Retroviral Transduction in Fetal Thymic Organ Culture Bronwyn M Owens Robert G Hawley and Lisa M Spain

Summary

T-cell development requires cytokines and intimate contact with stromal cells provided exclusively by the thymus. Consequently, an in vitro model of thymocyte differentiation, fetal thymic organ culture (FTOC), has been developed. FTOC recapitulates the normal development of T-cells derived from both mouse and human progenitor populations, providing a more rapid means to study T-cell development compared with alternative in vivo approaches. Furthermore, FTOC is easily amenable to genetic manipulation using retroviral gene transfer. In this chapter, we outline the basic FTOC technique and describe several applications, including retroviral transduction of mouse thymocyte subsets and human CD34+ stem/progenitor cells.

Key Words: Thymic microenvironment; T-cell development; FTOC; gene transfer; hematopoietic stem/progenitor cell; thymocyte progenitor; retroviral transduction; CD34+ cells; cord blood; human/mouse chimeras; double negative thymocyte.

1. Introduction

The differentiation of mature T-cells from hematopoietic stem and progenitor cells occurs almost exclusively in the thymus. The thymic microenvironment provides many factors important for T-cell development. Even the three-dimensional structure of the thymus itself is functionally important because thymic stromal cells do not substitute for the thymic microenvironment unless they are allowed to reaggregate into organoids (reviewed in ref. 1). However, fetal thymic organs can be very easily cultured ex vivo and will support the sequential differentiation of T-cell progenitors from CD4-CD8-double negative (DN) to CD4+CD8+double positive (DP) and finally to CD4+CD8-CD3+ and CD4-CD8+CD3+ single positive (SP) populations (reviewed in ref. 2).

Fetal thymic organ culture (FTOC) has been used to investigate the mechanisms of positive and negative selection, which tolerize the T-cell repertoire to self (reviewed in ref. 3). FTOC has also proved to be a useful method for elucidating the molecular and cellular mechanisms of T-cell development (1,4). In this latter case, gene transfer into developing T-cell precursors in FTOC provides a faster and more economical

From: Methods in Molecular Medicine, Vol. 105: Developmental Hematopoiesis: Methods and Protocols. Edited by: M. H. Baron © Humana Press Inc., Totowa, NJ

method (relative to germ line transgenics or bone marrow transplantation in live animals) to explore gene function during T-cell development (5-10).

Although direct retrovirus-mediated gene transfer to thymus in FTOC has been reported (5,11), in our hands transduction of hematopoietic stem/progenitor cells followed by thymic reconstitution in vitro is more efficient and reproducible. It has been shown previously that mouse FTOC can be used for the study of the development of human thymocytes from CD34+ progenitor cell populations (12-14). We extend those results and provide a detailed method here for retroviral gene transduction in human/ mouse chimeric FTOCs.

2. Materials

1. Retroviral expression plasmid with fluorescent protein marker, for example MIEV (11,15).

2. 293T Cells (ATCC CRL-1573), a human embryonic kidney cell line derived from primary human embryonal kidney transformed with adenovirus E1a carrying the SV40 large T antigen.

3. Phoenix™ eco cells (Orbigen), a 293T-derived cell line with stably transduced gag-pol and ecotropic envelope constructs.

4. HT1080 cells (ATCC CCL-121), a human fibrocarcinoma cell line.

5. NIH3T3 cells (ATCC CRL-1658).

6. Recombinant murine interleukin-3 (IL-3; 20 ng/mL) and recombinant murine stem cell factor (SCF; 100 ng/mL; Peprotech). Transfection reagents: CaCl2 solution (2.5 M); 2X HEPES-buffered saline (HeBS), pH 7.05. Store reagents at -20°C

7. Mouse breeder pairs (C57BL/6, BALB/cByJ, etc., Jackson or Taconic Laboratories).

8. Fetal liver and FTOC culture medium: RPMI culture medium (Invitrogen) supplemented with 10 to 15% heat-inactivated fetal bovine serum (FBS; BioWhittaker; see Note 1), 1% penicillin-streptomycin (Invitrogen), 10 mM HEPES, and 50 ^M |3-mercaptoethanol.

