In this article, a method to isolate cardiac mesenchymal stromal cells from endomyocardial bioptic samples of arrhythmogenic cardiomyopathy patients is provided. Their characterization and the protocol to boost their adipogenic differentiation are described.
A normal adult heart is composed of several different cell types, among which cardiac mesenchymal stromal cells represent an abundant population. The isolation of these cells offers the possibility of studying their involvement in cardiac diseases, and, in addition, provides a useful primary cell model to investigate biological mechanisms.
Here, the method for the isolation of C-MSC from arrhythmogenic cardiomyopathy patients' bioptic samples is described. The endomyocardial biopsy sampling is guided in the right ventricular areas adjacent to the scar visualized by electro-anatomical mapping. The digestion of the biopsies in collagenase and their plating on a plastic dish in culture medium to allow C-MSC growth is described. The isolated cells can be expanded in culture for several passages. To confirm their mesenchymal phenotype, the description of immuno-phenotypical characterization is provided. C-MSC are able to differentiate into several cell types like adipocytes, chondrocytes, and osteoblasts: in the context of ACM, characterized by adipocyte deposits in patients' hearts, the protocols for the adipogenic differentiation of C-MSC and the characterization of lipid droplet accumulation are described.
Mesenchymal Stromal Cells (MSC) are adult cells with an important supportive function in many tissues1. Bone marrow represents the historical source of MSC, but they can be isolated from different tissues including placenta, adipose tissue, cord blood, liver, and heart1,2.
In 2006, the International Society for Cellular Therapy (ISCT) specified, for the first time, the minimal criteria to define human MSC3. In particular, MSC must have the ability to adhere to plastic. They express specific surface antigens: the positivity for CD44, CD105, CD29, and CD90 mesenchymal markers, and the negativity for CD14, CD45, CD34, CD31 hematopoietic and endothelial markers characterize MSC. Due to the lack expression of HLA-DR, MSC are unable to trigger alloreactivity. Moreover, they are multipotent cells with the potential to differentiate toward adipogenic, chondrocyte, and osteoblast lineages1,3.
Focusing on cardiac cellular composition, cardiac mesenchymal stromal cells (C-MSC) are abundant in a normal adult heart4,5. They play a critical role both in the normal cardiac function and pathological conditions. While, physiologically, C-MSC provide a microenvironment that supports the structural and functional integrity of the myocardium, in heart diseases they are activated in response to heart injury participating in the wound healing and fibrotic remodeling6,7.
Recently, the involvement of C-MSC in arrhythmogenic cardiomyopathy (ACM) adipose substitution has been demonstrated8. In particular, ACM is a genetic disorder mainly caused by mutations in desmosomal genes that lead to myocardial fibro-fatty replacement, mainly in the right ventricle9. This substitution, extending from epicardium to endocardium, creates a non-conductive substrate that elicits progressive heart failure and worsens ventricular arrhythmias, which can lead, in severe cases, to sudden death. Sommariva et al. demonstrated the mesenchymal origin of the pre-adipocytes present in ACM patients' explanted heart sections. Furthermore, C-MSC isolated from endomyocardial biopsies of ACM hearts express desmosomal genes and therefore can be affected by their mutations. In particular, under adipogenic conditions, ACM C-MSC accumulate more lipids than those from control hearts. This evidence leads to the conclusion that C-MSC both have an active role in the disease pathogenesis and represent a valid cell model to study ACM.
To facilitate future research in this context or in other heart diseases for which a cardiac biopsy is indicated, a detailed protocol is presented for the isolation of C-MSC from small fragments of endomyocardial tissue, their expansion, their immuno-phenotypical characterization, and adipogenic differentiation.
This approach complies with the Declaration of Helsinki and the collection of right ventricular samples was approved (07/06/2012) by "Centro Cardiologico Monzino IRCCS" Ethics Committee.
1. Solutions
2. Isolation of Cardiac Mesenchymal Stromal Cells
3. Cell Expansion
Note: Before starting make sure to have TMES medium ready.
