博士YoderはEndGenitor Technologies社とRimedion Technologies社の取締役会のメンバーへのコンサルタントです。

<ol>
	<li>Carmeliet, P., Jain, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+and+clinical+applications+of+angiogenesis.">Molecular mechanisms and clinical applications of angiogenesis.</a> Nature. 473, 298-307 (2011).</li><li>Asahara, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+putative+progenitor+endothelial+cells+for+angiogenesis.">Isolation of putative progenitor endothelial cells for angiogenesis.</a> Science. 275, 964-967 (1997).</li><li>Urbich, C., Dimmeler, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+progenitor+cells:+characterization+and+role+in+vascular+biology.">Endothelial progenitor cells: characterization and role in vascular biology.</a> Circ. Res. 95, 343-353 (2004).</li><li>Critser, P. J., Voytik-Harbin, S. L., Yoder, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolating+and+defining+cells+to+engineer+human+blood+vessels.">Isolating and defining cells to engineer human blood vessels.</a> Cell. Prolif. 44, 15-21 (2011).</li><li>Matthias, M., David, N., Josef, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+bench+to+bedside:+what+physicians+need+to+know+about+endothelial+progenitor+cells.">From bench to bedside: what physicians need to know about endothelial progenitor cells.</a> Am. J. Med. 124, 489-4897 (2011).</li><li>Ingram, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vessel+wall-derived+endothelial+cells+rapidly+proliferate+because+they+contain+a+complete+hierarchy+of+endothelial+progenitor+cells.">Vessel wall-derived endothelial cells rapidly proliferate because they contain a complete hierarchy of endothelial progenitor cells.</a> Blood. 105, 2783-276 (2005).</li><li>Ingram, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+novel+hierarchy+of+endothelial+progenitor+cells+using+human+peripheral+and+umbilical+cord+blood.">Identification of a novel hierarchy of endothelial progenitor cells using human peripheral and umbilical cord blood.</a> Blood. 104, 2752-2760 (2004).</li><li>Yoder, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Redefining+endothelial+progenitor+cells+via+clonal+analysis+and+hematopoietic+stem/progenitor+cell+principals.">Redefining endothelial progenitor cells via clonal analysis and hematopoietic stem/progenitor cell principals.</a> Blood. 109, 1801-1809 (2007).</li><li>Reinisch, A., Strunk, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+Animal+Serum+Free+Expansion+of+Human+Umbilical+Cord+Derived+Mesenchymal+Stromal+Cells+(MSCs)+and+Endothelial+Colony+Forming+Progenitor+Cells+(ECFCs.">Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells (MSCs) and Endothelial Colony Forming Progenitor Cells (ECFCs.</a> J. Vis. Exp. (32), e1525 (2009).</li><li>Au, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+in+vivo+potential+of+endothelial+progenitor+cells+from+human+umbilical+cord+blood+and+adult+peripheral+blood+to+form+functional+long-lasting+vessels.">Differential in vivo potential of endothelial progenitor cells from human umbilical cord blood and adult peripheral blood to form functional long-lasting vessels.</a> Blood. 111, 1302-135 (2008).</li><li>Critser, P. J., Kreger, S. T., Voytik-Harbin, S. L., Yoder, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collagen+matrix+physical+properties+modulate+endothelial+colony+forming+cell-derived+vessels+in+vivo.">Collagen matrix physical properties modulate endothelial colony forming cell-derived vessels in vivo.</a> Microvasc. Res. 80, 23-30 (2010).</li><li>Melero-Martin, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+robust+and+functional+vascular+networks+in+vivo+with+human+adult+and+cord+blood-derived+progenitor+cells.">Engineering robust and functional vascular networks in vivo with human adult and cord blood-derived progenitor cells.</a> Circ. Res. 103, 194-202 (2008).</li><li>Melero-Martin, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+vasculogenic+potential+of+human+blood-derived+endothelial+progenitor+cells.">In vivo vasculogenic potential of human blood-derived endothelial progenitor cells.</a> Blood. 109, 4761-4768 (2007).</li><li>Hofmann, N. A., Reinisch, A., Strunk, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+Large+Scale+Expansion+of+Adult+Human+Endothelial+Colony+Forming+Progenitor+Cells.">Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells.</a> J. Vis. Exp. (32), e1524 (2009).</li><li>Lin, Y., Weisdorf, D. J., Solovey, A., Hebbel, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Origins+of+circulating+endothelial+cells+and+endothelial+outgrowth+from+blood.">Origins of circulating endothelial cells and endothelial outgrowth from blood.</a> J. Clin. Invest. 105, 71-77 (2000).</li><li>Witting, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+Large+Volume+Lentiviral+Vector+Production+Using+Flow+Electroporation.">Efficient Large Volume Lentiviral Vector Production Using Flow Electroporation.</a> Hum. Gene. Ther. , (2011).</li><li>Yoon, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synergistic+neovascularization+by+mixed+transplantation+of+early+endothelial+progenitor+cells+and+late+outgrowth+endothelial+cells:+the+role+of+angiogenic+cytokines+and+matrix+metalloproteinases.">Synergistic neovascularization by mixed transplantation of early endothelial progenitor cells and late outgrowth endothelial cells: the role of angiogenic cytokines and matrix metalloproteinases.</a> Circulation. , 112-1618 (2005).</li><li>Dubois, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+effects+of+progenitor+cell+populations+on+left+ventricular+remodeling+and+myocardial+neovascularization+after+myocardial+infarction.">Differential effects of progenitor cell populations on left ventricular remodeling and myocardial neovascularization after myocardial infarction.</a> J. Am. Coll. Cardiol. 55, 2232-2243 (2010).</li><li>Medina, R. J., O'Neill, C. L., Humphreys, M. W., Gardiner, T. A., Stitt, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Outgrowth+endothelial+cells:+characterization+and+their+potential+for+reversing+ischemic+retinopathy.">Outgrowth endothelial cells: characterization and their potential for reversing ischemic retinopathy.</a> Invest. Ophthalmol. Vis. Sci. 51, 5906-5913 (2010).</li></ol>

