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Abstract

  1. Top of page
  2. Abstract
  3. Which Stem Cells?
  4. Bone Marrow-Derived Stem Cells
  5. Resident Cardiac Stem Cells
  6. Heart Stem Cell Niches
  7. Engraftment
  8. Bioengineering
  9. Conclusions
  10. References

The authors extend their coverage of recent developments in stem cell-based therapy for repairing the heart to cover the basic questions of what stem cells should be used and how best to favor their survivability within the injured heart. The authors focus their attention on those adult stem/progenitor cells that have been best investigated in animal studies for repairing the infarcted heart and are the focus of completed or ongoing clinical trials. In addition, they discuss the promise that resident cardiac stem cells offer and the recent identification of specialized architecturally defined niches within the heart to nurse their development. Bioengineering approaches employing off-the-shelf mesenchymal stem cell patches may soon provide a way to recreate these niches in the scarred heart. Conceivably, these patches might also be seeded with prescribed mixtures of culturally expanded autologous stem/progenitor cells that would lead to new blood vessel and cardiac myocyte formation. The convergence of bioengineering and molecular biology on stem cell therapy would seem to make what was once unimaginable, cardiac regeneration, a clinical reality in less than one generation.

In our preceding article,1 we discussed some of the fundamental issues that need to be resolved before cytokine-based cell therapy for repairing the heart becomes a clinical reality. These include establishing the best time to initiate treatment, optimizing mobilization and homing of cells to the injured heart, and determining which route of delivery works best. Even more basic are the questions of which stem cells should be used and what factors favor their functional engraftment into the myocardium. Those issues are dealt with here. Unlike in part A, we will not overtly deal with granulocyte colony-stimulating factor (G-CSF)-based therapies, but bear in mind that G-CSF and other cytokines will play an important role in any approach involving stem cells.

Which Stem Cells?

  1. Top of page
  2. Abstract
  3. Which Stem Cells?
  4. Bone Marrow-Derived Stem Cells
  5. Resident Cardiac Stem Cells
  6. Heart Stem Cell Niches
  7. Engraftment
  8. Bioengineering
  9. Conclusions
  10. References

In the quest to attain cardiac regeneration through cell-based strategies, embryonic stem cells, a number of adult stem/progenitor cells, and skeletal myoblasts have been used. An accounting of the pros and cons of each is beyond the scope of this article, and the reader is referred to some excellent recent reviews that cover this expansive topic in great detail.2–8 We restrict our focus to just some of the adult stem/progenitor cells that have been considered for heart repair, namely bone marrow-derived and cardiac resident stem/progenitor cells. A word of clarification first: stem cells by definition are undifferentiated cells with the property of being self-renewing and capable of differentiating into more than one specific type of mature cell.9 Because in many cases with adult “stem cells,” these features have not been rigorously established, the less restrictive classification, progenitor cells, is more appropriate. With that noted and for simplicity, we will herein simply use the term “adult stem cell.”

Bone Marrow-Derived Stem Cells

  1. Top of page
  2. Abstract
  3. Which Stem Cells?
  4. Bone Marrow-Derived Stem Cells
  5. Resident Cardiac Stem Cells
  6. Heart Stem Cell Niches
  7. Engraftment
  8. Bioengineering
  9. Conclusions
  10. References

Adult bone marrow stem cells can be divided into 2 groups based on whether they express the cell surface marker CD34.10 The CD34+ hematopoietic stem/progenitor cells give rise to blood and endothelial cells. Mobilized endothelial progenitor cells (EPCs) contribute to postnatal neovascularization, reendothelialization, and angiogenesis via paracrine actions.10,11 The CD34 group includes the mesenchymal stem cells (MSCs). A synopsis of the current thinking on the potential value of each cell type in cardiac repair/regeneration follows (Table I).

Table I.  Key Features of Studies Using Bone Marrow-Derived Stem Cells to Repair the Heart
 Hematopoietic Stem CellsEndothelial Progenitor CellsMesenchymal Stem Cells
Relevant markersCD34+/c-kit+CD34+/c-kit+/VEGFRCD34
Homing to infarct+++
Engraftmenta
   Short-termModestModerateModerate
   Long-termReducedReducedNo/reduced
Cardiac myocyte neogenesis in vivoinline imageLowLow
Improved function
   Animal+++
   Human+b,cinline image
aSome if not all engraftment attributed to cell fusion. bFrom Numaguchi et al.27cFrom Tatsumi et al.28

Stem Cells for Cardiac Repair/Regeneration

Embryonic

Adult

  • Umbilical cord (Cord blood/Wharton's jelly)
  • Skeletal myoblasts
  • Mesenchymal (bone marrow/adipose tissue)
  • Hematopoietic
  • Endothelial progenitor cells
  • Cardiac

