SP cells have distinct morphology and express ABC transporters.

<p>A) SP cells and non-SP cells were isolated by FACS after Hoechst 33342 staining. The SP was not detected in the presence of verapamil. B) Hematoxylin and eosin staining of SP and non-SP cells. The majority of SP cells (arrow) had abundant, strongly eosinophilic cytoplasm; a minority were small with little cytoplasmic space (arrowhead). C) Total RNA from cells was analysed for the expression of MDR1 (ABCB1 transporter), BCRP1 (ABCG2 transporter) and β-actin by RT-PCR. D) Immunofluorescence staining of SP and non-SP cells with rat anti-mouse ABCG2 monoclonal antibody. The control was staining with goat anti-rat Ig-PE.</p

The proportion of SP cells and their colony forming potential decreases with age.

<p>A) Pancreas cells from littermates aged 5 days to 40 weeks were stained with Hoechst 33342 dye to quantitate the SP by flow cytometry (n = 7–15 per group). B) Colonies formed by SP and non-SP cells isolated from pancreas of littermates aged 3 to 12 weeks were quantified (n = 7–10 per group). C) Two colony types were observed after 10 days of culture on Matrigel.</p

SP and non-SP cells are not distinguished by markers of stem cells or pancreatic duct antigens.

<p>A) Expression of CD45 was compared for SP (green line) and non-SP (black dotted line) cells. (B–H) Expression of c-kit, Thy1, CD24, CD26, CD29, CD49f, Sca-1 by CD45⁻ SP cells. The solid black line shows staining by isotype control antibodies. (I) Expression of Sca-1 was analysed in CD45⁻ SP cells from Pdx1-GFP⁺ pancreas. (J–L) Expression of CD133, DBA lectin and CD326 in adult mouse pancreas was confirmed by immunofluoresence staining. (M–O) Expression of CD133, DBA lectin and CD326 by CD45⁻ SP cells. The solid black line shows staining by isotype control antibodies, except for DBA lectin where it represents staining by streptavidin-PE only. (P) Expression of CD326 was analysed in CD45⁻ SP cells from Pdx1-GFP⁺ pancreas.</p

Pdx1 expression reflects proliferation and CFP of SP cells.

<p>A,C) Pancreas cells from Pdx1-GFP mice were stained with Hoechst 33342 and then Pyronin Y and analysed by flow cytometry. B,D) Each Pdx1-GFP subpopulation was analysed for size (FSC) and intracellular complexity (SSC). Colony formation in each subpopulation was quantified in 10-day cultures.</p

Pdx1 exhibits markedly different expression patterns in SP and non-SP cells.

<p>A) Pdx1 expression in SP and non-SP cells from 1–12 week-old Pdx1-GFP mice was measured by flow cytometry. B) Data obtained as in (A) were plotted to demonstrate Pdx1-GFP⁺ expression in SP and non-SP cells from 1–21 weeks of age.</p

SP cells expand after β cell damage or partial pancreatectomy.

<p>A) The SP in four mouse models of β cell damage and diabetes, and B) colony formation in two of these models, were quantified (non-diabetic mice □, diabetic mice ▪, n = 4 mice/group. * p = 0.05). C) Proportion of SP cells and D) colony formation by SP and non-SP cells were quantified in Pdx1-GFP mice starting 2 weeks after splenectomy (□ ) or splenectomy + partial pancreatectomy (▪ ) (n = 4 mice/group. * p = 0.05).</p

Growth factor combinations.

<p>Growth factor combinations.</p

SP cells proliferate and express islet hormones in defined culture conditions.

