Publications by authors named "Edouard G Stanley"

104 Publications

Human yolk sac-like haematopoiesis generates , and/or dependent blood and -positive endothelium.

Development 2020 10 29;147(20). Epub 2020 Oct 29.

Murdoch Children's Research Institute, The Royal Children's Hospital, Flemington Road, Parkville, Victoria 3052, Australia

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 model of human yolk-sac development. We identified SOX17CD34CD43 endothelial cells at day 2 of blast colony development, as a haemangioblast-like branch point from which SOX17CD34CD43 blood cells and SOX17CD34CD43 endothelium subsequently arose. Most human blood cell development was dependent on RUNX1. Deletion of 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 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.
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http://dx.doi.org/10.1242/dev.193037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7648599PMC
October 2020

CRISPR/Cas9 gene editing of a SOX9 reporter human iPSC line to produce two TRPV4 patient heterozygous missense mutant iPSC lines, MCRIi001-A-3 (TRPV4 p.F273L) and MCRIi001-A-4 (TRPV4 p.P799L).

Stem Cell Res 2020 10 3;48:101942. Epub 2020 Aug 3.

Murdoch Children's Research Institute, Australia; Department of Paediatrics, University of Melbourne, Australia.

To produce in vitro models of human chondrodysplasias caused by dominant missense mutations in TRPV4, we used CRISPR/Cas9 gene editing to introduce two heterozygous patient mutations (p.F273L and p.P799L) into an established control human iPSC line. This control line expressed a fluorescent reporter (tdTomato) at the SOX9 locus to allow real-time monitoring of cartilage differentiation by SOX9 expression. Both TRPV4 mutant iPSC lines had normal karyotypes, expressed pluripotency markers, and could differentiate into cells representative of the three embryonic germ layers. These iPSC lines, with the parental isogenic control, will be used to study TRPV4 chondrodysplasia mechanisms and explore therapeutic approaches.
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http://dx.doi.org/10.1016/j.scr.2020.101942DOI Listing
October 2020

Expression of RUNX1-ETO Rapidly Alters the Chromatin Landscape and Growth of Early Human Myeloid Precursor Cells.

Cell Rep 2020 05;31(8):107691

Institute for Cancer and Genomic Sciences, College of Medicine and Dentistry, University of Birmingham, Birmingham B15 2TT, UK. Electronic address:

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.
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http://dx.doi.org/10.1016/j.celrep.2020.107691DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7262600PMC
May 2020

Generation of a heterozygous COL2A1 (p.R989C) spondyloepiphyseal dysplasia congenita mutation iPSC line, MCRIi001-B, using CRISPR/Cas9 gene editing.

Stem Cell Res 2020 05 11;45:101843. Epub 2020 May 11.

Murdoch Children's Research Institute, Australia; Department of Paediatrics, University of Melbourne, Australia.

To produce an in vitro model of the human chondrodysplasia, spondyloepiphyseal dysplasia congenita, we used CRISPR/Cas9 gene editing to generate a heterozygous patient COL2A1 mutation in an established control human iPSC line. The gene-edited heterozygous COL2A1 p.R989C line had a normal karyotype, expressed pluripotency markers, and could differentiate into cells representative of the three embryonic germ layers. When differentiated into cartilage this cell line and the parental isogenic control may be used to explore disease mechanisms and evaluate therapeutic approaches.
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http://dx.doi.org/10.1016/j.scr.2020.101843DOI Listing
May 2020

Multipotent RAG1+ progenitors emerge directly from haemogenic endothelium in human pluripotent stem cell-derived haematopoietic organoids.

Nat Cell Biol 2020 01 6;22(1):60-73. Epub 2020 Jan 6.

Murdoch Children's Research Institute, Parkville, Victoria, Australia.

