Name: protocol for microrna transfer into adult bone marrow-derived hematopoietic stem cells to enable cell engineering combined with magnetic targeting
Uploaded: 2018-06-18T13:00:00.000+00:00
Duration: 11 min 37 s
Description: Watch this Scientific Journal Video about Protocol for MicroRNA Transfer into Adult Bone Marrow-derived Hematopoietic Stem Cells to Enable Cell Engineering Combined with Magnetic Targeting at JoVE.com

この仕事は、連邦教育省とドイツ研究 (FKZ 0312138A、FKZ 316159)、EU 構造基金 (ESF/IVWM-B34-0030/10 と ESF/IVBM-B35-0010/12) と状態 Mecklenburg 西部の Pomerania、DFG (DA1296/2-1) に支えられ、ドイツ心臓財団 (F/01/12)、(VIP + 00240) BMBF と湿気がある財団。さらに、f. H. と午後は、FORUN プログラムのロストック大学医療センター (889001) でサポートされます。

著者は利益相反を宣言しません。

近年では、CD133+ SCs 有望な細胞集団として登場した SC 基づかせていた療法のいくつかによって証明されるようフェーズ I、II、および III の臨床43,44,45,46,47,48,49,50,5...

CD133+ SCs は再生医療の可能性を有望な異種幹・前駆細胞の人口を表します。CD133 を有効に、造血、血管内皮、筋分化ポテンシャル1,2,3 +の細胞、例えば、差別化による血管新生のプロセスに貢献するには新しく成形容器とプロ新生パラクライン機構4,5,6,7シグナル伝達の活性化。
以上 30 承認された臨床試験 (ClinicalTrails.gov) に示す彼らの高可能性にもかかわらずその治療成績は物議を醸す議論4中です。確かに、SCs の臨床応用は、関心と大規模な初期細胞死5,8,9器官の低保持によって妨げられています。CD133 のエンジニア リング追加+ SCs 事前移植は、これらの課題を克服を助けることができます。
効率的な細胞療法のための 1 つの前提条件を治療関連細胞10の生着を強化する大規模な初期細胞死の減少となります。現在研究が最初 1-2 h、移植細胞型の独立の間に高い脳や心臓など臓器の 90-99% の巨大なセル損失を実証またはアプリケーション ルート11,12,13 、14,15,16,17,18,19,20,21。関心22,23,24,25,26 のサイトに標的細胞に革新的な非侵襲的戦略により、SC は、磁性ナノ粒子 (MNPs) を使用したラベリングと同時に携帯の MRI27と磁性粒子 (MPI) をイメージングを使用して監視できます。生体内で最も効率的な静脈注射23,24,28 後セル指導に優先してローカル投与後に使用されるセルの保持を対象と適用の磁化細胞を研究します。.したがって、私たちのグループには、超常磁性酸化鉄ナノ粒子の29から成る配信システムが設計されています。この手法では、CD133 で+ SCs とひと臍帯静脈血管内皮細胞 (Huvec) をターゲット効率的に、体外30,31の試みによって示されるように。
SC 療法のもう一つのハードルは、影響を受ける組織の敵対的な炎症性環境を初期細胞死32に貢献、移植後です。いくつかの前処理研究に加えて治療関連 miRs のアプリケーションはテスト33;抗アポトーシス miRs体外アポトーシスを阻害させ、体内細胞の生着33正常に実証されています。20-25 のヌクレオチドの構成これらの小さい分子はメッセンジャー Rna (Mrna) の転写調節因子として重要な役割を果たすし、幹細胞の運命や行動34影響を及ぼします。また、miRs の外因性の導入は望ましくない安定したホストのゲノム34統合を避けてください。
プライマリ SCs に核酸 (NAs) の効率的な導入のため現在試行は組換えウイルス8,35に大抵基づいています。にもかかわらず高いトランスフェクション効率組換えウイルス操作提示ベンチ-ベッドサイド翻訳、例えば、手に負えない遺伝子発現、病原性、免疫原性、および挿入変異35 の主要な障害 ,36。したがって、ポリマー ベースの構成などの非ウイルスの伝達システムの開発にとって重要です。中でもポリエチレンイミン (PEI) は、miRs 細胞取り込みの劣化から保護するために NA 結露などのメリットを提供する有効な配信手段を表し、エンドソームを介して細胞内放出脱出37,38。さらに、ミール PEI 錯体は、臨床試験第39高い生体親和性を示した。したがって、当社の配信システムから成って、ビオチン標識ストレプトアビジン コーティング MNP コア30,31,40行きの分岐 25 kDa プリンスエド ワード島。
本稿では, (i) CD133 の手動分離を記述する包括的なプロトコルを提案する+ SC SC 製品および (ii) の効率的かつ穏やかなトランスフェクション戦略の詳細な特性解析とひと骨髄 (BM) 寄付金から、CD133 の遺伝子工学のための磁気的非ウイルス性ポリマー ベース配信システム+ SCs miRs を使用します。CD133+ SCs 隔離され、人間胸骨 BM 吸引、表面抗体を用いた磁気活性化細胞選別 (MAC) システムを使用して磁気濃縮しました。その後、細胞と細胞の純度によっては、フローサイトメトリー分析されます。ミール/プリンスエド ワード島/MNP 錯体の調製後、CD133+ SCs が transfected。トランスフェクション後 18 h、吸収効率とトランスフェクション SC マーカーの発現と細胞の生存率に与える影響を分析します。また、トランスフェクションの複雑な化合物の細胞内の分布の評価は、4 色ラベルと構造化照明顕微鏡を用いて (SIM) を使用して実行されます。

