A human iPSC line capable of differentiating into functional macrophages expressing ZsGreen: a tool for the study and in vivo tracking of therapeutic cells

We describe the production of a human induced pluripotent stem cell (iPSC) line, SFCi55-ZsGr, that has been engineered to express the fluorescent reporter gene, ZsGreen, in a constitutive manner. The CAG-driven ZsGreen expression cassette was inserted into the AAVS1 locus and a high level of expression was observed in undifferentiated iPSCs and in cell lineages derived from all three germ layers including haematopoietic cells, hepatocytes and neurons. We demonstrate efficient production of terminally differentiated macrophages from the SFCi55-ZsGreen iPSC line and show that they are indistinguishable from those generated from their parental SFCi55 iPSC line in terms of gene expression, cell surface marker expression and phagocytic activity. The high level of ZsGreen expression had no effect on the ability of macrophages to be activated to an M(LPS + IFNγ), M(IL10) or M(IL4) phenotype nor on their plasticity, assessed by their ability to switch from one phenotype to another. Thus, targeting of the AAVS1 locus in iPSCs allows for the production of fully functional, fluorescently tagged human macrophages that can be used for in vivo tracking in disease models. The strategy also provides a platform for the introduction of factors that are predicted to modulate and/or stabilize macrophage function. This article is part of the theme issue ‘Designer human tissue: coming to a lab near you’

Manipulating transcription factors in human induced pluripotent cell-derived cells to enhance the production and the maturation of red blood cells

The most widely transfused blood component is red blood cells (RBCs), and voluntary donation is the main resource for RBC transfusion. In the UK, 7,000 units of RBCs are transfused daily but this life-saving cell therapy is completely dependent on donors and there are persistent problems associated with transfusion transmitted infections and in blood group compatibility. Furthermore, the quality, safety and efficiency of donated RBCs gradually decrease with storage time. A number of novel sources of RBCs are being explored including the production of RBCs from adult haematopoietic progenitor cells, erythroid progenitor cell lines and induced pluripotent stem cells (iPSCs). The iPSC source could essentially provide a limitless supply and a route to producing cells that are matched to the recipient. A number of protocols have been described to produce mature RBCs from human pluripotent stem cells but they are relatively inefficient and would be difficult to scale up to the levels required for clinical translation. We tested and evaluated a defined feeder- and serum-free differentiation protocol for deriving erythroid cells from hiPSCs. RBC production was not efficient, the cells that were produced did not enucleate efficiently and they expressed embryonic rather than adult globin. We hypothesised that the production of RBCs from iPSCs could be enhanced by enforced expression of erythroid-specific transcription factors (TFs). Previous studies had demonstrated that Krüppel-like factor 1 (KLF1) plays an important role in RBC development and maturation so we generated iPSC lines expressing a tamoxifen-inducible KLF1-ERT2 fusion protein. Using zinc finger nuclease technology, we targeted the expression cassette to the AAVS1 locus to ensure consistent expression levels and to avoid integration site specific effects and/or silencing. These iKLF1 iPSCs were applied to our defined RBC differentiation protocol and the activity of KLF1 was induced by adding tamoxifen. Activation of KLF1 from day 10 accelerated erythroid differentiation and maturation with an increase in the proportion of erythroblasts, a higher level of expression of erythroid genes associated with maturation and an apparently more robust morphology. However, KLF1 activation had an anti-proliferation effect resulting in significantly less cell generated overall and HPLC analysis demonstrated that KLF1-activated cells expressed higher levels of embryonic globin compared to control iPSCs-derived cells. Many of the effects that were observed when KLF1 was activated from day 10 were not observed when activated from day 18. We therefore concluded that activation of exogenous KLF1 is able to promote erythroid cell production and maturation in progenitors (day 10) but not at the later stage of erythropoiesis (day 18). We hypothesised that KLF1 might require a co-factor to regulate RBC maturation and adult globin expression at the later stage of erythropoiesis. The TF, B-cell lymphoma/leukaemia 11a (BCL11A), plays a key role in the suppression of foetal globin expression, thereby completing globin switching to adult globin. Preliminary data showed that iPSC-derived erythroid cells were able to express adult globin when transduced with a BCL11A-expressing lentiviral-vector. Based on that finding we then generated an iPSC line expressing tamoxifen-inducible BCL11AERT2 and KLF1-ERT2 fusion proteins, applied this iBK iPSC line to our differentiation protocol. Activation of both TFs from day 18 slightly increased the expression of genes associated with RBC maturation and the inclusion of BCL11A appeared to eliminate the anti-proliferation effect of KLF1. Most importantly, activation of both BCL11A and KLF1 from day 18 of the differentiation protocol increased the production of α- globin (foetal / adult globin) indicating that some definitive-like erythroid cells might be generated by activation of both TFs at the later stage of erythroid differentiation. Collectively, these findings demonstrate that enforced expression of erythroid TFs could be a useful strategy to enhance RBC maturation from iPSCs

