Publications by authors named "Thorsten M Schlaeger"

49 Publications

Genome sequencing unveils a regulatory landscape of platelet reactivity.

Nat Commun 2021 06 15;12(1):3626. Epub 2021 Jun 15.

Division of Intramural Research, Population Sciences Branch, National Heart, Lung and Blood Institute, Bethesda, MD, USA.

Platelet aggregation at the site of atherosclerotic vascular injury is the underlying pathophysiology of myocardial infarction and stroke. To build upon prior GWAS, here we report on 16 loci identified through a whole genome sequencing (WGS) approach in 3,855 NHLBI Trans-Omics for Precision Medicine (TOPMed) participants deeply phenotyped for platelet aggregation. We identify the RGS18 locus, which encodes a myeloerythroid lineage-specific regulator of G-protein signaling that co-localizes with expression quantitative trait loci (eQTL) signatures for RGS18 expression in platelets. Gene-based approaches implicate the SVEP1 gene, a known contributor of coronary artery disease risk. Sentinel variants at RGS18 and PEAR1 are associated with thrombosis risk and increased gastrointestinal bleeding risk, respectively. Our WGS findings add to previously identified GWAS loci, provide insights regarding the mechanism(s) by which genetics may influence cardiovascular disease risk, and underscore the importance of rare variant and regulatory approaches to identifying loci contributing to complex phenotypes.
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http://dx.doi.org/10.1038/s41467-021-23470-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8206369PMC
June 2021

A recurrent, homozygous EMC10 frameshift variant is associated with a syndrome of developmental delay with variable seizures and dysmorphic features.

Genet Med 2021 06 2;23(6):1158-1162. Epub 2021 Feb 2.

Centogene AG, Rostock, Germany.

Purpose: The endoplasmic reticulum membrane complex (EMC) is a highly conserved, multifunctional 10-protein complex related to membrane protein biology. In seven families, we identified 13 individuals with highly overlapping phenotypes who harbor a single identical homozygous frameshift variant in EMC10.

Methods: Using exome, genome, and Sanger sequencing, a recurrent frameshift EMC10 variant was identified in affected individuals in an international cohort of consanguineous families. Multiple families were independently identified and connected via Matchmaker Exchange and internal databases. We assessed the effect of the frameshift variant on EMC10 RNA and protein expression and evaluated EMC10 expression in normal human brain tissue using immunohistochemistry.

Results: A homozygous variant EMC10 c.287delG (Refseq NM_206538.3, p.Gly96Alafs*9) segregated with affected individuals in each family, who exhibited a phenotypic spectrum of intellectual disability (ID) and global developmental delay (GDD), variable seizures and variable dysmorphic features (elongated face, curly hair, cubitus valgus, and arachnodactyly). The variant arose on two founder haplotypes and results in significantly reduced EMC10 RNA expression and an unstable truncated EMC10 protein.

Conclusion: We propose that a homozygous loss-of-function variant in EMC10 causes a novel syndromic neurodevelopmental phenotype. Remarkably, the recurrent variant is likely the result of a hypermutable site and arose on distinct founder haplotypes.
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http://dx.doi.org/10.1038/s41436-021-01097-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8187145PMC
June 2021

Derivation of Airway Basal Stem Cells from Human Pluripotent Stem Cells.

Cell Stem Cell 2021 01 23;28(1):79-95.e8. Epub 2020 Oct 23.

Center for Regenerative Medicine, Boston University and Boston Medical Center, Boston, MA 02118, USA; The Pulmonary Center and Department of Medicine, Boston University and Boston Medical Center, Boston, MA 02118, USA. Electronic address:

The derivation of tissue-specific stem cells from human induced pluripotent stem cells (iPSCs) would have broad reaching implications for regenerative medicine. Here, we report the directed differentiation of human iPSCs into airway basal cells ("iBCs"), a population resembling the stem cell of the airway epithelium. Using a dual fluorescent reporter system (NKX2-1;TP63), we track and purify these cells as they first emerge as developmentally immature NKX2-1 lung progenitors and subsequently augment a TP63 program during proximal airway epithelial patterning. In response to primary basal cell medium, NKX2-1/TP63 cells display the molecular and functional phenotype of airway basal cells, including the capacity to self-renew or undergo multi-lineage differentiation in vitro and in tracheal xenografts in vivo. iBCs and their differentiated progeny model perturbations that characterize acquired and genetic airway diseases, including the mucus metaplasia of asthma, chloride channel dysfunction of cystic fibrosis, and ciliary defects of primary ciliary dyskinesia.
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http://dx.doi.org/10.1016/j.stem.2020.09.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7796997PMC
January 2021

Mitochondrial and Redox Modifications in Huntington Disease Induced Pluripotent Stem Cells Rescued by CRISPR/Cas9 CAGs Targeting.

Front Cell Dev Biol 2020 22;8:576592. Epub 2020 Sep 22.

CNC-Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal.

Mitochondrial deregulation has gained increasing support as a pathological mechanism in Huntington's disease (HD), a genetic-based neurodegenerative disorder caused by CAG expansion in the gene. In this study, we thoroughly investigated mitochondrial-based mechanisms in HD patient-derived iPSC (HD-iPSC) and differentiated neural stem cells (NSC) control cells, as well as in cells subjected to CRISPR/Cas9-CAG repeat deletion. We analyzed mitochondrial morphology, function and biogenesis, linked to exosomal release of mitochondrial components, glycolytic flux, ATP generation and cellular redox status. Mitochondria in HD cells exhibited round shape and fragmented morphology. Functionally, HD-iPSC and HD-NSC displayed lower mitochondrial respiration, exosomal release of cytochrome c, decreased ATP/ADP, reduced PGC-1α and complex III subunit expression and activity, and were highly dependent on glycolysis, supported by pyruvate dehydrogenase (PDH) inactivation. HD-iPSC and HD-NSC mitochondria showed ATP synthase reversal and increased calcium retention. Enhanced mitochondrial reactive oxygen species (ROS) were also observed in HD-iPSC and HD-NSC, along with decreased UCP2 mRNA levels. CRISPR/Cas9-CAG repeat deletion in HD-iPSC and derived HD-NSC ameliorated mitochondrial phenotypes. Data attests for intricate metabolic and mitochondrial dysfunction linked to transcriptional deregulation as early events in HD pathogenesis, which are alleviated following CAG deletion.
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http://dx.doi.org/10.3389/fcell.2020.576592DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7536317PMC
September 2020

An induced pluripotent stem cell model of Fanconi anemia reveals mechanisms of p53-driven progenitor cell differentiation.

