Name: three-dimensional angiogenesis assay system using co-culture spheroids formed by endothelial colony forming cells and mesenchymal stem cells
Uploaded: 2019-09-18T12:30:00
Description: Watch this Scientific Journal Video about Three-dimensional Angiogenesis Assay System using Co-culture Spheroids Formed by Endothelial Colony Forming Cells and Mesenchymal Stem Cells at JoVE.com

This research was supported by a grant (17172MFDS215) from Ministry of Food and Drug Safety, the National Research Foundation of Korea(NRF) grant funded by the Korea government(MSIP) (2017R1A2B4005463), and the Basic Science Research Program through the National Research Foundation of Korea(NRF) funded by the Ministry of Education (2016R1A6A1A03007648).

Human ECFCs were isolated from human peripheral blood as described in a previous report27. Briefly, the mononuclear cell layer was separated from the whole blood using the Ficoll-Paque Plus, and cultured in the proper medium until the endothelial-like colonies were appeared. Colonies were collected and ECFCs were isolated using CD31-coated magnetic beads. MSCs were isolated from the adherent mononuclear cell (MNC) fraction of human adult bone marrow. The study protocol was approved by the institut...

<ol>
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The present study introduce an improved in vitro angiogenesis assay system utilizing co-culture spheroids formed by two human vascular cell progenitors, ECFCs and MSCs. Co-culture spheroid system can mimic in vivo vascular sprout formation, which is accomplished by interaction and incorporation between endothelial cells and pericytes. Compared to other in vitro angiogenesis assays that reflect only ECs-mediated angiogenesis, this co-culture assay system is more representative of the multistep cascade of physiologic angio...

Approximately 500 million people worldwide are expected to benefit from angiogenesis-modulating therapy for vascular malformation-associated diseases such as rheumatoid arthritis, oculopathy, cardiovascular diseases, and tumor metastasis1. Thus, the development of drugs that control angiogenesis has become an important research area in the pharmaceutical industry. During the drug development process, in vivo animal study is necessary to explore the effects of drug candidates on physiologic functions and systemic interactions between organs. However, ethical and cost issues have increased the concerns regarding animal experiments2. Therefore, improved in vitro assay systems are needed to obtain more accurate and predictable data leading to the better decision-making before animal experiments. Current in vitro angiogenesis assays usually measure proliferation, invasion, migration, or tubular structure formation of endothelial cells (ECs) seeded in two-dimensional (2D) culture plates3. These 2D angiogenesis assays are quick, simple, quantitative, and cost-effective, and have significantly contributed to the discovery of angiogenesis-modulating drugs. However, several issues remain to be improved.
Such 2D in vitro assay systems cannot reflect complex multi-step events of angiogenesis that occurs in in vivo physiologic conditions, leading to inaccurate results that cause discrepancies between in vitro assay data and clinical trial outcomes4. 2D culture conditions also induce the change of cellular phenotypes. For example, after proliferation in 2D culture plates, ECs have a weak cellular phenotype as manifested by reduced expression of CD34 and several signals that govern cellular responses5,6. To overcome the limitations of 2D culture-based angiogenesis assay systems, three-dimensional (3D) spheroid angiogenesis assay systems have been developed. Sprouting followed by tubular structure formation from spheroids formed by ECs reflect in vivo neo-vascularization processes7,8. Thus, the 3D spheroid angiogenesis assay has been considered an effective assay system for screening potential pro- or anti-angiogenesis drugs.
Most 3D spheroid angiogenesis assays utilize only ECs, mainly human umbilical vein endothelial cells (HUVECs) or human dermal microvascular endothelial cells (HDMECs) to focus on the cellular response of ECs during angiogenesis. However, blood capillaries are composed of two cell types: ECs and pericytes. Elaborating bi-directional interaction between ECs and pericytes is critical for proper vascular integrity and function. Several diseases, such as hereditary stroke, diabetic retinopathy, and venous malformation, are associated with altered pericyte density or decreased pericyte attachment to the endothelium9. Pericytes are also known as a key element of the angiogenic process. Pericytes are recruited to stabilize newly-formed vessel structures by ECs. In this regard, mono-culture spheroid angiogenesis assay does not incorporate pericytes7,10. Therefore, co-culture spheroids formed by ECs and pericytes may provide a valuable approach to more closely mimic physiologic angiogenic events.
The present study aimed to develop a 3D co-culture spheroid angiogenesis assay with a combination of human endothelial colony forming cells (ECFCs) and mesenchymal stem cells (MSCs) to more closely reflect in vivo angiogenesis. Co-culture spheroid system as an in vitro representation assembly of a normal blood vessel was first established by Korff et al. in 200111. They combined HUVECs and human umbilical artery smooth muscle cells (HUSMCs), and demonstrated that co-culture of two mature vascular cells decreased the sprouting potential. Mature ECs (HUVECs) are known to progressively lose their ability to proliferate and differentiate, which negatively affects their angiogenesis responses12,13. Mature perivascular cells (HUSMCs) can cause endothelial cell inactivation through the abrogation of the vascular endothelial growth factor (VEGF) responsiveness11. The main difference between Korff&#8217;s and our co-culture spheroid system is the cell types used. We applied two vascular precursors, ECFCs and MSCs, to establish a proper angiogenesis assay system to screen and investigate pro-or-anti-angiogenic agents. ECFCs are the precursor of ECs. ECFCs have robust proliferation capacity compared with mature ECs14, which enable to overcome the limitation of ECs. ECFCs contribute to new vessel formation in many post-natal pathophysiologic conditions15,16,17. MSCs are pluripotent stem cells that have the capacity to differentiate into pericytes, thereby contributing to angiogenesis18,19.
In previous reports, ECFCs and MSCs showed synergistic effects on in vitro tube formation20, in vivo neo-vascularization21,22, and improved reperfusion of ischemic tissues23,24. In the present study, ECFCs and MSCs were used to form co-culture spheroids and embedded in type I collagen gel to reflect an in vivo 3D environment. Collagen is considered as a major constituents of the extracellular matrix (ECM) surrounding ECs25. The ECM plays a critical role in regulating cell behavior26. The assay protocol proposed here can be easily and quickly carried out within two days using common laboratory techniques. For effective cell tracking during the sprouting process, each cell type can be fluorescently labeled and monitored using a real-time cell recorder. The newly-established 3D co-culture spheroid angiogenesis assay system is designed to increase sensitivity for evaluating potential angiogenic modulators and to provide predictable information in advance of in vivo study.