A role for mospd1 in mesenchymal stem cell proliferation and differentiation

Mesenchymal stem cells (MSCs) isolated from many tissues including bone marrow and fat can be expanded in vitro and can differentiate into a range of different cell types such as bone, cartilage, and adipocytes. MSCs can also exhibit immunoregulatory properties when transplanted but, although a number of clinical trials using MSCs are in progress, the molecular mechanisms that control their production, proliferation, and differentiation are poorly understood. We identify MOSPD1 as a new player in this process. We generated MOSPD1‐null embryonic stem cells (ESCs) and demonstrate that they are deficient in their ability to differentiate into a number of cell lineages including osteoblasts, adipocytes, and hematopoietic progenitors. The self‐renewal capacity of MOSPD1‐null ESCs was normal and they exhibited no obvious defects in early germ layer specification nor in epithelial to mesenchymal transition (EMT), indicating that MOSPD1 functions after these key steps in the differentiation process. Mesenchymal stem cell (MSC)‐like cells expressing CD73, CD90, and CD105 were generated from MOSPD1‐null ESCs but their growth rate was significantly impaired implying that MOSPD1 plays a role in MSC proliferation. Phenotypic deficiencies exhibited by MOSPD1‐null ESCs were rescued by exogenous expression of MOSPD1, but not MOSPD3 indicating distinct functional properties of these closely related genes. Our in vitro studies were supported by RNA‐sequencing data that confirmed expression of Mospd1 mRNA in cultured, proliferating perivascular pre‐MSCs isolated from human tissue. This study adds to the growing body of knowledge about the function of this largely uncharacterized protein family and introduces a new player in the control of MSC proliferation and differentiation. Stem Cells 2015;33:3077–308

Enforced Expression of HOXB4 in Human Embryonic Stem Cells Enhances the Production of Hematopoietic Progenitors but Has No Effect on the Maturation of Red Blood Cells

We have developed a robust, Good Manufacturing Practice-compatible differentiation protocol capable of producing scalable quantities of red blood cells (RBCs) from human pluripotent stem cells (hPSCs). However, translation of this protocol to the clinic has been compromised because the RBCs produced are not fully mature; thus, they express embryonic and fetal, rather than adult globins, and they do not enucleate efficiently. Based on previous studies, we predicted that activation of exogenous HOXB4 would increase the production of hematopoietic progenitor cells (HPCs) from hPSCs and hypothesized that it might also promote the production of more mature, definitive RBCs. Using a tamoxifen-inducible HOXB4-ERT2 expression system, we first demonstrated that activation of HOXB4 does increase the production of HPCs from hPSCs as determined by colony-forming unit culture activity and the presence of CD43+CD34+ progenitors. Activation of HOXB4 caused a modest, but significant, increase in the proportion of immature CD235a+/CD71+ erythroid cells. However, this did not result in a significant increase in more mature CD235a+/CD71− cells. RBCs produced in the presence of enhanced HOXB4 activity expressed embryonic (ε) and fetal (γ) but not adult (β) globins, and the proportion of enucleated cells was comparable to that of the control cultures. We conclude that programming with the transcription factor HOXB4 increases the production of hematopoietic progenitors and immature erythroid cells but does not resolve the inherent challenges associated with the production of mature adult-like enucleated RBCs