利害の衝突が宣言されません。

推定される内皮前駆細胞の表現型および機能解析は、クローンと連続して再メッキ文化の中で可能である善意の ECFCsを識別し、in vivoでの耐久性と機能的な植込み型血管を生じさせることが重要です。ヒト臍帯血はECFCsと老化や病気の10のこれらの循環細胞の減少の濃度で濃縮されています。最近の研究では、ECFCは、血管損傷、心筋梗塞、網膜症17から19までの状?...

<table border="1"><tbody><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ番号 </td></tr><tr><td>ヘパリンナトリウム注射液、USP </td><td> APPファーマシューティカル</td><td> 504031 </td></tr><tr><td>フィコール - Pague </td><td>アマシャムバイオサイエンス</td><td> 17-1440-03 </td></tr><tr><td>ミキシングカニューレ</td><td>マースク医療</td><td> 500.11.012 </td></tr><tr><td> EGM-2 </td><td>ロンザ</td><td> CC-3162 </td></tr><tr><td>定義されたFBS </td><td>ハイクローン</td><td> SH30070.03 </td></tr><tr><td> TrypLE急行</td><td>ギブコ</td><td> 12605 </td></tr><tr><td>ラットI型コラーゲン</td><td> BDバイオサイエンス</td><td> 354236 </td></tr><tr><td>マトリゲル</td><td> BDバイオサイエンス</td><td> 356234 </td></tr><tr><td> FcRをブロック</td><td>ミルテニーバイオテク</td><td> 130-059-901 </td></tr><tr><td> hCD31、FITC抱合テッド</td><td> BD Pharmingen社</td><td> 555445 </td></tr><tr><td> hCD45、FITCコンジュゲート</td><td> BD Pharmingen社</td><td> 555482 </td></tr><tr><td> hCD14、FITCコンジュゲート</td><td> BD Pharmingen社</td><td> 555397 </td></tr><tr><td> hCD144、PEコンジュゲート</td><td>ベイバイオサイエンス</td><td> 12-1449-80 </td></tr><tr><td> hCD146、PEコンジュゲート</td><td> BD Pharmingen社</td><td> 550315 </td></tr><tr><td> hCD105、PEコンジュゲート</td><td>インビトロジェン</td><td> MHCD10504 </td></tr><tr><td> MSのIgG 1、K抗体、FITCコンジュゲート</td><td> BD Pharmingen社</td><td> 555748 </td></tr><tr><td> MSのIgG 1、K抗体、PEコンジュゲート</td><td> BD Pharmingen社</td><td> 559320 </td></tr><tr><td> MSのIgG 2aは、K抗体、FITCコンジュゲート</td><td> BD Pharmingen社</td><td> 555573 </td></tr><tr><td>抗ヒトCD31 </td><td> Dako社</td><td>クローンJC70 / </td></tr><tr><td>アンチ覚書電子CD31 </td><td> BD Pharmingen社</td><td> 553370 </td></tr><tr><td> 0.22-μmの真空ろ過システム</td><td>ミリポア</td><td> SCGPU05RE </td></tr><tr><td>氷酢酸、17.4N </td><td>フィッシャー</td><td> A38-500 </td></tr><tr><td>抗生物質抗真菌</td><td>インビトロジェン</td><td> 15240-062 </td></tr><tr><td>ウシ胎児血清（FBS） </td><td>ハイクローン</td><td> SH30070.03 </td></tr><tr><td> IHC亜鉛固定液</td><td> BDバイオサイエンス</td><td> 550523 </td></tr><tr><td> SYTOXグリーン試薬</td><td>インビトロジェン</td><td> S33025 </td></tr><tr><td>滅菌クローニングシリンダー、 </td><td>フィッシャー·サイエンティフィック</td><td> 07-907-10 </td></tr></tbody></table>

