Geographic variation and speciation in Rough-winged swallows (Aves: Hirundinidae: Stelgidopteryx)

This study is an examination of geographic variation and evolutionary history of Stelgidopteryx swallows. These swallows comprise two recognized species: Northern Rough-winged Swallow (S. serripennis) and Southern Rough-winged Swallow (S. ruficollis). A third species, Yucatan Rough-winged Swallow (S. ridgwayi)is also commonly recognized. The species are largely allopatric, except for a contact zone in Costa Rica. Using plumage and molecular variation, I examined the likelihood of two (or three) different species of rough-winged swallows, the genetic interrelationships among taxa, and their biogeographic history. Geographic plumage variation reveals a latitudinal cline across the genus from North to South America. Specimens from throughout the range of Stelgidopteryx show that most published subspecies should be synonymized as clinal variants. Molecular data (mitochondrial cytochrome-b sequences, microsatellite allele frequencies) show a pattern consistent with a species-level division between S. serripennis and S. ruficollis . The data also suggest that the Yucatan swallow is distinct. Phylogenetic trees from the sequence data divide Stelgidopteryx into northern and southern clades consistent with S. serripennis and S. ruficollis. The Yucatan clade, S. ridgwayi, is sister to the northern group. Microsatellite data indicate allelic frequency differences between the groups, but none fixed. Population genetic analyses among individuals within S. serripennis and S. ruficollis reveal genetic structure possibly worthy of taxonomic recognition. Measures of the population parameter theta indicate high allelic diversity in each group and suggest S. ruficollis is the younger population. The Northern Rough-winged Swallow exhibits broad clinal variation, but little subspecific subdivision. The Yucatan Rough-winged Swallow, is phenotypically and genetically dissimilar to the other groups, further supporting species-status. The Southern Rough-winged Swallow contains at least two subspecies, including decolor on the Pacific slope of Central America, and ruficollis throughout the rest of its range. The widespread distribution and contact zone in Costa Rica are consistent with secondary contact between the northern and southern swallows. The emergence of the Panamanian landbridge could have contributed to the formation of the contact zone. Pleistocene climatic shifts could also have played a role in isolating birds on opposite slopes of Central America allowing divergence between S. r. uropygialis and S. r. decolor

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 50–100 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

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

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

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

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