INTRODUCTION:A mouse model of progeria derived by insertion of the human mutant LMNA gene (mLMNA), producing mutant lamin A, shows loss of smooth muscle cells in the media of the ascending aorta. We hypothesized that high shear stress, in the presence of mutant lamin A, induces this vasculopathy and tried to define the molecular and cellular basis for aortic vasculopathy.METHODS:Ascending and descending aortas from wild type (WT) and mLMNA+ mice were compared using proteomics, Western blots, PCR and immunostaining. To determine whether high fluidic shear stress, known to occur in the ascending aorta, contributed to the vasculopathy, we exposed descending aortas of mLMNA+ mice, with no apparent vasculopathy, to 75 dynes/cm2 shear stress for 30 minutes using a microfluidic system.RESULTS:When the mice were one year of age, expression of several mechanotransduction proteins in the ascending aorta, including vimentin, decreased in mLMNA+ mice but no decrease occurred in the descending aorta. High fluidic shear stress produced a significant reduction in vimentin of mLMNA+ mice but not in similarly treated WT mice.CONCLUSIONS:The occurrence of mutant lamin A and high shear stress correlate with a reduction in the level of mechanotransduction proteins in smooth muscle cells of the media. Reduction of these proteins may contribute over time to development of vasculopathy in the ascending aorta in progeria syndrome.

HSC were harvested from transgenic mice expressing green fluorescent protein (GFP) and injected in the region bordering the infarct, 3-5 hours after coronary artery occlusion in mice. A band of closely packed cells was identified 7 to 17 days later in nearly 50% of HSC injected hearts, between the endocardial and epicardial surface of the infarcted ventricle. This band occupied 50 75% of the damaged portion of the wall. c-kit/GFP positive HSC were found in the infarcted area shortly after coronary ligation and were still detectable at 7 days. c-kit stained HSC were not labeled by markers of myocytes, a-sarcomeric actin and myosin, endothelial cells, factor VIII, and smooth muscle cells, smooth muscle actin. The band of tissue included in the infarcted zone was constituted 75% by GFP, a-sarcomeric actin, myosin and a-actinin positive cardiac muscle cells. Other GFP-positive cell populations were endothelial cells and smooth muscle cells, organized in nascent capillary structures and arterioles. Proliferating myocytes were small with partially aligned myofibrils and resembled late fetal-neonatal cells. GFP-positive replicating myocytes, endothelial cells and smooth muscle cells were c-kit negative. Infarcted mice were injected with BrdU, once a day for 4 days, to establish the extent of cell proliferation in the regenerating myocardium: 28% myocytes, 17% endothelial cells and 12% smooth muscle cells were BrdU positive. These three levels of BrdU labeling were statistically different. Additionally, the percentages of cells positive to Ki67 were measured to evaluate the fraction of cycling cells at this stage of repair: 18% myocytes, 10% endothelial cells and 8% smooth muscle cells were in G1-G2M. In conclusion, HSC, when injected in the heart, rapidly differentiate into myocardium resulting in significant recovery of muscle mass after infarction.

Rationale: The ability of the human heart to regenerate large quantities of myocytes remains controversial, and the extent of myocyte renewal claimed by different laboratories varies from none to nearly 20% per year.Objective: To address this issue, we examined the percentage of myocytes, endothelial cells, and fibroblasts labeled by iododeoxyuridine in postmortem samples obtained from cancer patients who received the thymidine analog for therapeutic purposes. Additionally, the potential contribution of DNA repair, polyploidy, and cell fusion to the measurement of myocyte regeneration was determined.Methods and Results: The fraction of myocytes labeled by iododeoxyuridine ranged from 2.5% to 46%, and similar values were found in fibroblasts and endothelial cells. An average 22%, 20%, and 13% new myocytes, fibroblasts, and endothelial cells were generated per year, suggesting that the lifespan of these cells was approximately 4.5, 5, and 8 years, respectively. The newly formed cardiac cells showed a fully differentiated adult phenotype and did not express the senescence-associated protein p16(INK4a). Moreover, measurements by confocal microscopy and flow cytometry documented that the human heart is composed predominantly of myocytes with 2n diploid DNA content and that tetraploid and octaploid nuclei constitute only a small fraction of the parenchymal cell pool. Importantly, DNA repair, ploidy formation, and cell fusion were not implicated in the assessment of myocyte regeneration.Conclusions: Our findings indicate that the human heart has a significant growth reserve and replaces its myocyte and nonmyocyte compartment several times during the course of life. (Circ Res. 2010; 107:305-315.)