phenotypic and functional characterization of endothelial colony forming cells derived from human umbilical cord blood

血管新生、脈管形成、および動脈形成を介して新しい血管の形成の長年のビューは、最近1日されています。内皮前駆細胞（EPC）の循環の存在が最初に麻原らが成人ヒト末梢血で同定された1997年2血管の再生と修理のための新しい仮説と戦略の注入をもたらすインチEPCは血管形成または血管リモデリングのサイトへのホームという血液の循環まれではあるが、通常のコンポーネントであり、主にセル3派生した既存の血管壁のパラクリン刺激を介して出生後の脈管形成、血管新生、または動脈形成のいずれかを容易にします。 EPCを識別するために、特定のマーカーが同定されず、フィールドの現時点での状態は、血管新生、造血幹細胞や前駆細胞を含め、その多くの細胞タイプを理解することであり、新生細胞、Tie2の+単球、骨髄前駆セルを循環されていますlsは、腫瘍関連マクロファージ、M2活性化マクロファージは、前臨床動物モデル系の様々な、多くの病気の状態4、5、人間の被験者の血管新生のプロセスを刺激するに参加しています。内皮コロニー形成細胞（ECFCs）堅牢なクローン増殖能、播種時に能力を形成する第二級および第三級コロニー、および免疫不全マウスへの移植時に6月8日 in vivoで血管内に本質的な形成する能力によって特徴付けまれな循環可能な内皮細胞である。 ECFCsが正常健康成人被験者、健康な新生児の臍帯血（CB）、および多数の人間の動脈および静脈の血管6-9の血管壁の末梢血から単離されているが、CBはECFCs 7の最高周波数を有していることを表示最も堅牢なクローン増殖能および in vivo 8、10-13 のフォーム耐久性と機能的な血管を。 whileの導出成人の末梢血からECFCここでは、我々が導出するための方法論を記述し、14、15を提示されており、クローニング、拡張、およびin vitroで同様にヒト臍帯CBからECFCs の in vivo特性のように 。

Watch this Scientific Journal Video about Phenotypic and Functional Characterization of Endothelial Colony Forming Cells Derived from Human Umbilical Cord Blood at JoVE.com

Phenotypic and Functional Characterization of Endothelial Colony Forming Cells Derived from Human Umbilical Cord Blood

内皮コロニー形成細胞（ECFCs）組み込み表示されていること、堅牢なクローン増殖能と血管内皮細胞を循環している<emin vivoでの&gt;</em&gt;船が能力を形成する。 CBから派生した副産物内皮細胞の表現型および機能解析を識別し、分離することが重要です。<em善意&gt;善意</em損傷した組織を修復するの潜在的な臨床応用のために&gt; ECFCs。

