Baiming Sun

Direct comparison of different stem cell types and subpopulations reveals superior paracrine potency and myocardial repair efficacy with cardiosphere-derived cells

OBJECTIVES The goal of this study was to conduct a direct head-to-head comparison of different stem cell types in vitro for various assays of potency and in vivo for functional myocardial repair in the same mouse model of myocardial infarction. BACKGROUND Adult stem cells of diverse origins (e.g., bone marrow, fat, heart) and antigenic identity have been studied for repair of the damaged heart, but the relative utility of the various cell types remains unclear. METHODS Human cardiosphere-derived cells (CDCs), bone marrow-derived mesenchymal stem cells, adipose tissue-derived mesenchymal stem cells, and bone marrow mononuclear cells were compared. RESULTS CDCs revealed a distinctive phenotype with uniform expression of CD105, partial expression of c-kit and CD90, and negligible expression of hematopoietic markers. In vitro, CDCs showed the greatest myogenic differentiation potency, highest angiogenic potential, and relatively high production of various angiogenic and antiapoptotic-secreted factors. In vivo, injection of CDCs into the infarcted mouse hearts resulted in superior improvement of cardiac function, the highest cell engraftment and myogenic differentiation rates, and the least-abnormal heart morphology 3 weeks after treatment. CDC-treated hearts also exhibited the lowest number of apoptotic cells. The c-kit(+) subpopulation purified from CDCs produced lower levels of paracrine factors and inferior functional benefit when compared with unsorted CDCs. To validate the comparison of cells from various human donors, selected results were confirmed in cells of different types derived from individual rats. CONCLUSIONS CDCs exhibited a balanced profile of paracrine factor production and, among various comparator cell types/subpopulations, provided the greatest functional benefit in experimental myocardial infarction.

Cardiomyocyte proliferation and progenitor cell recruitment underlie therapeutic regeneration after myocardial infarction in the adult mouse heart

Cardiosphere‐derived cells (CDCs) have been shown to regenerate infarcted myocardium in patients after myocardial infarction (MI). However, whether the cells of the newly formed myocardium originate from the proliferation of adult cardiomyocytes or from the differentiation of endogenous stem cells remains unknown. Using genetic fate mapping to mark resident myocytes in combination with long‐term BrdU pulsing, we investigated the origins of postnatal cardiomyogenesis in the normal, infarcted and cell‐treated adult mammalian heart. In the normal mouse heart, cardiomyocyte turnover occurs predominantly through proliferation of resident cardiomyocytes at a rate of ∼1.3–4%/year. After MI, new cardiomyocytes arise from both progenitors as well as pre‐existing cardiomyocytes. Transplantation of CDCs upregulates host cardiomyocyte cycling and recruitment of endogenous progenitors, while boosting heart function and increasing viable myocardium. The observed phenomena cannot be explained by cardiomyocyte polyploidization, bi/multinucleation, cell fusion or DNA repair. Thus, CDCs induce myocardial regeneration by differentially upregulating two mechanisms of endogenous cell proliferation.

Macrophages mediate cardioprotective cellular postconditioning in acute myocardial infarction

Ischemic injury in the heart induces an inflammatory cascade that both repairs damage and exacerbates scar tissue formation. Cardiosphere-derived cells (CDCs) are a stem-like population that is derived ex vivo from cardiac biopsies; they confer both cardioprotection and regeneration in acute myocardial infarction (MI). While the regenerative effects of CDCs in chronic settings have been studied extensively, little is known about how CDCs confer the cardioprotective process known as cellular postconditioning. Here, we used an in vivo rat model of ischemia/reperfusion (IR) injury-induced MI and in vitro coculture assays to investigate how CDCs protect stressed cardiomyocytes. Compared with control animals, animals that received CDCs 20 minutes after IR had reduced infarct size when measured at 48 hours. CDCs modified the myocardial leukocyte population after ischemic injury. Specifically, introduction of CDCs reduced the number of CD68+ macrophages, and these CDCs secreted factors that polarized macrophages toward a distinctive cardioprotective phenotype that was not M1 or M2. Systemic depletion of macrophages with clodronate abolished CDC-mediated cardioprotection. Using both in vitro coculture assays and a rat model of adoptive transfer after IR, we determined that CDC-conditioned macrophages attenuated cardiomyocyte apoptosis and reduced infarct size, thereby recapitulating the beneficial effects of CDC therapy. Together, our data indicate that CDCs limit acute injury by polarizing an effector macrophage population within the heart.

Expansion of human cardiac stem cells in physiological oxygen improves cell production efficiency and potency for myocardial repair.