The incidence, aetiology and outcome of acute seizures in children admitted to a rural Kenyan district hospital

<p>Abstract</p> <p>Background</p> <p>Acute seizures are a common cause of paediatric admissions to hospitals in resource poor countries and a risk factor for neurological and cognitive impairment and epilepsy. We determined the incidence, aetiological factors and the immediate outcome of seizures in a rural malaria endemic area in coastal Kenya.</p> <p>Methods</p> <p>We recruited all children with and without seizures, aged 0–13 years and admitted to Kilifi District hospital over 2 years from 1^stDecember 2004 to 30^thNovember 2006. Only incident admissions from a defined area were included. Patients with epilepsy were excluded. The population denominator, the number of children in the community on 30^thNovember 2005 (study midpoint), was modelled from a census data.</p> <p>Results</p> <p>Seizures were reported in 900/4,921(18.3%) incident admissions and at least 98 had status epilepticus. The incidence of acute seizures in children 0–13 years was 425 (95%CI 386, 466) per 100,000/year and was 879 (95%CI 795, 968) per 100,000/year in children <5 years. This incidence data may however be an underestimate of the true incidence in the community. Over 80% of the seizures were associated with infections. Neonatal infections (28/43 [65.1%]) and falciparum malaria (476/821 [58.0%]) were the main diseases associated with seizures in neonates and in children six months or older respectively. Falciparum malaria was also the main illness (56/98 [57.1%]) associated with status epilepticus. Other illnesses associated with seizures included pyogenic meningitis, respiratory tract infections and gastroenteritis. Twenty-eight children (3.1%) with seizures died and 11 surviving children (1.3%) had gross neurological deficits on discharge. Status epilepticus, focal seizures, coma, metabolic acidosis, bacteraemia, and pyogenic meningitis were independently associated with mortality; while status epilepticus, hypoxic ischaemic encephalopathy and pyogenic meningitis were independently associated with neurological deficits on discharge.</p> <p>Conclusion</p> <p>There is a high incidence of acute seizures in children living in this malaria endemic area of Kenya. The most important causes are diseases that are preventable with available public health programs.</p

The FAIR Guiding Principles for scientific data management and stewardship

There is an urgent need to improve the infrastructure supporting the reuse of scholarly data. A diverse set of stakeholders—representing academia, industry, funding agencies, and scholarly publishers—have come together to design and jointly endorse a concise and measureable set of principles that we refer to as the FAIR Data Principles. The intent is that these may act as a guideline for those wishing to enhance the reusability of their data holdings. Distinct from peer initiatives that focus on the human scholar, the FAIR Principles put specific emphasis on enhancing the ability of machines to automatically find and use the data, in addition to supporting its reuse by individuals. This Comment is the first formal publication of the FAIR Principles, and includes the rationale behind them, and some exemplar implementations in the community

SplitAx:A novel method to assess the function of engineered nucleases

Engineered nucleases have been used to generate knockout or reporter cell lines and a range of animal models for human disease. These new technologies also hold great promise for therapeutic genome editing. Current methods to evaluate the activity of these nucleases are time consuming, require extensive optimization and are hampered by readouts with low signals and high background. We have developed a simple and easy to perform method (SplitAx) that largely addresses these issues and provides a readout of nuclease activity. The assay involves splitting the N-terminal (amino acid 1-158) coding region of GFP and an out-of-frame of C-terminal region with a nuclease binding site sequence. Following exposure to the test nuclease, cutting and repair by error prone non-homologous end joining (NHEJ) restores the reading frame resulting in the production of a full length fluorescent GFP protein. Fluorescence can also be restored by complementation between the N-terminal and C-terminal coding sequences in trans. We demonstrate successful use of the SplitAx assay to assess the function of zinc finger nucleases, CRISPR hCAS9 and TALENS. We also test the activity of multiple gRNAs in CRISPR/hCas9/D10A systems. The zinc finger nucleases and guide RNAs that showed functional activity in the SplitAx assay were then used successfully to target the endogenous AAVS1, SOX6 and Cfms loci. This simple method can be applied to other unrelated proteins such as ZsGreen1 and provides a test system that does not require complex optimization

Functional validation of the GFP-AAVS1 SplitAx reporter assay with zinc fingers and CRISPR/CAS9 system.

