Teratoma Formation Pluripotency Analysis Report
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1 Teratoma Formation Pluripotency Analysis Report Project and Customer Information Service Title: Teratoma formation pluripotency analysis Customer: PepGel LLC PI/Contact: Susan, Service Descriptions Cell type: Human ipscs Cell line & passage: ASC ipsc-13 Mouse type: Fox Chase SICD-beige, male, 8 weeks old, from Charles River Cell quantity for inoculation: 1.5 million/site, in 50% Pepgel (0.5% PGmatrix solution) Control: same cells, same concentration, in 30% Matrigel Injection date: March 21, 2016 Report date: May 13, 2016 Project manager: Qi Zheng Contact: Qi Zheng Mouse A (Pepgel) Mouse B (Matrigel) Injection sites kidney capsule, testis kidney capsule, testis Tissue harvested One kidney tumor One kidney tumor, one testis tumor Days post-injection H&E Histology Procedure Histology: Tumor tissues were fixed with 10% Formalin over night, embedded in paraffin, cut into 5- m serial sections, H&E stained Three embryonic germ cell layers: endoderm, mesoderm and ectoderm Endoderm: digestive system (includes liver and pancreas), respiratory system, most glands Mesoderm: muscle, blood vessels, much of the genital-urinary system, skeletal system Ectoderm: skin, hair, nails, sweat and mammary glands, nervous system (including hypothalamus and both lobes of the pituitary gland), oral and nasal cavities, portions of the vagina, vestibule, penis and clitoris
2 1. Tumor collection Kidney tumors, testis tumor or testis were collected at each injection sites from two mice. 2. Identification of germ layers from kidney tumors derived from the ASC ipsc-13 cells in Pepgel (H&E staining) EN: Gland (200x) ME: Cartilage (200x) EC: Neuronal Rosette (200x) ME: Blood vessel (200x)
3 EC: Pigment cells (200x) EC: Neuronal rosette (200x) Figure 1. Histological analyses of kidney tumors injected with the ASC ipsc-13 cells/pepgel. Variously differentiated tissues that represent the three germ layers are shown and indicated by arrow heads. Abbreviations: EN, endoderm; ME, mesoderm; EC, ectoderm. 3. Identification of germ layers from kidney tumors derived from the ASC ipsc-13 cells in Matrigel (Control, H&E staining) EN: Gland (200x) ME: Cartilage (200x)
4 EC: Pigment cells (200x) EC: Epidermal cells (200x) ME: Bone (200x) ME: Blood vessel (200x) EC: Neuronal rosette (200x)
5 Figure 2. Histological analyses of kidney and testis tumors injected with the ASC ipsc-13 cells/matrigel. Variously differentiated tissues that represent the three germ layers are shown and indicated by arrow heads. Abbreviations: EN, endoderm; ME, mesoderm; EC, ectoderm. Summary Forty-four days post inoculation, the tumors were big enough for harvest. One kidney tumor was collected from kidney injection sites in SCID mouse injected with ASC ipsc-13 cells/pepgel; one kidney and one testis tumor were harvested from the injection sites injected with ASC ipsc-13 cells/martrigel. Histology results indicated that within the each tumor, there was well differentiated teratoma composed of scattered regions of differentiated cells and a large population of undifferentiated neoplastic cells. The tissues listed above indicate small areas of differentiated cells, which represent all three germ layers. Overall, there was some degree of differentiation of these cells with organized structures, suggesting that some of these cells are pluripotent. There was no obvious difference for teratoma formation between Pepgel and Matrigel. Project manager Signature: Date: 05/13/2016 Qi Zheng, Ph.D. Senior Scientist Reviewed and proved by Signature: Date: 05/13/2016 Jinling Li, Ph.D. VP, Operation Management
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