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Cell culture SiHa cells were obtained from the American
Fig. 1. Preparation of GFP-FA3-hiPS cells for in vivo vascularized tissue engineering chambers. (A) Representative bright field micrograph of an undifferentiated GFP-FA3-hiPS cells colony cultured on human fibroblast feeder cell layer. (B) Pieces of undifferentiated GFP-FA3-hiPS cells following manual dissection. (C) Differentiated EBs derived from GFP-FA3-hiPS cells. (D) Representative micrograph of OCT3/4-stained section demonstrating pluripotent cells engrafted in the tissue construct and in close proximity to blood vessels. Representative tissue construct generated at 4 weeks post-implantation with GFP-FA3-hiPS cells stained with H&E (E), Masson’s trichrome (F) and GFP immunohistochemistry (G). Scale bar = 500 μm.Figure optionsDownload full-size imageDownload as PowerPoint slide2.2. AnimalsExperimental procedures were approved by the Animal and Human Ethics Committees of St. Vincent’s Hospital (Melbourne, Australia) and were conducted in accordance with the Australian National Health and Medical Research Council guidelines for the care and maintenance of animals. Nude rats (CBH-rnu) were purchased from Animal Resources Centre (Perth, Western Australia) and maintained with a 12 h dark/light cycle and given water and chow ad libitum.2.3. In vivo vascularized tissue engineering chamber and experimental groupsAn arteriovenous loop (AVL) was constructed in the groin region of adult male nude rats weighing between 200 and 250 g as previously described [9] and [11]. Briefly, rats were anesthetized with isoflurane inhalation (4% induction and 2% maintenance). A vein graft was dissected from the left femoral vein and interposed between the right femoral artery and right femoral vein with microanastomoses. The AVL was laid onto the base of a polycarbonate chamber (0.5 ml internal volume, 1.3 cm internal diameter and 0.5 cm height; Department of Chemical Engineering, The University of Melbourne, Melbourne, Australia), which was sutured into the underlying muscle fascia. 250 μL of Matrigel (BD Biosciences Pharmingen) containing GFP-iPS cells was placed into the well of the chamber and allowed to solidify before the lid of the chamber was attached. Skin wounds were then closed in two layers and the animals were allowed to recover. Animals were randomized to receive chambers containing undifferentiated GFP-FA3-hiPS cells (approximately 60 pieces containing 1,200,000 cells, n = 5) or differentiated GFP-FA3-hiPS cells (approximately 30 intact EBs containing 700,000 cells, n = 4).2.4. Harvest of chamber tissue and histological analysisAt 4 weeks postoperatively, rats were anesthetized and the tissue constructs formed in the chambers were harvested. The tissue constructs were fixed in 4% paraformaldehyde for 24 h, then divided into serial 2 mm thick transverse slices and all slices from each construct embedded into the same block for histology and immunohistochemistry. Paraffin-embedded 5 μm-thick sections were stained for (1) hematoxylin and eosin (H&E), (2), Masson’s trichrome, (3) toluidine blue, (4) OCT3/4 (1:200, mouse monoclonal IgG, sc-5279; Santa Cruz, CA, USA), (5) desmin (1:100, mouse anti-human monoclonal IgG, M0760; Dako, Denmark), (6) GFP (1:400, rabbit polyclonal IgG, A11122; Invitrogen), (7) α-fetoprotein (1:500, rabbit anti-human polyclonal IgG, A0008; Dako), ( cool nestin (1:100, rabbit monoclonal IgG, ab105389; Abcam, MA, USA), and (9) troponin T (1:500, mouse monoclonal IgG, ab8295; Abcam). For immunohistochemistry, sections were then incubated with the appropriate secondary antibodies: rabbit anti-mouse (1:200, E0464; Dako) or goat anti-rabbit (1:200, BA-1000; Vector, CA, USA). Peroxidase activity was visualized with diaminobenzidine (Thermo Scientific, Rockford, IL, USA) and sections were counterstained with hematoxylin before mounting. The appropriate IgG BX-795 (rabbit or mouse) were used as negative controls. The incidence of teratoma in the tissue constructs was evaluated by a pathologist (JS) in a blinded fashion.3. ResultsAll 9 rats implanted with GFP-FA3-hiPS cells remained healthy throughout the 4 weeks experimental duration. GFP-FA3-hiPS cells implanted in the in vivo vascularized chamber survived through 4 weeks post-implantation and resulted in teratoma formation in 7 of 9 chambers. The tissue constructs formed all had patent vasculature, including the original implanted arteriovenous loop and the microcirculation which forms off it and fills the chamber within the first week. Substantial tissue volumes were formed ( Fig. 1E and F) by cells from the host rat and integrated within them were cells of human origin, indicated by constitutive GFP [removed]( Fig. 1G). Although tumour formation was observed after implanting either undifferentiated (as freshly dissected pieces, Fig. 1B) or differentiated (as intact EBs, Fig. 1C) GFP-FA3-hiPS cells, chambers with differentiated GFP-FA3-hiPS cells had a higher incidence of teratoma formation (4 out of 4 chambers) compared to undifferentiated GFP-FA3-hiPS cells (3 out of 5 chambers).Detailed immunohistochemical analysis of the teratoma tissue constructs revealed human cells representing all three germ layers, including mesoderm (Fig. 2A–F), endoderm (Fig. 2G–K) and ectoderm (Fig. 2L), as well as residual undifferentiated hiPS cells expressing pluripotency markers, OCT3/4, in close proximity to blood vessels (Fig. 1D). These pluripotent cells were retained through implantation and incorporated into tissues in the chamber by 4 weeks. Interestingly, OCT3/4 positive cells were detected in all chamber constructs including the two constructs