Defining the ontogeny of the human adaptive immune system during embryogenesis has implications for understanding childhood diseases including leukaemias and autoimmune conditions. Using RAG1:GFP human pluripotent stem cell reporter lines, we examined human T-cell genesis from pluripotent-stem-cell-derived haematopoietic organoids. Under conditions favouring T-cell development, RAG1+ cells progressively upregulated a cohort of recognized T-cell-associated genes, arresting development at the CD4+CD8+ stage. Sort and re-culture experiments showed that early RAG1+ cells also possessed B-cell, myeloid and erythroid potential. Flow cytometry and single-cell-RNA-sequencing data showed that early RAG1+ cells co-expressed the endothelial/haematopoietic progenitor markers CD34, VECAD and CD90, whereas imaging studies identified RAG1+ cells within CD31+ endothelial structures that co-expressed SOX17+ or the endothelial marker CAV1. Collectively, these observations provide evidence for a wave of human T-cell development that originates directly from haemogenic endothelium via a RAG1+ intermediate with multilineage potential.
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http://dx.doi.org/10.1038/s41556-019-0445-8DOI Listing
January 2020

Generation of a SOX9-tdTomato reporter human iPSC line, MCRIi001-A-2, using CRISPR/Cas9 editing.

Stem Cell Res 2020 01 19;42:101689. Epub 2019 Dec 19.

Murdoch Children's Research Institute, Australia; Department of Paediatrics, University of Melbourne, Australia.

To develop an iPSC SOX9 reporter line for monitoring differentiation into SOX9 expressing cells such as chondrocytes, cranial neural crest and Sertoli cells, we used gene editing to introduce sequences encoding the tdTomato fluorescent protein into the SOX9 locus. The gene-edited line had a normal karyotype, expressed pluripotency markers and differentiated into cells representative of the three embryonic germ layers. Endogenous SOX9 expression was undisturbed and the tdTomato fluorescent reporter mirrored SOX9 mRNA expression. This iPSC line will be useful for assessing iPSC differentiation into SOX9-expressing cells and enrichment by cell sorting.
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http://dx.doi.org/10.1016/j.scr.2019.101689DOI Listing
January 2020

Publisher Correction: The role of cardiac transcription factor NKX2-5 in regulating the human cardiac miRNAome.

Sci Rep 2019 Dec 27;9(1):20269. Epub 2019 Dec 27.

Murdoch Children's Research Institute, Royal Children's Hospital, Flemington Road, Parkville, Victoria, 3052, Australia.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41598-019-55970-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6934776PMC
December 2019

The role of cardiac transcription factor NKX2-5 in regulating the human cardiac miRNAome.

Sci Rep 2019 11 4;9(1):15928. Epub 2019 Nov 4.

Murdoch Children's Research Institute, Royal Children's Hospital, Flemington Road, Parkville, Victoria, 3052, Australia.

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 NKX2-5 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.
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http://dx.doi.org/10.1038/s41598-019-52280-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6828809PMC
November 2019

Induced pluripotent stem cell macrophages present antigen to proinsulin-specific T cell receptors from donor-matched islet-infiltrating T cells in type 1 diabetes.

Diabetologia 2019 12 12;62(12):2245-2251. Epub 2019 Sep 12.

Murdoch Children's Research Institute, Flemington Road, Parkville, VIC, 3052, Australia.

Aims/hypothesis: 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.

Methods: Induced pluripotent stem cell (iPSC) lines derived from the above donor were differentiated into CD14 macrophages and tested for their capacity to present antigen to T cell receptors (TCRs) derived from islet-infiltrating CD4 T cells from the same donor.

Results: The iPSC macrophages displayed typical macrophage morphology, surface markers (CD14, CD86, CD16 and CD11b) and were phagocytic. In response to IFNγ treatment, iPSC macrophages upregulated expression of HLA class II, a characteristic that correlated with their capacity to present epitopes derived from proinsulin C-peptide to a T cell line expressing TCRs derived from islet-infiltrating CD4 T cells of the original donor. T cell activation was specifically blocked by anti-HLA-DQ antibodies but not by antibodies directed against HLA-DR.

Conclusions/interpretation: This study provides a proof of principle for the use of iPSC-derived immune cells for modelling key cellular interactions in human type 1 diabetes.
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http://dx.doi.org/10.1007/s00125-019-04988-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6861360PMC
December 2019

The use of simultaneous reprogramming and gene correction to generate an osteogenesis imperfecta patient COL1A1 c. 3936 G>T iPSC line and an isogenic control iPSC line.

Stem Cell Res 2019 07 4;38:101453. Epub 2019 May 4.

