The authors wish to thank Shonna Johnston, Claire Cryer, Fiona Rossi and Will Ramsay at the University of Edinburgh and Alison Logar and Megan Blanchard at the University of Pittsburgh for their expert assistance with flow cytometry. We also wish to thank Anne Saunderson and Lindsay Mock for their help with obtaining human tissues. Human adult and fetal heart tissue samples were procured with full ethics permission of the NHS Scotland Tayside Committee on Medical Research Ethics and the NHS Lothian Research Ethics Committee (REC08/S1101/1) respectively. This work was supported by grants from the Medical Research Council (BP), British Heart Foundation (BP), Commonwealth of Pennsylvania (BP), Children's Hospital of Pittsburgh (BP), National Institute of Health R01AR49684 (JH) and R21HL083057 (BP), and the Henry J. Mankin Endowed Chair at University of Pittsburgh (JH). JEB was supported by a British Heart Foundation Centre of Research Excellence doctoral training award (RE/08/001/23904). WC was supported in part by an American Heart Association predoctoral fellowship (11PRE7490001).

1. Processing of Human Cardiac Sample
<ol>
	<li>Ensure that all fluids, containers, instruments, and the dedicated operational area are sterile.</li>
	<li>Place the cardiac tissue sample (procured by the tissue bank or surgical team) in storage medium composed of chilled Dulbecco&#39;s modified Eagle medium (DMEM) containing 20% fetal bovine serum (FBS) and 1% penicillin-streptomycin (P/S) on ice for transportation3.</li>
	<li>Remove the cardiac sample from the storage medium and wash with a washing medium composed of phosphate-buffered saline (PBS) supplemented with 2% FBS and 1% P/S. When handling the sample, use fine tipped forceps to grasp large vessels or pericardium and minimise crushing of the myocardium.</li>
	<li>Submerge the sample in washing medium in a petri dish and remove the pericardium, endocardium and large blood vessels with sterilized iris scissors and fine-tipped forceps as described3.</li>
	<li>Cut the remaining myocardium into small pieces of approximately 1 mm3 using a single side razor blade or a pair of sharp iris scissors. Note: Highest cell yields will be obtained if samples are processed immediately. If a delay is unavoidable, storage of the processed cardiac tissue in the fresh storage medium (&#62;5 ml per 1 cm3 tissue) at 4 &#176;C for up to 72 hr is possible, however presumably with an associated decline in the cell yield.</li>
</ol>
2. Digestion of the Tissue and Isolation of Cells
<ol>
	<li>Freshly make up the digestion solution comprising 1.5 ml each of collagenase I, collagenase II, and collagenase IV (all at 0.5 mg/ml in DMEM) and warm to 37 &#176;C in a waterbath. 
	Note:Volume and concentration of collagenases are suitable for samples of approximately 1 cm3.</li>
	<li>Filter the suspended tissue pieces through a 100 &#181;m strainer and wash twice with PBS. Transfer the tissue pieces to a sterile 30 ml container with a tightly sealed lid. 
	Note: Short wide pots rather than tall narrow tubes give better results. Alternatively, put tissue pieces in a sterile 50 ml tube and place horizontally if a 30 ml container is not available.</li>
	<li>Add all the digestion solutions (4.5 ml total) and place the container within a sealed plastic bag in a 37 &#176;C shaking water bath set to 120 rpm. Alternatively, seal the container with paraffin film and place in a heated orbital shaker set to 120 rpm.</li>
	<li>After 15 min, remove and invert the pot three times before replacing for a further 15 min. Remove and quench the collagenases with 5 ml of DMEM supplemented with 20% FBS and 1% P/S.</li>
	<li>Triturate the digest by gently pipetting ten to twenty times with a 10 ml serological pipette to break up any tissue clumps. Filter the suspension sequentially through 100 &#181;m, 70 &#181;m and finally 40 &#181;m cell strainers to remove undigested materials and obtain a single cell suspension. 
	Note: Do not rinse between cell strainers to avoid cell debrisgenerated by the enzyme digestion process.</li>
	<li>Centrifuge the cell suspension at 200 x g for 4 min, carefully decant the supernatant and re-suspend the pellet in 1 ml of red cell lysing buffer for 2 min at room temperature before diluting with 4 ml of washing medium.</li>
	<li>Repeat the centrifugation step and re-suspend the pellet in 1 ml of washing medium. Mix well and take 10 &#181;l of the cell suspension for cell counting.</li>
	<li>Mix 10 &#181;l of the cell suspension with 10 &#181;l of Trypan blue stain and place 10 &#181;l of the mixture on a standard hemocytometer for cell counting. Count the number of bright, round cells present in the four corner squares (each subdivided into sixteen small squares) and divide by 4 to get the mean per corner square. Multiply the mean by 2 to account for the stain dilution factor and then by 104 to obtain the total number of cells per 1 ml of sample.</li>