RATIONALE:The ability of the human heart to regenerate large quantities of myocytes remains controversial, and the extent of myocyte renewal claimed by different laboratories varies from none to nearly 20% per year. OBJECTIVE:To address this issue, we examined the percentage of myocytes, endothelial cells, and fibroblasts labeled by iododeoxyuridine in postmortem samples obtained from cancer patients who received the thymidine analog for therapeutic purposes. Additionally, the potential contribution of DNA repair, polyploidy, and cell fusion to the measurement of myocyte regeneration was determined. METHODS AND RESULTS:The fraction of myocytes labeled by iododeoxyuridine ranged from 2.5% to 46%, and similar values were found in fibroblasts and endothelial cells. An average 22%, 20%, and 13% new myocytes, fibroblasts, and endothelial cells were generated per year, suggesting that the lifespan of these cells was approximately 4.5, 5, and 8 years, respectively. The newly formed cardiac cells showed a fully differentiated adult phenotype and did not express the senescence-associated protein p16(INK4a). Moreover, measurements by confocal microscopy and flow cytometry documented that the human heart is composed predominantly of myocytes with 2n diploid DNA content and that tetraploid and octaploid nuclei constitute only a small fraction of the parenchymal cell pool. Importantly, DNA repair, ploidy formation, and cell fusion were not implicated in the assessment of myocyte regeneration. CONCLUSIONS:Our findings indicate that the human heart has a significant growth reserve and replaces its myocyte and nonmyocyte compartment several times during the course of life.

Aims Bone marrow (BM)-derived endothelial progenitor cells (EPCs) in the circulation replace damaged vascular endothelium. We assessed the hypothesis that a BM transplant from healthy animals would restore normal arterial endothelium and prevent hypertension in young endothelial nitric oxide synthase-deficient (eNOS(-/-)) mice.Methods and results Radiation or busulfan-induced BM ablation in eNOS(-/-) mice on day 6, day 14, or day 28 was followed by a BM transplant consisting of enhanced green fluorescent protein positive (EGFP(+)) cells from C57BL/6J mice. Peripheral blood cell chimerism was always greater than 85% at 4 months after BM transplant. Molecular assays of heart, kidney, and liver revealed low-level chimerism in all treatment groups, consistent with residual circulating EGFP+ blood cells. When aorta, coronary, renal, hepatic, and splenic arteries in BM-transplanted eNOS(-/-) mice were examined by confocal microscopy, there were no EGFP- or eNOS-positive endothelial cells detected in these vessels in any of the treatment groups. Likewise, telemetry did not detect any reduction in blood pressure. Thus, no differences were observed in our measurements using several different treatment protocols.Conclusion We found no evidence for BM-derived EPIC renewal of endothelium in this eNOS-deficient mouse model of a chronic vascular disease or in wild-type mice during postnatal growth. Hence, renewal of chronic dysfunctional endothelium and endothelial homeostasis may be dependent on resident vascular progenitor cells.

Adult bone marrow consists of several populations of stem cells that are the focus of investigations into their potential to regenerate nonhematopoietic tissues. According to this hypothesis, bone marrow stem cells display a plasticity not previously recognized. Although data supporting bone marrow stem cell plasticity is extensive, many researchers dispute this concept. One of the most controversial aspects of stem cell plasticity relates to regeneration of heart muscle following acute myocardial infarction (MI). When experimentally induced MIs in rodents were analyzed for regeneration of the heart tissues, it was reported that cardiomyocyte renewal was achieved as a result of bone marrow stem cell infiltration of the damaged tissue. Evidence continues to accumulate in support of and against the potential for myocardial regeneration, indicating the need for a better scientific basis for the possible involvement of bone marrowderived stem cells in myocardial regeneration. In order to achieve a higher level of acceptance for myocardial regeneration, researchers must develop more exacting methodologies to monitor repair over time at the cellular level. A major effort must be undertaken to identify the signals required for stem cell mobilization and trafficking to infarcted cardiac tissue and to define the genetic mechanisms involved in stem cell plasticity. To answer these questions it will be necessary to establish the exact identity of the stem cell population involved. Controversies regarding myocardial regeneration in rodent models will require additional experiments using large animal models, with an emphasis on tracking of labeled donor cells. These preclinical experiments will also enable testing celldelivery devices and noninvasive modalities for detecting improvement in regional and global myocardial function. If key questions relating to transdifferentiation potential, cell survival, and function can be resolved, we may one day be able to fully exploit the potential of stem cells for myocardial repair.

