Name: isolating mesangiogenic progenitor cells (mpcs) from human bone marrow
Uploaded: 2016-07-15T15:00:00.000+00:00
Duration: 9 min 53 s
Description: Watch this Scientific Journal Video about Isolating Mesangiogenic Progenitor Cells MPCs from Human Bone Marrow at JoVE.com

Die Autoren möchten sich besonders Dr. Paolo Parchi, Abteilung für Chirurgische, medizinische und molekulare Pathologie und Critical Care Medicine, Universität von Pisa, für die Bereitstellung von Knochenmarkproben und sein Know-how in der menschlichen Osteo-Vorläufern zu danken

HINWEIS: Nach der schriftlichen Zustimmung HBM Proben wurden während der orthopädischen Chirurgie für Hüftoperation erhalten. Unmittelbar nach dem Oberschenkelhals Osteotomie und vor femoralis eine 20-ml-Spritze, die 500 UI von Heparin Aufreiben, verwendet wurde frisch BM abzusaugen. Das Protokoll ist allgemein anwendbar auf jede BM Quelle betrachtet werden. 1. Isolierung von menschlichem Knochenmark mononukleären Zellen (HBM-MNU) <ol><li> Verdünnte von 5 bis 10 ml frischem BM bis 50 ml, Aufbringen Dulbeccos phosphatgepufferter Salzlösung (D-PBS) und durch Invertieren mischen. verteilen Ebenso 25 ml in zwei neue 50 ml konische Röhrchen, 25 ml D-PBS zu jedem Röhrchen und mischen durch Umdrehen. </li><li> Ermöglichen die Röhrchen für 10 min bei Raumtemperatur stehen, zur Trennung mineralischer Knochenfragmenten und Fett aus der Lösung. </li><li> Entfernen Sie vorsichtig mit einer sterilen Pasteur-Pipette und Filterzellsuspension durch 70 &amp; mgr; m Filter ohne Störung des Mineralknochenfragment Pellet Floating Fett. </li> &lt;li&gt; Stellen Sie vier 50-ml-Röhrchen mit 15 ml Medium für diskontinuierliche Dichtegradientenzentrifugation (1,077 g / ml). Stellen Sie sicher, dass dieses Medium bei Raumtemperatur ist. </li><li> Sanft legen 20 bis 25 ml verdünntem BM oben auf Dichtegradientenmedium. Diese Operation sorgfältig Zellsuspension trickle an den Rohrwänden zu lassen Mischungsschichten zu verhindern. </li><li> Durchführung Dichtegradienten Zentrifugation bei 400 xg für 30 min bei Raumtemperatur mit Bremse deaktiviert. </li><li> Sammeln Sie die weißlich Ring zwischen den beiden Phasen befindlichen Zellen mit einer sterilen Pasteurpipette und übertragen sie in ein frisches 50-ml-Tube. </li><li> Wasche die Zellen mit frischem Kulturmedium: Phenolrot-frei, niedrige Glukose (1000 mg / l) nach Dulbecco modifiziertem Eagle-Medium (DMEM), 10% (v / v) gepooltem humanem AB-Typ-Serum (PhAbs), 2 mM L-Glutamin und Antibiotika (DMEM / 10% PhAbs). Zentrifuge bei 400 × g für 5 min. </li><li> Überstand entfernen und wieder auszusetzen Pellet in 5-10 ml frischem DMEM / 10% PhAbs. </li><li> Fahren Sie mit Zellzahl.Bestimmen Sie die Anzahl der weißen Blutkörperchen durch 1: 1-Verdünnung in Trypanblau. Wenden Sie diese auf die Zählkammer und beobachten unter einem Phasenkontrastmikroskop. Ausschließen klein und perfekt abgerundeten Erythrozyten und blau gefärbten toten Zellen aus der Zellzahl. HINWEIS: Es wird dringend für ihre Leistung in MPC Recovery Bildschirm PhAbs Chargen empfohlen. PhAbs aus den USA Quellen haben die besten Ergebnisse, während die meisten Seren unterschiedlicher Herkunft in MPC Kulturen mit höheren Anteilen von MSC-ähnlichen Zellen resultierte gegeben. </li></ol> 2. Isolierung von MPL von HBM-MNU <ol><li> Set hydrophobe T-75 - Kolben mit 15 ml frischem DMEM / 10% PhAbs und lassen pH und Temperatur durch Vorinkubation in 5% CO 2 für 30 min bei 37 ° C äquilibrieren. </li><li> Seed 4 - 6 x 10 7 HBM-MNU pro Kolben und Inkubieren bei 37 ° C in 5% CO 2 für 48 Stunden. </li><li> Absaugen und Medium und nicht haftenden Zellen aus den Kolben zu verwerfen. 15 ml frischem DMEM / 10% PhAbs und Inkubation bei 37 ° Cin 5% CO 2. Pflegen Kulturen für 6 - 8 Tage Mediumwechsel alle 48 Stunden. OPTIONAL: MPC Rendite, nicht haftenden Zellen von 2,3 zu erhöhen, erneut plattiert in einem neuen Kulturflasche und gepflegt für die Primärkulturen wie beschrieben werden könnte. </li><li> Absaugen und entsorgen Medium aus den Kolben, waschen mit frischem DMEM und 2 ml freier Protease Tier Ablösen Lösung. bei 37 ° C inkubieren 5 bis 15 min (Vermeidung längerer Inkubation). </li><li> 10 ml frischem DMEM / 10% PhAbs absaugen Zellsuspension und Zentrifuge bei 400 xg für 5 min </li><li> Absaugen und Überstand verwerfen und erneut zu suspendieren Pellet in 1 bis 2 ml frischem DMEM / 10% PhAbs. Fahren Sie mit Zellzahl, wie in Schritt 1.10 beschrieben. HINWEIS: Verwenden Sie Trypsin / EDTA nicht als Reagenz löst. MPCs sind Trypsin resistent. Morphologische Screening der Kulturen, vor der Zellernte, wird dringend empfohlen, um das Vorhandensein von spindelförmigen MSC-ähnlichen Zellen zu bewerten. Im Falle beträchtliche Menge an MSC-ähnlichen Zellen sind detected, erhöhen die Reinheit des Zellprodukt durch selektives die verunreinigten Zellen zu entfernen. Dazu kann Trypsinverdaus vor durchgeführt werden, um MPC Ernte, durch Zugabe von 2 ml Trypsin / EDTA 0,05% für 2 min. Wasch Kulturen zweimal mit 5 ml DMEM / 10% PhAbs, dann gehen die Behandlung wie oben Protease ist. </li></ol> 3. Zell Characterization <ol><li> Durchflusszytometrie <ol><li> Einrichten Doppelproben von 10 5 frisch abgelösten Zellen in Waschlösung D-PBS ergänzt mit 0,5% (v / v) Rinderserumalbumin (BSA) und 0,02% (w / v) Natriumazid. Zentrifuge bei 400 × g für 5 min. ACHTUNG: Natriumazid ist giftig. </li><li> Re-suspendieren Pellets in 200 ul Waschlösung und fügen anti-CD90, anti-CD45, anti-CD73 und anti-CD31-Antikörper, konjugiert mit fluoreszierendem Farbstoffen ( &quot;Test&quot;); parallel Isotypkontrollen einrichten ( &quot;CTRL&quot;). HINWEIS: Die Mengen Antikörper-Färbung sollte durch Titration oder accor bestimmt werdending den Anweisungen des Herstellers. </li><li> Inkubieren Proben bei 4 ° C für 30 min. </li><li> Zentrifuge bei 400 × g für 5 min. Re-suspendieren Zellen in 500 &amp; mgr; l Waschlösung und erwerben mindestens 5 x 10 4 Ereignissen auf Mehrfarben Durchflusszytometer 7 9 10. </li><li> Analysieren Ergebnisse durch Dot-Plots und mit &quot;CTRL&quot; aufgezeichneten Ereignisse Quadranten zu setzen. HINWEIS: Um die Kultur als MPC Kultur der Anteil der CD73 neg CD90 neg CD45 + CD31 + Zellen zu definieren , sollte mehr als 95% betragen. Für einige spezifische Anwendungen, dh Genexpressionsanalyse hat der Wiederherstellungs cut-off bis 97 erhöht werden - 98%. </li></ol></li><li> Nestin Erkennung und F-Actin-Organisation