Somatic stem cells and myocardial regeneration: A ‘cure’ for heart failure?

Jason Ali, Addenbrooke's Hospital, Hills Road, Cambridge, CB2 0SP

It is well recognised that cardiovascular disease is one of the leading causes of mortality in western societies. The British Heart Foundation in 2004 [1] stated that 1-3 people per 500 per annum suffer myocardial infarction. The natural history of this pathological process is the development of a fibrotic, non-contracting region of the ventricular wall, which physiologically manifests as reduced ventricular function. The success of the acute management of these patients in reducing mortality, combined with the ageing nature of western populations has contributed to the significant increase in the prevalence of chronic heart failure [2]. In this group of patients, the ventricular myocardium becomes dilated and hypertrophied – or ‘remodelled’ which leads to progressive diastolic and systolic dysfunction. This occurs because of cardiomyocyte loss. Of all current treatments, only cardiac transplantation addresses this loss. However, due to limited donor availability much work has recently concentrated on alternative ‘biological’ strategies to re-power the failing heart.

Cellular Transplantation

Post infarction of myocardial tissue, a process of remodelling occurs which has been found to involve cardiomyocyte apoptosis and replacement by fibrous tissue [3]. Currently, it is believed cardiac tissue cannot undergo regeneration, as many believe cardiac myocytes are terminally differentiated and unable to enter the cell cycle [4]. Cellular transplantation has emerged as a possible revolutionary means of myocardial regeneration – replacing damaged myocardium with the potential for preventing or treating cardiac failure in this large population.

Somatic stem cells

Injury to many organs is sensed by stem cells, which are recruited and undergo differentiation, promoting structural and functional repair. In the past, it has been believed that somatic stem cells could only differentiate into cells characteristic of the tissue they resided. However, work has suggested this not to be the case, and stem cells can be guided to differentiate into cells other than those of the tissue of origin in-vitro and in animal models [5]. It was the identification of this developmental plasticity which initiated work into the potential use of stem cells for regeneration of injured myocardium. However, it is unclear whether human somatic stem cells can demonstrate this pluri-potency to any useful extent in-vivo.

The most widely studied somatic stem cells are those of the bone marrow: haematopoietic stem cells (HSCs) which give rise to cells of the blood and immune systems. These cells are well characterised, relatively easy to isolate and have high plasticity [6]. The HSCs are surrounded by stromal cells within the bone marrow. This stroma includes another population of stem cells known as the mesenchymal stem cells (MSCs). These too are multipotent cells that grow as adherent cells in culture and have been shown to differentiate into diverse mesenchymal tissues including: cartilage, fat, tendon and skeletal muscle4. It is important to emphasise that both HSCs and MSCs are not single cell types, but comprise different cell populations which may have different characteristics.

It was the pioneering experiments of Orlic et al [7] which stimulated interest into the use of bone marrow derived cells as a source for myocardial regeneration. Orlic studied a mouse model of myocardial infarction and was the first to show that injection of a phenotypically well defined population of bone marrow cells (lin- c-kit+) after coronary artery ligation led to engraftment of transplanted bone marrow cells, repopulating the infarct areas. It was demonstrated that these cells had undergone differentiation into both cardiomyocytes and endothelial cells reducing the infarcted area, and led to improved cardiac haemodynamics. However, the actual extent of differentiation into cardiomyocytes is disputed, and these results have not been reproduced [8].

Once it was identified that HSCs are unable to differentiate into cardiomyocytes [8], work turned to the MSCs, which have been shown in vitro to be able to differentiate into cardiomyocytes [9]. Studies have identified that incubation with 5-azacytidine leads to the expression of a cardiomyocyte phenotype [10]. Functional cardiomyocytes have been created in vitro from MSCs.

Tomita et al [11] tested these cells in-vivo using a rat cryoinjury model of cardiac injury, and injected MSCs that had been isolated from bone marrow and incubated with 5-azacytidine. Transplanted MSC’s which had undergone differentiation to cardiomyocytes were identified in the scar at 8 weeks by immunohistochemistry. In the transplanted group there was found to be a significant reduction in scar size and reduced thinning of the scar. Additionally, an improvement in cardiac contractility was found with reduced remodelling. Interestingly increased angiogenesis was identified in those that were transplanted, opening the possibility that the MSCs not only differentiate into cardiomyocytes, but also other stromal lineages, or alternatively, lead to the induction of angiogenesis by the secretion of growth factors.