<p>A) Total RNA isolated from fresh SP cells (lane 1) and from SP cells cultured for 3 weeks (lane 2) was analysed by RT-PCR for <i>PDX1, INSULIN, GLUCAGON, SOMATOSTATIN</i> and <i>β-ACTIN</i> expression. B) SP and non-SP cells were isolated by FACS, cytospun and stained for insulin expression. SP cells had the unique phenotype of low nuclear staining by Hoechst 33342 dye and were negative for insulin expression (left panel). In contrast, non-SP cells had high nuclear staining by Hoechst 33342 and included insulin-positive cells (right panel). C) SP cells were cultured on Matrigel-coated slides in serum-free conditions for 3 weeks, with BrdU for the first 72 h. Growth factors present for the first week were replaced with differentiation factors for the subsequent 2 weeks (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048977#s2" target="_blank">Methods</a>). BrdU uptake into proliferating cells and proinsulin/insulin expression in differentiated cells were identified by indirect immunofluorescence (left panel). Proinsulin/insulin was not detected when the anti-insulin antibody was pre-incubated with a mixture of human proinsulin + insulin (10 mg/L each) (right panel). D) SP cells were cultured on Matrigel-coated slides in serum-free conditions with defined factors for 3 weeks. Indirect immunofluorescence staining for glucagon (left panel) and the secretory vesicle protein, synaptophysin (right panel). E) Colonies derived from 5×10³ SP cells cultured in triplicate wells in growth-differentiation conditions for 3 weeks were washed then incubated with 1 mM glucose for 3 h followed by 20 mM glucose for 3 h. Media collected in each period were assayed for insulin and the results compared by paired t test (*p = 0.02). F) Colonies derived from 3-week SP cell cultures in growth-differentiation conditions were transplanted inside vascularised chambers (10⁴ cells per chamber) in NOD/SCID/IL-2rγnull mice rendered diabetic by streptozotocin injection. Chamber contents were recovered 2 weeks later and stained by indirect immunofluorescence for proinsulin/insulin in the absence (top panel) or presence (bottom panel) of human proinsulin + insulin.</p

The Closely Related CD103⁺ Dendritic Cells (DCs) and Lymphoid-Resident CD8⁺ DCs Differ in Their Inflammatory Functions

<div><p>Migratory CD103⁺ and lymphoid-resident CD8⁺ dendritic cells (DCs) share many attributes, such as dependence on the same transcription factors, cross-presenting ability and expression of certain surface molecules, such that it has been proposed they belong to a common sub-lineage. The functional diversity of the two DC types is nevertheless incompletely understood. Here we reveal that upon skin infection with herpes simplex virus, migratory CD103⁺ DCs from draining lymph nodes were more potent at inducing Th17 cytokine production by CD4⁺ T cells than CD8⁺ DCs. This superior capacity to drive Th17 responses was also evident in CD103⁺ DCs from uninfected mice. Their differential potency to induce Th17 differentiation was reflected by higher production of IL-1β and IL-6 by CD103⁺ DCs compared with CD8⁺ DCs upon stimulation. The two types of DCs from isolated lymph nodes also differ in expression of certain pattern recognition receptors. Furthermore, elevated levels of GM-CSF, typical of those found in inflammation, substantially increased the pool size of CD103⁺ DCs in lymph nodes and skin. We argue that varied levels of GM-CSF may explain the contrasting reports regarding the positive role of GM-CSF in regulating development of CD103⁺ DCs. Together, we find that these two developmentally closely-related DC subsets display functional differences and that GM-CSF has differential effect on the two types of DCs.</p></div

CD103⁺ DCs and CD8⁺ DCs differ in expression of costimulatory molecules, inflammasomes and TLR.

<p>(<b>A</b>) Cells of pooled cutaneous LNs from Langerin-EGFP mice and Langerin-EGFP/<i>CD103−/−</i> mice were analyzed. CD103⁺ DCs were identified as CD326⁻CD205⁺langerin⁺ within migratory DCs (mDC, CD11c^intMHC II^high); CD8⁺ DCs were identified as CD205⁺ CD8⁺ within cDCs (CD11c^highMHC II^int). Histograms show the expression of CD103 and langerin-EGFP by CD8⁺ and CD103⁺ DCs. For CD103 expression, CD8⁺ DCs (grey dot line) and CD205⁺CD11b⁻ migratory DCs (equivalent of CD103⁺ DCs, black dot line) from CD103−/− mice were included. (B) CD8⁺ and CD103⁺ DCs from B6 mice were analyzed for the expression of costimulatory molecules. (C&D) CD8⁺ and CD103⁺ DCs from B6 mice were sorted. RT-qPCR was performed for the indicated transcripts with 3 reference genes as controls. One of three repeated experiments is shown.</p