Selectins for Cardiosphere Culture: The “E's” Have It!
Authors: Darryl R Davis, Duncan J Stewart
Journal: Molecular Therapy (2012)

For many years, the translation of autologous cellular cardiac therapy has been frustrated by the need for prolonged culture of rare cardiac cell types to generate therapeutically relevant dosages. In a forthcoming issue of Molecular Therapy, Cho and colleagues1Cho H Lee H Youn S Koh S Won J Chung Y et al.Secondary sphere formation enhances the functionality of cardiac progenitor cells.Mol Ther. 2012; (e-pub ahead of print 19 June 2012 doi10.1038/mt.2012.109)Google Scholar report their finding that E-selectin–ERK/Sp1–VEGF autostimulation induced by cardiosphere culture underlies the ability of expanded cardiac stem cells (CSCs) to undergo prolonged culture under these conditions while retaining therapeutic potential. Rather than using small molecules or genetic manipulation, which could lead to activation of off-target malignant genes, the authors used straightforward sphere-culture practices to upregulate production of vascular endothelial growth factor (VEGF) within expanded CSC sources that ultimately provided enhanced outcomes following transplantation. Before the end of the twentieth century, the adult mammalian heart was thought to be incapable of repair after injury. It has since become widely accepted that the heart contains small reservoirs of stem cells2Beltrami AP Barlucchi L Torella D Baker M Limana F Chimenti S et al.Adult cardiac stem cells are multipotent and support myocardial regeneration.Cell. 2003; 114: 763-776Abstract Full Text Full Text PDF PubMed Scopus (2987) Google Scholar,3Oh H Bradfute SB Gallardo TD Nakamura T Gaussin V Mishina Y et al.Cardiac progenitor cells from adult myocardium: homing, differentiation, and fusion after infarction.Proc Natl Acad Sci USA. 2003; 100: 12313-12318Crossref PubMed Scopus (1508) Google Scholar and that cardiomyocytes renew with a 0.5–1% yearly turnover that results in fewer than 50% of cardiomyocytes being exchanged during a normal life span.4Bergmann O Bhardwaj RD Bernard S Zdunek S Barnabe-Heider F Walsh S et al.Evidence for cardiomyocyte renewal in humans.Science. 2009; 324: 98-102Crossref PubMed Scopus (2260) Google Scholar The origin of this turnover is unclear, but emerging evidence suggests that, in the absence of invasive genetic manipulation,5Qian L Huang Y Spencer CI Foley A Vedantham V Liu L et al.In vivo reprogramming of murine cardiac fibroblasts into induced cardiomyocytes.Nature. 2010; (e-pub ahead of print 18 April 2012)Google Scholar,6Riley PR Converting scar to muscle in situ in the injured heart.Mol Ther. 2012; 20: 1294-1296Abstract Full Text Full Text PDF PubMed Scopus (1) Google Scholar committed adult cardiac cells are unable to dedifferentiate into functional myocytes.7Porrello ER Mahmoud AI Simpson E Hill JA Richardson JA Olson EN et al.Transient regenerative potential of the neonatal mouse heart.Science. 2011; 331: 1078-1080Crossref PubMed Scopus (1675) Google Scholar Because these stem cells reside within the heart, it follows that if their number and activity could be increased they would be natural candidates to repopulate damaged hearts. To this end, we and others have shown that distinct subpopulations of CSCs can be isolated directly from cultured cardiac tissue.8Davis DR Kizana E Terrovitis J Barth AS Zhang Y Smith RR et al.Isolation and expansion of functionally-competent cardiac progenitor cells directly from heart biopsies.J Mol Cell Cardiol. 2010; 49: 312-321Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar This technique provides a complementary collection of cardiac, endothelial, and mesenchymal progenitor cells while simplifying cell culture practices through focus on the primary product from cardiac samples, without the need for antigenic subselection or prolonged culture. When samples of minced cardiac tissue are cultured, a lawn of flat cells emigrates spontaneously from the plated cardiac tissue. Within that lawn, clusters of CSCs emerge and proliferate. Using mild enzymatic dissociation, loosely adherent cells surrounding the explant can be serially harvested. When injected into murine models of cardiac ischemia, these cells provide long-term functional benefits through direct cardiomyocyte and vascular transdifferentiation, in combination with indirect