Patient-Specific Embryonic Stem Cells Derived from Human SCNT Blastocysts

doi:10.1126/science.1112286

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Originally published in Science Express on 19 May 2005
Science 17 June 2005:
Vol. 308. no. 5729, pp. 1777 - 1783
DOI: 10.1126/science.1112286

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Patient-Specific Embryonic Stem Cells Derived from Human SCNT Blastocysts

Woo Suk Hwang¹^,2^*, Sung Il Roh³, Byeong Chun Lee¹, Sung Keun Kang¹, Dae Kee Kwon¹, Sue Kim¹, Sun Jong Kim³, Sun Woo Park¹, Hee Sun Kwon¹, Chang Kyu Lee², Jung Bok Lee³, Jin Mee Kim³, Curie Ahn⁴, Sun Ha Paek⁴, Sang Sik Chang⁵, Jung Jin Koo⁵, Hyun Soo Yoon⁶, Jung Hye Hwang⁶, Youn Young Hwang⁶, Ye Soo Park⁶, Sun Kyung Oh⁴, Hee Sun Kim⁴, Jong Hyuk Park⁷, Shin Yong Moon⁴ and Gerald Schatten⁷^*

¹ College of Veterinary Medicine, Seoul National University, Seoul 151-742, Korea.
² School of Agricultural Biotechnology, Seoul National University, Seoul 151-742, Korea.
³ Medical Research Center, MizMedi Hospital, Seoul 135-280, Korea.
⁴ College of Medicine, Seoul National University, Seoul 110-744, Korea.
⁵ Hanna Women's Clinic, Seoul 137-872, Korea.
⁶ School of Medicine, Hanyang University, Seoul 471-701, Korea.
⁷ Pittsburgh Development Center, Magee-Womens Research Institute, Department of Obstetrics, Gynecology, and Reproductive Sciences and Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA.

Table 2. Summary of patient-specific NT-hESC lines. ZF-blast, zona-free blastocyst; ImmS, immunosurgery; Plurip, pluripotent. The check marks denote pluripotency demonstrated by both EBs and teratomas. Normal karyotypes have been shown for each line. Lines derived from male patients are shown in blue; lines derived from female patients are in pink. [View Larger Version of this Image (23K GIF file)]

Fig. 1. hESC lines established from NT blastocysts using patient somatic cells (NT-hESCs) are pluripotent with normal karyotypes. The cell line for male NT-hESC-2 is shown in the left columns, and the cell line for female NT-hESC-3 is shown in the right columns. (A and B) Phase contrast imaging. (C to L) Pluripotency marker detections. Alkaline phosphatase(AP), (C) and (D); SSEA-1(notdetected), (E) and (F); SSEA-4, (G) and (H); TRA-1-81, (I) and (J); and Oct-4, (K) and (L) are shown. Magnification, x100; scale bars, 100 µm. (M and N) G-banded karyotyping (7) shows that NT-hESC-2 and -3 have normal XY and XX karyotypes, respectively. [View Larger Version of this Image (99K GIF file)]

Fig. 2. DNA fingerprinting analysis of 3 of 11 NT-hESCs cell lines (-2 to -4) demonstrates genetic identities with donor patient nuclei (DNA fingerprinting of NT-hESC-5 through -12 are shown in fig. S2A). Isogenic analysis in loci amelogenin (chromosome location: X, p22.1 to 22.3; Y, p11.2); D5S818 (chromosome location 5p22 to 31); and FGA (chromosome location 4q28) is shown. The boxed numbers and corresponding peaks represent locations of polymorphisms for each short tandem-repeat marker at loci amelogenin (peak: X, Y); D5S818 (peak no. 9 to 13); and FGA (peak no. 18 to 26). Figure S2 provides additional DNA fingerprinting evidence. [View Larger Version of this Image (19K GIF file)]

Fig. 3. Patient-specific human NT-hESCs differentiate into tissues from all three germ cell layers in vivo in teratomas (first three rows) and in vitro in EBs (bottom row). NT-hESC-2 (A to H), -3 (I to P), and -4 (Q to X) differentiated into all of the following somatic tissue types: skin [(A), (I), and (R)]; primitive neuroepithelium (B); striated muscle [(C) and (K)]; cartilage [(D), (L), and (T)]; renal tissues [(E) and (U)]; gastrointestinal epithelium [(F), (M), and (V)]; retina and primitive neuroepithelium [(J) and (Q)]; smooth muscle and respiratory epithelium [(G), (P), and (X)]; colon epithelium (H); mucosa gland (N); smooth muscle [(O) and (W)]; and bone (S). Immunohistochemical staining for EBs was performed for Pax3/7 (Y), MAP-2 (Z), GFAP (a), ANP (b), CD34 (c), desmin (d), and MHC (e). EBs also differentiated into all three germ layers expressing ectodermal [(Y), (Z), and (a)], mesodermal [(b) to (d)], and endodermal (e) marker genes. Figure S3 shows differentiation details for all NT-hESC lines. Magnification: x100, (A) to (R); x200, (S) to (Y). Scale bars, 100 µm. [View Larger Version of this Image (84K GIF file)]