Dr. Yoder é consultor de Tecnologias EndGenitor, Inc. e membro do conselho de Rimedion Technologies, Inc.

<ol>
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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>

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Caracterização fenotípica e funcional dos putativos células progenitoras endoteliais é importante para identificar os ECFCs autênticas que são capazes de clonalmente e serialmente re-plaqueamento em cultura e dar origem a duráveis ​​e funcional vasos sanguíneos implantáveis ​​in vivo. O sangue do cordão umbilical humano é enriquecido com ECFCs ea concentração desses declínios células circulantes com o envelhecimento ou a doença de 10. Estudos recentes sugerem que ECF...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td></tr><tr><td> Injeção de heparina sódica, USP </td><td> APP Pharmaceuticals </td><td> 504031 </td></tr><tr><td> Ficoll-Pague </td><td> Amersham Biosciences </td><td> 17-1440-03 </td></tr><tr><td> Cânula de mistura </td><td> Maersk Medical </td><td> 500.11.012 </td></tr><tr><td> EGM-2 </td><td> Lonza </td><td> CC-3162 </td></tr><tr><td> FBS definido </td><td> Hyclone </td><td> SH30070.03 </td></tr><tr><td> TrypLE expressa </td><td> Gibco </td><td> 12605 </td></tr><tr><td> Tipo Rat colágeno I </td><td> BD Biosciences </td><td> 354236 </td></tr><tr><td> Matrigel </td><td> BD Biosciences </td><td> 356234 </td></tr><tr><td> FcR Bloco </td><td> Miltenyi Biotech </td><td> 130-059-901 </td></tr><tr><td> hCD31, FITC Conjugated </td><td> BD Pharmingen </td><td> 555445 </td></tr><tr><td> hCD45, conjugado com FITC </td><td> BD Pharmingen </td><td> 555482 </td></tr><tr><td> hCD14, conjugado com FITC </td><td> BD Pharmingen </td><td> 555397 </td></tr><tr><td> hCD144, PE conjugado </td><td> eBioscience </td><td> 12-1449-80 </td></tr><tr><td> hCD146, PE conjugado </td><td> BD Pharmingen </td><td> 550315 </td></tr><tr><td> hCD105, PE conjugado </td><td> Invitrogen </td><td> MHCD10504 </td></tr><tr><td> Ms IgG 1, o anticorpo k, conjugado com FITC </td><td> BD Pharmingen </td><td> 555748 </td></tr><tr><td> Ms IgG 1, o anticorpo k, PE conjugado </td><td> BD Pharmingen </td><td> 559320 </td></tr><tr><td> Ms IgG 2a, o anticorpo k, conjugado com FITC </td><td> BD Pharmingen </td><td> 555573 </td></tr><tr><td> Anti-CD31 humano </td><td> Dako </td><td> clone JC70 / A </td></tr><tr><td> Anti-mouse CD31 </td><td> BD Pharmingen </td><td> 553370 </td></tr><tr><td> 0,22 mícrons sistema de filtração de vácuo </td><td> Millipore </td><td> SCGPU05RE </td></tr><tr><td> Ácido acético glacial, 17.4N </td><td> Pescador </td><td> A38-500 </td></tr><tr><td> Antibiótico-antimicótica </td><td> Invitrogen </td><td> 15240-062 </td></tr><tr><td> Soro fetal de bovino (FBS) </td><td> Hyclone </td><td> SH30070.03 </td></tr><tr><td> IHC zinco Fixação </td><td> BD Biosciences </td><td> 550523 </td></tr><tr><td> Sytox reagente verde </td><td> Invitrogen </td><td> S33025 </td></tr><tr><td> Cilindros de clonagem, estéreis </td><td> Fisher Scientific </td><td> 07-907-10 </td></tr></tbody></table>