Analyse <ol><li> Teller frisch isolierten MPCs auf Kulturkammer Dias (20.000 / cm 2). Erlauben Zellen durch Inkubation über Nacht bei 37 ° C in 5% CO 2 anhaften. </li><li> Wash-Zellen in Waschlösung und fix in 4% (w / v) Paraformaldehyd bei Raumtemperatur für 15 min. Zum Entfernen Fixiermittel Waschlösung hinzufügen, Inkubation für 2 Minuten und gießen. </li><li> Wiederholen Waschen zweimal. </li><li> Permeabilisieren Zellen in D-PBS, supplementiert mit 0,05% (v / v) Triton X-100 für 15 min bei RT. </li><li> Stopp-Reaktion durch Protein freien Signalverstärker (30 min bei RT) oder Standard-Blockierungslösung (D-PBS, ergänzt mit 3% (w / v) BSA). </li><li> Entfernen Sie Signalverstärker / Blocking-Lösung. </li><li> ADD 7 &amp; mgr; g / ml (w / v) von anti-humanen Nestin primären Antikörper und Inkubieren bei 4 ° C über Nacht in einer befeuchteten Kammer. Parallel dazu verwenden Isotypiekontrolle Antikörper keine spezifischen Fluoreszenzsignale zu bewerten. </li><li> Waschen Sie gleitet durch D-PBS Zugabe, lassen für 2 Minuten und gießen. Zweimal wiederholen. </li><li> Fügen Sie 2 &amp; mgr; g / ml (w / v) der Fluoreszenzfarbstoff konjugierten sekundären Antikörper und Inkubieren bei 4 ° C für 1 Stunde im Dunkeln. </li><li> Wash gleitet wie oben. </li><li> In fluoreszierenden Phalloidin (5 UI / ml), lassen bei RT für 30 min im Dunkeln und wasche 3mal in D-PBS. </li><li> Entfernen Sie die Kammerwände und montieren Folien in wässrigen Eindeckmittel mit Antifade Reagenz ergänzt und 4 &#39;, 6-diamidino-2-phenylindole (DAPI) für Kerne Erkennung. Gehen Sie 7 9 10 bis Bildgebung. </li></ol></li></ol> 4. Mesengenic Differenzierung von MPL <ol><li> Plate 2 x 10 4 / cm 2 frisch isolierten MPCs in TC-behandelt T75 Kulturflaschen und lassen Zellen anhaften über Nacht in DMEM / 10% PhAbs bei 37 ° C in 5% CO 2. </li><li> Ersetzen DMEM / 10% PhAbs mit Standard reduzierten Serummedium, etwa 200 &amp; mgr; l / cm 2, bestimmt für mesenchymale Stromazellen Expansion (MSC-RS - Medium). Zellen wachsen zu Konfluenz (P1-MSCs), typischerweise von 7 bis 10 Tagen von der Induktion. Aktualisieren Medium alle 2 Tage. </li><li> Absaugen und entsorgen Medium aus den Kolben, waschen mit frischem MSC-RS-Medium und 2 ml freier Protease Tier Ablösen Lösung. Inkubieren bei 3776; C für 5 bis 15 min (Vermeidung längerer Inkubation). </li><li> 10 ml frisch MSC-RS Medium absaugen Zellsuspension und Zentrifuge bei 400 xg für 5 min </li><li> Überstand entfernen und wieder auszusetzen Pellet in 1 bis 2 ml frischem MSC-RS Medium. Fahren Sie mit Zellzahl wie in Schritt 1.10 </li><li> Gehen Sie zur Subkultur sie durch Impfen von 3 bis 5 x 10 3 Zellen / cm 2. Wachsen Zellen bis zur Konfluenz (P2-MSCs). </li><li> Ernten Sie die Zellen durch Protease Verdauung von Schritt beschrieben 4.3 4.5 zu Schritt und Charakterisierung gehen Sie wie in Abschnitt 3 beschrieben. HINWEIS: Ein bestimmter Prozentsatz von CD73 + CD90 + CD45 neg CD31 neg Zellen weniger als 95% würden nur eine partielle Differenzierung zeigen und eine weitere Kulturpassage erfordern. </li><li> Platte P2-MSCs bei 2 x 10 4 / cm 2 in sechs gut TC-behandelten Platten und wachsen in MSC-RS - Medium bis zur Konfluenz. </li><li> Mark zwei Brunnen als &quot;No Diff&quot; und aktualisieren Sie MSC-RS Medium. </li><li> Mark zwei guts als &quot;Osteo&quot; und ersetzen Medium mit 200 &amp; mgr; l / cm 2 von Standard osteogenen Medium, die speziell für die Differenzierung MSC konzipiert. </li><li> Mark zwei Brunnen als &quot;Adipo&quot; und ersetzen Medium mit 200 &amp; mgr; l / cm 2 von Standard adipogenetische Medium, das speziell für die MSC - Differenzierung. </li><li> Pflegen Kulturen bei 37 ° C in 5% CO 2 von der gesamten Medien jede 48 Stunden ändern. HINWEIS: Es ist sehr kommerziell erhältlichen Standard Differenzierungsmedien für Test Reproduzierbarkeit empfohlen zu verwenden. Nach 2/3 Wochen unter Differenzierungsbedingungen, erscheinen Kalziumablagerungen in den Kulturen osteogenen induziert, während die intrazelluläre Akkumulation Lipidtröpfchen in adipogenetische induzierten Zellen zu erkennen ist. </li><li> Absaugen und entsorgen Kulturmedien waschen dann mit D-PBS. </li><li> Fix die Kulturen durch Zugabe von 1 ml von 4% (w / v) Paraformaldehyd für 15 min bei Raumtemperatur. </li><li> Zum Entfernen Fixiermittel hinzufügen 2 min D-PBS, Inkubation und gießen. Wiederholen Waschenzweimal. </li><li> Stain ein &quot;Nein Diff&quot; zusammen mit den beiden &quot;Osteo&quot; Brunnen markiert in Hydroxyapatit spezifische fluoreszierende Lösung und ein &quot;Nein Diff&quot; zusammen mit den beiden &quot;Adipo&quot; Brunnen markiert in 200 nM Nile Red-Lösung. Inkubieren für 30 Minuten bei Raumtemperatur 11,12. </li><li> Entfernen Sie Lösungen Färbung und waschen in D-PBS zweimal. </li><li> Entfernen D-PBS, fügen D-PBS , ergänzt mit 50% (v / v) Glycerin und fahren Sie mit Imaging - 7 9 10. </li></ol> 5. MPC Sphäroid Sprouting Assay <ol><li> Zur Herstellung von 3D - Sphäroiden, lag 20 ul Tropfen frisch isolierten MPC - Suspension (1,5 x 10 4 Zellen / Tropfen) auf der inneren Oberfläche der Petrischale mit Deckel. HINWEIS: Handhabung Tropfen zu ihren Bruch führen könnten, ist es sehr empfehlenswert ist, sie über zu legen. </li><li> Verwenden Sie den Deckel vorsichtig eine Petrischale mit D-PBS zu rekapitulieren hängenden Tropfen Verdunstung zu verhindern. Inkubieren bei 37 ° C in 5% CO 2über Nacht Zellen zu ermöglichen in 3D Sphäroide zu aggregieren. </li><li> Legen Sie ein dickes Gel von murinen extrazellulären Matrix (ECM) Proteine ​​durch Zugabe von 300 ul Aliquots von Standard-ECM-Proteine ​​in einem vorgekühlte 24-Well-Kulturplatte, und bei 37 ° C für 30 min. </li><li> Vorsichtig die Schalendeckel Petri umkippen und sanft die Sphäroiden mit einer sterilen Pasteur-Pipette abholen. </li><li> Lay Sphäroiden auf das ECM - Protein - Gel, fügen 700 ul Aliquots von Standard - VEGF-reiche endothelialen Zellwachstumsmedium und Inkubation bei 37 ° C in 5% CO 2. </li><li> Nach 24 Stunden und 7 Tage der Kultur, machen Sie Fotos von der 3D-Kulturen bei Vergrösserungs 4X. Auswerten von Sphäroiden sprießen die Anwendung der Bildanalyse-Software durch den radialen Abstand zwischen dem letzten eindringenden Zelle und dem Sphäroid Kante zu messen. Wiederholungsmaßnahmen entlang mindestens 20 verschiedenen Richtungen. Der mittlere Abstand wird als positiv angesehen, wenn 50 &amp; mgr; m oder mehr beträgt. </li></ol>