Current myeloablative conditioning regimens for hematopoietic stem cell (HSC) transplantation are associated with significant morbidity and mortality. Thus, alternative strategies to promote engraftment of infused HSCs with increased safety warrant investigation. Using parabiotic mice, we determined that, after mobilization with AMD3100 (a CXCR4 antagonist), HSCs exited from marrow, transited blood, and engrafted in open niches in partner marrow. We then hypothesized that mobilization before transplantation might vacate niches and improve HSC engraftment. When PeP3(b) mice were treated with AMD3100 at 2 hours before the transplantation of 4 x 10(7) marrow cells, donor cell engraftment was higher (4.6% +/- 1.1%) than in control animals (no AMD3100; 1.0% +/- 0.24%, P < .001). When mice received weekly injections of AMD3100 on 3 consecutive weeks and marrow cells were transplanted 2 hours after each mobilization, donor cell engraftment further increased (9.1% +/- 1.7%, P = .001). In contrast, in similar experiments with Balb/cByJ mice that mobilize poorly, there was no difference between the donor cell engraftment of AMD3100-treated and control recipients. These results indicate that the number of available niches regulates the number of HSCs. In addition, mobilization with AMD3100 may provide a safer preparative approach for HSC transplantation in genetic and other nonmalignant disorders.

AMD3100, a bicyclam antagonist of the chemokine receptor CXCR4, has been shown to induce rapid mobilization of CD34(+) hematopoietic cells in mice, dogs, and humans, offering an alternative to G-CSF mobilization of peripheral-blood hematopoietic stem cells. In this study, AMD3100-mobilized CD34(+) cells were phenotypically analyzed, marked with Neo(R)-containing retroviral vectors, and subsequently transplanted into myeloablated rhesus macaques. We show engraftment of transduced AMD3100-mobilized CD34(+) cells with Neo(R) gene marked myeloid and lymphoid cells up to 32 months after transplantation, demonstrating the ability of AMD3100 to mobilize true long-term repopulating hematopoietic stem cells. More AMD3100-mobilized CD34(+) cells are in the G(1) phase of the cell cycle and more cells express CXCR4 and VLA-4 compared with G-CSF-mobilized CD34(+) cells. In vivo gene marking levels obtained with AMD3100-mobilized CD34(+) cells were better than those obtained using CD34(+) cells mobilized with G-CSF alone. Overall, these results indicate that AMD3100 mobilizes a population of hematopoietic stem cells with intrinsic characteristics different from those of hematopoietic stem cells mobilized with G-CSF, suggesting fundamental differences in the mechanism of AMD3100-mediated and G-CSF-mediated hematopoietic stem cell mobilization. Thus, AMD3100-mobilized CD34(+) cells represent an alternative source of hematopoietic stem cells for clinical stem cell transplantation and genetic manipulation with integrating retroviral vectors.