Murdoch Children's Research Institute, Australia; Department of Biochemistry and Molecular Biology, University of Melbourne, Australia. Electronic address:

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.
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http://dx.doi.org/10.1016/j.scr.2019.101453DOI Listing
July 2019

Generation of a heterozygous COL1A1 (c.3969_3970insT) osteogenesis imperfecta mutation human iPSC line, MCRIi001-A-1, using CRISPR/Cas9 editing.

Stem Cell Res 2019 05 23;37:101449. Epub 2019 Apr 23.

Murdoch Children's Research Institute, University of Melbourne, Australia; Department of Biochemistry and Molecular Biology, University of Melbourne, Australia. Electronic address:

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.
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http://dx.doi.org/10.1016/j.scr.2019.101449DOI Listing
May 2019

Kidney micro-organoids in suspension culture as a scalable source of human pluripotent stem cell-derived kidney cells.

Development 2019 03 7;146(5). Epub 2019 Mar 7.

Murdoch Children's Research Institute, Flemington Rd, Parkville, Victoria 3052, Australia

Kidney organoids have potential uses in disease modelling, drug screening and regenerative medicine. However, novel cost-effective techniques are needed to enable scaled-up production of kidney cell types We describe here a modified suspension culture method for the generation of kidney micro-organoids from human pluripotent stem cells. Optimisation of differentiation conditions allowed the formation of micro-organoids, each containing six to ten nephrons that were surrounded by endothelial and stromal populations. Single cell transcriptional profiling confirmed the presence and transcriptional equivalence of all anticipated renal cell types consistent with a previous organoid culture method. This suspension culture micro-organoid methodology resulted in a three- to fourfold increase in final cell yield compared with static culture, thereby representing an economical approach to the production of kidney cells for various biological applications.
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http://dx.doi.org/10.1242/dev.172361DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6432662PMC
March 2019

Generation of iPSC lines from peripheral blood mononuclear cells from 5 healthy adults.

Stem Cell Res 2019 01 27;34:101380. Epub 2018 Dec 27.

Murdoch Children's Research Institute, The Royal Children's Hospital, Melbourne, Victoria 3052, Australia; Department of Paediatrics, The University of Melbourne, Melbourne, Victoria 3052, Australia; Department of Anatomy and Developmental Biology, Monash University, Clayton, Victoria 3800, Australia.

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.
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http://dx.doi.org/10.1016/j.scr.2018.101380DOI Listing
January 2019

NKX2-5 regulates human cardiomyogenesis via a HEY2 dependent transcriptional network.

Nat Commun 2018 04 10;9(1):1373. Epub 2018 Apr 10.

Murdoch Childrens Research Institute, Royal Children's Hospital, Flemington Road, Parkville, VIC, 3052, Australia.

Congenital heart defects can be caused by mutations in genes that guide cardiac lineage formation. Here, we show deletion of NKX2-5, a critical component of the cardiac gene regulatory network, in human embryonic stem cells (hESCs), results in impaired cardiomyogenesis, failure to activate VCAM1 and to downregulate the progenitor marker PDGFRα. Furthermore, NKX2-5 null cardiomyocytes have abnormal physiology, with asynchronous contractions and altered action potentials. Molecular profiling and genetic rescue experiments demonstrate that the bHLH protein HEY2 is a key mediator of NKX2-5 function during human cardiomyogenesis. These findings identify HEY2 as a novel component of the NKX2-5 cardiac transcriptional network, providing tangible evidence that hESC models can decipher the complex pathways that regulate early stage human heart development. These data provide a human context for the evaluation of pathogenic mutations in congenital heart disease.
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http://dx.doi.org/10.1038/s41467-018-03714-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5893543PMC
April 2018

Generation of RAB39B knockout isogenic human embryonic stem cell lines to model RAB39B-mediated Parkinson's disease.

Stem Cell Res 2018 04 21;28:161-164. Epub 2018 Feb 21.

Bruce Lefroy Centre for Genetic Health Research, Murdoch Children's Research Institute, Melbourne, Victoria, 3052, Australia; Department of Paediatrics, The University of Melbourne, Melbourne, Victoria, 3052, Australia. Electronic address:

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.
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http://dx.doi.org/10.1016/j.scr.2018.02.015DOI Listing
April 2018

WNT9A Is a Conserved Regulator of Hematopoietic Stem and Progenitor Cell Development.