Blood Adv 2020 10;4(19):4679-4692

Stem Cell Program, Boston Children's Hospital, Boston, MA.

Fanconi anemia (FA) is a disorder of DNA repair that manifests as bone marrow (BM) failure. The lack of accurate murine models of FA has refocused efforts toward differentiation of patient-derived induced pluripotent stem cells (IPSCs) to hematopoietic progenitor cells (HPCs). However, an intact FA DNA repair pathway is required for efficient IPSC derivation, hindering these efforts. To overcome this barrier, we used inducible complementation of FANCA-deficient IPSCs, which permitted robust maintenance of IPSCs. Modulation of FANCA during directed differentiation to HPCs enabled the production of FANCA-deficient human HPCs that recapitulated FA genotoxicity and hematopoietic phenotypes relative to isogenic FANCA-expressing HPCs. FANCA-deficient human HPCs underwent accelerated terminal differentiation driven by activation of p53/p21. We identified growth arrest specific 6 (GAS6) as a novel target of activated p53 in FANCA-deficient HPCs and modulate GAS6 signaling to rescue hematopoiesis in FANCA-deficient cells. This study validates our strategy to derive a sustainable, highly faithful human model of FA, uncovers a mechanism of HPC exhaustion in FA, and advances toward future cell therapy in FA.
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http://dx.doi.org/10.1182/bloodadvances.2020001593DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7556119PMC
October 2020

Transcriptome Dynamics of Hematopoietic Stem Cell Formation Revealed Using a Combinatorial Runx1 and Ly6a Reporter System.

Stem Cell Reports 2020 05 16;14(5):956-971. Epub 2020 Apr 16.

Division of Pediatric Hematology/Oncology, Boston Children's Hospital and Dana Farber Cancer Institute, Harvard Medical School, Boston, MA 02115, USA; Stem Cell Program, Boston Children's Hospital, Harvard Medical School, Boston, MA 02115, USA; Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA; Harvard Stem Cell Institute, Harvard University, Boston, MA, USA. Electronic address:

Studies of hematopoietic stem cell (HSC) development from pre-HSC-producing hemogenic endothelial cells (HECs) are hampered by the rarity of these cells and the presence of other cell types with overlapping marker expression profiles. We generated a Tg(Runx1-mKO2; Ly6a-GFP) dual reporter mouse to visualize hematopoietic commitment and study pre-HSC emergence and maturation. Runx1-mKO2 marked all intra-arterial HECs and hematopoietic cluster cells (HCCs), including pre-HSCs, myeloid- and lymphoid progenitors, and HSCs themselves. However, HSC and lymphoid potential were almost exclusively found in reporter double-positive (DP) cells. Robust HSC activity was first detected in DP cells of the placenta, reflecting the importance of this niche for (pre-)HSC maturation and expansion before the fetal liver stage. A time course analysis by single-cell RNA sequencing revealed that as pre-HSCs mature into fetal liver stage HSCs, they show signs of interferon exposure, exhibit signatures of multi-lineage differentiation gene expression, and develop a prolonged cell cycle reminiscent of quiescent adult HSCs.
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http://dx.doi.org/10.1016/j.stemcr.2020.03.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7220988PMC
May 2020

Xeno-Free Reprogramming of Peripheral Blood Mononuclear Erythroblasts on Laminin-521.

Curr Protoc Stem Cell Biol 2020 03;52(1):e103

Stem Cell Core Facility, Boston Children's Hospital, Stem Cell Program, Boston, Massachusetts.

Translating human induced pluripotent stem cell (hiPSC)-derived cells and tissues into the clinic requires streamlined and reliable production of clinical-grade hiPSCs. This article describes an entirely animal component-free procedure for the reliable derivation of stable hiPSC lines from donor peripheral blood mononuclear cells (PBMCs) using only autologous patient materials and xeno-free reagents. PBMCs are isolated from a whole blood donation, from which a small amount of patient serum is also generated. The PBMCs are then expanded prior to reprogramming in an animal component-free erythroblast growth medium supplemented with autologous patient serum, thereby eliminating the need for animal serum. After expansion, the erythroblasts are reprogrammed using either cGMP-grade Sendai viral particles (CytoTune™ 2.1 kit) or episomally replicating reprogramming plasmids (Epi5™ kit), both commercially available. Expansion of emerging hiPSCs on a recombinant cGMP-grade human laminin substrate is compatible with a number of xeno-free or chemically defined media (some available as cGMP-grade reagents), such as E8, Nutristem, Stemfit, or mTeSR Plus. hiPSC lines derived using this method display expression of expected surface markers and transcription factors, loss of the reprogramming agent-derived nucleic acids, genetic stability, and the ability to robustly differentiate in vitro to multiple lineages. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Isolating peripheral blood mononuclear cells using CPT tubes Support Protocol 1: Removal of clotting factors to produce serum from autologous plasma collected in Basic Protocol 1 Basic Protocol 2: PBMC expansion in an animal-free erythroblast expansion medium containing autologous serum Basic Protocol 3: Reprogramming of expanded PBMCs with Sendai viral reprogramming particles Alternate Protocol: Reprogramming of expanded PBMCs with episomal plasmids Basic Protocol 4: Picking, expanding, and cryopreserving hiPSC clones Support Protocol 2: Testing Sendai virus kit-reprogrammed hiPSC for absence of Sendai viral RNA Support Protocol 3: Testing Epi5 kit-reprogrammed hiPSC for absence of episomal plasmid DNA Support Protocol 4: Assessing the undifferentiated state of human pluripotent stem cell cultures by multi-color immunofluorescent staining and confocal imaging Support Protocol 5: Coating plates with extracellular matrices to support hiPSC attachment and expansion.
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http://dx.doi.org/10.1002/cpsc.103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7176073PMC
March 2020

Morphological and Molecular Defects in Human Three-Dimensional Retinal Organoid Model of X-Linked Juvenile Retinoschisis.

