The authors thank Katharina Schallmoser for providing pHPL, Eva Rohde and Nicole Hofmann for useful comments to the manuscript, Daniela Thaler and Margaretha Fr hwirth for technical assistance, Monica Farrell for linguistic editing, Uwe Lang, Margit Holzapfel and Sandra Eppich at Dept. of Obstetrics for providing the cords.
This work has been supported by the Austrian Research Foundation (FWF, grant N211-NAN to DS), the Austrian Research Promotion Agency (FFG, grant N200 to DS) and by The Adult Stem Cell Research Foundation (A.R.). A.R is a fellow of the PhD program Molecular Medicine of the Medical University of Graz.

Part 1: Setting up
1. Preparation of cell culture medium
Before starting the medium preparation collect all the materials and tools you will need. 
 Thaw 2 x 56 ml aliquots of pooled human platelet lysate (pHPL, self-made: reference 1 &amp; JoVE #1523), 10 ml of a 100x Penicillin/Streptomycin solution, 2 x 5 mL of L-Glutamine (both Sigma).
Thaw the cytokine and growth factor aliquots (VEGF, bFGF, EGF, IGF, hydrocortisone, ascorbic acid, Heparin, Amphotericin provided as 'single quots') adjusted for supplementing 1 bottle (500 mL) of EGM-2 medium (Lonza).
From the fridge take one 500 ml bottle each of (i) alpha-modified minimal essential medium (a-MEM) and (ii) endothelial basal medium (EBM).
All of the following steps are performed in a laminar flow tissue culture hood under sterile conditions.
Prepare preservative-free heparin (e.g., Biochrom) by dissolving the powder to a final concentration of 1000 IU/mL with sterile water.
MSC-Medium:
Use 500 mL of a-MEM, add 56 mL of thawed pHPL (see also reference 1 for further details) and 2 IU/mL (=224 &mu;l of stock solution) of preservative-free Heparin (avoids coagulation of the medium through clumping of the fibrinogen in the plasma) to reach a final concentration of 10% pHPL. Additionally add Penicillin (100U/mL) /Streptomycin (100&mu;g/mL) solution and 2mM of L-Glutamin (both Sigma).
Filter the medium through a 20 &mu;m-pore size vacuum filter (Millipore). Label the bottle appropriately (content, date).
ECFC-Medium:
Use one bottle (500 mL) of EBM, add the cytokine-aliquots, 56 mL of pHPL, 10 IU/ml (=1120&mu;l of stock solution) of preservative-free Heparin, Penicillin (100U/mL) /Streptomycin (100 g/mL) solution and 2mM of L-Glutamin to the basal medium and filter with a 20 &mu;l-pore size vacuum filter (Millipore). Label the bottle appropriately (content, date).
2. Sterilization of surgical instruments
Forceps, sharp pair of surgical scissors and scalpel holder must be heat sterilized ore sterile disposables.
3. Preparation of tubes for cord collection
Add 500 IU of preservative - free heparin to 50 ml polypropylene tubes (Falcon) and adjust to a final volume of 20 mL with PBS. Transfer the tubes into the delivery room.
4. Informed consent (confirmed by your local ethical committee or IRB).
Ask the parents prior to delivery if they would like to donate a piece of the cord and adequately inform them of the purposes it will serve.
5. Cord donation
After delivery and inspection of placenta and cord cut off a 10 cm piece and immediately transfer it to your tube prefilled with heparin to avoid coagulation of the remaining blood inside the cord vessels. Continue on with cell isolation as soon as possible. 
Part 2: Cell isolation
<ol>
<li>Prepare laminar flow tissue culture hood by cleaning surfaces with Incidin or 70% EtOH. Place sterile 150 cm2 cell culture plates, 75 cm cell culture flasks (both Corning), PBS, sterilized surgical instruments, cell scrapers, tube holder, 25 mL stripettes and 5 ml syringes equipped with blunt end needles appropriately in your cleaned hood.</li>
<li>Pre-warm your prepared &alpha;-MEM and EGM-2 cell culture medium to 37&deg;C in a water bath.</li>
<li>Bring the cord from the delivery room to your lab equipped with the cell culture hood. After transferring the cord into the laminar flow wash it twice in PBS (by transferring to a new plate) to get rid of contaminating blood cells. Rinse the umbilical vein after canulating it on one side with a sterile 5mL syringe equipped with a blunt end needle with PBS until the fluid coming out the other end of the cord becomes completely transparent (reddish indicates blood contamination).</li>
</ol>
ECFCs:
<ol start="4">
<li>Prepare a 75 cm2 flask (Corning), label it and fill in 15-20 mL of pre-warmed EGM-2 medium.</li>
<li>Place the cord into a new plate filled with sterile PBS. 
Take the surgical scissors and cut the umbilical cord into two pieces of about 5 cm in length (short pieces are easier to process, because the umbilical vessels are twisted along their axis).</li>
<li>Try to find the umbilical vein (large vessel), insert one blade of your surgical scissors and cut the vein longitudinally. At this point it could be helpful if an assistant can hold the already cut vein with two forceps while you cut further.</li>
<li>Place the cord piece with the cut vein in a new plate without PBS, take the cell scraper and scrape of the inner (endothelial) surface of the umbilical vein by rubbings from one end to the other. Transfer the cells to your prepared flask by cleaning the tip of the scraper in the medium. Repeat this procedure at least 10 times.</li>
<li>Close your flask and put it into an incubator (37&deg;C, 5% CO2, 95% Humidity).</li>
</ol>
MSCs:
<ol start="4">
<li>Prepare a 150 cm2 culture plate (Corning) and label it.</li>
