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Bone Marrow Mononuclear Cells Have Neurovascular Tropism and Improve Diabetic Neuropathy
Kim, Hyongbum,Park, Jong-seon,Choi, Yong Jin,Kim, Mee-Ohk,Huh, Yang Hoon,Kim, Sung-Whan,Han, Ji Woong,Lee, JiYoon,Kim, Sinae,Houge, Mackenzie A.,Ii, Masaaki,Yoon, Young-sup Wiley (John WileySons) 2009 Stem Cells Vol.27 No.7
<P>Bone marrow-derived mononuclear cells (BMNCs) have been shown to effectively treat ischemic cardiovascular diseases. Because diabetic neuropathy (DN) is causally associated with impaired angiogenesis and deficiency of angiogenic and neurotrophic factors in the nerves, we investigated whether DN can be ameliorated by local injection of BMNCs. Severe peripheral neuropathy, characterized by a significant decrease in the motor and sensory nerve conduction velocities (NCVs), developed 12 weeks after the induction of diabetes with streptozotocin in rats. The injection of BMNCs restored motor and sensory NCVs to normal levels and significantly improved vascular density and blood flow in diabetic nerves over 4 weeks. Fluorescent microscopic observation revealed that DiI-labeled BMNCs preferentially engrafted in sciatic nerves. Whole-mount fluorescent imaging and confocal microscopic evaluation demonstrated that many of the BMNCs localized following the course of the vasa nervorum in close proximity to blood vessels without incorporation into vasa nervorum as endothelial cells at a detectable level. Real-time reverse transcription-polymerase chain reaction analysis showed that the levels of angiogenic and neurotrophic factors were significantly increased in the nerves by BMNC injection. Local transplantation of BMNCs improved experimental DN by augmenting angiogenesis and increasing angiogenic and neurotrophic factors in peripheral nerves. These findings suggest that BMNC transplantation may represent a novel therapeutic option for treating DN.</P>
Seo, Jung Hwa,Kim, Hyongbum,Park, Eun Sook,Lee, Jong Eun,Kim, Dong Wook,Kim, Hyun Ok,Im, Sang Hee,Yu, Ji Hea,Kim, Ji Yeon,Lee, Min-Young,Kim, Chul Hoon,Cho, Sung-Rae Pergamon Press ; Elsevier Science Ltd ; Elsevier S 2013 Cell transplantation Vol.22 No.9
<P>We investigated the effects of environmental enrichment (EE) on the function of transplanted adipose stem cells (ASCs) and the combined effect of EE and ASC transplantation on neurobehavioral function in an animal model of chronic hypoxic-ischemic (HI) brain injury. HI brain damage was induced in 7-day-old mice by unilateral carotid artery ligation and exposure to hypoxia (8% O2 for 90 min). At 6 weeks of age, the mice were randomly injected with either ASCs or PBS into the striatum and were randomly assigned to either EE or standard cages (SC), comprising ASC-EE (n=18), ASC-SC (n=19), PBS-EE (n=12), PBS-SC (n=17), and untreated controls (n=23). Rotarod, forelimb-use asymmetry, and grip strength tests were performed to evaluate neurobehavioral function. The fate of transplanted cells and the levels of endogenous neurogenesis, astrocyte activation, and paracrine factors were also measured. As a result, EE and ASC transplantation synergistically improved rotarod latency, forelimb-use asymmetry, and grip strength compared to those of the other groups. The number of engrafted ASCs and βIII-tubulin(+) neurons derived from the transplanted ASCs was significantly higher in mice in EE than those in SC. EE and ASC transplantation also synergistically increased BrdU(+)βIII-tubulin(+) neurons, GFAP(+) astrocytic density, and fibroblast growth factor 2 (FGF2) level but not the level of CS-56(+) glial scarring in the striatum. In conclusion, EE and ASC transplantation synergistically improved neurobehavioral functions. The underlying mechanisms of this synergism included enhanced repair processes such as higher engraftment of the transplanted ASCs, increased endogenous neurogenesis and astrocytic activation coupled with upregulation of FGF2.</P>
Genome Editing using CRISPR-Cas9 and -Cpf1
Hyongbum (Henry) Kim 한국동물생명공학회(구 한국동물번식학회) 2017 Reproductive & Developmental Biology(Supplement) Vol.41 No.2
Programmable nucleases, which include zinc-finger nucleases, transcriptional activatorlike effector nucleases, and RNA-guided engineered nucleases derived from the prokaryotic CRISPR/Cas system, enable targeted genetic modifications in cultured cells, animals, and plants, tools of great value in research, medicine, and biotechnology. Cpf1 is a recently reported effector endonuclease protein of the class 2 CRISPR-Cas system. Cpf1 has several differences from Cas9: cleavage with 5’ overhangs, shorter guide RNA, and a longer distance between the seed sequence and cleavage site, which could provide potential advantages for some cases of genome editing such as nonhomologous end joining-based gene insertion and efficient genome editing using homology- directed repair. However, limited information is available about Cpf1 activity profiles in mammalian cells, precluding its wide use for genome editing. Here, we performed en masse evaluation of guide RNA and Cpf1 activity using synthetic target sequences and deep sequencing. Using this approach, we determined target sequence- dependent activity profiles and protospacer adjacent motif sequences of Cpf1. We found that sequence features of high activity AsCpf1 guide RNAs are distinct from those of SpCas9. Evaluation of activity at mismatched target sequences showed that Cpf1 target sequences can be divided into seed, trunk, and promiscuous regions depending on mismatch tolerability. The Cpf1 characterization profile will greatly facilitate Cpf1-based genome editing.