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The authors have no competing financial interests or other conflicts of interest.

In the last decades, MSCs have been extensively researched and pre-clinically evaluated for possible application in the treatment of various bone/articular, immunological, neurological, cardiovascular, gastrointestinal and hematological disorders14,15. The easy and inexpensive isolation of multipotent MSCs, from many different tissues, together with their lack of significant immunogenicity16, contribute to make these cells one of the most interesting cell population to be applied in cell based therapies. Nonetheless, the very low frequency in the tissue of origin represents a great limitation to the MSCs application in clinics, forcing the expansion of these cells, in vitro, before the infusion or transplantation. 
Expanded MSC cultures have revealed high grades of heterogeneity and variability17-19 making it difficult to reach a consensus about MSC production and characterization protocols. Moreover, recent investigations suggested the presence of multiple in vivo MSC ancestors in a wide range of tissues, which contribute to culture heterogeneity10,20. In fact, it has been proposed that particular culture conditions possibly select or simply promote specific sub-populations of MSCs progenitors present, in various percentages, in "crude" and unprocessed samples like bone marrow (hBM-MNCs) or adipose tissues (stromal vascular fraction)2. Thus, the variability in MSC-initiating cell populations together with the great number of different enrichment/isolation and culture protocols applied, represent a great obstacle to the definition of feasible MSC-based therapies.
A crucial factor affecting heterogeneity of MSC cultures is serum supplementation21. In our hands replacement of FBS with PhABS in primary cultures from hBM-MNCs, combined with high density seeding on hydrophobic plastics, led to the isolation of a novel highly adherent cell population with distinct biological features named MPCs5,6. We observed that the addition of small percentages of PhABS to FBS primary cultures also allowed MPC isolation, suggesting the presence of MPC inducing agents in the human serum6. At the moment, the MPC isolation/characterization protocol is a unique method available to obtain almost pure MPCs. The protocol has been carefully adjusted and it is highly reproducible for quality screening of MPC preparations before further applications.
MPCs could be used as a source for MSC production, thus limiting the variability introduced by use of unfractionated starting material. The precise definition of the multiple steps characterizing MPC mesengenic differentiation reported9 would allow synchronized mesenchymal cell expansion. Nonetheless, this latest condition could be realized exclusively applying highly purified MPC population, as a consequence the characterization of the cell products obtained by the protocol described here, results of crucial importance. This isolating method has been reported allowing MPC recovery with purity generally around 95%. However, donor/patient variability together with the variability related to the different batches of human pooled serum applied, could lead to a significant percentage of MSC-like cells co-isolated together with MPCs, under selective conditions. 
It is not clear if these "contaminating" MSC-like cells could arise from the other different in vivo progenitors described in bone marrow22 or from uncontrolled and spontaneous MPC differentiation. In any case, a consistent percentage of MSC-like cells in the MPC products nullify the possibility to applying these cells as homogeneous starting material for the MSC expansion. Thus, here it has been suggested a simple and inexpensive method, based on the MPC resistance to trypsin digestion, increasing the purity of the MPC products. Similar or even better results in purifying MPC cultures could be achieved by fluorescent or magnetic cell sorting performing CD73 and/or CD90 depletion, but significantly prolonging the process time and increasing the costs.
Moreover, MPCs showed expression of pluripotency-associated markers and Nestin, all rapidly lost during mesengenic differentiation7. Sprouting assay revealed MPC ability to invade murine ECM protein gel. Taken together these results indicate that MPCs have to be considered a more immature progenitor, retaining angiogenic potential. Nonetheless, the initial enthusiasm about mesodermal differentiation potential of MPCs is actually waning. In fact, after more than 7 years of studies on MPCs, mesengenic and angiogenic potential have been extensively described5-9, but differentiation toward any other cells of mesodermal origin is still lacking. Thus, here we propose a new, and more rigorous, definition of these cells as "Mesangiogenic Progenitor Cells", maintaining the acronym MPCs.
We also believe that most controversies about MSC angiogenic potential could be related to the heterogeneous composition of expanded cultures consisting of sub-populations of MPCs and MSCs in variable percentages23.
Finally, MPCs could also play a crucial role for the implementation of CBMPs applicable for tissue reconstruction, as these cells could also support the neo-vascularization. In fact, future studies on regeneration should take in consideration that the newly formed tissue growth should be supported by concomitant neo-angiogenesis. The co-existence of mesengenic and angiogenic potential in MPCs could significantly improve the regeneration potential of new therapeutic approaches that involve these interesting cells.