<li>Take the piece of cord (after scraping off the endothelial layer) and cut the whole cord into very small pieces by using sterile scissors and sterile scalpel. Maximum size of the cord pieces should be 1-2 mm.</li>
<li>Transfer your cord pieces with the sterile forceps into your pre-labeled culture plate. By leaving the plate open for 5 minutes before filling up with medium, the attachment of the pieces to the plastic surface is strengthened.</li>
<li>Once the cord pieces are connected appropriately to the plastic, gently add in 30-35 mL of pre-warmed alpha modified MEM. Try to use the lowest speed to be sure that all pieces remain attached.</li>
<li>Close the plate and put it into an incubator (37&deg;C, 5% CO2, 95% Humidity).</li>
</ol>
Part 3: Cell expansion and confirmation of immune-phenotype by flow cytometry
Cell Expansion
EFCFs:
<ol><li>Check the attachment of endothelial cells the next day on an inverted microscope and exchange whole EGM-2 medium to get rid of all non-attached cells and particles.</li>
<li>Expand the cells until confluence and exchange one third of the medium twice a week. When confluence is reached, detach the cells with 0.05% Trypsin/0.7 mM EDTA and transfer them to new culture vessels. Depending on the number of cells within your T75 flask, you should choose the size and number of your new flasks to guarantee low seeding density for further expansion.</li>
<li>Exchange one third of the medium twice a week during the expansion of the cells.</li>
</ol>
MSCs:
<ol>
<li>The outgrowth of MSCs from the cord will take more time, so you can wait at least 3-4 days till you check on an inverted microscope for the appearance of spindle-shaped cells in the surroundings of the attached tissue pieces. When there is sufficient cell out-growth, the pieces tend to detach because they lose contact with the plastic.</li>
<li>After 10-12 days remove the tissue pieces, detach the MSCs from the plastic with 0.05% Trypsin/0.7 mM EDTA and transfer them to new culture vessels. Depending on the amount of cells within your plate you should decide the size and number of your new flasks to guarantee low seeding density for further expansion.</li>
<li>Exchange one third of the medium twice a week during the expansion of the cells.</li>
</ol>
Flow cytometry
Equipment needed:
Flow cytometer (BD FACSCalibur), micronic tubes (Corning), tube holder, sheep serum (blocking reagent), Eppifuge (Eppendorf), monoclonal antibodies 
 MSCs: CD45, CD14, CD19, HLA-DR, CD31, CD73, CD90, CD105, isotype controls 
 ECFCs: CD45, CD14, CD19, HLA-DR, CD31, CD34, CD90, CD105, CD144, CD146, isotype controls
<ol>
<li>After reaching confluence, detach the cell with 0.05% Trypsin/0.7 mM EDTA and centrifuge at 300g for 7 min at 4&deg;C.</li>
<li>Resuspend the cells to a concentration of 1x106 cells/mL in modified PBS and block unspecific antibody binding with 10% v/v sheep serum for 20 min at 4&deg;C.</li>
<li>Label the micronic tubes and add the appropriate concentration of fluorescent labeled antibodies.</li>
<li>Add 50 &mu;l of cell suspension (blocked) to each tube and incubate for 30 min at 4&deg;C.</li>
<li>Wash the cells by adding modified PBS to remove non-binding antibodies.</li>
<li>Go with your labeled cell to the flow cytometer and perform the analysis.</li>
</ol>
Part 4: ECFC colony growth, fixation, staining and hierarchy analysis
<ol>
 <li>Seed the virtually pure harvested ECFCs at very low plating density of 3 or 10 cells per square centimeter in cell culture plates to allow for single colony growth.</li>
<li>Exchange one third of the medium twice a week.</li>
 <li>After 14 days (end of culture) remove medium, wash twice with PBS and fix the colonies with ice cold fixation solution (Acetone : Methanol = 3 : 2) for 15 minutes in the refrigerator. </li>
<li>Discard the fixation solution and leave the plates open to dry for 10 minutes. </li>
 <li>Rehydrate the cells with distilled water for 10 minutes. </li>
<li>Add Harris Hematoxylin solution and stain the fixed colonies for 12 minutes. </li>
 <li>Remove H matoxylin solution and rinse the plates with tap water to get rid of the remaining staining solution. </li>
<li>Take photos of each colony by using a stereomicroscope and import *.jpg or *.tif format files onto your computer. </li>
 <li>Open the photos with ImageJ software and analyze the accurate cell number of each colony as described in the animation. </li>
 <li>Open the ImageJ software and import a colony image using the &#x2018;File\Open&#x2019; function in the pull down menu. </li>
 <li>Proceed by using the &#x2018;Process\Subtract Background&#x2019; function</li>
 <li>Use the &#x2018;*elliptical* or brush selection&#x2019; function in the ImageJ menu to mark the colony margin.</li>
 <li>Clear the surrounding image using the &#x2018;Edit\Clear Outside&#x2019; function and crop the picture by selecting &#x2018;Image\Crop&#x2019;. </li>
 <li>Adjust the threshold manually using the &#x2018;Image\Adjust\Threshold&#x2019; function, so that all cells are completely colored in red. </li>
<li>Count the cell number of the colony by selecting &#x2018;Analyze\Analyze Particles&#x2019;. Choose appropriate settings (optimized for each cell type) and select &#x2018;OK&#x2019;. The results box, including cell count and a new image containing the corresponding outlines of the counted cells will appear. 
<img src="/files/ftp_upload/1525/1525op2.jpg" alt="Figure 1" /></li>
</ol>