</ol>
3. Cell Labelling and Sorting
<ol>
	<li>Add 50 &#181;l of mouse serum to the cell suspension and incubate at 4 &#176;C for 20 min to block non-specific antibody binding.</li>
	<li>Centrifuge the cell suspension with mouse serum at 200 x g for 4 min, remove supernatant, and re-suspend in washing medium at a concentration of 1 x 106 cells per 100 &#181;l.</li>
	<li>Aliquot 50 &#181;l each of the cell suspension for the isotype and unstained controls into two polystyrene round bottom flow cytometry tubes then place the remaining volume into a third tube for multi-color staining.</li>
	<li>Add CD34-PE, CD45-APC-Cy7, CD56-PE-Cy7, CD144-PerCP-Cy5.5, and CD146-AF647 antibodies (all 1:100) to the single cell suspension for multi-color staining. For the isotype control, add equivalent volumes of PE-, APC-Cy7-, PE-Cy7-, PerCP-Cy5.5- and AF647-conjugated isotype antibodies. Gently pipette to mix the antibodies with the cells and incubate all tubes at 4 &#176;C for 20 min in the dark.</li>
	<li>Prepare compensation control beads by adding one drop of positive beads to 100 &#181;l of washing medium in five polystyrene round bottom flow-cytometry tubes. Add CD34-PE, CD45-APC-Cy7, CD56-PE-Cy7, CD144-PerCP-Cy5.5, and CD146-AF647 antibodies (all 1:100) individually to each of the 5 tubes. Incubate all tubes at 4 &#176;C for 20 min in the dark.</li>
	<li>During the incubation, prepare cell collection tubes each with 500 &#181;l of Endothelial Growth Medium-2 (EGM-2) culture medium and place at 4 &#176;C.</li>
	<li>Following antibody incubation, add 5 ml of washing medium to wash cells and beads in order to remove unbound antibody. Centrifuge all tubes at 200 x g for 4 min. Repeat the washing and centrifugation step. Carefully decant the supernatant and pipette gently when re-suspending cells.</li>
	<li>Re-suspend in washing medium at a concentration of approximately 1 x 106 cells per 250 &#181;l. Re-suspend the beads in 100 &#181;l of washing medium.</li>
	<li>Transport all cell and bead suspensions to the cell sorter on ice in the dark. Add 1 drop of negative beads to the positive bead compensation control tubes and use these to set the fluorescence compensation.</li>
	<li>Run unstained control cells according to manufacturer&#39;s instructions to establish the background fluorescence and set the voltages to the following: FSC 150V; SSC 290V; V450/50 350V; V710/50 620V; B530/30 400V; B710/50 530V; YG780/60 460; R670/30 480V; R780/60 460V. Run isotypes controls according to manufacturer&#39;s instructions to establish the background fluorescence thresholds related to non-specific binding. Run the multi-color stained sample and collect the pericyte and adventitial cell populations into the collection tubes (Figure 1). 
	NOTE: Optimal viable cell yields are approximately 3% of the total live cell dissociation for pericytes and 4% for adventitial cells.</li>
</ol>
4. Cell Culture
<ol>
	<li>Select suitable size culture plates according to the outcome of the FACS sorting. Add 100 &#181;l of sterile 0.2% gelatin solution per cm2 of growth area and agitate manually to coat the entire wells. Incubate plates at 4 &#176;C for 10 min and remove gelatin solution completely. Keep collected cells on ice whilst preparing culture plates. 
	Note: Freshly sorted pericytes and adventitial cells grow well when seeded at a density of 30,000 to 40,000 cells/cm2 on gelatin-coated culture surface.</li>
	<li>Centrifuge freshly collected cells at 200 x g for 4 min and gently re-suspend the cell pellet in an appropriate amount of EGM-2.Seed the cells on gelatin-coated plates. 
	Note: The actual number of cells to be seeded per well and the amount of EGM-2 to be added depend on the plate selection. For example, 0.5 ml and 1 ml EGM-2 are needed per well for 48- and 24-well plates respectively.</li>
	<li>Exchange EGM-2 for perivascular cell growth medium (DMEM supplemented with 20% FBS and 1% P/S) for both pericytes and adventitial cells once cells have settled and adhered to the plate (after at least 72 hr). Change the media every 72 hr from then on. Carry out initial and all subsequent incubations in 5% CO2 and at 37 &#176;C.</li>
	<li>Dissociate cells using 0.05% Trypsin EDTA when pericytes and adventitial cells reach 80 to 90% confluence. Quench with 20% FBS in PBS, centrifuge at 200 x g for 4 min, re-suspend in perivascular cell growth medium, and then passage the cells at a 1:3 ratio onto uncoated polystyrene culture plates. From Passage 2 onwards, passage cells at a 1:5 ratio (or at approximately 7,000 cells/cm2) to uncoated polystyrene culture plates or flasks.</li>
</ol>