Mesenchymale Stromazellen (MSCs) sind von relevanten klinischen Wert für ihre Multilinien Differenzierungsfähigkeit und ihre Fähigkeit , die Blutbildung zu unterstützen, zu sezernieren Wachstumsfaktoren / Zytokine sowie eine Rolle in der Immunregulation 1 spielen. In der Definition von MSC-basierte Therapien Zellproduktion und Anwendung haben sich die Aufgabe gestellt umfangreichen klinischen und präklinischen Forschung 2, mit besonderem Augenmerk auf spezifische internationale Regelung für die Sicherheit und Wirksamkeit von zellbasierten Arzneimittel (CBMP) Behandlungen 3. Menschliche MSCs sind ausführlich in Medien kultiviert Ergänzungsmittel und Reagenzien von tierischen Ursprungs, wie fötalem Rinderserum (FBS) und Rinder-Trypsin enthält. Daher neben mit Infektionsrisiken im Zusammenhang mit Zellmanipulation, Patienten auch Prion-Exposition sowie immunologische Risiken im Zusammenhang mit Proteinen, Peptiden oder anderen Biomolekülen tierischen Ursprungs stellen, die nach der Zellernte und TRANS bestehen könntetation 4. Um das Problem zu umgehen, kultiviert wir dem menschlichen Knochenmark (HBM) abgeleitetes MSCs in tierfreien Medium, ersetzt FBS mit gepoolten menschlichen Typ AB-Serum (PhAbs). Unter diesen Bedingungen neben wachsenden MSCs identifizierten wir eine neuartige Zellpopulation. Diese Zellen wurden morphologisch und phänotypisch unterschiedlich von MSCs und zeigte eine ausgeprägte Genexpressionsprofil sowie charakteristische Anbau / Haftungseigenschaften. Sie behielten beide mesengenic und angiogene Potential und wurden daher mesodermalen Progenitorzellen (MPL) 5 genannt. Anschließend konnten wir selektive und reproduzierbare Kulturbedingungen zu definieren MPCs bei hohen Grad von Reinheit 6 zu erzeugen. Wir untersuchten weiter die morphologischen und biologischen Eigenschaften von MPL. Die MPL zeigte durch lange Telomere 5 gekennzeichnet Nestin-positiv, langsam im Radsport, Ki-67-negativ, und mit den Chromosomen zu sein. Sie drückten Pluripotency-assoziierten Transkriptionsfaktoren Oct-4 und Nanog statt MSC Master Regler Runx2 und Sox9 7. Phenotypisch, ausgedrückt MPCs Endoglin (CD105) auf einer niedrigeren Ebene als MSCs während mesenchymalen Marker CD73, CD90, CD166 fehlt. MPCs zeigte auch ein charakteristisches Muster von Adhäsionsmolekülen durch konsequente Expression von PECAM (CD31) , dadurch gekennzeichnet, Integrine &amp; alpha; L (CD11a), &amp; alpha; M (CD11b), &amp; agr; X (CD11c) sowie Integrin β2 (CD18) , die podosome artige Strukturen spezifisch aufrecht 8 . Im Standard - MSC Expansion Medien, MPCs prompt in MSCs durch eine Zwischenstufe differenziert mit der Aktivierung von Wnt5 / Calmodulin Zellsignal 9. MPCs behielt auch angiogene Eigenschaften, wie durch ihre Fähigkeit unter Beweis gestellt von Sphäroiden in murine extrazellulären Matrix (ECM) Protein 3D-Kulturen zu sprießen. Die angiogene Potential wurde schnell nach MPC Differenzierung entlang der mesengenic Linie verloren. Hier präsentieren wir proProtokolle optimiert zu isolieren und zu hochgereinigten MPCs von HBM Blutproben zu charakterisieren. Reproduzierbare Protokolle für MPC mesengenic und angiogene Differenzierung werden ebenfalls beschrieben.