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		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">0.05 % Trypsin EDTA (1X)</td>
			<td style="width:123px;">Gibco</td>
			<td style="width:161px;">25300-054</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Bevacizumab</td>
			<td style="width:123px;">Roche</td>
			<td style="width:161px;">NA</td>
			<td>Commercial name: Avastin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Dulbecco Modified Eagle Medium</td>
			<td style="width:123px;">Gibco</td>
			<td style="width:161px;">11885-084</td>
			<td>DMEM</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:259px;">Dulbeco&#39;s Phosphate buffered saline (10X)</td>
			<td style="width:123px;">Gibco</td>
			<td style="width:161px;">21600-010</td>
			<td>PBS (10X)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:259px;">Dulbeco&#39;s Phosphate buffered saline (1X)</td>
			<td style="width:123px;">Corning</td>
			<td style="width:161px;">21-031-CVR</td>
			<td>PBS (1X)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:259px;">Endothelial cell Growth medium MV2 kit</td>
			<td style="width:123px;">Promocell</td>
			<td style="width:161px;">C-22121</td>
			<td>ECGM-MV2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal bovine serum (FBS)</td>
			<td style="width:123px;">Atlas</td>
			<td style="width:161px;">FP-0500A</td>
			<td>FBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Gelatin</td>
			<td style="width:123px;">BD Sciences</td>
			<td style="width:161px;">214340</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">L-Glutamine&#8211;Penicillin&#8211;Streptomycin</td>
			<td style="width:123px;">Gibco</td>
			<td style="width:161px;">10378-016</td>
			<td>GPS</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:259px;">Mesenchymal stem cell growth medium-2</td>
			<td style="width:123px;">Promocell</td>
			<td style="width:161px;">C-28009</td>
			<td>MSCGM-2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Methyl cellulose</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:161px;">M0512</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">PKH26 Fluorescent Cell Linker Kits</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:161px;">MINI26</td>
			<td>PKH26</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">PKH67 Fluorescent Cell Linker Kits</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:161px;">MINI67</td>
			<td>PKH67</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Sodium Hydroxide</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:161px;">S5881</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Type I collagen gel</td>
			<td style="width:123px;">Corning</td>
			<td style="width:161px;">354236</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Vascular endothelial growth factor A</td>
			<td style="width:123px;">R&#38;D</td>
			<td style="width:161px;">293-VE-010</td>
			<td>VEGF</td>
		</tr>
	</tbody>
</table>

