Meera Arumugam

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 × 105 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.

Orthosilicic Acid Increases Collagen Type I mRNA Expression in Human Bone-Derived Osteoblasts In Vitro

The effect of 0, 5 μM, 10μM, 20μM and 50μM orthosilicic acid on collagen type I gene expression in cultured human trabecular bone-derived osteoblasts was inve stigated. Collagen type I messenger RNA (mRNA) was quantified using real–time reverse t ranscriptase-polymerase chain reaction (RT-PCR). After 20 hours incubation a significant increa se in collagen type I mRNA was seen in cells cultured with 5 μM, 10μM and 50μM orthosilicic acid ( p<0.05) compared to untreated controls. This study suggests that silicon has a stimulatory ef fect on collagen type I mRNA in human osteoblasts.

The Effect Orthosilicic Acid on Collagen Type I, Alkaline Phosphatase and Osteocalcin mRNA Expression in Human Bone-Derived Osteoblasts In Vitro

The object of this study was to investigate the effect of the concentration of orthosilicic acid (0, 0.5, 1, 5 and 10µM) on gene expression in human osteoblast cells isolated from trabecular bone. This was measured using reverse transcriptase-polymerase chain reaction (RT-PCR) to quantify messenger RNA (mRNA) levels for collagen type I, alkaline phosphatase and osteocalcin. Results showed that while collagen type I mRNA expression was increased by the addition of up to 10µM orthosilicic acid, ALP message was suppressed over time and osteocalcin levels were decreased.

The effect of particle size on the in vivo degradation of poly(d,l-lactide-co-glycolide)/α-tricalcium phosphate micro- and nanocomposites.

This paper reports the effect of particle size within a resorbable composite on the in vivo degradation rate and host response. Resorbable composites based on poly(d,l-lactide-co-glycolide) (PLGA) reinforced with tricalcium phosphate (TCP) have shown suitable degradation, biological and mechanical properties for bone repair. Composites with nano-sized TCP particles degrade more homogenously in vitro than equivalent composites with micro-sized particles. In this study, PLGA and PLGA/TCP composites containing micro- or nano-sized α-TCP particles were implanted into an ovine distal femoral condyle defect and harvested at 6, 12, 18 and 24weeks. An intimate interface was observed between the new bone tissue and degrading implants. Visual scoring of histological images and semi-automated segmentation of X-ray images were used to quantify implant degradation and the growth of new bone tissue in the implant site. Bone growth into the implant site occurred at a similar rate for both composites and the PLGA control. However, the in vivo degradation rate of the nanocomposite was slower than that of the microcomposite and consequently more closely matched the rate of bone growth. For the first 6weeks, the rate of in vivo degradation matched that of in vitro degradation, but lagged significantly at longer time points. These results point to the potential use of ceramic particle size in controlling composite degradation whilst maintaining good bone formation. STATEMENT OF SIGNIFICANCE This paper concerns degradable composites for orthopaedic application. The effect of particle size on implant degradation in vivo is not yet well characterised and these results give the first opportunity to directly compare in vitro and in vivo degradation rates for composites with micro- and nano-sized particles. This type of data is vital for the validation of models of composite degradation behaviour, which will lead to the design and manufacture of composites with a tailored, predictable degradation profile. The trainable segmentation tool can be used for future studies where X-rays of partially degraded implants (which have complicated greyscales and morphologies) need to be quantified without bias.

Structurally graduated collagen scaffolds applied to the ex vivo generation of platelets from human pluripotent stem cell-derived megakaryocytes: Enhancing production and purity

Platelet transfusions are a key treatment option for a range of life threatening conditions including cancer, chemotherapy and surgery. Efficient ex vivo systems to generate donor independent platelets in clinically relevant numbers could provide a useful substitute. Large quantities of megakaryocytes (MKs) can be produced from human pluripotent stem cells, but in 2D culture the ratio of platelets harvested from MK cells has been limited and restricts production rate. The development of biomaterial cell supports that replicate vital hematopoietic micro-environment cues are one strategy that may increase in vitro platelet production rates from iPS derived Megakaryocyte cells. In this paper, we present the results obtained generating, simulating and using a novel structurally-graded collagen scaffold within a flow bioreactor system seeded with programmed stem cells. Theoretical analysis of porosity using micro-computed tomography analysis and synthetic micro-particle filtration provided a predictive tool to tailor cell distribution throughout the material. When used with MK programmed stem cells the graded scaffolds influenced cell location while maintaining the ability to continuously release metabolically active CD41 + CD42 + functional platelets. This scaffold design and novel fabrication technique offers a significant advance in understanding the influence of scaffold architectures on cell seeding, retention and platelet production.

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

This corrects the article DOI: 10.1038/ncomms11208.

Research data supporting "The effect of particle size on the in vivo degradation of poly(D,L-lactide-co-glycolide)/α-tricalcium phosphate micro- and nanocomposites"

Microtomography data, scanning electron microscopy images and thermogravimetric analysis data analysed and reported in the associated publication.

Incorporation of Human Osteoblast Cells and Osteoblast-Like Cells into Porous Hydroxyapatite Scaffolds

The object of this study was to investigate methods of seeding cells onto porous hydroxyapatite granules with the aim of optimising cell attachment. Two cells types were used; an osteosarcoma cell line, MG63, and human osteoblasts (HOBs) isolated from trabecular bone. Several conditions were investigated to determine their effect on cell attachment. These included varying the initial seeding concentration, pre-adsorption of the granules with the adhesion protein fibronectin and the use of mechanical agitation. Human osteoblasts and MG63 osteosarcoma cells attached to both dense and porous HA granules but with a low seeding efficiency while seeding was not significantly improved by pre-coating scaffolds with fibronectin or by introducing fluid flow.