From bench to bedside

Although animal studies remained controversial, with increasing data from animal models, the transplantation of bone marrow derived stem cells moved to human clinical trials. The first randomised controlled trial was the BOOST study [12] (Bone marrow transfer to enhance ST-elevation infarct regeneration). In this study, which involved 60 patients, patients were randomised to best medical therapy vs transplantation of autologous bone marrow cells. The trial was not placebo controlled for ethical reasons. In those receiving transplantation, bone marrow biopsy was undertaken and cells prepared in vitro. All patients received best medical therapy and percutaneous coronary intervention (PCI), but in addition, the treatment group underwent intra-coronary transplantation of autologous bone marrow cells at 4.8 days post PCI. Results at 6 months showed promise. There was a significant increase in left ventricular ejection fraction, and increased wall motion in the border zone, but not the infarct region. Disappointingly there appeared not to be signs of reverse remodelling, but reassuringly, the technique proved safe, with no adverse outcomes. There were no differences in stent re-stenosis nor arrhythmia as determined by Holter monitoring, between treatment and control groups.

The excitement and possibilities that had been raised by this study were soon dampened by the publication of the 18 month follow up results [13]. Although the improvement in ejection fraction was sustained in the treatment group, there was late recovery in the control group such that there was no longer statistical significance in the difference between treatment and control groups. These results were disappointing, and the authors questioned whether transplanted cells only transiently enhance cardiac contractility without promoting structural repair, or if they merely accelerated an endogenous regenerative process. It is important to consider that if the effects of transplanted cells are transient, then the impact on long term end points such as the incidence of heart failure, or long term survival would likely be limited.

Recently, a Cochrane systematic review was published on stem cell treatment for acute myocardial infarction [14]. The authors felt that there was insufficient evidence to fully assess the clinical effects of this treatment. They concluded that stem cell treatment ‘may lead to a moderate improvement in left ventricular function’, noting a positive correlation with dose of cells transplanted. They did, however, comment that the procedure appears safe, but issue a note of caution regarding the lack of long term follow-up data.

For completeness it is important to mention the earliest experience of stem cell transplantation into humans – with skeletal myoblasts (non bone-marrow derived) in the 1990’s [15]. Although some success was reported, it was noted that there was an increase in arrhythmic events. Indeed, these cells can no longer be transplanted without prophylactic implantable cardioverter defibrillator implantation.

Cardiac progenitor cells?

The heart has been considered a terminally differentiated organ, excluding the presence of a tissue specific cardiac stem cell. This has been challenged recently by the finding of cycling myocytes undergoing mitosis and cytokinesis in pathological conditions such as infarction [16]. The clinical significance of these mitoses has been questioned. However, the implication is that there may be cardiac progenitor cells which differentiate into cardiomyocytes and cardiac stromal tissue. Many questions remain including whether these cells are resident in the myocardium or if they derive from the circulation – and ultimately questions regarding their existence. If such a population is identified then this could provide a new option for autologous transplantation.

Embryonic stem cells

Embryonic stem cells (ESCs) have emerged as an attractive alternative cell population for regenerating cardiac tissue as it is widely appreciated that ESCs have cardiomyocyte potential [17]. Work is at an earlier stage, and no human trials have yet occurred as there are several hurdles that still remain, including optimising yield of cardiomyocytes from ESC culture; managing the possibility of immunological rejection in allogeneic transplantation; and the risk of arrhythmogenesis as ESC populations are heterogenous with cardiomyocytes possessing atrial, pacemaker and ventricular action potentials.

Issues remain unanswered

There remain several challenges in cell transplantation therapy that must be addressed before this therapy becomes a mainstream therapy for heart failure.

- Optimal delivery of cells

It is recognised that delivery of cells to the myocardium is inefficient. One author reported only 1.3%-2.6% bone marrow derived stem cells remained in the myocardium after intra-coronary infusion [18]. The phenomenon of ‘cell washout’ was described, whereby transplanted cardiomyocytes were identified in tissues throughout the body [19]. As a result, cell retention is variable, and thus the graft size is unpredictable.

- Survival of transplanted cells

The poor survival of transplanted cells in the host environment has become a major impediment. It is seen that there is a high death rate in these cells over the early hours and days post-transplantation. Several factors have been proposed to explain this including: hypoxia due to poor vascularity of scar tissue, increased apoptosis, and enhanced immune recognition due to the inflammatory conditions post infarction [20]. Ensuring cells transplanted at the optimal time and cell culture conditions are optimal is where work is focused currently. The possibility of transferring anti-apoptotic genes has also been considered to promote survival.