Stem Cell Reports 2019 11 24;13(5):906-923. Epub 2019 Oct 24.

Department of Medical Research, Taipei Veterans General Hospital, Taipei 11217, Taiwan; Institute of Pharmacology, National Yang-Ming University, Taipei 11221, Taiwan; School of Medicine, National Yang-Ming University, Taipei 11221, Taiwan; Department of Ophthalmology, Taipei Veterans General Hospital, Taipei 11217, Taiwan; Genomic Research Center, Academia Sinica, Taipei 11529, Taiwan. Electronic address:

X-linked juvenile retinoschisis (XLRS), linked to mutations in the RS1 gene, is a degenerative retinopathy with a retinal splitting phenotype. We generated human induced pluripotent stem cells (hiPSCs) from patients to study XLRS in a 3D retinal organoid in vitro differentiation system. This model recapitulates key features of XLRS including retinal splitting, defective retinoschisin production, outer-segment defects, abnormal paxillin turnover, and impaired ER-Golgi transportation. RS1 mutation also affects the development of photoreceptor sensory cilia and results in altered expression of other retinopathy-associated genes. CRISPR/Cas9 correction of the disease-associated C625T mutation normalizes the splitting phenotype, outer-segment defects, paxillin dynamics, ciliary marker expression, and transcriptome profiles. Likewise, mutating RS1 in control hiPSCs produces the disease-associated phenotypes. Finally, we show that the C625T mutation can be repaired precisely and efficiently using a base-editing approach. Taken together, our data establish 3D organoids as a valid disease model.
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http://dx.doi.org/10.1016/j.stemcr.2019.09.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6895767PMC
November 2019

Author Correction: Epoxyeicosatrienoic acids enhance embryonic haematopoiesis and adult marrow engraftment.

Nature 2019 Sep;573(7772):E1

Stem Cell Program and Division of Haematology/Oncology, Boston Children's Hospital and Dana-Farber Cancer Institute, Howard Hughes Medical Institute, Harvard Stem Cell Institute, Harvard Medical School, Boston, MA, 02115, USA.

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/s41586-019-1489-4DOI Listing
September 2019

Applying Deep Neural Network Analysis to High-Content Image-Based Assays.

SLAS Discov 2019 09 8;24(8):829-841. Epub 2019 Jul 8.

2 Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA, USA.

The etiological underpinnings of many CNS disorders are not well understood. This is likely due to the fact that individual diseases aggregate numerous pathological subtypes, each associated with a complex landscape of genetic risk factors. To overcome these challenges, researchers are integrating novel data types from numerous patients, including imaging studies capturing broadly applicable features from patient-derived materials. These datasets, when combined with machine learning, potentially hold the power to elucidate the subtle patterns that stratify patients by shared pathology. In this study, we interrogated whether high-content imaging of primary skin fibroblasts, using the Cell Painting method, could reveal disease-relevant information among patients. First, we showed that technical features such as batch/plate type, plate, and location within a plate lead to detectable nuisance signals, as revealed by a pre-trained deep neural network and analysis with deep image embeddings. Using a plate design and image acquisition strategy that accounts for these variables, we performed a pilot study with 12 healthy controls and 12 subjects affected by the severe genetic neurological disorder spinal muscular atrophy (SMA), and evaluated whether a convolutional neural network (CNN) generated using a subset of the cells could distinguish disease states on cells from the remaining unseen control-SMA pair. Our results indicate that these two populations could effectively be differentiated from one another and that model selectivity is insensitive to batch/plate type. One caveat is that the samples were also largely separated by source. These findings lay a foundation for how to conduct future studies exploring diseases with more complex genetic contributions and unknown subtypes.
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http://dx.doi.org/10.1177/2472555219857715DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6710615PMC
September 2019

The Lin28/let-7 Pathway Regulates the Mammalian Caudal Body Axis Elongation Program.

Dev Cell 2019 02 17;48(3):396-405.e3. Epub 2019 Jan 17.

Division of Pediatric Hematology/Oncology, Children's Hospital Boston, Boston, MA 02115, USA; Howard Hughes Medical Institute, Boston, MA 02115, USA; Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA; Harvard Stem Cell Institute, Boston, MA 02115, USA. Electronic address:

The heterochronic genes Lin28a/b and let-7 regulate invertebrate development, but their functions in patterning the mammalian body plan remain unexplored. Here, we describe how Lin28/let-7 influence caudal vertebrae number during body axis formation. We found that FoxD1-driven overexpression of Lin28a strikingly increased caudal vertebrae number and tail bud cell proliferation, whereas its knockout did the opposite. Lin28a overexpression downregulated the neural marker Sox2, causing a pro-mesodermal phenotype with a decreased proportion of neural tissue relative to nascent mesoderm. Manipulating Lin28a and let-7 led to opposite effects, and manipulating Lin28a's paralog, LIN28B caused similar yet distinct phenotypes. These findings suggest that Lin28/let-7 play a role in the regulation of tail length through heterochrony of the body plan. We propose that the Lin28/let-7 pathway controls the pool of caudal progenitors during tail development, promoting their self-renewal and balancing neural versus mesodermal cell fate decisions.
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http://dx.doi.org/10.1016/j.devcel.2018.12.016DOI Listing
February 2019

The ESCRT-III Protein CHMP1A Mediates Secretion of Sonic Hedgehog on a Distinctive Subtype of Extracellular Vesicles.

