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In vitro co-culture systems for studying molecular basis of cellular interaction between Aire-expressing medullary thymic epithelial cells and fresh thymocytes.

Yamaguchi Y, Kudoh J, Yoshida T, Shimizu N - Biol Open (2014)

Bottom Line: Thus, these Aire(+) cells appear to behave like differentiating mTECs as if they pass through the developmental stages from intermediate state toward mature state.Surprisingly, an in vitro co-culture system consisting of Aire(+) cells and fractionated sub-populations of fresh thymocytes implied the possible existence of two distinct subtypes of thymocytes (named as CD4(+) killer and CD4(-) rescuer) that may determine the fate (dead or alive) of the differentiating Aire(+)mTECs.Thus, our in vitro co-culture system appears to mimic a part of "in vivo thymic crosstalk".

View Article: PubMed Central - PubMed

Affiliation: Advanced Research Center for Genome Super Power, Keio University, 2 Okubo, Tsukuba, Ibaraki 300-2611, Japan.

No MeSH data available.


Related in: MedlinePlus

Morphology and comparative analysis of increasing gene expression of Aire, TSAs and components for IS and IPSM in semi-confluent and confluent conditions.(A) Cell morphology. Three Aire+ cell lines (TEC1, TE2 and DC) were grown to semi-confluency (SC) (0.75×106 cells/dish for Aire+TEC1 and Aire+TEC2, 2.0×106 cells/dish for Aire+DC) and to confluency (C) (3.5×106 cells/dish for Aire+TEC1 and Aire+TEC2, 1.4×107 cells/dish for Aire+DC). Pictures were taken by a Nikon phase-contrast microscope ECLIPSE Ti-U. Scale bar, 100 µm. (B) Aire-mRNA expression. Aire-mRNA in total RNAs was quantified by qRT-PCR analysis, the amount of Aire-mRNA was normalized to β-Actin and the expression was compared between two cell densities (SC and C). Average of three separate experiments was shown with standard deviation (bar). (C) Expression of TSA-mRNAs. TSA-mRNA in total RNAs was quantified by qRT-PCR analysis with primers specific for 5 different TSAs (Csna, Cyp17, Fabp2, InsII and Tgn) and β-Actin, the amount of TSA-mRNA was normalized to β-Actin and the expression was compared between two cell densities (SC and C). Average of three separate experiments was shown with standard deviation (bar). The expression levels at SC were set as 1.00. Note the scale difference in the vertical axis. (D) Expression of mRNAs for subunits of PSM and IPSM. The subunit (Sub)-specific mRNA in total RNA was quantified by qRT-PCR analysis with primers specific for various subunits of PSM and IPSM (Table 1) and β-Actin. The amount of Sub-mRNA was normalized to β-Actin and the expression was compared between two cell densities (SC and C). Average of three separate experiments was shown with standard deviation (bar). The expression levels at SC were arbitrarily set as 1.00. Note the scale difference in the vertical axis. Upper panel: mRNAs for PSM-subunits (β1, β2, β5), Middle panel: mRNAs for IPSM-subunits (β1i, β2i β5i), Bottom panel: mRNAs for IPSM-subunits (PA28α, PA28β and PA28γ). (E) Expression of IS-components. Aire+ cell lines (5,000 cells each) at different cell density (SC and C) were stained with fluorescent antibodies specific to IS-components (CD40, MHC-II, CD80 and CD86) and the surface fluorescence was measured by a flow cytometry system Guava (Millipore). The background fluorescence by the same animal IgG was subtracted. The X-Arithmetic means of four separate experiments were plotted with standard deviation (bar). (F) Flow cytometry analysis of surface markers: Aire+TEC1, TEC2 and DC cells at semi-confluent and confluent conditions were analyzed with specific antibodies (CD40, MHC class II, CD80 and CD86) for cell surface marker. Each panel shows the distribution of the markers by fluorescent intensity vs cell count. The histograms were compared among control IgG (gray), semi-confluent (blue) and confluent condition (red).
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f03: Morphology and comparative analysis of increasing gene expression of Aire, TSAs and components for IS and IPSM in semi-confluent and confluent conditions.(A) Cell morphology. Three Aire+ cell lines (TEC1, TE2 and DC) were grown to semi-confluency (SC) (0.75×106 cells/dish for Aire+TEC1 and Aire+TEC2, 2.0×106 cells/dish for Aire+DC) and to confluency (C) (3.5×106 cells/dish for Aire+TEC1 and Aire+TEC2, 1.4×107 cells/dish for Aire+DC). Pictures were taken by a Nikon phase-contrast microscope ECLIPSE Ti-U. Scale bar, 100 µm. (B) Aire-mRNA expression. Aire-mRNA in total RNAs was quantified by qRT-PCR analysis, the amount of Aire-mRNA was normalized to β-Actin and the expression was compared between two cell densities (SC and C). Average of three separate experiments was shown with standard deviation (bar). (C) Expression of TSA-mRNAs. TSA-mRNA in total RNAs was quantified by qRT-PCR analysis with primers specific for 5 different TSAs (Csna, Cyp17, Fabp2, InsII and Tgn) and β-Actin, the amount of TSA-mRNA was normalized to β-Actin and the expression was compared between two cell densities (SC and C). Average of three separate experiments was shown with standard deviation (bar). The expression levels at SC were set as 1.00. Note the scale difference in the vertical axis. (D) Expression of mRNAs for subunits of PSM and IPSM. The subunit (Sub)-specific mRNA in total RNA was quantified by qRT-PCR analysis with primers specific for various subunits of PSM and IPSM (Table 1) and β-Actin. The amount of Sub-mRNA was normalized to β-Actin and the expression was compared between two cell densities (SC and C). Average of three separate experiments was shown with standard deviation (bar). The expression levels at SC were arbitrarily set as 1.00. Note the scale difference in the vertical axis. Upper panel: mRNAs for PSM-subunits (β1, β2, β5), Middle panel: mRNAs for IPSM-subunits (β1i, β2i β5i), Bottom panel: mRNAs for IPSM-subunits (PA28α, PA28β and PA28γ). (E) Expression of IS-components. Aire+ cell lines (5,000 cells each) at different cell density (SC and C) were stained with fluorescent antibodies specific to IS-components (CD40, MHC-II, CD80 and CD86) and the surface fluorescence was measured by a flow cytometry system Guava (Millipore). The background fluorescence by the same animal IgG was subtracted. The X-Arithmetic means of four separate experiments were plotted with standard deviation (bar). (F) Flow cytometry analysis of surface markers: Aire+TEC1, TEC2 and DC cells at semi-confluent and confluent conditions were analyzed with specific antibodies (CD40, MHC class II, CD80 and CD86) for cell surface marker. Each panel shows the distribution of the markers by fluorescent intensity vs cell count. The histograms were compared among control IgG (gray), semi-confluent (blue) and confluent condition (red).