AIMS the ex vivo expansion of cardiac stem cells from minimally invasive human heart biopsies yields tens of millions of cells within 3-4 weeks, but chromosomal abnormalities were frequently detected in preliminary production runs. Here we attempt to avoid aneuploidy and improve cell quality by expanding human cardiac stem cells in physiological low-oxygen (5% O(2)) conditions, rather than in traditional culture in a general CO(2) incubator (20% O(2)). METHODS AND RESULTS human heart biopsies (n = 16) were divided and processed in parallel to expand cardiac stem cells under 5% or 20% O(2). Compared with 20% O(2), 5% O(2) culture doubled the cell production and markedly diminished the frequency of aneuploidy. Cells expanded in 5% O(2) showed lower intracellular levels of reactive oxygen species, less cell senescence, and higher resistance to oxidative stress than those grown in 20% O(2), although the expression of stem cell antigens and adhesion molecules was comparable between groups, as was the paracrine secretion of growth factors into conditioned media. In vivo, the implantation of 5% O(2) cells into infarcted hearts of mice resulted in greater cell engraftment and better functional recovery than with conventionally cultured cells. CONCLUSION the expansion of human adult cardiac stem cells in low oxygen increased cell yield, and the resulting cells were superior by various key in vitro and in vivo metrics of cell quality. Physiological oxygen tensions in culture facilitate the ex vivo expansion of healthy, biologically potent stem cells.

Functional performance of human cardiosphere-derived cells delivered in an in situ polymerizable hyaluronan-gelatin hydrogel

The vast majority of cells delivered into the heart by conventional means are lost within the first 24 h. Methods are needed to enhance cell retention, so as to minimize loss of precious material and maximize effectiveness of the therapy. We tested a cell-hydrogel delivery strategy. Cardiosphere-derived cells (CDCs) were grown from adult human cardiac biopsy specimens. In situ polymerizable hydrogels made of hyaluronan and porcine gelatin (Hystem(®)-C™) were formulated as a liquid at room temperature so as to gel within 20 min at 37 °C. CDC viability and migration were not compromised in Hystem-C™. Myocardial infarction was created in SCID mice and CDCs were injected intramyocardially in the infarct border zone. Real-time PCR revealed engraftment of CDCs delivered in Hystem-C™ was increased by nearly an order of magnitude. LVEF (left ventricular ejection fraction) deteriorated in the control (PBS only) group over the 3-week time course. Hystem-C™ alone or CDCs alone preserved LVEF relative to baseline, while CDCs delivered in Hystem-C™ resulted in a sizable boost in LVEF. Heart morphometry revealed the greatest attenuation of LV remodeling in the CDC + Hystem-C™ group. Histological analysis suggested cardiovascular differentiation of the CDCs in Hystem-C™. However, the majority of functional benefit is likely from paracrine mechanisms such as tissue preservation and neovascularization. A CDC/hydrogel formulation suitable for catheter-based intramyocardial injection exhibits superior engraftment and functional benefits relative to naked CDCs.

Allogeneic Cardiospheres Safely Boost Cardiac Function and Attenuate Adverse Remodeling After Myocardial Infarction in Immunologically Mismatched Rat Strains

OBJECTIVES We sought to characterize the immunologic profile of allogeneic cardiospheres, which are 3-dimensional, self-assembling, cardiac-derived microtissues, and to evaluate their safety and efficacy in repairing ischemic heart tissue. BACKGROUND Intramyocardial injection of autologous cardiospheres ameliorates remodeling and improves global function in infarcted myocardium. It is as yet unknown whether allogeneic cardiospheres are similarly effective without eliciting deleterious immune reactions. METHODS We expanded cardiospheres from male Wistar Kyoto rat hearts and injected them surgically in the peri-infarct zone of Wistar Kyoto (syngeneic group, n = 28) and Brown Norway female rats (allogeneic group, n = 29). Female rats from both strains (n = 37) injected with normal saline served as controls. RESULTS In vitro, cardiospheres expressed a low immunogenic profile and inhibited proliferation of alloreactive T cells. In vivo, cell engraftment was similar in the syngeneic and allogeneic groups 1 week and 3 weeks after transplantation. Reductions in scar size and scar collagen content and increases in viable mass in the risk region were accompanied by improvements in left ventricular function and attenuation of left ventricle remodeling that were sustained during 6 months of follow up. Transplantation of allogeneic cardiospheres increased tissue expression of the regenerative growth factors vascular endothelial growth factor, hepatocyte growth factor, and insulin-like growth factor-1, stimulating angiogenesis. Syngeneic and allogeneic cardiospheres attenuated the inflammatory response observed histologically in the peri-infarct region. CONCLUSIONS Allogeneic cardiospheres increase viable myocardium, decrease scar, improve function, and attenuate adverse remodeling in the infarcted rat heart, without deleterious immunological sequelae. These observations lay the groundwork for developing cardiospheres as a novel off-the-shelf microtissue product for myocardial regeneration.

Magnetic enhancement of cell retention, engraftment, and functional benefit after intracoronary delivery of cardiac-derived stem cells in a rat model of ischemia/reperfusion.