ヒト臍帯血由来血管内皮コロニー形成細胞の表現型および機能解析

Longstanding views of new blood vessel formation via angiogenesis, vasculogenesis, and arteriogenesis have been recently reviewed1. The presence of ...

phenotypic-functional-characterization-endothelial-colony-forming

Research

JoVE Journal

Biology

16.2K ビュー.  Indiana University School of Medicine. 内皮コロニー形成細胞（ECFCs）組み込み表示されていること、堅牢なクローン増殖能と血管内皮細胞を循環している船が能力を形成する。 CBから派生した副産物内皮細胞の表現型および機能解析を識別し、分離することが重要です。善意 ECFCs。

ビデオ: Phenotypic and Functional Characterization of Endothelial Colony Forming Cells Derived from Human Umbilical Cord Blood

Colony Forming Cell (CFC) Assay for Human Hematopoietic Cells

Human hematopoietic stem/progenitor cells are usually obtained from bone marrow, cord blood, or peripheral blood and are used to study hematopoiesis and leukemogenesis. They have the capacity to differentiate into lymphoid and myeloid lineages. The colony forming cell (CFC) assay is used to study the proliferation and differentiation pattern of hematopoietic progenitors by their ability to form colonies in a semisolid medium. The number and the morphology of the colonies formed by a fixed number of input cells provide preliminary information about the ability of progenitors to differentiate and proliferate. Cells can be harvested from individual colonies or from the whole plate to further assess their numbers and differentiation states using flow cytometry and morphologic evaluation of Giemsa-stained slides. This assay is useful for assessing myeloid but not lymphoid differentiation. The term myeloid in this context is used in its wider sense to encompass granulocytic, monocytic, erythroid, and megakaryocytic lineages.

We have used this assay to assess the effects of oncogenes on the differentiation of primary human CD34+ cells derived from peripheral blood. For this purpose cells are transduced with either control retroviral construct or a construct expressing the oncogene of interest, in this case NUP98-HOXA9. We employ a commonly used retroviral vector, MSCV-IRES-GFP, that expresses a bicistronic mRNA that produces the gene of interest and a GFP marker. Cells are pre-activated by growing in the presence of cytokines for two days prior to retroviral transduction. After another two days, GFP+ cells are isolated by fluorescence-activated cell sorting (FACS) and mixed with a methylcellulose-containing semisolid medium supplemented with cytokines and incubated till colonies appear on the surface, typically 14 days. The number and morphology of the colonies are documented. Cells are then removed from the plates, washed, counted, and subjected to flow cytometry and morphologic examination. Flow cytometry with antibodies specific to the cell surface markers expressed during hematopoiesis provides information about lineage and maturation stage. Morphological studies of individual cells under a microscope after Wright- Giemsa staining provide further information with regard to lineage and maturation. Comparison of cells transduced with control empty vector to those transduced with an oncogene reveals the effects of the oncogene on hematopoietic differentiation.

Colony Forming Cell CFC Assay for Human Hematopoietic Cells

The colony forming cell (CFC) assay is an in vitro assay in which hematopoietic progenitors form colonies in a semi-solid medium. A combination of colony morphology, cell morphology, and flow cytometry are used to assess the ability of the progenitors to proliferate and differentiate along the different hematopoietic lineages.

ヒト造血細胞のためのコロニー形成細胞（CFC）アッセイ

Human hematopoietic stem/progenitor cells are usually obtained from bone marrow, cord blood, or peripheral blood and are used to study hematopoiesis and ...

免疫・感染学

Isolation of Murine Embryonic Hemogenic Endothelial Cells

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, and is necessary for the emergence of definitive HSPC from the murine extra embryonic yolk sac, placenta, umbilical vessels, and the embryonic aorta-gonad-mesonephros (AGM) region. The transient nature and small size of this cell population renders its reproducible isolation for careful quantification and experimental applications technically difficult. We have established a fluorescence-activated cell sorting (FACS)-based protocol for simultaneous isolation of hemogenic endothelial cells and HSPC during their peak generation times in the yolk sac and AGM. We demonstrate methods for dissection of yolk sac and AGM tissues from mouse embryos, and we present optimized tissue digestion and antibody conjugation conditions for maximal cell survival prior to identification and retrieval via FACS. Representative FACS analysis plots are shown that identify the hemogenic endothelial cell and HSPC phenotypes, and describe a methylcellulose-based assay for evaluating their blood forming potential on a clonal level.