<ol>
	<li>Schallmoser, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+platelet+lysate+can+replace+fetal+bovine+serum+for+clinical-scale+expansion+of+functional+mesenchymal+stromal+cells.">Human platelet lysate can replace fetal bovine serum for clinical-scale expansion of functional mesenchymal stromal cells.</a> Transfusion. 47, 1436-1446 (2007).</li><li>Reinisch, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Humanized+system+to+propagate+cord+blood-derived+multipotent+mesenchymal+stromal+cells+for+clinical+application.">Humanized system to propagate cord blood-derived multipotent mesenchymal stromal cells for clinical application.</a> Regen Med. 2, 371-382 (2007).</li><li>Reinisch, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Humanized+large-scale+expanded+endothelial+colony-forming+cells+function+in+vitro+and+in+vivo.">Humanized large-scale expanded endothelial colony-forming cells function in vitro and in vivo.</a> Blood. 113, 6716-6725 (2009).</li><li>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=Phenotypic+characterization+and+preclinical+production+of+human+lineage-negative+cells+for+regenerative+stem+cell+therapy.">Phenotypic characterization and preclinical production of human lineage-negative cells for regenerative stem cell therapy.</a> Transfusion. 45, 315-326 (2005).</li></ol>

The authors don't have any financial conflicts of interest to declare.

There is a variety of sources from which to get MSCs or ECFCs including bone marrow, adipose tissue, cord blood, etc.
It is necessary to get both types of progenitors from the same source to directly compare the involvement of the different cell types in processes like tissue and vessel regeneration or contribution to tumor progression.
The ease with which to isolate and subsequently study autologous pairs of ECFCs and MSCs from the same donor makes this method ad...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>alpha modified MEM</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>L-GLutamin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin/Streptomycin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>EBM</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Single cytokine quots</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>75 cm2 flasks</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>150 cm2 plates</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Cell scraper</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin/EDTA</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Monoclonal antibodies</td>
<td>&nbsp;</td>
<td>Becton Dickenson</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>PBS</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>50 ml tubes</td>
<td>&nbsp;</td>
<td>Falcon</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>500 mL filter 20 &mu;m pore size </td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Preservative free heparin</td>
<td>&nbsp;</td>
<td>Biochrom</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
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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.