<table><tbody><tr><td>Matrigel Basement Membrane Matrix</td><td>BD Bioscience (San Jose, CA-USA)</td><td>354230</td><td>Murine ECM proteins&lt;br/&gt; Stock Concentration: 100% (9 - 12 mg/ml)&lt;br/&gt; Final Concentration: 100%</td></tr><tr><td>Dulbecco&#039;s Phosphate-Buffered Saline (D-PBS)</td><td>Sigma (St. Louis, MO, USA)</td><td>D8537</td><td></td></tr><tr><td>70 &amp;mu;m Filters</td><td>Miltenyi Biotec (BergischGladbach, Germany)</td><td>130-095-823</td><td></td></tr><tr><td>Ficoll-Paque PREMIUM&amp;nbsp;</td><td>GE Healthcare (Uppsala, Sweden)</td><td>17-5442-03</td><td>medium for discontinuos density gradient centrifugation</td></tr><tr><td>Pooled human AB type serum (PhABS)</td><td>LONZA (Walkersville MD-USA)</td><td>14-490E</td><td>Final Concentration: 10%</td></tr><tr><td>Glutamax-I</td><td>ThermoFisher (Waltham,&amp;nbsp; MA USA)</td><td>35050-038</td><td>Stabilized L-Glutamine&lt;br/&gt; Stock Concentration: 100x&lt;br/&gt; Final Concentration: 2 mM</td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Sigma (St. Louis, MO, USA)</td><td>A8412</td><td>Stock Concentration: 7.5%&lt;br/&gt; Final Concentration: 0.5%</td></tr><tr><td>Sodium Azide</td><td>Sigma (St. Louis, MO, USA)</td><td>S8032</td><td>Final Concentration: 0.02%</td></tr><tr><td>Penicillin/Streptomycin (Pen Strep)</td><td>Gibco (Grand Island, NY, USA)</td><td>15070-063</td><td>Antibiotics&lt;br/&gt; Stock Concentration: 5,000 UI/ml penicillin, 5,000 &amp;#956;g/ml Streptomycin&lt;br/&gt; Final Concentration: 50 UI/ml penicillin, 50 &amp;#956;g/ml Streptomycin</td></tr><tr><td>T-75 culture flask for suspension cultures</td><td>Greiner Bio-one&amp;nbsp; (Frickenhausen, Germany)</td><td>658 190</td><td></td></tr><tr><td>T-75 culture flask TC treated</td><td>Greiner Bio-one&amp;nbsp; (Frickenhausen, Germany)</td><td>658170</td><td></td></tr><tr><td>TrypLE Select</td><td>ThermoFisher (Waltham,&amp;nbsp; MA USA)</td><td>12563-011</td><td>Animal-free proteases detaching solution&lt;br/&gt; Stock Concentration: 1x&lt;br/&gt; Final Concentration: 1x</td></tr><tr><td>Trypsin/EDTA</td><td>ThermoFisher (Waltham,&amp;nbsp; MA USA)</td><td>15400-054</td><td>Phenol red free&lt;br/&gt; Stock Concentration: 0.5%&lt;br/&gt; Final Concentration: 0.25%</td></tr><tr><td>anti-CD90 APC antibody&amp;nbsp; (CD90)</td><td>MiltenyiBiotec (BergischGladbach, Germany)</td><td>130-095-402</td><td>Final Concentration: 1:40</td></tr><tr><td>anti-CD45 APC-Vio770 antibody (CD45)</td><td>MiltenyiBiotec (BergischGladbach, Germany)</td><td>130-096-609</td><td>Final Concentration: 1:40</td></tr><tr><td>anti-CD73 PE antibody (CD73)</td><td>MiltenyiBiotec (BergischGladbach, Germany)</td><td>130-095-182</td><td>Final Concentration: 1:40</td></tr><tr><td>anti-CD31 PE Vio-770 antibody (CD31)</td><td>MiltenyiBiotec (BergischGladbach, Germany)</td><td>130-105-260</td><td>Final Concentration: 1:40</td></tr><tr><td>Mouse IgG1 APC antibody</td><td>MiltenyiBiotec (BergischGladbach, Germany)</td><td>130-098-846</td><td>Final Concentration: 1:40</td></tr><tr><td>Mouse IgG2a APC Vio770 antibody</td><td>MiltenyiBiotec (BergischGladbach, Germany)</td><td>130-096-637</td><td>Final Concentration: 1:40</td></tr><tr><td>Mouse IgG1 PE antibody</td><td>MiltenyiBiotec (BergischGladbach, Germany)</td><td>130-098-845</td><td>Final Concentration: 1:40</td></tr><tr><td>Mouse IgG1 PE Vio-770 antibody</td><td>MiltenyiBiotec (BergischGladbach, Germany)</td><td>130-098-563</td><td>Final Concentration: 1:40</td></tr><tr><td>Low Glucose Dulbecco&#039;s Modified Eagle Medium&amp;nbsp; (DMEM)</td><td>ThermoFisher (Waltham,&amp;nbsp; MA USA)</td><td>13-1331-82</td><td>Phenol red-free minimal essential medium&lt;br/&gt; Stock Concentration: 1,000 mg/L glucose</td></tr><tr><td>Fetal Bovine Serum&amp;nbsp; (FBS)</td><td>ThermoFisher (Waltham,&amp;nbsp; MA USA)</td><td>10500</td><td>Stock Concentration:0.2 mg/ml&lt;br/&gt; Final Concentration: 2 &amp;mu;g/ml</td></tr><tr><td>Prolong Gold antifade reagent with 4&amp;rsquo;,6-diamidino-2-phenylindole</td><td>Invitrogen (Waltham, MA,&amp;nbsp; USA)</td><td>P-36931</td><td>Aqueous mounting medium + DAPI&lt;br/&gt; Final Concentration: 1x</td></tr><tr><td>Paraformaldehyde</td><td>Sigma (St. Louis, MO, USA)</td><td>P6148</td><td>Fixative&lt;br/&gt; Final Concentration: 4%</td></tr><tr><td>LAB-TEK two-well chamber slides</td><td>Sigma (St. Louis, MO, USA)</td><td>C6682</td><td></td></tr><tr><td>Anti-Nestin antibody [clone 10C2]</td><td>Abcam (Cambridge, UK)</td><td>ab2035</td><td>Stock Concentration: 1 mg/ml&lt;br/&gt; Final Concentration: 7 &amp;mu;g/ml</td></tr><tr><td>Alexa Fluor 555 Phalloidin</td><td>ThermoFisher (Waltham,&amp;nbsp; MA USA)</td><td>A34055</td><td>Stock Concentration: 200 UI/ml&lt;br/&gt; Final Concentration: 5 UI/ml</td></tr><tr><td>Triton X-100</td><td>Euroclone (Milan, Italy)</td><td>EMR237500</td><td>Final Concentration: 0.05%</td></tr><tr><td>MesenPRO RS Medium&amp;nbsp; (MSC-RS medium)</td><td>ThermoFisher (Waltham,&amp;nbsp; MA USA)</td><td>12746-012</td><td></td></tr><tr><td>Alexa Fluor 488 anti-mouse SFX kit</td><td>ThermoFisher (Waltham,&amp;nbsp; MA USA)</td><td>A31619</td><td>Goat anti-mouse secondary antibody + Signal enhancer&lt;br/&gt; Stock Concentration: 2 mg/ml&lt;br/&gt; Final Concentration: 2 &amp;mu;g/ml</td></tr><tr><td>Pasteur Pipette</td><td>Kartell Labware&amp;nbsp; (Noviglio (MI), ITALY )</td><td>329</td><td></td></tr><tr><td>StemMACS AdipoDiff&amp;nbsp; Media</td><td>MiltenyiBiotec (BergischGladbach, Germany)</td><td>130-091-679</td><td></td></tr><tr><td>StemMACS OsteoDiff&amp;nbsp; Media</td><td>MiltenyiBiotec (BergischGladbach, Germany)</td><td>130-091-678</td><td></td></tr><tr><td>Osteoimage Bone mineralization Assay</td><td>LONZA (Walkersville MD-USA)</td><td>PA-1503</td><td>Hydroxyapatite specific fluorescent staining solution</td></tr><tr><td>50 ml Polystyrene conical tube</td><td>Greiner bio-one (Kremsm&amp;uuml;nster&lt;br/&gt; Austria)</td><td>227261</td><td></td></tr><tr><td>Nile Red</td><td>ThermoFisher (Waltham,&amp;nbsp; MA USA)</td><td>N1142</td><td>Fluorescent staining solution for lipids&lt;br/&gt; Stock Concentration: 100 mM&lt;br/&gt; Final Concentration: 200 Nm</td></tr><tr><td>Glycerin</td><td>Sigma (St. Louis, MO, USA)</td><td>G2289</td><td>Final Concentration: 50%</td></tr><tr><td>Polistirene Petri dishes</td><td>Sigma (St. Louis, MO, USA)</td><td>P5606</td><td></td></tr><tr><td>24-well plates TC-treated</td><td>Greiner Bio-one GmbH (Frickenhausen, Germany)</td><td>662160</td><td></td></tr><tr><td>Endothelial Growth Medium, EGM-2 BulletKit&amp;nbsp; (EGM-2)</td><td>LONZA (Walkersville MD-USA)</td><td>CC-3162</td><td>VEGF-rich endothelial cell growth medium</td></tr><tr><td>Leica Qwin Image Analisys Software</td><td>Leica (Wetzlar, Germany)</td><td></td><td>Image analysis software</td></tr></tbody></table>