4. Characterization of Cardiac Mesenchymal Stromal Cells by Flow Cytometry
Note: Before starting, make sure to have the cell dissociation reagent, washing buffer, and the specific antibodies prepared.
5. Adipogenic Differentiation of Cardiac Mesenchymal Stromal Cells
Note: Before starting, make sure to have prepared T. ADIPO medium and ORO working solution.
Cardiac stromal cells isolation: The C-MSC isolation from endomyocardial biopsies procedure is summarized in Figure 1.
Cardiac stromal cells mesenchymal characterization: As established by the International Society for Cellular Therapy (ISCT), the minimal criteria for defining multipotent mesenchymal stromal cells includes their immuno-phenotypic characterization3. In particular, to confirm their mesenchymal lineage, cells are incubated with appropriate FITC/PE/APC-conjugated antibodies and analyzed by flow cytometry. All of the antibodies used in the C-MSC characterization are listed in Table of Materials.
As illustrated in Figure 2, C-MSC obtained from biopsies are positive for the specific mesenchymal surface antigens CD29, CD44, and CD105. The percentage of CD90 positive cells is variable, as reported previously11. The endothelial (CD31, CD34), monocyte/macrophage (CD14), hematopoietic (CD45) markers, and major histocompatibility complex (HLA-DR) are not expressed (Figure 2).
Cardiac mesenchymal stromal cells adipogenic differentiation: To prompt their adipogenic differentiation, C-MSC obtained from patients affected by ACM and HC have to be cultured in T. ADIPO medium (see Solutions section). The cells are maintained in culture for 72 h or 1 week, replacing the medium twice a week.
The accumulation of intracellular lipid droplets is evidenced by ORO staining (Figure 3). Differences in the ability, as well as in the degree of differentiation, can be observed between the cells obtained from ACM and HC. As shown in the representative images, ACM C-MSC accumulates more lipid droplets than HC C-MSC after 72 h of culture in adipogenic medium (Figure 3). These differences are maintained also when the cells are exposed to adipogenic differentiation conditions for a longer period (1 week) (Figure 3).
Video 1: Integration of electroanatomical map and intracardiac echocardiography. Endocardial unipolar electroanatomical maps of the right ventricle (left anterior oblique and right anterior oblique views) in a patient who undergoes endomyocardial biopsy (lower panels). Limited areas of low voltage (red/green) are visible at the apex. Endomyocardial bioptic samples (tagged as circle) are collected in correspondence to the diseased myocardium and the interventricular septum. Real-time intracardiac echocardiography allows checking the correct position of the bioptome at the target area (upper panel). Please click here to view this video. (Right-click to download.)
Video 2: Fluoroscopy video in right anterior oblique view. The bioptome is inserted through a long-deflectable sheath and advanced into the ventricle. After a careful check of the bioptome contact with the myocardium, the jaws are opened and then firmly closed to collect the sample. Please click here to view this video. (Right-click to download.)
Figure 1: Procedure draft. The endomyocardial bioptic sample is collected using a bioptome catheter, a small pincer-shaped cutting instrument. The weight of the obtained biopsy is approximately 5 mg (A). The endomyocardial bioptic sample is minced into 0.5 – 1 mm3 fragments, with sterile scissors (B), and collagenase solution is added. The sample is placed in a 37 °C incubator on a rotating platform mixer for 1.5 h of digestion (C). The digested solution is centrifuged and plated in TMES in a plastic dish (D). C-MSC are obtained thanks to their plastic adherence properties either in single cells or clones, or sprouting from small undigested bioptic fragments (E). C-MSC are then characterized by flow cytometry (F). C-MSC are plated in adipogenic medium and lipid droplet accumulation is tested by Oil Red O staining (G). Scale bars indicate 50 µm. Please click here to view a larger version of this figure.
