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I.R.F. / Aging news / Diseases / 04010701

Bone Marrow Stem Cells – a New Hope for Patients
Posted on: January 7, 2004

Myocardial infarction is, by nature, an irreversible injury. Regional systolic function and regional metabolism decrease within a few heartbeats of a sudden decrease in myocardial perfusion. In some patients, impaired diastolic relaxation may precede global systolic abnormalities. Irreversible cardiomyocyte injury begins after ~15 to 20 minutes of coronary artery occlusion. The subendocardial myocardium has high metabolic needs and thus is most vulnerable to ischemia. The extent of the infarction depends on the duration and severity of the perfusion defect. However, the extent of infarction is also modulated by a number of factors including collateral blood supply, medications, and ischemic preconditioning. Beyond contraction and fibrosis of myocardial scar, progressive ventricular remodeling of nonischemic myocardium can further reduce cardiac function in the weeks to months after the initial event.

Many of the therapies available to clinicians today can significantly improve the prognosis of patients with acute myocardial infarction. Although angioplasty and thrombolytic agents can relieve the cause of the infarction, the time from onset of occlusion to reperfusion determines the degree of irreversible myocardial injury. No medication or procedure used clinically has shown efficacy in replacing myocardial scar with functioning contractile tissue. There is need for new therapeutics to regenerate normal cardiomyocytes.

Recent attempts to repair experimentally induced acute myocardial infarctions have provided encouraging but limited success in a number of animal models. The most promising results have been obtained after transplantation and mobilization of bone marrow cells to the area of infarction.

Adult Stem Cells Engage in Normal Tissue Regeneration
Stem cells are the ancestors of the specialized cells that impart function to tissues and organs. Throughout postnatal life, stem cells regenerate tissues that continually lose cells through maturation and senescence. These include the epithelial layers in skin; intestinal and pulmonary mucosal linings; and connective tissues such as bone, cartilage, muscle, blood, and bone marrow. Although previously considered to be postmitotic organs, recent evidence is persuading us to include brain and heart on the list of adult tissues with regenerative capacity. The extremely low rate of neural and myocardial cell turnover may explain why renewing cells were not previously detected in these organs.

The origin of stem cells in regenerating adult tissues is now being called into question. It can no longer be assumed that tissue-specific stem cells are self-sustaining throughout life or that they are responsible for regenerating tissues damaged by radiation or chemotherapy treatments. Bone marrow cells appear to have the capacity to repopulate many nonhematopoietic tissues. Thus, bone marrow may serve as a central repository for the primitive stem cells that can repopulate somatic tissues.

Potentiality of Bone Marrow Steam Cells (BMSCs)
Adult Mesenchymal Stem Cells (MSCs) can be derived from adult bone marrow and in vitro appear to have multilineage differentiation capacity. In culture, MSCs can maintain an undifferentiated, stable phenotype over many generations. Preclinical models have shown the ability of undifferentiated human MSCs to undergo site-specific differentiation into a functional cardiac muscle phenotype after injection into sheep. Thus, they seem to avoid detection by the host immune system. Allogeneic bone marrow MSCs may therefore have potential clinical utility because of their lack of immunogenicity and relative ease of culture. As such they can be harvested and cryopreserved ready for infusion immediately after myocardial infarction.

Another subset of bone marrow stromal cells referred to as mesodermal is progenitor cells or multipotent adult progenitor cells. Multipotent adult progenitor cells copurify with MSCs. They proliferate extensively and differentiate in vitro into cells of all three germ layers. When injected in vivo they reconstitute bone marrow, liver, gut, lungs, and endothelium.

Two categories of blood-forming stem cells exist in adult bone marrow. One population can provide permanent long - term reconstitution of the entire hematopoietic system. These cells referred to as Hematopoietic Stem Cells (HSCs) are rare, perhaps as few as 1:10 000 bone marrow cells. As few as 20 to 100 highly purified mouse bone marrow HSCs can reconstitute the entire lymphohematopoietic system in myeloablated adult mice. They can self-renew and can differentiate into the more mature progenitor cells in bone marrow.

