The search for the most suitable cell for cardiac repair continues

The search for the most suitable cell for cardiac repair continues. Bone marrow is an easy accessible source of heterogenous population of stem and progenitor cells with high potential for cardiac repair and regeneration. Among cells residing in bone marrow are stem and progenitor cells as well as already committed cells, primarily contained in the hematopoietic compartment. In most clinical trials the non-selected mix of bone marrow-derived cells was used in order to achieve the structural and functional improvement, however the benefits of such treatment are modest [1] [2] [3] [4]. Therefore there is still a need for further research to identify the optimal population of cells which can be used for studies of cardiac repair. The ideal population of cells would be easily accessible, expandable and have a high potential of differentiation. 

Types and hierarchy of bone marrow cells

In addition to committed lineages such as endothelial progenitor cells (EPC) and hematopoietic stem cells (HSC), numerous populations of cells with a higher differentiation potential were identified in adult bone marrow - multipotent mesenchymal stromal cells (MSC), multipotent adult progenitor cells (MAPC) and very small embryonic-like stem cells (VSEL) [5] [6] .

The pivotal property of stem cells is self-renewal as well as the ability to differentiate into tissue-committed cells. The pool of stem cells is hierarchical from the most to the least primitive cells :

• most primitive totipotent stem cells can give rise to embryo and placenta (zygote, first balstomeres)
• pluripotent stem cells - population with high degree of developmental potential Pluripotent stem cells can differentiate into cells from all three germ layers, but cannot form the trophoblast. These cells are derived from the inner cell mass of the blastocyst and in vitro from the embryonic cell lines.
• multipotent stem cells form one of three germ layers: endo, ecto or mesoderm
• monopotent (tissue-committed stem cells) give rise to cells of one lineage  e.g., hematopoietic stem cells, neural stem cells, skeletal muscle stem cells [7].

Pluripotent stem cells

a) The developmental potency of pluripotent stem cells can be compared to embryonic stem cells

Pluripotent stem cells are present during embryonic development, however recent data from animal and human studies showed that adult bone marrow as well as solid organs (kidneys, liver, thymus, heart) can contain a limited number of these primitive cells. The developmental potency of pluripotent stem cells can be compared to embryonic stem cells. Since the potential use of embryonic stem cells is limited due to ethical and legal reasons the perspective of obtaining pluripotent stem cells from an easily accessible source such as bone marrow and their potency of the differentiation into all cell lineages would be of great advantage for the regenerative cardiology [7] [8].

b) Oct-4 cells are pluripotent cells

The pluripotency of the cells can be assessed using several criteria. First of all the cells express markers typical of pluripotent stem cells, i.e transcription factors Oct-4+ and Nanog which are present in cells of the developing blastocyst, surface marker of early embryonic stem cells SSEA (SSEA-1 in murine cells, SSEA-3/4 in human cells). So far the presence of Oct-4 is the most frequently used marker of pluripotent cells in adult tissues. Oct-4 plays important role in the formation of mature blastocysts and is down-regulated during development, and the fact that Oct-4 had been identified in some rare cells present in adult tissues suggests that some embryonic stem cells may persist into adulthood. The most convincing evidence that these cells are in fact pluripotent would be to demonstrate that they can complement the development of the blastocyst, however it remains to be proved [9] [7].

c) Very small embryonic-like stem cells (VSEL) display the features of pluripotent stem cells

Cells displaying the features of pluripotent stem cells are good candidates for further studies in this area. VSEL (very small embryonic-like stem cells) can be easily isolated from bone marrow using live cell sorting. In addition, murine VSEL can be expanded and differentiated into cardiomyocytes. A similar population of cells was identified in humans, so this type of pluripotent cell is a promising candidate for clinical studies in patients with myocardial infarction and/or ischemic cardiomyopathy.