<table><tbody><tr><td>7-AAD</td><td>BD Biosciences</td><td>559925</td><td></td></tr><tr><td>Acetic Acid with Methylene Blue</td><td>Stemcell Technologies</td><td>7060</td><td>3%</td></tr><tr><td>anti-CD133/2-PE (clone: 293C3)</td><td>Miltenyi Biotec GmbH</td><td>130-090-853</td><td></td></tr><tr><td>anti-CD34-FITC (clone: AC136)</td><td>Miltenyi Biotec GmbH</td><td>130-081-001</td><td></td></tr><tr><td>anti-CD45-APC-H7 (clone: 2D1)</td><td>BD Biosciences</td><td>560178</td><td></td></tr><tr><td>rhodamine dye; Atto 565 dye conjugated to biotin</td><td>ATTO-TEC GmbH</td><td>AD 565-71</td><td></td></tr><tr><td>BD FACS LSRII flow cytometer</td><td>BD Biosciences</td><td></td><td></td></tr><tr><td>BD FACSDiva Software 6.1.2</td><td>BD Biosciences</td><td></td><td></td></tr><tr><td>BSA</td><td>Sigma-Aldrich GmbH</td><td>A7906</td><td></td></tr><tr><td>CD133 antibody-linked superparamagnetic iron dextran particles; CD133 MicroBead Kit</td><td>Miltenyi Biotec GmbH</td><td>130-097-049</td><td></td></tr><tr><td>collagenase B</td><td>Roche Diagnostics GmbH</td><td>11088831001</td><td></td></tr><tr><td>counting chamber</td><td>Paul Marienfeld GmbH &amp;amp; Co. KG</td><td></td><td></td></tr><tr><td>Cyanine 3 dye labelled precursor miR; Cy3 Dye-Labeled Pre-miR Negative Control #1</td><td>Ambion</td><td>AM17120</td><td></td></tr><tr><td>Cyanine 5 dye miR labelling kit; Cy5 dye Label IT miRNA Labeling Kit</td><td>Mirus Bio</td><td>MIR 9650</td><td></td></tr><tr><td>DNAse I</td><td>Roche Diagnostics GmbH</td><td>10104159001</td><td>(100 U/mL)</td></tr><tr><td>ELYRA PS.1 LSM 780 confocal microscope</td><td>Carl Zeiss Jena GmbH</td><td></td><td></td></tr><tr><td>FcR Blocking Reagent, human</td><td>Miltenyi Biotec GmbH</td><td>130-059-901</td><td></td></tr><tr><td>bright green protein labeling kit; Oregon Green 488 Protein Labeling Kit</td><td>Thermo Fisher Scientific</td><td>O10241</td><td></td></tr><tr><td>aqueous mounting medium; Fluoroshield</td><td>Sigma-Aldrich GmbH</td><td>F6182</td><td></td></tr><tr><td>density gradient centrifugation tube; Leukosep Centrifuge Tube</td><td>Greiner Bio-One</td><td>89048-932</td><td></td></tr><tr><td>MACS magnet holder; MACS MultiStand</td><td>Miltenyi Biotec GmbH</td><td>130-042-303</td><td></td></tr><tr><td>MACS pre-separation filter</td><td>Miltenyi Biotec GmbH</td><td>130-041-407</td><td>30 &amp;micro;m</td></tr><tr><td>MACS separation column (MS / LS)</td><td>Miltenyi