isolating mesangiogenic progenitor cells (mpcs) from human bone marrow

In a research study aimed to isolate human bone marrow (hBM)-derived Mesenchymal Stromal Cells (MSCs) for clinical applications, we identified a novel cell population specifically selected for growth in human serum supplemented medium. These cells are characterized by morphological, phenotypic, and molecular features distinct from MSCs and we named them Mesodermal Progenitor Cells (MPCs). MPCs are round, with a thick highly refringent core region; they show strong, trypsin resistant adherence to plastic. Failure to expand MPCs directly revealed that they are slow in cycling. This is as also suggested by Ki-67 negativity. On the other hand, culturing MPCs in standard medium designed for MSC expansion, gave rise to a population of exponentially growing MSC-like cells. Besides showing mesenchymal differentiation capacity MPCs retained angiogenic potential, confirming their multiple lineage progenitor nature. Here we describe an optimized highly reproducible protocol to isolate and characterize hBM-MPCs by flow cytometry (CD73, CD90, CD31, and CD45), nestin expression, and F-actin organization. Protocols for mesengenic and angiogenic differentiation of MPCs are also provided. Here we also suggest a more appropriate nomenclature for these cells, which has been re-named as "Mesangiogenic Progenitor Cells".

Die selektiven Kulturbedingungen hier beschrieben haben , die Isolierung eines neuartigen haftend und fast monomorphic Zellpopulation als 1,0% des HBM-MNU erlaubt (0,5-2,0 x 10 6 HBM-MNU von 5 bis 10 ml frischem BM Proben) 5,6 . Wir identifizierten diese große (40 - 60 &amp; mgr; m im Durchmesser), gerundet, Ruhe, Ki-67-negativen Zellen als MPL 5. Morphologisch, werden sie durch eine ausgeprägte Spiegelei-Form mit einem dicken Kernbereich durch einen flachen , dünnen Rand zeigt viele Filopodien bei höheren Vergrößerungsleistung (weiße Pfeile in Abbildung 1 A.1) umgeben gekennzeichnet. Polar Dehnung der äußeren Zellgrenze wird häufig beobachtet (schwarze Pfeile in Abbildung 1 A.1). Solche Morphologie ist deutlich verschieden von der typischen spindelförmigen Aussehen mesenchymalen Stromazellen in Standard MSC Kulturen berichtet. Durchfluss-Zytometrie zeigte über 95% der frisch isolierten MPCs CD31 und CD45, während m auszudrückenesenchymal assoziierte Marker CD90 und CD73 13 waren nicht nachweisbar (Abbildung 1 A.2). Wir betrachten dies eingeschränkten Satz von vier Antigene für die MPL als Hinweis. Weitere Unterscheidungsmerkmale der MPCs sind punktierte F-Aktin Verteilung eine Anzahl von podosome artigen Strukturen (rot in Abbildung 1 A.3) und intensive Expression von Nestin (grün in Abbildung 1 A.3) offenbart, das nicht in den Zellen festgestellt wird gefärbt mit Isotypiekontrolle Antikörper (Daten nicht gezeigt). Die Kultivierung MPCs in Standard - RS - Medium konzipiert für MSC Expansion führt zu einer schnellen Differenzierung in exponentiell wachsenden MSC-ähnlichen Zellen (Abbildung 1 B.1). Nach zwei Passagen Zellen schließlich ihren Phänotyp von CD73 neg CD90 neg CD45 + CD31 + CD73 + zu CD90 + CD45 neg CD31 neg (Abbildung 1 B.2) wechseln. In dem Prozess, MPCs Wieder organize F-Aktin in Stressfasern während nestin Ausdruck zu einigen seltenen Zellen (Abbildung 1 B.3) beschränkt wird. MPC mesengenic Differenzierung in eine bestimmte MSC-ähnlichen Phänotyp erfolgt durch zwei verschiedene Schritte, die von verschiedenen Zell Morphologien enthüllt. Nach einer Woche in MSC RS Medium eine Restpopulation von MPC-ähnlichen Zellen noch nachweisbar innerhalb einer konfluenten Schicht von flachen, polygonalen mehrfach verzweigten Zellen (P1-MSCs in Abbildung 2 A). Ein weiterer Durchgang erforderlich , um eine fast monomorphe Kultur von spindelförmigen MSC-ähnliche Zellen (P2-MSCs in Figur 2 A) zu erhalten. Diese exponentiell wachsende Zellen können leicht unterscheiden zu Osteoblasten oder Adipozyten wenn sie für mindestens 2 Wochen in Selektionsmedien überführt, wodurch deren MSC Art bestätigt. In osteogenen induzierten Kulturen, entweder durch kolorimetrische Alizarin-S - Färbung oder spezifische fluoreszierende Farbstoffe (grün in Abbildung 2 B) erfasst werden , um Ablagerungen Kalzium kann. Nach adipogenetische Induktions Zellenzeigen Lipidtröpfchen Akkumulation als entweder durch kolorimetrische Oil Red oder fluoreszierende Nile Red - Färbung (rot in Abbildung 2 B) ergab. MPC Typisierung wurde durch Sprossung Angiogenese-Test bestätigt. MPCs zeigte ihre Fähigkeit , von 3D - Sphäroiden nach 24 Stunden VEGF-Stimulus (Abbildung 2 C) (mehr als 50 &amp; mgr; m) murine ECM - Protein - Gel einzudringen. 600 &amp; mgr; m Abstand - Nach einer Woche eindringenden Zellen wurden bei 300 festgestellt. Im Gegensatz dazu wurde Invasion Kapazität nach mesengenic Differenzierung in P2-MSCs verloren (Abbildung 2 D). <img alt="Abbildung 1" src="/files/ftp_upload/54225/54225fig1.jpg" /> Abbildung 1. Frisch isolierte MPCs haben Besondere Merkmale. Die Anzucht HBM-MNU in DMEM / 10% PhAbs für sieben Tage gibt Anlass zu einer Bevölkerung von Ruhe MPL (A) leicht von MSCs (B) </strong&gt; in Bezug auf die Morphologie (A.1, B.1, Maßstabsbalken = 100 &amp; mgr; m), Phänotyp (A.2, B.2), F-Actin-Verteilung (rot in A.3, B.3) und nestin Ausdruck (grün in A.3, B.3, Maßstabsbalken = 20 &amp; mgr; m). <a href="https://www.jove.com/files/ftp_upload/54225/54225fig1large.jpg" target="_blank">Bitte hier klicken</a> , um <a href="https://www.jove.com/files/ftp_upload/54225/54225fig1large.jpg" target="_blank">eine größere Version dieser Figur zu sehen.</a> <img alt="Figur 2" src="/files/ftp_upload/54225/54225fig2.jpg" /> Abbildung 2. MPCs Differenzieren in Standard - MSCs und zeigen Sprießen Angiogenesis in vitro. Ersatz von DMEM / PhAbs mit handelsüblichen RS - Medium konzipiert für MSC Expansion löst mesengenic Induktion von MPL. Nach einer Woche in Kultur sind wenige Rest MPL noch nachweisbar (P1-MSCs) , während eine weitere Passage in MSC-RS Medium führt zu einer Population von konfluenten MSC-ähnlichen Zellen (P2-MSCs, ein Maßstabsbalken = 100 &amp; mgr; m). P2-MSCs unterscheiden terminal in Osteozyten oder Adipozyten unter geeigneten Stimuli , wie durch Kalkablagerung (grün in B) und Lipidtröpfchen Ansammlung (Rot in B, Maßstabsbalken = 200 &amp; mgr; m) ergab, respectively. MPCs zeigen konsistente Sprießen von Sphäroiden in murine ECM - Protein - Gel (C) bei einer Differenz mit P2-MSCs (D, Maßstabsbalken = 1,0 mm). <a href="https://www.jove.com/files/ftp_upload/54225/54225fig2large.jpg" target="_blank">Bitte hier klicken</a> , um <a href="https://www.jove.com/files/ftp_upload/54225/54225fig2large.jpg" target="_blank">eine größere Version dieser Figur zu sehen.</a>

