-quality reagents and disposable plastic ware in all procedures is highly recommended.

The techniques in this chapter incorporate a labor intensive, specialized subculture routine requiring manual colony manipulation, slicing, and microdissection under stereo optics. The key objective of this subculture routine is the passage of cells in clusters of approx 400-500 cells and the elimination of differentiated cells from the subculture. An obvious drawback of this method is the difficulty in generating sufficient numbers of cells for clinical application and some large-scale experiments.

Nevertheless, we feel that this technique will be particularly useful for the expansion of critical very early passage hES cell stock and for propagating hES cell lines that are not amenable to bulk culture protocols. In addition, manual colony slicing results in the growth of larger colonies, thus making colony selection and harvesting for experiments easier. It is also possible when harvesting hES cells for experiments to slice into the peripheral regions of the hES colony to avoid harvesting feeder cells and degenerating cells at the core.

Enzymatic dispersion and the bulk-culture of hES lines on human feeders is also possible (see Subheading 3.5.2.). Interestingly, this often leads to an acceleration of growth and an overall decrease in colony differentiation. A recent study has reported karyotypic instabilities in mid to late passage hES cell lines subcultured in this manner on MEFs (9). It remains to be seen to what extent culture conditions can influence and affect the karyotypic stability of these lines.

The procedures for the handling and subculturing of hES cells described in this chapter are conservative and it is likely that with future refinements, a less labor-intensive approach enabling the routine and massive scale-up of cells in defined culture conditions as well as the low-density clonal growth of cells will be possible.

3.2. hES Colony Morphology and Grading hES colonies on human feeder layers are spindle-shaped with straight edges and appear to be thinner than the rounder hES colonies, which typically form on MEF feeders (Fig. 1A-F) (3). Mature 6- to 8-d-old hES colonies approx 1-2 mm in diameter are serially passaged by slicing and microdissection. Colonies passaged by manual slicing are considerably larger than bulk cultured, enzymatically passaged colonies and often appear to contain a central darker "blob" of cells (Fig. 1A,C,E). This darker central region of the colony represents piled up differentiating hES cells and should be avoided during serial passage. Good undifferentiated regions of the hES colony are generally found at the colony periphery adjacent to the colony-feeder layer boundary (Fig. 1G).

With a little practice, it is easy to differentiate between morphologically differentiated and undifferentiated regions of the hES colony. Undifferentiated individual hES cells have a high nucleus:cytoplasm ratio (Fig. 1H), which is visible under high magnification. Undifferentiated cells are tightly packed together with very little intercellular space between cells (Fig. 1H). Differentiating cells are morphologically diverse, but in culture conditions containing serum, an epithelioid cell type appears rapidly during hES cell differentiation (Fig. 2A,B).

Good undifferentiated hES colonies have sharp defined colony boundaries (Fig. 1G), whereas colonies on the onset of differentiation have diffused and "fuzzy" colony boundaries (Fig. 2C). For serial passage harvest, the outer perimeter of colonies that appear at least 50% undifferentiated. A small amount of spontaneous differentiation approx 5-10% is usually present even in well-maintained hES cultures.

3.3. Serial Passaging and Transfer of hES Colonies on Human Feeder Layers

3.3.1. Microdissection and Slicing of hES Colonies Under Stereo Optics

1. A sterile 30-G hypodermic needle affixed to a 1-mL syringe is used for slicing hES colonies. Work with x2.0-3.0 magnification under a stereomicroscope. Because colony slicing takes time, work on a 37°C warmed surface in a laminar flow hood to keep cells warm (see Note 8).

2. Remove hES media from organ culture dish and wash cells twice with warm PBS (+) solution.

3. Perform colony microdissection/slicing with 30-G needle under microscope control with the left hand holding the dish. It helps to support the wrist of the right hand with the needle on the bench top.