Hematopoietic stem and progenitor cells (HSPC) derive from specialized (hemogenic) endothelial cells during development, yet little is known about the process by which some endothelial cells specify to become blood forming. We demonstrate a flow-cytometry based method allowing simultaneous isolation of hemogenic endothelial cells and HSPC from murine embryonic tissues.

マウス胚性造血内皮細胞の単離

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, ...

発生生物学

Isolation of Endothelial Progenitor Cells from Human Umbilical Cord Blood

The existence of endothelial progenitor cells (EPCs) in peripheral blood and its involvement in vasculogenesis was first reported by Ashara and colleagues1. Later, others documented the existence of similar types of EPCs originating from bone marrow2,3. More recently, Yoder and Ingram showed that EPCs derived from umbilical cord blood had a higher proliferative potential compared to ones isolated from adult peripheral blood4,5,6. Apart from being involved in postnatal vasculogenesis, EPCs have also shown promise as a cell source for creating tissue-engineered vascular and heart valve constructs7,8. Various isolation protocols exist, some of which involve the cell sorting of mononuclear cells (MNCs) derived from the sources mentioned earlier with the help of endothelial and hematopoietic markers, or culturing these MNCs with specialized endothelial growth medium, or a combination of these techniques9. Here, we present a protocol for the isolation and culture of EPCs using specialized endothelial medium supplemented with growth factors, without the use of immunosorting, followed by the characterization of the isolated cells using Western blotting and immunostaining.

The goal of this protocol is to isolate endothelial progenitor cells from umbilical cord blood. Some of the applications include using these cells as a biomarker for identifying patients with cardiovascular risk, treating ischemic diseases, and creating tissue-engineered vascular and heart valve constructs.

ひと臍帯血由来の血管内皮前駆細胞の分離

The existence of endothelial progenitor cells (EPCs) in peripheral blood and its involvement in vasculogenesis was first reported by Ashara and ...

Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult human peripheral blood within a mean of 12 days. After large scale expansion &gt;1x108 ECFCs can be obtained for further tests. Advantageously by using pHPL the contact of human cells with bovine serum antigens can be excluded. By flow cytometry and immunohistochemistry the isolated cells can be characterized as ECFC and their in vitro functionality to form vascular like structures can be tested in a matrigel assay. Further these cells can be subcutaneously injected in a mouse model to form functional, perfused vessels in vivo. After long term expansion and cryopreservation proliferation, function and genomic stability appear to be preserved. 3,4
This animal-protein free isolation and expansion method is easily applicable to generate a large quantity of ECFCs.

Endothelial colony forming progenitor cells (ECFCs) are a promising tool to study vascular homeostasis and repair.1,2 This paper introduces a novel animal-serum free method for isolation and expansion of ECFC from heparinised adult human peripheral blood with pooled human platelet lysate (pHPL) diminishing the risk of anti-bovine immunisation.

分離と成人ヒト内皮コロニー形成前駆細胞の大規模拡張

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult ...

生物学

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells (MSCs) and Endothelial Colony Forming Progenitor Cells (ECFCs)

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal stem cells, MSCs) and endothelial colony forming progenitor cells (ECFCs). Both cell types are key players in maintaining the integrity of tissue and are probably also involved in regenerative processes and tumor formation.
To study their biology and function in a comparative manner it is important to have both cells types available from the same donor. It may also be beneficial for regenerative purposes to derive MSCs and ECFCs from the same tissue.
Because cellular therapeutics should eventually find their way from bench to bedside we established a new method to isolate and further expand progenitor cells without the use of animal protein. Pooled human platelet lysate (pHPL) replaced fetal bovine serum in all steps of our protocol to completely avoid contact of the cells to xenogeneic proteins.
This video demonstrates a methodology for the isolation and expansion of progenitor cells from one umbilical cord.
All materials and procedures will be described.

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells MSCs and Endothelial Colony Forming Progenitor Cells ECFCs

This protocol describes the isolation and subsequent expansion of mesenchymal stromal cells and endothelial colony forming cells without the use of animal serum to generate autologous pairs for experimental transplantation purposes.