three-dimensional angiogenesis assay system using co-culture spheroids formed by endothelial colony forming cells and mesenchymal stem cells

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

Comparison experiments were performed using mono-culture spheroids (ECFCs-only) and co-culture spheroids (ECFCs+MSCs) to examine whether MSCs play a considerable role in ECFCs-mediated angiogenesis. Sprouting formation from each spheroid was monitored for 24 h by a real-time cell recorder that could capture the progression of angiogenic sprouting from spheroids. Angiogenic potential was quantified by counting the number of sprouts and measuring the cumulative length of sprouts generated from individual spheroids. Five ra...

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

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

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

This 3D co-culture spheroid system is designed to mimic physiologic angiogenesis. It will be effective for evaluating potential angiogenic modulators and provides predictable information in advance of in vivo studies. This system utilizes co-culture spheroids formed by two vascular progenitor cells and can be used to investigate cellular interactions, sprouting, and the tubular maturation of physiologic angiogenesis.If we can use patient cells to form co-culture spheroids, we can use this system to develop personalized treatments for abnormal angiogenesis-related diseases, such as cancer. This assay system can increase the sensitivity and efficiency of studies of angiogenesis and drug development. Begin by using 05%trypsin-EDTA to detach ECFCs and MSCs from 80 to 90%confluent cell cultures for three or five minutes in a cell culture incubator, respectively.Inactivate trypsin with fresh complete DMEM medium, and pipette the cells a few times to generate a single cell suspension. Collect the cells by centrifugation, followed by two washes with serum-free medium. After the second wash, dilute the ECFCs to a three times 10 to the six cells per milliliter of medium concentration and the MSCs to a two times 10 to the six cells per millimeter of medium concentration in individual two-milliliter microcentrifuge tubes.Pellet the cells by centrifugation, and carefully aspirate all but the last 15 to 25 microliters of supernatant from each cell sample. Next, resuspend each pellet in 250 microliters of diluent C from a fluorescent dye kit, and gently pipette the cell suspensions to ensure a complete dispersion. Add 250 microliters of freshly prepared 20-micromolar PKH67 to the tube of ECFCs.Add 250 microliters of 12-micromolar PKH26 to the tube of MSCs. Immediately mix both cell suspensions. After five minutes at room temperature with shaking, protected from light, stop the staining process with a one-minute incubation in 0.5 milliliters of FBS per tube.At the end of the incubation, collect the cells by centrifugation, and carefully remove the supernatant, followed by two washes in two milliliters of fresh complete medium per wash. Then suspend the cells in respective medium in 15-milliliter tubes. For spheroid generation, after the second wash, first dilute the cell suspensions in the appropriate concentration of endothelial cell growth medium MV2 supplemented with 20%methyl cellulose solution and 5%fetal bovine serum concentration.Next, add each cell suspension into a sterilized polystyrene rectangular reservoir. Use a multichannel pipette to add approximately 125-microliter droplets of cells onto the cover of a 150-millimeter culture plate. When all of the cells have been added, invert the cover over the bottom of a dish containing 15 milliliters of PBS, and place the plates in the cell culture incubator for 24 hours.The next day, rinse the dish covers with five milliliters of PBS into one 50-milliliter tube per dish. Rinse the covers with an additional five milliliters of PBS to collect any remaining spheroids, and pellet the spheroids by centrifugation. Carefully discard all but the last 100 to 200 microliters of supernatant, and gently tap on the wall of the tube so that the spheroids are freely suspended in the remaining supernatant.Add the appropriate volume of endothelial cell growth medium MV2 supplemented with 5%fetal bovine serum and 40%methyl cellulose solution to each tube to resuspend the spheroids at a 100 spheroids per milliliter of medium concentration. Use a one-milliliter pipette tip cut to a three-to five-millimeter diameter to gently mix the spheroid suspensions. Use a new wide-tip, one-milliliter pipette to mix the spheroid suspension solution with a neutralized type I collagen