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

Vistas de longa data de formação de novos vasos sanguíneos através de angiogênese, vasculogênese e arteriogênese foram avaliados recentemente 1. A presença de circulação de células progenitoras endoteliais (EPCs) foram primeiramente identificadas no sangue periférico humano adulto por Asahara et al. Em 1997 2 trazendo uma infusão de novas hipóteses e estratégias para a regeneração e reparação vascular. EPCs são raras, mas os componentes normais do sangue circulante de que o lar de locais de formação dos vasos sanguíneos ou remodelação vascular, e facilitar quer vasculogênese pós-natal, a angiogênese, ou arteriogênese em grande parte através da estimulação parácrina de parede do vaso existente derivadas de células 3. Nenhum marcador específico para identificar um EPC foi identificado, e no presente estado do campo é entender que tipos de células-tronco hematopoiéticas, incluindo numerosos proangiogenic e células progenitoras, células circulantes angiogênicas, TIE2 + de monócitos, cel progenitor mielóidels, tumorais macrófagos associados, e M2 macrófagos activados participar em estimular o processo angiogénico em uma variedade de sistemas de modelos animais e pré-clínicos em seres humanos em numerosos estados de doença 4, 5. Células endoteliais que formam colônias (ECFCs) são raras circulantes células endoteliais viáveis ​​caracterizadas por potencial robusto clonal proliferativa, colônia secundária e terciária capacidade de formação sobre repique, e capacidade de formar intrínseca em vasos in vivo sobre o transplante em ratos imunodeficientes 6-8. Enquanto ECFCs foram isoladas com sucesso a partir do sangue periférico de indivíduos adultos saudáveis, o sangue do cordão umbilical (CB) de recém-nascidos saudáveis ​​e parede do reservatório de numerosos humanos vasos arteriais e venosos 6-9, CB possui a maior freqüência de ECFCs 7 que exibição o mais robusto potencial clonal proliferativa e forma os vasos sanguíneos duráveis ​​e funcionais in vivo 8, 10-13. Enquanto a derivação deECFC a partir de sangue periférico adulto tem sido apresentada 14, 15, aqui descrever as metodologias para a derivação, a clonagem, a expansão, e in vitro em bem como em caracterização in vivo das ECFCs do umbilical humano CB.

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

Células endoteliais que formam colônias (ECFCs) estão circulando células endoteliais com potencial clonal proliferativa robusto que exibe intrínseco<em&gt; In vivo</emNavio&gt; capacidade de formação. Caracterização fenotípica e funcional das células endoteliais excrescência derivados de CB são importantes para identificar e isolar<em&gt; Bona fide</em&gt; ECFCs potencial para aplicação clínica na reparação de tecidos danificados.

Caracterização fenotípica e funcional das células endoteliais Formadoras de Colônias derivado do sangue do cordão umbilical humano

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 visualizações.  Indiana University School of Medicine. Células endoteliais que formam colônias (ECFCs) estão circulando células endoteliais com potencial clonal proliferativa robusto que exibe intrínseco In vivo capacidade de formação. Caracterização fenotípica e funcional das células endoteliais excrescência derivados de CB são importantes para identificar e isolar Bona fide ECFCs potencial para aplicação clínica na reparação de tecidos danificados.

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

Isolation of Human Umbilical Vein Endothelial Cells (HUVEC)

Angiogenesis is a complex multi-step process, where in response to angiogenic stimuli, new vessels are created from the existing vasculature. These steps include: degradation of the basement membrane, proliferation and migration (sprouting) of endothelial cells (EC) into the extracellular matrix, alignment of EC into cords, lumen formation, anastomosis, and formation of a new basement membrane. Many in vitro assays have been developed to study this process, but most only mimic certain stages of angiogenesis, and morphologically the vessels often do not resemble vessels in vivo. Here we demonstrate an optimized in vitro angiogenesis assay that utilizes human umbilical vein EC and fibroblasts. This model recapitulates all of the key early stages of angiogenesis, and importantly the vessels display patent intercellular lumens surrounded by polarized EC. Vessels can be easily observed by phase-contrast and time-lapse microscopy, and recovered in pure form for downstream applications.

Isolation of Human Umbilical Vein Endothelial Cells HUVEC

This video protocol illustrates the isolation and culture of human umbilical vein endothelial cells (HUVEC) from human umbilical cord. Once isolated these cells can be used for in vitro angiogenesis assays like the Optimized Fibrin Gel Bead Assay also demonstrated by the Hughes lab.

Isolamento de células endoteliais da veia umbilical humana (HUVEC)

Angiogenesis is a complex multi-step process, where in response to angiogenic stimuli, new vessels are created from the existing vasculature. These steps ...

Biologia

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.

Isolamento e expansão em grande escala de Adulto Humanos endotelial Colony Células Progenitoras Forming

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

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.

