WAO9 P-32 August 1, 2008 Bank Characterization Report

WAO9 P-32 August 1, 2008 Bank Characterization Report

Cell Line description 3 Karyotype.. 4 5 Fluorescent in Situ Hybridization 6 7 Teratoma Assay 8 10 Flow Cytometry.. 11 Post Thaw Recovery 12 2

Cell Line Description Species Cell Line Passage Date Frozen Embryonic Stem Cell Line Human WA09 32 August 1,2008 3

Texas Children s Hospital Clinical and Research Cytogenetic Laboratory 1102 Bates Street, Suite C1070.20 Houston, Texas 77030-2399 Phone: (832)-824-4457 Fax: (832)-825-4846 Email: Cytogenetics@TexasChildrensHospital.org CYTOGENETIC RESEARCH REPORT SPECIMEN: Human Embryonic Cell Lines TECHNIQUES EMPLOYED: G-Banding; fluorescence in situ hybridization (FISH) RESULTS: Cell line MCBWA09p32Aug-01-08 Karyotype 46,XX[20] INTERPRETATION: Cytogenetic evaluation of harvested cell pellets from the cell lines revealed 46,XX normal female karyotypes in all examined cells. In addition, FISH evaluation of cell lines - MCBWA09p32Aug-01-08 using probes specific for chromosome 2, 12 and 17 centromeres revealed a normal pattern in 200 interphase nuclei and 10 metaphase spreads for each probe and a cell line. Low-level mosaicism and subtle chromosomal abnormalities may not be detectable by the techniques employed in the present study. Interpretation By: Pulivarthi Rao, Ph.D., Associate Director of Research Clinical and Research Cytogenetics/Molecular Laboratory 4

G-banded Karyotype Texas Children s Hospital MCBWA09 P32 August 1, 2008 5

Texas Children s Hospital Clinical and Research Cytogenetic Laboratory 1102 Bates Street, Suite C1070.20 Houston, Texas 77030-2399 Phone: (832)-824-4457 Fax: (832)-825-4846 Email: Cytogenetics@TexasChildrensHospital.org SPECIMEN: Human Embryonic Cell Lines CYTOGENETIC RESEARCH REPORT TECHNIQUES EMPLOYED: G-Banding; fluorescence in situ hybridization (FISH) RESULTS: Cell line MCBWA09p32Aug-01-08 Karyotype 46,XX[20] INTERPRETATION: Cytogenetic evaluation of harvested cell pellets from the cell lines revealed 46,XX normal female karyotypes in all examined cells. In addition, FISH evaluation of cell lines -MCBWA09p32Aug-01-08 using probes specific for chromosome 2, 12 and 17 centromeres revealed a normal pattern in 200 interphase nuclei and 10 metaphase spreads for each probe and a cell line. Low-level mosaicism and subtle chromosomal abnormalities may not be detectable by the techniques employed in the present study. Interpretation By: Pulivarthi Rao, Ph.D., Associate Director of Research Clinical and Research Cytogenetics/Molecular Laboratory 6

Fluorescent in Situ Hybridization Texas Children s Hospital MCBWA09p32Aug-01-08 probes specific for chromosome 2, 12 and 17 7

Investigator: Ana Crane Dept: GAGT-Star Species: Mouse Strain:SCID ID: WA09 Date Reported: January 26, 2009 Slide 1 Teratoma Formation Assay Animal Pathology Report An infiltrative tumor is found within skeletal muscle. The infiltrative tumor is in multiple sites, and has multiple foci of mesenchymal tissue that differentiates into cartilage. There are numerous rosettes of neuroectoderm and variably sized cysts lined by a single layer of columnar epithelium. This primitive tumor in an implant from a human cell line and is consistent with a teratoma Slide 2 An infiltrative tumor is found within skeletal muscle. This tumor is in multiple sites and has numerous cysts that are sometimes filled with a pale pink acellular secretion, and lined by columnar epithelium. There are multiple foci of primitive mesenchymal cells that sometimes differentiate into fibroblasts. There are multiple rosettes of neuroectoderm that sometimes have numerous mitotic figures. This primitive tumor is an implant from a human cell line and is consistent with a teratoma. 8

Investigator: Ana Crane Dept: GAGT-Star Species: Mouse Strain:SCID ID: WA09 Date Reported: January 26, 2009 Slide 3 Teratoma Formation Assay Animal Pathology Report An infiltrative tumor is found within skeletal muscle. The tumor is in multiple sites and has numerous cysts lined by columnar epithelium. There are multiple foci of primitive mesenchymal cells that tend to be elongate. There is at least one cyst lined by endothelium and filled with erythrocytes. There are numerous rosettes of neuroectoderm. This primitive tumor is an implant from a human cell line and is consistent with a teratoma Slide 5 An infiltrative tumor is found within skeletal muscle. The tumor is in multiple sites and occasionally has cysts lined by columnar epithelium. There are numerous sites of primitive mesenchymal cells that sometimes have streaming fibroblasts and islands of cartilage. There are numerous rosettes of neuroectoderm. This primitive tumor is an implant from a human cell line and is consistent with a teratoma. 9

Investigator: Ana Crane Dept: GAGT-Star Species: Mouse Strain:SCID ID: WA09 Date Reported: January 26, 2009 Teratoma Formation Assay Animal Pathology Report Slide 6 An infiltrative tumor is found within skeletal muscle. The tumor is in multiple sites and has an occasional cyst lined by columnar epithelium. There are multiple foci of primitive, elongate mesenchymal cells that produce a pale foamy matrix and sometimes differentiate into fibroblasts. There are numerous rosettes of neuroectoderm. This primitive tumor is an implant from a human cell line and is consistent with a teratoma. Alan J Herron, DVM Head Comparative Pathology, Baylor College of Medicine 10

Flow Cytometry Oct 3/4 SSEA-4 Double Labeled 11

WA09 Cell Bank Alkaline Phosphatase Stain 5 Days post Thaw 12