Cell Rep 2018 07;24(4):973-986.e8

Division of Genetics and Genomics and Howard Hughes Medical Institute, Boston Children's Hospital, Departments of Pediatrics and Neurology, Harvard Medical School, Boston, MA 02115, USA. Electronic address:

Endosomal sorting complex required for transport (ESCRT) complex proteins regulate biogenesis and release of extracellular vesicles (EVs), which enable cell-to-cell communication in the nervous system essential for development and adult function. We recently showed human loss-of-function (LOF) mutations in ESCRT-III member CHMP1A cause autosomal recessive microcephaly with pontocerebellar hypoplasia, but its mechanism was unclear. Here, we show Chmp1a is required for progenitor proliferation in mouse cortex and cerebellum and progenitor maintenance in human cerebral organoids. In Chmp1a null mice, this defect is associated with impaired sonic hedgehog (Shh) secretion and intraluminal vesicle (ILV) formation in multivesicular bodies (MVBs). Furthermore, we show CHMP1A is important for release of an EV subtype that contains AXL, RAB18, and TMED10 (ART) and SHH. Our findings show CHMP1A loss impairs secretion of SHH on ART-EVs, providing molecular mechanistic insights into the role of ESCRT proteins and EVs in the brain.
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http://dx.doi.org/10.1016/j.celrep.2018.06.100DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6178983PMC
July 2018

Nonintegrating Human Somatic Cell Reprogramming Methods.

Adv Biochem Eng Biotechnol 2018;163:1-21

Stem Cell Program, Boston Children's Hospital, Karp RB09213, 1 Blackfan Circle, Boston, MA, 02446, USA.

Traditional biomedical research and preclinical studies frequently rely on animal models and repeatedly draw on a relatively small set of human cell lines, such as HeLa, HEK293, HepG2, HL60, and PANC1 cells. However, animal models often fail to reproduce important clinical phenotypes and conventional cell lines only represent a small number of cell types or diseases, have very limited ethnic/genetic diversity, and either senesce quickly or carry potentially confounding immortalizing mutations. In recent years, human pluripotent stem cells have attracted a lot of attention, in part because these cells promise more precise modeling of human diseases. Expectations are also high that pluripotent stem cell technologies can deliver cell-based therapeutics for the cure of a wide range of degenerative and other diseases. This review focuses on episomal and Sendai viral reprogramming modalities, which are the most popular methods for generating transgene-free human induced pluripotent stem cells (hiPSCs) from easily accessible cell sources. Graphical Abstract.
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http://dx.doi.org/10.1007/10_2017_29DOI Listing
September 2018

A chemical screen in zebrafish embryonic cells establishes that Akt activation is required for neural crest development.

Elife 2017 08 23;6. Epub 2017 Aug 23.

Stem Cell Program and Hematology/Oncology, Children's Hospital Boston, Howard Hughes Medical Institute, Boston, United States.

The neural crest is a dynamic progenitor cell population that arises at the border of neural and non-neural ectoderm. The inductive roles of FGF, Wnt, and BMP at the neural plate border are well established, but the signals required for subsequent neural crest development remain poorly characterized. Here, we conducted a screen in primary zebrafish embryo cultures for chemicals that disrupt neural crest development, as read out by expression. We found that the natural product caffeic acid phenethyl ester (CAPE) disrupts neural crest gene expression, migration, and melanocytic differentiation by reducing Sox10 activity. CAPE inhibits FGF-stimulated PI3K/Akt signaling, and neural crest defects in CAPE-treated embryos are suppressed by constitutively active Akt1. Inhibition of Akt activity by constitutively active PTEN similarly decreases expression and Sox10 activity. Our study has identified Akt as a novel intracellular pathway required for neural crest differentiation.
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http://dx.doi.org/10.7554/eLife.29145DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5599238PMC
August 2017

Using CRISPR-Cas9 to Generate Gene-Corrected Autologous iPSCs for the Treatment of Inherited Retinal Degeneration.

Mol Ther 2017 09 12;25(9):1999-2013. Epub 2017 Jun 12.

Stephen A. Wynn Institute for Vision Research and Department of Ophthalmology and Visual Sciences, Carver College of Medicine, University of Iowa, Iowa City, IA 52241, USA. Electronic address:

Patient-derived induced pluripotent stem cells (iPSCs) hold great promise for autologous cell replacement. However, for many inherited diseases, treatment will likely require genetic repair pre-transplantation. Genome editing technologies are useful for this application. The purpose of this study was to develop CRISPR-Cas9-mediated genome editing strategies to target and correct the three most common types of disease-causing variants in patient-derived iPSCs: (1) exonic, (2) deep intronic, and (3) dominant gain of function. We developed a homology-directed repair strategy targeting a homozygous Alu insertion in exon 9 of male germ cell-associated kinase (MAK) and demonstrated restoration of the retinal transcript and protein in patient cells. We generated a CRISPR-Cas9-mediated non-homologous end joining (NHEJ) approach to excise a major contributor to Leber congenital amaurosis, the IVS26 cryptic-splice mutation in CEP290, and demonstrated correction of the transcript and protein in patient iPSCs. Lastly, we designed allele-specific CRISPR guides that selectively target the mutant Pro23His rhodopsin (RHO) allele, which, following delivery to both patient iPSCs in vitro and pig retina in vivo, created a frameshift and premature stop that would prevent transcription of the disease-causing variant. The strategies developed in this study will prove useful for correcting a wide range of genetic variants in genes that cause inherited retinal degeneration.
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http://dx.doi.org/10.1016/j.ymthe.2017.05.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5589061PMC
September 2017

Drug discovery for Diamond-Blackfan anemia using reprogrammed hematopoietic progenitors.

Sci Transl Med 2017 02;9(376)

Division of Hematology/Oncology, Boston Children's Hospital and Dana Farber Cancer Institute, Boston, MA 02115, USA.