Hematopoietic Stem Cells. Most of the early animal and clinical studies used nonfractionated bone marrow stem cells, which are largely hematopoietic stem cells (HSCs). HSCs normally are mobilized following infarction and home to the injured myocardium.10 Unfortunately, once in the heart their numbers apparently decrease substantially over time. Transdifferentiation of these cells into cardiac myocytes in vitro has not been definitively proven; however, several studies in mice have shown that HSCs do differentiate in vivo into smooth muscle, endothelial cells, and cardiac myocytes.6,9 The reported numbers of newly formed cardiac myocytes are for the most part extremely low, and transdifferentiation of HSCs into cardiac myocytes may be a process that only occurs to any measurable extent in nonprimates.8 Whether these engrafted stem cells expressing myocyte structural proteins go on to become mature cardiac myocytes, functionally integrated into the myocardium, is unknown.8 Over the past several years, there has been much heated controversy as to whether HSCs actually transdifferentiate to cardiac myocytes in vivo or whether the observed presence of cardiac myocyte-specific proteins in these stem cells is due to their fusion with a mature muscle cell.4,8 In any event, both camps seem to agree that HSCs improve cardiac function in infarcted mouse hearts, with those on the side of the cell fusion argument postulating a beneficial paracrine effect of the HSCs on endogenous cardiac myocytes.4,10 In hindsight, the entire issue seems rather misdirected given the disappointing outcome of recent controlled clinical trials on the efficacy of improving cardiac function post-myocardial infarction (MI) with mobilized bone marrow-derived stem cells.1

Endothelial Progenitor Cells. EPCs are a very small subpopulation of hematopoietic stem cells. They are a heterogeneous group but have in common the ability to differentiate into endothelial cells. Like HSCs, EPCs express CD34, CD133, c-kit, and vascular endothelial growth factor receptor 2 (ie, flk-1 or kinase insert domain receptor). They also express CD38, but unlike HSC generally also express vascular endothelial-cadherin and fibroblast growth factor receptor.11 Vascular injuries, such as MI, result in their mobilization from the bone marrow and remarkably efficient homing to the site of injury. In animals, EPCs were shown to engraft in the infarct zone and cause formation of new blood vessels (vasculogenesis) as well as to enhance the branching of preexisting vessels (angiogenesis), presumably via the secretion of growth factors.9 Left ventricular function is improved as well. A hurdle that needs to be overcome to make clinical use of EPCs practical in heart treatments is their low circulating numbers, which is further reduced by atherosclerosis and age.1,8,11 As previously discussed,1 molecular strategies represent a logical approach to improve the number, mobilization, homing, and survivability/engraftment of autologous EPCs in the injured heart. In vitro, expansion of EPCs in culture is a complementary strategy that is also being undertaken. Last, delivery route is a potential issue with EPCs because they might contribute to in-stent restenosis or destabilize atherosclerotic plaques.12

Mesenchymal Stem Cells. MSCs are found not only in the bone marrow but probably in all postnatal organs and tissues, including adipose tissue, dental pulp, periosteum, and muscle.13 Three subpopulations have been described.5 The recycling stem (RS) cells are the group that consists of the smallest, most rapidly dividing cells with a defined multilineage differentiation potential. In addition to not expressing hematopoietic cell surface markers, RS cells do not express c-kit. The other 2 groups, the multipotent adult progenitor cells and the human bone marrow-derived multipotent stem cells, are distinguished from RS cells by both the expression of c-kit as well as being able to differentiate into all 3 germ layer cell types.5 When repairing the infracted heart, MSCs exhibit many of the same advantages and drawbacks as HSCs and EPCs (Table I). In animal studies, MSCs home to the infracted myocardium, engraft, and produce beneficial effects on cardiac function.4,5,12,13 MSCs will transdifferentiate into cardiac myocytes in culture when seeded on top of neonatal rat ventricular myocytes14 or when treated with the DNA demethylating agent 5-azacytidine15; however, in vivo transdifferentiation into cardiac myocytes is low and a significant contribution of cellular fusion cannot be excluded. In addition, engraftment declines over time and the sustainability of cardiac myocyte transdifferentiation has yet to be established. Evidence suggests that MSCs stimulate neovascularization in the heart through paracrine actions,5,15 which likely underlines the improvement in cardiac function. Significant myocardium regeneration with MSCs does not occur. Notably, MSCs offer 2 advantages over either HSCs or EPCs. MSCs are easily purified and grow extremely well in culture.14 In addition, MSCs possess the property of being both immunosuppressive, which raises the possibility of using them “off-shelf,”5 and anti-inflammatory.13