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W., Corselli, M., Andriolo, G., Lazzari, L., P&#233;ault, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perivascular+multipotent+progenitor+cells+in+human+organs.">Perivascular multipotent progenitor cells in human organs.</a> Annals of the New York Academy of Sciences. 1176, 118-123 (2009).</li><li>Kang, S. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+perivascular+localization+of+mesenchymal+stem+cells+from+mouse+brain.">Isolation and perivascular localization of mesenchymal stem cells from mouse brain.</a> Neurosurgery. 67 (3), 711-720 (2010).</li><li>Chen, C. 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The authors have no conflict to disclose.

Increasing evidence supports a limited regenerative capacity of the adult human heart after injury. Identification and characterization of native precursor cells responsible for such regenerative responses in injured hearts are critical for both the understanding of associated mechanisms and signalling pathways and the development of approaches to utilize these cells therapeutically.
Previous protocols have described the isolation of perivascular precursor cell subsets from human skeletal musc...

The heart has long been considered a post-mitotic organ. However, recent studies have demonstrated the presence of limited cardiomyocyte turnover in adult human hearts1. Native stem/progenitor cells with cardiomyocyte differentiation potential have also been identified within the myocardium in adult rodent and human hearts, including Sca-1+, c-kit+, cardiosphere-forming, and most recently, perivascular precursor cells2,3. These cells represent attractive candidates for therapies aimed at enhancing cardiac repair/regeneration through cell transplantation or stimulation of in-situ proliferation.
Mesenchymal stem/stromal cells (MSC) have been isolated from almost every human tissue4,5 Clinical trials of the therapeutic applications of MSC have been carried out for multiple pathological conditions such as cardiovascular repair6, graft-versus-host-disease7, and liver cirrhosis8. Beneficial effects have been attributed to the ability of MSCs to: home to sites of inflammation9; differentiate into different cell types10; secrete pro-reparative molecules11; and modulate host immune responses12. The isolation of MSCs has traditionally relied on their preferential adherence to plastic substrates. However, the resulting population of cells is typically markedly heterogenous13. By using fluorescent activated cell sorting (FACS) with a combination of key perivascular cell markers, we have been able to isolate and purify a multipotent MSC-like precursor population (CD146+/CD31-/CD34-/CD45-/CD56-) from multiple human tissues including adult skeletal muscle and white fat14.
Perivascular cell populations in various non-cardiac tissues have been shown to have stem/progenitor cell properties and are being investigated for clinical use in the cardiovascular setting. Pericytes, one of the most well-known perivascular cell subsets, are a heterogeneous population that play several pathophysiological roles including in the development of new vessels15, the regulation of blood pressure16, and maintenance of vascular integrity17,18. As shown in multiple tissues, specific subsets of pericytes natively express MSC antigens and sustain their MSC-like phenotypes in primary culture after FACS purification14. Moreover, these cells stably maintain their long-term phenotypes within culture and exhibit multi-lineage differentiation potential, similar to MSCs19,20. These results suggest that pericytes are one of the origins of the elusive MSC14. The therapeutic potential of pericytes has been demonstrated with a reduction in myocardial scarring and enhanced cardiac function following transplantation into ischemically injured hearts21. Recently, we successfully purified pericytes from the human myocardium and demonstrated their MSC-like phenotypes and multipotency (adipogenesis, chondrogenesis and osteogenesis) with the absence of skeletal myogenesis3. In addition, myocardial pericytes exhibited differential cardiomyogenic potential and angiogenic capacities when compared with counterparts purified from other organs.