Figure 2: Representative cyto-fluorimetric profile of C-MSC. Each histogram shows the cell count vs. the intensity of fluorescence in the indicated channel (PE: phycoerythrin; APC: allophycocyanin; FITC: fluorescein-isothyocyanate). In each graph the isotype control (white) and a sample conjugated with the specific cell surface marker antibody (red) are shown. C-MSC are positive for the mesenchymal surface antigens CD29, CD44, CD105 and, partially, for CD90, whereas they do not express CD31, CD34, CD14, CD45, and HLA-DR. Please click here to view a larger version of this figure.
Figure 3: Adipogenic differentiation of C-MSC. Representative images of ACM and HC C-MSC, cultured in adipogenic medium for 72 h and 1 week, stained with ORO (left pictures; n = 3). In the right graphs, quantification of the luminance of the 255-red channel staining is reported: intensity is expressed in arbitrary units (A.U.). ACM C-MSC accumulate more lipid droplets than controls. Student's T-test was used, *: p < 0.05. Scale bars indicate 50 µm. Please click here to view a larger version of this figure.
MSC and C-MSC: MSC are multipotent cells resident in the stromal fraction of different adult tissues, such as bone marrow, adipose tissue, cartilage, brain, skin, fetal annexes, and heart12. Different studies have been performed to isolate and characterize them for potential applications in basic and translational research12,13.
In healthy conditions, MSC are quiescent, self-renewing at low rates14. Since they are exposed to environmental pathological changes, they react fostering tissue remodeling through either direct transdifferentiation, matrix deposition, or the paracrine effect14.
The cardiac MSC (C-MSC) represent a large non-myocyte cell population of the heart4. They originate from the epicardium and migrate into the myocardium undergoing the process of epithelial-to-mesenchymal transition15. They contribute to the mechanical and electrical integrity of the cardiac structure, both in physiological and in pathological states, through interactions with cardiomyocytes and extracellular matrix homeostasis7,16. However, the broad range of C-MSC functions is still not completely understood. A deeper knowledge of their role both in physiological and pathological conditions can be facilitated by in vitro studies performed after their isolation.
C-MSC have been obtained from different districts of the human heart, such as the atrial appendage2,17 and right ventricle18.
Recently, C-MSC from human right ventricular endomyocardial bioptic samples have been obtained8, demonstrating that the source tissue could be as little as 3-5 mg.
Possible applications: The method outlined in this manuscript allows obtaining cells with few simple passages, such as digestion and selection for plastic adherence, from very small heart specimens.
C-MSC can be considered a cell model, since they are easy to amplify and maintain in vitro, and are able to differentiate into cells of mesenchymal lineage (endothelium, osteocytes, and adipocytes). Moreover, the possibility of obtaining cells directly from patients constitutes a great in vitro tool for mechanistic studies in the context of personalized/precision medicine. Indeed, these cells carry the genetic background and eventually specific mutations of the donors, and are influenced by the specific patients' characteristics, such as clinical conditions, age, sex, lifestyle, and medications. Moreover, the possibility of sorting them for different markers may allow the study of specific C-MSC subsets19.
C-MSC are known to be active players in different cardiovascular diseases, mostly characterized by adverse remodeling of the heart. Therefore, they represent candidate targets for novel therapeutic strategies to counteract heart diseases8,20.
C-MSC stem-like properties and their lack of significant immunogenicity suggests their potential application in cell-therapy for cardiac regenerative medicine. Indeed, like MSC from bone marrow or other sources, C-MSC could be potentially used both in autologous and in allogenic settings, without the need for matching between donor and recipient21.
Moreover, C-MSC, being isolated directly from heart tissue, have the advantage of being preconditioned by the cardiac micro-environment and epigenetic profile. In the context of cardiac regenerative medicine, this could be particularly important to obtain successful results.
To date, preclinical studies of regenerative medicine identified useful therapeutic potential in the C-MSC and their paracrine activity18,22,23. Importantly, clinical trials in which the cell source is the heart are underway either with cardiosfere-derived cells or with subpopulations of C-MSC13,24,25. However, as for bone-marrow-derived MSC, different protocols may be necessary to obtain clinical grade C-MSC26.