Genes (Basel) 2018 Jan 29;9(2). Epub 2018 Jan 29.

Department of Cellular and Molecular Medicine, University of California, San Diego, CA 92037, USA.

Hematopoietic stem cells (HSCs) differentiate into all cell types of the blood and can be used therapeutically to treat hematopoietic cancers and disorders. Despite decades of research, it is not yet possible to derive therapy-grade HSCs from pluripotent precursors. Analysis of HSC development in model organisms has identified some of the molecular cues that are necessary to instruct hematopoiesis in vivo, including Wnt9A, which is required during an early time window in zebrafish development. Although bona fide HSCs cannot be derived in vitro, it is possible to model human hematopoietic progenitor development by differentiating human pluripotent stem cells to hematopoietic cells. Herein, we modulate WNT9A expression during the in vitro differentiation of human embryonic stem cells to hematopoietic progenitor cells and demonstrate that WNT9A also regulates human hematopoietic progenitor cell development in vitro. Overexpression of WNT9A only impacts differentiation to CD34⁺/CD45⁺ cells during early time windows and does so in a dose-dependent manner. The cells that receive the Wnt signal-not the cells that secrete WNT9A-differentiate most efficiently to hematopoietic progenitors; this mimics the paracrine action of Wnt9a during in vivo hematopoiesis. Taken together, these data indicate that WNT9A is a conserved regulator of zebrafish and human hematopoietic development.
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http://dx.doi.org/10.3390/genes9020066DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5852562PMC
January 2018

Regulatory networks specifying cortical interneurons from human embryonic stem cells reveal roles for CHD2 in interneuron development.

Proc Natl Acad Sci U S A 2017 12 11;114(52):E11180-E11189. Epub 2017 Dec 11.

Department of Developmental Biology, Washington University School of Medicine, St. Louis, MO 63110;

Cortical interneurons (cINs) modulate excitatory neuronal activity by providing local inhibition. During fetal development, several cIN subtypes derive from the medial ganglionic eminence (MGE), a transient ventral telencephalic structure. While altered cIN development contributes to neurodevelopmental disorders, the inaccessibility of human fetal brain tissue during development has hampered efforts to define molecular networks controlling this process. Here, we modified protocols for directed differentiation of human embryonic stem cells, obtaining efficient, accelerated production of MGE-like progenitors and MGE-derived cIN subtypes with the expected electrophysiological properties. We defined transcriptome changes accompanying this process and integrated these data with direct transcriptional targets of NKX2-1, a transcription factor controlling MGE specification. This analysis defined NKX2-1-associated genes with enriched expression during MGE specification and cIN differentiation, including known and previously unreported transcription factor targets with likely roles in MGE specification, and other target classes regulating cIN migration and function. NKX2-1-associated peaks were enriched for consensus binding motifs for NKX2-1, LHX, and SOX transcription factors, suggesting roles in coregulating MGE gene expression. Among the NKX2-1 direct target genes with cIN-enriched expression was , which encodes a chromatin remodeling protein mutated to cause human epilepsies. Accordingly, CHD2 deficiency impaired cIN specification and altered later electrophysiological function, while CHD2 coassociated with NKX2-1 at -regulatory elements and was required for their transactivation by NKX2-1 in MGE-like progenitors. This analysis identified several aspects of gene-regulatory networks underlying human MGE specification and suggested mechanisms by which NKX2-1 acts with chromatin remodeling activities to regulate gene expression programs underlying cIN development.
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http://dx.doi.org/10.1073/pnas.1712365115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5748186PMC
December 2017

Overcoming Monocarboxylate Transporter 8 (MCT8)-Deficiency to Promote Human Oligodendrocyte Differentiation and Myelination.

EBioMedicine 2017 Nov 19;25:122-135. Epub 2017 Oct 19.

