Nonhuman primate parthenogenetic stem cells

1. 

   View larger version:

   • In this window
   • In a new window

   • Download as PowerPoint Slide

   Fig. 7.

   Immunological profile of Cyno-1 cells. PBLs and Cyno-1-derived neural cells were analyzed by flow cytometry to quantitate expression of M. fasicularis class I (anti-HLA-A,-B,-C) and class II (anti-HLA-DR) antigens and compared with cells stained with isotype-matched control antibodies (shaded curves). PBLs express both class I and class II (DR) antigens whereas differentiated Cyno-1-derived neurons do not express either class of antigen unless treated with IFN-γ.

   
   
2. 

   View larger version:

   • In this window
   • In a new window

   • Download as PowerPoint Slide

   Fig. 1.

   Characterization of parthenogenetic embryos and derived cell lines. (A) Parthenogenetically activated eggs at day 8 of development before ICM isolation. (B) Phase contrast of Cyno-1 stem cells growing on top of mitotically inactivated mouse feeder layer (mef). (C) Alkaline phosphatase staining. (D) Stage-specific embryonic antigen 4. (E) Tumor rejection antigen 1-60. (F) Tumor rejection antigen 1-81 staining. (G) RT-PCR octamer-binding transcription factor 4 expression in undifferentiated Cyno-1 cells. (Scale bars = 50 μm in A, 10 μm in B and D-F, and 4 mm in C.)

   
   
3. 

   View larger version:

   • In this window
   • In a new window

   • Download as PowerPoint Slide

   Fig. 2.

   In vivo differentiation of Cyno-1 cells. Cells were injected i.p. in severe combined immunodeficient mice. Eight and 15 weeks after injection, teratomas 12 and 30 mm in diameter, respectively, were isolated, fixed with 10% paraformaldehyde, and paraffin-embedded. Sections were stained with hematoxylin/eosin. The following complex structures were observed: gut (A), intestinal epithelium with typical goblet cells (gc) and smooth muscle (sm) (B), neuronal tissue with melanocytes (C), hair follicle complex with evident hair (h) and sebaceous gland (sg) (D), skin (E), cartilage (F), ganglion cells (G), and bone (H). (Scale bars = 40 μmin A,10 μmin B and D-H, and 20 μmin C.)

   
   
4. 

   View larger version:

   • In this window
   • In a new window

   • Download as PowerPoint Slide

   Fig. 3.

   Telomerase activity. (A) Cyno-1 cells, maintained in the undifferentiated state on mouse feeder layers, express telomerase activity that diminishes to undetectable levels in differentiated Cyno-1 cells. (B) RT-PCR to detect expression of the paternally expressed imprinted gene Snrpn in Cyno-1 cells (lane 1) and in adult fibroblasts (lane 2) from the same species. The housekeeping gene Gapdh is used as a control to demonstrate that equal amounts of mRNA were used.

   
   
5. 

   View larger version:

   • In this window
   • In a new window

   • Download as PowerPoint Slide

   Fig. 4.

   Nestin-positive neural precursors derived from Cyno-1 cells. (A) Neural precursors stained for nestin (green). The nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (blue). (B) Phase contrast of nestin precursors.

   
   
6. 

   View larger version:

   • In this window
   • In a new window

   • Download as PowerPoint Slide

   Fig. 5.

   Neural differentiation of Cyno ES cells in vitro. (A) Phase contrast microscopy of proliferating Cyno1-derived neural precursors at day 1 in vitro (DIV1). (B) Same cluster of precursors shown at DIV9. The total cell number has increased by 5- to 8-fold over a 9-day period. (Inset) One of many mitotic figures. Immunohistochemical analyses after 5 days of neural differentiation in the absence of bFGF and epidermal growth factor and the presence of ascorbic acid revealed positive staining for glial fibrillary acidic protein (GFAP), an astrocytic marker seen in C, and TUJ1, a neuronal marker seen in D. (E) Sequential exposure to sonic hedgehog, FGF8b, and ascorbic acid yielded an average of 25% TUJ1+ neurons coexpressing tyrosine-hydroxylase (TH), a marker for dopamine neurons. (F) HPLC revealing the release of dopamine (DA) and serotonin (Ser).

   
   
7. 

   View larger version:

   • In this window
   • In a new window

   • Download as PowerPoint Slide

   Fig. 6.

   Single-cell electrophysiology. (A) Neurons derived from Cyno-1 express voltage-dependent inward currents that are blocked by tetrodotoxin. Currents were elicited by membrane depolarizations to 0 mV every 15 s from a holding potential of -70 mV. Application of 0.5 μM tetrodotoxin inhibited >90% of these currents. (B) Inhibition was complete within 30 s of tetrodotoxin application and washed completely in <1 min.