4. Avoid harvesting morphologically differentiated colony sections and the central region of the colony that contains piled up cells. As a rule of thumb, harvest only colony edges with good undifferentiated hES cell outgrowths. See Fig. 1 for examples of undifferentiated and Fig. 2 for examples of differentiated hES colonies.

Fig. 1. Undifferentiated hES colonies on human and MEF feeder layers. (A) x5 phase contrast image of a hES cell colony on a Detroit 551 human fetal feeder layer. (B) x5 phase contrast image of a hES cell colony on a MEF feeder layer. (C) Bright field image of a hES cell colony on Detroit 551. (D) Bright field image of a hES cell colony on a MEF feeder layer. (E) Five 6-d-old hES cell colonies growing on Detroit 551. (F) Six 6-d-old hES cell colonies growing on MEFs. (G) x20 phase contrast image of an undifferentiated well-defined hES cell colony edge on Detroit 551. (H) x40 phase contrast image of individual undifferentiated hES cells in a colony on D551. Scale bars: A-D, 500 |im; G, 100 |im; H, 50 |im.

Fig. 1. Undifferentiated hES colonies on human and MEF feeder layers. (A) x5 phase contrast image of a hES cell colony on a Detroit 551 human fetal feeder layer. (B) x5 phase contrast image of a hES cell colony on a MEF feeder layer. (C) Bright field image of a hES cell colony on Detroit 551. (D) Bright field image of a hES cell colony on a MEF feeder layer. (E) Five 6-d-old hES cell colonies growing on Detroit 551. (F) Six 6-d-old hES cell colonies growing on MEFs. (G) x20 phase contrast image of an undifferentiated well-defined hES cell colony edge on Detroit 551. (H) x40 phase contrast image of individual undifferentiated hES cells in a colony on D551. Scale bars: A-D, 500 |im; G, 100 |im; H, 50 |im.

Fig. 2. Differentiating hES colonies on human fibroblast feeder layers. (A) x10 phase contrast image of differentiated cells in a partially differentiated hES cell colony on Detroit 551. (B) x20 phase contrast high magnification image of a common differentiated cell type. (C) x5 phase contrast image of a whole partially differentiated hES cell colony on Detroit 551. Arrow indicates colony region that is differentiated; this area lacks a well-defined colony-feeder layer boundary. (D) x10 phase contrast image of a hES cell colony showing extensive differentiation. More than two differentiated cell types are visible, and all cells have lost the high nucleus to cytoplasm ratio that is typical of hES cells. Scale bars: A,D, 250 pm; B, 100 pm; C, 500 pm.

Fig. 2. Differentiating hES colonies on human fibroblast feeder layers. (A) x10 phase contrast image of differentiated cells in a partially differentiated hES cell colony on Detroit 551. (B) x20 phase contrast high magnification image of a common differentiated cell type. (C) x5 phase contrast image of a whole partially differentiated hES cell colony on Detroit 551. Arrow indicates colony region that is differentiated; this area lacks a well-defined colony-feeder layer boundary. (D) x10 phase contrast image of a hES cell colony showing extensive differentiation. More than two differentiated cell types are visible, and all cells have lost the high nucleus to cytoplasm ratio that is typical of hES cells. Scale bars: A,D, 250 pm; B, 100 pm; C, 500 pm.

5. Make cuts along the perimeter of undifferentiated colony edges well into the feeder layer then slice inwards into the colony.

6. After slicing is completed, aspirate off PBS (+) and add 1 mL of warm Dispase solution.

7. Incubate for approx 2-5 min at 37°C. Observe the dish carefully.

8. During the incubation period, you should be able to see the feeder layer disintegrating and retreating away from the hES colony fragments. After the feeder cells have detached from the colony, the sliced hES fragments also begin to round up and detach from the plastic; you will be able to see the hES fragments curl up slightly at the edges and begin to lift off from the dish. At this point, use a Gilson P20 to blow gently at the edge of a hES colony fragment. The fragment should lift off the dish very easily.