Biotec GmbH</td><td>130-042-201 / 130-042-401</td><td></td></tr><tr><td>MACS permanent magnet; MACS Separator</td><td>Miltenyi Biotec GmbH</td><td>130-042-302</td><td></td></tr><tr><td>Millex-HV PVDF Filter</td><td>Merck</td><td>SLHV013SL</td><td>0.45 &amp;mu;m</td></tr><tr><td>mouse IgG 2b-PE</td><td>Miltenyi Biotec GmbH</td><td>130-092-215</td><td></td></tr><tr><td>amine reactive dye; Near-IR LIVE/DEAD Fixable Dead Cell Stain Kit</td><td>Thermo Fisher Scientific</td><td>L10119</td><td></td></tr><tr><td>human lymphocyte separating medium; Pancoll</td><td>Pan Biotech GmbH</td><td>P04-60500</td><td>density: 1.077 g/mL</td></tr><tr><td>PBS</td><td>Pan Biotech GmbH</td><td>P04-53500</td><td>without Ca and Mg</td></tr><tr><td>PEI</td><td>Sigma-Aldrich GmbH</td><td>408727</td><td>branched; 25 kDa</td></tr><tr><td>Penicillin/Streptomycin</td><td>Thermo Fisher Scientific</td><td>15140122</td><td>100 U/mL, 100 &amp;mu;g/mL</td></tr><tr><td>PFA</td><td>Merck Schuchardt OHG</td><td>1040051000</td><td></td></tr><tr><td>unlabelled precursor miR; Pre-miR miRNA Precursor Negative Control #1</td><td>Ambion</td><td>AM17110</td><td></td></tr><tr><td>RBC lysis buffer</td><td>eBioscience</td><td>00-4333-57</td><td></td></tr><tr><td>RNAse decontamination solution; RNaseZap</td><td>Thermo Fisher Scientific</td><td>AM9780</td><td></td></tr><tr><td>human lymphocyte medium; Roswell Park Memorial Institute (RPMI) 1640 medium</td><td>Pan Biotech GmbH</td><td>P04-16500</td><td></td></tr><tr><td>recombinant human cytokine supplement; StemSpan CC100</td><td>Stemcell Technologies</td><td>2690</td><td></td></tr><tr><td>serum-free haematopoietic cell expansion medium; StemSpan H3000</td><td>Stemcell Technologies</td><td>9800</td><td></td></tr><tr><td>Streptavidin MagneSphere Paramagnetic Particles</td><td>Promega Corporation</td><td>Z5481</td><td></td></tr><tr><td>Trypan Blue solution</td><td>Sigma-Aldrich GmbH</td><td>T8154</td><td>0.4 %</td></tr><tr><td>UltraPure EDTA</td><td>Thermo Fisher Scientific</td><td>15575020</td><td>0.5 M; pH 8.0</td></tr><tr><td>ZEN2011 software</td><td>Carl Zeiss Jena GmbH</td><td></td><td></td></tr><tr><td>NanoDrop 1000 Spectrophotometer</td><td>Thermo Fisher Scientific</td><td></td><td></td></tr><tr><td>Sonorex RK 100 SH sonicating water bath</td><td>Bandelin electronic</td><td></td><td>Ultrasonic nominal output: 80 W; Ultrasonic frequency: 35 kHz</td></tr></tbody></table>

