Multiorgan engraftment and differentiation of human cord blood CD34 + Lin − cells in goats assessed by gene expression profiling

Zeng et al. 10.1073/pnas.0602646103.

Supporting Information

Files in this Data Supplement:

Supporting Figure 5
Supporting Figure 6
Supporting Text

Fig. 5. Detection of human GFP⁺ cells in liver of the MIG-transplant goats after perfusion. (Upper) Liver cells under fluorescence microscopy. (Left) Normal goat in which no fluorescent signal occurs. (Right) MIG goat in which the liver cells show strong fluorescence. (Magnification: ´400.) (Lower) Light microscopy for a section of perfused MIG liver (Left), visible light plus fluorescent light (Center), and fluorescent light only (Right). (+)→, cells expressing GFP; (–)→, GFP-negative cell. (Magnification: ´400.)

Supporting Figure 6

Fig. 6. Southern blot analysis of human genes in the genome of the MIG-transplant goats. (Top) Southern blot of liver DNA showing the presence of human a-satellite sequence using the p17H8 probe. Lane 1, DNA from human; lane 2, normal goat; lanes 3–5, three MIG goats; lanes 6 and 7, TG goats. (Middle) Southern blot for human Y chromosome-specific DNA using the pY3.4 probe. Lanes 1 and 11, DNA samples from blood of human males; lanes 2 and 10, human female blood; lanes 3–5, liver of three MIG goats; lane 7, sorted GFP⁺ cells; lanes 8 and 9, liver of TG goats. All of the human donor cells for the MIG (including GFP⁺ cells) and TG goats are originated from human males. (Bottom) Southern blot analysis of the PCR products from liver DNA samples using the human IGF-1 probe. Lanes 1–3, PCR products from MIG goats; lanes 4–6, normal goats; lanes 7–9, humans.

Supporting Materials and Methods

Transduction of Human CD34⁺Lin^– Cells with MSCV-IRES-GFP Vector. Virus preparations were obtained by transfecting amphotropic Phoenix packaging cells cultured in DMEM plus 10% FCS (StemCell Technologies) by using CaPO₄. Virus-containing medium (VCM) was harvested 32-72 h later, filtered through a 0.45-mm filter, and used immediately for transduction. Titers of MSCV-IRES-GFP (MIG) virus were 5-9 × 10⁵ infectious units/ml, respectively, as assessed by GFP expression in NIH 3T3 cells 2 days after their exposure to limiting dilutions of VCM. Cells to be transduced were cultured at 2 × 10⁵ per ml for 48 h in Iscove’s minimal essential Dulbecco’s medium (IMDM, StemCell Technologies) supplemented with complete serum-free medium (SFM) containing the following five purified recombinant human growth factors: Flt-3 ligand (100 ng/ml; Immunex), Steel Factor (100 ng/ml; StemCell Technologies), IL-3 (20 ng/ml; Novartis), IL-6 (20 ng/ml; Cangene), and G-CSF (20 ng/ml; StemCell Technologies). The SFM consisted of IMDM supplemented with a combination of BSA, insulin, and transferrin (BIT) (StemCell Technologies), 40 mg/ml low-density lipoproteins (Sigma), and 10 mM 2-mercaptoethanol (Sigma). The cells were then resuspended in filtered VCM supplemented with the same five growth factors and 5 mg/ml protamine sulfate (Sigma) and incubated at 37°C for 12 h in fibronectin-coated Petri dishes previously "loaded" twice with VCM (each time for 30 min). This infection procedure was repeated twice, and the cells were finally transferred to fresh SFM plus the five growth factors from above (without VCM) and incubated for another 48 h before harvesting and further analysis.

Analysis of GFP⁺ Cell Distributions in Tissues of Transplant Goats. Suspensions of single cells were prepared as follows. The tissues were thoroughly washed with saline solution to remove blood traces and homogenized in PBS containing 1 mM EDTA on ice. The cells were then passed through gauze to remove tissue clumps. Liver cells were incubated with a final concentration of 0.002% collagenase type II (Worthington) and 10 units/ml DNase I (Worthington) for 20 min at 37°C, and washed with PBS. The cell suspension was filtered with a 70-mm filter and then were centrifuged at low speed (50 g) for 5 min. The cells were again passed through a 40-mm filter. A small portion of the cells were dyed with 0.4% trypan blue to differentiate live from dead cells and observed under the microscope to monitor the dispersion into a suspension of single cells.

PCR. The PCR primers for GFP amplification were 5'-CAC ATG AAG CAG CAC GAC TTC T-3' and 5'-AAC TCC AGC AGG ACC ATG TGA T-3'. The PCR products were further confirmed by DNA sequencing with an ABI 377 sequencer (Applied Biosystems).