9. Dislodge all sliced fragments this way. As a rule of thumb, try minimizing the incubation period with Dispase.

10. Wash sliced colony fragments twice by picking up all colony fragments from the Dispase treated plate and transferring them to two new one-well dishes filled with warm PBS (+) sequentially.

11. Place four to five sliced fragments on a new human fibroblast feeder dish (Fig. 1E). After fragments have settled on the feeder layer, transfer dish very carefully to a humidified incubator at equilibrated at 37°C and 5% CO2.

12. hES cells should be transferred to fresh feeder layers every 6-8 d; this is the duration of one serial passage.

13. Feeder layers perform optimally 24-48 h after mitomycin C treatment, so hES cells should be subcultured onto freshly inactivated feeders within this period.

3.3.2. Enzymatic Serial Bulk Culture of hES Cells on Human Feeder Layers

For bulk culture of hES colonies, cells are cultured in six-well dishes in a medium containing 20% knockout serum replacement, 80% knockout DMEM, 1X

nonessential amino acids, 1 mM L-glutamine, 1X insulin-transferrin-selenium, and

0.1 mM P-mercaptoethanol supplemented with 6 ng/mL basic fibroblast factor.

3.3.2.1. Initiation of Bulk Cultured hES Cultures

1. Choose 10 colonies from dishes with microdissected colonies that are at least 85% undifferentiated.

2. Dissect colony peripheries; exclude harvesting the central colony core.

3. Detach sliced colony fragments from human feeder layer with Dispase treatment.

4. Wash hES colony fragments once in PBS (+).

5. Transfer fragment to a sterile 1.5-mL Eppendorf tube taking as little PBS (+) as possible.

6. Add 1 mL of warm Collagenase IV solution (1 mg/mL prepared in serum-free media-F12 DMEM).

7. Incubate Eppendorf tube at 37°C for 5 min.

8. After 5 min, dissociate colony slices into fragments of approx 30-60 cells with a 1-mL Gilson pipet. Triturate collagenase solution five times to aid fragment dissociation.

9. To dilute and inactivate collagenase enzymic activity, transfer cell suspension to 6 mL of bulk culture media.

10. Centrifuge at 170g (1000 rpm), remove supernatant, and resuspend pellet in 2 mL media.

11. Before plating hES suspension, aspirate media from feeders and seed entire 2 mL into one well of a six-well dish.

3.3.2.2. hES Bulk Culture Subculture Protocol

1. Bulk cultured cells grown in six-well dishes.

2. Human feeders are seeded at 180,000 mitomycin C treated cells per well of a six-well dish or 18,750 cells/cm2.

3. Six-well dishes are coated with 0.5% gelatin before plating feeders.

5. Add 1 mL of warm collagenase IV per well (1 mg/mL in serum-free F12 media, filtered sterile), return to incubator for 5-6 min.

6. Feeders may be detached from plastic and in cell suspension after incubation period.

7. Scrape with 5-mL serological pipet in a grid-like pattern.

8. Scrape monolayer with a cell scraper. Pay attention to edges of the well, colonies in this area are hard to remove.

9. Triturate large cell clumps with 1-mL Gilson set at 900 |L with a filtered tip and pipet up and down no more than five times.

10. Add 2 mL KO media per well to wash well and transfer cell clumps to a 15-mL Falcon. Add 1 mL more of KO media per well's worth of cells to effectively dilute collagenase activity.

11. Centrifuge at 170g (1000 rpm), remove supernatant, and resuspend pellet in appropriate volume of media. The optimal split ratio is usually 1:2 or 1:3.

12. Before plating hES-feeder suspension on fresh feeders, wash feeder layer 1X with KO and aspirate.

13. Resuspend pellet in appropriate volume (e.g., if pellet splits into four wells, resuspend in 8 mL KO).

14. Cells should be split every 5-7 d.

3.4. Characterization of hES Colonies hES cells should be tested for markers of pluripotency, the ability to form teratomas, and karyotyped regularly.