protocol for microrna transfer into adult bone marrow-derived hematopoietic stem cells to enable cell engineering combined with magnetic targeting

一方、CD133+造血幹細胞 (SCs) は、再生医療の分野で高い可能性を提供するために証明されている、負傷者の組織として観察された大規模な細胞の死亡率への注入後、低定着率が非常につながる制限された治療効果。これらの制限を克服するために我々 は、投与前に適切な細胞工学のための非ウイルス性ベースのプロトコルを確立しようと思う。人間の CD133 の変更+吸収効率と安全性、細胞のターゲットの可能性に関して対処されたマイクロ Rna (ミール) 読み込まれた磁気 polyplexes を利用した SCs を表現します。我々 のプロトコルに依存している、CD133 80-90% の高いミール取り込み率を達成できる+幹細胞特性に影響はありません。さらに、これらの変更されたセルは、磁気ターゲットのオプションを提供しています。我々 は CD133 の修正のため安全かつ非常に効率的な手順をここに記述する+ SCs。幹細胞の治療効果の最適化および磁気共鳴画像 (MRI) を介して投与細胞製品のモニターの標準技術を提供するためにこのアプローチを見込んでいます。

提案するプロトコルは、手動分離とひと骨髄由来 CD133 の磁気濃縮について説明します+ SCs エンジニア リング戦略、体外細胞操作とのための非侵襲的技術としてその後ウイルス独立セルvivo監視ツール。
この 3 つのステップの分離技術は、密度勾配遠心法による事前消化胸骨 BM から多国籍企業の分離?...

Watch this Scientific Journal Video about Protocol for MicroRNA Transfer into Adult Bone Marrow-derived Hematopoietic Stem Cells to Enable Cell Engineering Combined with Magnetic Targeting at JoVE.com

Protocol for MicroRNA Transfer into Adult Bone Marrow-derived Hematopoietic Stem Cells to Enable Cell Engineering Combined with Magnetic Targeting

このプロトコルは、CD133 を変更する安全で効率的な手順を示しています+造血幹細胞。提示された非ウイルス性、磁気 polyplex ベースのアプローチ治療幹細胞効果だけでなく磁気共鳴イメージ投射を介して投与細胞製品を監視の最適化のための基礎があります。

成人骨髄由来造血幹細胞が細胞工学磁気ターゲッティングと結合を有効にするにマイクロ Rna の転送のためのプロトコル

While CD133+ hematopoietic stem cells (SCs) have been proven to provide high potential in the field of regenerative medicine, their low retention rates ...

protocol-for-microrna-transfer-into-adult-bone-marrow-derived

Research

JoVE Journal

Bioengineering

6.6K ビュー.  Rostock University Medical Center. このプロトコルは、CD133 を変更する安全で効率的な手順を示しています+造血幹細胞。提示された非ウイルス性、磁気 polyplex ベースのアプローチ治療幹細胞効果だけでなく磁気共鳴イメージ投射を介して投与細胞製品を監視の最適化のための基礎があります。

ビデオ: Protocol for MicroRNA Transfer into Adult Bone Marrow-derived Hematopoietic Stem Cells to Enable Cell Engineering Combined with Magnetic Targeting

Isolation, Characterization and MicroRNA-based Genetic Modification of Human Dental Follicle Stem Cells

To date, several stem cell types at different developmental stages are in the focus for the treatment of degenerative diseases. Yet, certain aspects, such as initial massive cell death and low therapeutic effects, impaired their broad clinical translation. Genetic engineering of stem cells prior to transplantation emerged as a promising method to optimize therapeutic stem cell effects. However, safe and efficient gene delivery systems are still lacking. Therefore, the development of suitable methods may provide an approach to resolve current challenges in stem cell-based therapies.
The present protocol describes the extraction and characterization of human dental follicle stem cells (hDFSCs) as well as their non-viral genetic modification. The postnatal dental follicle unveiled as a promising and easily accessible source for harvesting adult multipotent stem cells possessing high proliferation potential. The described isolation procedure presents a simple and reliable method to harvest hDFSCs from impacted wisdom teeth. Also this protocol comprises methods to define stem cell characteristics of isolated cells. For genetic engineering of hDFSCs, an optimized cationic lipid-based transfection strategy is presented enabling highly efficient microRNA introduction without causing cytotoxic effects. MicroRNAs are suitable candidates for transient cell manipulation, as these small translational regulators control the fate and behavior of stem cells without the hazard of stable genome integration. Thus, this protocol represents a safe and efficient procedure for engineering of hDFSCs that may become important for optimizing their therapeutic efficacy.