The efficiency of stem cell transplantation is limited by low cell retention. Intracoronary (IC) delivery is convenient and widely used but exhibits particularly low cell retention rates. We sought to improve IC cell retention by magnetic targeting. Rat cardiosphere-derived cells labeled with iron microspheres were injected into the left ventricular cavity of syngeneic rats during brief aortic clamping. Placement of a 1.3 Tesla magnet ~1 cm above the heart during and after cell injection enhanced cell retention at 24 h by 5.2–6.4-fold when 1, 3, or 5 × 105 cells were infused, without elevation of serum troponin I (sTnI) levels. Higher cell doses (1 or 2 × 106 cells) did raise sTnI levels, due to microvascular obstruction; in this range, magnetic enhancement did not improve cell retention. To assess efficacy, 5 × 105 iron-labeled, GFP-expressing cells were infused into rat hearts after 45 min ischemia/20 min reperfusion of the left anterior coronary artery, with and without a superimposed magnet. By quantitative PCR and optical imaging, magnetic targeting increased cardiac retention of transplanted cells at 24 h, and decreased migration into the lungs. The enhanced cell engraftment persisted for at least 3 weeks, at which time left ventricular remodeling was attenuated, and therapeutic benefit (ejection fraction) was higher, in the magnetic targeting group. Histology revealed more GFP+ cardiomyocytes, Ki67+ cardiomyocytes and GFP-/ckit+ cells, and fewer TUNEL+ cells, in hearts from the magnetic targeting group. In a rat model of ischemia/reperfusion injury, magnetically enhanced intracoronary cell delivery is safe and improves cell therapy outcomes.

Stimulation of endogenous cardioblasts by exogenous cell therapy after myocardial infarction

Controversy surrounds the identity, origin, and physiologic role of endogenous cardiomyocyte progenitors in adult mammals. Using an inducible genetic labeling approach to identify small non‐myocyte cells expressing cardiac markers, we find that activated endogenous cardioblasts are rarely evident in the normal adult mouse heart. However, myocardial infarction results in significant cardioblast activation at the site of injury. Genetically labeled isolated cardioblasts express cardiac transcription factors and sarcomeric proteins, exhibit spontaneous contractions, and form mature cardiomyocytes in vivo after injection into unlabeled recipient hearts. The activated cardioblasts do not arise from hematogenous seeding, cardiomyocyte dedifferentiation, or mere expansion of a preformed progenitor pool. Cell therapy with cardiosphere‐derived cells amplifies innate cardioblast‐mediated tissue regeneration, in part through the secretion of stromal cell‐derived factor 1 by transplanted cells. Thus, stimulation of endogenous cardioblasts by exogenous cells mediates therapeutic regeneration of injured myocardium.

Magnetic antibody-linked nanomatchmakers for therapeutic cell targeting

Stem cell transplantation is a promising strategy for therapeutic cardiac regeneration, but current therapies are limited by inefficient interaction between potentially beneficial cells (either exogenously transplanted or endogenously recruited) and the injured tissue. Here we apply targeted nanomedicine to achieve in vivo cell-mediated tissue repair, imaging and localized enrichment without cellular transplantation. Iron nanoparticles are conjugated with two types of antibodies (one against antigens on therapeutic cells and the other directed at injured cells) to produce magnetic bifunctional cell engager (MagBICE). The antibodies link the therapeutic cells to the injured cells, whereas the iron core of MagBICE enables physical enrichment and imaging. We treat acute myocardial infarction by targeting exogenous bone marrow-derived stem cells (expressing CD45) or endogenous CD34-positive cells to injured cardiomyocytes (expressing myosin light chain. Targeting can be further enhanced by magnetic attraction, leading to augmented functional benefits. MagBICE represents a generalizable platform technology for regenerative medicine.

Therapeutic efficacy of cardiosphere-derived cells in a transgenic mouse model of non-ischaemic dilated cardiomyopathy

AIM Cardiosphere-derived cells (CDCs) produce regenerative effects in the post-infarct setting. However, it is unclear whether CDCs are beneficial in non-ischaemic dilated cardiomyopathy (DCM). We tested the effects of CDC transplantation in mice with cardiac-specific Gαq overexpression, which predictably develop progressive cardiac dilation and failure, with accelerated mortality. METHODS AND RESULTS Wild-type mouse CDCs (10(5) cells) or vehicle only were injected intramyocardially in 6-, 8-, and 11-week-old Gαq mice. Cardiac function deteriorated in vehicle-treated mice over 3 months of follow-up, accompanied by oxidative stress, inflammation and adverse ventricular remodelling. In contrast, CDCs preserved cardiac function and volumes, improved survival, and promoted cardiomyogenesis while blunting Gαq-induced oxidative stress and inflammation in the heart. The mechanism of benefit is indirect, as long-term engraftment of transplanted cells is vanishingly low. CONCLUSIONS Cardiosphere-derived cells reverse fundamental abnormalities in cell signalling, prevent adverse remodelling, and improve survival in a mouse model of DCM. The ability to impact favourably on disease progression in non-ischaemic heart failure heralds new potential therapeutic applications of CDCs.