3.4.1. Immunohistochemical Characterization of hES Cell Colonies With SSEA-3, SSEA-4, Tra-1-60, Tra-1-81, and Oct-4 Cell Surface Pluripotency Markers

1. For demonstration of stem cell surface markers, Tra-1-60, Tra-1-81, SSEA-3, and SSEA-4 hES colonies in four-well slide flasks are fixed with 4% paraformaldehyde in PBS for 15-20 min.

2. Wash three times with PBS (+) and let the slide dry.

3. For intracellular antigens, fixed cells can be permeabilized with 0.1% Triton X-100/PBS (+) for 15 min at room temperature. Wash three times with PBS (+).

4. Fixed cells are blocked with 5% normal goat serum in PBS (+) for 20 min on an orbital shaker or nutator.

5. Primary antibodies (MC-631, MC-813-70, MAB-4360, MAB-4381, and SC-5279) are diluted in PBS (+) according to manufacturer's instructions and incubated overnight or 2 h (minimum) on an orbital shaker or nutator.

7. Antibody localization is performed using rabbit anti-mouse immunoglobulin secondary antibody conjugated to fluorescein isothiocyanate for Oct-4, SSEA-4 (Fig. 3A-C), Tra-1-60 (Fig. 3F,H), and Tra-1-81 (Fig. 3E,G).

8. SSEA-3 antibody localization is performed with anti-goat immunoglobulin secondary antibody conjugated to fluorescein isothiocyanate.

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Fig. 3. Immunohistochemical cell surface marker detection of hES colonies on human fibroblast feeders. (A) High magnification picture of SSEA-4 cell surface antigen staining localized with FITC labelled secondary antibody. (B) DAPI counter stain of A. (C) Two SSEA-4-stained hES colonies. (D) Alkaline phosphatase activity visualized with rhodamine filters. (E) Tra-1-81 cell surface antigen staining localized with FITC-labeled secondary antibody. (F) Tra-1-60 cell surface antigen staining localized with FITC labeled secondary antibody. (G) High magnification picture of E. (H) High magnification picture of F. Scale bars: A,B, 250 |im; C,E,F, 1000 |im; D, 500 |im; G,H, 250 |im. (Please see the companion CD for the color version of this figure.)

9. Dilute secondary antibody according to manufacturer's specifications in PBS (+) and incubate for 30-60 min at room temperature in the dark on an orbital shaker or nutator.

10. Remove secondary antibody and wash once with PBS (+).

11. Cells can be counterstained with DAPI or Hoechst dye at this point if desired.

13. Cells should be covered in PBS (+) for visualization or mounted on a slide with an anti-fade mountant if cells are stained on a cover slip.

3.4.2. Alkaline Phosphatase Activity Detection

Alkaline phosphatase activity is easily detected with the Vector Red Alkaline Phosphatase Substrate Kit I using the manufacturer's recommended protocol and viewed under bright field optics or with rhodamine excitation and emission filters (Fig. 3D).

3.4.3. Teratoma Formation in SCID Mice

Morphologically undifferentiated regions of postwarmed HES-2, HES-3, and HES-4 colonies grown on D551/CCL110 were sliced into clumps of approx 300-400 cells each. About 1 x 106 cells are injected with a sterile 27-G needle into the thigh muscle of anesthetized SCID mice (see Note 9). Tumors usually become palpable or visible by the 6th week and mice are sacrificed at the 8th or 12th week. Mice are sacrificed by cervical dislocation, tumors dissected and fixed overnight in 4% paraformaldehyde, then embedded in paraffin and examined histologically after hematoxylin and eosin staining (Fig. 4).

3.4.4. Karyotyping

Although general karyotyping protocols are straightforward, getting sufficient numbers of good metaphase spreads with hES cells is not easy. This is partly because hES cells have a low mitotic index. Thus metaphase spread preparation and karyotype interpretation is best done by a qualified cytogeneti-cist. To reduce ambiguity in interpreting karyotype data, it is best to karyotype a male hES cell line supported on a female human fibroblast feeder.