Department of Medicine, Central Clinical School, Monash University, Prahran, Victoria 3004, Australia. Electronic address:

Cell membrane thyroid hormone (TH) transport can be facilitated by the monocarboxylate transporter 8 (MCT8), encoded by the solute carrier family 16 member 2 (SLC16A2) gene. Human mutations of the gene, SLC16A2, result in the X-linked-inherited psychomotor retardation and hypomyelination disorder, Allan-Herndon-Dudley syndrome (AHDS). We posited that abrogating MCT8-dependent TH transport limits oligodendrogenesis and myelination. We show that human oligodendrocytes (OL), derived from the NKX2.1-GFP human embryonic stem cell (hESC) reporter line, express MCT8. Moreover, treatment of these cultures with DITPA (an MCT8-independent TH analog), up-regulates OL differentiation transcription factors and myelin gene expression. DITPA promotes hESC-derived OL myelination of retinal ganglion axons in co-culture. Pharmacological and genetic blockade of MCT8 induces significant OL apoptosis, impairing myelination. DITPA treatment limits OL apoptosis mediated by SLC16A2 down-regulation primarily signaling through AKT phosphorylation, driving myelination. Our results highlight the potential role of MCT8 in TH transport for human OL development and may implicate DITPA as a promising treatment for developmentally-regulated myelination in AHDS.
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http://dx.doi.org/10.1016/j.ebiom.2017.10.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5704066PMC
November 2017

Inhibition of DYRK1A disrupts neural lineage specificationin human pluripotent stem cells.

Elife 2017 09 8;6. Epub 2017 Sep 8.

Department of Anatomy and Neuroscience, University of Melbourne, Victoria, Australia.

Genetic analysis has revealed that the dual specificity protein kinase DYRK1A has multiple roles in the development of the central nervous system. Increased gene dosage, such as occurs in Down syndrome, is known to affect neural progenitor cell differentiation, while haploinsufficiency of 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 , we show here that chemical inhibition or genetic knockdown of 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 on early mammalian development, and reveal a requirement for in the acquisition of competence for differentiation in human pluripotent stem cells.
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http://dx.doi.org/10.7554/eLife.24502DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5656431PMC
September 2017

Fusion of Regionally Specified hPSC-Derived Organoids Models Human Brain Development and Interneuron Migration.

Cell Stem Cell 2017 09 27;21(3):383-398.e7. Epub 2017 Jul 27.

Department of Genetics, Yale Stem Cell Center, Yale School of Medicine, New Haven, CT 06520, USA. Electronic address:

Organoid techniques provide unique platforms to model brain development and neurological disorders. Whereas several methods for recapitulating corticogenesis have been described, a system modeling human medial ganglionic eminence (MGE) development, a critical ventral brain domain producing cortical interneurons and related lineages, has been lacking until recently. Here, we describe the generation of MGE and cortex-specific organoids from human pluripotent stem cells that recapitulate the development of MGE and cortex domains, respectively. Population and single-cell RNA sequencing (RNA-seq) profiling combined with bulk assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) analyses revealed transcriptional and chromatin accessibility dynamics and lineage relationships during MGE and cortical organoid development. Furthermore, MGE and cortical organoids generated physiologically functional neurons and neuronal networks. Finally, fusing region-specific organoids followed by live imaging enabled analysis of human interneuron migration and integration. Together, our study provides a platform for generating domain-specific brain organoids and modeling human interneuron migration and offers deeper insight into molecular dynamics during human brain development.
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http://dx.doi.org/10.1016/j.stem.2017.07.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5720381PMC
September 2017

Human haematopoietic stem cell development: from the embryo to the dish.

Development 2017 07;144(13):2323-2337

Institute for Stem Cell Research, MRC Centre for Regenerative Medicine, University of Edinburgh, Edinburgh EH16 4UU, UK

Haematopoietic stem cells (HSCs) emerge during embryogenesis and give rise to the adult haematopoietic system. Understanding how early haematopoietic development occurs is of fundamental importance for basic biology and medical sciences, but our knowledge is still limited compared with what we know of adult HSCs and their microenvironment. This is particularly true for human haematopoiesis, and is reflected in our current inability to recapitulate the development of HSCs from pluripotent stem cells In this Review, we discuss what is known of human haematopoietic development: the anatomical sites at which it occurs, the different temporal waves of haematopoiesis, the emergence of the first HSCs and the signalling landscape of the haematopoietic niche. We also discuss the extent to which differentiation of human pluripotent stem cells recapitulates bona fide human developmental haematopoiesis, and outline some future directions in the field.
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http://dx.doi.org/10.1242/dev.134866DOI Listing
July 2017

Efficiently Specified Ventral Midbrain Dopamine Neurons from Human Pluripotent Stem Cells Under Xeno-Free Conditions Restore Motor Deficits in Parkinsonian Rodents.