Diamond-Blackfan anemia (DBA) is a congenital disorder characterized by the failure of erythroid progenitor differentiation, severely curtailing red blood cell production. Because many DBA patients fail to respond to corticosteroid therapy, there is considerable need for therapeutics for this disorder. Identifying therapeutics for DBA requires circumventing the paucity of primary patient blood stem and progenitor cells. To this end, we adopted a reprogramming strategy to generate expandable hematopoietic progenitor cells from induced pluripotent stem cells (iPSCs) from DBA patients. Reprogrammed DBA progenitors recapitulate defects in erythroid differentiation, which were rescued by gene complementation. Unbiased chemical screens identified SMER28, a small-molecule inducer of autophagy, which enhanced erythropoiesis in a range of in vitro and in vivo models of DBA. SMER28 acted through autophagy factor ATG5 to stimulate erythropoiesis and up-regulate expression of globin genes. These findings present an unbiased drug screen for hematological disease using iPSCs and identify autophagy as a therapeutic pathway in DBA.
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http://dx.doi.org/10.1126/scitranslmed.aah5645DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5501179PMC
February 2017

Developmental Vitamin D Availability Impacts Hematopoietic Stem Cell Production.

Cell Rep 2016 10;17(2):458-468

Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA; Harvard Stem Cell Institute, Cambridge, MA 02138, USA. Electronic address:

Vitamin D insufficiency is a worldwide epidemic affecting billions of individuals, including pregnant women and children. Despite its high incidence, the impact of active vitamin D3 (1,25(OH)D3) on embryonic development beyond osteo-regulation remains largely undefined. Here, we demonstrate that 1,25(OH)D3 availability modulates zebrafish hematopoietic stem and progenitor cell (HSPC) production. Loss of Cyp27b1-mediated biosynthesis or vitamin D receptor (VDR) function by gene knockdown resulted in significantly reduced runx1 expression and Flk1cMyb HSPC numbers. Selective modulation in vivo and in vitro in zebrafish indicated that vitamin D3 acts directly on HSPCs, independent of calcium regulation, to increase proliferation. Notably, ex vivo treatment of human HSPCs with 1,25(OH)D3 also enhanced hematopoietic colony numbers, illustrating conservation across species. Finally, gene expression and epistasis analysis indicated that CXCL8(IL-8) was a functional target of vitamin D3-mediated HSPC regulation. Together, these findings highlight the relevance of developmental 1,25(OH)D3 availability for definitive hematopoiesis and suggest potential therapeutic utility in HSPC expansion.
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http://dx.doi.org/10.1016/j.celrep.2016.09.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5338633PMC
October 2016

Epoxyeicosatrienoic acids enhance embryonic haematopoiesis and adult marrow engraftment.

Nature 2015 Jul;523(7561):468-71

1] Stem Cell Program and Division of Haematology/Oncology, Boston Children's Hospital and Dana-Farber Cancer Institute, Howard Hughes Medical Institute, Harvard Stem Cell Institute, Harvard Medical School, Boston, Massachuestts 02115, USA [2] Chemical Biology Program, Harvard University, Cambridge, Massachusetts 02138, USA.

Haematopoietic stem and progenitor cell (HSPC) transplant is a widely used treatment for life-threatening conditions such as leukaemia; however, the molecular mechanisms regulating HSPC engraftment of the recipient niche remain incompletely understood. Here we develop a competitive HSPC transplant method in adult zebrafish, using in vivo imaging as a non-invasive readout. We use this system to conduct a chemical screen, and identify epoxyeicosatrienoic acids (EETs) as a family of lipids that enhance HSPC engraftment. The pro-haematopoietic effects of EETs were conserved in the developing zebrafish embryo, where 11,12-EET promoted HSPC specification by activating a unique activator protein 1 (AP-1) and runx1 transcription program autonomous to the haemogenic endothelium. This effect required the activation of the phosphatidylinositol-3-OH kinase (PI(3)K) pathway, specifically PI(3)Kγ. In adult HSPCs, 11,12-EET induced transcriptional programs, including AP-1 activation, which modulate several cellular processes, such as migration, to promote engraftment. Furthermore, we demonstrate that the EET effects on enhancing HSPC homing and engraftment are conserved in mammals. Our study establishes a new method to explore the molecular mechanisms of HSPC engraftment, and discovers a previously unrecognized, evolutionarily conserved pathway regulating multiple haematopoietic generation and regeneration processes. EETs may have clinical application in marrow or cord blood transplantation.
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http://dx.doi.org/10.1038/nature14569DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4754787PMC
July 2015

Flow-induced protein kinase A-CREB pathway acts via BMP signaling to promote HSC emergence.

J Exp Med 2015 May 13;212(5):633-48. Epub 2015 Apr 13.

Division of Pediatric Hematology/Oncology, Boston Children's Hospital and Dana Farber Cancer Institute; Howard Hughes Medical Institute, Harvard Stem Cell Institute; Stem Cell Transplantation Program, Division of Pediatric Hematology/Oncology, Boston Children's Hospital and Dana Farber Cancer Institute; and Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115 Division of Pediatric Hematology/Oncology, Boston Children's Hospital and Dana Farber Cancer Institute; Howard Hughes Medical Institute, Harvard Stem Cell Institute; Stem Cell Transplantation Program, Division of Pediatric Hematology/Oncology, Boston Children's Hospital and Dana Farber Cancer Institute; and Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115 Division of Pediatric Hematology/Oncology, Boston Children's Hospital and Dana Farber Cancer Institute; Howard Hughes Medical Institute, Harvard Stem Cell Institute; Stem Cell Transplantation Program, Division of Pediatric Hematology/Oncology, Boston Children's Hospital and Dana Farber Cancer Institute; and Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115

Fluid shear stress promotes the emergence of hematopoietic stem cells (HSCs) in the aorta-gonad-mesonephros (AGM) of the developing mouse embryo. We determined that the AGM is enriched for expression of targets of protein kinase A (PKA)-cAMP response element-binding protein (CREB), a pathway activated by fluid shear stress. By analyzing CREB genomic occupancy from chromatin-immunoprecipitation sequencing (ChIP-seq) data, we identified the bone morphogenetic protein (BMP) pathway as a potential regulator of CREB. By chemical modulation of the PKA-CREB and BMP pathways in isolated AGM VE-cadherin(+) cells from mid-gestation embryos, we demonstrate that PKA-CREB regulates hematopoietic engraftment and clonogenicity of hematopoietic progenitors, and is dependent on secreted BMP ligands through the type I BMP receptor. Finally, we observed blunting of this signaling axis using Ncx1-null embryos, which lack a heartbeat and intravascular flow. Collectively, we have identified a novel PKA-CREB-BMP signaling pathway downstream of shear stress that regulates HSC emergence in the AGM via the endothelial-to-hematopoietic transition.
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http://dx.doi.org/10.1084/jem.20141514DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4419355PMC
May 2015

Adenosine signaling promotes hematopoietic stem and progenitor cell emergence.

