Publications by authors named "In-Hyun Park"

108 Publications

How well do brain organoids capture your brain?

iScience 2021 Feb 19;24(2):102063. Epub 2021 Jan 19.

Department of Genetics, Yale Stem Cell Center, Yale Child Study Center, Yale School of Medicine, New Haven, CT 06520, USA.

Brain organoids closely recapitulate many features and characteristics of brain tissue. This technology in turn allows unprecedented possibilities to investigate brain development and function in the dish. Several brain organoid protocols have been established, and the studies have focused on validating the architecture, cellular composition, and function of the organoids. In future, the improved and advanced organoid models will enable us to understand cellular and molecular features of the developing brain. However, several obstacles, such as the quality of the organoids, 3D structural analysis, and measurement of the neural connectivity need to be improved. In this perspective, we will provide an overview of the current state of the art of the brain organoid field, with a focus on protocols and organoid characterization. Additionally, we will address the current limitations of this evolving field and provide an understanding of the current brain organoid landscape and insight toward the next steps.
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http://dx.doi.org/10.1016/j.isci.2021.102063DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7856464PMC
February 2021

Genes causing congenital hydrocephalus: Their chromosomal characteristics of telomere proximity and DNA compositions.

Exp Neurol 2021 Jan 4;335:113523. Epub 2020 Nov 4.

Department of Biomedical Engineering, Marshall University, Huntington, WV 25755, USA. Electronic address:

Congenital hydrocephalus (CH) is caused by genetic mutations, but whether factors impacting human genetic mutations are disease-specific remains elusive. Given two factors associated with high mutation rates, we reviewed how many disease-susceptible genes match with (i) proximity to telomeres or (ii) high adenine and thymine (A + T) content in human CH as compared to other disorders of the central nervous system (CNS). We extracted genomic information using a genome data viewer. Importantly, 98 of 108 genes causing CH satisfied (i) or (ii), resulting in >90% matching rate. However, such a high accordance no longer sustained as we checked two factors in Alzheimer's disease (AD) and/or familial Parkinson's disease (fPD), resulting in 84% and 59% matching, respectively. A disease-specific matching of telomere proximity or high A + T content predicts causative genes of CH much better than neurodegenerative diseases and other CNS conditions, likely due to sufficient number of known causative genes (n = 108) and precise determination and classification of the genotype and phenotype. Our analysis suggests a need for identifying genetic basis of both factors before human clinical studies, to prioritize putative genes found in preclinical models into the likely (meeting at least one) and more likely candidate (meeting both), which predisposes human genes to mutations.
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http://dx.doi.org/10.1016/j.expneurol.2020.113523DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7750280PMC
January 2021

Generation of Regionally Specified Human Brain Organoids Resembling Thalamus Development.

STAR Protoc 2020 Jun 4;1(1). Epub 2020 Jun 4.

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

Thalamus is a critical information relay hub in the cortex; its malfunction causes multiple neurological and psychiatric disorders. However, there are no model systems to study the development and function of human thalamus. Here, we present a protocol to generate regionally specified human brain organoids that recapitulate the development of the thalamus using human pluripotent stem cells (hPSCs). Thalamic organoids can be used to study human thalamus development, to model related diseases, and to discover potential therapeutics. For complete information on human thalamic organoids and their application, please refer to the paper by Xiang et al. (2019).
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http://dx.doi.org/10.1016/j.xpro.2019.100001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7580078PMC
June 2020

Intracerebral Transplants of GMP-Grade Human Umbilical Cord-Derived Mesenchymal Stromal Cells Effectively Treat Subacute-Phase Ischemic Stroke in a Rodent Model.

Front Cell Neurosci 2020 25;14:546659. Epub 2020 Sep 25.

Department of Biomedical Science, CHA Stem Cell Institute, CHA University, Seongnam-si, South Korea.

In subacute and chronic phases of the stroke, there are no therapeutics available at present to promote functional recovery. Human umbilical cord-derived mesenchymal stromal cells (hUC-MSCs) are one of the candidate cell types for treating subacute-phase stroke. The benefits of cell-based therapy largely depend on the migratory capacity of products administered, as well as their potential for engraftment in targeted tissues and paracrine activities. Timing and delivery modes may also influence the outcomes of stem-cell therapy. Still, the functional recuperative effects of differing hUC-MSC delivery modes, about cell replacement and cell-to-cell paracrine activity levels, have yet to be clarified in subacute phases of stroke.This study was conducted to compare the therapeutic effects of various delivery routes when administering Good Manufacturing Practice (GMP)-grade hUC-MSCs in a rodent model of subacute-phase stroke. Cell aliquots (1 × 10) were given to rats as intravenous (IV) injections or intracerebral (IC) transplants 1 week after middle cerebral artery occlusion (MCAo). Transplanted rats were examined up to 7 weeks later using various behavioral tests and immunohistochemical analyses. Most IC-transplanted cells survived for short periods (i.e., <4 weeks after receipt) and gradually disappeared, whereas IV-injected cells were undetectable in the brain at the same time points (i.e., 3 days, 4 weeks, or 7 weeks after injection). Although short-lived, IC-transplanted cells effectively improved behavioral deficits, serving to reduce infarct volumes and glial scar formation, increase subventricular counts of proliferating neuroblasts, and promote cerebrovascular ingrowth in ischemic penumbra regions. IV injection, however, failed to improve behavioral function or histologic parameters during the same 7-week time frame. These findings overall suggest that IC transplantation is preferable to IV injection for delivery of hUC-MSCs during subacute phases of stroke.
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http://dx.doi.org/10.3389/fncel.2020.546659DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7546889PMC
September 2020

Reprogramming progressive cells display low CAG promoter activity.