A second population of multipotent perivascular stem/progenitor cells, the adventitial cell, has been isolated from human saphenous veins on the basis of positive CD34 expression22. Venous adventitial cells have been shown to have clonogenic potential, mesodermal differentiation capacity and proangiogenic potential in vitro. Transplantation of these cells into the ischemically injured hearts of mice resulted in a reduction in interstitial fibrosis, an increase in angiogenesis and myocardial blood flow, reduced ventricular dilation, and increased cardiac ejection fraction23. Interestingly, adipose adventitial cells have been shown to lose CD34 expression and upregulate CD146 expression in culture in response to angiopoietin II treatment, suggesting the adoption of a pericyte phenotype with stimulation24. Within the heart, however, the adventitial cell population has not yet been prospectively purified by FACS and/or well characterized. Utilizing the cell isolation procedures described in the following sections, we are currently characterizing myocardial adventitial cells and investigating their potential for regenerative applications.
Herein we describe a method to isolate and purify two subpopulations of perivascular stem/progenitor cells from human fetal or adult myocardium. This prospective cell isolation method will enable researchers to obtain isogenic perivascular stem/progenitor cell subsets from human heart biopsies for comparative studies and further explore their therapeutic potential in various cardiac pathological conditions.

<table><tbody><tr><td>AbC Anti-mouse Bead Kit</td><td>Molecular Probes</td><td>A-10344</td><td></td></tr><tr><td>Collagenase I</td><td>Gibco</td><td>17100-017</td><td>Reconstitute powder as required and filter sterilise</td></tr><tr><td>Collagenase II</td><td>Gibco</td><td>17101-015</td><td></td></tr><tr><td>Collagenase IV</td><td>Gibco</td><td>17104-019</td><td></td></tr><tr><td>anti-human CD34-PE</td><td>BD Pharmingen</td><td>555822</td><td>Keep sterile</td></tr><tr><td>anti-human CD45-APC-Cy7</td><td>BD Pharmingen</td><td>557833</td><td>Keep sterile</td></tr><tr><td>anti-human CD56-PE-Cy7</td><td>BD Pharmingen</td><td>557747</td><td>Keep sterile</td></tr><tr><td>anti-human CD144-PerCP-Cy5.5</td><td>BD Pharmingen</td><td>561566</td><td>Keep sterile</td></tr><tr><td>anti-human CD146-AF647</td><td>AbD Serotec</td><td>MCA2141A647</td><td>Keep sterile</td></tr><tr><td>EGM2-BulletKit</td><td>Lonza</td><td>CC-3162</td><td>For collection of cells and culture until adhered</td></tr><tr><td>DMEM, high glucose, GlutaMAX without sodium pyruvate</td><td>ThermoFischer Scientific</td><td>10566-016</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>ThermoFischer Scientific</td><td>10500-064</td><td>Freeze in aliquots and keep sterile</td></tr><tr><td>Gelatin</td><td>Sigma Aldrich</td><td>G1393</td><td>Dilute with sterile water</td></tr><tr><td>IgG1k-PE</td><td>BD Pharmingen</td><td>559320</td><td>Keep sterile</td></tr><tr><td>IgG1k-APC-Cy7</td><td>BD Pharmingen</td><td>557873</td><td>Keep sterile</td></tr><tr><td>IgG1k-PE-Cy7</td><td>BD Pharmingen</td><td>557872</td><td>Keep sterile</td></tr><tr><td>IgG1k-PerCP-Cy5.5</td><td>BD Pharmingen</td><td>561566</td><td>Keep sterile</td></tr><tr><td>IgG1k-647</td><td>AbD Serotec</td><td>MCA1209A647</td><td>Keep sterile</td></tr><tr><td>Mouse serum</td><td>Sigma Aldrich</td><td>M5905</td><td>Keep sterile</td></tr><tr><td>Paraffin Film - Parafilm M</td><td>Sigma Aldrich</td><td>P7793</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Gibco</td><td>15979-063</td><td>Freeze in aliquots and keep sterile</td></tr><tr><td>Phosphate buffered saline pH 7.4</td><td>ThermoFischer Scientific</td><td>10010-023</td><td>Keep sterile</td></tr><tr><td>Red Blood Cell Lysing Buffer Hybri-Max</td><td>Sigma Aldrich</td><td>R7757</td><td>Keep sterile</td></tr><tr><td>Trypan Blue Solution</td><td>Sigma Aldrich</td><td>T8154</td><td></td></tr><tr><td>Trypsin-EDTA 0.5%(10x)</td><td>Invitrogen</td><td>15400-054</td><td></td></tr><tr><td>&amp;nbsp;FACSARIA FUSION</td><td>BD Pharmingen</td><td></td><td>Fluorescence Activated Cell Sorter</td></tr></tbody></table>