1. In general, hES colonies are incubated with 50 |g/mL Colcemid solution for 0.5-2.5 hr at 37°C and in a 5% CO2 in air atmosphere.

2. Cells are removed from the feeder layer, trypsinized, washed with PBS, and pellets resuspended and incubated with 0.075 M KCl for 30 min at 37°C.

3. After treatment with hypotonic solution, cells are fixed with freshly prepared 3:1 methanol:glacial acetic acid three times and dropped onto precleaned chilled glass slides.

4. Chromosome spreads are digested with bacto-trypsin and banded with Leishman's or Giemsa stain and photographed digitally under x100 magnification (see Note 10).

Fig. 4. Ectodermal, endodermal, and mesodermal components of teratomas derived from SCID mice injected with hES cells grown on human fibroblast feeders. (A) x20 magnification of developing bone. (B) x10 magnification of cartilage and glandular structures. (C) x5 magnification showing developing gut and intestines. (D) x10 magnification of hyaline cartilage. (E) x10 magnification of a cluster of neural rosettes. (F) x20 magnification of a cluster of pigmented epithelial cells. Scale bars: A,F, 100 |im; B,D, 250 |im; C, 1000 |im; E, 1000 |im.

Fig. 4. Ectodermal, endodermal, and mesodermal components of teratomas derived from SCID mice injected with hES cells grown on human fibroblast feeders. (A) x20 magnification of developing bone. (B) x10 magnification of cartilage and glandular structures. (C) x5 magnification showing developing gut and intestines. (D) x10 magnification of hyaline cartilage. (E) x10 magnification of a cluster of neural rosettes. (F) x20 magnification of a cluster of pigmented epithelial cells. Scale bars: A,F, 100 |im; B,D, 250 |im; C, 1000 |im; E, 1000 |im.

5. At least 20 metaphase spreads and 5 banded karyotypes at 200-band resolution should be evaluated for chromosomal rearrangements (Fig. 5).

3.4.5. Semiquantitative RT-PCR Detection of Pluripotent Markers hES cells can be tested with semiquantitative RT-PCR for several molecular markers of the pluripotent phenotype. This is in contrast to the limited array of antibodies against immunochemical cell surface markers.

Fig. 5. Normal 46, XY karyotype of a mid-passage hES cell line grown on human fibroblast feeder layers.

1. Total RNA is routinely extracted from hES cells with TRIzol reagent following the manufacturer's protocol and treated with DNAse I using the DNA-free reagent from Ambion.

2. First-strand synthesis is performed using the SuperScript II first-strand synthesis system for RT-PCR. One microliter of first-strand reaction was used for each 50 |L PCR reaction together with 50 pmol of forward and reverse primers.

3. Initial denaturation was carried out at 94°C for 2 min, and followed by 35 cycles of PCR (94°C for 30 s, 55°C for 30 s, 72°C for 1 min) and a final extension cycle at 72°C for 5 min.

4. One-tenth of each PCR reaction was loaded on a 1.5% 1X TAE agarose gel and size fractionated (Fig. 6).

Primers used: Gene Forward primer

ACTB 5'-tggcaccacacctttctacaat-gagc-3'

NANOG 5'-ggcaaacaacccacttctgc-3' DNMT3B 5'-ctcttaccttaccatcgacc-3' REX1 5'-tctagtagtgctcacagtcc-3' SOX2 5'-ccgcatgtacaacatgatgg-3' OCT4 5'-cgrgaagctggagaagga-gaagctg-3'

Reverse primer Band size (bp)

5'-gcacagcttctccttaatgt- 400 cacgc-3'

5'-tgttccaggcctgattgttc-3' 493

5'-ctccagagcatggtacatgg-3' 433

5'-tctttaggtattccaaggact-3' 418

5'-cttcttcatgagcgtcttgg-3' 370

5'-caagggccgcagcttaca- 247 catgttc-3'

Fig. 6. Semiquantitative RT-PCR analysis for markers of pluripotency. Lane 1 = 100 bp DNA ladder, lane 2 = ACTB, lane 3 = NANOG, lane 4 = DNMT3B, lane 5 = REX1, lane 6 = SOX2, and lane 7 = OCT4. One-tenth of the PCR mix was loaded into each lane of a 1.2% 1 X TAE agarose gel and run at 100 V for 1 h.