<p>Schematic of the GFP cDNA with the N-terminus and C-terminus separated by the <i>AAVS1</i> binding site. The DNA sequence of the <i>AAVS1</i> binding site is shown and the location of zinc finger left (ZF L), Zinc finger right (ZF R), <i>AAVS1</i> guide RNAs T1 and T2 underlined (a). The translated DNA sequence of the <i>AAVS1</i> binding site with stop codons (-) (b). The translated DNA after genome editing. In this case a 1 bp deletion removes the stop codons and allows in frame of translation of the C-terminal GFP resulting in fluorescence (c). Representative flow cytometry plots of 293FT cells 44–48 hours after transfection with GFP-AAVS1 SplitAx only (d), GFP-AAVS1 SplitAx with single AAVS1 Zinc Finger Left (Zn L) (e), GFP-AAVS1 SplitAx with AAVS1 single Zinc Finger Right (Zn R) (f), and GFP-AAVS1 SplitAx with both AAVS1 Zinc Finger Lefand/Zinc Finger Right (Zn L, Zn R) (g). Quantification of flow cytometry data for the GFP-AAVS1 SplitAx with the AAVS1 Zinc Fingers (+), cells not transfected with a plasmid (-). Data shown as mean +/- SD (n = 3) (h). Representative flow cytometry plots of 293FT cells 44–48 hours after transfection with GFP-AAVS1 SplitAx only (i) GFP-AAVS1 SplitAx and hCAS9 (j), GFP-AAVS1 SplitAx, hCAS9 CRISPR and gRNA_AAVS1-T1 (k), GFP-AAVS1 SplitAx, hCAS9 CRISPR and gRNA_AAVS1-T2 (l). Quantification of flow cytometry data for the GFP-AAVS1 SplitAx with the CRIPSR gRNA_AAVS1- T1 or T2 and hCAS9 (+), cells not transfected with a plasmid (-). Data shown as +STDev (n = 3) (m).</p

Functional validation of the ZsGreen1-Cfms-SplitAx reporter assay with Cfms gRNAs and hCAS9 or D10A nickase.

<p>Schematic diagram of the 5’ and 3’end of Zs Green1 separated by the <i>Cfms</i> binding site. The DNA sequence of the Cfms binding site is shown and the location of the gRNA_Cfms-8a, 8b and 9b are underlined (a). Representative flow cytometry plots of 293FT cells 44–48 hours after transfection with ZsGreen1-Cfms-SplitAx with hCAS9 (b), ZsGreen1-Cfms-SplitAx, hCAS9 with gRNA_Cfms-8a (c), ZsGreen1-Cfms-SplitAx, hCAS9 with gRNA_Cfms-8b (d), ZsGreen1-Cfms-SplitAx, hCAS9 with gRNA_Cfms-9b (e). Quantification of flow cytometry data for the ZsGreen1-Cfms- SplitAx and hCAS9 with the gRNAs_Cfms (+), cells not transfected with a plasmid (-). Data shown as mean +/- SD (n = 3) (f). Representative flow cytometry plots of 293FT cells 44–48 hours after transfection with ZsGreen1-Cfms-SplitAx only (g), ZsGreen1-Cfms-SplitAx with D10A nickase (h), ZsGreen1-Cfms-SplitAx, D10A nickase with gRNA_Cfms-8a and8b (i), ZsGreen1-Cfms-SplitAx, D10A nickase with gRNA_Cfms-8a and 8b (j). Graphical representation of flow cytometry data for the ZsGreen1-Cfms- SplitAx and D10A nickase with the gRNAs_Cfms (+), cells not transfected with a plasmid (-). Data shown as mean +/- SD (n = 3) (k).</p