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.

Single cells were distinguished from debris and doublets on the basis of forward and side scatter distributions. Live cells were identified by their failure to take up the DAPI dye. The gating strategy was chosen on the basis of isotype control labelling of this live, whole-cardiac cell dissociation (Figure 1). From the live cells, CD45+ cells were first gated out, followed by CD56+ cells. CD144+ endothelial cells were then removed from th...

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Isolation of Perivascular Multipotent Precursor Cell Populations from Human Cardiac Tissue

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.

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

isolation-perivascular-multipotent-precursor-cell-populations-from

Research

JoVE Journal

Developmental Biology

7.3K Views.  University of Edinburgh.  Menselijk hartweefsel herbergt multipotente perivasculaire precursor celpopulaties die geschikt zijn voor myocard regeneratie kunnen zijn. De hier beschreven techniek maakt de gelijktijdige isolatie en zuivering van twee multipotente stromale cel populaties geassocieerd met natieve bloedvat, dat wil zeggen CD146 + CD34 - pericyten en CD34 + CD146 - adventitia-cellen van de menselijke hartspier. 

Video: Isolation of Perivascular Multipotent Precursor Cell Populations from Human Cardiac Tissue

Isolation of Cardiomyocyte Nuclei from Post-mortem Tissue

Identification of cardiomyocyte nuclei has been challenging in tissue sections as most strategies rely only on cytoplasmic marker proteins1. Rare events in cardiac myocytes such as proliferation and apoptosis require an accurate identification of cardiac myocyte nuclei to analyze cellular renewal in homeostasis and in pathological conditions2. Here, we provide a method to isolate cardiomyocyte nuclei from post mortem tissue by density sedimentation and immunolabeling with antibodies against pericentriolar material 1 (PCM-1) and subsequent flow cytometry sorting. This strategy allows a high throughput analysis and isolation with the advantage of working equally well on fresh tissue and frozen archival material. This makes it possible to study material already collected in biobanks. This technique is applicable and tested in a wide range of species and suitable for multiple downstream applications such as carbon-14 dating3, cell-cycle analysis4, visualization of thymidine analogues (e.g. BrdU and IdU)4, transcriptome and epigenetic analysis.

Cardiac nuclei are isolated via density sedimentation and immunolabeled with antibodies against pericentriolar material 1 (PCM-1) to identify and sort cardiomyocyte nuclei by flow cytometry.

Identification of cardiomyocyte nuclei has been challenging in tissue sections as most strategies rely only on cytoplasmic marker proteins1. Rare events ...