ヒト臍帯由来間葉系幹細胞（MSCs）と血管内皮コロニー形成前駆細胞（ECFCs）の単離および動物血清自由膨張

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal ...

Hemogenic Endothelium Differentiation from Human Pluripotent Stem Cells in A Feeder- and Xeno-free Defined Condition

Deriving functional hematopoietic stem and progenitor cells (HSPCs) from human pluripotent stem cells (PSCs) have been an elusive goal for autologous transplants to treat hematological and malignant disorders. Hemogenic endothelium (HE) is an amenable platform to produce functional HSPCs by transcription factors or to induce lymphocytes in a robust manner. However, current methods to derive HE are either costly or variable in yield. In this protocol, we established a cost-effective and precise method to derive HE within 4 days in a feeder- and xeno-free defined condition. The resultant HE underwent an endothelial-to-hematopoietic transition and produced CD34+CD45+ clonogenic hematopoietic progenitors. The potential capacity of our platform for generating functional HSPCs will have broad applications in disease modeling and screening for therapeutic compounds.

Here, we present a protocol to precisely form hemogenic endothelium from human pluripotent stem cells in a feeder- and xeno-free condition. This method will have broad applications in disease modeling and screening for therapeutic compounds.

フィーダーとゼノフリーの定義条件におけるヒト多能性幹細胞からの血原性内皮分化

Deriving functional hematopoietic stem and progenitor cells (HSPCs) from human pluripotent stem cells (PSCs) have been an elusive goal for autologous ...

Pan-myeloid Differentiation of Human Cord Blood Derived CD34+ Hematopoietic Stem and Progenitor Cells

Ex vivo differentiation of human hematopoietic stem cells is a widely used model for studying hematopoiesis. The protocol described here is for cytokine induced differentiation of CD34+ hematopoietic stem and progenitor cells to the four myeloid lineage cells. CD34+ cells are isolated from human umbilical cord blood and co-cultured with MS-5 stromal cells in the presence of cytokines. Immunophenotypic characterization of the stem and progenitor cells, and the differentiated myeloid lineage cells are described. Using this protocol, CD34+ cells may be incubated with small molecules or transduced with lentiviruses to express myeloid disease mutations to investigate their impact on myeloid differentiation.

Pan-myeloid Differentiation of Human Cord Blood Derived CD34+ Hematopoietic Stem and Progenitor Cells

Here, we present a protocol for immunophenotypic characterization and cytokine induced differentiation of cord blood derived CD34+ hematopoietic stem and progenitor cells to the four myeloid lineages. The applications of this protocol include investigations on the effect of myeloid disease mutations or small molecules on myeloid differentiation of the CD34+ cells.

ヒト臍帯血由来CD34+造血幹細胞および前駆細胞のパンミエロイド分化

Ex vivo differentiation of human hematopoietic stem cells is a widely used model for studying hematopoiesis. The protocol described here is for cytokine ...

Three-dimensional Angiogenesis Assay System using Co-culture Spheroids Formed by Endothelial Colony Forming Cells and Mesenchymal Stem Cells

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more than 50 different pathological conditions, such as rheumatoid arthritis, oculopathy, cardiovascular diseases, and tumor metastasis. During angiogenesis drug development, it is crucial to use in vitro assay systems with appropriate cell types and proper conditions to reflect the physiologic angiogenesis process. To overcome limitations of current in vitro angiogenesis assay systems using mainly endothelial cells, we developed a 3-dimensional (3D) co-culture spheroid sprouting assay system. Co-culture spheroids were produced by two human vascular cell precursors, endothelial colony forming cells (ECFCs) and mesenchymal stem cells (MSCs) with a ratio of 5 to 1. ECFCs+MSCs spheroids were embedded into type I collagen matrix to mimic the in vivo extracellular environment. A real-time cell recorder was utilized to continuously observe the progression of angiogenic sprouting from spheroids for 24 h. Live cell fluorescent labeling technique was also applied to tract the localization of each cell type during sprout formation. Angiogenic potential was quantified by counting the number of sprouts and measuring the cumulative length of sprouts generated from the individual spheroids. Five randomly-selected spheroids were analyzed per experimental group. Comparison experiments demonstrated that ECFCs+MSCs spheroids showed greater sprout number and cumulative sprout length compared with ECFCs-only spheroids. Bevacizumab, an FDA-approved angiogenesis inhibitor, was tested with the newly-developed co-culture spheroid assay system to verify its potential to screen anti-angiogenic drugs. The IC50 value for ECFCs+MSCs spheroids compared to the ECFCs-only spheroids was closer to the effective plasma concentration of bevacizumab obtained from the xenograft tumor mouse model. The present study suggests that the 3D ECFCs+MSCs spheroid angiogenesis assay system is relevant to physiologic angiogenesis, and can predict an effective plasma concentration in advance of animal experiments.

