While Cas9 nucleases permit rapid and efficient generation of gene-edited cell lines, the CRISPR-Cas9 system can introduce undesirable "on-target" mutations within the second allele of successfully modified cells via non-homologous end joining (NHEJ). To address this, we fused the Streptococcus pyogenes Cas9 (SpCas9) nuclease to a peptide derived from the human Geminin protein (SpCas9-Gem) to facilitate its degradation during the G1 phase of the cell cycle, when DNA repair by NHEJ predominates. We also use mRNA transfection to facilitate low and transient expression of modified and unmodified versions of Cas9. Although the frequency of homologous recombination was similar for SpCas9-Gem and SpCas9, we observed a marked reduction in the capacity for SpCas9-Gem to induce NHEJ-mediated indels at the target locus. Moreover, in contrast to native SpCas9, we demonstrate that transient SpCas9-Gem expression enables reliable generation of both knockin reporter cell lines and genetically repaired patient-specific induced pluripotent stem cell lines free of unwanted mutations at the targeted locus.

We describe the generation and characterization of 5 human induced pluripotent stem cell (iPSC) lines derived from peripheral blood mononuclear cells (PBMCs) of healthy adult individuals. The PBMCs were reprogrammed using non-integrating Sendai viruses containing the reprogramming factors POU5F1 (OCT4), SOX2, KLF4 and MYC. The iPSC lines exhibited a normal karyotype, expressed pluripotency markers and differentiated into cells representative of the three embryonic germ layers. These iPSC lines can be used as controls in studying disease mechanisms.

Mutations in RAB39B are a known cause of X-linked early onset Parkinson's disease. Isogenic human embryonic stem cell lines carrying two independent deletions of RAB39B were generated using CRISPR/Cas9 genome editing tool. The deletions were confirmed by PCR and direct sequence analysis in two edited stem cell lines. Both cell lines showed pluripotency and displayed a normal karyotype. Further, they were able to form embryoid bodies in vitro, and express markers indicative of differentiation to the three germ layers.

Human pluripotent stem cell (hPSC) differentiation typically yields heterogeneous populations. Knowledge of signals controlling embryonic lineage bifurcations could efficiently yield desired cell types through exclusion of alternate fates. Therefore, we revisited signals driving induction and anterior-posterior patterning of definitive endoderm to generate a coherent roadmap for endoderm differentiation. With striking temporal dynamics, BMP and Wnt initially specified anterior primitive streak (progenitor to endoderm), yet, 24 hr later, suppressed endoderm and induced mesoderm. At lineage bifurcations, cross-repressive signals separated mutually exclusive fates; TGF-β and BMP/MAPK respectively induced pancreas versus liver from endoderm by suppressing the alternate lineage. We systematically blockaded alternate fates throughout multiple consecutive bifurcations, thereby efficiently differentiating multiple hPSC lines exclusively into endoderm and its derivatives. Comprehensive transcriptional and chromatin mapping of highly pure endodermal populations revealed that endodermal enhancers existed in a surprising diversity of "pre-enhancer" states before activation, reflecting the establishment of a permissive chromatin landscape as a prelude to differentiation.

Diminished mitochondrial function is causally related to some heart diseases. Here, we developed a human disease model based on cardiomyocytes from human embryonic stem cells (hESCs), in which an important pathway of mitochondrial gene expression was inactivated. Repression of PGC-1α, which is normally induced during development of cardiomyocytes, decreased mitochondrial content and activity and decreased the capacity for coping with energetic stress. Yet, concurrently, reactive oxygen species (ROS) levels were lowered, and the amplitude of the action potential and the maximum amplitude of the calcium transient were in fact increased. Importantly, in control cardiomyocytes, lowering ROS levels emulated this beneficial effect of PGC-1α knockdown and similarly increased the calcium transient amplitude. Our results suggest that controlling ROS levels may be of key physiological importance for recapitulating mature cardiomyocyte phenotypes, and the combination of bioassays used in this study may have broad application in the analysis of cardiac physiology pertaining to disease.

