Human iPS Cells for Clinical Applications and Cellular Products.

Human induced pluripotent stem cells (iPSCs), since their discovery in 2007, have rapidly become a starting cell type of choice for the differentiation of many mature cell types. Their flexibility, amenability to gene editing and functional equivalence to embryonic stem cells ensured their subsequent adoption by many manufacturing processes for cellular products. In this chapter, we will discuss the process whereby iPSCs are generated, key quality control steps which should be considered during manufacturing, the application of good manufacturing practice to production processes and iPSC-derived cellular products which are already undergoing clinical trials. iPSCs provide a new avenue for the next generation of cellular therapeutics and by combining new differentiation protocols, quality control and reproducible manufacturing, iPSC-derived cellular products could provide treatments for many currently untreatable diseases, allowing the large-scale manufacture of high-quality cell therapies.

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

Platelet deficiency, known as thrombocytopenia, can cause hemorrhage 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 five surface markers that reproducibly purify MKPs, allowing us insight into their transcriptional and epigenetic profiles. Last, we performed culture optimization, increasing MKP production. Together, 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 toward the clinic.

CRLF3 plays a key role in the final stage of platelet genesis and is a potential therapeutic target for thrombocythemia.

The process of platelet production has so far been understood to be a 2-stage process: megakaryocyte maturation from hematopoietic stem cells followed by proplatelet formation, with each phase regulating the peripheral blood platelet count. Proplatelet formation releases into the bloodstream beads-on-a-string preplatelets, which undergo fission into mature platelets. For the first time, we show that preplatelet maturation is a third, tightly regulated, critical process akin to cytokinesis that regulates platelet count. We show that deficiency in cytokine receptor-like factor 3 (CRLF3) in mice leads to an isolated and sustained 25% to 48% reduction in the platelet count without any effect on other blood cell lineages. We show that Crlf3-/- preplatelets have increased microtubule stability, possibly because of increased microtubule glutamylation via the interaction of CRLF3 with key members of the Hippo pathway. Using a mouse model of JAK2 V617F essential thrombocythemia, we show that a lack of CRLF3 leads to long-term lineage-specific normalization of the platelet count. We thereby postulate that targeting CRLF3 has therapeutic potential for treatment of thrombocythemia.

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.

Transfer to the clinic: refining forward programming of hPSCs to megakaryocytes for platelet production in bioreactors.

The production of in vitro-derived platelets has great potential for transfusion medicine. Here, we build on our experience in the forward programming (FoP) of human pluripotent stem cells (hPSCs) to megakaryocytes (MKs) and address several aspects of the complex challenges to bring this technology to the bedside. We first identify clinical-grade hPSC lines that generate MKs efficiently. We design a bespoke media to maximize both production and maturity of MKs and improve platelet output. Crucially, we transition the lentiviral-based FoP of hPSCs to a nonviral inducible system. We also show how small molecules promote a definitive hematopoiesis phenotype during the differentiation process, thereby increasing the quality of the final product. Finally, we generate platelets using a bioreactor designed to reproduce the physical cues that promote platelet production in the bone marrow. We show that these platelets are able to contribute to both thrombus formation in vitro and have a hemostatic effect in thrombocytopenic mice in vivo.

ZMYM2 inhibits NANOG-mediated reprogramming.

Background: NANOG is a homeodomain-containing transcription factor which forms one of the hubs in the pluripotency network and plays a key role in the reprogramming of somatic cells and epiblast stem cells to naïve pluripotency.  Studies have found that NANOG has many interacting partners and some of these were shown to play a role in its ability to mediate reprogramming. In this study, we set out to analyse the effect of NANOG interactors on the reprogramming process. Methods: Epiblast stem cells and somatic cells were reprogrammed to naïve pluripotency using MEK/ERK inhibitor PD0325901, GSK3ß inhibitor CHIR99021 and Leukaemia Inhibitory Factor (together termed 2i Plus LIF). Zmym2 was knocked out using the CRISPR/Cas9 system or overexpressed using the PiggyBac system. Reprogramming was quantified after ZMYM2 deletion or overexpression, in diverse reprogramming systems. In addition, embryonic stem cell self renewal was quantified in differentiation assays after ZMYM2 removal or overexpression. Results: In this work, we identified ZMYM2/ZFP198, which physically associates with NANOG as a key negative regulator of NANOG-mediated reprogramming of both epiblast stem cells and somatic cells. In addition, ZMYM2 impairs the self renewal of embryonic stem cells and its overexpression promotes differentiation. Conclusions: We propose that ZMYM2 curtails NANOG's actions during the reprogramming of both somatic cells and epiblast stem cells and impedes embryonic stem cell self renewal, promoting differentiation.

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 ß2-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 megakaryopoiesis 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.

Lateral Thinking: How Histone Modifications Regulate Gene Expression.

The DNA of each cell is wrapped around histone octamers, forming so-called 'nucleosomal core particles'. These histone proteins have tails that project from the nucleosome and many residues in these tails can be post-translationally modified, influencing all DNA-based processes, including chromatin compaction, nucleosome dynamics, and transcription. In contrast to those present in histone tails, modifications in the core regions of the histones had remained largely uncharacterised until recently, when some of these modifications began to be analysed in detail. Overall, recent work has shown that histone core modifications can not only directly regulate transcription, but also influence processes such as DNA repair, replication, stemness, and changes in cell state. In this review, we focus on the most recent developments in our understanding of histone modifications, particularly those on the lateral surface of the nucleosome. This region is in direct contact with the DNA and is formed by the histone cores. We suggest that these lateral surface modifications represent a key insight into chromatin regulation in the cell. Therefore, lateral surface modifications form a key area of interest and a focal point of ongoing study in epigenetics.

NANOG-dependent function of TET1 and TET2 in establishment of pluripotency.

Molecular control of the pluripotent state is thought to reside in a core circuitry of master transcription factors including the homeodomain-containing protein NANOG, which has an essential role in establishing ground state pluripotency during somatic cell reprogramming. Whereas the genomic occupancy of NANOG has been extensively investigated, comparatively little is known about NANOG-associated proteins and their contribution to the NANOG-mediated reprogramming process. Using enhanced purification techniques and a stringent computational algorithm, we identify 27 high-confidence protein interaction partners of NANOG in mouse embryonic stem cells. These consist of 19 previously unknown partners of NANOG that have not been reported before, including the ten-eleven translocation (TET) family methylcytosine hydroxylase TET1. We confirm physical association of NANOG with TET1, and demonstrate that TET1, in synergy with NANOG, enhances the efficiency of reprogramming. We also find physical association and reprogramming synergy of TET2 with NANOG, and demonstrate that knockdown of TET2 abolishes the reprogramming synergy of NANOG with a catalytically deficient mutant of TET1. These results indicate that the physical interaction between NANOG and TET1/TET2 proteins facilitates reprogramming in a manner that is dependent on the catalytic activity of TET1/TET2. TET1 and NANOG co-occupy genomic loci of genes associated with both maintenance of pluripotency and lineage commitment in embryonic stem cells, and TET1 binding is reduced upon NANOG depletion. Co-expression of NANOG and TET1 increases 5-hydroxymethylcytosine levels at the top-ranked common target loci Esrrb and Oct4 (also called Pou5f1), resulting in priming of their expression before reprogramming to naive pluripotency. We propose that TET1 is recruited by NANOG to enhance the expression of a subset of key reprogramming target genes. These results provide an insight into the reprogramming mechanism of NANOG and uncover a new role for 5-methylcytosine hydroxylases in the establishment of naive pluripotency.