with no evidence of teratoma tissues.Fig. 2. GFP-FA3-hiPS cells differentiate into mesoderm (A–E), endoderm (G–J) and ectoderm (K) lineages. Mesoderm: (A) Representative micrograph of desmin-stained section demonstrating skeletal muscle with sarcomeric striation pattern. GFP-stained sections showing GFP-FA3-hiPS cells derived blood vessels (arrow heads in B) and adipose tissue (arrow heads in C). Representative micrographs of H&E (D and E) and toluidine blue (F) stained sections demonstrating chondrogenic and osteogenic differentiation of GFP-FA3-hiPS cells in the chambers. Some cartilaginous areas were undergoing calcification and ossification (? in D), connected by a bundle of smooth muscle cells (arrow in D) and containing chondrocytes (arrow heads in E). Endoderm: Representative micrograph of H&E (G–I) and α-fetoprotein (J) stained sections. (G) Glands in myxoid stroma (m) with epithelial structures (e) that contained secretory products and cellular debris within the lumen (?). (H) Columnar epithelium resembling hair follicles (h) and with sebaceous glands (s) in close proximity. (I) Gland structures with epithelial cells arranged in a columnar pattern (arrowheads) or in a reticular pattern (arrows) within oedematous stroma. (J) Glomeruloid structures with polypoid projections into the lumen that contained mature erythrocytes (arrow heads) and immature nucleated red blood cells (arrows). The gland structure was lined with epithelial cells with vacuolated areas within the cytoplasm. (K) Gland structures were stained positively for α-fetoprotein. Ectoderm: (L) Representative micrographs of nestin-stained sections demonstrating positive cells with filamentous extensions. Scale bar = 50 μm.Figure optionsDownload full-size imageDownload as PowerPoint slideMesoderm-derived cells included skeletal muscle (Fig. 2A), GFP-positive blood vessels (Fig. 2B), GFP-positive adipose tissues (Fig. 2C), as well as chondrocytes within developed cartilage (Fig. 2D–F). Some of the cartilaginous areas were undergoing calcification and ossification (Fig. 2D) and toluidine blue staining indicated that there was proteoglycan-containing extracellular matrix in the tumour tissues (Fig. 2F). Staining with cardiac specific marker troponin T did not detect any spontaneous cardiomyocyte differentiation in the tissue construct (data not shown). H&E staining also revealed many glandular elements lined with columnar epithelial cells (Fig. 2G–J) throughout the teratoma tissues indicating a supportive environment for endoderm differentiation of hiPS cells in the in vivo vascularized chambers. Some teratomas also contained extra-embryonic endodermal tissue where the glandular structures were arranged in a reticular pattern within the edematous stroma ( Fig. 2I) and others contained epithelial cells with vacuolated areas within the cytoplasm ( Fig 2J). The endoderm lineage was further shown by positive staining with α-fetoprotein ( Fig. 2K). Ectoderm lineage of implanted GFP-FA3-hiPS cells was shown by nestin positive structures resembling neuronal cells, with clear filamentous extensions ( Fig. 2L).4. DiscussionIn the present study, we showed for the first time that a vascularized tissue engineering chamber supports survival and differentiation of implanted GFP-FA3-hiPS cells in vivo. Implanting GFP-FA3-hiPS cells into the chamber has a high success rate in producing teratomas with all three germ cell lineages and multiple tissue types within 4 weeks. Furthermore, all animals in this study survived and remained healthy throughout the experimental period, indicating that the in vivo vascularized chamber offers a tumor-prone environment for pluripotency and safety evaluation without aggravating the welfare of host animals.Teratoma formation in immunodeficient animals has been regarded as the gold standard assay to evaluate pluripotentiality of human pluripotent stem cells and may be further developed to assess their safety prior to clinical translation into human therapies [12]. The standard teratoma assay may involve in vivo administration of pluripotent cells as a single cell suspension or small pieces. Dissociating GFP-FA3-hiPS cells into single cell suspension significantly reduces cell viability and they may not survive the implantation process. Therefore, in the present study, GFP-FA3-hiPS cells were implanted as either pieces of 2D culture or EBs to maintain their cell-cell contact to prevent anoikis and thus reduce cell death. Additionally, GFP-FA3-hiPS cells were also implanted with Matrigel to further enhance cell survival and growth [7]. Interestingly, implantation of partially differentiated GFP-FA3-hiPS cells in the form of EBs resulted in a higher incidence of teratoma formation in the chamber compared to undifferentiated GFP-FA3-hiPS cells in the form of pieces. This observation seems unlikely to be explained by the pluripotency of implanted cells because previous findings demonstrated that teratoma formation from pluripotent stem cells was positively correlated with the residual presence of undifferentiated cells [13]. Another possible explanation would be survival through implantation of higher cell numbers, as suggested by previous studies reporting that efficiency of teratoma formation in the heart was dependent on implanted cell number [8] and [14] and the site of delivery [8]. Refinements to the present model, such as the development of a modified chamber which removes the technical requirements for microanastomoses and identification of the minimum number of cells required for a successful teratoma assay in this model are the subject of ongoing studies.





 
 
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