Directed pancreatic differentiation of CyT49 in suspension culture.

<p>(A) Schematic representation of aggregation and the four-stage differentiation protocol from hESC (ES) to mesendoderm (ME), definitive endoderm (DE), primitive gut tube (PG), posterior foregut (PF), and a mixed population comprising primarily of pancreatic endoderm (PE) and endocrine precursor/endocrine cells (EP). Culture conditions, timing and rotation speeds are indicated. Markers used to identify the different stages are shown.*TBI: TGF-β RI kinase Inhibitor IV, first day of Stage-2 only. **ITS: Insulin-Transferrin-Selenium used at different concentrations in Stage-1 and -2. HrgB, heregulin 1β; D/F12, DMEM/F12; TT, TTNPB; CYC, cyclopamine; NOG, noggin. (B,C,D) Hematoxylin and eosin staining of sections of CyT49 aggregates after (B) culture in StemPro medium for 2 days (paraffin section), or (C) at d5, or (D) at d12 of differentiation (frozen sections). Scale bars, 50 µm. (E) Immunofluorescence analysis of sections of d5 aggregates stained with OCT4/DAPI, or (F) FOXA2/DAPI. A single cluster of ∼four OCT4⁺ nuclei within the field of view is indicated (arrow). Imaged with a 20× objective. Immunofluorescence analysis of d12 aggregates for (G) NKX6-1, PDX1 and CHGA expression, and (H) FOXA2, PDX1 and NKX2-2 expression. Imaged with a 40× objective. DAPI, 4′,6-diamidino-2-phenylindole.</p

Digital mRNA profiling of scaled pancreatic differentiation runs.

<p>The dynamics of gene expression demonstrated that undifferentiated cells (POU5F1) were directed through mesendoderm (Brachyury: T), definitive endoderm (SOX17, CXCR4), primitive gut tube (FOXA1), posterior foregut (PDX1), to form pancreatic epithelium (NKX6-1, PTF1A), and endocrine cells (NEUROG3, NKX2-2). Precise temporal control and consistency between manufacturing runs indicated a reproducible and robust specification of each lineage. The plots are ordered according to CyT49 cell bank (left to right): black bar (MCB4: Expt #18–21), grey bar (RCB-Dw: Expt #25–30), open grey bar (WCB4B: Expt #35–37). The average and standard deviation of three biological replicates are plotted. Additional data is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037004#pone.0037004.s006" target="_blank">Figs. S6</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037004#pone.0037004.s007" target="_blank">S7</a>.</p

Schematic representation of the manufacturing process for pancreatic progenitors.

<p>Scaled and high-density single-cell master (MCB) and working cell banks (WCB) of CyT49 cells were prepared with cGMP and serve as a virtually unlimited source of starting material for differentiation. Cryopreserved vials of 10⁷ CyT49 cells from a qualified working cell bank are thawed and expanded in adherent culture conditions for 4 or 5 passages over a 2-week period. A single cell suspension is harvested and aggregated in rotational culture in 6-well trays. After 24 hrs the hESC aggregates are differentiated en masse with the 4-stage protocol to a population of pancreatic progenitors. For clinical development, scaled lots of differentiated aggregates will be produced with cGMP and cryopreserved, enabling a proportion of each lot to be tested for safety, efficacy and other regulatory considerations. Qualified lots of differentiated pancreatic progenitors will be thawed, recovered, formulated, loaded into a durable immunoisolation device and delivered for preclinical or clinical studies. The manufacturing process is amenable to further scaling via additional passages in adherent culture, as well as aggregation and differentiation in larger vessels.</p

Histological and immunofluorescence analyses of CyT49-derived neo-pancreatic grafts at 18 weeks post-implant.

<p>(A) Hematoxylin and eosin staining of a graft cross-section, and (B) higher magnification of boxed area. (C, D) GCG, SST and INS staining in a cross-section of a graft, demonstrating single-pancreatic hormone expression and large clusters of INS⁺ cells. (E) Co-expression of NKX6-1, PDX1 and INS. (F) TRY, INS and DAPI staining. (G) CK19, PDX1 and HuNU staining. GCG, glucagon; SST, somatostatin; INS, insulin; TRY, trypsin; HuNU, human nuclear antigen; CK19, cytokeratin 19. Grafts were from expt #18 (A, B), and #20 (C–G). These representative mice exhibited fasting human C-peptide levels of 229–865 pM, and maximum GSIS (30 or 60 min stimulation) of 2614–3485 pM at week 15. An additional ten representative grafts are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037004#pone.0037004.s010" target="_blank">Figs. S10</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037004#pone.0037004.s011" target="_blank">S11</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037004#pone.0037004.s012" target="_blank">S12</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037004#pone.0037004.s013" target="_blank">S13</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037004#pone.0037004.s014" target="_blank">S14</a>. Scale bars: 3 mm (A), 300 µm (B), 500 µm (C), 200 µm (D, F, G), 50 µm (E).</p

Colony cluster banks, single cell research-, master-, and working-cell banks of CyT49.

<p>p#: passage number from derivation of the line. Hrv: total cells harvested. #V: number of vials frozen. #/vial: cells/vial. T%: thaw viability (%). na: not available. ^C: bank of colony clusters. ^G: cGMP manufacture. ^Ω: derived from MCB1. ^¶: derived from WCB1. Karyotype analyses indicates the thaw number (left column), and the number of nuclei (bracketed) for each class of result. M: multiple thaws. *: non-clonal, deemed technical.</p