Expression of CD200, SSEA4 and CD140a during MSC differentiation.

<p>(A) Clonally derived conditionally immortalized MSCs were differentiated into osteocytes (upper panel, Alizarin Red staining) or adipocytes (lower panel, Oil Red staining). Undifferentiated state, early time point and late differentiation were analyzed (0, 3, 10 days of osteogenic induction, and 0, 3, 5 days of adipogenic induction). (B) Flow cytometry results of the expression of CD200, SSEA4 and CD140a in the representative OA clone during differentiation time course (0, 3, 10 days of osteogenic treatment and 0, 3, 5 days of adipogenic treatment). (C) Dynamics of markers expression in the MSC progenitors during osteogenic differentiation (O and OA clones, blue and blue patterned bars, respectively), and adipogenic differentiation (the same OA and A clone, red patterned and red bars), shown as Mean Fluorescence Intensity from flow cytometry measurements. CD200 was upregulated during osteogenic induction, while SSEA4 was downregulated in all differentiation conditions. CD140a reached a high level during adipogenesis in the OA clone, and then dropped, and in the A clone CD140a downregulated from high level during differentiation.</p

Immunophenotype of conditionally immortalized mouse BM MSCs.

*<p>reported for mouse BM MSCs.</p><p>+/−reported as inconsistent level of expression between MSC preparations.</p><p><b>N</b>-not reported for expression in MSCs before.</p

Characterization of conditionally immortalized mouse bone marrow MSCs.

<p>(A) An improved tet-inducible system for conditional expression of Large T-antigen. In the presence of Dex/Dox irtTA-GBD* fusion protein can be translocated to the nucleus and activate transcription of Large T. (B) MSCs were isolated from bone marrow of transgenic mice and expanded in the immortalization conditions (Phase Contrast, 20×). (C) Western blot showing induction of T-antigen in primary MSCs (“−“ cells before induction, “+DD” induced cells, “−DD” cells deinduced for 3 days). (D) Conditionally immortalized MSCs maintained differentiation potential <i>in vitro</i> into osteo-, adipo- and chondrogenic lineages. (E) Flow cytometry results for the markers highly expressed in MSCs, as shown by the staining with BD Lyoplate Antibody Screening Panel. Abbreviations: Dex – Dexamethasone, Dox – Doxycycline, irtTA – improved reverse tetracycline transactivator, GBD* - mutated glucocorticoid-binding domain, TAg – Large T-antigen, CAGGs and PGK – constitutively active promoters, tet-tk – tetracycline operator and minimal promoter, IRES – internal ribosome binding site, Puro and Hygro – puromycin and hygromycin resistance genes.</p

Characterization of clonally derived bone marrow MSC progenitors.

<p>(A) Cellular cloning of conditionally immortalized MSCs was performed by limiting dilutions, and the resulted single-cell derived subpopulations were cultured in 96-well plates and screened for the potential for osteogenic and adipogenic differentiation. (B) Differentiation assay revealed the bipotential clones capable of osteo- and adipogenesis “OA”, and monopotential osteo- and adipogenic clones, “O” and “A”. (C) Summary of screening of two independent MSC lines. (D) Flow cytometry results of staining of clonally derived MSC progenitors with OA, O and A potential, for the differentially expressed markers CD200, SSEA4 and CD140a. The percent of cells in the positive gate is indicated on the histogram. (E) Comparison of levels of differentially expressed markers CD200, SSEA4 and CD140a shown by Mean Fluorescence Intensity of flow cytometry measurement, between OA, O and A progenitors. 4 clones of each type were used for experiment, statistical significance is indicated.</p

<i>In vivo</i> immune response induced by de-iniDCs and BM-DCs.

<p>(A) 48 hours after intratracheal application of cells, BAL fluid was collected and cells were counted in a haemocytometer. (B) The percentage of the CD66a⁺ neutrophils in the BAL fluid was analyzed by flow cytometry. (C) Percentage of CD3⁺ T cells in the BAL fluid of provoked mice were analyzed by flow cytometry. (D) Numbers of F4/80⁺ macrophages in the BAL fluid were analyzed by flow cytometry. (E) T cell cytokine secretion was measured in the BAL fluid by CBA. (F) May-Grünwald-Giemsa stained cytospin preparations demonstrate recruited eosinophils. (G) Paraffin-embedded lung sections were stained with Hematoxylin and Eosin. Results are expressed as mean ± SEM from 5 mice per group. Statistical significance is indicated, *(P<0.05) and **(P<0.01).</p

Morphology, cell cycle and proliferation.