The study of human cardiogenesis would benefit from a detailed cell lineage fate map akin to that established for the haematopoietic lineages. Here we sought to define cell lineage relationships based on the expression of NKX2-5 and the cell surface markers VCAM1, SIRPA and CD34 during human cardiovascular development. Expression of NKX2-5(GFP) was used to identify cardiac progenitors and cardiomyocytes generated during the differentiation of NKX2-5(GFP/w) human embryonic stem cells (hESCs). Cardiovascular cell lineages sub-fractionated on the basis of SIRPA, VCAM1 and CD34 expression were assayed for differentiation potential and gene expression. The NKX2-5(pos)CD34(pos) population gave rise to endothelial cells that rapidly lost NKX2-5 expression in culture. Conversely, NKX2-5 expression was maintained in myocardial committed cells, which progressed from being NKX2-5(pos)SIRPA(pos) to NKX2-5(pos)SIRPA(pos)VCAM1(pos). Up-regulation of VCAM1 was accompanied by the expression of myofilament markers and reduced clonal capacity, implying a restriction of cell fate potential. Combinatorial expression of NKX2-5, SIRPA, VCAM1 and CD34 can be used to define discrete stages of cardiovascular cell lineage differentiation. These markers identify specific stages of cardiomyocyte and endothelial lineage commitment and, thus provide a scaffold for establishing a fate map of early human cardiogenesis.

We investigated the role of canonical WNT signaling in mesoderm and hematopoietic development from human embryonic stem cells (hESCs) using a recombinant human protein-based differentiation medium (APEL). In contrast to prior studies using less defined culture conditions, we found that WNT3A alone was a poor inducer of mesoderm. However, WNT3A synergized with BMP4 to accelerate mesoderm formation, increase embryoid body size, and increase the number of hematopoietic blast colonies. Interestingly, inclusion of WNT3A or a GSK3 inhibitor in methylcellulose colony-forming assays at 4 days of differentiation abrogated blast colony formation but supported the generation of mesospheres that expressed genes associated with mesenchymal lineages. Mesospheres differentiated into cells with characteristics of bone, fat, and smooth muscle. These studies identify distinct effects for WNT3A, supporting the formation of hematopoietic or mesenchymal lineages from human embryonic stem cells, depending upon differentiation stage at the time of exposure.

Genetic analysis has revealed that the dual specificity protein kinase DYRK1A has multiple roles in the development of the central nervous system. Increased DYRK1A gene dosage, such as occurs in Down syndrome, is known to affect neural progenitor cell differentiation, while haploinsufficiency of DYRK1A is associated with severe microcephaly. Using a set of known and newly synthesized DYRK1A inhibitors, along with CRISPR-mediated gene activation and shRNA knockdown of DYRK1A, we show here that chemical inhibition or genetic knockdown of DYRK1A interferes with neural specification of human pluripotent stem cells, a process equating to the earliest stage of human brain development. Specifically, DYRK1A inhibition insulates the self-renewing subpopulation of human pluripotent stem cells from powerful signals that drive neural induction. Our results suggest a novel mechanism for the disruptive effects of the absence or haploinsufficiency of DYRK1A on early mammalian development, and reveal a requirement for DYRK1A in the acquisition of competence for differentiation in human pluripotent stem cells.

Timed exposure of pluripotent stem cell cultures to exogenous molecules is widely used to drive differentiation towards desired cell lineages. However, screening differentiation conditions in conventional static cultures can become impractical in large parameter spaces, and is intrinsically limited by poor spatiotemporal control of the microenvironment that also makes it impossible to determine whether exogenous factors act directly or through paracrine-dependent mechanisms. We detail here the development of a continuous flow microbioreactor array platform that combines full-factorial multiplexing of input factors with progressive accumulation of paracrine factors through serially-connected culture chambers, and further, the use of this system to explore the combinatorial parameter space of both exogenous and paracrine factors involved in human embryonic stem cell (hESC) differentiation to a MIXL1-GFP(+) primitive streak-like population. We show that well known inducers of primitive streak (BMP, Activin and Wnt signals) do not simply act directly on hESC to induce MIXL1 expression, but that this requires accumulation of surplus, endogenous factors; and, that conditioned medium or FGF-2 supplementation is able to offset this. Our approach further reveals the presence of a paracrine, negative feedback loop to the MIXL1-GFP(+) population, which can be overcome with GSK-3β inhibitors (BIO or CHIR99021), implicating secreted Wnt inhibitory signals such as DKKs and sFRPs as candidate effectors. Importantly, modulating paracrine effects identified in microbioreactor arrays by supplementing FGF-2 and CHIR in conventional static culture vessels resulted in improved differentiation outcomes. We therefore demonstrate that this microbioreactor array platform uniquely enables the identification and decoding of complex soluble factor signalling hierarchies, and that this not only challenges prevailing strategies for extrinsic control of hESC differentiation, but also is translatable to conventional culture systems.