As discussed in detail elsewhere,4,5 several clinical studies on the applicability of MSCs for cardiac repair have been completed or are currently under way. The results of 2 published studies in post-MI patients support the safety and feasibility of intracoronary autologous MSC delivery, as well as the ability of MSCs to reduce infarct size. Currently, 3 studies registered with ClinicalTrials.gov deal with the use of MSC in heart patients (Table II). One, closed to recruitment, is designed to assess the safety and effectiveness of MSCs in treating acute MI. A second, currently recruiting study is a double-blind trial assessing whether combined coronary artery bypass graft surgery and MSC transplantation offers any additional advantages over coronary artery bypass graft surgery alone in patients with ischemic coronary heart disease and heart failure. The third study, which has not yet started recruiting, will assess the safety and efficacy of autologous MSC therapy in patients with severe chronic myocardial ischemia.

Table II.  Heart-Related MSC Clinical Trials Registered With ClinicalTrials.gov
IdentifierSponsorTargetPhasePrimary Outcome Measure(s)Status
NCT00418418Helsinki UniversityPatients with low left ventricular ejection fraction scheduled for coronary bypassIIImprovement in ejection fraction measured with magnetic resonance imagingRecruiting
NCT00260338Rigshospitalet, DenmarkSafety and efficacy study in patients with severe chronic myocardial ischemiaI/IIImprovement in myocardial perfusion measured by single photon emission computed tomographyNot yet open
NCT00114452Osiris TherapeuticsSafety (and efficacy) study of patients with acute myocardial infarctionITreatment adverse event ratesClosed

Resident Cardiac Stem Cells

  1. Top of page
  2. Abstract
  3. Which Stem Cells?
  4. Bone Marrow-Derived Stem Cells
  5. Resident Cardiac Stem Cells
  6. Heart Stem Cell Niches
  7. Engraftment
  8. Bioengineering
  9. Conclusions
  10. References

Four resident cardiac stem cell (CSC) populations have been identified in various species with at least 2 being present in the adult human heart.6,9,16 What, if any, relationship exists among these populations is presently unknown. Nor is it known how cardiac and bone marrow-derived stem cells relate, although there is evidence that at least one pool of resident CSCs is replenished in the infarcted myocardium by bone marrow-derived stem cells.17 The 2 populations found in the adult human heart have been shown to have the capacity to differentiate into cardiac myocytes and endothelial cells in vitro and in vivo and into smooth muscle at least in vitro.6,9

Resident Cardiac Stem Cells

Population Human Adult Heart c-kit + SP + Isl-1 inline image Sca1 −

Both EPCs and MSCs have been shown to enhance the recruitment of endogenous CSCs to damaged regions of the heart.8,18 CSCs, however, are readily obtained from small biopsies of human myocardium and can be expanded in culture under conditions that maintain their differentiation potential, purity, and ability to engraft and migrate into the infracted zone.19–21 Human CSCs expanded in culture and cryopreserved have been shown to enhance myocardial regeneration and improve left ventricular function when injected into the border zone of acute myocardial infarcts of immunodeficient mice 4 hours after reperfusion.21

Heart Stem Cell Niches

  1. Top of page
  2. Abstract
  3. Which Stem Cells?
  4. Bone Marrow-Derived Stem Cells
  5. Resident Cardiac Stem Cells
  6. Heart Stem Cell Niches
  7. Engraftment
  8. Bioengineering
  9. Conclusions
  10. References

Stem cells require a protective microenvironment that allows for their slow cycling and self-renewal and fosters differentiation. Last year, breathtakingly beautiful images of stem cell niches in the adult mouse heart were published.22 These niches were found to be architecturally defined spaces in which CSCs divide mostly asymmetrically, giving rise to one daughter stem cell and one daughter cell committed to becoming an endothelial cell, smooth muscle cell, or cardiac myocyte. Mature cardiac myocytes and fibroblasts form the structural elements of the niches and communicate with CSCs at the outer niche surface via gap and adherens junctions. Based on available evidence, it seems reasonable to speculate that effective stem cell engraftment in the heart may be enhanced by their traversing first through these niches (Figure).

image

Figure Figure. A somewhat whimsical illustration of the key role that stem cell niches likely make to cardiac regeneration. Circulating stem cells, such as hematopoietic stem cells (HSCs) and endothelial progenitor cells (EPCs), home to the infarcted zone of the heart. Engraftment is low, however, and declines over time. Within the heart are specialized microenvironments or niches that nurture and protect cardiac stem cells (CSCs), thereby facilitating their engraftment and eventual migration to the injured region. These niches may be in dynamic equilibrium with circulating stem cells, facilitating their engraftment and survivability as well. Finally, the surgical attachment of mesenchymal stem cells (MSCs) may stimulate cardiac repair by artificially creating niches in the injured heart.