Isolamento e animais de Expansão de soro livre de cordão umbilical humano Derivado células mesenquimais estromais (MSCs) e endoteliais Células Progenitoras Colony Forming (ECFCs)

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

Isolation and Enrichment of Rat Mesenchymal Stem Cells (MSCs) and Separation of Single-colony Derived MSCs

MSCs are a population of adult stem cells that is a promising source for therapeutic applications. These cells can be isolated from the bone marrow and can be easily separated from the hematopoietic stem cells (HSCs) due to their plastic adherence. This protocol describes how to isolate MSCs from rat femurs and tibias. The isolated cells were further enriched against two MSCs surface markers CD54 and CD90 by magnetic cell sorting. Expression of surface markers CD54 and CD90 were then confirmed by flow cytometry analysis. HSC marker CD45 was also included to check if the sorted MSCs were depleted of HSCs. MSCs are naturally quite heterogeneous. There are subpopulations of cells that have different shapes, proliferation and differentiation abilities. These subpopulations all express the known MSCs markers and no unique marker has yet been identified for the different subpopulations. Therefore, an alternative approach to separate out the different subpopulations is using cloning cylinders to separate out single-colony derived cells. The cells derived from the single-colonies can then be cultured and evaluated separately.

Isolation and Enrichment of Rat Mesenchymal Stem Cells MSCs and Separation of Single-colony Derived MSCs

Rat MSCs were isolated from femurs and tibias and then enriched by magnetic cell sorting. Sorted cells were confirmed for the expression of surface markers by flow cytometry. These cells were also cultured at clonal density to form single colonies and then these colonies were separated by cloning cylinders.

Isolamento e Enriquecimento de células-tronco mesenquimais Rat (MSC) e Separação de-colônia Único Derivado MSCs

MSCs are a population of adult stem cells that is a promising source for therapeutic applications. These cells can be isolated from the bone marrow and ...

Isolation of Human Umbilical Arterial Smooth Muscle Cells (HUASMC)

The human umbilical cord (UC) is a biological sample that can be easily obtained just after birth. This biological sample is, most of the time, discarded and their collection does not imply any added risk to the newborn or mother s health. Moreover no ethical concerns are raised. The UC is composed by one vein and two arteries from which both endothelial cells (ECs) 1 and smooth muscle cells (SMCs) 2, two of the main cellular components of blood vessels, can be isolated. In this project the SMCs were obtained after enzymatic treatment of the UC arteries accordingly the experimental procedure previously described by Jaffe et al 3. After cell isolation they were kept in t-flash with DMEM-F12 supplemented with 5% of fetal bovine serum and were cultured for several passages. Cells maintained their morphological and other phenotypic characteristics in the different generations. The aim of this study was to isolate smooth muscle cells in order to use them as models for future assays with constrictor drugs, isolate and structurally characterize L-type calcium channels, to study cellular and molecular aspects of the vascular function 4 and to use them in tissue engineering.

Isolation of Human Umbilical Arterial Smooth Muscle Cells HUASMC

The umbilical cords are used to isolate smooth muscle cells by different ways. In this work we used the enzymatic treatment to isolated smooth muscle cells.

Isolamento da Human Umbilical células musculares arteriais (HUASMC)

The human umbilical cord (UC) is a biological sample that can be easily obtained just after birth. This biological sample is, most of the time, discarded ...

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells (HUVECs) have shown to be convenient, easy to obtain and culture, and thus are the most widely studied endothelial cells. However, for research focused on processes like angiogenesis, permeability or many others, microvascular endothelial cells (ECs) are a much more physiologically relevant model to study 2. Furthermore, ECs isolated from knockout mice provide a useful tool for analysis of protein function ex vivo. Several approaches to isolate and culture microvascular ECs of different origin have been reported to date 3-7, but consistent isolation and culture of pure ECs is still a major technical problem in many laboratories. Here, we provide a step-by-step protocol on a reliable and relatively simple method of isolating and culturing mouse lung endothelial cells (MLECs). In this approach, lung tissue obtained from 6- to 8-day old pups is first cut into pieces, digested with collagenase/dispase (C/D) solution and dispersed mechanically into single-cell suspension. MLECS are purified from cell suspension using positive selection with anti-PECAM-1 antibody conjugated to Dynabeads using a Magnetic Particle Concentrator (MPC). Such purified cells are cultured on gelatin-coated tissue culture (TC) dishes until they become confluent. At that point, cells are further purified using Dynabeads coupled to anti-ICAM-2 antibody. MLECs obtained with this protocol exhibit a cobblestone phenotype, as visualized by phase-contrast light microscopy, and their endothelial phenotype has been confirmed using FACS analysis with anti-VE-cadherin 8 and anti-VEGFR2 9 antibodies and immunofluorescent staining of VE-cadherin. In our hands, this two-step isolation procedure consistently and reliably yields a pure population of MLECs, which can be further cultured. This method will enable researchers to take advantage of the growing number of knockout and transgenic mice to directly correlate in vivo studies with results of in vitro experiments performed on isolated MLECs and thus help to reveal molecular mechanisms of vascular phenotypes observed in vivo.