This protocol describes the transient genetic engineering of dental stem cells extracted from the human dental follicle. The applied non-viral modification strategy may become a basis for the improvement of therapeutic stem cell products.

分離の特性と人間の歯小嚢細胞のマイクロ Rna を用いた遺伝子組み換え

To date, several stem cell types at different developmental stages are in the focus for the treatment of degenerative diseases. Yet, certain aspects, such ...

遺伝学

Cell Labeling and Targeting with Superparamagnetic Iron Oxide Nanoparticles

Targeted delivery of cells and therapeutic agents would benefit a wide range of biomedical applications by concentrating the therapeutic effect at the target site while minimizing deleterious effects to off-target sites. Magnetic cell targeting is an efficient, safe, and straightforward delivery technique. Superparamagnetic iron oxide nanoparticles (SPION) are biodegradable, biocompatible, and can be endocytosed into cells to render them responsive to magnetic fields. The synthesis process involves creating magnetite (Fe3O4) nanoparticles followed by high-speed emulsification to form a poly(lactic-co-glycolic acid) (PLGA) coating. The PLGA-magnetite SPIONs are approximately 120 nm in diameter including the approximately 10 nm diameter magnetite core. When placed in culture medium, SPIONs are naturally endocytosed by cells and stored as small clusters within cytoplasmic endosomes. These particles impart sufficient magnetic mass to the cells to allow for targeting within magnetic fields. Numerous cell sorting and targeting applications are enabled by rendering various cell types responsive to magnetic fields. SPIONs have a variety of other biomedical applications as well including use as a medical imaging contrast agent, targeted drug or gene delivery, diagnostic assays, and generation of local hyperthermia for tumor therapy or tissue soldering.

Targeted cell delivery is useful in a variety of biomedical applications. The goal of this protocol is to use superparamagnetic iron oxide nanoparticles (SPION) to label cells and thereby enable magnetic cell targeting approaches for a high degree of control over cell delivery and localization.

細胞標識および超常磁性酸化鉄ナノ粒子とターゲティング

Targeted delivery of cells and therapeutic agents would benefit a wide range of biomedical applications by concentrating the therapeutic effect at the ...

生物工学

3D Magnetic Stem Cell Aggregation and Bioreactor Maturation for Cartilage Regeneration

Cartilage engineering remains a challenge due to the difficulties in creating an in vitro functional implant similar to the native tissue. An approach recently explored for the development of autologous replacements involves the differentiation of stem cells into chondrocytes. To initiate this chondrogenesis, a degree of compaction of the stem cells is required; hence, we demonstrated the feasibility of magnetically condensing cells, both within thick scaffolds and scaffold-free, using miniaturized magnetic field sources as cell attractors. This magnetic approach was also used to guide aggregate fusion and to build scaffold-free, organized, three-dimensional (3D) tissues several millimeters in size. In addition to having an enhanced size, the tissue formed by magnetic-driven fusion presented a significant increase in the expression of collagen II, and a similar trend was observed for aggrecan expression. As the native cartilage was subjected to forces that influenced its 3D structure, dynamic maturation was also performed. A bioreactor that provides mechanical stimuli was used to culture the magnetically seeded scaffolds over a 21-day period. Bioreactor maturation largely improved chondrogenesis into the cellularized scaffolds; the extracellular matrix obtained under these conditions was rich in collagen II and aggrecan. This work outlines the innovative potential of magnetic condensation of labeled stem cells and dynamic maturation in a bioreactor for improved chondrogenic differentiation, both scaffold-free and within polysaccharide scaffolds.