paracrine-mediated tissue preservation and/or recruitment of endogenous progenitors.9Chimenti I Smith RR Li TS Gerstenblith G Messina E Giacomello A et al.Relative roles of direct regeneration versus paracrine effects of human cardiosphere-derived cells transplanted into infarcted mice.Circ Res. 2010; 106: 971-980Crossref PubMed Scopus (520) Google Scholar Unfortunately, application of cells cultured from the direct outgrowth of cardiac samples is limited by the amount of cell product that can be derived from the modest amount of cardiac tissue acquired/plated using clinically acceptable techniques. This has prompted the use of "enrichment" strategies using sphere-culture conditions followed by expansion into a single-cell product for intracoronary delivery.10Messina E De Angelis L Frati G Morrone S Chimenti S Fiordaliso F et al.Isolation and expansion of adult cardiac stem cells from human and murine heart.Circ Res. 2004; 95: 911-921Crossref PubMed Scopus (1225) Google Scholar,11Smith RR Barile L Cho HC Leppo MK Hare JM Messina E et al.Regenerative potential of cardiosphere-derived cells expanded from percutaneous endomyocardial biopsy specimens.Circulation. 2007; 115: 896-908Crossref PubMed Scopus (949) Google Scholar,12Li TS Cheng K Lee ST Matsushita S Davis D Malliaras K et al.Cardiospheres recapitulate a niche-like microenvironment rich in stemness and cell–matrix interactions, rationalizing their enhanced functional potency for myocardial repair.Stem Cells. 2010; 28: 2088-2098Crossref PubMed Scopus (189) Google Scholar This cell product, termed cardiosphere-derived cells, has been shown to improve functional outcomes and directly regenerate injured cardiac tissue.13Makkar RR Smith RR Cheng K Malliaras K Thomson LE Berman D et al.Intracoronary cardiosphere-derived cells for heart regeneration after myocardial infarction (CADUCEUS): a prospective, randomised phase 1 trial.Lancet. 2012; 379: 895-904Abstract Full Text Full Text PDF PubMed Scopus (1122) Google Scholar Although early publications hinted that the intermediate cardiosphere stage might provide greater functional benefits than single-cell preparations, proof has been lacking.10Messina E De Angelis L Frati G Morrone S Chimenti S Fiordaliso F et al.Isolation and expansion of adult cardiac stem cells from human and murine heart.Circ Res. 2004; 95: 911-921Crossref PubMed Scopus (1225) Google Scholar In this light, we recently demonstrated that three-dimensional sphere culture enhances expression of markers of "stemness," adhesion molecules, and outcomes of in vitro assays of potency.12Li TS Cheng K Lee ST Matsushita S Davis D Malliaras K et al.Cardiospheres recapitulate a niche-like microenvironment rich in stemness and cell–matrix interactions, rationalizing their enhanced functional potency for myocardial repair.Stem Cells. 2010; 28: 2088-2098Crossref PubMed Scopus (189) Google Scholar These benefits were preserved when expanded monolayer-cultured cells underwent sphere culture (secondary cardiospheres). We proposed that dissociation of cardiospheres into single cells decreased the expression of extracellular matrix and adhesion molecules while increasing vulnerability to oxidative stress and reducing in vivo functional benefits. Cho and colleagues confirm these findings, but they were unable to demonstrate a difference in post-infarct benefits between monolayer-cultured cells and primary or secondary cardiospheres. This result may be related to culture practices and media formulations that are dissimilar to those in the published literature (i.e., fetal bovine serum concentrations, monolayer culture in cardiosphere growth media, and hanging-drop culture of CSCs). These details are important because it has been shown that variations in culture methods may have a profound impact on final culture output.14Davis DR Zhang Y Smith RR Cheng K Terrovitis J Malliaras K et al.Validation of the cardiosphere method to culture cardiac progenitor cells from myocardial tissue.PLoS ONE. 2009; 4: e7195Crossref PubMed Scopus (236) Google Scholar However, the authors recognize these limitations and draw a conscious distinction between their cell product and those under clinical investigation using surface-antigen profiling and clearly dissimilar terminology (sphere-derived cells vs. cardiosphere-derived cells). Cho et al. also show that hanging-drop culture may