Fig. 6. Semiquantitative RT-PCR analysis for markers of pluripotency. Lane 1 = 100 bp DNA ladder, lane 2 = ACTB, lane 3 = NANOG, lane 4 = DNMT3B, lane 5 = REX1, lane 6 = SOX2, and lane 7 = OCT4. One-tenth of the PCR mix was loaded into each lane of a 1.2% 1 X TAE agarose gel and run at 100 V for 1 h.

3.4.6. Real-Time Quantitative RT-PCR Detection of Pluripotent Markers Quantitative real-time PCR can be performed using TaqMan (see Note 11).

1. Total RNA is extracted from hES colonies using TRIzol reagent and first-strand synthesis performed using the SuperScript III first-strand synthesis system for RT-PCR.

2. Primer-probe sets for these target genes can be obtained from ABI's Assay on Demand service. Gene expression is usually normalized to endogenous housekeeping genes such as 18S rRNA, GAPDH, or ACTB levels (see Note 12).

3. Equal amounts of input cDNA (25 ng) must be used per reaction and all PCR reactions performed in triplicate (see Note 13).

4. Real-time PCR analysis is conducted using the ABI PRISM 7000 Sequence Detection System (ABI).

5. After an initial denaturation for 10 min at 95°C, real-time PCR was carried out using 40 cycles of PCR (95°C for 15 s, 60°C for 1 min).

6. Changes in gene expression levels are calculated using the AACT method (10).

3.5. Classification of Human Feeder Layers

3.5.1. Supportive Human Feeder Layers

In a ranking study, we found that human fetal muscle fibroblasts derived from 14-wk human abortuses were the best supportive human feeders (4). The human fetal muscle feeder layer was capable of supporting both the prolonged undifferentiated growth of existing hES cell lines for more than 60 weekly serial passages and the derivation of a new hES line as well (4). A commercially available fetal skin fibroblast cell line Detroit 551 (CCL-110) from ATCC

(American Type Culture Collection, Manassas, VA) also performed very well as a supportive human feeder and was ranked second. Human fibroblast feeders derived from two adult skin biopsy samples from different patients also supported undifferentiated hES growth and were ranked third (4). Other groups have reported that various isolates of human foreskin fibroblasts (5,7) and even human adult marrow cells (6) are capable of supporting undifferentiated hES cell growth.

3.5.2. Nonsupportive Human Feeder Layers

Not all human fibroblasts support the growth of hES cells. In particular, two widely used and well characterized human fetal lung fibroblast cell lines MRC-5 and WI-38 performed dismally (4). The BJ foreskin fibroblast line also did not perform optimally (4). Interestingly, data from our group and other published reports suggest that supportive human fibroblast feeders may be obtained from various isolates of fetal, neonatal, or adult skin.

It is important to note that not all human fibroblasts survive mitomycin C treatment well; this could be a chief consideration in evaluating the efficacy of a particular human fibroblast feeder for hES support. We have noticed that the plating efficiencies of some fibroblasts are dramatically decreased after mito-mycin C treatment; this is often accompanied by extensive cell death. It is strongly recommended that different batches of human fibroblast feeder cells be tested for hES cell support before use.