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Engraftment

  1. Top of page
  2. Abstract
  3. Which Stem Cells?
  4. Bone Marrow-Derived Stem Cells
  5. Resident Cardiac Stem Cells
  6. Heart Stem Cell Niches
  7. Engraftment
  8. Bioengineering
  9. Conclusions
  10. References

All data to date indicate that engraftment, both short- and long-term, is a limiting factor for stem cell therapy in repairing the heart. After all, the infracted myocardium represents a hostile environment for stem and progenitor cell survivability and maturation. On top of that is the underevaluated possibility that drugs used to treat cardiac remodeling and heart failure may exert harmful effects on stem cells that have homed to the heart.7 Using molecular biology to harden stem cells is a possible solution.1 Stem cells may require a period of in situ nurturing, however, to enhance their survivability and transdifferentiation capacity. In common speak, some level of communication with the mature/differentiated cells of the heart may be necessary to program stem cells to make more heart and do it well. The recent findings and hypothesis of Mazhari and Hare18 touch on this possibility. These investigators found that allogeneic MSCs improved ejection fraction and reduced infarct size in a porcine model of MI. Moreover, a rim of new cardiac tissue was found where the MSCs were injected, although they were able to discount the possibility that the MSCs transdifferentiated into cardiac myocytes. Rather, these investigators found evidence suggesting that the MSCs had induced the proliferation of both mature cardiac myocytes and resident c-kit+ stem cells that express GATA4, which is a transcription factor important early on for differentiation of stem cells into cardiac myocytes.18 Replication of microvessels was also noted. To explain these findings, Mazhari and Hare propose the very exciting hypothesis that imported MSCs positively influence cardiac repair by recreating CSC niches in the injured heart, thereby establishing a nurturing environment for stem cells, analogous to the role in the bone marrow.

Bioengineering

  1. Top of page
  2. Abstract
  3. Which Stem Cells?
  4. Bone Marrow-Derived Stem Cells
  5. Resident Cardiac Stem Cells
  6. Heart Stem Cell Niches
  7. Engraftment
  8. Bioengineering
  9. Conclusions
  10. References

Advances in nanotechnology and biomaterials offer exciting possibilities for enhancing cardiac regeneration by applying bioengineering approaches to the delivery, engraftment, and survivability of circulating or harvested (from bone marrow or adipose tissue) stem cells in the heart or by enhancing the native regenerative capacity of the heart.23 Two recent reports illustrate the promise as well as the yet-to-be surmounted shortcomings of using bioengineering to repair the heart.24,25 Both studies applied MSCs to the scar area of the infarcted hearts of rats. One study used the more conventional approach of attaching a 3-dimensional tissue construct made with rat tail type I collagen (applied to the infarct 10 minutes post-coronary ligation).24 The other study harvested MSC monolayers grown at 37°C on culture dishes that became hydrophilic when the temperature was reduced to below 32°C, thereby detaching the cells (applied to infarct 4 weeks post-coronary ligation).25 When assessed 4 weeks after MSC transplant, both studies reported improvements in cardiac function, evidence of newly formed vessels (more so with the monolayers), and increased ventricular wall thickness. The latter was either due to a regenerative effect of the MSCs on residual noncardiac myocyte cells in the infracted zone24 or to a thickening of the MSC monolayer.25 Either by omission24 or by analysis,25 the studies reported few if any new cardiac myocytes within the area of repair.

Conclusions

  1. Top of page
  2. Abstract
  3. Which Stem Cells?
  4. Bone Marrow-Derived Stem Cells
  5. Resident Cardiac Stem Cells
  6. Heart Stem Cell Niches
  7. Engraftment
  8. Bioengineering
  9. Conclusions
  10. References

Despite the rapid increase in our understanding of stem cells, the goal to regenerate actual myocardium in the injured heart has yet to be realized. Only time will tell whether this goal can be attained. We now know things about the heart and its ability to repair itself that were unimaginable just 5 years ago. The heart has shown itself to be a more dynamic and resilient organ than once thought. Whether it is now simply a matter of assembling the pieces of the puzzle, such as figuring out the right combination of MSCs, EPCs, and CSCs, or whether an alternative strategy may ultimately prove successful, such as reprogramming the cell cycle of cardiac myocytes to allow for their proliferation,26 is anyone's guess. What does seem certain is that our new way of viewing this organ, which is still developing, will lead to novel and better ways to repair the injured and failing heart.

Relevant Web Sites

References

  1. Top of page
  2. Abstract
  3. Which Stem Cells?
  4. Bone Marrow-Derived Stem Cells
  5. Resident Cardiac Stem Cells
  6. Heart Stem Cell Niches
  7. Engraftment
  8. Bioengineering
  9. Conclusions
  10. References