The Progenitor Cells of bone marrow have a limited capacity for self-renewal and differentiation. They can only sustain hematopoiesis for 1 to 2 months and therefore are considered to be short-term repopulating stem cells. It is proposed that one subclass of mouse progenitor cells, the common lymphocytic progenitors (CLP), is restricted to the generation of B and T lymphocytes, whereas another subclass, the common myelocytic progenitors (CMP), is restricted to the generation of myelocytic cells. These CLP and CMP initiate hematopoietic activity by giving rise to precursor cells responsible for the formation of each blood cell lineage. Although in vivo assays for human CLP and CMP are not established, it is clear from in vitro studies that these progenitors exist in human bone marrow. In the treatment of blood disorders, it is now routine clinical practice to isolate and transplant stem cells. These include progenitor cells and HSCs that provide short-term and long-term hematopoietic reconstitution.

Emerging data suggest that BMSCs have the ability to differentiate into stem and progenitor cells that mature into functional cells in a variety of tissues including myocardium.

Adult Mouse BMSC Plasticity
In a series of reports, it has been suggested that adult BMSCs retain the capacity to produce cells of unrelated tissues. Based on evidence from several mouse models, tissues of all three germ layers can be derived from adult BMSCs (Figure 1). These include skeletal muscle, hepatocytes, neural cells, vascular endothelium, and epithelium of skin and several internal organs. Plasticity of transplanted BMSCs has been established by identifying specific cell surface markers or by fluorescence in situ hybridization identification of Y-positive nuclei in donor-derived cells that have acquired the capacity to synthesize specific protein in regenerating tissues.

Fig. 1. Proposed developmental pattern for adult BMSC transdifferentiation into multiple lineages. Exact identity of candidate BMSC (single or multiple) has not been established. There is evidence suggesting that marrow-derived HSCs, mesenchymal (stromal) stem cells, and/or cells with potential of embryonic hemangioblasts may be involved in transdifferentiation.

Environmental Factors, Involved in Stem Cell Migration
The homing of stem cells to areas of tissue injury may potentially occur via two or more distinct scenarios. One hypothesis suggests that cell necrosis following an injury such as myocardial infarction may cause the release of signals that circulate and induce mobilization of stem cells from the bone marrow pool. The injured tissue may express appropriate receptors or ligands to facilitate trafficking and adhesion of stem cells to the site of injury where initiation of a differentiation cascade results in the generation of cells of the appropriate lineage. An alternative hypothesis suggests that stem cells are continually circulating with constant trafficking through all tissues, but only at the time of injury do they exit the blood and begin to infiltrate the site of injury. Both concepts support the view that there are circulating stem cells that could originate from a common pool in bone marrow. This is further supported by findings that show that stem cells isolated from skeletal muscle retain hematopoietic activity and are itinerant cells derived from bone marrow.

It is still unclear what environmental cues initiate mobilization and homing of adult BMSCs to normal and injured tissue. Several receptor-ligand interactions that may regulate BMSC trafficking and that are the subject of intense investigation are as follow.

Central role in the mobilization of stem and progenitor cells from the bone marrow niche within hours of onset of myocardial necrosis play Stem Cell Factor (SCF), c-kit (a tyrosine kinase receptor) and matrix metalloproteinase-9. The intense inflammatory reaction that initiates healing after a left ventricular (LV) myocardial infarction causes a local accumulation of mast cells that are positive for CD117, the human equivalent of c-kit. They may migrate locally in response to macrophage secretion of SCF. This reinforces the idea that homing signals are released soon after myocardial injury.

CXCR4 and Stromal Cell-Derived Factor-1 (SDF-1) are important for lymphocyte trafficking and recruitment at sites of inflammation, e.g., after myocardial infarction.