Medicine

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading cause of mortality worldwide. Mesenchymal stem cells (MSCs) are a promising option for cell-based therapies to replace current, invasive techniques. MSCs can differentiate into mesenchymal lineages, including cardiac cell types, but complete differentiation into functional cells has not yet been achieved. Previous methods of differentiation were based on pharmacological agents or growth factors. However, more physiologically relevant strategies can also enable MSCs to undergo cardiomyogenic transformation. Here, we present a differentiation method using MSC aggregates on cardiomyocyte feeder layers to produce cardiomyocyte-like contracting cells.
Human umbilical cord perivascular cells (HUCPVCs) have been shown to have a greater differentiation potential than commonly investigated MSC types, such as bone marrow MSCs (BMSCs). As an ontogenetically younger source, we investigated the cardiomyogenic potential of first-trimester (FTM) HUCPVCs compared to older sources. FTM HUCPVCs are a novel, rich source of MSCs that retain their in utero immunoprivileged properties when cultured in vitro. Using this differentiation protocol, FTM and term HUCPVCs achieved significantly increased cardiomyogenic differentiation compared to BMSCs, as indicated by the increased expression of cardiomyocyte markers (i.e., myocyte enhancer factor 2C, cardiac troponin T, heavy chain cardiac myosin, signal regulatory protein &#945;, and connexin 43). They also maintained significantly lower immunogenicity, as demonstrated by their lower HLA-A expression and higher HLA-G expression. Applying aggregate-based differentiation, FTM HUCPVCs showed increased aggregate formation potential and generated contracting cells clusters within 1 week of co-culture on cardiac feeder layers, becoming the first MSC type to do so. 
Our results demonstrate that this differentiation strategy can effectively harness the cardiomyogenic potential of young MSCs, such as FTM HUCPVCs, and suggests that in vitro pre-differentiation could be a potential strategy to increase their regenerative efficacy in vivo.

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

Here, we present a method to efficiently harness the cardiac differentiation potential of young sources of human mesenchymal stem cells in order to generate functional, contracting, cardiomyocyte-like cells in vitro.

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading ...

Generation of First Heart Field-like Cardiac Progenitors and Ventricular-like Cardiomyocytes from Human Pluripotent Stem Cells

The generation of large amounts of functional human pluripotent stem cells-derived cardiac progenitors and cardiomyocytes of defined heart field origin is a pre-requisite for cell-based cardiac therapies and disease modeling. We have recently shown that Id genes are both necessary and sufficient to specify first heart field progenitors during vertebrate development. This differentiation protocol leverages these findings and uses Id1 overexpression in combination with Activin A as potent specifying cues to produce first heart field-like (FHF-L) progenitors. Importantly, resulting progenitors efficiently differentiate (~70&#8211;90%) into ventricular-like cardiomyocytes. Here we describe a detailed method to 1) generate Id1-overexpressing hPSCs and 2) differentiate scalable quantities of cryopreservable FHF-L progenitors and ventricular-like cardiomyocytes.

Here we describe a scalable method, using a simple combination of Activin A and lentivirus-mediated Id1-overexpression, to generate first heart field-like cardiac progenitors and ventricular-like cardiomyocytes from human pluripotent stem cells.

The generation of large amounts of functional human pluripotent stem cells-derived cardiac progenitors and cardiomyocytes of defined heart field origin is ...

Induction of Endothelial Differentiation in Cardiac Progenitor Cells Under Low Serum Conditions

Cardiac progenitor cells (CPCs) may have therapeutic potential for cardiac regeneration after injury. In the adult mammalian heart, intrinsic CPCs are extremely scarce, but expanded CPCs could be useful for cell therapy. A prerequisite for their use is their ability to differentiate in a controlled manner into the various cardiac lineages using defined and efficient protocols. In addition, upon in vitro expansion, CPCs isolated from patients or preclinical disease models may offer fruitful research tools for the investigation of disease mechanisms.
Current studies use different markers to identify CPCs. However, not all of them are expressed in humans, which limits the translational impact of some preclinical studies. Differentiation protocols that are applicable irrespective of the isolation technique and marker expression will allow for the standardized expansion and priming of CPCs for cell therapy purpose. Here we describe that the priming of CPCs under a low fetal bovine serum (FBS) concentration and low cell density conditions facilitates the endothelial differentiation of CPCs. Using two different subpopulations of mouse and rat CPCs, we show that laminin is a more suitable substrate than fibronectin for this purpose under the following protocol: after culturing for 2 - 3 days in medium including supplements that maintain multipotency and with 3.5% FBS, CPCs are seeded on laminin at &#60;60% confluence and cultured in supplement-free medium with low concentrations of FBS (0.1%) for 20 - 24 hours before differentiation in endothelial differentiation medium. Because CPCs are a heterogeneous population, serum concentrations and incubation times may need to be adjusted depending on the properties of the respective CPC subpopulation. Considering this, the technique can be applied to other types of CPCs as well and provides a useful method to investigate the potential and mechanisms of differentiation and how they are affected by disease when using CPCs isolated from respective disease models.