Here, we describe a protocol for isolation and culture of murine pulmonary endothelial cells. This method comprises mechanic and enzymatic lung tissue dissociation as well as a 2-step purification process using anti-PECAM-1 and anti-ICAM-2 antibodies conjugated to magnetic beads, which produces a pure endothelial cell population of mostly microvascular origin.

Isolamento e cultura de células endoteliais pulmonares de camundongos Neonatal

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells ...

A Quantitative Assay for Insulin-expressing Colony-forming Progenitors

The field of pancreatic stem and progenitor cell biology has been hampered by a lack of in vitro functional and quantitative assays that allow for the analysis of the single cell. Analyses of single progenitors are of critical importance because they provide definitive ways to unequivocally demonstrate the lineage potential of individual progenitors. Although methods have been devised to generate "pancreatospheres" in suspension culture from single cells, several limitations exist. First, it is time-consuming to perform single cell deposition for a large number of cells, which in turn commands large volumes of culture media and space. Second, numeration of the resulting pancreatospheres is labor-intensive, especially when the frequency of the pancreatosphere-initiating progenitors is low. Third, the pancreatosphere assay is not an efficient method to allow both the proliferation and differentiation of pancreatic progenitors in the same culture well, restricting the usefulness of the assay.
To overcome these limitations, a semi-solid media based colony assay for pancreatic progenitors has been developed and is presented in this report. This method takes advantage of an existing concept from the hematopoietic colony assay, in which methylcellulose is used to provide viscosity to the media, allowing the progenitor cells to stay in three-dimensional space as they undergo proliferation as well as differentiation. To enrich insulin-expressing colony-forming progenitors from a heterogeneous population, we utilized cells that express neurogenin (Ngn) 3, a pancreatic endocrine progenitor cell marker. Murine embryonic stem (ES) cell-derived Ngn3 expressing cells tagged with the enhanced green fluorescent protein reporter were sorted and as many as 25,000 cells per well were plated into low-attachment 24-well culture dishes. Each well contained 500 &mu;L of semi-solid media with the following major components: methylcellulose, Matrigel, nicotinamide, exendin-4, activin &beta;B, and conditioned media collected from murine ES cell-derived pancreatic-like cells. After 8 to 12 days of culture, insulin-expressing colonies with distinctive morphology were formed and could be further analyzed for pancreatic gene expression using quantitative RT-PCR and immunoflourescent staining to determine the lineage composition of each colony.
In summary, our colony assay allows easy detection and quantification of functional progenitors within a heterogeneous population of cells. In addition, the semi-solid media format allows uniform presentation of extracellular matrix components and growth factors to cells, enabling progenitors to proliferate and differentiate in vitro. This colony assay provides unique opportunities for mechanistic studies of pancreatic progenitor cells at the single cell level.

A three-dimensional clonogenic assay that allows pancreatic-like progenitors to differentiate into insulin-expressing colonies is described. This method takes advantage of semi-solid media containing methylcellulose, Matrigel and growth factors, in which single progenitors proliferate and differentiate in vitro, permitting quantification of the number of functional progenitors in a population.

Uma análise quantitativa para expressar Insulin-formadoras de colônias Progenitores

The field of pancreatic stem and progenitor cell biology has been hampered by a lack of in vitro functional and quantitative assays that allow for the ...

Manual Isolation of Adipose-derived Stem Cells from Human Lipoaspirates

In 2001, researchers at the University of California, Los Angeles, described the isolation of a new population of adult stem cells from liposuctioned adipose tissue that they initially termed Processed Lipoaspirate Cells or PLA cells. Since then, these stem cells have been renamed as Adipose-derived Stem Cells or ASCs and have gone on to become one of the most popular adult stem cells populations in the fields of stem cell research and regenerative medicine. Thousands of articles now describe the use of ASCs in a variety of regenerative animal models, including bone regeneration, peripheral nerve repair and cardiovascular engineering. Recent articles have begun to describe the myriad of uses for ASCs in the clinic. The protocol shown in this article outlines the basic procedure for manually and enzymatically isolating ASCs from large amounts of lipoaspirates obtained from cosmetic procedures. This protocol can easily be scaled up or down to accommodate the volume of lipoaspirate and can be adapted to isolate ASCs from fat tissue obtained through abdominoplasties and other similar procedures.