- Optimising graft blood supply

It has been shown that cultured cardiomyocytes that incorporate more vascular structures demonstrate significantly greater survival in vivo [4]. Investigators have identified two different approaches to neo-vascularisation of infarcted areas. 1) Delivery of angioblasts which will differentiate into vascular structures, have been shown to improve infarct vascularity and lead to improved cardiac function in rodent models4. 2) Cardiomyocyte over-expression of angiogenic growth factors by gene transfection.

Mechanism of action

Surprising as it may sound, there is no consensus on the mechanism by which engrafted cardiomyocytes lead to improved cardiac function. There are several theories though.

Re-muscularisation – The obvious mechanism of action for engrafted cells is that they lead to the formation of an electormechanically coupled muscle graft, replacing the infarcted muscle, which contracts in synchrony and therefore contribute to contractility and systolic function as force generating units. However, there is surprisingly little data backing this suggestion.

Indirect effects: Paracrine – Much work has turned to the possibility that the engrafted cells have an indirect effect on the myocardium promoting improvement in cardiac function. The idea is that the cells produce paracrine factors, such as cytokines and growth factors. Investigators have removed the supernatant from cell culture preparations and have delivered this alone. In one study this lead to increased capillary density, decreased infarct size and improved cardiac function [21].

Structural support – It is recognised that post infarction, the scar tissue that develops is mechanically passive and the thinned myocardium is able to dilate over time, or remodel. It is suggested that rather than having a direct effect on improving systolic function, the impact of engrafted cells is to thicken the scar which acts to stabilise the tissue, thus attenuating remodelling mechanically – which has positive effects on cardiac function (22) This effect is analogous to dynamic cardiac myoplasty.

Restoration of ventricular synchrony – In a similar manner to cardiac synchronisation therapy with biventricular pacing, a recent paper suggested that there was improved ventricular synchrony in their stem cell transplanted group which correlated positively with improvement in cardiac function. The authors propose this as the mechanism of reverse remodelling, as is seen in biventricular pacing [23].

Other theories – There are a multitude of other theories proposed, a sign that there remains uncertainty. These are comprehensively reviewed by Laflamme et al [22]. It is important that work continues looking at mechanism of action as it is likely that once this is more fully understood, greater steps will be taken towards providing the optimal preparation for cellular transplantation.

The future

It is clear that the potential for cellular transplantation as a biological therapy for heart failure is significant. Although early human trials have shown only modest effects, it is early days. There are still many unknowns, and with time, our understanding of the biological processes involved will increase, allowing for optimisation of the processes involved such as culturing the best cell type, delivering the cells by the most efficient route and ensuring long term survival of the engrafted cells. This will likely involve combining recent advances in stem cell biology, developmental biology and tissue engineering.

In order to become a significant player in the management of cardiac failure, larger, randomised controlled trials are required which look particularly at patient focused outcomes: adverse effects, improvement of clinical indices, as well as quality of life measures. It is noticeable that the effects of cellular transplantation have shown transience, and looking at long term outcomes, not only of cardiac function, but also morbidity and mortality, and keeping an eye out for unforeseen long term adverse effects.

As the time approaches when these techniques are ready for integration into clinical guidelines, it will be important that these treatments are more formally assessed against current best management. Additionally evaluation of the cost of cellular transplantation will become important.

References: 

1. British Heart Foundation. Coronary heart disease statistics: factsheet. www.bhf.org.uk/news/uploaded/fact_sheet2.pdf [accessed 25/01/2009] 2004.

2. Masoudi F et al. The burden of chronic heart failure in older persons: the magnitude and implications for policy and research. Heart Failure Reviews. 2002;7:9-16 http://dx.doi.org/10.1023/A:1013793621248

3. Colucci WS. Molecular and cellular mechanisms of myocardial failure. The American Journal of Cardiology. 1997;80:15L-25L http://www.sciencedirect.com/science/article/pii/S000291499700845X

4. Itescu S et al. New directions in strategies using cell therapy for heart disease. Journal of Molecular Medicine. 2003;81:288-96 http://dx.doi.org/10.1007/s00109-003-0432-0

