Large-scale production of megakaryocytes from human pluripotent stem cells by chemically defined forward programming.

The production of megakaryocytes (MKs)--the precursors of blood platelets--from human pluripotent stem cells (hPSCs) offers exciting clinical opportunities for transfusion medicine. Here we describe an original approach for the large-scale generation of MKs in chemically defined conditions using a forward programming strategy relying on the concurrent exogenous expression of three transcription factors: GATA1, FLI1 and TAL1. The forward programmed MKs proliferate and differentiate in culture for several months with MK purity over 90% reaching up to 2 × 10(5) mature MKs per input hPSC. Functional platelets are generated throughout the culture allowing the prospective collection of several transfusion units from as few as 1 million starting hPSCs. The high cell purity and yield achieved by MK forward programming, combined with efficient cryopreservation and good manufacturing practice (GMP)-compatible culture, make this approach eminently suitable to both in vitro production of platelets for transfusion and basic research in MK and platelet biology.This work was supported by the Leukemia and Lymphoma Society grant, the UK Medical Research Council (Roger Pedersen), the National Institute for Health Research (NIHR; RP-PG-0310-1002; Willem Ouwehand and Cedric Ghevaert) and a core support grant from the Wellcome Trust and MRC to the Wellcome Trust – Medical Research Council Cambridge Stem Cell Institute. Cedric Ghevaert is supported by the British Heart Foundation (FS/09/039); Marloes Tijssen is supported by the European Hematology Association (Research fellowship) and the British Heart Foundation (PG/13/77/30375). Catherine Hobbs was supported by the National Health Service Blood and Transplant. Matthew Trotter was supported by a Medical Research Council Centre grant (MRC Centre for Stem Cell Biology and Regenerative Medicine); since participation in the work described, Matthew Trotter has become an employee of Celgene Research SLU, part of Celgene Corporation. Nicole Soranzo's research and Sanger Institute affiliates are supported by the Wellcome Trust (WT098051 and WT091310), the EU FP7 (Epigenesys 257082 and Blueprint HEAL TH-F5-2011-282510). The Cambridge Biomedical Centre (BRC) hIPSCs core facility is funded by the NIHR.This is the final version of the article. It first appeared from Nature Publishing Group via https://doi.org/10.1038/ncomms1120

Upcycling cellulose waste textile into aerogel beads via prilling technique

International audienceThe goal is to demonstrate the possibility of upcycling waste textile into high value-added materials, aerogels. Prilling (or jet-vibration) method was used to make aerogel beads based on viscose textile and also on microcrystalline cellulose (MCC), the latter used as a reference. The solvent was ionic liquid 1,5-diazabicyclo[4.3.0]non-5-ene acetate ([DBNH][OAc]):DMSO = 50:50. The rheological properties of solutions were studied at temperatures 20–60 °C. Cellulose droplets formed under vibration were falling in ethanol coagulation bath forming alcogel beads, and the latter were dried with supercritical CO2 resulting in cellulose aerogel beads. Cellulose solution jet, droplet and alcogel bead diameters, as well as size distribution of MCC- and viscose-based aerogel beads were measured and evaluated as a function of processing conditions (nozzle size, frequency of vibration, distance between the nozzle and coagulation bath and its temperature). Correlation parameter was used to rate the impact of the processing conditions on aerogel bead roundness and aspect ratio. Highly spherical aerogel beads (aspect ratio 1.09–1.16 and roundness 94.6–96.2) based on viscose- and MCC-based solutions were obtained with the nozzle size 400 µm, vibration frequency 100 Hz and nozzle-to-bath distance 3.5 cm. Their density was 0.08–0.12 g.cm−3 and specific surface area around 400 m2.g−1. Cytotoxicity experiments demonstrated that both types of aerogels are harmless as tested on in vitro cultures

The impairment of osteogenesis in bone sialoprotein (BSP) knockout calvaria cell cultures is cell density dependent.

Bone sialoprotein (BSP) belongs to the "small integrin-binding ligand N-linked glycoprotein" (SIBLING) family, whose members interact with bone cells and bone mineral. BSP is strongly expressed in bone and we previously showed that BSP knockout (BSP-/-) mice have a higher bone mass than wild type (BSP+/+) littermates, with lower bone remodelling. Because baseline bone formation activity is constitutively lower in BSP-/- mice, we studied the impact of the absence of BSP on in vitro osteogenesis in mouse calvaria cell (MCC) cultures. MCC BSP-/- cultures exhibit fewer fibroblast (CFU-F), preosteoblast (CFU-ALP) and osteoblast colonies (bone nodules) than wild type, indicative of a lower number of osteoprogenitors. No mineralized colonies were observed in BSP-/- cultures, along with little/no expression of either osteogenic markers or SIBLING proteins MEPE or DMP1. Osteopontin (OPN) is the only SIBLING expressed in standard density BSP-/- culture, at higher levels than in wild type in early culture times. At higher plating density, the effects of the absence of BSP were partly rescued, with resumed expression of osteoblast markers and cognate SIBLING proteins, and mineralization of the mutant cultures. OPN expression and amount are further increased in high density BSP-/- cultures, while PHEX and CatB expression are differentiatlly regulated in a manner that may favor mineralization. Altogether, we found that BSP regulates mouse calvaria osteoblast cell clonogenicity, differentiation and activity in vitro in a cell density dependent manner, consistent with the effective skeletogenesis but the low levels of bone formation observed in vivo. The BSP knockout bone microenvironment may alter the proliferation/cell fate of early osteoprogenitors

Growth, attachment, proliferation and apoptosis in standard density calvaria cell cultures of BSP-/- and BSP+/+ mice.