Stem Cells Transl Med 2017 03 14;6(3):937-948. Epub 2016 Oct 14.

The Florey Institute of Neuroscience and Mental Health, University of Melbourne, Melbourne, Victoria, Australia.

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.
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http://dx.doi.org/10.5966/sctm.2016-0073DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5442782PMC
March 2017

Coculturing with endothelial cells promotes in vitro maturation and electrical coupling of human embryonic stem cell-derived cardiomyocytes.

J Heart Lung Transplant 2017 Jun 10;36(6):684-693. Epub 2017 Jan 10.

Stem Cell and Microenvironment Laboratory, Weill Cornell Medicine-Qatar, Doha, Qatar; Department of Genetic Medicine, Weill Cornell Medicine, New York, New York, USA. Electronic address:

Background: Pluripotent human embryonic stem cells (hESC) are a promising source of repopulating cardiomyocytes. We hypothesized that we could improve maturation of cardiomyocytes and facilitate electrical interconnections by creating a model that more closely resembles heart tissue; that is, containing both endothelial cells (ECs) and cardiomyocytes.

Methods: We induced cardiomyocyte differentiation in the coculture of an hESC line expressing the cardiac reporter NKX2.5-green fluorescent protein (GFP), and an Akt-activated EC line (E4ECs). We quantified spontaneous beating rates, synchrony, and coordination between different cardiomyocyte clusters using confocal imaging of Fura Red-detected calcium transients and computer-assisted image analysis.

Results: After 8 days in culture, 94% ± 6% of the NKX2-5GFP cells were beating when hESCs embryonic bodies were plated on E4ECs compared with 34% ± 12.9% for controls consisting of hESCs cultured on BD Matrigel (BD Biosciences) without ECs at Day 11 in culture. The spatial organization of beating areas in cocultures was different. The GFP cardiomyocytes were close to the E4ECs. The average beats/min of the cardiomyocytes in coculture was faster and closer to physiologic heart rates compared with controls (50 ± 14 [n = 13] vs 25 ± 9 [n = 8]; p < 0.05). The coculture with ECs led to synchronized beating relying on the endothelial network, as illustrated by the loss of synchronization upon the disruption of endothelial bridges.

Conclusions: The coculturing of differentiating cardiomyocytes with Akt-activated ECs but not EC-conditioned media results in (1) improved efficiency of the cardiomyocyte differentiation protocol and (2) increased maturity leading to better intercellular coupling with improved chronotropy and synchrony.
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http://dx.doi.org/10.1016/j.healun.2017.01.001DOI Listing
June 2017

Engineered human pluripotent-stem-cell-derived intestinal tissues with a functional enteric nervous system.

Nat Med 2017 01 21;23(1):49-59. Epub 2016 Nov 21.

Division of Developmental Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, USA.

The enteric nervous system (ENS) of the gastrointestinal tract controls many diverse functions, including motility and epithelial permeability. Perturbations in ENS development or function are common, yet there is no human model for studying ENS-intestinal biology and disease. We used a tissue-engineering approach with embryonic and induced pluripotent stem cells (PSCs) to generate human intestinal tissue containing a functional ENS. We recapitulated normal intestinal ENS development by combining human-PSC-derived neural crest cells (NCCs) and developing human intestinal organoids (HIOs). NCCs recombined with HIOs in vitro migrated into the mesenchyme, differentiated into neurons and glial cells and showed neuronal activity, as measured by rhythmic waves of calcium transients. ENS-containing HIOs grown in vivo formed neuroglial structures similar to a myenteric and submucosal plexus, had functional interstitial cells of Cajal and had an electromechanical coupling that regulated waves of propagating contraction. Finally, we used this system to investigate the cellular and molecular basis for Hirschsprung's disease caused by a mutation in the gene PHOX2B. This is, to the best of our knowledge, the first demonstration of human-PSC-derived intestinal tissue with a functional ENS and how this system can be used to study motility disorders of the human gastrointestinal tract.
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http://dx.doi.org/10.1038/nm.4233DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5562951PMC
January 2017

Differentiation of human embryonic stem cells to HOXA hemogenic vasculature that resembles the aorta-gonad-mesonephros.