gel at a one-to-one ratio on ice.Add 900 microliters of each spheroid-suspending collagen gel solution into the wells of a pre-warmed 24-well plate. Allow the suspensions to polymerize for 30 minutes in the cell culture incubator, before covering the spheroids with 100 microliters of endothelial cell growth medium MV2 supplemented with 2.5%FBS and 50 nanograms per milliliter of VEGF to the ECFC-only spheroids and medium supplemented with FBS only to the MSC and ECFC-plus-MSC spheroid cultures. Then place each plate in a real-time cell recorder installed in a cell culture incubator, and randomly focus on five to 10 spheroids at a 10x magnification to monitor the sprouting formation of each fluorescence-labeled spheroid every hour for 24 hours.To quantify the spheroid sprouting, import the image files to ImageJ, and measure the number and length of sprouts expressing the appropriate fluorescent signal from five randomly selected spheroids per experimental group. For ECFC-plus-MSC spheroids, the number of sprouts and cumulative sprout length are significantly higher compared to those of ECFC-only spheroids at all time points. MSCs-only spheroids do not form sprouts but demonstrate an individual migration of the MSC outside of spheroids.The labeling of ECFC with green-fluorescent dye and MSC with red-fluorescent dye before combining to generate ECFC-plus-MSC spheroids demonstrates that ECFC-mediated sprout structures are covered with MSCs, suggesting that combined MSCs function as perivascular cells during sprout formation, enhancing sprout stability and durability by the tight association between two vascular cells. ECFC-plus-MSC spheroids pretreated with an angiogenesis inhibitor show a decreased sprout number and a cumulative sprout length in a dose-dependent manner compared to control ECFC-plus-MSC spheroids. In parallel experiments, ECFC-only spheroids pretreated with the inhibitor followed by stimulation with VEGF also demonstrate a decreased growth factor-induced sprout number and cumulative sprout length in a dose-dependent manner.Of note, a higher concentration of angiogenesis inhibitor is needed to inhibit ECFC-plus-MSC spheroids compared to ECFC-only spheroids. Tumor growth is significantly inhibited in high dose angiogenesis inhibitor-treated animals compared to both control-treated animals and low dose angiogenesis inhibitor-treated animals in a human xenograft tumor mouse model. The plasma concentration of bevacizumab in high dose-treated mice was 568 plus or minus 40.62 micrograms per milliliter, which was closer to the IC50 value of bevacizumab for inhibiting cumulative sprout length in ECFC-plus-MSC spheroids rather than ECFC-only spheroids.This suggests that the ECFC-plus-MSC co-culture spheroid system is suitable for predicting effective plasma concentrations in advance of an animal study. Using a wide-tip, one-mL pipette to gently mix the spheroids while they are in collagen gel on ice is important for protecting the integrity of the spheroids. We can modify this system to introduce other cell types such as immune cells and other extracellular matrices to investigate cell-matrix crosstalk during angiogenesis.

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Developmental Biology

Three-dimensional Quantification of Dendritic Spines from Pyramidal Neurons Derived from Human Induced Pluripotent Stem Cells

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are distributed along the dendrites. Their morphology is largely dependent on neuronal activity, and they are dynamic. Dendritic spines express glutamatergic receptors (AMPA and NMDA receptors) on their surface and at the levels of postsynaptic densities. Each spine allows the neuron to control its state and local activity independently. Spine morphologies have been extensively studied in glutamatergic pyramidal cells of the brain cortex, using both in vivo approaches and neuronal cultures obtained from rodent tissues. Neuropathological conditions can be associated to altered spine induction and maturation, as shown in rodent cultured neurons and one-dimensional quantitative analysis 1. The present study describes a protocol for the 3D quantitative analysis of spine morphologies using human cortical neurons derived from neural stem cells (late cortical progenitors). These cells were initially obtained from induced&#160;pluripotent stem cells. This protocol allows the analysis of spine morphologies at different culture periods, and with possible comparison between induced&#160;pluripotent stem cells obtained from control individuals with those obtained from patients with psychiatric diseases.

