Nat Protoc. 2014 Jul;9(7):1662-81. doi: 10.1038/nprot.2014.113.

Isolation and characterization of resident endogenous c-Kit+ cardiac stem cells from the adult mouse and rat heart.

Smith AJ1, Lewis FC1, Aquila I2, Waring CD3, Nocera A4, Agosti V4, Nadal-Ginard B1, Torella D5, Ellison GM6.
  • 11] Centre of Human & Aerospace Physiological Sciences, School of Biomedical Sciences, King’s College London, London, UK. [2] Centre for Stem Cells and Regenerative Medicine, King’s College London, London, UK.
  • 2Laboratory of Molecular and Cellular Cardiology, Department of Medical and Surgical Sciences, Magna Graecia University, Catanzaro, Italy.
  • 3Stem Cell and Regenerative Biology Unit (Biostem), Liverpool John Moores University, Liverpool, UK.
  • 41] Laboratory of Molecular Oncology, Department of Experimental and Clinical Medicine, Magna Graecia University, Catanzaro, Italy. [2] CIS for Genomics and Molecular Pathology, Magna Graecia University, Catanzaro, Italy.
  • 51] Laboratory of Molecular and Cellular Cardiology, Department of Medical and Surgical Sciences, Magna Graecia University, Catanzaro, Italy. [2].
  • 61] Centre of Human & Aerospace Physiological Sciences, School of Biomedical Sciences, King’s College London, London, UK. [2] Centre for Stem Cells and Regenerative Medicine, King’s College London, London, UK. [3] Laboratory of Molecular and Cellular Cardiology, Department of Medical and Surgical Sciences, Magna Graecia University, Catanzaro, Italy. [4].

 

Abstract

This protocol describes the isolation of endogenous c-Kit (also known as CD117)-positive (c-Kit(+)), CD45-negative (CD45(-)) cardiac stem cells (eCSCs) from whole adult mouse and rat hearts. The heart is enzymatically digested via retrograde perfusion of the coronary circulation, resulting in rapid and extensive breakdown of the whole heart. Next, the tissue is mechanically dissociated further and cell fractions are separated by centrifugation. The c-Kit(+)CD45(-) eCSC population is isolated by magnetic-activated cell sorting technology and purity and cell numbers are assessed by flow cytometry. This process takes ∼4 h for mouse eCSCs or 4.5 h for rat eCSCs. We also describe how to characterize c-Kit(+)CD45(-) eCSCs. The c-Kit(+)CD45(-) eCSCs exhibit the defining characteristics of stem cells: they are self-renewing, clonogenic and multipotent. This protocol also describes how to differentiate eCSCs into three main cardiac lineages: functional, beating cardiomyocytes, smooth muscle, and endothelial cells. These processes take 17-20 d.

PMID: 24945383

 

Additional text

The presence of a population of endogenous cardiac stem cells (eCSCs) that are resident within the myocardium of the adult mammalian heart has provided an explanation for the formation of new cardiomyocytes within the human heart throughout adult life (1 – Bergmann et al., 2009). A range of markers have been used for these cells, with the first discovery of eCSCs achieved using the stem cell factor receptor c-kit as the population marker (2 – Beltrami et al., 2003). Although the potential of these cells has been clearly demonstrated (3 – Ellison et al., 2013), they are relatively few in number and scattered throughout the myocardium.

 BMF Figure 1

Figure 1. Summary of stages of eCSC isolation. Perfusion of tissue via the aorta and coronary circulation allows rapid and full digestion of the cardiac tissue (A). Following this, the cardiac small cells are separated from larger cells using centrifugation and filtration (B). Magnetic-activated cells sorting allows isolation of the CD45-negative, c-kit-positive cell population (C). Within the CD45-positive, c-kit-positive population are the tryptase positive cardiac mast cells. By selecting for CD45-negative, c-kit-positive cells the mast cells are eliminated (D). Adapted from Smith et al. (2014), Nature Protocols, 9:1662-1681.

We therefore optimised our methods for isolating a highly-enriched c-kit-positive, CD45-negative eCSC population directly from the whole heart of both mice and rats, with some modifications of the technique for each species (4 – Smith et al., 2014). This method allows researchers to obtain a maximum number of these cells for study, a valuable advantage considering their rarity and also to isolate a cell population with high purity, which is essential to carrying out studies of the bulk eCSC population. Another potentially significant difficulty that is avoided is the potential for an obligatory in vitro stage to alter the cells’ characteristics. Using the method described, cells can be obtained fresh, directly from tissue and characterised immediately to more accurately determine their status when in situ.