The production of human platelets from embryonic stem cells in a defined culture system is a prerequisite for the generation of platelets for therapeutic use. As an important step towards this goal, we report the differentiation of human embryonic stem cells (hESCs) towards the megakaryocyte (Mk) lineage using a 'spin embryoid body' method in serum-free differentiation medium.

Recent studies have shown evidence for the functional integration of human pluripotent stem cell (hPSC)-derived ventral midbrain dopamine (vmDA) neurons in animal models of Parkinson's disease. Although these cells present a sustainable alternative to fetal mesencephalic grafts, a number of hurdles require attention prior to clinical translation. These include the persistent use of xenogeneic reagents and challenges associated with scalability and storage of differentiated cells. In this study, we describe the first fully defined feeder- and xenogeneic-free protocol for the generation of vmDA neurons from hPSCs and utilize two novel reporter knock-in lines (LMX1A-eGFP and PITX3-eGFP) for in-depth in vitro and in vivo tracking. Across multiple embryonic and induced hPSC lines, this "next generation" protocol consistently increases both the yield and proportion of vmDA neural progenitors (OTX2/FOXA2/LMX1A) and neurons (FOXA2/TH/PITX3) that display classical vmDA metabolic and electrophysiological properties. We identify the mechanism underlying these improvements and demonstrate clinical applicability with the first report of scalability and cryopreservation of bona fide vmDA progenitors at a time amenable to transplantation. Finally, transplantation of xeno-free vmDA progenitors from LMX1A- and PITX3-eGFP reporter lines into Parkinsonian rodents demonstrates improved engraftment outcomes and restoration of motor deficits. These findings provide important and necessary advancements for the translation of hPSC-derived neurons into the clinic. Stem Cells Translational Medicine 2017;6:937-948.

The genetic regulatory network controlling early fate choices during human blood cell development are not well understood. We used human pluripotent stem cell reporter lines to track the development of endothelial and haematopoietic populations in an in vitro model of human yolk-sac development. We identified SOX17-CD34+CD43- endothelial cells at day 2 of blast colony development, as a haemangioblast-like branch point from which SOX17-CD34+CD43+ blood cells and SOX17+CD34+CD43- endothelium subsequently arose. Most human blood cell development was dependent on RUNX1. Deletion of RUNX1 only permitted a single wave of yolk sac-like primitive erythropoiesis, but no yolk sac myelopoiesis or aorta-gonad-mesonephros (AGM)-like haematopoiesis. Blocking GFI1 and/or GFI1B activity with a small molecule inhibitor abrogated all blood cell development, even in cell lines with an intact RUNX1 gene. Together, our data define the hierarchical requirements for RUNX1, GFI1 and/or GFI1B during early human haematopoiesis arising from a yolk sac-like SOX17-negative haemogenic endothelial intermediate.

MicroRNAs (miRNAs) are translational regulatory molecules with recognised roles in heart development and disease. Therefore, it is important to define the human miRNA expression profile in cardiac progenitors and early-differentiated cardiomyocytes and to determine whether critical cardiac transcription factors such as NKX2-5 regulate miRNA expression. We used an NKX2-5eGFP/w reporter line to isolate both cardiac committed mesoderm and cardiomyocytes. We identified 11 miRNAs that were differentially expressed in NKX2-5 -expressing cardiac mesoderm compared to non-cardiac mesoderm. Subsequent profiling revealed that the canonical myogenic miRNAs including MIR1-1, MIR133A1 and MIR208A were enriched in cardiomyocytes. Strikingly, deletion of NKX2-5 did not result in gross changes in the cardiac miRNA profile, either at committed mesoderm or cardiomyocyte stages. Thus, in early human cardiomyocyte commitment and differentiation, the cardiac myogenic miRNA program is predominantly regulated independently of the highly conserved NKX2-5 -dependant gene regulatory network.

We describe the generation and characterisation of five human induced pluripotent stem cell (iPSC) lines derived from peripheral blood mononuclear cells (PBMCs) of healthy adult individuals. The PBMCs were reprogrammed using non-integrating Sendai viruses containing the reprogramming factors POU5F1 (OCT4), SOX2, KLF4 and MYC. The iPSC lines exhibited a normal karyotype, and pluripotency was validated by flow cytometry and immunofluorescence of pluripotency markers, and their differentiation into cells representative of the three embryonic germ layers. These iPSC lines can be used as controls in studying disease mechanisms.