<p>(A) Microscopic images of BM-DCs, iniDCs and de-iniDCs 3-days after de-induction at 10× magnification. Both, BM-DCs and de-iniDCs show an adherent phenotype with the typical formation of dendrites. (B) Proliferation of iniDCs and de-iniDCs was analyzed by counting the cells in a haemocytometer over a time period of 6 days. (C) Percentage of dead cells counted over a time period of 6 days. (D) Apoptosis and necrosis of iniDCs, 3- and 5-days cultured de-iniDCs were analyzed using anti-AnnexinV-PE antibody and DAPI. Dot blots display AnnexinV and DAPI stained cells. (E) For cell cycle analysis, iniDCs, 3- and 5-days cultured de-iniDCs were stained with PI and analyzed by flow cytometry. Cell cycle stages G1 (left peak), S (middle) and G2 (right peak) were calculated with the Dean-Jett-Fox model using FlowJo software. Proliferation, apoptosis and cell cycle analyses were performed in three independent experiments. For apoptosis and cell cycle analysis the result of a representative experiment is given.</p

Lentiviral vector mediated transgene expression in iniDCs.

<p>(A) RFP expression level was measured in untransduced (grey dotted) and lentiviral vector particle-transduced iniDCs before (grey) and after (black) puromycin selection. (B) Expression level of maturation markers MHCII, CD40 and CD86 were determined in transduced iniDCs and after their deinduction (de-iniDCs) using flow cytometry. Transduced iniDCs and de-iniDCs (grey) and LPS-stimulated transduced iniDCs and de-iniDCs (black) are shown. Isotype controls are displayed as grey dotted lines. One representative experiment out of 3 is shown.</p

Antigen presentation of de-iniDC clone #1 and BM-DCs to T cells.

<p>De-iniDC clone #1 or BM-DCs were incubated with OVA (13.5 µg/mL) for 24 hours prior to co-culture with OTII/CD45.1 CD4⁺ T cells or OTI CD8⁺ T cells. (A) Proliferation of CD4⁺ T cells was measured using CFSE staining and analyzed by flow cytometry. (B) Secretion of IL-2 was measured with CBA. (C) CD4⁺ T cell secreted cytokines IFNγ, IL-13 and IL-17 were measured in the cell culture supernatant using CBA after 48 hours. (D) Proliferation of CD8⁺ T cells was measured using CFSE staining and flow cytometry. (E) CD8⁺ T cell secreted cytokines IL-2 and IFNγ were measured in the supernatant using CBA after 48 hours. Results of three to four independent experiments are given as mean ± SEM, (n.d.) not detectable. Statistical significance is indicated, *(P<0.05), **(P<0.01) and ***(P<0.001).</p

Dendritic cell surface marker expression.

<p>(A) BM-DCs, iniDCs and 3-days cultured de-iniDCs were stained with antibodies against the dendritic cell subset markers CD11c, CD8α, CD11b, B220 and Ly6C. CD11c⁺ cells (black curve) were further gated for CD8α and CD11b, Ly6C and B220 (contour blots). Gates for CD8α and CD11b, Ly6C and B220 were set on the respective unstained control (red). (B) Immature and mature BM-DCs, iniDCs and de-iniDCs were stained for MHCII, CD40, and CD86. Dead cells (DAPI staining) and cell doublets were excluded. Histograms show the isotype control (grey, dotted), immature cells (grey) and LPS-matured cells (black). The result of one representative experiment is given.</p

CD11c expression and IL-12 production in single cell clones.

<p>De-iniDC single cell clones were stimulated with LPS or left untreated for 24 hours in the presence of the protein transport inhibitor Monensin. Afterwards, cells were stained for the surface marker CD11c, permeabilized and stained for intracellular IL-12. (A) CD11c expression (black) of LPS stimulated cells is displayed. (B) Intracellular IL-12 expression level of CD11c⁺ LPS stimulated (black) and non-stimulated cells (grey) are shown. Isotype control is displayed as grey, dotted curve (A, B).</p