The PCR primers for IGF-1 were as follows: HI-1, 5'-TCTGCACGAGTTACCTGTTA-3'; HI-2, 5'CAATCTACCAACTCCAGGAC-3'; GI-1, 5'-CGAGTTACCTGTTCAACACC-3'; GI-2, 5'CCTGCTGAATGAATGTCAC-3'; I-1, 5'-GCTCTGCACGAGTTACCTGT-3'; I-2, 5'CTCCTAAAGACAATGTTGGAATG-3'.

PCR primers for LOC285292 (hnRNPA3) were 5'-TATGATGGTTACAATGAAGG-3' and 5'-TATGACAGTCCTGAACAAGA-3'; PCR primers for EPB41L2 were 5'-AAGTAGCAGAAAATCAGCAG-3' and 5'-TTCAACAACAGCTTGGGTAG-3'; PCR primers for SSR1 were 5'-TAGGAGTTGCCTTCTATGGT-3' and 5'-ACATGAGTTGGATTCCTCAG-3'. The primers for hHNF-3b were 5'-CCTACGCCAACATGAACTCC-3' and 5'-GTAGCAGCCGTTCTCGAACA-3'; for hALB they were 5'-CCGATTGGTGAGACCAGAG-3' and 5'-GCAGCATTCCGTGTGGACT-3', and for GAPDH (an internal control) they were 5'-GTCTTCACCACCATGGAGAAGG-3' and 5'-GCCTGCTTCACCACCTTCTTGA-3'.

The routine PCR reaction conditions were as follows: an initial denaturation at 95°C for 3 min, followed by 45 cycles of 95°C for 30 sec and 60°C for 30 sec. For real-time PCR analysis, reaction mixtures contained 1 ml of cDNA, 5 pmol of each primer, 2 mM MgCl₂, 200 nM dNTP, and 1 unit of Taq DNA polymerase and 1 ml of 1:1,000 SYBR Green I (Biosearch) in a final volume of 25 ml . The thermal profile for PCR was 94°C for 5 min, followed by 35–40 cycles of 30 sec at 94°C, 30 sec at 60°C, and 30 sec at 72°C. At the end of each cycle, the SYBR Green was measured spectrofluorometrically. An additional 10-min incubation at 72°C was included after completion of the last cycle.

Immunohistochemistry Assays. Tissue specimens were obtained from MIG goats and from normal goats, and the human positive controls were obtained with the formal consent of the victims’ relatives or POA of healthy individuals who died from traffic accidents. The polyclonal antibodies against human b-2 microglobulin (b-2M) (NovoCastra), human serum albumin (hALB) (DAKO), and GFP (Abcam), as well as monoclonal antibodies specific for human proliferating cell nuclear antigen (PCNA) (Santa Cruz Biotechnology) and human hepatocyte-specific antigen (HSA) (DAKO), were used to detect positively staining cells.

Gene-Expression Profile Analysis Using Microarrays and Real-Time Quantitative RT-PCR. MICROARRAY ANALYSIS SUITE 5.0 (Affymetrix) was used to quantify microarray signals with default analysis parameters, except that no normalization across tissue types was applied. Normalization methods are generally based on either global signal assuming most genes are unchanged or unaltered internal housekeeping controls, but neither approach is valid for these cross-species assays. Spiked hybridization controls indicated very little technical variance for array processing steps. Quality control parameters for all human samples were within the following ranges: scale factor 0.9–3.5, background 52–83, percent genes detected 31–49%, and GAPDH 3'/5' signal ratio 1.2–8.7. GeneChip tabular data for all samples are available at the Gene Expression Omnibus, www.ncbi.nlm.nih.gov/geo. The MICROARRAY ANALYSIS SUITE output was loaded into GENESPRING 5.1 (Silicon Genetics) to identify genes with low or no signal in normal goat samples but 2.5-fold or greater signal in transplant goats and normal human samples.

Three transcripts were further confirmed by using traditional RT-PCR, real-time quantitative PCR, and sequencing analysis. Real-time quantitative RT-PCR was conducted with SYBR Green I. External controls were constructed, consisting of cDNA plasmid standards to obtain standardized quantitative results. Melting curve analyses were conducted after completion of the cycling process with the aid of a temperature ramp (from 50°C to 99°C at 1°C/5 sec) and continuous fluorescence monitoring. All reactions were independently repeated in duplicate to ensure the reproducibility of the results. Data were viewed and analyzed by using the Rotor-Gene's real-time analysis software (Rotor-Gene 3000; Corbett Life Science, Sydney). Amplification plots and the corresponding dissociation curves were examined for each sample.