Watch this Scientific Journal Video about Isolating Mesangiogenic Progenitor Cells MPCs from Human Bone Marrow at JoVE.com

Isolating Mesangiogenic Progenitor Cells MPCs from Human Bone Marrow

Here we describe an optimized, highly reproducible protocol to isolate Mesodermal Progenitor Cells (MPCs) from human bone marrow (hBM). MPCs were characterized by flow cytometry and nestin expression. They showed the ability to give rise to exponentially growing MSC-like cell cultures while retaining their angiogenic potential.

Isolieren von Mesangiogenic Progenitorzellen (MPL) aus menschlichem Knochenmark

In a research study aimed to isolate human bone marrow (hBM)-derived Mesenchymal Stromal Cells (MSCs) for clinical applications, we identified a novel ...

isolating-mesangiogenic-progenitor-cells-mpcs-from-human-bone-marrow

Research

JoVE Journal

Developmental Biology

Isolating Mesangiogenic Progenitor Cells (MPCs) from Human Bone Marrow

8.0K Aufrufe.  University of Pisa. Here we describe an optimized, highly reproducible protocol to isolate Mesodermal Progenitor Cells (MPCs) from human bone marrow (hBM). MPCs were characterized by flow cytometry and nestin expression. They showed the ability to give rise to exponentially growing MSC-like cell cultures while retaining their angiogenic potential.

Serie: Isolating Mesangiogenic Progenitor Cells MPCs from Human Bone Marrow

An Enzymatic Method to Rescue Mesenchymal Stem Cells from Clotted Bone Marrow Samples

Mesenchymal stem cells (MSCs) - usually obtained from bone marrow - often require expansion culture. Our protocol uses clinical grade urokinase to degrade clots in the bone marrow and release MSCs for further use. This protocol provides a rapid and inexpensive alternative to bone marrow resampling. Bone marrow is a major source of MSCs, which are interesting for tissue engineering and autologous stem cell therapies. Upon withdrawal bone marrow may clot, as it comprises all of the hematopoietic system. The resulting clots contain also MSCs that are lost for expansion culture or direct stem cell therapy. We experienced that 74% of canine bone marrow samples contained clots and yielded less than half of the stem cell number expected from unclotted samples. Thus, we developed a protocol for enzymatic digestion of those clots to avoid labor-intense and costly bone marrow resampling. Urokinase - a clinically approved and readily available thrombolytic drug &#8211; clears away the bone marrow clots almost completely. As a consequence, treated bone marrow aspirates yield similar numbers of MSCs as unclotted samples. Also, after urokinase treatment the cells kept their metabolic activity and the ability to differentiate into chondrogenic, osteogenic and adipogenic lineages. Our protocol salvages clotted blood and bone marrow samples without affecting the quality of the cells. This obsoletes resampling, considerably reduces sampling costs and enables the use of clotted samples for research or therapy.

Mesenchymal stem cells are usually obtained from bone marrow and require expansion culture. When samples clot before processing, a protocol using the (enzymatic) thrombolytic drug urokinase can be applied to degrade the clot. Thus, cells are released and available for expansion culture. This protocol provides a rapid and inexpensive alternative to resampling.

Ein enzymatisches Verfahren zur mesenchymalen Stammzellen aus Knochenmarkproben Clotted Rettung

Mesenchymal stem cells (MSCs) - usually obtained from bone marrow - often require expansion culture. Our protocol uses clinical grade urokinase to degrade ...

Forschung

Entwicklungsbiologie

Isolation of Perivascular Multipotent Precursor Cell Populations from Human Cardiac Tissue

Multipotent mesenchymal stem/stromal cells (MSC) were conventionally isolated, through their plastic adherence, from primary tissue digests whilst their anatomical tissue location remained unclear. The recent discovery of defined perivascular and MSC cell marker expression by perivascular cells in multiple tissues by our group and other researchers has provided an opportunity to prospectively isolate and purify specific homogenous subpopulations of multipotent perivascular precursor cells. We have previously demonstrated the use of fluorescent activated cell sorting (FACS) to purify microvascular CD146+CD34- pericytes and vascular CD34+CD146- adventitial cells from human skeletal muscle. Herein we describe a method to simultaneously isolate these two perivascular cell subsets from human myocardium by FACS, based on the expression of a defined set of cell surface markers for positive and negative selections. This method thus makes available two specific subpopulations of multipotent cardiac MSC-like precursor cells for use in basic research and/or therapeutic investigations.

Human cardiac tissue harbours multipotent perivascular precursor cell populations that may be suitable for myocardial regeneration. The technique described here allows for the simultaneous isolation and purification of two multipotent stromal cell populations associated with native blood vessels, i.e. CD146+CD34- pericytes and CD34+CD146- adventitial cells, from the human myocardium.

Isolierung von Perivaskuläre multipotenten Vorläuferzellpopulationen aus menschlichem Herzgewebe

Multipotent mesenchymal stem/stromal cells (MSC) were conventionally isolated, through their plastic adherence, from primary tissue digests whilst their ...

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.

Isolierung der endothelialen Vorläuferzellen aus menschlichen Nabelschnurblut

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

Isolation, Culture, and Differentiation of Bone Marrow Stromal Cells and Osteoclast Progenitors from Mice

Bone marrow stromal cells (BMSCs) constitute a cell population routinely used as a representation of mesenchymal stem cells in vitro. They reside within the bone marrow cavity alongside hematopoietic stem cells (HSCs), which can give rise to red blood cells, immune progenitors, and osteoclasts. Thus, extractions of cell populations from the bone marrow results in a very heterogeneous mix of various cell populations, which can present challenges in experimental design and confound data interpretation. Several isolation and culture techniques have been developed in laboratories in order to obtain more or less homogeneous populations of BMSCs and HSCs invitro. Here, we present two methods for isolation of BMSCs and HSCs from mouse long bones: one method that yields a mixed population of BMSCs and HSCs and one method that attempts to separate the two cell populations based on adherence. Both methods provide cells suitable for osteogenic and adipogenic differentiation experiments as well as functional assays.