Stem Cells 2021 Jan 4;39(1):43-54. Epub 2020 Nov 4.

Department of Cell Biology, Yale University, New Haven, Connecticut, USA.

There is wide variability in the propensity of somatic cells to reprogram into pluripotency in response to the Yamanaka factors. How to segregate these variabilities to enrich for cells of specific traits that reprogram efficiently remains challenging. Here we report that the variability in reprogramming propensity is associated with the activity of the MKL1/SRF transcription factor and concurs with small cell size as well as rapid cell cycle. Reprogramming progressive cells can be prospectively identified by their low activity of a widely used synthetic promoter, CAG. CAG cells arise and expand during cell cycle acceleration in the early reprogramming culture of both mouse and human fibroblasts. Our work illustrates a molecular scenario underlying the distinct reprogramming propensities and demonstrates a convenient practical approach for their enrichment.
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http://dx.doi.org/10.1002/stem.3295DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7821215PMC
January 2021

Mural Cell-Specific Deletion of Cerebral Cavernous Malformation 3 in the Brain Induces Cerebral Cavernous Malformations.

Arterioscler Thromb Vasc Biol 2020 09 9;40(9):2171-2186. Epub 2020 Jul 9.

From the Interdepartmental Program in Vascular Biology and Therapeutics, Department of Pathology (K.W., H.Z., Y.H., Q.J., J.S.P., W.M., H.J.Z.), Yale University School of Medicine, New Haven, CT.

Objective: Cerebral cavernous malformations (CCM), consisting of dilated capillary channels formed by a single layer of endothelial cells lacking surrounding mural cells. It is unclear why CCM lesions are primarily confined to brain vasculature, although the 3 CCM-associated genes (, , and ) are ubiquitously expressed in all tissues. We aimed to determine the role of gene in brain mural cell in CCM pathogenesis. Approach and Results: -Cre was used to drive a specific deletion of in mural cells, including pericytes and smooth muscle cells (Ccm3smKO). Ccm3smKO mice developed CCM lesions in the brain with onset at neonatal stages. One-third of Ccm3smKO mice survived upto 6 weeks of age, exhibiting seizures, and severe brain hemorrhage. The early CCM lesions in Ccm3smKO neonates were loosely wrapped by mural cells, and adult Ccm3smKO mice had clustered and enlarged capillary channels (caverns) formed by a single layer of endothelium lacking mural cell coverage. Importantly, CCM lesions throughout the entire brain in Ccm3smKO mice, which more accurately mimicked human disease than the current endothelial cell-specific deletion models. Mechanistically, loss in brain pericytes dramatically increased paxillin stability and focal adhesion formation, enhancing ITG-β1 (integrin β1) activity and extracellular matrix adhesion but reducing cell migration and endothelial cell-pericyte associations. Moreover, CCM3-wild type, but not a paxillin-binding defective mutant, rescued the phenotypes in CCM3-deficient pericytes.

Conclusions: Our data demonstrate for the first time that deletion of a gene in the brain mural cell induces CCM pathogenesis.
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http://dx.doi.org/10.1161/ATVBAHA.120.314586DOI Listing
September 2020

Deconstructing and reconstructing the human brain with regionally specified brain organoids.

Semin Cell Dev Biol 2021 Mar 15;111:40-51. Epub 2020 Jun 15.

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

Brain organoids, three-dimensional neural cultures recapitulating the spatiotemporal organization and function of the brain in a dish, offer unique opportunities for investigating the human brain development and diseases. To model distinct parts of the brain, various region-specific human brain organoids have been developed. In this article, we review current approaches to produce human region-specific brain organoids, developed through the endeavor of many researchers. We highlight the applications of human region-specific brain organoids, especially in reconstructing regional interactions in the brain through organoid fusion. We also outline the existing challenges to drive forward further the brain organoid technology and its applications for future studies.
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http://dx.doi.org/10.1016/j.semcdb.2020.05.023DOI Listing
March 2021

Dysregulation of BRD4 Function Underlies the Functional Abnormalities of MeCP2 Mutant Neurons.

Mol Cell 2020 07 10;79(1):84-98.e9. Epub 2020 Jun 10.

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

Rett syndrome (RTT), mainly caused by mutations in methyl-CpG binding protein 2 (MeCP2), is one of the most prevalent intellectual disorders without effective therapies. Here, we used 2D and 3D human brain cultures to investigate MeCP2 function. We found that MeCP2 mutations cause severe abnormalities in human interneurons (INs). Surprisingly, treatment with a BET inhibitor, JQ1, rescued the molecular and functional phenotypes of MeCP2 mutant INs. We uncovered that abnormal increases in chromatin binding of BRD4 and enhancer-promoter interactions underlie the abnormal transcription in MeCP2 mutant INs, which were recovered to normal levels by JQ1. We revealed cell-type-specific transcriptome impairment in MeCP2 mutant region-specific human brain organoids that were rescued by JQ1. Finally, JQ1 ameliorated RTT-like phenotypes in mice. These data demonstrate that BRD4 dysregulation is a critical driver for RTT etiology and suggest that targeting BRD4 could be a potential therapeutic opportunity for RTT.
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http://dx.doi.org/10.1016/j.molcel.2020.05.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7375197PMC
July 2020

Implantation of the clinical-grade human neural stem cell line, CTX0E03, rescues the behavioral and pathological deficits in the quinolinic acid-lesioned rodent model of Huntington's disease.