5. Smits AM et al. The role of stem cells in cardiac regeneration. Journal of Cellular and Molecular Medicine. 2005;9:25-36 http://dx.doi.org/10.1111/j.1582-4934.2005.tb00334.x

6. Bonnet D. Haematopoietic stem cells. Journal of Pathology. 2002;197:430-440 http://dx.doi.org/10.1002/path.1153

7. Orlic D et al. Bone marrow cells regenerate infarcted myocardium. Nature. 2001;410:701-5 http://dx.doi.org/10.1038/35070587

8. Murry CE et al. Haematopoietic stem cells do not transdifferentiate into cardiac myocytes in myocardial infarcts. Nature. 2004;428:664-68 http://dx.doi.org/10.1038/nature02446

9. Heng BC et al. Strategies for directing the differentiation of stem cells into the cadiomyogenic lineage in vitro. Cardiovascular Research. 2004;62:34-42 http://dx.doi.org/10.1016/j.cardiores.2003.12.022

10. Makino S et al.Cardiomyocytes can be generated from marrow stromal cells in vitro. Journal of Clinical Investigation. 1999;103:697-705 http://dx.doi.org/10.1172/JCI5298

11. Tomita S et al. Autologous transplantation of bone marrow cells improves damaged heart function. Circulation. 1999;100:II245-56 http://circ.ahajournals.org/content/100/suppl_2/II-247.full?sid=d052bddc...

12. Wollert KC et al. Intracoronary autologous bone-marrow cell transfer after myocardial infarction: the BOOST randomised controlled clinical trial. Lancet. 2004;364:141-48 http://www.thelancet.com/journals/lancet/article/PIIS0140-6736%2804%2916...

13. Meyer GP et al. Intracoronary bone marrow cell transfer after myocardial infarction: eighteen months’ follow-up data from the randomised, controlled BOOST (Bone marrow transfer to enhance ST-elevation infarct regeneration) Trial. Circulation 2006;113:1287-94 http://dx.doi.org/10.1161/CIRCULATIONAHA.105.575118

14. Martin-Rendon E et al. Stem cell treatment for acute myocardial infarction (Review) Cochrane Database of Systematic Reviews. 2008;Issue 4. http://dx.doi.org/10.1002/14651858.CD006536.pub2

15. Menasche P, Hagege AA, Vilquinn JT, et al. Autologous skeletal myoblast transplantation for severe postinfarction left ventricular dysfunction. Journal of the American College of Cardiology. 2003;41(7):1078-83 http://www.sciencedirect.com/science/article/pii/S0735109703000925

16. Anversa P et al. Myocyte renewal and ventricular remodelling. Nature. 2002;415:240-43 http://dx.doi.org/10.1038/415240a

17. Kehat I et al. Human embryonic stem cells can differentiate into myocytes and structural and functional properties of cardiomyocytes. Journal of Clinical Investigation. 2001;108:407-14 http://dx.doi.org/10.1172/JCI12131

18. Barbash IM et al. Myocardial regeneration by adult stem cells. The Israel Medical Association Journal. 2006;8:283-7 http://www.ima.org.il/IMAJ/ViewArticle.aspx?aId=1399

19. Dow I et al. Washout of transplanted cells from the heart: A potential new hurdle for cell transplantation therapy. Cardiovascular Research 2005;67:301-7 http://dx.doi.org/10.1016/j.cardiores.2005.04.011

20. Menasche P. Stem cells for clinical use in cardiovascular medicine. Thrombosis and Haemostasis. 2005; 94:697-701 http://dx.doi.org/10.1160/TH05-03-0218

21. Takahashi M et al. Cytokines produced by bone marrow cells can contribute to functional improvement of the infarcted heart by protecting cardiomyocytes from ischemic injury. American journal of physiology. Heart and Circulatory Physiology. 2006;291:H886–93 http://dx.doi.org/10.1152/ajpheart.00142.2006

22. Laflamme MA et al. Cell-based therapy for myocardial ischaemia and infarction: pathophysiological mechanisms. Annual Reviews of Pathology: Mechanisms of Disease. 2007;2:307-39 http://dx.doi.org/10.1146/annurev.pathol.2.010506.092038

23. Chang SA et al. Restoration of left ventricular synchronous contraction after acute myocardial infarction by stem cell therapy: new insights into the therapeutic implication of stem cell therapy for acute myocardial infarction. Heart. 2008; 94:995-1001 http://dx.doi.org/10.1136/hrt.2007.124701