To develop a disease model for the human 'brittle bone' disease, osteogenesis imperfecta, we have used gene editing to produce a facsimile of the patient heterozygous COL1A1 mutation in an established control iPSC line. The gene-edited line had a normal karyotype, expressed pluripotency markers and differentiated into cells representative of the three embryonic germ layers. This iPSC line and the isogenic parental iPSC line will be of use in exploring osteogenesis imperfecta disease mechanisms and therapeutic approaches in vitro.

To develop a disease model for the human 'brittle bone' disease, osteogenesis imperfecta, we used a simultaneous reprogramming and CRISPR-Cas9 genome editing method to produce an iPSC line with the heterozygous patient mutation (COL1A1 c. 3936 G>T) along with an isogenic gene-corrected control iPSC line. Both IPSC lines had a normal karyotype, expressed pluripotency markers and differentiated into cells representative of the three embryonic germ layers. This osteogenesis imperfecta mutant and isogenic iPSC control line will be of use in exploring disease mechanisms and therapeutic approaches in vitro.

Type 1 diabetes is an autoimmune disorder characterised by loss of insulin-producing beta cells of the pancreas. Progress in understanding the cellular and molecular mechanisms underlying the human disease has been hampered by a dearth of appropriate human experimental models. We previously reported the characterisation of islet-infiltrating CD4+ T cells from a deceased organ donor who had type 1 diabetes.

Thymic epithelial cells (TECs) play a critical role in T cell maturation and tolerance induction. The generation of TECs from in vitro differentiation of human pluripotent stem cells (PSCs) provides a platform on which to study the mechanisms of this interaction and has implications for immune reconstitution. To facilitate analysis of PSC-derived TECs, we generated hESC reporter lines in which sequences encoding GFP were targeted to FOXN1, a gene required for TEC development. Using this FOXN1 (GFP/w) line as a readout, we developed a reproducible protocol for generating FOXN1-GFP(+) thymic endoderm cells. Transcriptional profiling and flow cytometry identified integrin-β4 (ITGB4, CD104) and HLA-DR as markers that could be used in combination with EpCAM to selectively purify FOXN1(+) TEC progenitors from differentiating cultures of unmanipulated PSCs. Human FOXN1(+) TEC progenitors generated from PSCs facilitate the study of thymus biology and are a valuable resource for future applications in regenerative medicine.

Mixl1 is a homeodomain transcription factor required for mesoderm and endoderm patterning during mammalian embryogenesis. Despite its crucial function in development, co-factors that modulate the activity of Mixl1 remain poorly defined. Here we report that Mixl1 interacts physically and functionally with the T-box protein Brachyury and related members of the T-box family of transcription factors. Transcriptional and protein analyses demonstrated overlapping expression of Mixl1 and Brachyury during embryonic stem cell differentiation. In vitro protein interaction studies showed that the Mixl1 with Brachyury associated via their DNA-binding domains and gel shift assays revealed that the Brachyury T-box domain bound to Mixl1-DNA complexes. Furthermore, luciferase reporter experiments indicated that association of Mixl1 with Brachyury and related T-box factors inhibited the transactivating potential of Mixl1 on the Gsc and Pdgfrα promoters. Our results indicate that the activity of Mixl1 can be modulated by protein-protein interactions and that T-box factors can function as negative regulators of Mixl1 activity.

Acute myeloid leukemia (AML) is a hematopoietic malignancy caused by recurrent mutations in genes encoding transcriptional, chromatin, and/or signaling regulators. The t(8;21) translocation generates the aberrant transcription factor RUNX1-ETO (RUNX1-RUNX1T1), which by itself is insufficient to cause disease. t(8;21) AML patients show extensive chromatin reprogramming and have acquired additional mutations. Therefore, the genomic and developmental effects directly and solely attributable to RUNX1-ETO expression are unclear. To address this, we employ a human embryonic stem cell differentiation system capable of forming definitive myeloid progenitor cells to express RUNX1-ETO in an inducible fashion. Induction of RUNX1-ETO causes extensive chromatin reprogramming by interfering with RUNX1 binding, blocks differentiation, and arrests cellular growth, whereby growth arrest is reversible following RUNX1-ETO removal. Single-cell gene expression analyses show that RUNX1-ETO induction alters the differentiation of early myeloid progenitors, but not of other progenitor types, indicating that oncoprotein-mediated transcriptional reprogramming is highly target cell specific.