provide a superior means of replicating the niche-like properties of the cardiosphere. By departing from the electrostatic inhibition between poly-d-lysine and CSCs that underlies traditional cardiosphere and neurosphere culture, the investigators translated classic embryonic stem cell culture techniques to CSCs by confining cells within a drop of medium suspended by gravity and hydrostatic tension. Secondary cardiospheres grown using this new method had greater persistent functional benefits. The mechanism underlying this finding is unclear but deserves to be explored in future studies. If validated, this observation may significantly alter the manner in which CSCs are "enhanced" before delivery. By far the most important aspect of this report is the data defining the mechanism of sphere formation. VEGF has long been known to play important roles in the survival of neural stem cells, and several publications have explored this in the world of neural stem cells or "neurospheres."15Campos LS Leone DP Relvas JB Brakebusch C Fassler R Suter U et al.Beta1 integrins activate a MAPK signalling pathway in neural stem cells that contributes to their maintenance.Development. 2004; 131: 3433-3444Crossref PubMed Scopus (272) Google Scholar,16Ishii S Okada Y Kadoya T Matsuzaki Y Shimazaki T Okano H Stromal cell-secreted factors promote the survival of embryonic stem cell–derived early neural stem/progenitor cells via the activation of MAPK and PI3K-Akt pathways.J Neurosci Res. 2010; 88: 722-734PubMed Google Scholar Cho et al. are the first to develop our understanding of sphere mechanisms in CSCs. To this end, they contrast transcriptome profiling of primary cardiospheres with monolayer-cultured cells (or sphere-derived cells). The rationale for selecting E-selectin as opposed to other adhesion molecules identified using their microarray—such as intercellular adhesion molecule 1 (ICAM1) or β-catenin (CTNNB1)—as a target for investigation is not entirely clear. The data from E-selectin knockout mice or short interfering RNA knockdown provides convincing proof that this molecule plays an important role in CSC sphere formation. The report also demonstrates that ERK/Sp1 signaling, downstream from the E-selectin–matrix interactions, plays a role in sphere growth by enhancing VEGF production and leading to auto/paracrine stimulation of sphere maturation. However, these findings deserve more attention because it is unclear what cells within the aggregate population of sphere-derived cells express E-selectin with downstream effects on ERK/Sp1. Drawing on our previous work, it is possible that the cardiac progenitor cell population is stimulated by this E-selectin–ERK/Sp1–VEGF loop, but further study is warranted.12Li TS Cheng K Lee ST Matsushita S Davis D Malliaras K et al.Cardiospheres recapitulate a niche-like microenvironment rich in stemness and cell–matrix interactions, rationalizing their enhanced functional potency for myocardial repair.Stem Cells. 2010; 28: 2088-2098Crossref PubMed Scopus (189) Google Scholar In summary, the study by Cho and colleagues is a welcome addition to the field because it provides new methods for enhancing the potency of expanded autologous cells as well as insights into the fundamental mechanisms of this effect. As with all good studies, the findings raise many new questions and opportunities. If these techniques are translatable to CSC sources applied in clinical trials,13Makkar RR Smith RR Cheng K Malliaras K Thomson LE Berman D et al.Intracoronary cardiosphere-derived cells for heart regeneration after myocardial infarction (CADUCEUS): a prospective, randomised phase 1 trial.Lancet. 2012; 379: 895-904Abstract Full Text Full Text PDF PubMed Scopus (1122) Google Scholar,17Bolli R Chugh AR D'Amario D Loughran JH Stoddard MF Ikram S et al.Cardiac stem cells in patients with ischaemic cardiomyopathy (SCIPIO): initial results of a randomised phase 1 trial.Lancet. 2011; 378: 1847-1857Abstract Full Text Full Text PDF PubMed Scopus (1106) Google Scholar they may provide a safe, nontoxic means of easily enhancing cell products before delivery. Alternatively, the fundamental pathways highlighted by this study may eventually pinpoint targets for specific small molecules to drive CSC proliferation forward while avoiding undesirable effects such as phenotypic drift or malignant transformation.