A guide to genome engineering with programmable nucleases
Kim, Hyongbum,Kim, Jin-Soo Nature Publishing Group, a division of Macmillan P 2014 Nature reviews. Genetics Vol.15 No.5
Programmable nucleases — including zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and RNA-guided engineered nucleases (RGENs) derived from the bacterial clustered regularly interspaced short palindromic repeat (CRISPR)–Cas (CRISPR-associated) system — enable targeted genetic modifications in cultured cells, as well as in whole animals and plants. The value of these enzymes in research, medicine and biotechnology arises from their ability to induce site-specific DNA cleavage in the genome, the repair (through endogenous mechanisms) of which allows high-precision genome editing. However, these nucleases differ in several respects, including their composition, targetable sites, specificities and mutation signatures, among other characteristics. Knowledge of nuclease-specific features, as well as of their pros and cons, is essential for researchers to choose the most appropriate tool for a range of applications.
In vivo high-throughput profiling of CRISPR–Cpf1 activity
Kim, Hui K,Song, Myungjae,Lee, Jinu,Menon, A Vipin,Jung, Soobin,Kang, Young-Mook,Choi, Jae W,Woo, Euijeon,Koh, Hyun C,Nam, Jin-Wu,Kim, Hyongbum Nature Publishing Group, a division of Macmillan P 2017 Nature methods Vol.14 No.2
<P>CRISPR from Prevotella and Francisella 1 (Cpf1) is an effector endonuclease of the class 2 CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins) gene editing system. We developed a method for evaluating Cpf1 activity, based on target sequence composition in mammalian cells, in a high-throughput manner. A library of >11,000 target sequence and guide RNA pairs was delivered into human cells using lentiviral vectors. Subsequent delivery of Cpf1 into this cell library induced insertions and deletions (indels) at the integrated synthetic target sequences, which allowed en masse evaluation of Cpfl activity by using deep sequencing. With this approach, we determined protospacer-adjacent motif sequences of two Cpf1 nucleases, one from Acidaminococcus sp. BV3L6 (hereafter referred to as AsCpf1) and the other from Lachnospiraceae bacterium ND2006 (hereafter referred to as LbCpfl). We also defined target-sequence-dependent activity profiles of AsCpf1, which enabled the development of a web tool that predicts the indel frequencies for given target sequences (http://big.hanyang.ac.kr/cindel). Both the Cpf1 characterization profile and the in vivo high-throughput evaluation method will greatly facilitate Cpf1-based genome editing.</P>
Kim, Myung-Sun,Yu, Ji Hea,Kim, Chul Hoon,Choi, Jae Yong,Seo, Jung Hwa,Lee, Min-Young,Yi, Chi Hoon,Choi, Tae Hyun,Ryu, Young Hoon,Lee, Jong Eun,Lee, Bae Hwan,Kim, Hyongbum,Cho, Sung-Rae SAGE Publications 2016 Journal of cerebral blood flow and metabolism Vol.36 No.12
<P> Environmental enrichment (EE) with a complex combination of physical, cognitive and social stimulations enhances synaptic plasticity and behavioral function. However, the mechanism remains to be elucidated in detail. We aimed to investigate dopamine-related synaptic plasticity underlying functional improvement after EE. For this, six-week-old CD-1 mice were randomly allocated to EE or standard conditions for two months. EE significantly enhanced behavioral functions such as rotarod and ladder walking tests. In a [<SUP>18</SUP>F]FPCIT positron emission tomography scan, binding values of striatal DAT were significantly decreased approximately 18% in the EE mice relative to the control mice. DAT inhibitor administrated to establish the relationship of the DAT down-regulation to the treatment effects also improved rotarod performances, suggesting that DAT inhibition recapitulated EE-mediated treatment benefits. Next, EE-induced internalization of DAT was confirmed using a surface biotinylation assay. In situ proximity ligation assay and immunoprecipitation demonstrated that EE significantly increased the phosphorylation of striatal DAT as well as the levels of DAT bound with protein kinase C (PKC). In conclusion, we suggest that EE enables phosphorylation of striatal DAT via a PKC-mediated pathway and causes DAT internalization. This is the first report to suggest an EE-mediated mechanism of synaptic plasticity by internalization of striatal DAT. </P>