Nat Biotechnol 2016 Nov 17;34(11):1168-1179. Epub 2016 Oct 17.

Murdoch Childrens Research Institute, The Royal Children's Hospital, Parkville, Victoria, Australia.

The ability to generate hematopoietic stem cells from human pluripotent cells would enable many biomedical applications. We find that hematopoietic CD34 cells in spin embryoid bodies derived from human embryonic stem cells (hESCs) lack HOXA expression compared with repopulation-competent human cord blood CD34 cells, indicating incorrect mesoderm patterning. Using reporter hESC lines to track the endothelial (SOX17) to hematopoietic (RUNX1C) transition that occurs in development, we show that simultaneous modulation of WNT and ACTIVIN signaling yields CD34 hematopoietic cells with HOXA expression that more closely resembles that of cord blood. The cultures generate a network of aorta-like SOX17 vessels from which RUNX1C blood cells emerge, similar to hematopoiesis in the aorta-gonad-mesonephros (AGM). Nascent CD34 hematopoietic cells and corresponding cells sorted from human AGM show similar expression of cell surface receptors, signaling molecules and transcription factors. Our findings provide an approach to mimic in vitro a key early stage in human hematopoiesis for the generation of AGM-derived hematopoietic lineages from hESCs.
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http://dx.doi.org/10.1038/nbt.3702DOI Listing
November 2016

GAPTrap: A Simple Expression System for Pluripotent Stem Cells and Their Derivatives.

Stem Cell Reports 2016 09 1;7(3):518-526. Epub 2016 Sep 1.

Murdoch Childrens Research Institute, Flemington Road, Parkville, VIC 3052, Australia; Department of Paediatrics, University of Melbourne, Parkville, VIC 3050, Australia; Department of Anatomy and Developmental Biology, Monash University, Clayton, VIC 3800, Australia. Electronic address:

The ability to reliably express fluorescent reporters or other genes of interest is important for using human pluripotent stem cells (hPSCs) as a platform for investigating cell fates and gene function. We describe a simple expression system, designated GAPTrap (GT), in which reporter genes, including GFP, mCherry, mTagBFP2, luc2, Gluc, and lacZ are inserted into the GAPDH locus in hPSCs. Independent clones harboring variations of the GT vectors expressed remarkably consistent levels of the reporter gene. Differentiation experiments showed that reporter expression was reliably maintained in hematopoietic cells, cardiac mesoderm, definitive endoderm, and ventral midbrain dopaminergic neurons. Similarly, analysis of teratomas derived from GT-lacZ hPSCs showed that β-galactosidase expression was maintained in a spectrum of cell types representing derivatives of the three germ layers. Thus, the GAPTrap vectors represent a robust and straightforward tagging system that enables indelible labeling of PSCs and their differentiated derivatives.
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http://dx.doi.org/10.1016/j.stemcr.2016.07.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5032031PMC
September 2016

A Cas9 Variant for Efficient Generation of Indel-Free Knockin or Gene-Corrected Human Pluripotent Stem Cells.

Stem Cell Reports 2016 09 4;7(3):508-517. Epub 2016 Aug 4.

Murdoch Childrens Research Institute, The Royal Children's Hospital, Flemington Road, Parkville, VIC 3052, Australia; Department of Paediatrics, Faculty of Medicine, Dentistry and Health Sciences, University of Melbourne, Parkville, VIC 3052, Australia; Department of Anatomy and Developmental Biology, Faculty of Medicine, Nursing and Health Sciences, Monash University, Clayton, VIC 3800, Australia.

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.
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http://dx.doi.org/10.1016/j.stemcr.2016.07.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5031952PMC
September 2016

Spatio-temporal re-organization of replication foci accompanies replication domain consolidation during human pluripotent stem cell lineage specification.

Cell Cycle 2016 Sep 19;15(18):2464-75. Epub 2016 Jul 19.

a Department of Biological Science , Florida State University , Tallahassee , FL , USA.