3.5.3. Factors Secreted by Human Feeder Cells

An exogenous supply of the cytokine leukemia inhibitory factor (LIF) is sufficient to keep many mouse ES cell lines undifferentiated in culture (11,12). hES cells, on the other hand, appear not to have a perceivable response to LIF (1,2). Indeed, LIF appears not to elicit the same response of self-renewal in hES cells as it does in mouse ES cells (1-3). Many hES cell lines have been derived and propagated without LIF supplementation as well (3). Some recent reports also indicate that the LIF receptor and its cognate receptor, GP130 are expressed at very low levels in hES cells, suggesting that the LIF pathway may be inactive in hES cells (13-15).

The identity of the growth factor or group of growth factors secreted by supportive human feeder layers or MEFs, which promotes the self-renewal and undifferentiated expansion of hES cells, is still unknown. Several transcription profiling studies have suggested that members of the Wnt, BMP4/transforming growth factor-P, and FGF family may act synergistically in providing the extracellular cues necessary for hES cells to divide and self-renew (13-17). The role of direct hES cell and feeder contact though clearly of importance is also not well understood and warrants further study.

3.5.4. Thawing and Expansion of Human Fibroblast Feeders

1. Remove a vial from liquid nitrogen storage and let nitrogen vapor disperse for 30 s before performing a quick thaw in a 37°C water bath (see Note 7).

2. Carefully swirl the vial in a 37°C water bath, being careful not to immerse vial above the level of the cap.

3. When ice crystals have thawed, swab vial with 70% isopropanol, remove cells, and transfer to a 15-mL conical tube.

4. Add 10 mL human feeder media slowly dropwise to reduce osmotic shock.

5. Centrifuge cells at 250g for 5 min.

6. Remove supernatant and resuspend cell pellet in 5 mL media, transfer to a T75 flask with warm 15 mL media, and place in an incubator calibrated at 37°C, 5% CO2.

7. Change media the next day and split cells in approx 2 or 3 d, when they become 90% confluent.

4. Notes

1. Make sure white precipitate visible when L-glutamine is thawed is completely dissolved before use. To do this warm L-glutamine for about 5-10 min in a 37°C water bath.

2. Mitomycin C solid is not light sensitive. Mitomycin C solution, on the other hand, is extremely light sensitive and aliquots should be wrapped in aluminum foil and stored at 4°C.

3. All stock solutions should be aliquoted and stored in the appropriate conditions as listed in Subheading 2.

4. Gloves and safety glasses should be worn when handling mitomycin C; it is carcinogenic and highly cytotoxic. Appropriate procedures for the disposal of this hazardous compound should be strictly followed.

5. If a black precipitate develops, discard the stock solution and prepare a fresh batch; the precipitate is toxic to cells. Mitomycin C solutions need not be sterile filtered.

6. Gelatin is used to coat one-well organ culture dishes before plating feeder cells to aid feeder cell and hES cell attachment.

7. Always wear protective face shield and cryogloves when handling liquid nitrogen. Nitrogen vapors escaping from cryovials at high pressure may cause occasional cryovial explosions. It is recommended that cryovial caps be loosened immediately to release pressure after removal from N2W storage then screwed tight again. Alternatively, wait 5 min for N2 vapors to disperse before plunging into 37°C water bath.

8. All laminar flow cabinets in our laboratory have a heated base and a mounted stereomicroscope with a small footprint.

9. It is best to inject at least two SCID mice per cell line per experiment.

10. Karyotyping protocols need to be optimized for different hES cell lines at different passages.

11. We favor the use of robust TaqMan chemistry together with predesigned primerprobe sets from ABI, which are guaranteed to be optimally efficient (18). This saves time in designing PCR amplicons and optimizing reaction conditions. Firststrand synthesis should be performed with random hexamers if using ABI designed TaqMan probes because most ABI TaqMan probes are designed to anneal in the 59 UTR of target genes.

12. Examples of markers of pluripotency include OCT4, SOX2, NANOG, and REX1; examples of early markers of differentiation include AFF, ND1, and BMP4.

13. Accurate pipetting is crucial to the success of the experiment and it is recommended that a dedicated set of pipets be reserved for quantitative PCR.