This protocol describes an endothelial differentiation technique for cardiac progenitor cells. It particularly focuses on how serum concentration and cell-seeding density affect the endothelial differentiation potential.

Cardiac progenitor cells (CPCs) may have therapeutic potential for cardiac regeneration after injury. In the adult mammalian heart, intrinsic CPCs are ...

In Vitro Generation of Heart Field-specific Cardiac Progenitor Cells

Pluripotent stem cells offer great potential for understanding heart development and disease and for regenerative medicine. While recent advances in developmental cardiology have led to generating cardiac cells from pluripotent stem cells, it is unclear if the two cardiac fields - the first and second heart fields (FHF and SHF) &#8212; are induced in pluripotent stem cells systems. To address this, we generated a protocol for in vitro specification and isolation of heart field-specific cardiac progenitor cells. We used embryonic stem cells lines carrying Hcn4-GFP and Tbx1-Cre; Rosa-RFP reporters of the FHF and the SHF, respectively, and live cell immunostaining of the cell membrane protein Cxcr4, a SHF marker. With this approach, we generated progenitor cells which recapitulate the functional properties and transcriptome of their in vivo counterparts. Our protocol can be utilized to study early specification and segregation of the two heart fields and to generate chamber-specific cardiac cells for heart disease modelling. Since this is an in vitro organoid system, it may not provide precise anatomical information. However, this system overcomes the poor accessibility of gastrulation-stage embryos and can be upscaled for high-throughput screens.

The purpose of this method is to generate heart field-specific cardiac progenitor cells in vitro in order to study the progenitor cell specification and functional properties, and to generate chamber specific cardiac cells for heart disease modelling.

Pluripotent stem cells offer great potential for understanding heart development and disease and for regenerative medicine. While recent advances in ...

Generation and Expansion of Human Cardiomyocytes from Patient Peripheral Blood Mononuclear Cells

Generating patient-specific cardiomyocytes from a single blood draw has attracted tremendous interest in precision medicine on cardiovascular disease. Cardiac differentiation from human induced pluripotent stem cells (iPSCs) is modulated by defined signaling pathways that are essential for embryonic heart development. Numerous cardiac differentiation methods on 2-D and 3-D platforms have been developed with various efficiencies and cardiomyocyte yield. This has puzzled investigators outside the field as the variety of these methods can be difficult to follow. Here we present a comprehensive protocol that elaborates robust generation and expansion of patient-specific cardiomyocytes from peripheral blood mononuclear cells (PBMCs). We first describe a high-efficiency iPSC reprogramming protocol from a patient&#39;s blood sample using non-integration Sendai virus vectors. We then detail a small molecule-mediated monolayer differentiation method that can robustly produce beating cardiomyocytes from most human iPSC lines. In addition, a scalable cardiomyocyte expansion protocol is introduced using a small molecule (CHIR99021) that could rapidly expand patient-derived cardiomyocytes for industrial- and clinical-grade applications. At the end, detailed protocols for molecular identification and electrophysiological characterization of these iPSC-CMs are depicted. We expect this protocol to be pragmatic for beginners with limited knowledge on cardiovascular development and stem cell biology.

Here, we present a protocol to robustly generate and expand human cardiomyocytes from patient peripheral blood mononuclear cells.

Generating patient-specific cardiomyocytes from a single blood draw has attracted tremendous interest in precision medicine on cardiovascular disease. ...