Chondrogenesis from stem cells requires fine tuning the culture conditions. Here, we present a magnetic approach for condensing cells, an essential step to initiate chondrogenesis. In addition, we show that dynamic maturation in a bioreactor applies mechanical stimulation to the cellular constructs and enhances cartilaginous extracellular matrix production.

軟骨再生のための3次元磁気幹細胞凝集およびバイオリアクター成熟

Cartilage engineering remains a challenge due to the difficulties in creating an in vitro functional implant similar to the native tissue. An approach ...

Identification and Isolation of Oligopotent and Lineage-committed Myeloid Progenitors from Mouse Bone Marrow

Myeloid progenitors that yield neutrophils, monocytes and dendritic cells (DCs) can be identified in and isolated from the bone marrow of mice for hematological and immunological analyses. For example, studies of the cellular and molecular properties of myeloid progenitor populations can reveal mechanisms underlying leukemic transformation, or demonstrate how the immune system responds to pathogen exposure. Previously described flow cytometry strategies for myeloid progenitor identification have enabled significant advances in many fields, but the fractions they identify are very heterogeneous. The most commonly used gating strategies define bone marrow fractions that are enriched for the desired populations, but also contain large numbers of &#34;contaminating&#34; progenitors. Our recent studies have resolved much of this heterogeneity, and the protocol we present here permits the isolation of 6 subpopulations of oligopotent and lineage-committed myeloid progenitors from 2 previously described bone marrow fractions. The protocol describes 3 stages: 1) isolation of bone marrow cells, 2) enrichment for hematopoietic progenitors by magnetic-activated cell sorting (lineage depletion by MACS), and 3) identification of myeloid progenitor subsets by flow cytometry (including fluorescence-activated cell sorting, FACS, if desired). This approach permits progenitor quantification and isolation for a variety of in vitro and in vivo applications, and has already yielded novel insight into pathways and mechanisms of neutrophil, monocyte, and DC differentiation.

We demonstrate how to identify and isolate 6 subsets of myeloid progenitors from murine bone marrow using a combination of magnetic and fluorescence sorting (MACS and FACS). This protocol can be used for in vitro culture assays (methylcellulose or liquid cultures), in vivo adoptive transfer experiments, and RNA/protein analyses.

寡能性と系列拘束されたマウスの骨髄から骨髄前駆細胞の分離と同定

Myeloid progenitors that yield neutrophils, monocytes and dendritic cells (DCs) can be identified in and isolated from the bone marrow of mice for ...

免疫・感染学

Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs constitute a minute cell population of pluripotent cells capable of generating all blood cell lineages for a life-time1. The molecular dissection of HSCs homeostasis in the bone marrow has important implications in hematopoiesis, oncology and regenerative medicine. We describe the labeling protocol with fluorescent antibodies and the electronic gating procedure in flow cytometry to score hematopoietic progenitor subsets and HSCs distribution in individual mice (Fig. 1). In addition, we describe a method to extensively enrich hematopoietic progenitors as well as long-term (LT) and short term (ST) reconstituting HSCs from pooled bone marrow cell suspensions by magnetic enrichment of cells expressing c-Kit. The resulting cell preparation can be used to sort selected subsets for in vitro and in vivo functional studies (Fig. 2).
Both trabecular osteoblasts2,3 and sinusoidal endothelium4 constitute functional niches supporting HSCs in the bone marrow. Several mechanisms in the osteoblastic niche, including a subset of N-cadherin+ osteoblasts3 and interaction of the receptor tyrosine kinase Tie2 expressed in HSCs with its ligand angiopoietin-15 concur in determining HSCs quiescence. "Hibernation" in the bone marrow is crucial to protect HSCs from replication and eventual exhaustion upon excessive cycling activity6. Exogenous stimuli acting on cells of the innate immune system such as Toll-like receptor ligands7 and interferon-&alpha;6 can also induce proliferation and differentiation of HSCs into lineage committed progenitors. Recently, a population of dormant mouse HSCs within the lin- c-Kit+ Sca-1+ CD150+ CD48- CD34- population has been described8. Sorting of cells based on CD34 expression from the hematopoietic progenitors-enriched cell suspension as described here allows the isolation of both quiescent self-renewing LT-HSCs and ST-HSCs9. A similar procedure based on depletion of lineage positive cells and sorting of LT-HSC with CD48 and Flk2 antibodies has been previously described10. In the present report we provide a protocol for the phenotypic characterization and ex vivo cell cycle analysis of hematopoietic progenitors, which can be useful for monitoring hematopoiesis in different physiological and pathological conditions. Moreover, we describe a FACS sorting procedure for HSCs, which can be used to define factors and mechanisms regulating their self-renewal, expansion and differentiation in cell biology and signal transduction assays as well as for transplantation.