Stem Cells 2020 08 6;38(8):936-947. Epub 2020 May 6.

Department of Biomedical Science, CHA Stem Cell Institute, CHA University, Seongnam-si, Gyeonggi-do, Republic of Korea.

Huntington's disease (HD) is a devastating, autosomal-dominant neurodegenerative disease, for which there are currently no disease-modifying therapies. Clinical trials to replace the damaged striatal medium spiny neurons (MSNs) have been attempted in the past two decades but have met with only limited success. In this study, we investigated whether a clonal, conditionally immortalized neural stem cell line (CTX0E03), which has already shown safety and signals of efficacy in chronic ischemic stroke patients, could rescue deficits seen in an animal model of HD. After CTX0E03 transplantation into the quinolinic acid-lesioned rat model of HD, behavioral changes were measured using the rotarod, stepping, and staircase tests. In vivo differentiation and neuronal connections of the transplanted CTX0E03 cells were evaluated with immunohistochemical staining and retrograde tracing with Fluoro-Gold. We found that transplantation of CTX0E03 gave rise to a significant behavioral improvement compared with the sham- or fibroblast-transplanted group. Transplanted CTX0E03 formed MSNs (DARPP-32) and GABAergic neurons (GABA, GAD65/67) with BDNF expression in the striatum, while cortically transplanted cells formed Tbr1-positive neurons. Using a retrograde label, we also found stable engraftment and connection of the transplanted cells with host brain tissues. CTX0E03 transplantation also reduced glial scar formation and inflammation, as well as increasing endogenous neurogenesis and angiogenesis. Overall, our results demonstrate that CTX0E03, a clinical-grade neural stem cell line, is effective for preclinical test in HD, and, therefore, will be useful for clinical development in the treatment of HD patients.
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http://dx.doi.org/10.1002/stem.3191DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7496241PMC
August 2020

Synthetic Analyses of Single-Cell Transcriptomes from Multiple Brain Organoids and Fetal Brain.

Cell Rep 2020 02;30(6):1682-1689.e3

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

Human brain organoid systems offer unprecedented opportunities to investigate both neurodevelopmental and neurological disease. Single-cell-based transcriptomics or epigenomics have dissected the cellular and molecular heterogeneity in the brain organoids, revealing a complex organization. Similar but distinct protocols from different labs have been applied to generate brain organoids, providing a large resource to perform a comparative analysis of brain developmental processes. Here, we take a systematic approach to compare the single-cell transcriptomes of various human cortical brain organoids together with fetal brain to define the identity of specific cell types and differentiation routes in each method. Importantly, we identify unique developmental programs in each protocol compared to fetal brain, which will be a critical benchmark for the utility of human brain organoids in the future.
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http://dx.doi.org/10.1016/j.celrep.2020.01.038DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7043376PMC
February 2020

Engineering of human brain organoids with a functional vascular-like system.

Nat Methods 2019 11 7;16(11):1169-1175. Epub 2019 Oct 7.

Department of Genetics, Yale Stem Cell Center, Yale School of Medicine, New Haven, CT, USA.

Human cortical organoids (hCOs), derived from human embryonic stem cells (hESCs), provide a platform to study human brain development and diseases in complex three-dimensional tissue. However, current hCOs lack microvasculature, resulting in limited oxygen and nutrient delivery to the inner-most parts of hCOs. We engineered hESCs to ectopically express human ETS variant 2 (ETV2). ETV2-expressing cells in hCOs contributed to forming a complex vascular-like network in hCOs. Importantly, the presence of vasculature-like structures resulted in enhanced functional maturation of organoids. We found that vascularized hCOs (vhCOs) acquired several blood-brain barrier characteristics, including an increase in the expression of tight junctions, nutrient transporters and trans-endothelial electrical resistance. Finally, ETV2-induced endothelium supported the formation of perfused blood vessels in vivo. These vhCOs form vasculature-like structures that resemble the vasculature in early prenatal brain, and they present a robust model to study brain disease in vitro.
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http://dx.doi.org/10.1038/s41592-019-0586-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6918722PMC
November 2019

The critical role of persistent sodium current in hippocampal gamma oscillations.

Neuropharmacology 2020 01 21;162:107787. Epub 2019 Sep 21.

Department of Neurology, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA; Department of Neuroscience, University of Kentucky, Lexington, KY 40536, USA; Center for Translational Neuroscience, Department of Neurobiology and Developmental Sciences, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA. Electronic address:

Gamma network oscillations in the brain are fast rhythmic network oscillations in the gamma frequency range (~30-100 Hz), playing key roles in the hippocampus for learning, memory, and spatial processing. There is evidence indicating that GABAergic interneurons, including parvalbumin-expressing basket cells (PVBCs), contribute to cortical gamma oscillations through synaptic interactions with excitatory cells. However, the molecular, cellular, and circuit underpinnings underlying generation and maintenance of cortical gamma oscillations are largely elusive. Recent studies demonstrated that intrinsic and synaptic properties of GABAergic interneurons and excitatory cells are regulated by a slowly inactivating or non-inactivating sodium current (i.e., persistent sodium current, I), suggesting that I is involved in gamma oscillations. Here, we tested whether I plays a role in hippocampal gamma oscillations using pharmacological, optogenetic, and electrophysiological approaches. We found that I blockers, phenytoin (40 μM and 100 μM) and riluzole (10 μM), reduced gamma oscillations induced by optogenetic stimulation of CaMKII-expressing cells in CA1 networks. Whole-cell patch-clamp recordings further demonstrated that phenytoin (100 μM) reduced I and firing frequencies in both PVBCs and pyramidal cells without altering threshold and amplitude of action potentials, but increased rheobase in both cell types. These results suggest that I in pyramidal cells and PVBCs is required for hippocampal gamma oscillations, supporting a pyramidal-interneuron network gamma model. Phenytoin-mediated modulation of hippocampal gamma oscillations may be a mechanism underlying its anticonvulsant efficacy, as well as its contribution to cognitive impairments in epilepsy patients.
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http://dx.doi.org/10.1016/j.neuropharm.2019.107787DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6952064PMC
January 2020

The RNA exosome nuclease complex regulates human embryonic stem cell differentiation.

J Cell Biol 2019 08 15;218(8):2564-2582. Epub 2019 Jul 15.

Department of Cell Biology, Yale School of Medicine, New Haven, CT

A defining feature of embryonic stem cells (ESCs) is the ability to differentiate into all three germ layers. Pluripotency is maintained in part by a unique transcription network that maintains expression of pluripotency-specific transcription factors and represses developmental genes. While the mechanisms that establish this transcription network are well studied, little is known of the posttranscriptional surveillance pathways that degrade differentiation-related RNAs. We report that the surveillance pathway mediated by the RNA exosome nuclease complex represses ESC differentiation. Depletion of the exosome expedites differentiation of human ESCs into all three germ layers. LINE-1 retrotransposons and specific miRNAs, lncRNAs, and mRNAs that encode developmental regulators or affect their expression are all bound by the exosome and increase in level upon exosome depletion. The exosome restrains differentiation in part by degrading transcripts encoding FOXH1, a transcription factor crucial for mesendoderm formation. Our studies establish the exosome as a regulator of human ESC differentiation and reveal the importance of RNA decay in maintaining pluripotency.
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http://dx.doi.org/10.1083/jcb.201811148DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6683745PMC
August 2019

Generation and Fusion of Human Cortical and Medial Ganglionic Eminence Brain Organoids.

Curr Protoc Stem Cell Biol 2018 11 11;47(1). Epub 2018 Sep 11.

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

Three-dimensional (3D) brain organoid culture has become an essential tool for investigating human brain development and modeling neurological disorders during the past few years. Given the specific regionalization during brain development, it is important to produce distinct brain organoids that reproduce different brain regions and their interaction. The authors' laboratory recently established the platform to generate brain organoids resembling the medial ganglionic eminence (MGE), a specific brain region responsible for interneurogenesis, and found when fusing with organoid resembling the cortex, the fused organoids enabled modeling of interneuron migration in the brain. This unit describes four basic protocols that have been successfully applied in the authors' laboratory, covering the generation of embryonic body (EB) with neuroectodermal fate, the production of MGE organoids (hMGEOs) and cortical organoids (hCOs), and the fusion of the two organoids.
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http://dx.doi.org/10.1002/cpsc.61DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6402040PMC
November 2018

hESC-Derived Thalamic Organoids Form Reciprocal Projections When Fused with Cortical Organoids.

Cell Stem Cell 2019 03 21;24(3):487-497.e7. Epub 2019 Feb 21.

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

Human brain organoid techniques have rapidly advanced to facilitate investigating human brain development and diseases. These efforts have largely focused on generating telencephalon due to its direct relevance in a variety of forebrain disorders. Despite its importance as a relay hub between cortex and peripheral tissues, the investigation of three-dimensional (3D) organoid models for the human thalamus has not been explored. Here, we describe a method to differentiate human embryonic stem cells (hESCs) to thalamic organoids (hThOs) that specifically recapitulate the development of thalamus. Single-cell RNA sequencing revealed a formation of distinct thalamic lineages, which diverge from telencephalic fate. Importantly, we developed a 3D system to create the reciprocal projections between thalamus and cortex by fusing the two distinct region-specific organoids representing the developing thalamus or cortex. Our study provides a platform for understanding human thalamic development and modeling circuit organizations and related disorders in the brain.
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http://dx.doi.org/10.1016/j.stem.2018.12.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6853597PMC
March 2019

Correction to: New Advances in Human X Chromosome Status from a Developmental and Stem Cell Biology.

Tissue Eng Regen Med 2018 02 19;15(1):125. Epub 2018 Jan 19.

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

[This corrects the article DOI: 10.1007/s13770-017-0096-4.].
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http://dx.doi.org/10.1007/s13770-017-0111-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6171639PMC
February 2018

Uhrf1 regulates active transcriptional marks at bivalent domains in pluripotent stem cells through Setd1a.

Nat Commun 2018 07 3;9(1):2583. Epub 2018 Jul 3.

Department of Genetics, Yale Stem Cell Center, Yale Child Study Center, Yale School of Medicine, New Haven, CT, 06520, USA.