<p>(A) Time-course of <i>in vitro</i> cell growth in BSP+/+ and BSP-/- MCC cultures N = 12 dishes/time point. (B) Number of adherent BSP-/- and BSP+/+ MCC 30 min, 1 hr and 2 hrs after plating. N = 4 dishes/group. (C and D) <i>In vitro</i> proliferation in BSP+/+ and BSP-/- MCC cultures assayed by BrdU incorporation (C, N = 12 dishes/group) and KI67 labelling (D, N = 6 coverslips/genotype/time-point, 10 fields analysed/coverslip, results expressed as number of labelled cells/1000). (E) <i>In vitro</i> apoptosis in BSP+/+ and BSP-/- MCC cultures assayed by TUNEL at D6. **: p< 0.01, ***: p< 0.001 vs BSP+/+; Mann-Whitney U Test and (D) contingency table with χ₂ test.</p

<i>In vitro</i> osteoblast differentiation in mouse bone marrow stromal cell (A-C) and mouse calvaria cell (MCC, D-F) cultures.

<p>(A) micrographs and (B) quantification of unmineralized, ALP+ colonies (CFU-ALP) in bone marrow stromal cell cultures (D12) from BSP-/- and +/+ mice. N = 13 dishes/genotype. (C) Time-course of <i>in vitro</i> cell growth in BSP+/+ and BSP-/- bone marrow cultures. N = 12 wells/time point. (D) Quantification of total colony forming units-fibroblasts (CFU-F) and CFU-ALP in low density cultures (50 cells/cm²) of BSP+/+ and BSP-/- MCC at D15. N = 5 dishes/group. (E) Micrographs of ALP+ and Von Kossa (VK) stained standard density (5000 cells/cm²) MCC cultures at D20. (F) Quantification of mineralized colonies (CFU-OB) in standard density BSP-/- and BSP+/+ MCC cultures at D20. N = 3 dishes/group. *: p<0.05, **: p< 0.01 vs BSP+/+; Mann-Whitney U Test.</p

Effects of BSP deletion on the time-course of osteoblast marker and SIBLING expression in standard density MCC cultures.

<p>(A-D) Time-course of expression of osteoblast-associated gene: osteocalcin (OCN), type 1 collagen (Col1α1), alkaline phosphatase (ALP), osterix (OSX) and Runx2 and SIBLING genes: osteopontin (OPN), bone sialoprotein (BSP), DMP1 and MEPE at successive time-points of BSP+/+ (A, C) and BSP-/- (B, D) MCC cultures. The arrow marks the appearance of bone nodules. N = 3 dishes/group. *: p< 0.05 vs time-matched BSP+/+; #: p<0.05 vs D3 to D14. Mann-Whitney U Test.</p

Expression of OPN protein in standard density MCC cultures.

<p>(A) OPN amounts were assayed in the cell layer and culture medium of BSP+/+ and BSP-/- MCC cultures at D3, 6, 14 and 17. Results are normalized on total cell layer proteins. N = 6 dishes/time point. *: p<0.05, **: p<0.01 vs time-matched BSP+/+, Mann-Whitney U Test. (B) BSP+/+ and BSP-/- MCC cultures were arrested at D10 and immunolabeled for OPN. Nuclei were labeled with DAPI and the same fields were imaged in phase contrast microscopy. White bar = 40μm.</p

Time-course expression of PHEX (A) and cathepsin B (CatB, B) in BSP+/+ and-/- MCC cultures plated at standard and high density.

<p>N = 3 dishes/group. * p< 0.05 vs time-matched BSP+/+. (C) Quantification and (D) micrography of mineralized colonies at D17 in standard BSP-/- and BSP+/+ MCC cultures treated from D2 to D17 with 1μg/ml protease inhibitor mix (Leu.+Pep. = leupeptin + pepstatin A). N = 6 dishes/group. **: p< 0.01 vs genotype-matched control; ##: p<0.01 vs BSP+/+. Mann-Whitney U Test.</p

<i>In vitro</i> osteoblast differentiation in standard and high density BSP+/+ and BSP-/- MCC cultures.

<p>(A) Micrographs (Insets: higher magnification) and (B) quantification of total mineralized colonies formed at D14 in BSP+/+ and BSP-/- MCC cultures grown at standard (5000 cells/cm²) and high density (25000 cells/cm²). N = 3 dishes/group. Expression of osteoblast-associated (C and D) and SIBLING genes (E and F) at D14 in BSP+/+ (C, E) and BSP-/- (D, F) cultures. N = 3 dishes/group. Quantification of OPN in the cell layer (G) and the culture medium (H) of BSP+/+ and BSP-/- standard and high density cultures. Results are normalized on total cell layer proteins. N = 6 dishes/group. *: p< 0.05, **: p<0.01 vs BSP+/+; #: p< 0.05, ##: p<0.01 vs standard density. Mann-Whitney U Test.</p