A method to analyse the distribution of bone marrow hematopoietic progenitors in flow cytometry as well as to efficiently isolate highly purified hematopoietic stem cells (HSCs) is described. The isolation procedure is essentially based on magnetic enrichment of c-Kit+ cells and cell sorting to purify HSCs for cellular and molecular studies.

マウス造血幹細胞およびリネージュコミット前駆細胞の表現型の解析と分離

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs ...

生物学

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene and cell therapies are highly promising alternative approaches for CVD treatment. However, the broad clinical application of gene therapy is greatly limited by the lack of suitable gene delivery systems. The development of appropriate gene delivery vectors can provide a solution to current challenges in cell therapy. In particular, existing drawbacks, such as limited efficiency and low cell retention in the injured organ, could be overcome by appropriate cell engineering (i.e., genetic) prior to transplantation. The presented protocol describes the efficient and safe transient modification of endothelial cells using a polyethyleneimine superparamagnetic magnetic nanoparticle (PEI/MNP)-based delivery vector. Also, the algorithm and methods for cell characterization are defined. The successful intracellular delivery of microRNA (miR) into human umbilical vein endothelial cells (HUVECs) has been achieved without affecting cell viability, functionality, or intercellular communication. Moreover, this approach was proven to cause a strong functional effect in introduced exogenous miR. Importantly, the application of this MNP-based vector ensures cell magnetization, with accompanying possibilities of magnetic targeting and non-invasive MRI tracing. This may provide a basis for magnetically guided, genetically engineered cell therapeutics that can be monitored non-invasively with MRI.

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

This manuscript describes the efficient, non-viral delivery of miR to endothelial cells by a PEI/MNP vector and their magnetization. Thus, in addition to genetic modification, this approach allows for magnetic cell guidance and MRI detectability. The technique can be used to improve the characteristics of therapeutic cell products.

準備と<em&gt;インビトロ</em磁化のmiR修飾内皮細胞の&gt;キャラクタリゼーション

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene ...

医学

Lentiviral CRISPR/Cas9-Mediated Genome Editing for the Study of Hematopoietic Cells in Disease Models

Manipulating genes in hematopoietic stem cells using conventional transgenesis approaches can be time-consuming, expensive, and challenging. Benefiting from advances in genome editing technology and lentivirus-mediated transgene delivery systems, an efficient and economical method is described here that establishes mice in which genes are manipulated specifically in hematopoietic stem cells. Lentiviruses are used to transduce Cas9-expressing lineage-negative bone marrow cells with a guide RNA (gRNA) targeting specific genes and a red fluorescence reporter gene (RFP), then these cells are transplanted into lethally-irradiated C57BL/6 mice. Mice transplanted with lentivirus expressing non-targeting gRNA are used as controls. Engraftment of transduced hematopoietic stem cells are evaluated by flow cytometric analysis of RFP-positive leukocytes of peripheral blood. Using this method, ~90% transduction of myeloid cells and ~70% of lymphoid cells at 4 weeks after transplantation can be achieved. Genomic DNA is isolated from RFP-positive blood cells, and portions of the targeted site DNA are amplified by PCR to validate the genome editing. This protocol provides a high-throughput evaluation of hematopoiesis-regulatory genes and can be extended to a variety of mouse disease models with hematopoietic cell involvement.

Described are protocols for the highly efficient genome editing of murine hematopoietic stem and progenitor cells (HSPC) by the CRISPR/Cas9 system to rapidly develop mouse model systems with hematopoietic system-specific gene modifications.