Mentions: We then examined if cell growth states of Aire+ cell affects expression levels of Aire gene. To test this, we prepared Aire+ cells (Aire+TEC1, Aire+TEC2 and Aire+DC) at two different growth states: rapidly growing state at semi-confluency (SC) and resting state at confluency (C) (Fig. 3). Aire+ cells at SC exhibited characteristic shapes of thymic epithelial cells or dendritic cells, whereas all Aire+ cells at C formed monolayers of tightly packed cells with cell–cell contact (Fig. 3A).


In vitro co-culture systems for studying molecular basis of cellular interaction between Aire-expressing medullary thymic epithelial cells and fresh thymocytes.

Yamaguchi Y, Kudoh J, Yoshida T, Shimizu N - Biol Open (2014)

Morphology and comparative analysis of increasing gene expression of Aire, TSAs and components for IS and IPSM in semi-confluent and confluent conditions.(A) Cell morphology. Three Aire+ cell lines (TEC1, TE2 and DC) were grown to semi-confluency (SC) (0.75×106 cells/dish for Aire+TEC1 and Aire+TEC2, 2.0×106 cells/dish for Aire+DC) and to confluency (C) (3.5×106 cells/dish for Aire+TEC1 and Aire+TEC2, 1.4×107 cells/dish for Aire+DC). Pictures were taken by a Nikon phase-contrast microscope ECLIPSE Ti-U. Scale bar, 100 µm. (B) Aire-mRNA expression. Aire-mRNA in total RNAs was quantified by qRT-PCR analysis, the amount of Aire-mRNA was normalized to β-Actin and the expression was compared between two cell densities (SC and C). Average of three separate experiments was shown with standard deviation (bar). (C) Expression of TSA-mRNAs. TSA-mRNA in total RNAs was quantified by qRT-PCR analysis with primers specific for 5 different TSAs (Csna, Cyp17, Fabp2, InsII and Tgn) and β-Actin, the amount of TSA-mRNA was normalized to β-Actin and the expression was compared between two cell densities (SC and C). Average of three separate experiments was shown with standard deviation (bar). The expression levels at SC were set as 1.00. Note the scale difference in the vertical axis. (D) Expression of mRNAs for subunits of PSM and IPSM. The subunit (Sub)-specific mRNA in total RNA was quantified by qRT-PCR analysis with primers specific for various subunits of PSM and IPSM (Table 1) and β-Actin. The amount of Sub-mRNA was normalized to β-Actin and the expression was compared between two cell densities (SC and C). Average of three separate experiments was shown with standard deviation (bar). The expression levels at SC were arbitrarily set as 1.00. Note the scale difference in the vertical axis. Upper panel: mRNAs for PSM-subunits (β1, β2, β5), Middle panel: mRNAs for IPSM-subunits (β1i, β2i β5i), Bottom panel: mRNAs for IPSM-subunits (PA28α, PA28β and PA28γ). (E) Expression of IS-components. Aire+ cell lines (5,000 cells each) at different cell density (SC and C) were stained with fluorescent antibodies specific to IS-components (CD40, MHC-II, CD80 and CD86) and the surface fluorescence was measured by a flow cytometry system Guava (Millipore). The background fluorescence by the same animal IgG was subtracted. The X-Arithmetic means of four separate experiments were plotted with standard deviation (bar). (F) Flow cytometry analysis of surface markers: Aire+TEC1, TEC2 and DC cells at semi-confluent and confluent conditions were analyzed with specific antibodies (CD40, MHC class II, CD80 and CD86) for cell surface marker. Each panel shows the distribution of the markers by fluorescent intensity vs cell count. The histograms were compared among control IgG (gray), semi-confluent (blue) and confluent condition (red).
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Related In: Results  -  Collection