Dendritic spines of pyramidal neurons are the sites of most excitatory synapses in mammalian brain cortex. This method describes a 3D quantitative analysis of spine morphologies in human cortical pyramidal glutamatergic neurons derived from induced&#160;pluripotent stem cells.

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are ...

The Production of Pluripotent Stem Cells from Mouse Amniotic Fluid Cells Using a Transposon System

Induced pluripotent stem (iPS) cells are generated from mouse and human somatic cells by forced expression of defined transcription factors using different methods. Here, we produced iPS cells from mouse amniotic fluid cells, using a non-viral-based transposon system. All obtained iPS cell lines exhibited characteristics of pluripotent cells, including the ability to differentiate toward derivatives of all three germ layers in vitro and in vivo. This strategy opens up the possibility of using cells from diseased fetuses to develop new therapies for birth defects.

In this study, we generate induced pluripotent stem cells from mouse amniotic fluid cells, using a non-viral-based transposon system.

Induced pluripotent stem (iPS) cells are generated from mouse and human somatic cells by forced expression of defined transcription factors using ...

Isolation and Characterization of Mesenchymal Stromal Cells from Human Umbilical Cord and Fetal Placenta

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly gained attention because they do not pose any ethical or moral concerns. Current methods to isolate MSCs from UC yield low amounts of cells with variable proliferation potentials. Since UC is an anatomically-complex organ, differences in MSC properties may be due to the differences in the anatomical regions of their isolation. In this study, we first dissected the cord/placenta samples into three discrete anatomical regions: UC, cord-placenta junction (CPJ), and fetal placenta (FP). Second, two distinct zones, cord lining (CL) and Wharton&#39;s jelly (WJ), were separated. The explant culture technique was then used to isolate cells from the four sources. The time required for the primary culture of cells from the explants varied depending on the source of the tissue. Outgrowth of the cells occurred within 3 - 4 days of the CPJ explants, whereas growth was observed after 7 - 10 days and 11 - 14 days from CL/WJ and FP explants, respectively. The isolated cells were adherent to plastic and displayed fibroblastoid morphology and surface markers, such as CD29, CD44, CD73, CD90, and CD105, similarly to bone marrow (BM)-derived MSCs. However, the colony-forming efficiency of the cells varied, with CPJ-MSCs and WJ-MSCs showing higher efficiency than BM-MSCs. MSCs from all four sources differentiated into adipogenic, chondrogenic, and osteogenic lineages, indicating that they were multipotent. CPJ-MSCs differentiated more efficiently in comparison to other MSC sources. These results suggest that the CPJ is the most potent anatomical region and yields a higher number of cells, with greater proliferation and self-renewal capacities in vitro. In conclusion, the comparative analysis of the MSCs from the four sources indicated that CPJ is a more promising source of MSCs for cell therapy, regenerative medicine, and tissue engineering.

Here, we present a protocol for the dissection of human umbilical cord (UC) and fetal placenta sample into cord lining (CL), Wharton&#39;s jelly (WJ), cord-placenta junction (CPJ), and fetal placenta (FP) for the isolation and characterization of mesenchymal stromal cells (MSCs) using the explant culture technique.

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly ...

Transfer of Mammary Gland-forming Ability Between Mammary Basal Epithelial Cells and Mammary Luminal Cells via Extracellular Vesicles/Exosomes