9. 293T and HT1080 culture medium: Dulbecco's modified Eagle's medium (DMEM; Invitrogen) supplemented with 10% heat-inactivated FBS, 1% penicillin-streptomycin (Invitrogen), and 10 mM HEPES.

10. Human CD34+ cell culture medium: X-VIVO 15 (BioWhittaker) containing 10% bovine serum albumin, insulin, and human transferrin serum substitute (BIT, StemCell Technologies), 100 ^M P-mercaptoethanol, 100 ng/mL each SCF and Flt-3 ligand, and 20 ng/ mL each IL-3, IL-6, and thrombopoietin (Recombinant human growth factors all from Peprotech).

11. Dissecting tools (Roboz): curved forceps, Roboz cat. no. RS-5135; fine forceps, Roboz cat. no. RS-5040; rat-tooth forceps, Roboz cat. no. RS-5248; blunt-tip scissors, Roboz cat. no. S-5962; disposable scalpels, no. 11, Fisher cat. no. S17800: Microfuge tube tissue grinder; Fisher cat. no. 05-559-26.

12. Bel-Art dissecting microscope.

13. Polybrene and protamine sulfate (Sigma).

14. Terasaki dishes (Nunc).

15. Nuclepore® Polycarbonate Track Etch Membranes, pore size 8 ^m (Whatman), wrapped in foil pouches and sterilized by autoclaving.

16. Gelfoam gelatin sponge (Pharmacia Upjohn).

17. Tissue culture dishes: 100 mm, six-well (Costar).

18. Fluorochrome-labeled antibodies, for example, PE, PerCP, APC, available from many sources, for example, PharMingen, Caltag.

19. Staining media: Hank's buffered saline or phosphate-buffered saline with 2 to 3% FBS and 0.01% sodium azide. Sodium azide should be omitted if flow sorting will be performed.

20. Flow cytometer capable of four-color analysis, for example, BD FACSCalibur, BD LSR.

21. Magnetic-bead-labeled anti-human CD34, anti-mouse CD4 and anti-mouse-CD8 antibodies and separation apparatus (Miltenyi Biotec).

3. Methods

In this chapter, we describe general approaches to the FTOC technique. The primary method involves the stimulation and retroviral transduction of fetal liver cells, followed by the repopulation of irradiation-depleted fetal thymic lobes. We also describe methods, based on studies by Yasuda et al. (16), for the isolation and trans-duction of thymocyte progenitors as an alternative to whole fetal liver. In addition to murine cells, human fetal liver and cord blood-derived CD34+ cells have been used to study human T-cell differentiation and the effects of specific transgenes introduced using retroviral vectors (17). Although many previous studies have used SCID or NOD-SCID mouse thymi for repopulations, one study showed that human thymocyte differentiation can be achieved if immunocompentent thymi are first depleted using 2-deoxyguanosine (12). We show here that irradiation is also effective for depletion of thymocytes in human/mouse chimeras. Retroviral gene transfer into human CD34+ cord blood cells can be achieved using amphotropic retroviral vectors or retroviral vectors pseudotyped with envelope proteins from the gibbon ape leukemia virus, the feline endogenous virus RD114 or the vesicular stomatitis virus G (VSV-G) glycoprotein (18). Here, we describe the use of VSV-G-pseudotyped retroviral vectors.

3.1. Retroviral Expression Plasmids

Efficient transduction and transgene expression in murine hematopoietic cells has been attained using retroviral vectors (18). We use here MSCV-based vectors containing an enhanced green fluorescent protein (GFP) gene as a FACS-selectable reporter (19-21). The gene of interest can be inserted into the vectors via unique restriction sites using standard molecular biology techniques.

3.2. Production of Ecotropic Retroviral Supernatants

1. Phoenix-eco cells should be kept subconfluent and passaged every 2 to 3 d to maximize transfection efficiency, (see Orbigen data sheet for further details).

