Questo lavoro è stato sostenuto da una borsa di studio presso l&#39;Istituto Nazionale di artrite e malattie della pelle muscolo-scheletrico, NIH RO1AR054458.

1. L&#39;etichettatura di hMSCs con Endorem <ol><li> Le cellule sono coltivate a 80% confluenza di almeno 18 ore prima di etichettatura. </li><li> Durante questo periodo, i media di etichettatura si prepara aggiungendo Endorem al campione ad una dose di 100 ug Fe / ml di siero-media liberi. </li><li> Dopo che le cellule sono coltivate a confluenza, la cultura media è aspirato e le cellule vengono lavate con PBS 1x o senza siero dei media. </li><li> Quindi, la soluzione di risciacquo viene aspirato e dei media etichettatura precedentemente preparato viene aggiunto. Le cellule vengono poi incubate a 37 ° C, 5% di CO 2 per 4 ore. </li><li> Dopo 4 ore di incubazione, l&#39;etichettatura dei media è aspirato e le cellule vengono sciacquati con PBS e trypsinized come al solito. </li><li> Una volta trypsinized, le cellule vengono lavate 3 volte con PBS e centrifugati a 400rcf per 5 minuti. </li><li> Dopo questa fase di lavaggio, le cellule sono risospese, contati, valutati per la viabilità, e utilizzato come necessario per la sperimentazione. </li></ol> 2. Preparazione agarosio <ol><li> Dopo che le cellule sono state etichettate è tempo di preparare la soluzione agarosio. </li><li> Su una scala bene, 80 mg di polvere di agarosio sono pesati e aggiunti 2 ml di PBS. </li><li> Questa soluzione è poi delicatamente scossa e sterilizzati in autoclave. </li><li> Dopo circa 40 minuti, la soluzione di agarosio fluido può essere tolto dal autoclave. Il volume deve essere misurato e regolato di conseguenza, preriscaldato PBS. </li><li> Allora la soluzione è raffreddata a 42 ° C </li></ol> 3. Creazione MASI <ol><li> Quando la soluzione di agarosio ha raggiunto la temperatura desiderata, le celle sono contati e centrifugati per l&#39;ultima volta in un tubo Eppendorf 1,5 ml. </li><li> Dopo la centrifugazione, il surnatante viene eliminato e le cellule sono mescolate con le DMEM di raggiungere una concentrazione di 30 x 10 6 cellule / ml. </li><li> Ora, le cellule sono mescolate con i 42 ° C caldo agarosio. </li><li> Un puntale freddo può causare agarosio a solidificare all&#39;interno della punta, quindi si consiglia di preriscaldare la punta pipettando caldo PBS sterile su e giù </li><li> Ora, lo stesso volume di agarosio come media è ripreso e miscelare con la cellula-media-sospensione in modo che una sospensione omogenea viene creato. </li><li> Tenere il tubo Eppendorf all&#39;interno di un bagno d&#39;acqua preriscaldata mescolando in modo che l&#39;agarosio non si raffredda abbastanza per solidificare. </li><li> Quando la sospensione è omogenea, le cellule vengono impiantate nel difetto. </li></ol><img src="/files/ftp_upload/1793/1793fig1.jpg" alt="Figura 1" /> Figura 1. Coronale T2 pesate SE (TR 4000 ms / TE 18,27 ms) di un campione rotula con hMSCs impiantati in difetti della cartilagine.

<ol>
	<li>Mauck, R. L., Yuan, X., Tuan, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chondrogenic+differentiation+and+functional+maturation+of+bovine+mesenchymal+stem+cells+in+long-term+agarose+culture.">Chondrogenic differentiation and functional maturation of bovine mesenchymal stem cells in long-term agarose culture.</a> Osteoarthritis Cartilage. 14, 179-189 (2006).</li><li>Pittenger, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multilineage+potential+of+adult+human+mesenchymal+stem+cells.">Multilineage potential of adult human mesenchymal stem cells.</a> Science. 284, 143-147 (1999).</li><li>Rahfoth, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+of+allograft+chondrocytes+embedded+in+agarose+gel+into+cartilage+defects+of+rabbits.">Transplantation of allograft chondrocytes embedded in agarose gel into cartilage defects of rabbits.</a> Osteoarthritis Cartilage. 6, 50-65 (1998).</li><li>Henning, T. D., Boddington, S., Daldrup-Link, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Labeling+hESCs+and+hMSCs+with+iron+oxide+nanoparticles+for+non-invasive+in+vivo+tracking+with+MR+imaging.">Labeling hESCs and hMSCs with iron oxide nanoparticles for non-invasive in vivo tracking with MR imaging.</a> J Vis Exp. , (2008).</li></ol>

Il protocollo descritto fornisce un modo riproducibile per etichettare MSC con nanoparticelle di ossido di ferro e di impianto tali etichettati MSCs in difetti della cartilagine. Questa tecnica permette non invasiva rappresentazione dei trapianti di cellule staminali a difetti della cartilagine con la RM, che consente una diagnosi precoce di fallimento MASI. Una lussazione o efflusso delle cellule etichettate può essere diagnosticata sulla base di una scomparsa del marchio dal sito trapianto sulle immagini MR. Una diag...