Embryonic stem cells (ESCs) maintain pluripotency through unique epigenetic states. When ESCs commit to a specific lineage, epigenetic changes in histones and DNA accompany the transition to specialized cell types. Investigating how epigenetic regulation controls lineage specification is critical in order to generate the required cell types for clinical applications. Uhrf1 is a widely known hemi-methylated DNA-binding protein, playing a role in DNA methylation through the recruitment of Dnmt1 and in heterochromatin formation alongside G9a, Trim28, and HDACs. Although Uhrf1 is not essential in ESC self-renewal, it remains elusive how Uhrf1 regulates cell specification. Here we report that Uhrf1 forms a complex with the active trithorax group, the Setd1a/COMPASS complex, to maintain bivalent histone marks, particularly those associated with neuroectoderm and mesoderm specification. Overall, our data demonstrate that Uhrf1 safeguards proper differentiation via bivalent histone modifications.
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http://dx.doi.org/10.1038/s41467-018-04818-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6030064PMC
July 2018

New Advances in Human X chromosome status from a Developmental and Stem Cell Biology.

Tissue Eng Regen Med 2017 Dec 22;14(6):643-652. Epub 2017 Nov 22.

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

Recent advances in stem cell biology have dramatically increased the understanding of molecular and cellular mechanism of pluripotency and cell fate determination. Additionally, pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), arose as essential resources for disease modeling and cellular therapeutics. Despite these advancements, the epigenetic dysregulation in pluripotency such as the imprinting status, and X chromosome dosage compensation, and its consequences on future utility of PSCs yet remain unresolved. In this review, we will focus on the X chromosome regulation in human PSCs (hPSCs). We will introduce the previous findings in the dosage compensation process on mouse model, and make comparison with those of human systems. Particularly, the biallelic X chromosome activation status of human preimplantation embryos, and the regulation of the active X chromosome by human specific lincRNA, XACT, will be discussed. We will also discuss the recent findings on higher order X chromosome architecture utilizing Hi-C, and abnormal X chromosome status in hPSCs.
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http://dx.doi.org/10.1007/s13770-017-0096-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5738034PMC
December 2017

Enhanced Therapeutic and Long-Term Dynamic Vascularization Effects of Human Pluripotent Stem Cell-Derived Endothelial Cells Encapsulated in a Nanomatrix Gel.

Circulation 2017 Nov 29;136(20):1939-1954. Epub 2017 Sep 29.

Division of Cardiology, Department of Medicine, Emory University School of Medicine, Atlanta, GA (S.-J.L., Y.-D.S., A.A., S.K., J.B., J.W.H., Y.-S.Y.).

Background: Human pluripotent stem cell (hPSC)-derived endothelial cells (ECs) have limited clinical utility because of undefined components in the differentiation system and poor cell survival in vivo. Here, we aimed to develop a fully defined and clinically compatible system to differentiate hPSCs into ECs. Furthermore, we aimed to enhance cell survival, vessel formation, and therapeutic potential by encapsulating hPSC-ECs with a peptide amphiphile (PA) nanomatrix gel.

Methods: We induced differentiation of hPSCs into the mesodermal lineage by culturing on collagen-coated plates with a glycogen synthase kinase 3β inhibitor. Next, vascular endothelial growth factor, endothelial growth factor, and basic fibroblast growth factor were added for endothelial lineage differentiation, followed by sorting for CDH5 (VE-cadherin). We constructed an extracellular matrix-mimicking PA nanomatrix gel (PA-RGDS) by incorporating the cell adhesive ligand Arg-Gly-Asp-Ser (RGDS) and a matrix metalloproteinase-2-degradable sequence. We then evaluated whether the encapsulation of hPSC-CDH5 cells in PA-RGDS could enhance long-term cell survival and vascular regenerative effects in a hind-limb ischemia model with laser Doppler perfusion imaging, bioluminescence imaging, real-time reverse transcription-polymerase chain reaction, and histological analysis.

Results: The resultant hPSC-derived CDH5 cells (hPSC-ECs) showed highly enriched and genuine EC characteristics and proangiogenic activities. When injected into ischemic hind limbs, hPSC-ECs showed better perfusion recovery and higher vessel-forming capacity compared with media-, PA-RGDS-, or human umbilical vein EC-injected groups. However, the group receiving the PA-RGDS-encapsulated hPSC-ECs showed better perfusion recovery, more robust and longer cell survival (> 10 months), and higher and prolonged angiogenic and vascular incorporation capabilities than the bare hPSC-EC-injected group. Surprisingly, the engrafted hPSC-ECs demonstrated previously unknown sustained and dynamic vessel-forming behavior: initial perivascular concentration, a guiding role for new vessel formation, and progressive incorporation into the vessels over 10 months.

Conclusions: We generated highly enriched hPSC-ECs via a clinically compatible system. Furthermore, this study demonstrated that a biocompatible PA-RGDS nanomatrix gel substantially improved long-term survival of hPSC-ECs in an ischemic environment and improved neovascularization effects of hPSC-ECs via prolonged and unique angiogenic and vessel-forming properties. This PA-RGDS-mediated transplantation of hPSC-ECs can serve as a novel platform for cell-based therapy and investigation of long-term behavior of hPSC-ECs.
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http://dx.doi.org/10.1161/CIRCULATIONAHA.116.026329DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5685906PMC
November 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

Development of an inducible platform for intercellular protein delivery.

Int J Pharm 2017 Apr 28;522(1-2):1-10. Epub 2017 Feb 28.