In this article, we present methods to isolate and differentiate bone marrow stromal cells and hematopoietic stem cells from mouse long bones. Two different protocols are presented yielding different cell populations suitable for expansion and differentiation into osteoblasts, adipocytes, and osteoclasts.

Isolation, Kultur und Differenzierung von Stromazellen Knochenmarkzellen und Osteoklasten-Vorläufer von Mäusen

Bone marrow stromal cells (BMSCs) constitute a cell population routinely used as a representation of mesenchymal stem cells in vitro. They reside within ...

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.

Isolation und tierisches Serum Freie Expansion der menschlichen Nabelschnur Derived mesenchymale Stromazellen (MSCs) und Endothelial Colony Forming Progenitorzellen (ECFCs)

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

Biologie

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

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

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

Identifizierung und Isolierung von Oligopotent und Lineage-committed myeloischen Vorläuferzellen aus Maus Knochenmark

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

Immunologie und Infektion

Isolation and Culture of Bone Marrow Mesenchymal Stem Cells from the Human Mandible

Human mesenchymal stem cells (hMSCs) have shown great potential in bone regeneration, immune modulation, and treating refractory chronic diseases. Various origins have been found to obtain hMSCs recently, while bone marrow was still considered the main source. Bone marrow-derived MSCs (BMSCs) from different donor bone sites have distinct characteristics due to microenvironmental factors. Studies have shown that BMSCs from maxillofacial bone may have greater proliferative and osteogenic capacities than BMSCs from long bones or the iliac crest. And maxillofacial BMSCs were considered more suitable for stem cell therapy in the maxillofacial tissues. The mandible, especially the ascending ramal area with sufficient marrow, was a feasible donor site for harvesting BMSCs. This study described a protocol for harvesting, isolating, and culturing human mandibular bone marrow-derived MSCs (hmBMSCs). Furthermore, immunophenotyping of hmBMSCs, proliferation assays, and in vitro induction of osteogenic, adipogenic, and chondrogenic differentiation was performed to identify the cultured cells. Applying this protocol can help the researchers successfully obtain enough high-quality hmBMSCs, which is necessary for further studies of the biological function, microenvironmental effects, and clinical applications.

The present protocol describes an efficient procedure for isolating and culturing of human mandibular bone marrow-derived mesenchymal stem cells using the whole bone marrow adherence method. The cultured cells were identified by cell proliferation assays, flow cytometry, and multilineage differentiation induction.

Human mesenchymal stem cells (hMSCs) have shown great potential in bone regeneration, immune modulation, and treating refractory chronic diseases. Various ...

Biotechnik

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.

Isolierung von Blutgefäß abgeleiteten multi Vorläufer von menschlichen Skelettmuskel

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

Isolation and Identification of Porcine Bone Marrow Mesenchymal Stem Cells and their Derived Extracellular Vesicles

With the development of stem cell therapy in translational research and regenerative medicine, bone marrow mesenchymal stem cells (BM-MSCs), as a kind of pluripotent stem cells, are favored for their instant availability and proven safety. It has been reported that transplantation of BM-MSCs is of great benefit to repairing injured tissues in various diseases, which might be related to modulating the immune and inflammatory responses via paracrine mechanisms. Extracellular vesicles (EVs), featuring a double-layer lipid membrane structure, are considered to be the main mediators of the paracrine effects of stem cells. Recognized for their crucial roles in cell communication and epigenetic regulation, EVs have already been applied in vivo for immunotherapy. However, similar to its maternal cells, most of the studies on the efficacy of transplantation of EVs still remain at the level of small animals, which is not enough to provide essential evidence for clinical translation. Here, we use density-gradient centrifugation to isolate bone marrow cells (BMC) from porcine bone marrow at first, and get porcine BM-MSCs (pBM-MSCs) by cell culture subsequently, identified by the results of observation under the microscope, induced differentiation assay, and flow cytometry. Furthermore, we isolate EVs derived from pBM-MSCs in cell supernatant by ultracentrifugation, proved by the techniques of transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and western blotting successfully. Overall, pBM-MSCs and their derived EVs can be isolated and identified effectively by the following protocols, which might be widely used in pre-clinical studies on the transplantation efficacy of BM-MSCs and their derived EVs.

This article elaborates a method to isolate and identify porcine bone marrow mesenchymal stem cells (pBM-MSCs) and extracellular vesicles (EVs) derived from them, providing a methodological basis for the pre-clinical evaluation of transplantation efficacy of BM-MSCs and their derived EVs.

With the development of stem cell therapy in translational research and regenerative medicine, bone marrow mesenchymal stem cells (BM-MSCs), as a kind of ...

Medizin

Nischenbasierte Isolierung von mesenchymalen Stammzellen aus dem Kieferknochenmark der Ratte

Technique for Isolation and Culture of Rat Jaw Bone Marrow Mesenchymal Stem Cells

Bone marrow mesenchymal stem cells (BMMSCs) are a type of stem cell with multi-directional differentiation potential. Compared with BMMSCs derived from appendicular bones, BMMSCs derived from the jaw have greater proliferative and osteogenic differentiation ability, gradually becoming important seed cells for jaw defect repair. However, the mandible has a complex bony structure and less cancellous content than appendicular bones. It is difficult to acquire a large number of high-quality jaw-derived marrow mesenchymal stem cells using traditional methods. This study presents a &#39;niche-based approach on stemness&#39; for isolating and culturing rat jaw bone marrow mesenchymal stem cells (JBMMSCs). Primary rat JBMMSCs were isolated and cultured using the whole bone marrow adherent method combined with the bone slice digestion method. The isolated cells were identified as JBMMSCs through cell morphology observation, detection of cell surface markers, and multi-directional differentiation induction. The cells extracted by this method exhibit a &#39;fibroblast-like&#39; spindle shape. The cells are long, spindle-shaped and fibroblast-like. The flow cytometry analysis shows these cells are positive for CD29, CD44, and CD90 but negative for CD11b/c, CD34, and CD45, which is congruent with BMMSCs characteristics. The cells show strong proliferation capacity and can undergo osteogenic, adipogenic, and chondrogenic differentiation. This study provides an effective and stable method for obtaining enough high-quality JBMMSCs with strong differentiation ability in a short time, which could facilitate further studies of the exploration of biological function, regenerative medicine, and related clinical applications.

Autor im Rampenlicht: Anwendung von hochreinen mesenchymalen Stammzellen aus dem Kieferknochenmark im Tissue Engineering

Mesenchymal stem cells from the jaw bone marrow have significant functions in diverse differentiation, self-renewal, and immune modulation. They have emerged as a crucial reservoir of precursor cells in gene therapy, tissue engineering, and regenerative medicine. Here, we present a unique method for isolating jaw bone marrow mesenchymal stem cells in rats.

Technik zur Isolierung und Kultivierung von mesenchymalen Stammzellen aus dem Kieferknochenmark von Ratten

Bone marrow mesenchymal stem cells (BMMSCs) are a type of stem cell with multi-directional differentiation potential. Compared with BMMSCs derived from ...