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f03: Morphology and comparative analysis of increasing gene expression of Aire, TSAs and components for IS and IPSM in semi-confluent and confluent conditions.(A) Cell morphology. Three Aire+ cell lines (TEC1, TE2 and DC) were grown to semi-confluency (SC) (0.75×106 cells/dish for Aire+TEC1 and Aire+TEC2, 2.0×106 cells/dish for Aire+DC) and to confluency (C) (3.5×106 cells/dish for Aire+TEC1 and Aire+TEC2, 1.4×107 cells/dish for Aire+DC). Pictures were taken by a Nikon phase-contrast microscope ECLIPSE Ti-U. Scale bar, 100 µm. (B) Aire-mRNA expression. Aire-mRNA in total RNAs was quantified by qRT-PCR analysis, the amount of Aire-mRNA was normalized to β-Actin and the expression was compared between two cell densities (SC and C). Average of three separate experiments was shown with standard deviation (bar). (C) Expression of TSA-mRNAs. TSA-mRNA in total RNAs was quantified by qRT-PCR analysis with primers specific for 5 different TSAs (Csna, Cyp17, Fabp2, InsII and Tgn) and β-Actin, the amount of TSA-mRNA was normalized to β-Actin and the expression was compared between two cell densities (SC and C). Average of three separate experiments was shown with standard deviation (bar). The expression levels at SC were set as 1.00. Note the scale difference in the vertical axis. (D) Expression of mRNAs for subunits of PSM and IPSM. The subunit (Sub)-specific mRNA in total RNA was quantified by qRT-PCR analysis with primers specific for various subunits of PSM and IPSM (Table 1) and β-Actin. The amount of Sub-mRNA was normalized to β-Actin and the expression was compared between two cell densities (SC and C). Average of three separate experiments was shown with standard deviation (bar). The expression levels at SC were arbitrarily set as 1.00. Note the scale difference in the vertical axis. Upper panel: mRNAs for PSM-subunits (β1, β2, β5), Middle panel: mRNAs for IPSM-subunits (β1i, β2i β5i), Bottom panel: mRNAs for IPSM-subunits (PA28α, PA28β and PA28γ). (E) Expression of IS-components. Aire+ cell lines (5,000 cells each) at different cell density (SC and C) were stained with fluorescent antibodies specific to IS-components (CD40, MHC-II, CD80 and CD86) and the surface fluorescence was measured by a flow cytometry system Guava (Millipore). The background fluorescence by the same animal IgG was subtracted. The X-Arithmetic means of four separate experiments were plotted with standard deviation (bar). (F) Flow cytometry analysis of surface markers: Aire+TEC1, TEC2 and DC cells at semi-confluent and confluent conditions were analyzed with specific antibodies (CD40, MHC class II, CD80 and CD86) for cell surface marker. Each panel shows the distribution of the markers by fluorescent intensity vs cell count. The histograms were compared among control IgG (gray), semi-confluent (blue) and confluent condition (red).
Mentions: We then examined if cell growth states of Aire+ cell affects expression levels of Aire gene. To test this, we prepared Aire+ cells (Aire+TEC1, Aire+TEC2 and Aire+DC) at two different growth states: rapidly growing state at semi-confluency (SC) and resting state at confluency (C) (Fig. 3). Aire+ cells at SC exhibited characteristic shapes of thymic epithelial cells or dendritic cells, whereas all Aire+ cells at C formed monolayers of tightly packed cells with cell–cell contact (Fig. 3A).

Bottom Line: Thus, these Aire(+) cells appear to behave like differentiating mTECs as if they pass through the developmental stages from intermediate state toward mature state.Surprisingly, an in vitro co-culture system consisting of Aire(+) cells and fractionated sub-populations of fresh thymocytes implied the possible existence of two distinct subtypes of thymocytes (named as CD4(+) killer and CD4(-) rescuer) that may determine the fate (dead or alive) of the differentiating Aire(+)mTECs.Thus, our in vitro co-culture system appears to mimic a part of "in vivo thymic crosstalk".

View Article: PubMed Central - PubMed

Affiliation: Advanced Research Center for Genome Super Power, Keio University, 2 Okubo, Tsukuba, Ibaraki 300-2611, Japan.

No MeSH data available.


Related in: MedlinePlus