Watch this Scientific Journal Video about 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

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 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 ...

isolation-animal-serum-free-expansion-human-umbilical-cord-derived

Research

JoVE Journal

Biology

15.4K Views.  Medical University of Graz, Austria. Questo protocollo descrive l'isolamento e la successiva espansione delle cellule stromali mesenchimali e le cellule endoteliali che formano colonie senza l'uso di siero animale per generare le coppie a fini di trapianto autologo sperimentali.

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

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.

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

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.

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

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.

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.

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 ...

Feeder-Free Adaptation, Culture and Passaging of Human IPS Cells using Complete KnockOut Serum Replacement Feeder-Free Medium

The discovery in 2006 that human and mouse fibroblasts could be reprogrammed to generate iPS cells 1-3 with qualities remarkably similar to embryonic stem cells has created a valuable new source of pluripotent cells for drug discovery, cell therapy, and basic research.
GIBCO media and reagents have been at the forefront of pluripotent stem cell research for years. Knockout DMEM supplemented with Knockout Serum Replacement is the media of choice for embryonic stem cell growth and now iPS cell culture 3-9. This gold standard media system can now be used for feeder-free culture with the addition of Knockout SR Growth Factor Cocktail.
Traditional human ES and iPS cell culture methods require the use of mouse or human fibroblast feeder layers, or feeder-conditioned medium. These culture methods are labor-intensive, hard to scale and it is difficult to maintain hiPS cells undifferentiated due to the undefined conditions. Invitrogen has developed Knockout SR Growth Factor Cocktail to allow you to easily transition your hiPS cell cultures to feeder-free while still maintaining your use of Knockout SR.

The following protocol provides instruction for adapting human induced Pluripotent Stem (iPS) Cells to feeder-free culture using complete KnockOut Serum Replacement Feeder-Free medium (KSR-FF). Once adapted, instructions for continual maintenance are also provided.

The discovery in 2006 that human and mouse fibroblasts could be reprogrammed to generate iPS cells 1-3 with qualities remarkably similar to embryonic stem ...

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

Longstanding views of new blood vessel formation via angiogenesis, vasculogenesis, and arteriogenesis have been recently reviewed1. The presence of circulating endothelial progenitor cells (EPCs) were first identified in adult human peripheral blood by Asahara et al. in 1997 2 bringing an infusion of new hypotheses and strategies for vascular regeneration and repair. EPCs are rare but normal components of circulating blood that home to sites of blood vessel formation or vascular remodeling, and facilitate either postnatal vasculogenesis, angiogenesis, or arteriogenesis largely via paracrine stimulation of existing vessel wall derived cells3. No specific marker to identify an EPC has been identified, and at present the state of the field is to understand that numerous cell types including proangiogenic hematopoietic stem and progenitor cells, circulating angiogenic cells, Tie2+ monocytes, myeloid progenitor cells, tumor associated macrophages, and M2 activated macrophages participate in stimulating the angiogenic process in a variety of preclinical animal model systems and in human subjects in numerous disease states4, 5. Endothelial colony forming cells (ECFCs) are rare circulating viable endothelial cells characterized by robust clonal proliferative potential, secondary and tertiary colony forming ability upon replating, and ability to form intrinsic in vivo vessels upon transplantation into immunodeficient mice6-8. While ECFCs have been successfully isolated from the peripheral blood of healthy adult subjects, umbilical cord blood (CB) of healthy newborn infants, and vessel wall of numerous human arterial and venous vessels 6-9, CB possesses the highest frequency of ECFCs7 that display the most robust clonal proliferative potential and form durable and functional blood vessels in vivo8, 10-13. While the derivation of ECFC from adult peripheral blood has been presented14, 15, here we describe the methodologies for the derivation, cloning, expansion, and in vitro as well as in vivo characterization of ECFCs from the human umbilical CB.

Endothelial colony forming cells (ECFCs) are circulating endothelial cells with robust clonal proliferative potential that display intrinsic in vivo vessel forming ability. Phenotypic and functional characterization of outgrowth endothelial cells derived from CB are important to identify and isolate bona fide ECFCs for potential clinical application in repairing damaged tissues.

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

Isolation and Differentiation of Stromal Vascular Cells to Beige/Brite Cells

Brown adipocytes have the ability to uncouple the respiratory chain in mitochondria and dissipate chemical energy as heat. Development of UCP1-positive brown adipocytes in white adipose tissues (so called beige or brite cells) is highly induced by a variety of environmental cues such as chronic cold exposure or by PPAR&gamma; agonists, therefore, this cell type has potential as a therapeutic target for obesity treatment. Although most immortalized adipocyte lines cannot recapitulate the process of "browning" of white fat in culture, primary adipocytes isolated from stromal vascular fraction in subcutaneous white adipose tissue (WAT) provide a reliable cellular system to study the molecular control of beige/brite cell development. Here we describe a protocol for effective isolation of primary preadipocytes and for inducing differentiation to beige/brite cells in culture. The browning effect can be assessed by the expression of brown fat-selective markers such as UCP1.