Department of Molecular Medicine, Institute of Basic Medical Sciences, Faculty of Medicine, University of Oslo, PO Box 1112 Blindern, Oslo 0317, Norway; Norwegian Center for Stem Cell Research, PO Box 1112 Blindern, Oslo 0317, Norway; Institute of Immunology, Oslo University Hospital-Rikshospitalet, PO Box 4950 Nydalen, Oslo 0424, Norway. Electronic address:

A challenge to protein based therapies is the ability to produce biologically active proteins and their ensured delivery. Various approaches have been utilised including fusion of protein transduction domains with a protein or biomolecule of interest. A compounding issue is lack of specificity, efficiency and indeed whether the protein fusions are actually translocated into the cell and not merely an artefact of the fixation process. Here we present a novel platform, allowing the inducible export and uptake of a protein of interest. The system utilises a combination of the Tetracyline repressor system, combined with a fusion protein containing the N-terminal signal peptide from human chorionic gonadotropin beta-subunit, and a C-terminal poly-arginine domain for efficient uptake by target cells. This novel platform was validated using enhanced green fluorescent protein as the gene of interest. Doxycycline efficiently induced expression of the fusion protein. The human chorionic gonadotropin beta-subunit facilitated the export of the fusion protein into the cell culture media. Finally, the fusion protein was able to efficiently enter into neighbouring cells (target cells), mediated by the poly-arginine cell penetrating peptide. Importantly we have addressed the issue of whether the observed uptake is an artefact of the fixation process or indeed genuine translocation. In addition this platform provides a number of potential applications in diverse areas such as stem cell biology, immune therapy and cancer targeting therapies.
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http://dx.doi.org/10.1016/j.ijpharm.2017.02.067DOI Listing
April 2017

Bisulfite-independent analysis of CpG island methylation enables genome-scale stratification of single cells.

Nucleic Acids Res 2017 Jun;45(10):e77

Department of Genetics, Yale School of Medicine, New Haven, CT 06520, USA.

Conventional DNA bisulfite sequencing has been extended to single cell level, but the coverage consistency is insufficient for parallel comparison. Here we report a novel method for genome-wide CpG island (CGI) methylation sequencing for single cells (scCGI-seq), combining methylation-sensitive restriction enzyme digestion and multiple displacement amplification for selective detection of methylated CGIs. We applied this method to analyzing single cells from two types of hematopoietic cells, K562 and GM12878 and small populations of fibroblasts and induced pluripotent stem cells. The method detected 21 798 CGIs (76% of all CGIs) per cell, and the number of CGIs consistently detected from all 16 profiled single cells was 20 864 (72.7%), with 12 961 promoters covered. This coverage represents a substantial improvement over results obtained using single cell reduced representation bisulfite sequencing, with a 66-fold increase in the fraction of consistently profiled CGIs across individual cells. Single cells of the same type were more similar to each other than to other types, but also displayed epigenetic heterogeneity. The method was further validated by comparing the CpG methylation pattern, methylation profile of CGIs/promoters and repeat regions and 41 classes of known regulatory markers to the ENCODE data. Although not every minor methylation differences between cells are detectable, scCGI-seq provides a solid tool for unsupervised stratification of a heterogeneous cell population.
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http://dx.doi.org/10.1093/nar/gkx026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5605247PMC
June 2017

Single cell transcriptomics reveals unanticipated features of early hematopoietic precursors.

Nucleic Acids Res 2017 02;45(3):1281-1296

Department of Genetics, Yale School of Medicine, New Haven, CT, USA.

Molecular changes underlying stem cell differentiation are of fundamental interest. scRNA-seq on murine hematopoietic stem cells (HSC) and their progeny MPP1 separated the cells into 3 main clusters with distinct features: active, quiescent, and an un-characterized cluster. Induction of anemia resulted in mobilization of the quiescent to the active cluster and of the early to later stage of cell cycle, with marked increase in expression of certain transcription factors (TFs) while maintaining expression of interferon response genes. Cells with surface markers of long term HSC increased the expression of a group of TFs expressed highly in normal cycling MPP1 cells. However, at least Id1 and Hes1 were significantly activated in both HSC and MPP1 cells in anemic mice. Lineage-specific genes were differently expressed between cells, and correlated with the cell cycle stages with a specific augmentation of erythroid related genes in the G2/M phase. Most lineage specific TFs were stochastically expressed in the early precursor cells, but a few, such as Klf1, were detected only at very low levels in few precursor cells. The activation of these factors may correlate with stages of differentiation. This study reveals effects of cell cycle progression on the expression of lineage specific genes in precursor cells, and suggests that hematopoietic stress changes the balance of renewal and differentiation in these homeostatic cells.
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http://dx.doi.org/10.1093/nar/gkw1214DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5388401PMC
February 2017

Direct Reprogramming of Human Dermal Fibroblasts Into Endothelial Cells Using ER71/ETV2.

Circ Res 2017 Mar 21;120(5):848-861. Epub 2016 Dec 21.

From the Department of Pharmacology, College of Medicine, University of Illinois at Chicago (C.P., E.J.K.); Department of Pediatrics, Children's Heart Research and Outcomes Center, Emory University School of Medicine, Atlanta, GA (C.P., J.Y.K., S.Y.O.); Division of Cardiology, Department of Medicine, Emory University School of Medicine, Atlanta, GA (S.L., J.W.H., K.C., S.K., H.J.A., C.M.S., S.S., Y.-s.Y.); Department of Genetics, Yale Stem Cell Center, Yale School of Medicine, New Haven, CT (Y.T., I.-H.P.); and Division of Cardiology, Department of Medicine, Severance Biomedical Science Institute, Yonsei University College of Medicine, Seoul, Korea (S.-J.L., Y.-s.Y.)