Cells can communicate via exosomes, ~100-nm extracellular vesicles (EVs) that contain proteins, lipids, and nucleic acids. Non-adherent/mesenchymal mammary epithelial cell (NAMEC)-derived extracellular vesicles can be isolated from NAMEC medium via differential ultracentrifugation. Based on their density, EVs can be purified via ultracentrifugation at 110,000 x g. The EV preparation from ultracentrifugation can be further separated using a continuous density gradient to prevent contamination with soluble proteins. The purified EVs can then be further evaluated using nanoparticle-tracking analysis, which measures the size and number of vesicles in the preparation. The extracellular vesicles with a size ranging from 50 to 150 nm are exosomes. The NAMEC-derived EVs/exosomes can be ingested by mammary epithelial cells, which can be measured by flow cytometry and confocal microscopy. Some mammary stem cell properties (e.g., mammary gland-forming ability) can be transferred from the stem-like NAMECs to mammary epithelial cells via the NAMEC-derived EVs/exosomes. Isolated primary EpCAMhi/CD49flo luminal mammary epithelial cells cannot form mammary glands after being transplanted into mouse fat pads, while EpCAMlo/CD49fhi basal mammary epithelial cells form mammary glands after transplantation. Uptake of NAMEC-derived EVs/exosomes by EpCAMhi/CD49flo luminal mammary epithelial cells allows them to generate mammary glands after being transplanted into fat pads. The EVs/exosomes derived from stem-like mammary epithelial cells transfer mammary gland-forming ability to EpCAMhi/CD49flo luminal mammary epithelial cells.

This protocol describes methods for purifying, quantitating, and characterizing extracellular vesicles (EVs)/exosomes from non-adherent/mesenchymal mammary epithelial cells and for using them to transfer mammary gland-forming ability to luminal mammary epithelial cells. EVs/exosomes derived from stem-like mammary epithelial cells can transfer this cell property to cells that ingest the EVs/exosomes.

Cells can communicate via exosomes, ~100-nm extracellular vesicles (EVs) that contain proteins, lipids, and nucleic acids. Non-adherent/mesenchymal ...

The Aortic Ring Co-culture Assay: A Convenient Tool to Assess the Angiogenic Potential of Mesenchymal Stromal Cells In Vitro

Angiogenesis is a complex, highly regulated process responsible for providing and maintaining adequate tissue perfusion. Insufficient vasculature maintenance and pathological malformations can result in severe ischemic diseases, while overly abundant vascular development is associated with cancer and inflammatory disorders. A promising form of pro-angiogenic therapy is the use of angiogenic cell sources, which can provide regulatory factors as well as physical support for newly developing vasculature.
Mesenchymal Stromal Cells (MSCs) are extensively investigated candidates for vascular regeneration due to their paracrine effects and their ability to detect and home to ischemic or inflamed tissues. In particular, first trimester human umbilical cord perivascular cells (FTM HUCPVCs) are a highly promising candidate due to their pericyte-like properties, high proliferative and multilineage potential, immune-privileged properties, and robust paracrine profile. To effectively evaluate potentially angiogenic regenerative cells, it is a requisite to test them in reliable and &#34;translatable&#34; pre-clinical assays. The aortic ring assay is an ex vivo angiogenesis model that allows for easy quantification of tubular endothelial structures, provides accessory supportive cells and extracellular matrix (ECM) from the host, excludes inflammatory components, and is fast and inexpensive to set up. This is advantageous when compared to in vivo models (e.g., corneal assay, Matrigel plug assay); the aortic ring assay can track the administered cells and observe intercellular interactions while avoiding xeno-immune rejection.
We present a protocol for a novel application of the aortic ring assay, which includes human MSCs in co-cultures with developing rat aortic endothelial networks. This assay allows for the analysis of the MSC contribution to tube formation and development through physical pericyte-like interactions and of their potency for actively migrating to sites of angiogenesis, and for evaluating their ability to perform and mediate ECM processing. This protocol provides further information on changes in MSC phenotype and gene expression following co-culture.

The Aortic Ring Co-culture Assay: A Convenient Tool to Assess the Angiogenic Potential of Mesenchymal Stromal Cells In Vitro

Here, we present a novel application of the aortic ring assay where prelabelled mesenchymal cells are co-cultured with rat aorta-derived endothelial networks. This novel method allows visualization of Mesenchymal Stromal Cells (MSCs) homing and integration with endothelial networks, quantification of network properties, and evaluation of MSC immunophenotypes and gene expression.

Angiogenesis is a complex, highly regulated process responsible for providing and maintaining adequate tissue perfusion. Insufficient vasculature ...