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<td>D-MEM High Glucose</td>
<td>Reagent</td>
<td>Sigma</td>
<td>D5648</td>
<td>&nbsp;</td>
<tr>
<td>PBS (Ca++, Mg++ free)</td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>14190-144</td>
<td>&nbsp;</td>
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<td>Trypsin-EDTA 0.05%</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>25399-120</td>
<td>&nbsp;</td>
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<td>FBS</td>
<td>&nbsp;</td>
<td>Hyclone</td>
<td>SH30071.03</td>
<td>&nbsp;</td>
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<td>Penicillin/Streptomycin</td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>15140-122</td>
<td>&nbsp;</td>
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<td>Ferumoxides (Endorem)</td>
<td>
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<td>Guerbet</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Agarose Typ VII</td>
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<td>A9045</td>
<td>Dilute Agarose in PBS to final concentration of 4%</td>
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implantation of ferumoxides labeled human mesenchymal stem cells in cartilage defects

Il campo della ingegneria dei tessuti integra i principi della biologia cellulare ingegneria e medicina verso la rigenerazione di cellule specifiche e tessuti funzionali. Matrice associata impianti di cellule staminali (MASI) mirano a rigenerare i difetti della cartilagine a causa di lesioni alle articolazioni artritiche o traumatica. Cellule staminali mesenchimali (MSCs) hanno la capacità di differenziarsi in cellule della linea condrogenico e hanno mostrato risultati promettenti per cellulari basati su tecnologie di riparazione della cartilagine articolare. Autologo di cellule staminali mesenchimali possono essere isolate da una varietà di tessuti, può essere ampliato in colture cellulari, senza perdere il loro potenziale di differenziazione, e hanno dimostrato condrogenico differenziazione in vitro e in vivo 1, 2. Al fine di garantire la conservazione locale e la vitalità delle cellule staminali mesenchimali trapiantate in difetti della cartilagine, un ponteggio è necessario, che supporta anche la differenziazione e la proliferazione successive. L&#39;architettura del tessuto patibolo formazione di guide e permette la matrice extracellulare, prodotto dalle cellule staminali, ad espandersi. Indagini precedenti hanno mostrato che un 2% di agarosio impalcatura può sostenere lo sviluppo della cartilagine ialina stabile e non induce risposte immunitarie 3. Conservazione a lungo termine delle cellule staminali trapiantate nel MASI è fondamentale per la rigenerazione della cartilagine. L&#39;etichettatura di MSC con nanoparticelle di ossido di ferro permette di lungo termine il monitoraggio in vivo con non invasive tecniche di imaging RM 4. Questa presentazione dimostrerà le tecniche per MSC etichettatura con nanoparticelle di ossido di ferro, la generazione di cellule-agarosio costrutti e l&#39;impianto di questi costrutti nei difetti della cartilagine. I costrutti etichettati possono essere monitorati in modo non invasivo con MR-imaging.

Watch this Scientific Journal Video about Implantation of Ferumoxides Labeled Human Mesenchymal Stem Cells in Cartilage Defects at JoVE.com

Implantation of Ferumoxides Labeled Human Mesenchymal Stem Cells in Cartilage Defects

Obiettivo della presentazione è quello di dimostrare un metodo altamente riproducibile per generare matrice associata impianti di cellule staminali a difetti della cartilagine, che può essere visualizzato con la RM. Le cellule staminali sono etichettati con FDA ha approvato Ferumoxides, mescolato con agarosio, impiantati in difetti della cartilagine e ripreso con uno scanner 7T MR.

L&#39;impianto di Ferumoxides etichetta cellule staminali mesenchimali in difetti della cartilagine

The field of tissue engineering integrates the principles of engineering, cell biology and medicine towards the regeneration of specific cells and ...

implantation-ferumoxides-labeled-human-mesenchymal-stem-cells

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JoVE Journal

Biology

10.5K Views.  Medical Center, University of California San Francisco. Obiettivo della presentazione è quello di dimostrare un metodo altamente riproducibile per generare matrice associata impianti di cellule staminali a difetti della cartilagine, che può essere visualizzato con la RM. Le cellule staminali sono etichettati con FDA ha approvato Ferumoxides, mescolato con agarosio, impiantati in difetti della cartilagine e ripreso con uno scanner 7T MR.