References

1 Thompson, J. A., Itskovitz-Eldor, J., Shapiro, S. S., et al. (1998) Embryonic stem cell lines derived from human blastocysts. Science 282, 1145-1147.

2 Reubinoff, B. E., Pera, M. F., Fong, C. Y., Trounson, A., and Bongso, A. (2000) Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Nat. Biotechnol. 18, 399-404.

3 Richards, M., Fong, C. Y., Chan, W. K., Wong, P. C., and Bongso, A. (2002) Human feeders support prolonged undifferentiated growth of human inner cell masses and embryonic stem cells. Nat. Biotechnol. 20, 933-936.

4 Richards, M., Tan, S., Fong, C. Y., Biswas, A., Chan, W. K., and Bongso, A. (2003) Comparative evaluation of various human feeders for prolonged undifferentiated growth of human embryonic stem cells. Stem Cells 21, 546-556.

5 Amit, M., Margulets, V., Segev, H., et al. (2003) Human feeder layers for human embryonic stem cells. Biol. Reprod. 68, 2150-2156.

6 Cheng, L., Hammond, H., Ye, Z., Zhan, X., and Dravid, G. (2003) Human adult marrow cells support prolonged expansion of human embryonic stem cells in culture. Stem Cells 21, 131-142.

7. Hovatta, O., Mikkola, M., Gertow, K., et al. (2003) A culture system using human foreskin fibroblasts as feeder cells allows production of human embryonic stem cells. Hum. Reprod. 18, 1404-1409.

8. Schuldiner, M., Yanuka, O., Itskovitz-Eldor, J., Melton, D. A., and Benvenisty, N. (2000) Effects of eight growth factors on the differentiation of cells derived from human embryonic stem cells. Proc. Natl. Acad. Sci. USA 97,11,307-11,312.

9 Draper, J. S., Smith, K., Gokhale, P., et al. (2004) Recurrent gain of chromosomes 17q and 12 in cultured human embryonic stem cells. Nat. Biotechnol. 22, 53-54.

10 Livak, K. J. and Schmittgen, T. D. (2001) Analysis of relative gene expression using quantitative PCR and the 2-AACT method. Methods 25, 402-408.

11 Smith, A. G., Heath, J. K., Donaldson, D. D., et al. (1988) Inhibition of pluripotent embryonic stem cell differentiation by purified polypeptides. Nature 336, 688-690.

12 Williams, R. L., Hilton, D. J., Pease, S., et al. (1988) Myeloid leukaemia inhibitory factor maintains the developmental potential embryonic stem cells. Nature 336, 684-687.

13 Sperger, J. M., Chen, X., Draper, J. S., et al. (2003) Gene expression patterns in human embryonic stem cells and human pluripotent germ cell tumors. Proc. Natl. Acad. Sci. USA 100, 13,350-13,355.

14 Richards, M., Tan, S. P., Tan, J. H., Chan, W. K., and Bongso, A. (2004) The tran-scriptome profile of human embryonic stem cells as defined by SAGE. Stem Cells 22, 51-64.

15 Bhattacharya, B., Miura, T., Brandenberg, R., et al. (2004) Gene expression in human embryonic stem cell lines: unique molecular signature. Blood 103, 2956-2964.

16 Sato, N., Meijer, L., Skaltsounis, L., Greengard, P., and Brivanlou, A. H. (2004) Maintenance of pluripotency in human and mouse embryonic stem cells through activation of Wnt signaling by a pharmacological GSK-3-specific inhibitor. Nat. Med. 10, 55-63.

17 Sato, N., Sanjuan, I. M., Heke, M., Uchida, M., Naef, F., and Brivanlou, A. H. (2003) Molecular signature of human embryonic stem cells and its comparison with the mouse. Dev Biol. 260, 404-413.

18 Applied Biosystems, Foster City, CA, application note. "Amplification Efficiency of TaqMan, Assay on Demand Gene Expression Products." January 2004.

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