Three-dimensional co-culture spheroid angiogenesis assay system is designed to mimic the physiologic angiogenesis. Co-culture spheroids are formed by two human vascular cell precursors, ECFCs and MSCs, and embedded in collagen gel. The new system is effective for evaluating angiogenic modulators, and provides more relevant information to the in vivo study.

内皮コロニー形成細胞と間葉系幹細胞によって形成された共培養スフェロイドを用いた3次元血管新生アッセイ系

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more ...

Directed Differentiation of Hemogenic Endothelial Cells from Human Pluripotent Stem Cells

Blood vessels are ubiquitously distributed within all tissues of the body and perform diverse functions. Thus, derivation of mature vascular endothelial cells, which line blood vessel lumens, from human pluripotent stem cells is crucial for a multitude of tissue engineering and regeneration applications. In vivo, primordial endothelial cells are derived from the mesodermal lineage and are specified toward specific subtypes, including arterial, venous, capillary, hemogenic, and lymphatic. Hemogenic endothelial cells are of particular interest because, during development, they give rise to hematopoietic stem and progenitor cells, which then generate all blood lineages throughout life. Thus, creating a system to generate hemogenic endothelial cells in vitro would provide an opportunity to study endothelial-to-hematopoietic transition, and may lead to ex vivo production of human blood products and reduced reliance on human donors. While several protocols exist for the derivation of progenitor and primordial endothelial cells, generation of well-characterized hemogenic endothelial cells from human stem cells has not been described. Here, a method for the derivation of hemogenic endothelial cells from human embryonic stem cells in approximately 1 week is presented: a differentiation protocol with primitive streak cells formed in response to GSK3&#946; inhibitor (CHIR99021), then mesoderm lineage induction mediated by bFGF, followed by primordial endothelial cell development promoted by BMP4 and VEGF-A, and finally hemogenic endothelial cell specification induced by retinoic acid. This protocol yields a well-defined population of hemogenic endothelial cells that can be used to further understand their molecular regulation and endothelial-to-hematopoietic transition, which has the potential to be applied to downstream therapeutic applications.

Presented here is a simple protocol for the directed differentiation of hemogenic endothelial cells from human pluripotent stem cells in approximately 1 week.

ヒト多能性幹細胞からの血原性内皮細胞の指向性分化

Blood vessels are ubiquitously distributed within all tissues of the body and perform diverse functions. Thus, derivation of mature vascular endothelial ...

Characterization of Blood Outgrowth Endothelial Cells (BOEC) from Porcine Peripheral Blood

The endothelium is a dynamic integrated structure that plays an important role in many physiological functions such as angiogenesis, hemostasis, inflammation, and homeostasis. The endothelium also plays an important role in pathophysiologies such as atherosclerosis, hypertension, and diabetes. Endothelial cells form the inner lining of blood and lymphatic vessels and display heterogeneity in structure and function. Various groups have evaluated the functionality of endothelial cells derived from human peripheral blood with a focus on endothelial progenitor cells derived from hematopoietic stem cells or mature blood outgrowth endothelial cells (or endothelial colony-forming cells). These cells provide an autologous resource for therapeutics and disease modeling. Xenogeneic cells may provide an alternative source of therapeutics due to their availability and homogeneity achieved by using genetically similar animals raised in similar conditions. Hence, a robust protocol for the isolation and expansion of highly proliferative blood outgrowth endothelial cells from porcine peripheral blood has been presented. These cells can be used for numerous applications such as cardiovascular tissue engineering, cell therapy, disease modeling, drug screening, studying endothelial cell biology, and in vitro co-cultures to investigate inflammatory and coagulation responses in xenotransplantation.