2. Trypsinize Phoenix-eco cells and plate at 4 x 106 per 10-cm dish in DMEM + 10 % FBS the morning of the transfection. (Cells and reagents can be scaled up or down according to requirements.)

3. In the afternoon, mix the retroviral construct (10-15 ^g) with sterile water to a total volume of 400 ^L in a sterile tube.

4. Add 100 ^L of 2.5 M CaCl2 dropwise to the tube.

5. To a separate 15-mL tube, add 500 ^L of 2X HeBS. Then add DNA/CaCl2 mixture dropwise while blowing bubbles using another pipet or by vortexing the mixture.

6. Incubate at room temperature for 20 min.

7. Add 1 mL of calcium phosphate mixture to the dish of producer cells.

8. The following day, change media on producer cells (6.5 mL).

9. One day after changing the medium, collect the retroviral supernatant and pass through a 0.45-^m filter. Store at -70°C. Replace the medium on the producer cells.

10. Twenty-four h later, collect retroviral supernatants and filter as in step 9. Discard 293T-cells. Retroviral supernatants can be concentrated (see Note 2).

11. To titrate the virus, plate 2 x 105 NIH3T3 cells per well in six-well plates. Allow the cells to attach.

12. Make 10-fold dilutions of the viral supernatant and replace the medium on the NIH3T3 cells with 1 mL of diluted retroviral supernatants plus 6 ^g/mL of polybrene.

13. Twenty-four h later, replace the viral supernatant with fresh medium.

14. After a further 24 h, trypsinize the cells and analyze on a flow cytometer for GFP expression. Retroviral infectious units per mL of the supernatants can be calculated as follows:

% GFP+ x dilution factor x starting cell number per well

3.3. Production of VSV-G Pseudotyped Retroviral Supernatants

1. Trypsinize and plate 4 x 106 293T-cells per 10-cm dish in DMEM + 10 % FBS the morning of the transfection.

2. In the afternoon, mix retroviral construct (10-15 ^g), packaging construct pEQPAM3-E (10 ^g; ref. 22), and VSV-G envelope construct pMD.G (6.7 ^g; ref. 23) and sterile water to a total volume of 400 ^L.

3. Add 100 ^L of 2.5 M CaCl2 dropwise to the tube.

4. To a separate 15-mL tube, add 500 ^L of 2X HeBS. Then add DNA/CaCl2 mixture dropwise while blowing bubbles using another pipet or vortexing the mixture.

5. Proceed as described in step 6 of Subheading 3.2.

6. Viral titers can be determined using the HT1080 cell line as described for titration of retroviral supernatants on NIH3T3 cells (from step 11 of Subheading 3.2.).

3.4. Collections of Fetal Livers

1. Place mice together for breeding in the evening.

2. The following morning, check for vaginal plugs using a polished capillary. If females are plugged, separate from the males and date cages. Count this time point as d 0.5.

3. Dissections can be performed on the benchtop on ethanol-soaked paper towels supported by a polystyrene platform. For collection of fetal livers, sacrifice pregnant females (method determined by institutional guidelines) on d 14 of gestation (E14). Prepare the dissection site by soaking fur with 70% ethanol. Using sterile dissecting scissors and forceps, remove uterus containing fetuses and place in a 6-cm dish of complete RPMI on ice (see Note 3).

4. Remove the fetuses from the uterus and place them into a fresh dish of medium.

5. Pin down a fetus with abdomen facing upward and, using a dissection microscope, carefully slice open the abdomen with a scalpel to expose the liver. Remove the liver using curved fine tweezers and transfer to a well of a 24-well dish that contains medium. Alternatively, using fine jeweler's forceps, make a small horizontal slit in the abdomen just above the umbilicus. Holding the forceps slightly open, push down on either side of the slit: this will force the liver out, whereupon it can be pinched off with the forceps and transferred to a culture dish.