Primary white preadipocytes isolated from white adipose tissues in mice can be differentiated into beige/brite cells. Presented here is a reliable cellular model system to study the molecular regulation of "browning" of white fat.

Brown adipocytes have the ability to uncouple the  respiratory chain in mitochondria and dissipate chemical energy as heat.  Development of UCP1-positive ...

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.

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

Isolation of Murine Valve Endothelial Cells

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and surrounded by a monolayer of valve endothelial cells (VECs). VECs play essential roles in establishing the valve structures during embryonic development, and are important for maintaining life-long valve integrity and function. In contrast to a continuous endothelium over the surface of healthy valve leaflets, VEC disruption is commonly observed in malfunctioning valves and is associated with pathological processes that promote valve disease and dysfunction. Despite the clinical relevance, focused studies determining the contribution of VECs to development and disease processes are limited. The isolation of VECs from animal models would allow for cell-specific experimentation. VECs have been isolated from large animal adult models but due to their small population size, fragileness, and lack of specific markers, no reports of VEC isolations in embryos or adult small animal models have been reported. Here we describe a novel method that allows for the direct isolation of VECs from mice at embryonic and adult stages. Utilizing the Tie2-GFP reporter model that labels all endothelial cells with Green Fluorescent Protein (GFP), we have been successful in isolating GFP-positive (and negative) cells from the semilunar and atrioventricular valve regions using fluorescence activated cell sorting (FACS). Isolated GFP-positive VECs are enriched for endothelial markers, including CD31 and von Willebrand Factor (vWF), and retain endothelial cell expression when cultured; while, GFP-negative cells exhibit molecular profiles and cell shapes consistent with VIC phenotypes. The ability to isolate embryonic and adult murine VECs allows for previously unattainable molecular and functional studies to be carried out on a specific valve cell population, which will greatly improve our understanding of valve development and disease mechanisms.

The ability to isolate heart valve endothelial cells (VECs) is critical for understanding mechanisms of valve development, maintenance, and disease. Here we describe the isolation of VECs from embryonic and adult Tie2-GFP mice using FACS that will allow for studies determining the contribution of VECs in developmental and disease processes.

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and ...

Isolation of CD146+ Resident Lung Mesenchymal Stromal Cells from Rat Lungs

Mesenchymal stromal cells (MSCs) are increasingly recognized for their therapeutic potential in a wide range of diseases, including lung diseases. Besides the use of bone marrow and umbilical cord MSCs for exogenous cell therapy, there is also increasing interest in the repair and regenerative potential of resident tissue MSCs. Moreover, they likely have a role in normal organ development, and have been attributed roles in disease, particularly those with a fibrotic nature. The main hurdle for the study of these resident tissue MSCs is the lack of a clear marker for the isolation and identification of these cells. The isolation technique described here applies multiple characteristics of lung resident MSCs (L-MSCs). Upon sacrifice of the rats, lungs are removed and rinsed multiple times to remove blood. Following mechanical dissociation by scalpel, the lungs are digested for 2-3 hr using a mix of collagenase type I, neutral protease and DNase type I. The obtained single cell suspension is subsequently washed and layered over density gradient medium (density 1.073 g/ml). After centrifugation, cells from the interphase are washed and plated in culture-treated flasks. Cells are cultured for 4-7 days in physiological 5% O2, 5% CO2 conditions. To deplete fibroblasts (CD146-) and to ensure a population of only L-MSCs (CD146+), positive selection for CD146+ cells is performed through magnetic bead selection. In summary, this procedure reliably produces a population of primary L-MSCs for further in vitro study and manipulation. Because of the nature of the protocol, it can easily be translated to other experimental animal models.

Isolation of CD146+ Resident Lung Mesenchymal Stromal Cells from Rat Lungs

This protocol describes an isolation technique for obtaining primary lung resident mesenchymal stromal cells from rats, through the use of enzymatic digestion, density gradient separation, plastic adherence and CD146+ magnetic bead selection.

Mesenchymal stromal cells (MSCs) are increasingly recognized for their therapeutic potential in a wide range of diseases, including lung diseases. Besides ...