In 2001, researchers at UCLA described the isolation of a population of adult stem cells, termed Adipose-derived Stem Cells or ASCs, from adipose tissue. This article outlines the isolation of ASCs from lipoaspirates using a manual, enzymatic digestion protocol using collagenase.

Isolamento manual de células-tronco adiposo-derivadas de Lipoaspirates Humanos

In 2001, researchers at the University of  California, Los Angeles, described the isolation of a new population of adult  stem cells from liposuctioned ...

Isolation of Blood-vessel-derived Multipotent Precursors from Human Skeletal Muscle

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective isolation in culture. Recently, using fluorescence-activated cell sorting (FACS), we and other researchers prospectively identified and purified three subpopulations of multipotent precursor cells associated with the vasculature of human skeletal muscle. These three cell populations: myogenic endothelial cells (MECs), pericytes (PCs), and adventitial cells (ACs), are localized respectively to the three structural layers of blood vessels: intima, media, and adventitia. All of these human blood-vessel-derived stem cell (hBVSC) populations not only express classic MSC markers but also possess mesodermal developmental potentials similar to typical MSCs. Previously, MECs, PCs, and ACs have been isolated through distinct protocols and subsequently characterized in separate studies. The current isolation protocol, through modifications to the isolation process and adjustments in the selective cell surface markers, allows us to simultaneously purify all three hBVSC subpopulations by FACS from a single human muscle biopsy. This new method will not only streamline the isolation of multiple BVSC subpopulations but also facilitate future clinical applications of hBVSCs for distinct therapeutic purposes.

Blood vessels within human skeletal muscle harbor several multi-lineage precursor populations that are ideal for regenerative applications. This isolation method allows simultaneous purification of three multipotent precursor cell populations respectively from three structural layers of blood vessels: myogenic endothelial cells from intima, pericytes from media, and adventitial cells from adventitia.

Isolamento de Blood-derivado do navio Multipotentes Precursores do músculo-esquelético humano

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective ...

Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta

Calcium is a very important regulator of many physiological processes in vascular tissues. Most endothelial and smooth muscle functions highly depend on changes in intracellular calcium ([Ca2+]i) and nitric oxide (NO). In order to understand how [Ca2+]i, NO and downstream molecules are handled by a blood vessel in response to vasoconstrictors and vasodilators, we developed a novel technique that applies calcium-labeling (or NO-labeling) dyes with two photon microscopy to measure calcium handling (or NO production) in isolated blood vessels. Described here is a detailed step-by-step procedure that demonstrates how to isolate an aorta from a rat, label calcium or NO within the endothelial or smooth muscle cells, and image calcium transients (or NO production) using a two photon microscope following physiological or pharmacological stimuli. The benefits of using the method are multi-fold: 1) it is possible to simultaneously measure calcium transients in both endothelial cells and smooth muscle cells in response to different stimuli; 2) it allows one to image endothelial cells and smooth muscle cells in their native setting; 3) this method is very sensitive to intracellular calcium or NO changes and generates high resolution images for precise measurements; and 4) described approach can be applied to the measurement of other molecules, such as reactive oxygen species. In summary, application of two photon laser emission microscopy to monitor calcium transients and NO production in the endothelial and smooth muscle cells of an isolated blood vessel has provided high quality quantitative data and promoted our understanding of the mechanisms regulating vascular function.

Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta

Vascular cell functiondepends on activity of intracellular messengers. Described here is an ex vivo two photon imaging method that allows the measurement of intracellular calcium and nitric oxide levels in response to physiological and pharmacological stimuli in individual endothelial and smooth muscle cells of an isolated aorta.

Dois fótons de imagem de cálcio intracelular<sup&gt; 2+</sup&gt; Manuseio e produção de óxido nítrico no endotélio e músculo liso células de uma aorta de rato isolada

Calcium is a very important regulator of many physiological processes in vascular tissues. Most endothelial and smooth muscle functions highly depend on ...