Video: Implantation of Ferumoxides Labeled Human Mesenchymal Stem Cells in Cartilage Defects

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 di espansione degli animali siero gratuito di cordone ombelicale umano derivato cellule stromali mesenchimali (MSC) e cellule endoteliali progenitrici formanti colonia (ECFCs)

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

Ricerca

Biologia

Peptides from Phage Display Library Modulate Gene Expression in Mesenchymal Cells and Potentiate Osteogenesis in Unicortical Bone Defects

Two novel synthetic peptides accelerate bone formation and can be delivered using a collagen matrix. The aim of this study was to investigate the effects on bone repair in a unicortical defect model. Treatment of mesenchymal cells produced an increase in alkaline phosphatase activity, showed nodule formation by the cells, and increased the expression of genes for runx2, osterix, bone sialoprotein, and osteocalcin. A collagen sponge soaked with peptide promoted repair of bone defects, whereas the control was less effective. The results from this study demonstrated that mesenchymal cells treated with peptide in vitro differentiate towards osteogenesis, and, that peptides delivered in vivo using a collagen sponge promote the repair of unicortical defects.

A phage display library was used to identify peptide sequences that target bone. The objective was to investigate the effect of these peptides on mesenchymal cell differentiation and to determine their effect on bone regeneration.

Peptidi dalla libreria phage display modulare l&#39;espressione genica nelle cellule mesenchimali e potenziare Osteogenesi in difetti ossei Unicortical

Two novel synthetic peptides accelerate bone formation and can be delivered using a collagen matrix. The aim of this study was to investigate the effects ...

Isolation &#38; Characterization of Hoechstlow CD45negative Mouse Lung Mesenchymal Stem Cells

Tissue resident mesenchymal stem cells (MSC) are important regulators of tissue repair or regeneration, fibrosis, inflammation, angiogenesis and tumor formation. Taken together these studies suggest that resident lung MSC play a role during pulmonary tissue homeostasis, injury and repair during diseases such as pulmonary fibrosis (PF) and arterial hypertension (PAH). Here we describe a technology to define a population of resident lung MSC. The definition of this population in vivo pulmonary tissue using a define set of markers facilitates the repeated isolation of a well-characterized stem cell population by flow cytometry and the study of a specific cell type and function.

Isolation &amp; Characterization of Hoechstlow CD45negative Mouse Lung Mesenchymal Stem Cells

In this article we demonstrate the isolation of murine resident lung mesenchymal stem cells (lung MSC), their expansion, characterization and analysis of immunomodulatory properties.

Isolamento e caratterizzazione di Hoechst Basso CD45 Negativo Mouse cellule staminali mesenchimali del polmone

Tissue resident mesenchymal stem cells (MSC) are important regulators of tissue repair or regeneration, fibrosis, inflammation, angiogenesis and tumor ...

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells (iPSCs) Using Retroviral Vector with GFP

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has been previously shown that human somatic cells can be reprogrammed to pluripotency by ectopic expression of four transcription factors (Oct4, Sox2, Klf4 and Myc) and become induced pluripotent stem cells (iPSCs)2-4 . Like hESCs, human iPSCs are pluripotent and a potential source for autologous cells. Here we describe the protocol to reprogram human fibroblast cells with the four reprogramming factors cloned into GFP-containing retroviral backbone4. Using the following protocol, we generate human iPSCs in 3-4 weeks under human ESC culture condition. Human iPSC colonies closely resemble hESCs in morphology and display the loss of GFP fluorescence as a result of retroviral transgene silencing. iPSC colonies isolated mechanically under a fluorescence microscope behave in a similar fashion as hESCs. In these cells, we detect the expression of multiple pluripotency genes and surface markers.

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells iPSCs Using Retroviral Vector with GFP

A method to generate human induced pluripotent stem cells (iPSCs) via retrovirus-mediated ectopic expression of OCT4, SOX2, KLF4 and MYC is described. A practical way to identify human iPSC colonies based on GFP expression is also discussed.

Riprogrammare cellule somatiche umane in cellule staminali pluripotenti indotte (iPSCs) Utilizzo vettore retrovirale con GFP

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

Induzione e Analisi dei epiteliale a Mesenchymal Transizione

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal&#39;s life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.

The continuously growing mouse incisor provides a model for studying renewal of dental tissues from dental epithelial stem cells (DESCs). A robust system for consistently and reliably obtaining these cells from the incisor and expanding them in vitro is reported here.