Rationale: Direct conversion or reprogramming of human postnatal cells into endothelial cells (ECs), bypassing stem or progenitor cell status, is crucial for regenerative medicine, cell therapy, and pathophysiological investigation but has remained largely unexplored.

Objective: We sought to directly reprogram human postnatal dermal fibroblasts to ECs with vasculogenic and endothelial transcription factors and determine their vascularizing and therapeutic potential.

Methods And Results: We utilized various combinations of 7 EC transcription factors to transduce human postnatal dermal fibroblasts and found that ER71/ETV2 (ETS variant 2) alone best induced endothelial features. KDR (kinase insert domain receptor) cells sorted at day 7 from ER71/ETV2-transduced human postnatal dermal fibroblasts showed less mature but enriched endothelial characteristics and thus were referred to as early reprogrammed ECs (rECs), and did not undergo maturation by further culture. After a period of several weeks' transgene-free culture followed by transient reinduction of ER71/ETV2, early rECs matured during 3 months of culture and showed reduced ETV2 expression, reaching a mature phenotype similar to postnatal human ECs. These were termed late rECs. While early rECs exhibited an immature phenotype, their implantation into ischemic hindlimbs induced enhanced recovery from ischemia. These 2 rECs showed clear capacity for contributing to new vessel formation through direct vascular incorporation in vivo. Paracrine or proangiogenic effects of implanted early rECs played a significant role in repairing hindlimb ischemia.

Conclusions: This study for the first time demonstrates that ER71/ETV2 alone can directly reprogram human postnatal cells to functional, mature ECs after an intervening transgene-free period. These rECs could be valuable for cell therapy, personalized disease investigation, and exploration of the reprogramming process.
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http://dx.doi.org/10.1161/CIRCRESAHA.116.309833DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5336520PMC
March 2017

Dnmt1 regulates the myogenic lineage specification of muscle stem cells.

Sci Rep 2016 10 18;6:35355. Epub 2016 Oct 18.

Department of Genetics, Yale Stem Cell Center, Yale School of Medicine, 10 Amistad, 201B, New Haven, CT, 06520.

DNA methylation is an important epigenetic mark that regulates gene expression. Dnmt1 plays an important role in maintaining DNA methylation patterns on daughter DNA strands. Studies have shed light into the functional role of Dnmt1 regulation in the hematopoietic and epidermal systems. Here we show that Dnmt1 is required for myogenesis. Loss of Dnmt1 results in reduced expression of myogenic genes and defects in myogenic differentiation. We have utilized a conditional knockout mouse approach to examine the functional consequences of Dnmt1 depletion specifically in the developing muscle. These mice were born runted, with smaller body weights, and reduced ability to form myotubes in vitro. We show that expression of Id-1, a negative regulator of myogenesis, is enhanced in Dnmt1-deficient cultures, leading to enhanced transdifferentiation of myoblasts toward the osteogenic lineage. Thus, these studies demonstrate that Dnmt1 influences cellular identity and determines lineage fidelity.
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http://dx.doi.org/10.1038/srep35355DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5082760PMC
October 2016

Modeling and correction of structural variations in patient-derived iPSCs using CRISPR/Cas9.

Nat Protoc 2016 Nov 6;11(11):2154-2169. Epub 2016 Oct 6.

Department of Physiology, Yonsei University College of Medicine, Seoul, Korea.

Genome engineering technology using engineered nucleases has been rapidly developing, enabling the efficient correction of simple mutations. However, the precise correction of structural variations (SVs) such as large inversions remains limited. Here we describe a detailed procedure for the modeling or correction of large chromosomal rearrangements and short nucleotide repeat expansions using engineered nucleases in human induced pluripotent stem cells (hiPSCs) from a healthy donor and patients with SVs. This protocol includes the delivery of engineered nucleases with no donor template to hiPSCs, and genotyping and derivation/characterization of gene-manipulated hiPSC clones. With engineered nucleases, genomic inversions, reversions, and deletions of short nucleotide expansions can be identified in 2 weeks, and desired clones can be generated in as little as 3-4 weeks. This protocol enables the correction of large inverted segments and short nucleotide repeat expansions in diseases such as hemophilia A, fragile X syndrome, Hunter syndrome, and Friedreich's ataxia.
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http://dx.doi.org/10.1038/nprot.2016.129DOI Listing
November 2016

Regulation of the DNA Methylation Landscape in Human Somatic Cell Reprogramming by the miR-29 Family.

Stem Cell Reports 2016 07 30;7(1):43-54. Epub 2016 Jun 30.

Department of Genetics, Yale Stem Cell Center, Yale School of Medicine, 10 Amistad, 201B, New Haven, CT 06520, USA. Electronic address:

Reprogramming to pluripotency after overexpression of OCT4, SOX2, KLF4, and MYC is accompanied by global genomic and epigenomic changes. Histone modification and DNA methylation states in induced pluripotent stem cells (iPSCs) have been shown to be highly similar to embryonic stem cells (ESCs). However, epigenetic differences still exist between iPSCs and ESCs. In particular, aberrant DNA methylation states found in iPSCs are a major concern when using iPSCs in a clinical setting. Thus, it is critical to find factors that regulate DNA methylation states in reprogramming. Here, we found that the miR-29 family is an important epigenetic regulator during human somatic cell reprogramming. Our global DNA methylation and hydroxymethylation analysis shows that DNA demethylation is a major event mediated by miR-29a depletion during early reprogramming, and that iPSCs derived from miR-29a depletion are epigenetically closer to ESCs. Our findings uncover an important miRNA-based approach to generate clinically robust iPSCs.
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http://dx.doi.org/10.1016/j.stemcr.2016.05.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4945581PMC
July 2016

Neural Stem Cells Restore Hair Growth Through Activation of the Hair Follicle Niche.