6. In a tissue culture hood, dissociate the fetal liver (FL) cells by pipetting and transfer to a 15-mL conical tube.

7. Bring the volume of the FL cells to 10 mL and allow any large tissue particles to settle to the bottom of the tube (5 min).

8. Transfer cells to a fresh tube and wash once with medium.

9. FL cells from two to three livers may be pooled and frozen in 90% FBS + 10% DMSO in liquid nitrogen for future use, or used immediately for retroviral transduction.

3.5. Collection of Fetal Thymic Lobes

1. Set up timed matings for plugged females as described in Subheading 3.4.

2. For fetal thymic lobes, sacrifice pregnant females according to institutional guidelines on d 15 or 16 of gestation (E15 or E16), remove fetuses and place each one in medium in a well of a 24-well plate (see Note 4).

3. Pin down the fetus at head, feet, and arms, with abdomen facing upward.

4. Using a scalpel and a dissecting microscope, carefully open the thorax to reveal the thy-mic lobes, which are two white organs located above the heart.

5. Using sterile fine straight forceps, tweeze out the individual lobes and place pairs of lobes into media in a well of a 24-well dish on ice. Transfer lobes to the tissue culture hood.

6. To prepare organ culture conditions, place pieces of Gelfoam sponge (0.5 x 0.5 cm) into wells of a six-well dish containing 2.5 mL of complete RPMI and completely soak the Gelfoam sponge in medium by squeezing it with a pipet tip. Place a Nuclepore® membrane shiny side up on each sponge.

7. Place up to four thymic lobes on each membrane. Thymi can be kept for up to 1 mo before use. Media should be changed weekly.

3.6. Retroviral Transduction

1. Thaw FL cells, wash and resuspend cells at 2 x 106 cells/mL in complete RPMI plus 20 ng/mL IL-3 and 100 ng/mL SCF for 24 to 48 h in a 37°C humidified incubator, 5 % CO2. (Retain some GFP-negative FL cells that can be used as controls for immunophenotypic analysis as described in Subheading 3.10.)

2. On the day of transduction, quickly thaw retroviral supernatants in a 37°C water bath, place on ice.

3. Replate the FL cells at a concentration of 1 x 106 cells/mL in ecotropic retroviral supernatant plus 20 ng/mL IL-3, 100 ng/mL SCF, and 6 ^g/mL polybrene in a multiwell tissue culture dish and "spinoculate" (centrifuge) cells at approx 500g for 1 h.

4. Place the cells in an incubator overnight. The following day, repeat spinoculation with additional ecotropic supernatant and incubate overnight.

5. After a 2-d transduction protocol, the transduced cells may be used directly in FTOC or sorted for purity based on GFP expression and then used in FTOC.

1. Thymic lobes must be irradiated prior to incubation with transduced/sorted FL cells. Using a sterile forceps, place Gelfoam sponge with lobes into a 6-cm dish and irradiate at 20002500 rads in a y irradiator (FTOC scheme is outlined in Fig. 1). Add 2 to 3 mL of complete RMPI to the dish until lobes are required.

2. Collect transduced FL cells, centrifuge at 500g for 5 min, and resuspend in 35 ^L of complete RPMI medium per lobe. Typically, 2-5 x 105 cells are used but fewer cells are sufficient to reconstitute thymi. However, there may be a delay in reconstitution and the development of different thymocyte subsets if fewer cells are used (dose dependence of thymic reconstitution is shown in Fig. 2).

3. Place 35 ^L of transduced cell suspension per well of a Terasaki dish. Space the drops of cells so that there is minimal risk of overflow between wells and use a separate Terasaki dish for each retroviral construct being used. Set up some untransduced cells for FTOC as negative controls.

4. Place one lobe in each aliquot of cells. Carefully invert the plate such that a hanging drop forms. Place the dishes into a humidified container, such as a Tupperware® with moist-

Fetal Thymic Organ Culture

Fig. 1. Flow diagram of fetal thymic organ culture model. E14 fetal liver (FL) hematopoietic progenitor cells are incubated in cytokines IL-3 and SCF for 24 to 48 h. Retrovirally transduced cells can be sorted based on GFP expression, mixed with E16 irradiated fetal thymi (FT), and inverted in hanging drops in Terasaki dishes. After incubating for 24 to 72 h, thymi are placed in organ culture until analysis.