Isolamento e Cultura della Dental epiteliali cellule staminali del topo adulto Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs include modeling pathogenesis of human genetic diseases, autologous cell therapy after gene correction, and personalized drug screening by providing a source of patient-specific and symptom relevant cells. However, there are several hurdles to overcome, such as eliminating the remaining reprogramming factor transgene expression after human iPSCs production. More importantly, residual transgene expression in undifferentiated human iPSCs could hamper proper differentiations and misguide the interpretation of disease-relevant in vitro phenotypes. With this reason, integration-free and/or transgene-free human iPSCs have been developed using several methods, such as adenovirus, the piggyBac system, minicircle vector, episomal vectors, direct protein delivery and synthesized mRNA. However, efficiency of reprogramming using integration-free methods is quite low in most cases.
Here, we present a method to isolate human iPSCs by using Sendai-virus (RNA virus) based reprogramming system. This reprogramming method shows consistent results and high efficiency in cost-effective manner.

Here, we present our established method to reprogram human somatic cells into transgene-free human iPSCs with Sendai virus, which shows consistent outcome and enhanced efficiency.

Efficiente generazione indotta umane pluripotenti cellule staminali da cellule somatiche umane con Sendai-virus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs ...

Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different lineages. A single cell transcriptome assay can be a new approach for dissecting individual variation. We have developed the single cell qRT-PCR method, and confirmed that this method works well in several gene expression profiles. In single cell level, each human embryonic stem cell, sorted by OCT4::EGFP positive cells, has high expression in OCT4, but a different level of NANOG expression. Our single cell gene expression assay should be useful to interrogate population heterogeneities.

Single cell gene expression assay is needed for understanding stem cell heterogeneities.

Profiling cellule staminali embrionali umane individuale mediante RT-PCR

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different ...

A Simple Benchtop Filtration Method to Isolate Small Extracellular Vesicles from Human Mesenchymal Stem Cells

The ultracentrifugation-based process is considered the common method for small extracellular vesicles (sEVs) isolation. However, the yield from this isolation method is relatively lower, and these methods are inefficient in separating sEV subtypes. This study demonstrates a simple benchtop filtration method to isolate human umbilical cord-derived MSC small extracellular vesicles (hUC-MSC-sEVs), successfully separated by ultrafiltration from the conditioned medium of hUC-MSCs. The size distribution, protein concentration, exosomal markers (CD9, CD81, TSG101), and morphology of the isolated hUC-MSC-sEVs were characterized with nanoparticle tracking analysis, BCA protein assay, western blot, and transmission electron microscope, respectively. The isolated hUC-MSC-sEVs&#39; size was 30-200 nm, with a particle concentration of 7.75 &#215; 1010 particles/mL and a protein concentration of 80 &#181;g/mL. Positive bands for exosomal markers CD9, CD81, and TSG101 were observed. This study showed that hUC-MSC-sEVs were successfully isolated from hUC-MSCs conditioned medium, and characterization showed that the isolated product fulfilled the criteria mentioned by Minimal Information for Studies of Extracellular Vesicles 2018 (MISEV 2018).

This protocol demonstrates how to isolate human umbilical cord-derived mesenchymal stem cells&#39; small extracellular vesicles (hUC-MSC-sEVs) with a simple lab-scale benchtop setting. The size distribution, protein concentration, sEVs markers, and morphology of isolated hUC-MSC-sEVs are characterized by nanoparticle tracking analysis, BCA protein assay, western blot, and transmission electron microscope, respectively.

Un semplice metodo di filtrazione da banco per isolare piccole vescicole extracellulari da cellule staminali mesenchimali umane

The ultracentrifugation-based process is considered the common method for small extracellular vesicles (sEVs) isolation. However, the yield from this ...

Isolation, Characterization, and Therapeutic Application of Extracellular Vesicles from Cultured Human Mesenchymal Stem Cells

Extracellular vesicles (EVs) are heterogeneous membrane nanoparticles released by most cell types, and they are increasingly recognized as physiological regulators of organismal homeostasis and important indicators of pathologies; in the meantime, their immense potential to establish accessible and controllable disease therapeutics is emerging. Mesenchymal stem cells (MSCs) can release large amounts of EVs in culture, which have shown promise to jumpstart effective tissue regeneration and facilitate extensive therapeutic applications with good scalability and reproducibility. There is a growing demand for simple and effective protocols for collecting and applying MSC-EVs. Here, a detailed protocol is provided based on differential centrifugation to isolate and characterize representative EVs from cultured human MSCs, exosomes, and microvesicles for further applications. The adaptability of this method is shown for a series of downstream approaches, such as labeling, local transplantation, and systemic injection. The implementation of this procedure will address the need for simple and reliable MSC-EVs collection and application in translational research.

The present protocol describes the differential centrifugation for isolating and characterizing representative EVs (exosomes and microvesicles) from cultured human MSCs. Further applications of these EVs are also explained in this article.

Isolamento, caratterizzazione e applicazione terapeutica di vescicole extracellulari da cellule staminali mesenchimali umane in coltura

Extracellular vesicles (EVs) are heterogeneous membrane nanoparticles released by most cell types, and they are increasingly recognized as physiological ...