Cell Transplant 2016 22;25(8):1439-51. Epub 2016 Apr 22.

School of Biosystem and Biomedical Science, College of Health Science, Korea University, Anam-dong, Seongbuk-gu, Seoul, Republic of Korea.

Several types of hair loss result from the inability of hair follicles to initiate the anagen phase of the hair regeneration cycle. Modulating signaling pathways in the hair follicle niche can stimulate entry into the anagen phase. Despite much effort, stem cell-based or pharmacological therapies to activate the hair follicle niche have not been successful. Here, we set out to test the effect of neural stem cell (NSC) extract on the hair follicle niche for hair regrowth. NSC extracts were applied to the immortalized cell lines HaCaT keratinocytes and dermal papilla cells (DPCs) and the shaven dorsal skin of mice. Treatment with NSC extract dramatically improved the growth of HaCaT keratinocytes and DPCs. In addition, NSC extract enhanced the hair growth of the shaven dorsal skin of mice. In order to determine the molecular signaling pathways regulated by NSCs, we evaluated the expression levels of multiple growth and signaling factors, such as insulin-like growth factor-1 (IGF-1), hepatocyte growth factor (HGF), keratinocyte growth factor (KGF), vascular endothelial growth factor (VEGF), transforming growth factor-β (TGF-β), and bone morphogenetic protein (BMP) family members. We found that treatment with an NSC extract enhanced hair growth by activating hair follicle niches via coregulation of TGF-β and BMP signaling pathways in the telogen phase. We also observed activation and differentiation of intrafollicular hair follicle stem cells, matrix cells, and extrafollicular DPCs in vivo and in vitro. We tested whether activation of growth factor pathways is a major effect of NSC treatment on hair growth by applying the growth factors to mouse skin. Combined growth factors, including TGF-β, significantly increased the hair shaft length and growth rate. DNA damage and cell death were not observed in skin cells of mice treated with the NSC extract for a prolonged period. Overall, our data demonstrate that NSC extract provides an effective approach for promoting hair growth by directly regulating hair follicle niches through TGF-β and BMP signaling pathways as well as induction of core growth factors.
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http://dx.doi.org/10.3727/096368916X691466DOI Listing
October 2017

Histone Deacetylases Positively Regulate Transcription through the Elongation Machinery.

Cell Rep 2015 Nov 5;13(7):1444-1455. Epub 2015 Nov 5.

Department of Biological Sciences and Center for Systems Biology, The University of Texas at Dallas, Richardson, TX 75080, USA. Electronic address:

Transcription elongation regulates the expression of many genes, including oncogenes. Histone deacetylase (HDAC) inhibitors (HDACIs) block elongation, suggesting that HDACs are involved in gene activation. To understand this, we analyzed nascent transcription and elongation factor binding genome-wide after perturbation of elongation with small molecule inhibitors. We found that HDACI-mediated repression requires heat shock protein 90 (HSP90) activity. HDACIs promote the association of RNA polymerase II (RNAP2) and negative elongation factor (NELF), a complex stabilized by HSP90, at the same genomic sites. Additionally, HDACIs redistribute bromodomain-containing protein 4 (BRD4), a key elongation factor involved in enhancer activity. BRD4 binds to newly acetylated sites, and its occupancy at promoters and enhancers is reduced. Furthermore, HDACIs reduce enhancer activity, as measured by enhancer RNA production. Therefore, HDACs are required for limiting acetylation in gene bodies and intergenic regions. This facilitates the binding of elongation factors to properly acetylated promoters and enhancers for efficient elongation.
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http://dx.doi.org/10.1016/j.celrep.2015.10.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4934896PMC
November 2015

Tgif1 Counterbalances the Activity of Core Pluripotency Factors in Mouse Embryonic Stem Cells.

Cell Rep 2015 Oct 24;13(1):52-60. Epub 2015 Sep 24.

Department of Molecular Biosciences, the University of Texas at Austin, Austin, TX 78712, USA; Institute for Cellular and Molecular Biology, Center for Systems and Synthetic Biology, the University of Texas at Austin, Austin, TX 78712, USA. Electronic address:

Core pluripotency factors, such as Oct4, Sox2, and Nanog, play important roles in maintaining embryonic stem cell (ESC) identity by autoregulatory feedforward loops. Nevertheless, the mechanism that provides precise control of the levels of the ESC core factors without indefinite amplification has remained elusive. Here, we report the direct repression of core pluripotency factors by Tgif1, a previously known terminal repressor of TGFβ/activin/nodal signaling. Overexpression of Tgif1 reduces the levels of ESC core factors, whereas its depletion leads to the induction of the pluripotency factors. We confirm the existence of physical associations between Tgif1 and Oct4, Nanog, and HDAC1/2 and further show the level of Tgif1 is not significantly altered by treatment with an activator/inhibitor of the TGFβ/activin/nodal signaling. Collectively, our findings establish Tgif1 as an integral member of the core regulatory circuitry of mouse ESCs that counterbalances the levels of the core pluripotency factors in a TGFβ/activin/nodal-independent manner.
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http://dx.doi.org/10.1016/j.celrep.2015.08.067DOI Listing
October 2015