Horizontal Whole Mount: A Novel Processing and Imaging Protocol for Thick, Three-dimensional Tissue Cross-sections of Skin

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality microscopic images is not entirely dependent upon the quality of the microscope, but also upon the methods of tissue processing, which often involve multiple critical actions or steps. Furthermore, mesenchymal cell types in the skin and other tissues represent a new challenge for tissue preparation and imaging. Here, we present a complete process, from tissue harvest to microscopy. Our technique, called &#34;horizontal whole mount,&#34; is one that novices can quickly become proficient in and that allows for antigen preservation and detection in 60-300 &#181;m-thick sections cut with a cryostat. Sections of this thickness provide enhanced visualization of tissue microarchitecture in a three-dimensional environment. In addition, the protocol preserves mesenchymal cells in a manner that enhances image quality when compared to standard cryostat or paraffin sections, thereby increasing the efficacy and reliability of immunostaining. We believe that this protocol will benefit all laboratories that visualize skin, and possibly other tissues and organs.

This work presents a novel processing and imaging protocol for thick, three-dimensional tissue cross-section analysis that enables the full exploitation of confocal imaging modalities. This protocol preserves antigenicity and represents a robust system to analyze skin histology and potentially other tissue types.

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality ...

Single-cell Photoconversion in Living Intact Zebrafish

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause disease when disrupted. Scientists have developed clever techniques to investigate characteristics and natural dynamics of these cells within intact tissue by expressing fluorescent proteins in subsets of cells. However, at times, experiments require more selected visualization of cells within the tissue, sometimes at the single-cell or population-of-cells manner. To achieve this and visualize single cells within a population of cells, scientists have utilized single-cell photoconversion of fluorescent proteins. To demonstrate this technique, we show here how to direct UV light to an Eos-expressing cell of interest in an intact, living zebrafish. We then image those photoconverted Eos+ cells 24 h later to determine how they changed in the tissue. We describe two techniques: single cell photoconversion and photoconversions of populations of cell. These techniques can be used to visualize cell-cell interactions, cell-fate and differentiation, and cell migrations, making it a technique that is applicable in numerous biological questions.

Here, we present a protocol to show how cell photoconversion is achieved through UV exposure to specific areas expressing the fluorescent protein, Eos, in living animals.

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause ...

Identification of Skeletal Muscle Satellite Cells by Immunofluorescence with Pax7 and Laminin Antibodies

Immunofluorescence is an effective method that helps to identify different cell types on tissue sections. In order to study the desired cell population, antibodies for specific cell markers are applied on tissue sections. In adult skeletal muscle, satellite cells (SCs) are stem cells that contribute to muscle repair and regeneration. Therefore, it is important to visualize and trace the satellite cell population under different physiological conditions. In resting skeletal muscle, SCs reside between the basal lamina and myofiber plasma membrane. A commonly used marker for identifying SCs on the myofibers or in cell culture is the paired box protein Pax7. In this article, an optimized Pax7 immunofluorescence protocol on skeletal muscle sections is presented that minimizes non-specific staining and background. Another antibody that recognizes a protein (laminin) of the basal lamina was also added to help identify SCs. Similar protocols can also be used to perform double or triple labeling with Pax7 and antibodies for additional proteins of interest.

The precise identification of satellite cells is essential for studying their functions under various physiological and pathological conditions. This article presents a protocol to identify satellite cells on adult skeletal muscle sections by immunofluorescence-based staining.

Immunofluorescence is an effective method that helps to identify different cell types on tissue sections. In order to study the desired cell population, ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

Derivation and Differentiation of Canine Ovarian Mesenchymal Stem Cells

Interest in mesenchymal stem cells (MSCs) has increased over the past decade due to their ease of isolation, expansion, and culture. Recently, studies have demonstrated the wide differentiation capacity that these cells possess. The ovary represents a promising candidate for cell-based therapies due to the fact that it is rich in MSCs and that it is frequently discarded after ovariectomy surgeries as biological waste. This article describes procedures for the isolation, expansion, and differentiation of MSCs derived from the canine ovary, without the necessity of cell-sorting techniques. This protocol represents an important tool for regenerative medicine because of the broad applicability of these highly differentiable cells in clinical trials and therapeutic uses.

Herein, we describe a method for the isolation, expansion, and differentiation of mesenchymal stem cells from canine ovarian tissue.

Interest in mesenchymal stem cells (MSCs) has increased over the past decade due to their ease of isolation, expansion, and culture. Recently, studies ...