疾患モデルにおける血化細胞の研究のためのレンチウイルスCRISPR/Cas9媒介ゲノム編集

Manipulating genes in hematopoietic stem cells using conventional transgenesis approaches can be time-consuming, expensive, and challenging. Benefiting ...

Bone Marrow Transplantation Procedures in Mice to Study Clonal Hematopoiesis

Clonal hematopoiesis is a prevalent age-associated condition that results from the accumulation of somatic mutations in hematopoietic stem and progenitor cells (HSPCs). Mutations in driver genes, that confer cellular fitness, can lead to the development of expanding HSPC clones that increasingly give rise to progeny leukocytes harboring the somatic mutation. Because clonal hematopoiesis has been associated with heart disease, stroke, and mortality, the development of experimental systems that model these processes is key to understanding the mechanisms that underly this new risk factor. Bone marrow transplantation procedures involving myeloablative conditioning in mice, such as total-body irradiation (TBI), are commonly employed to study the role of immune cells in cardiovascular diseases. However, simultaneous damage to the bone marrow niche and other sites of interest, such as the heart and brain, is unavoidable with these procedures. Thus, our lab has developed two alternative methods to minimize or avoid possible side effects caused by TBI: 1) bone marrow transplantation with irradiation shielding and 2) adoptive BMT to non-conditioned mice. In shielded organs, the local environment is preserved allowing for the analysis of clonal hematopoiesis while the function of resident immune cells is unperturbed. In contrast, the adoptive BMT to non-conditioned mice has the additional advantage that both the local environments of the organs and the hematopoietic niche are preserved. Here, we compare three different hematopoietic cell reconstitution approaches and discuss their strengths and limitations for studies of clonal hematopoiesis in cardiovascular disease.

We describe three methods of bone marrow transplantation (BMT): BMT with total-body irradiation, BMT with shielded irradiation, and BMT method with no pre-conditioning (adoptive BMT) for the study of clonal hematopoiesis in mouse models.

クローン造造を研究するマウスにおける骨髄移植手順

Clonal hematopoiesis is a prevalent age-associated condition that results from the accumulation of somatic mutations in hematopoietic stem and progenitor ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

成体マウス頭蓋冠骨髄中の造血幹細胞および前駆細胞のin vivo 4次元トラッキングで

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

<meta charset="utf8"></head><body>生きたマウスの骨髄研究のための大腿内注射

Simplified Intrafemoral Injections Using Live Mice Allow for Continuous Bone Marrow Analysis

Despite the complexity of hematopoietic cell transplantation in humans, researchers commonly perform intravenous or intrafemoral (IF) injections in mice. In murine models, this technique has been adapted to enhance the seeding efficiency of transplanted hematopoietic stem and progenitor cells (HSPCs). This paper describes a detailed step-by-step technical procedure of IF injection and the following bone marrow (BM) aspiration in mice that allows for serial characterization of cells present in the BM. This method enables the transplantation of valuable samples with low cell numbers that are particularly difficult to engraft by intravenous injection. This procedure facilitates the creation of xenografts that are critical for pathological analysis. While it is easier to access peripheral blood (PB), the cellular composition of PB does not reflect the BM, which is the niche for HSPCs. Therefore, procedures providing access to the BM compartment are essential for studying hematopoiesis. IF injection and serial BM aspiration, as described here, allow for the prospective retrieval and characterization of cells enriched in the BM, such as HSPCs, without sacrificing the mice.

<meta charset="utf8"></head><body>生きたマウスを使用した簡略化された大腿内注射により、継続的な骨髄分析が可能

This protocol describes the intrafemoral injection of a few hematopoietic or leukemic stem cells, including gene-edited cells, in murine xenograft models, which will not only enable the quick and safe transplantation of cells but also serial analyses of the bone marrow.

生きたマウスを使用した簡略化された大腿内注射により、継続的な骨髄分析が可能

Despite the complexity of hematopoietic cell transplantation in humans, researchers commonly perform intravenous or intrafemoral (IF) injections in mice. ...