Fig. 1. Flow diagram of fetal thymic organ culture model. E14 fetal liver (FL) hematopoietic progenitor cells are incubated in cytokines IL-3 and SCF for 24 to 48 h. Retrovirally transduced cells can be sorted based on GFP expression, mixed with E16 irradiated fetal thymi (FT), and inverted in hanging drops in Terasaki dishes. After incubating for 24 to 72 h, thymi are placed in organ culture until analysis.

Retroviral Transduction

Fig. 2. Higher doses of reconstituting fetal liver cells increase the percentage of CD4+8+ thymocytes. Graded doses of fetal liver cells were delivered to irradiated fetal thymic lobes as described. The percentage of CD4-8- (hatched bars) and CD4+8+ (solid bars) thymocytes after 14 d was measured by flow cytometry. Shown are the average and standard deviation of at least three lobes per dose.

Fig. 2. Higher doses of reconstituting fetal liver cells increase the percentage of CD4+8+ thymocytes. Graded doses of fetal liver cells were delivered to irradiated fetal thymic lobes as described. The percentage of CD4-8- (hatched bars) and CD4+8+ (solid bars) thymocytes after 14 d was measured by flow cytometry. Shown are the average and standard deviation of at least three lobes per dose.

Facs Plot Thymocytes

Fig. 3. DN cells reconstitute thymi. CD4/CD8 DN thymocytes selected by several rounds of negative selection using magnetic bead separation were transduced with concentrated (10X) retroviral supernatants and enriched for GFP expression (upper plots). Total DN cells (104 cells per lobe) were used to reconstitute irradiated E16 thymi and immunophenotyped after 15 d for differentiation into DP and SP populations.

Fig. 3. DN cells reconstitute thymi. CD4/CD8 DN thymocytes selected by several rounds of negative selection using magnetic bead separation were transduced with concentrated (10X) retroviral supernatants and enriched for GFP expression (upper plots). Total DN cells (104 cells per lobe) were used to reconstitute irradiated E16 thymi and immunophenotyped after 15 d for differentiation into DP and SP populations.

ened paper towels that has been pre-equilibrated in the incubator. Incubate hanging drops for 24 to 72 h.

5. The following day, prepare organ culture conditions as described in Subheading 3.5. Change the media weekly until analysis.

3.8. Isolation and Transduction of DN Thymocytes

DN thymocytes can be isolated from fetal or young adult mice. Although the majority of cells in adult mice are CD4 and/or CD8 positive (>95%), the absolute numbers of DN cells is greater in adult than fetal mice and adults may therefore may be a preferable source of DN thymocytes.

1. Dissect thymi from 4- to 6-wk old mice and dissociate thymocytes using a sterile tissue grinder. Approximately 108 total cells can be isolated per mouse.

2. Label CD4 and CD8 cells with MicroBead-labeled antibodies and isolate CD4/CD8 DN cells by negative selection according to manufacturer's directions. Two rounds of negative selection may be required to ensure purified DN cells. Different DN populations can be further separated by sorting based on expression of CD25 and CD44 (Fig. 3).

3. Resuspend DN cells in ecotropic retroviral supernatant (neat or 10X concentrated; see Note 2) supplemented with polybrene (6 ^g/mL), IL-7 (10 ng/mL), and IL-2 (10 ng/mL) and spinoculate for 1 h at 1000g.

4. Repeat spinoculation with additional retroviral supernatant 24 h later.

5. Twenty-four hours later, analyze GFP expression on flow cytometer and either sort GFP+ DN populations or use cells directly in place of fetal liver cells in FTOC as in Subheading 3.7. For reconstitution, use 104 cells/thymus.

6. Change medium on FTOC weekly until analysis. CD4/CD8 double and single positive populations develop within 2 wk.

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