Mapping the biogenesis of forward programmed megakaryocytes from induced pluripotent stem cells

Platelet deficiency, known as thrombocytopenia, can cause haemorrhage and is treated with platelet transfusions. We developed a system for the production of platelet precursor cells, megakaryocytes, from pluripotent stem cells. These cultures can be maintained for >100 days, implying culture renewal by megakaryocyte progenitors (MKPs). However, it is unclear whether the MKP state in vitro mirrors the state in vivo, and MKPs cannot be purified using conventional surface markers. We performed single cell RNA sequencing throughout in vitro differentiation and mapped each state to its equivalent in vivo. This enabled the identification of 5 surface markers which reproducibly purify MKPs, allowing us an insight into their transcriptional and epigenetic profiles. Finally, we performed culture optimisation, increasing MKP production. Altogether, this study has mapped parallels between the MKP states in vivo and in vitro and allowed the purification of MKPs, accelerating the progress of in vitro-derived transfusion products towards the clinic

Using Genome Editing to Engineer Universal Platelets

Genome editing technologies such as Zinc Finger nucleases, TALENS and CRISPR/Cas9 have recently emerged as tools with the potential to revolutionise cellular therapy. This is particularly exciting for the field of regenerative medicine, where the large-scale, quality controlled editing of large numbers of cells could generate essential cellular products ready to move towards the clinic. This review details recent progress towards generating HLA Class-I null platelets using genome editing technologies for beta-2-microglobulin deletion, generating a universally transfusable cellular product. In addition, we discuss various methods for megakaryocyte (MK) production from human pluripotent stem cells and subsequent platelet production from the MKs. As well as simply producing platelets, differentiating MK cultures can enable us to understand megakaryopoeisis in vivo and take steps towards ameliorating bleeding disorders or deficiencies in MK maturation in patients. Thus by intersecting both these areas of research, we can produce optimised differentiation systems for the production of universal platelets, thus offering a stable supply of platelets for difficult-to-match patients and providing areas with transmissible disease concerns or an unpredictable supply of platelets with a steady supply of quality controlled platelet units.UK Regenerative Medicine Platform and the Pluripotent and Engineered Cell Hub. Research in the laboratory is supported by core funding from Wellcome and MRC to the Wellcome-MRC Cambridge Stem Cell Institut

In vitro-derived platelets: the challenges we will have to face to assess quality and safety.

Platelet transfusions are given to patients in hospital who have a low blood platelet count (thrombocytopenia) either because of major bleeding (following trauma or surgery) or because the bone marrow production of platelets is impaired often due to chemotherapy, infiltration with malignant cells, fibrosis or genetic disorders. We are currently entirely reliant on blood donors as a source of platelets in transfusion medicine. However, the demand for platelets continues to rise, driven by an aging population, advances in medical procedures and ever more aggressive cancer therapies, while the supply of blood donors continues to remain static. In recent years, several groups have made major advances toward the generation of platelets in vitro for human transfusion. Recent successes include results in both generating mature human megakaryocytes as well as in developing bioreactors for extracting platelets from these megakaryocytes. Platelets made in vitro could address several issues inherent to platelets derived from blood donors - the ability to scale up/down more flexibly according to demand and therefore less precarious supply line, reduction of the risk of exposure to infectious agents and finally the possibility of engineering stem cells to reduce immunogenicity. Here we define the quality control tools and suggest measures for implementation across the field for in vitro platelet genesis, to aid collaboration between laboratories and to aid production of the burdens of proof that will eventually be required by regulators for efficacy and biosafety. We will do this firstly, by addressing the quality control of the nucleated cells used to make the platelets with a particular emphasis to safety issues and secondly, we will look at how platelet function measurement are addressed particularly in the context of platelets derived in vitro.This work was supported by grants from the Rosetrees Trust (A1691), NHS Blood and Transplant and the European Union (SilkFusion: AMD-767309-3)

Beyond Clotting: A Role of Platelets in CNS Repair?

