Human mesenchymal and murine stromal cells support human lympho-myeloid progenitor expansion but not maintenance of multipotent haematopoietic stem and progenitor cells

<p>A major goal in haematopoietic stem cell (HSC) research is to define conditions for the expansion of HSCs or multipotent progenitor cells (MPPs). Since human HSCs/MPPs cannot be isolated, NOD/SCID repopulating cell (SRC) assays emerged as the standard for the quantification of very primitive haematopoietic cell. However, in addition to HSCs/MPPs, lympho-myeloid primed progenitors (LMPPs) were recently found to contain SRC activities, challenging this assay as clear HSC/MPP readout. Because our revised model of human haematopoiesis predicts that HSCs/MPPs can be identified as CD133⁺CD34⁺ cells containing erythroid potentials, we investigated the potential of human mesenchymal and conventional murine stromal cells to support expansion of HSCs/MPPs. Even though all stromal cells supported expansion of CD133⁺CD34⁺ progenitors with long-term myeloid and long-term lymphoid potentials, erythroid potentials were exclusively found within erythro-myeloid CD133^lowCD34⁺ cell fractions. Thus, our data demonstrate that against the prevailing assumption co-cultures on human mesenchymal and murine stromal cells neither promote expansion nor maintenance of HSCs and MPPs.</p

A call for the standardised reporting of factors affecting the exogenous loading of extracellular vesicles with therapeutic cargos

Extracellular vesicles (EVs) are complex nanoparticles required for the intercellular transfer of diverse biological cargoes. Unlike synthetic nanoparticles, EVs may provide a natural platform for the enhanced targeting and functional transfer of therapeutics across complex and often impenetrable biological boundaries (e.g. the blood–brain barrier or the matrix of densely organised tumours). Consequently, there is considerable interest in utilising EVs as advanced drug delivery systems for the treatment of a range of challenging pathologies. Within the past decade, efforts have focused on providing standard minimal requirements for conducting basic EV research. However, no standard reporting framework has been established governing the therapeutic loading of EVs for drug delivery applications. The purpose of this review is to critically evaluate progress in the field, providing an initial set of guidelines that can be applied as a benchmark to enhance reproducibility and increase the likelihood of translational outcomes

Introduction of a filtration step into the MVs isolation protocol significantly improves pureness of the isolates.

<p>A) Transmission electron micrographs of MVs isolated from serum starved HT29 supernatants by centrifugation without (a) or with (b) prior 0.8 µm filtration step. Samples were fixed, embedded in mold, cut in thin sections and observed using transmission electron microscopy. B) MVs filtered and isolated from serum starved HT29 cells were analyzed with the nanoparticle tracking analysis, allowing estimating the average size distribution of MVs.</p

CHO- and CHO-CEACAM1 derived MVs induce the CEACAM1-L tyrosine phosphorylation in confluent HT29 cells.

<p>CEACAM1 immunoprecipitates of confluent HT29 cells treated for 15 min with CHO- and CHO-CEACAM1 derived MVs were probed for tyrosine phosphorylation (upper panel) and CEACAM1 (lower panel) as control for equal loading. Untreated cells were used as negative control. Pervanadate was used to induce CEACAM1-L tyrosine phosphorylation. The data show one representative result of three independent repeats of the experiment.</p

CEACAM1-positive MVs significantly increase the anti-CD3 and anti-CD3/CD28 mAb triggered T-cell proliferation.

<p>Freshly isolated human PBMC were labeled with CFSE and cultured for 4 days in the presence of anti CD3 and anti CD3/CD28 with and without CHO- and CHO-CEACAM1 derived MVs (A). B) CFSE labeled PBMC were cultured for 4 days in the presence and absence of antiCD3 and antiCD3/CD28 with and without HT29-derived MVs. Untreated treated cells served as control. In indicated cases samples were co-cultured with antiCEACAM1 mAb18/20 (50 µg/ml) or isotype matched control IgG (50 µg/ml). Then PBMCs were analyzed utilizing the Accuri C6 flow cytometer system. The histograms depict PBMCs that have divided 1-3 times based on CFSE dilution peaks and reflex the cell proliferation rate given in %. The data shown are representative for three independent repeats of the experiment.</p

Pervanadate and peroxide treatment induce tyrosine phosphorylation in cells but not in corresponding MVs.

<p>Confluent HT29 cells and corresponding MVs were treated with pervanadate or H₂O₂, respectively, or left untreated. Western blot analyzes were performed using mAb 4G10 for detecting tyrosine phosphorylation in A) the CEACAM1 immunoprecipitates, and B) the whole cell and MVs lysates. In A) CEACAM1 detection and in B) beta-actin detection served as loading control. The data show one representative result of three independent repeats of the experiment.</p

MVs released by CEACAM expressing cell types reveal a parental cell like CEACAM expression pattern.

<p>Human epithelial cell (HT29, T102/3), human endothelial cells (AS-M.5), mouse endothelial cells (bEND3), CHO and CHO-CEACAM1 cells were serum starved for 48 h. Subsequently culture supernatants were filtered (0.8 µm) before MVs were isolation. Harvested cells and MVs were analyzed by flow cytometry (A). Cells were stained for CEACAM1 (thick line). Background fluorescence was determined by incubating the cells with control IgG antibody instead of anti-CEACAM1 antibody (thin line). (B) Parts of obtained samples were lysed and then analyzed by Western blot using anti-CEACAM1-, CEACAM5- and CEACAM6- specific mAbs. Beta-actin detection served as control for equally loading. The experiments shown are representative for three independent repeats.</p

Colon tumor epithelial cells release MVs in response to serum starvation.

<p>A) Transmission electron micrographs of HT29 cells grown under normal conditions (a) and serum starved (b) for 48 h. After culture, cells fixed, embedded in mold, cut in thin sections and analyzed by transmission electron microscopy. B) The amount of MVs released by HT29 cells cultured in media with or without serum was counted by flow cytometry (n=3).</p

CTC quantification.

<p>CTC quantification.</p

CTC subtypes were associated to clinical outcome.

<p>A) Kaplan-Meier test showed that the number of N-cadherin+/CK- cells was significantly associated to progression free survival (PFS) of mRCC patients during first-line treatment with anti-angiogenesis therapy (PFS; 7 vs. 15 months; p = 0.03; [HR] = 0.31; CI: 0.06–1.59). B) Mann-Whitney test revealed a significantly increased number of CD133+ cells in the presence of N-cadherin+ cells (p = 0.05).</p