C-MSC in ACM: The presented protocol is mostly suitable for the study of pathologies for which an endocardial biopsy is indicated. ACM patients undergo bioptic procedures for diagnostic purposes27. Their myocardium is gradually substituted by scar-tissue, an electrically inert tissue composed of adipocytes and fibrosis. In order to guide the bioptic sampling to the scar area, where the diagnostic yield is maximal, endomyocardial mapping is used10,28,29. The samples used in this protocol are taken in the border zone of the diseased myocardium.
Sommariva et al. has recently defined a pivotal role of C-MSC in the pathogenesis of ACM8, demonstrating that C-MSC are active players in ACM heart adipogenesis, since preadipocytes in those hearts are of mesenchymal origin. Moreover, C-MSC isolated with the present protocol from ACM patients' biopsies showed more propensity to both lipid accumulation and adipogenesis than controls. For this reason, these cells could be used to confirm some of the molecular mechanisms of ACM, proving their suitability as a cell model for mechanistic studies9.
Limitations and critical steps: Despite the advantages of obtaining C-MSC directly from patients (see the paragraph "Possible applications"), this protocol is subjected to different limitations.
First of all, the cardiac bioptic procedure is invasive and often avoided if not strictly necessary. Indeed, sampling cardiac tissue is both ethically and technically problematic. Reasons for performing a cardiac biopsy may be the achievement of a definite diagnosis in the context of cardiomyopathies in differential diagnosis, monitoring the status of cardiac transplants, or ascertaining the presence of a heart tumor30. Therefore, only patients for which an endomyocardial biopsy is indicated by consensus statement31 can be enrolled for research on C-MSC. Moreover, the cardiac bioptic procedure can have clinical complications, above all in cardiomyopathic hearts. Therefore, electrophysiologist's samplings are always cautious and bioptic samples could be very small, compromising the isolation of cells. Future experiments could overcome this issue by tuning collagenase concentration or timing of digestion.
C-MSC, as all primary human cells, show a high variability among different subjects in all phenotypes. Indeed, cells from different subjects are not only genetically different, but also subjected to variable environmental conditioning. Specifically, within this experiment, a high variability in cell isolation, growth, and adipogenic differentiation is observed.
Critical steps of the present protocol have to be acknowledged. If the bioptic sample includes capillaries, they must be removed to avoid the parallel isolation of endothelial cells, which may contaminate the C-MSC culture, and can be evidenced by the FACS analysis (positivity for CD31). To obtain an efficient adipogenic differentiation, cells must be in an active growth phase. The degree of confluence may also influence lipid accumulation.
Significance of the method: With respect to previous methods of isolation of mesenchymal stromal cells, this is the first time where the description of C-MSC obtainment directly from human ventricular bioptic samples is proposed in detail. Although this method is suggested for the processing of ACM patient samples, it is potentially applicable to all the patients for which a cardiac biopsy is indicated.
This protocol represents a useful implementation of previous methods for the obtainment of cells that required bigger cardiac samples32, which are often difficult to collect.
Moreover, the sample source constitutes an interesting innovation. While the ventricular biopsy is usually performed on the septum33, this protocol takes into account samples obtained from the right ventricular free wall. Cells derived from the diseased right ventricular district may be more representative of the pathologic status of diseases involving RV.
In addition, some of the reagents used in the present protocol are different with respect to other C-MSC isolation and differentiation methods32. For example, the type of collagenase proposed in this manuscript is a mix of class I and class II collagenases with a balanced ratio of proteolytic activities. Moreover, the digestion solution is composed of the collagenase mix dissolved in the same basal medium (IMDM) used for the preparation of C-MSC culture medium, allowing isolated C-MSC to adapt to future growth conditions.
In addition, though sorting procedures could standardize the cell batch, using the whole C-MSC population, isolated only through the plastic adherence property of these cells, constitutes a simplification without altering the immunophenotypic characteristics of C-MSC. The composition of T. ADIPO proposed in this manuscript is able to lead to adipogenic differentiation, avoiding the metabolic dysregulation induced by other components such as insulin.