This work was supported by the State Government of Salzburg, Austria, (Stifungsprofessur, and 20204-WISS/80/199-2014), through funding from the European Union's Seventh Framework Program (FP7/2007–2013) under grant agreements n° HEALTH-F2-2011-278850 (INMiND), n° HEALTH-F2-2011-279288 (IDEA), n° FP7-REGPOT-316120 (GlowBrain), the Austrian Science Fund FWF Special Research Program (SFB) F44 (F4413-B23) “Cell Signaling in Chronic CNS Disorders,” by the research funds from the Paracelsus Medical University PMU-FFF (Long-Term Fellowship L-12/01/001-RIV to FR and Stand Alone grant 2058).This is the final version of the article. It first appeared from Frontiers via http://dx.doi.org/10.3389/fncel.2015.00511

Process analysis of pluripotent stem cell differentiation to megakaryocytes to make platelets applying European GMP

Quality, traceability and reproducibility are crucial factors in the reliable manufacture of cellular therapeutics, as part of the overall framework of Good Manufacturing Practice (GMP). As more and more cellular therapeutics progress towards the clinic and research protocols are adapted to comply with GMP standards, guidelines for safe and efficient adaptation have become increasingly relevant. In this paper, we describe the process analysis of megakaryocyte manufacture from induced pluripotent stem cells with a view to manufacturing in vitro platelets to European GMP for transfusion. This process analysis has allowed us an overview of the entire manufacturing process, enabling us to pinpoint the cause and severity of critical risks. Risk mitigations were then proposed for each risk, designed to be GMP compliant. These mitigations will be key in advancing this iPS-derived therapy towards the clinic and have broad applicability to other iPS-derived cellular therapeutics, many of which are currently advancing towards GMP compliance. Taking these factors into account during protocol design could potentially save time and money, expediting the advent of safe, novel therapeutics from stem cells

Brain-derived neurotrophic factor in megakaryocytes

The biosynthesis of endogenous BDNF has thus far been examined in neurons where it is expressed at very low levels, in an activity-dependent fashion. In humans, BDNF has long been known to accumulate in circulating platelets, at levels far higher than in the brain. During the process of blood coagulation, BDNF is released from platelets which has led to its extensive use as a readily accessible biomarker, under the assumption that serum levels may somehow reflect brain levels. To identify the cellular origin of BDNF in platelets, we established primary cultures of megakaryocytes, the progenitors of platelets, and found that human and rat megakaryocytes express the BDNF gene. Surprisingly, the pattern of mRNA transcripts is similar to neurons. In the presence of thapsigargin and of external calcium, the levels of the mRNA species leading to efficient BDNF translation rapidly increase. Under these conditions, pro-BDNF, the obligatory precursor of biologically active BDNF, becomes readily detectable. Megakaryocytes store BDNF in α-granules, with more than 80% of them also containing platelet factor 4. By contrast, BDNF is undetectable in mouse megakaryocytes, in line with the absence of BDNF in mouse serum. These findings suggest that alterations of BDNF levels in human serum as reported in studies dealing with depression or physical exercise may primarily reflect changes occurring in megakaryocytes and platelets, including the ability of the latter to retain and release BDNF

