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Blood, Vol. 107, Issue 5, 2180-2183, March 1, 2006
Human embryonic stem cell–derived hematopoietic cells are capable of engrafting primary as well as secondary fetal sheep recipients
Blood Narayan et al.
107: 2180
Supplemental materials for: Narayan et al
Quantitative real-time PCR
Quantitative real time PCR was performed using an ABI Prism 7700 sequence detector, software version 1.7a (Applied Biosystems, Foster City, CA). Human microsatellite DNA was amplified using forward primer: 5’-ATT CAC GTC ACA AAC TGA ACA TTC-3’, and reverse primer: 5’-CGT TTG AAA TGT CCG TTT GTA GAT-3’. Forty cycles of three step (95°C for 15s, 60°C for 30s, and 72°C for 30s) reaction was carried out after an initial incubation at 95°C for 10 min. SYBR-green PCR Master Mix (Applied Biosystems) was used with 5 µM of each primer and 100 ng of DNA. Percentage of chimerism was calculated using human/mouse DNA controls with the minimal level of detection being 0.0001% human DNA. Data reported was confirmed with repeated PCR analyses. PCR Analyses
PCR was performed on a GeneAmp PCR System 9700 (PE Applied Biosystems, Foster City, CA). Fifty cycles of three step (94°C for 15s, 60°C for 15s, and 72°C for 15s) reaction was carried out after an initial incubation at 94°C for 4 min. Platinum Taq SuperMix kit (Invitrogen, Carlsbad, CA) was used according to manufacturer’s directions. The primers used were: Beta Actin forward: 5’-GTC CTC TCC CAA GTC CAC AC-3’, reverse: 5’-GGG AGA CCA AAA GCC TTC AT-3’; GAPDH forward: 5’-AGT CCC TGC CAC ACT CAG TC-3’, reverse: 5’-GCA CAG GGT ACT TTA TTG ATG G-3’; Gata2 forward: 5’-CTT CTC CAA GAC GCC ACT GC-3’, reverse: 5’-GGA AGA TGA GGC TGG AGA CG-3’; Nanog forward: 5’-CTT CTG CTG AGA TGC CTC AC-3’, reverse: 5’-GCT GAG GTT CAG GAT GTT GG 3’; Oct4 forward: 5’-GAT GTG GTC CGA GTG TGG TT-3’, reverse: 5’-CCG AGG AGT ACA GTG CAG TG-3’; Beta-2-microglobulin forward: 5’-GTG TCT GGG TTT CAT CAA TC-3’, reverse: 5’-GGC AGG CAT ACT CAT CTT TT-3’. Beta Actin amplified both human and sheep DNA while GAPDH and Beta-2-Microglobulin were human-specific.
Files in this Data Supplement:
- Table S1 (PDF, 11.2 KB) -
Chimerism in a secondary sheep (#2026) transplanted with 1 × 106 BM cells harvested from a primary animal that was transplanted with 100,000 CD34+/Lin— cells isolated from differentiated hESC. DNA samples were analyzed by real-time quantitative PCR. Limit of detection = 0.0001%.
- Figure S1 (JPG, 26.7 KB)
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PCR for human DNA in transplanted sheep BM samples. Lanes 1-8: transplanted sheep. Lane 9: Human DNA. Sheep #1757 (lane 4) was sacrificed and its BM harvested for transplanting into a secondary sheep.
- Figure S2 (JPG, 53.5 KB)
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RT-PCR profile of cells used for transplantation. Lane 1: H1 (passage 54) undifferentiated. Lane 2: H1 (passage 55) differentiated by co-culture on S17/day 17 cells. Lane 3: CD34+/Lin— isolated from lane 2 cells. Lane 4: Human peripheral blood.
- Figure S3 (JPG, 136 KB)
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Flowcytometry data comparing PB samples from a control sheep to a chimeric sheep. Both samples were analyzed side-by-side with the same parameters for data acquisition and analysis. 100,000 events were acquired and 7AAD was used for gating live cells as described under study design.
- Figure S4 (JPG, 12.5 KB)
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PCR for human beta-2-microglobulin (B2M) gene in methylcellulose colonies generated from a secondary sheep after a human-specific GM-CSF stimulation. Forty colonies were analyzed for B2M and for beta actin. Data shown is a repeat for the 12 positive (and 8 negative) colonies out of 40 total that were analyzed by PCR a second time for B2M.
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