12 research outputs found
A Scalable System for Production of Functional Pancreatic Progenitors from Human Embryonic Stem Cells
Development of a human embryonic stem cell (hESC)-based therapy for type 1 diabetes will require the translation of proof-of-principle concepts into a scalable, controlled, and regulated cell manufacturing process. We have previously demonstrated that hESC can be directed to differentiate into pancreatic progenitors that mature into functional glucose-responsive, insulin-secreting cells in vivo. In this study we describe hESC expansion and banking methods and a suspension-based differentiation system, which together underpin an integrated scalable manufacturing process for producing pancreatic progenitors. This system has been optimized for the CyT49 cell line. Accordingly, qualified large-scale single-cell master and working cGMP cell banks of CyT49 have been generated to provide a virtually unlimited starting resource for manufacturing. Upon thaw from these banks, we expanded CyT49 for two weeks in an adherent culture format that achieves 50â100 fold expansion per week. Undifferentiated CyT49 were then aggregated into clusters in dynamic rotational suspension culture, followed by differentiation en masse for two weeks with a four-stage protocol. Numerous scaled differentiation runs generated reproducible and defined population compositions highly enriched for pancreatic cell lineages, as shown by examining mRNA expression at each stage of differentiation and flow cytometry of the final population. Islet-like tissue containing glucose-responsive, insulin-secreting cells was generated upon implantation into mice. By four- to five-months post-engraftment, mature neo-pancreatic tissue was sufficient to protect against streptozotocin (STZ)-induced hyperglycemia. In summary, we have developed a tractable manufacturing process for the generation of functional pancreatic progenitors from hESC on a scale amenable to clinical entry
Dynamic Chromatin Remodeling Mediated by Polycomb Proteins Orchestrates Pancreatic Differentiation of Human Embryonic Stem Cells
Body weight affects behavioural indication of thermal nociceptive threshold in adult domestic cats (Felis catus)
Carbon dioxide (CO2 ) thermal lasers have previously been validated for the assessment of nociception in cats. This experiment sought to assess the potential impact of factors associated with age, sex, body weight and sterilisation upon nociceptive threshold as measured by latency to display a behavioural response. Cats (N = 113) were exposed to a CO2 thermal laser three times during a 45â60 min test period depending upon the interval between tests. A minimum of 15 min elapsed between consecutive tests on any one individual. Time to display either a skin twitch or withdrawal was measured. Intra-class correlations showed the three measurements to be repeatable across tests for any given cat (ICC = 0.482; P < 0.001). Males had a signiïŹcantly longer mean latency to respond than females (14.83 s and 12.59 s respectively; P = 0.028). Analyses of co-variance established that the body weight of females signiïŹcantly affected response threshold (P = 0.013) but for males this effect was marginal (P = 0.058). All other factors included in the analyses were non-signiïŹcant. A post hoc test for males and females with overlapping body weights found no signiïŹcant differences between the sexes (P = 0.721). The precise reason for the effect of body weight on latency to respond is unknown and further exploration is needed particularly as it relates to sub-cutaneous fat deposition and skin temperature. It is concluded that, for cats, the body weight of the subject should be standardised or included in any analyses for assessment of nociception. Inclusion of body weight data in analyses may also prove useful when using a CO2 laser protocol in other species
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-ÎČ 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 1ÎČ; 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 ”m. (E) Immunofluorescence analysis of sections of d5 aggregates stained with OCT4/DAPI, or (F) FOXA2/DAPI. A single cluster of âŒfour OCT4<sup>+</sup> nuclei within the field of view is indicated (arrow). Imaged with a 20Ă 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 40Ă objective. DAPI, 4âČ,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 #18â21), grey bar (RCB-Dw: Expt #25â30), open grey bar (WCB4B: Expt #35â37). 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 10<sup>7</sup> 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<sup>+</sup> 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 (CâG). These representative mice exhibited fasting human C-peptide levels of 229â865 pM, and maximum GSIS (30 or 60 min stimulation) of 2614â3485 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 ”m (B), 500 ”m (C), 200 ”m (D, F, G), 50 ”m (E).</p