Enabling human pluripotent stem cell derived megakaryocyte

Annually 4.5 million platelet units are transfused in Europe and the United States. These are obtained solely from allogeneic donations and have a shelf life of 5-7 days. To address the corresponding supply challenge, Moreau et al. have devised a novel process for producing megakaryocytes (MKs, the platelet precursor cell) in vitro. A transcription-factor driven, forward-programming approach converts human pluripotent stem cells into MKs. This strategy has the unique advantage of generating high yields of pure MKs in chemically defined medium through the establishment of 2-3 month long-term cultures. This could lead to the production of a consistent, reliable supply of platelets which overcomes the logistical, financial and biosafety challenges for health organisations worldwide. However to enable commercialisation of platelet manufacture, process optimisation and scale-up are essential. Medium can contribute a significant proportion of the cost of a cell based product. We have used tissue culture flasks to represent static culture and compared this to a scaled-down automated bioreactor system (ambr15, Sartorius) to evaluate feasibility and optimisation factors for the growth of forward programmed (FoP) MKs in scalable stirred-suspension culture. The medium supply and exchange strategy were analysed using high temporal resolution growth curves for three medium exchange regimes. We assessed the productivity of the medium, showing that approximately 1.3 million cells are produced per millilitre of medium. Common metabolites lactate and ammonium were unlikely to be limiting proliferation and only 20% of glucose was depleted. Using novel deterministic modelling software developed by our group, we have constructed a model of forward-programmed MKs growth. Based on inhibitor production, the model demonstrates the most efficient expansion strategy using the exchange strategies and observed growth characteristics of proliferating populations. Cell populations were identified using flow cytometry and phenotype analysis. This type of mechanistic modelling can be used to inform and optimise manufacturing strategy for scaled production of FoPMKs for platelet production and more generally for the manufacturing of cell based therapies

Platelet α-granules modulate the inflammatory response under systemic lipopolysaccharide injection in mice.

BACKGROUND: Beyond their role in hemostasis and thrombosis, platelets are also important mediators of inflammation by the release of hundreds of factors stored in their α-granules. Mutations in Nbeal2 cause gray platelet syndrome (GPS), characterized by the lack of platelet α-granules. This study aims to evaluate the immunological (proinflammatory) effects of platelet α-granules. STUDY DESIGN AND METHODS: We performed an experiment using Nbeal2-/- mice, the mouse model of GPS. Systemic inflammation was induced by intravenous injection of lipopolysaccharide (LPS). Inflammatory response was assessed by quantification of inflammatory soluble factors and platelet biological response modifiers. RESULTS: The lack of Nbeal2 (in Nbeal2 -/- mice, compared with controls) significantly reduced the recruitment of circulating neutrophils and monocytes. Moreover, after LPS injection, there was a significant increase in neutrophil and monocyte counts in control animals, compared with Nbeal2 -/- mice. The control of inflammation, evaluated by the production of anti-inflammatory cytokines, appeared to be greater in Nbeal2-/- mice compared with controls. Conversely, the production of certain inflammatory-soluble mediators known to characterize normal platelet secretion, such as soluble CD40 ligand (sCD40L), was decreased under experimental inflammation in Nbeal2 -/- mice. CONCLUSIONS: These results show that α-granules play a direct role in platelet-mediated inflammation balance, confirming the need to further investigate platelet-associated inflammatory pathophysiology and inflammatory adverse events related to blood transfusion.Supported by grants from the French Blood Establishment (Grant APR), France; the Agence Nationale de la Sécurité et du Médicament et des Produits de Santé (AAP‐2012‐011, Reference 2012S055); the French “Agence Nationale de la Recherche” (ANR‐12‐JSV1‐0012‐01); and the Association “Les Amis de Rémi” Savigneux, France

The placenta protects the fetal circulation from anxiety-driven elevations in maternal serum levels of brain-derived neurotrophic factor.

Brain-derived neurotrophic factor (BDNF) plays crucial roles in brain function. Numerous studies report alterations in BDNF levels in human serum in various neurological conditions, including mood disorders such as depression. However, little is known about BDNF levels in the blood during pregnancy. We asked whether maternal depression and/or anxiety during pregnancy were associated with altered serum BDNF levels in mothers (n = 251) and their new-born infants (n = 212). As prenatal exposure to maternal mood disorders significantly increases the risk of neurological conditions in later life, we also examined the possibility of placental BDNF transfer by developing a new mouse model. We found no association between maternal symptoms of depression and either maternal or infant cord blood serum BDNF. However, maternal symptoms of anxiety correlated with significantly raised maternal serum BDNF exclusively in mothers of boys (r = 0.281; P = 0.005; n = 99). Serum BDNF was significantly lower in male infants than female infants but neither correlated with maternal anxiety symptoms. Consistent with this observation, we found no evidence for BDNF transfer across the placenta. We conclude that the placenta protects the developing fetus from maternal changes in serum BDNF that could otherwise have adverse consequences for fetal development