Fabrication of 3D Cardiac Microtissue Arrays using Human iPSC-Derived Cardiomyocytes, Cardiac Fibroblasts, and Endothelial Cells

Generation of human cardiomyocytes (CMs), cardiac fibroblasts (CFs), and endothelial cells (ECs) from induced pluripotent stem cells (iPSCs) has provided a unique opportunity to study the complex interplay among different cardiovascular cell types that drives tissue development and disease. In the area of cardiac tissue models, several sophisticated three-dimensional (3D) approaches use induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) to mimic physiological relevance and native tissue environment with a combination of extracellular matrices and crosslinkers. However, these systems are complex to fabricate without microfabrication expertise and require several weeks to self-assemble. Most importantly, many of these systems lack vascular cells and cardiac fibroblasts that make up over 60% of the nonmyocytes in the human heart. Here we describe the derivation of all three cardiac cell types from iPSCs to fabricate cardiac microtissues. This facile replica molding technique allows cardiac microtissue culture in standard multi-well cell culture plates for several weeks. The platform allows user-defined control over microtissue sizes based on initial seeding density and requires less than 3 days for self-assembly to achieve observable cardiac microtissue contractions. Furthermore, the cardiac microtissues can be easily digested while maintaining high cell viability for single-cell interrogation with the use of flow cytometry and single-cell RNA sequencing (scRNA-seq). We envision that this in vitro model of cardiac microtissues will help accelerate validation studies in drug discovery and disease modeling.

Here, we describe an easy-to-use methodology to generate 3D self-assembled cardiac microtissue arrays composed of pre-differentiated human-induced pluripotent stem cell-derived cardiomyocytes, cardiac fibroblasts, and endothelial cells. This user-friendly and low cell requiring technique to generate cardiac microtissues can be implemented for disease modeling and early stages of drug development.

Generation of human cardiomyocytes (CMs), cardiac fibroblasts (CFs), and endothelial cells (ECs) from induced pluripotent stem cells (iPSCs) has provided ...

Bioengineering

Isolating Primary Melanocyte-like Cells from the Mouse Heart

We identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial arrhythmia triggers in mice. To investigate the electrical and biological properties of cardiac melanocytes we developed a procedure to isolate them from mouse hearts that we derived from those designed to isolate neonatal murine cardiomyocytes. In order to obtain healthier cardiac melanocytes suitable for more extensive patch clamp or biochemical studies, we developed a refined procedure for isolating and plating cardiac melanocytes based on those originally designed to isolate cutaneous melanocytes. The refined procedure is demonstrated in this review and produces larger numbers of healthy melanocyte-like cells that can be plated as a pure population or with cardiomyocytes.

In this protocol, we identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial arrhythmia triggers in mice.&#160;

We identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial ...

Biology

Isolation and Purification of Murine Cardiac Pericytes

Pericytes, perivascular cells of microvessels and capillaries, are known to play a part in angiogenesis, vessel stabilization, and endothelial barrier integrity. However, their tissue-specific functions in the heart are not well understood. Moreover, there is currently no protocol utilizing readily accessible materials to isolate and purify pericytes of cardiac origin. Our protocol focuses on using the widely used mammalian model, the mouse, as our source of cells. Using the enzymatic digestion and mechanical dissociation of heart tissue, we obtained a crude cell mixture that was further purified by fluorescence activating cell sorting (FACS) by a plethora of markers. Because there is no single unequivocal marker for pericytes, we gated for cells that were CD31-CD34-CD45-CD140b+NG2+CD146+. Following purification, these primary cells were cultured and passaged multiple times without any changes in morphology and marker expression. With the ability to regularly obtain primary murine cardiac pericytes using our protocol, we hope to further understand the role of pericytes in cardiovascular physiology and their therapeutic potential.

We have optimized a protocol to isolate and purify murine cardiac pericytes for basic research and investigation of their biology and therapeutic potential.

Pericytes, perivascular cells of microvessels and capillaries, are known to play a part in angiogenesis, vessel stabilization, and endothelial barrier ...

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.

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

Isolation of Perivascular Multipotent Precursor Cell Populations from Human Cardiac Tissue

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