Lineage specification of both mouse and human pluripotent stem cells (PSCs) is accompanied by spatial consolidation of chromosome domains and temporal consolidation of their replication timing. Replication timing and chromatin organization are both established during G1 phase at the timing decision point (TDP). Here, we have developed live cell imaging tools to track spatio-temporal replication domain consolidation during differentiation. First, we demonstrate that the fluorescence ubiquitination cell cycle indicator (Fucci) system is incapable of demarcating G1/S or G2/M cell cycle transitions. Instead, we employ a combination of fluorescent PCNA to monitor S phase progression, cytokinesis to demarcate mitosis, and fluorescent nucleotides to label early and late replication foci and track their 3D organization into sub-nuclear chromatin compartments throughout all cell cycle transitions. We find that, as human PSCs differentiate, the length of S phase devoted to replication of spatially clustered replication foci increases, coincident with global compartmentalization of domains into temporally clustered blocks of chromatin. Importantly, re-localization and anchorage of domains was completed prior to the onset of S phase, even in the context of an abbreviated PSC G1 phase. This approach can also be employed to investigate cell fate transitions in single PSCs, which could be seen to differentiate preferentially from G1 phase. Together, our results establish real-time, live-cell imaging methods for tracking cell cycle transitions during human PSC differentiation that can be applied to study chromosome domain consolidation and other aspects of lineage specification.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5026818PMC
http://dx.doi.org/10.1080/15384101.2016.1203492DOI Listing
September 2016

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.

Stem Cells Transl Med 2016 Aug 28;5(8):981-90. Epub 2016 Jun 28.

Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, United Kingdom

Unlabelled: : 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-ER(T2) 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.

Significance: As worldwide blood donations decrease and transfusable transmitted infections increase, intense interest has ensued in deriving red blood cells (RBCs) in vitro from alternative sources such as pluripotent stem cells. A translatable protocol was developed to generate RBCs; however, these RBCs have an immature phenotype. It was hypothesized that the transcription factor HOXB4 could enhance their production and maturation. Although HOXB4 increased the production of erythroid progenitors, it did not promote their maturation. Despite the remaining challenges, a robust system has been established to test other candidates and add to the knowledge base in this field.
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http://dx.doi.org/10.5966/sctm.2015-0324DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4954454PMC
August 2016

ALPK3-deficient cardiomyocytes generated from patient-derived induced pluripotent stem cells and mutant human embryonic stem cells display abnormal calcium handling and establish that ALPK3 deficiency underlies familial cardiomyopathy.

Eur Heart J 2016 Sep 22;37(33):2586-90. Epub 2016 Apr 22.

Bruce Lefroy Centre for Genetic Health Research, Murdoch Childrens Research Institute, Flemington Road, Parkville 3052, Victoria, Australia Department of Paediatrics, Faculty of Medicine, Dentistry and Health Sciences, The University of Melbourne, Parkville 3052, Victoria, Australia

Aims: We identified a novel homozygous truncating mutation in the gene encoding alpha kinase 3 (ALPK3) in a family presenting with paediatric cardiomyopathy. A recent study identified biallelic truncating mutations of ALPK3 in three unrelated families; therefore, there is strong genetic evidence that ALPK3 mutation causes cardiomyopathy. This study aimed to clarify the mutation mechanism and investigate the molecular and cellular pathogenesis underlying ALPK3-mediated cardiomyopathy.

Methods And Results: We performed detailed clinical and genetic analyses of a consanguineous family, identifying a new ALPK3 mutation (c.3792G>A, p.W1264X) which undergoes nonsense-mediated decay in ex vivo and in vivo tissues. Ultra-structural analysis of cardiomyocytes derived from patient-specific and human ESC-derived stem cell lines lacking ALPK3 revealed disordered sarcomeres and intercalated discs. Multi-electrode array analysis and calcium imaging demonstrated an extended field potential duration and abnormal calcium handling in mutant contractile cultures.

Conclusions: This study validates the genetic evidence, suggesting that mutations in ALPK3 can cause familial cardiomyopathy and demonstrates loss of function as the underlying genetic mechanism. We show that ALPK3-deficient cardiomyocytes derived from pluripotent stem cell models recapitulate the ultrastructural and electrophysiological defects observed in vivo. Analysis of differentiated contractile cultures identified abnormal calcium handling as a potential feature of cardiomyocytes lacking ALPK3, providing functional insights into the molecular mechanisms underlying ALPK3-mediated cardiomyopathy.
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http://dx.doi.org/10.1093/eurheartj/ehw160DOI Listing
September 2016