Moreover, the proposed method of lipid accumulation quantification, which is based on the evaluation of the ORO colorimetric intensity, provides more information about the quantity of the accumulated lipids, if compared with methods based only on the percentage of cells positive to the ORO staining. Often lipid accumulation is quantified by extracting the ORO incorporated by cells with isopropanol and measuring its absorbance. However, this method requires more passages and is subjected to variability due to isopropanol evaporation.
The authors have nothing to disclose.
This work was funded by Italian Ministry of Health, Ricerca Corrente to Centro Cardiologico Monzino-IRCCS.
IMDM | Gibco by Life Technologies | 12440053 | |
Fetal Bovine Serum (FBS) | Sigma-Aldrich | F2442 | |
PENICILLIN STREPTOMYCIN | Life Technologies Italia | 15140122 | |
Collagenase NB4 | Serva | 17454.02 | |
Basic fibroblast growth factor | Peprotech | 100-18B | |
L-glutammine | Sigma-Aldrich | G7513 | |
PBS | Lonza | 17-516F | |
Tryple select 1X (cell dissociation reagent) | Gibco | 12563029 | |
EDTA | Sigma-Aldrich | EDS | |
Trypsin-EDTA solution | Sigma-Aldrich | T6689 | |
Bovine Serum Albumin | Sigma-Aldrich | A2058 | |
3-Isobutyl-1-methylxanthine | Sigma-Aldrich | I5879 | |
Hydrocortisone | Sigma-Aldrich | H4001 | |
Indomethacin | Sigma-Aldrich | I7378 | |
Oil Red O | Sigma-Aldrich | O0625 | |
Isopropanol | Sigma-Aldrich | I9516 | |
Paraformaldehyde (PFA) Solution 4% in PBS | D.b.a. Italia S.r.l. | sc-281692 | |
Dimethyl Sulfoxide (DMSO) | Sigma-Aldrich | 276855 | |
Antibody CD14-FITC (MφP9) | Becton Dickinson | 345784 | |
Antibody Integrin β1 (CD29)-PE (clone MAR4) | Becton Dickinson | 561795 | |
Antibody PECAM-1 (CD31)- APC (clone 9G11) | R&D Systems | FAB3567A | |
Antibody Hematopoietic progenitor cell antigen (CD34)-APC (clone 581) | Thermo Fisher Scientific | CD34-581-05 | |
Antibody H-CAM (CD44)-PE (clone G44-26) | Becton Dickinson | 561858 | |
Antibody L-CA (CD45)-APC (clone HI30) | Thermo Fisher Scientific | MHCD4505-4 | |
Antibody THY1 (CD90)-FITC (clone 5E10) | Becton Dickinson | 561969 | |
Antibody Endoglin (CD105)-APC (clone 266) | Becton Dickinson | 562408 | |
Antibody HLA-DR-FITC (clone L243) | Becton Dickinson | 347400 |
|
Gima Quick Plus sterilizer | Gima | 35642 | |
Bench centrifuge Sigma 3-16K | Sigma Centrifuges | 10330 | |
Mr. Frosty Freezing Container | Thermo Fisher Scientific | 5100-0001 | |
AxioVert 200M microscope | Zeiss | B 40-080 e 03/01 | |
FACS Gallios | Beckman Coulter | 773231AF | |
ImageJ (image processing program) | NIH | ||
Stericup filter units | Merck S.P.A. | SCGPU05RE | |
Conical Tubes, 50 mL | Eppendorf | 30122178 | |
Conical Tubes, 15 mL | Eppendorf | 30122151 | |
Safe-Lock Tubes, 2 mL | Eppendorf | 30120094 | |
100 mm TC-Treated Cell Culture Dish | Corning | 430167 | |
60 mm TC-Treated Cell Culture Dish | Corning | 430166 | |
6 well TC-Treated Multiple Well Plates | Corning | 3516 | |
Polystyrene Round-Bottom Tubes, 5 mL | BD Falcon | 352058 | |
BRAND counting chamber BLAUBRAND Bürker-Türk | Sigma-Aldrich | BR719520 |