Granulocyte Colony-Stimulating Factor (G-CSF), the cytokine G-CSF, is widely used to mobilize stem/ progenitor cells that are harvested by leukapheresis, stored, and subsequently reinfused to support hematopoietic recovery in patients after chemotherapy or radiation treatment. How G-CSF mobilizes stem cells and progenitor cells from the bone marrow into the circulation is not clear because BMSCs do not generate G-CSF receptor. This suggests that an indirect mechanism may exist.

The roles of the Vascular Endothelial Growth Factor (VEGF)/Flk-1 isoforms and the tyrosine kinase VEGF receptors in endothelial cell proliferation and differentiation are well described.

Myocardial Regeneration in a Mouse Model
The data on regeneration in an adult mouse model of myocardial infarction demonstrate the ability of BMSCs to differentiate into cardiac myocytes, endothelial cells, and vascular smooth muscle cells. Ischemic injury to the myocardium of the left ventricle was produced by ligation of the descending branch of the left coronary artery (LCA) without reperfusion. The infarcts occupied as much as 70% of the free wall of the left ventricle with loss of myocytes and coronary vessels. When male BMSCs (labeled with green fluorescent protein (eGFP)) were injected within 5 hours after coronary ligation, a band of regenerating myocardium was seen at 9 days after surgery. This band consisted of cardiac myocytes and small coronary vessels.

Regeneration was not observed in hearts that were transplanted with the subpopulation of bone marrow cells known to be devoid of stem cells. Cardiomyocytes, smooth muscle cells, and endothelial cells were all eGFP positive. Early-acting cardiac-specific transcription factors were expressed in the developing cardiomyocytes as well as cardiac myosin, sarcomeric α-actin, and connexin 43. The immature myocytes were arranged into what appeared to be an early form of an integrated syncytium with some connexin 43 at the lateral border of adjacent cells. LV end-diastolic pressure and LV developed pressure improved 30% to 40% in hearts transplanted with BMSCs compared with negative control mice.

Myocardial regeneration was also examined in a mouse model in which BMSCs marked with the β-galactosidase gene were used to create chimeric bone marrow in adult mice. Subsequently, myocardial infarcts were induced, by ligation of the LCA. β-Galactosidase-positive BMSCs were found to migrate to the site of injury and to give rise to new cardiomyocytes and endothelium. Although the level of β-galactosidase-positive cells was only 0.02% of the total myocardial cells counted, this study demonstrated a natural but very inefficient response by the BMSCs to repair the damaged myocardium.

An extensive regeneration in the mouse myocardial infarction model was observed with cytokine-mobilized autologous BMSCs. After 5 daily injections of recombinant rat SCF and recombinant human G-CSF, the wave of circulating BMSCs reached a peak. Myocardial infarctions produced by ligation of the LCA showed a new band of myocardium. The new myocytes resembled fetal cardiac myocytes in size and gene expression. Their failure to mature and their continued proliferation remain unresolved issues. Numerous developing capillaries and arterioles were observed, and some contained red blood cells in their lumen. This suggested that anastomosis had occurred with the spared coronary vessels. Cytokine therapy improved hemodynamic functions, including ejection fraction, LV end-diastolic pressure, and LV end-systolic pressure, and resulted in markedly increased survival at 27 days.

These experiments demonstrated the capacity of adult BMSCs to give rise to new myocytes, endothelial cells, and smooth muscle cells in ischemic myocardium. However, they did not define whether one or more BMSC populations were responsible for the generation of these several myocardial cell types. Nor did they exclude the possibility that some stem cells with regenerative capability may have originated in other organs, including the heart.

Clinical Trials to Regenerate Myocardium in Human Ischemic Heart Disease
In 10 patients with acute myocardial infarction, autologous mononuclear bone marrow cells were transplanted via the infarct-related artery after angioplasty. At 3 months after transplant, the infarct area had decreased significantly compared with 10 patients not given cell therapy. The treated patients also showed improved LV end-systolic volume and contractibility. This demonstrates clinical feasibility of intracoronary infusion of bone marrow cells for myocardial repair.