J Exp Med 2015 May 13;212(5):649-63. Epub 2015 Apr 13.

Stem Cell Program, Division of Hematology/Oncology, Boston Children's Hospital and Dana-Farber Cancer Institute, Boston, MA 02115 Harvard Stem Cell Institute, Howard Hughes Medical Institute, and Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138 Harvard Stem Cell Institute, Howard Hughes Medical Institute, and Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138 Harvard Stem Cell Institute, Howard Hughes Medical Institute, and Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138

Hematopoietic stem cells (HSCs) emerge from aortic endothelium via the endothelial-to-hematopoietic transition (EHT). The molecular mechanisms that initiate and regulate EHT remain poorly understood. Here, we show that adenosine signaling regulates hematopoietic stem and progenitor cell (HSPC) development in zebrafish embryos. The adenosine receptor A2b is expressed in the vascular endothelium before HSPC emergence. Elevated adenosine levels increased runx1(+)/cmyb(+) HSPCs in the dorsal aorta, whereas blocking the adenosine pathway decreased HSPCs. Knockdown of A2b adenosine receptor disrupted scl(+) hemogenic vascular endothelium and the subsequent EHT process. A2b adenosine receptor activation induced CXCL8 via cAMP-protein kinase A (PKA) and mediated hematopoiesis. We further show that adenosine increased multipotent progenitors in a mouse embryonic stem cell colony-forming assay and in embryonic day 10.5 aorta-gonad-mesonephros explants. Our results demonstrate that adenosine signaling plays an evolutionary conserved role in the first steps of HSPC formation in vertebrates.
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http://dx.doi.org/10.1084/jem.20141528DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4419349PMC
May 2015

Notch1 acts via Foxc2 to promote definitive hematopoiesis via effects on hemogenic endothelium.

Blood 2015 Feb 13;125(9):1418-26. Epub 2015 Jan 13.

Stem Cell Program and Division of Hematology/Oncology, Children's Hospital Boston and Dana-Farber Cancer Institute, Howard Hughes Medical Institute, Harvard Medical School, Boston, MA; Stem Cell Transplantation Program, Manton Center for Orphan Disease Research, Children's Hospital Boston, Boston, MA; Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA; Harvard Stem Cell Institute, Boston, MA;

Hematopoietic and vascular development share many common features, including cell surface markers and sites of origin. Recent lineage-tracing studies have established that definitive hematopoietic stem and progenitor cells arise from vascular endothelial-cadherin(+) hemogenic endothelial cells of the aorta-gonad-mesonephros region, but the genetic programs underlying the specification of hemogenic endothelial cells remain poorly defined. Here, we discovered that Notch induction enhances hematopoietic potential and promotes the specification of hemogenic endothelium in differentiating cultures of mouse embryonic stem cells, and we identified Foxc2 as a highly upregulated transcript in the hemogenic endothelial population. Studies in zebrafish and mouse embryos revealed that Foxc2 and its orthologs are required for the proper development of definitive hematopoiesis and function downstream of Notch signaling in the hemogenic endothelium. These data establish a pathway linking Notch signaling to Foxc2 in hemogenic endothelial cells to promote definitive hematopoiesis.
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http://dx.doi.org/10.1182/blood-2014-04-568170DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4342355PMC
February 2015

A comparison of non-integrating reprogramming methods.

Nat Biotechnol 2015 Jan 1;33(1):58-63. Epub 2014 Dec 1.

1] Division of Pediatric Hematology/Oncology, Boston Children's Hospital, Dana-Farber Cancer Institute, Boston, Massachusetts, USA. [2] Harvard Stem Cell Institute, Cambridge, Massachusetts, USA. [3] Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts, USA. [4] Howard Hughes Medical Institute, Children's Hospital Boston and Dana Farber Cancer Institute, Boston, Massachusetts, USA.

Human induced pluripotent stem cells (hiPSCs) are useful in disease modeling and drug discovery, and they promise to provide a new generation of cell-based therapeutics. To date there has been no systematic evaluation of the most widely used techniques for generating integration-free hiPSCs. Here we compare Sendai-viral (SeV), episomal (Epi) and mRNA transfection mRNA methods using a number of criteria. All methods generated high-quality hiPSCs, but significant differences existed in aneuploidy rates, reprogramming efficiency, reliability and workload. We discuss the advantages and shortcomings of each approach, and present and review the results of a survey of a large number of human reprogramming laboratories on their independent experiences and preferences. Our analysis provides a valuable resource to inform the use of specific reprogramming methods for different laboratories and different applications, including clinical translation.
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http://dx.doi.org/10.1038/nbt.3070DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4329913PMC
January 2015

TMEM14C is required for erythroid mitochondrial heme metabolism.

J Clin Invest 2014 Oct 26;124(10):4294-304. Epub 2014 Aug 26.