Importantly, the entire heart is perfused via the coronary arteries (Figure 1A), first washing out blood with buffer, then digestive enzymes are perfused through the heart, to digest the heart tissue rapidly and fully. This allows the maximum number of eCSCs to be liberated from the myocardium and be made available for isolation. The cell suspension thus formed is centrifuged using a low braking speed to fraction the cells according to size: this allows the cardiac small cells (Figure 1B) to be separated for further selection of cell surface markers. At this stage, if required, the separated cardiomyocytes can be collected for purification and further use.

The cardiac small cells are then selected using magnetic-activated cell sorting (MACS) technology (Figure 1C), with two stages of selection necessary: the first is CD45 negative selection, to remove all haematopoietic lineage cells and tryptase positive cardiac mast cells (Figure 1D), the latter of which are c-kit-positive and would otherwise contaminate the c-kit-positive eCSC population (a particular problem as mast cells outnumber eCSCs in the heart by a ratio of ~3:1). Following this, the CD45-negative cell fraction undergoes a second round of MACS sorting for c-kit-positive selection, allowing the CD45-negative, c-kit-positive purified eCSC population to be obtained.

BMF Figure 2 pngFigure 2. Summary of eCSC characteristics. CD45-negative, c-kit-positive eCSCs are capable of self-renewal: forming clonal progeny, expressing pluripotency-associated genes, and able to generate spheres of primitive cells, analogous to neurospheres or embryoid bodies.

BMF Figure 3 png

Figure 3. Summary of eCSC differentiation potential. eCSCs can commit to the three main cell lineages of the heart, namely cardiomyocytes, endothelial cells and smooth muscle cells.

 

 

After CD45-negative, c-kit-positive eCSCs are isolated, they can be characterised immediately for their expression of surface marker proteins or pluripotency-associated genes, to determine their phenotype fresh from the in vivo environment. Alternatively, they may be placed in vitro for examination of their growth and their ability to generate clonal progeny or spheres of primitive cells in suspension. The eCSC population may also be expanded in culture, with their properties maintained over multiple passages (Figure 2).

We also document eCSC potential to differentiate into each of the three main lineages of the myocardium (Figure 3). We have identified techniques to maximise the in vitro generation of committed progenitors for cardiomyocytes, endothelial cells and smooth muscle cells, based around the sequential application of different combinations of growth factors. The most specialised differentiated cell type is the cardiomyocyte, and careful application of this stage-specific protocol to the eCSCs can generate spontaneously-beating cardiomyocyte precursors in vitro.

 

References

1 – Bergmann, O., Bhardwaj, R.D., Bernard, S., Zdunek, S., Barnabé-Heider, F., Walsh, S., Zupicich, J., Alkass, K., Buchholz, B.A., Druid, H., Jovinge, S. and Frisén, J. (2009). Evidence for cardiomyocyte renewal in humans. Science 324 (5923), 98-102.

2 – Beltrami, A.P., Barlucchi, L., Torella, D., Baker, M., Limana, F., Chimenti, S., Kasahara, H., Rota, M., Musso, E., Urbanek, K., Leri, A., Kajstura, J., Nadal-Ginard, B and Anversa, P. (2003). Adult cardiac stem cells are multipotent and support myocardial regeneration. Cell 114(6), 763-776.

3 – Ellison, G.M., Vicinanza, C., Smith, A.J., Aquila, I., Leone, A., Waring, C.D., Henning, B.J., Stirparo, G.G., Papait, R., Scarfò, M., Agosti, V., Viglietto, G., Condorelli, G., Indolfi, C., Ottolenghi, S., Torella, D. and Nadal-Ginard, B., (2013). Adult c-kitpos Cardiac Stem Cells Are Necessary and Sufficient for Functional Cardiac Regeneration and Repair. Cell 154(4), 827-42.

4 – Smith, A.J., Lewis, F.C., A, I., W, C.D., N, A., A, V., N-G, B., T, D., E, G.M., (2014) Isolation and characterisation of resident endogenous c-Kit+ cardiac stem cells from the adult mouse and rat heart. Nature Protocols 9(7), 1662-81.

 

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