Investigators recently reported the effects of intracoronary and systemic administration of granulocyte-macrophage colony-stimulating factor (GM-CSF) in patients with coronary artery disease. GM-CSF is a cytokine with effects on bone marrow similar to the more commonly utilized G-CSF, albeit with less potency for BMSC mobilization. Twenty-one patients who were not amenable to or refused coronary bypass surgery participated in this randomized, double blind, placebo-controlled study. Ten individuals received GM-CSF via intracoronary infusion into the vessel believed to subserve ischemic myocardium, followed by systemic administration of GM-CSF daily for 2 weeks. Analysis of mobilization of BMSCs was not performed in this study, but because the leukocyte counts were only twice the baseline values, it was suggested that only modest BMSC mobilization was achieved.

An invasive measure of collateral artery blood flow (estimated by coronary artery pressure distal to balloon occlusion) before and after administration of GM-CSF or placebo indicated improved collateral flow in the GM-CSF group at 2 weeks, but not in the placebo group, with reduced ECG signs of myocardial ischemia during coronary balloon occlusion. Because the quantity of BMSCs mobilized with GM-CSF was probably low, the coronary vascular benefit determined in this study may have resulted from direct effects of this cytokine on angiogenesis or on collateral vascular dilator tone with improved regional blood flow. No clinically relevant end points (e.g., exercise-induced myocardial ischemia or LV contractile response to stress) were assessed in this study.

These studies represent clinical attempts to regenerate myocardium after infarction. However, the empirical nature of these observations emphasizes the need for continued preclinical testing to determine the phenotype of the bone marrow cells involved in myocardial repair and to define the signaling required for their migration and differentiation into myocardial cells. When these questions are resolved, we can look to a time when transplanted or cytokine-mobilized stem cells may provide a new modality for the treatment of heart disease.

Predicted Role for BMSCs in Regenerative Medicine
It is now routine practice for patients about to undergo myeloablation to be treated with G-CSF to mobilize BMSCs into the circulation for the purpose of collection and subsequent use for bone marrow reconstitution. Hence, BMSC may have potential clinical utility in cardiac patients. In patients with refractory myocardial ischemia, safety feasibility studies have already begun. These studies are designed to determine whether BMSCs can traffic to the heart and develop into cardiomyocytes and coronary vessels.

Existing data indicate that BMSCs continually proliferate and enter the circulation. These circulating BMSCs appear to have an engraftment phenotype that fluctuates with the phase of cell cycle. Thus, BMSCs with different engraftment capabilities are continually passing through capillary networks in all tissues. Data showing production of a variety of bone marrow-derived cell types including hepatocytes and cardiomyocytes suggest that circulating BMSCs seed and differentiate into tissue-specific cells. It remains to be established whether this plasticity is attributed to a single subpopulation of BMSCs or to multiple subpopulations each being tissue restricted. At present, we can only acknowledge the ability of BMSCs to colonize skeletal muscle, skin, bone, liver, retina, and heart.

The differentiation of BMSCs that seed peripheral organs is regulated by exposure to local environmental factors. Thus, a BMSC or its progeny that in bone marrow would give rise to granulocytes, erythrocytes, and platelets will give rise to lymphocytes in the thymus, hepatocytes in the liver and cardiac myocytes in the heart. This has exciting clinical potential because BMSCs are self-renewing and can be easily harvested from bone marrow and peripheral blood. Furthermore, clinical experience shows that BMSC transplantation does not lead to neoplasia as may occur with other stem cell populations. The need to expand the scope of investigations using embryonic and fetal stem cells is of paramount importance and cannot be overstated, but adult BMSCs may offer the best near-term promise for tissue repair.

Tissue regeneration using adult BMSCs is now openly discussed among even the most conservative scientists and clinicians. If additional, encouraging preclinical data can be obtained, the next decade is likely to witness clinical trials aimed at testing the capacity of BMSCs to regenerate damaged tissues.

Source: Donald Orlic, Jonathan M Hill, Andrew E Arai.; Stem cells for myocardial regeneration; Circulation Research, Dec 13-27, 2002, Vol. 91, Iss. 12; pg. 1092-1102.
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