The transport and intracellular trafficking of heme biosynthesis intermediates are crucial for hemoglobin production, which is a critical process in developing red cells. Here, we profiled gene expression in terminally differentiating murine fetal liver-derived erythroid cells to identify regulators of heme metabolism. We determined that TMEM14C, an inner mitochondrial membrane protein that is enriched in vertebrate hematopoietic tissues, is essential for erythropoiesis and heme synthesis in vivo and in cultured erythroid cells. In mice, TMEM14C deficiency resulted in porphyrin accumulation in the fetal liver, erythroid maturation arrest, and embryonic lethality due to profound anemia. Protoporphyrin IX synthesis in TMEM14C-deficient erythroid cells was blocked, leading to an accumulation of porphyrin precursors. The heme synthesis defect in TMEM14C-deficient cells was ameliorated with a protoporphyrin IX analog, indicating that TMEM14C primarily functions in the terminal steps of the heme synthesis pathway. Together, our data demonstrate that TMEM14C facilitates the import of protoporphyrinogen IX into the mitochondrial matrix for heme synthesis and subsequent hemoglobin production. Furthermore, the identification of TMEM14C as a protoporphyrinogen IX importer provides a genetic tool for further exploring erythropoiesis and congenital anemias.
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http://dx.doi.org/10.1172/JCI76979DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4191016PMC
October 2014

Neurotrophin receptor TrkB promotes lung adenocarcinoma metastasis.

Proc Natl Acad Sci U S A 2014 Jul 30;111(28):10299-304. Epub 2014 Jun 30.

Stem Cell Program, Boston Children's Hospital, Boston MA 02115;Harvard Stem Cell Institute, Cambridge, MA 02138;Department of Genetics, Harvard Medical School, Boston, MA 02115;

Lung cancer is notorious for its ability to metastasize, but the pathways regulating lung cancer metastasis are largely unknown. An in vitro system designed to discover factors critical for lung cancer cell migration identified brain-derived neurotrophic factor, which stimulates cell migration through activation of tropomyosin-related kinase B (TrkB; also called NTRK2). Knockdown of TrkB in human lung cancer cell lines significantly decreased their migratory and metastatic ability in vitro and in vivo. In an autochthonous lung adenocarcinoma model driven by activated oncogenic Kras and p53 loss, TrkB deficiency significantly reduced metastasis. Hypoxia-inducible factor-1 directly regulated TrkB expression, and, in turn, TrkB activated Akt signaling in metastatic lung cancer cells. Finally, TrkB expression was correlated with metastasis in patient samples, and TrkB was detected more often in tumors that did not have Kras or epidermal growth factor receptor mutations. These studies demonstrate that TrkB is an important therapeutic target in metastatic lung adenocarcinoma.
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http://dx.doi.org/10.1073/pnas.1404399111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4104911PMC
July 2014

Iron regulatory protein-1 protects against mitoferrin-1-deficient porphyria.

J Biol Chem 2014 Mar 7;289(11):7835-43. Epub 2014 Feb 7.

From the Division of Hematology, Brigham and Women's Hospital; Division of Hematology-Oncology, Boston Children's Hospital and Harvard Medical School, Boston, Massachusetts 02115.

Mitochondrial iron is essential for the biosynthesis of heme and iron-sulfur ([Fe-S]) clusters in mammalian cells. In developing erythrocytes, iron is imported into the mitochondria by MFRN1 (mitoferrin-1, SLC25A37). Although loss of MFRN1 in zebrafish and mice leads to profound anemia, mutant animals showed no overt signs of porphyria, suggesting that mitochondrial iron deficiency does not result in an accumulation of protoporphyrins. Here, we developed a gene trap model to provide in vitro and in vivo evidence that iron regulatory protein-1 (IRP1) inhibits protoporphyrin accumulation. Mfrn1(+/gt);Irp1(-/-) erythroid cells exhibit a significant increase in protoporphyrin levels. IRP1 attenuates protoporphyrin biosynthesis by binding to the 5'-iron response element (IRE) of alas2 mRNA, inhibiting its translation. Ectopic expression of alas2 harboring a mutant IRE, preventing IRP1 binding, in Mfrn1(gt/gt) cells mimics Irp1 deficiency. Together, our data support a model whereby impaired mitochondrial [Fe-S] cluster biogenesis in Mfrn1(gt/gt) cells results in elevated IRP1 RNA-binding that attenuates ALAS2 mRNA translation and protoporphyrin accumulation.
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http://dx.doi.org/10.1074/jbc.M114.547778DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3953295PMC
March 2014

iPSC-derived neurons as a higher-throughput readout for autism: promises and pitfalls.

Trends Mol Med 2014 Feb 24;20(2):91-104. Epub 2013 Dec 24.

Center for Biomedical Informatics, Harvard Medical School, Boston, MA 02115, USA; Department of Pediatrics, Harvard Medical School, Boston, MA 02115, USA. Electronic address:

The elucidation of disease etiologies and establishment of robust, scalable, high-throughput screening assays for autism spectrum disorders (ASDs) have been impeded by both inaccessibility of disease-relevant neuronal tissue and the genetic heterogeneity of the disorder. Neuronal cells derived from induced pluripotent stem cells (iPSCs) from autism patients may circumvent these obstacles and serve as relevant cell models. To date, derived cells are characterized and screened by assessing their neuronal phenotypes. These characterizations are often etiology-specific or lack reproducibility and stability. In this review, we present an overview of efforts to study iPSC-derived neurons as a model for autism, and we explore the plausibility of gene expression profiling as a reproducible and stable disease marker.
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http://dx.doi.org/10.1016/j.molmed.2013.11.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4117413PMC
February 2014

Pluripotent stem cell models of Shwachman-Diamond syndrome reveal a common mechanism for pancreatic and hematopoietic dysfunction.

Cell Stem Cell 2013 Jun 18;12(6):727-36. Epub 2013 Apr 18.

Division of Pediatric Hematology/Oncology, Boston Children's Hospital, Dana-Farber Cancer Institute, Boston, MA 02115, USA.