Adult BM stem cells are being investigated for their potential to regenerate injured tissues by a process referred to as plasticity or transdifferentiation. Although data supporting stem cell plasticity is extensive, a controversy has emerged based on findings that propose cell-cell fusion as a more appropriate interpretation for this phenomenon. A major focus of this controversy is the claim that acutely infarcted myocardium in adult hearts can be regenerated by BM stem cells. Many researchers consider the adult heart to be a post-mitotic organ, whereas others believe that a low level of cardiomyocyte renewal occurs throughout life. If renewal occurs, it may be in response to cardiac stem cell activity or to stem cells that migrate from distant tissues. Post-mortem microscopic analysis of experimentally induced myocardial infarctions in several rodent models suggests that cardiomyocyte renewal is achieved by stem cells that infiltrate the damaged tissue. For a better understanding of the possible involvement of stem cells in myocardial regeneration, it is important to develop appropriate technologies to monitor myocardial repair over time with an emphasis on large animal models. Studies on non-human primate, swine and canine models of acute myocardial infarctions would enable investigators to utilize clinical quality cell-delivery devices, track labeled donor cells after precision transplantation and utilize non-invasive imaging for functional assays over time with clinical accuracy. In addition, if stem cell plasticity is to reach the next level of acceptance, it is important to identify the environmental cues needed for stem cell trafficking and to define the genetic and cellular mechanisms that initiate transdifferentiation. Only then will it be possible to determine if, and to what extent, BM stem cells are involved in myocardial regeneration and to begin to regulate precisely tissue repair.

AMD3100 (AMD) has recently been shown to rapidly mobilize primitive hematopoietic cells in mice and humans, but little is known about the properties of cells mobilized with this agent. We initiated a study to determine retroviral (RV) in vivo gene marking efficiency in AMD-mobilized CD34+ cells in rhesus macaques. CD34+ cells collected 3 hours after administration of AMD to 2 animals were transduced using RV vectors containing the Neo® gene. Animals were irradiated and cells reinfused immediately after transduction. By molecular analysis, the levels of PB MNC and granulocyte Neo® gene marking at steady-state (up to 12 months post-transplantation) was 1–2% in animal RC909 and 30–40% in RQ2851.

Hematopoietic stem cells (HSCs) circulate from the extraembryonic yolk sac (YS), the intraembryonic aorta-gonad mesonephros (AGM) region, and the liver during murine embryonic and fetal development, as they colonize organs active in postnatal hematopoiesis. In adult life, the ability of HSCs to enter the circulation, even in low numbers, may be an important aspect of hematopoietic homeostasis. Once released from its attachment, a stem cell undergoes transmural migration through openings in the basement membrane and endothelial lining of a blood sinus and enters the circulation. Mobilization has been achieved in both laboratory and clinical settings. Clarification of the interactions between HSCs and progenitor cells and the components of the marrow microenvironment are central to the understanding of stem cell mobilization and the converse process of “homing”. Homing is defined as migration of HSCs and progenitor cells specifically from the blood to the BM followed by “engraftment” in permissive niches of the microenvironment, where they survive, proliferate, and differentiate into multilineage cells.

AMD3100 (AMD) has recently been shown to rapidly mobilize primitive hematopoietic cells in mice and humans, but little is known about the properties of cells mobilized with this agent. We initiated a study to determine retroviral (RV) in vivo gene marking efficiency in AMD-mobilized CD34+ cells in rhesus macaques. CD34+ cells collected 3 hours after administration of AMD to 2 animals were transduced using RV vectors containing the NeoR gene. Animals were irradiated and cells reinfused immediately after transduction. By molecular analysis, the levels of PB MNC and granulocyte NeoR gene marking at steady-state (up to 12 months post-transplantation) was 1–2% in animal RC909 and 30–40% in RQ2851.

Recent studies have demonstrated the existence of several populations of primitive cells in mouse and human bone marrow that have the capacity, both in vitro and in vivo, to give rise to cells of all three germ layers. Mesenchymal/stromal stem cells and hematopoietic stem cells are the leading candidates for this activity that some believe may recapitulate the potential of embryonic stem cells. Very little is known about the molecular controls for this adult stem cell activity commonly referred to as transdifferentiation or plasticity. Regeneration of a large number of cell types and tissues has been investigated with one of the most extensively studied being the myocardium. These studies involved ligation of the left coronary artery in adult mice and the direct injection or mobilization of bone marrow stem cells. Using this protocol we, and others, have observed the generation of new cardiomyocytes and endothelial cells in the zone of ischemic myocardium. This approach has progressed to clinical trials at several academic institutions. Although the preliminary findings from these trials do not permit unequivocal conclusions, they do suggest that safety and feasibility are not significant problems that would argue against extending these trials in a large, randomized, double-blinded study. When considerations such as these are addressed, cell therapy may become a new modality in the treatment of heart patients.