Bone marrow-derived cells expressing markers of pluripotent stem cells differentiate into cardiac myocytes

Recently published studies showed that murine bone marrow contains cells capable of cardiogenic differentiation giving rise to the population of spontaneously contracting cardiomyocytes. The cardiomyogenic potential of bone marrow cells was confined to the rare population (0,05% of bone marrow mononuclear cells) of cells expressing pluripotent cell marker Oct-3/4. The cells were identified as non-hematopoietic cells expressing CXCR4 - receptor for chemokine SDF-1 and negative for stem cell markers Sca-1 and CD34 (Oct3/4+c-kit+CXCR4+Sca-1-CD34-CD45-) and were localised adjacent to the osteoblastic niche. The bone marrow cells in vitro differentiated into clusters of cells expressing cardiac structural proteins [α- and β-myosin heavy chain, cardiac troponin-T, (cTnT), α-sarcomeric actinin, α-cardiac actin, light chain ventricular myosin], beta-adrenergic receptors and connexins (Cx40, Cx43).

In addition, microelectrode studies showed the reactivity of bone marrow-derived cardiomyocytes to adrenergic stimulation. The pattern of cardiogenic differentiation including changes of the gene expression and cell morphology of bone marrow Oct-3/4+ cells was similar to the one observed in differentiation of embryonic stem cells into cardiomyoctyes. The presence of transcription factor Oct-3/4 is necessary for the early stages of cardiac differentiation, after which the expression is down-regulated similarly to murine embryonic stem cells [10]. 

This confirms studies showing that bone marrow cells can differentiate into cardiomyocytes and identifies the population of non-hematopoietic cells expre ssing pluripotent marker Oct-4 as the source of cells which have a high potential of differentiation. Since the number of cells in bone marrow is very small the possibility of cell expansion would be of great importance.

Recently published data confirmed that murine bone marrow contains non-hematopoietic (lin-Sca-1+CD45-) cells, which express several developmental markers characteristic for embryonic pluripotent stem cells and display a unique morphology. Based on their small size, morphology and presence of PSC markers Oct-4, Nanog, SSEA-1 and Rex-1 the cells were named very small embryonic-like (VSEL) stem cells. Electron microscopy analyses of sorted cells revealed that murine VSEL are very small (2-4µm) and morphologically resemble the pluripotent embryonic stem cells (narrow rim of cytoplasm, large nucleus, euchromatin). VSEL form rare population of BMMNC (0.02%) [9]. Murine VSEL in co-culture with C2C12 myoblastic cell line form spheres of primitive cells that express fetal alkaline phosphatase.

Importantly, cells isolated from these spheres differentiate in secondary tissue specific cultures into cells from all three germ layers, including mesoderm-derived cardiomyocytes. VSEL were also recently identified in human cord blood and in peripheral blood after acute myocardial infarction. This population of pluripotent cells can be isolated from the bone marrow and peripheral blood after mobilisation using multiparameter sterile live cell sorting according to their immunophenotype (Sca-1+lin-CD45-) [11]. 

The presence of Oct-4+ cells was also demonstrated in the adult murine myocardium. The profile of gene expression showed that these cells resemble embryonic stem cells [12]. In animal studies the number of Oct-4+ cells in the bone marrow is significantly (6-10-fold) reduced in 1-year old mice in comparison to 1-month old mice. Data form animal models showed that both younger and older mice had similar time course and number of circulating cells following acute MI, however the level of expression of pluripotent stem cells markers was significantly lower in older animals [13].

Trafficking of bone marrow-derived Oct-4+ cells

In the setting of myocardial ischemia Oct-4+ cells are mobilised from bone marrow and other putative niches. An increase in the number of circulating non-hematopoietic cells showing a high degree of differentiation potential coexist with overexpression of chemoattractant cytokines and growth factors which can facilitate cell homing into the damaged area [14] [13]. In addition, recent studies showed that the phenotype of bone marrow cells displaying the cardiomyogenic potential may be similar to resident cardiac stem cells. Bone marrow is also a potential source of cardiac stem cells and mobilisation from bone marrow may be the physiological pathway to replenish the population of CSC [15]. Therefore, bone marrow cells expressing markers of pluripotent stem cells may form one mobile compartment with resident cardiac stem cells.

The content of this article reflects the personal opinion of the author/s and is not necessarily the official position of the European Society of Cardiology.

Conclusion:

Adult bone marrow contains cells expressing the markers of pluripotent stem cells, such as Oct-4. Oct-4+ cells have high potency of cardiogenic differentiation, and undergo mobilization into peripheral blood in response to myocardial infarction. This population of cells is potentially good candidate for further studies involving myocardial regeneration.