Shwachman-Diamond syndrome (SDS), a rare autosomal-recessive disorder characterized by exocrine pancreatic insufficiency and hematopoietic dysfunction, is caused by mutations in the Shwachman-Bodian-Diamond syndrome (SBDS) gene. We created human pluripotent stem cell models of SDS through knockdown of SBDS in human embryonic stem cells (hESCs) and generation of induced pluripotent stem cell (iPSC) lines from two patients with SDS. SBDS-deficient hESCs and iPSCs manifest deficits in exocrine pancreatic and hematopoietic differentiation in vitro, enhanced apoptosis, and elevated protease levels in culture supernatants, which could be reversed by restoring SBDS protein expression through transgene rescue or by supplementing culture media with protease inhibitors. Protease-mediated autodigestion provides a mechanistic link between the pancreatic and hematopoietic phenotypes in SDS, highlighting the utility of hESCs and iPSCs in obtaining novel insights into human disease.
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http://dx.doi.org/10.1016/j.stem.2013.04.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3755012PMC
June 2013

Impaired intrinsic immunity to HSV-1 in human iPSC-derived TLR3-deficient CNS cells.

Nature 2012 Nov 28;491(7426):769-73. Epub 2012 Oct 28.

Center for Stem Cell Biology, Sloan-Kettering Institute for Cancer Research, New York, New York 10065, USA.

In the course of primary infection with herpes simplex virus 1 (HSV-1), children with inborn errors of toll-like receptor 3 (TLR3) immunity are prone to HSV-1 encephalitis (HSE). We tested the hypothesis that the pathogenesis of HSE involves non-haematopoietic CNS-resident cells. We derived induced pluripotent stem cells (iPSCs) from the dermal fibroblasts of TLR3- and UNC-93B-deficient patients and from controls. These iPSCs were differentiated into highly purified populations of neural stem cells (NSCs), neurons, astrocytes and oligodendrocytes. The induction of interferon-β (IFN-β) and/or IFN-λ1 in response to stimulation by the dsRNA analogue polyinosinic:polycytidylic acid (poly(I:C)) was dependent on TLR3 and UNC-93B in all cells tested. However, the induction of IFN-β and IFN-λ1 in response to HSV-1 infection was impaired selectively in UNC-93B-deficient neurons and oligodendrocytes. These cells were also much more susceptible to HSV-1 infection than control cells, whereas UNC-93B-deficient NSCs and astrocytes were not. TLR3-deficient neurons were also found to be susceptible to HSV-1 infection. The rescue of UNC-93B- and TLR3-deficient cells with the corresponding wild-type allele showed that the genetic defect was the cause of the poly(I:C) and HSV-1 phenotypes. The viral infection phenotype was rescued further by treatment with exogenous IFN-α or IFN-β ( IFN-α/β) but not IFN-λ1. Thus, impaired TLR3- and UNC-93B-dependent IFN-α/β intrinsic immunity to HSV-1 in the CNS, in neurons and oligodendrocytes in particular, may underlie the pathogenesis of HSE in children with TLR3-pathway deficiencies.
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http://dx.doi.org/10.1038/nature11583DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3527075PMC
November 2012

Induced pluripotent stem cells as a tool for gaining new insights into Fanconi anemia.

Cell Cycle 2012 Aug 24;11(16):2985-90. Epub 2012 Jul 24.

Division of Pediatric Hematology/Oncology, Boston Children's Hospital and Dana Farber Cancer Institute, Harvard Medical School, Boston, MA, USA.

Induced pluripotent stem cells (iPSC) hold significant promise for advancing biomedical research. In the case of monogenic diseases, patient-iPSC and their derivatives contain the disease-causing mutation, suggesting the possibility of recapitulating salient disease features in vitro. Fanconi anemia (FA) is the most common inherited bone marrow failure syndrome. The etiology of bone marrow failure in FA remains largely unclear, but limited studies on patient bone marrow cells indicate cell intrinsic defects as causative. We examined the feasibility of modeling FA in a system based on hematopoietic differentiation of patient-specific iPSC. An informative iPSC-based model is predicated on the ability to derive disease-specific (uncorrected) patient iPSC that contain the disease-causing mutation, are pluripotent, maintain a normal karyotype and are capable of hematopoietic differentiation. Careful analysis of hematopoietic differentiation of such iPSC holds the promise of uncovering new insights into bone marrow failure and may enable high-throughput screening with the goal of identifying compounds that ameliorate hematopoietic failure. Ultimately, genetic correction, molecular characterization and successful engraftment of iPSC-derived cells may provide an attractive alternative to current hematopoietic stem cell-targeted gene therapy in some monogenic diseases, including FA.
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http://dx.doi.org/10.4161/cc.21109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3442908PMC
August 2012

Live-cell immunofluorescence staining of human pluripotent stem cells.

Curr Protoc Stem Cell Biol 2011 Dec;Chapter 1:Unit 1C.12

Children's Hospital Boston, Boston, Massachusetts, USA.

Antibodies are instrumental tools in stem cell identification, purification, and analysis. Most commonly, cell samples are either dissociated to obtain a single-cell suspension suitable for FACS analysis or cell sorting, or fixed in situ for immunostaining and fluorescence microscopy imaging. This unit describes an alternative method in which live adherent cells are stained and imaged in situ without the need for cell dissociation, fixation, or fluorescent reporter genes. This minimally invasive method is particularly useful for identification and distinction of fully and partially reprogrammed induced pluripotent stem cells (iPSCs). The unit also describes the use of mCD49e and hCD29 antibodies in live-cell (vital) imaging. mCD49e strongly stains mouse embryonic fibroblast (MEF) feeder cells in human pluripotent stem cell cultures, whereas hCD29 recognizes an antigen expressed on undifferentiated and many differentiated cells. A distinguishing feature of hCD29 in live-cell staining is that its antigen is precluded from detection wherever cells have formed tight epithelial junctions (e.g., in the center but not the periphery of pluripotent stem cell colonies) due to basolateral location. A non-fluorescent fixed-cell staining protocol is also provided for medium- to high-throughput quantification of stem cell experiments without an automated microscope. The discussion addresses technical limitations, pitfalls, troubleshooting, and potential applications, such as identification of emerging bona fide human iPSC colonies in reprogramming experiments.
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http://dx.doi.org/10.1002/9780470151808.sc01c12s19DOI Listing
December 2011
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