Qiang Xiong

Cardiac Repair in a Porcine Model of Acute Myocardial Infarction with Human Induced Pluripotent Stem Cell-Derived Cardiovascular Cells

Human induced pluripotent stem cells (hiPSCs) hold promise for myocardial repair following injury, but preclinical studies in large animal models are required to determine optimal cell preparation and delivery strategies to maximize functional benefits and to evaluate safety. Here, we utilized a porcine model of acute myocardial infarction (MI) to investigate the functional impact of intramyocardial transplantation of hiPSC-derived cardiomyocytes, endothelial cells, and smooth muscle cells, in combination with a 3D fibrin patch loaded with insulin growth factor (IGF)-encapsulated microspheres. hiPSC-derived cardiomyocytes integrated into host myocardium and generated organized sarcomeric structures, and endothelial and smooth muscle cells contributed to host vasculature. Trilineage cell transplantation significantly improved left ventricular function, myocardial metabolism, and arteriole density, while reducing infarct size, ventricular wall stress, and apoptosis without inducing ventricular arrhythmias. These findings in a large animal MI model highlight the potential of utilizing hiPSC-derived cells for cardiac repair.

A fibrin patch-based enhanced delivery of human embryonic stem cell-derived vascular cell transplantation in a porcine model of postinfarction left ventricular remodeling

It is unknown how to use human embryonic stem cell (hESC) to effectively treat hearts with postinfarction left ventricular (LV) remodeling. Using a porcine model of postinfarction LV remodeling, this study examined the functional improvement of enhanced delivery of combined transplantation of hESC‐derived endothelial cells (ECs) and hESC‐derived smooth muscle cells (SMCs) with a fibrin three‐dimensional (3D) porous scaffold biomatrix. To facilitate tracking the transplanted cells, the hESCs were genetically modified to stably express green fluorescent protein and luciferase (GFP/Luc). Myocardial infarction (MI) was created by ligating the first diagonal coronary artery for 60 minutes followed by reperfusion. Two million each of GFP/Luc hESC‐derived ECs and SMCs were seeded in the 3D porous biomatrix patch and applied to the region of ischemia/reperfusion for cell group (MI+P+C, n = 6), whereas biomatrix without cell (MI+P, n = 5), or saline only (MI, n = 5) were applied to control group hearts with same coronary artery ligation. Functional outcome (1 and 4 weeks follow‐up) of stem cell transplantation was assessed by cardiac magnetic resonance imaging. The transplantation of hESC‐derived vascular cells resulted in significant LV functional improvement. Significant engraftment of hESC‐derived cells was confirmed by both in vivo and ex vivo bioluminescent imaging. The mechanism underlying the functional beneficial effects of cardiac progenitor transplantation is attributed to the increased neovascularization. These findings demonstrate a promising therapeutic potential of using these hESC‐derived vascular cell types and the mode of patch delivery. STEM CELLS 2011;29:367–375

Functional Consequences of Human Induced Pluripotent Stem Cell Therapy Myocardial ATP Turnover Rate in the In Vivo Swine Heart With Postinfarction Remodeling

Background— The use of cells derived from human induced pluripotent stem cells as cellular therapy for myocardial injury has yet to be examined in a large-animal model. Methods and Results— Immunosuppressed Yorkshire pigs were assigned to 1 of 3 groups: A myocardial infarction group (MI group; distal left anterior descending coronary artery ligation and reperfusion; n=13); a cell-treatment group (MI with 4×106 vascular cells derived from human induced pluripotent stem cells administered via a fibrin patch; n=14); and a normal group (n=15). At 4 weeks, left ventricular structural and functional abnormalities were less pronounced in hearts in the cell-treated group than in MI hearts (P<0.05), and these improvements were accompanied by declines in scar size (10.4±1.6% versus 8.3±1.1%, MI versus cell-treatment group, P<0.05). The cell-treated group displayed a significant increase in vascular density and blood flow (0.83±0.11 and 1.05±0.13 mL·min−1·g−1, MI versus cell-treatment group, P<0.05) in the periscar border zone (BZ), which was accompanied by improvements in systolic thickening fractions (infarct zone, −10±7% versus 5±5%; BZ, 7±4% versus 23±6%; P<0.05). Transplantation of vascular cells derived from human induced pluripotent stem cells stimulated c-kit+ cell recruitment to BZ and the rate of bromodeoxyuridine incorporation in both c-kit+ cells and cardiomyocytes (P<0.05). Using a magnetic resonance spectroscopic saturation transfer technique, we found that the rate of ATP hydrolysis in BZ of MI hearts was severely reduced, and the severity of this reduction was linearly related to the severity of the elevations of wall stresses (r=0.82, P<0.05). This decline in BZ ATP utilization was markedly attenuated in the cell-treatment group. Conclusions— Transplantation of vascular cells derived from human induced pluripotent stem cells mobilized endogenous progenitor cells into the BZ, attenuated regional wall stress, stimulated neovascularization, and improved BZ perfusion, which in turn resulted in marked increases in BZ contractile function and ATP turnover rate.

Quantitative Imaging of Energy Expenditure in Human Brain

Despite the essential role of the brain energy generated from ATP hydrolysis in supporting cortical neuronal activity and brain function, it is challenging to noninvasively image and directly quantify the energy expenditure in the human brain. In this study, we applied an advanced in vivo(31)P MRS imaging approach to obtain regional cerebral metabolic rates of high-energy phosphate reactions catalyzed by ATPase (CMR(ATPase)) and creatine kinase (CMR(CK)), and to determine CMR(ATPase) and CMR(CK) in pure gray mater (GM) and white mater (WM), respectively. It was found that both ATPase and CK rates are three times higher in GM than WM; and CMR(CK) is seven times higher than CMR(ATPase) in GM and WM. Among the total brain ATP consumption in the human cortical GM and WM, 77% of them are used by GM in which approximately 96% is by neurons. A single cortical neuron utilizes approximately 4.7 billion ATPs per second in a resting human brain. This study demonstrates the unique utility of in vivo(31)P MRS imaging modality for direct imaging of brain energy generated from ATP hydrolysis, and provides new insights into the human brain energetics and its role in supporting neuronal activity and brain function.

Bioenergetic and Functional Consequences of Cellular Therapy Activation of Endogenous Cardiovascular Progenitor Cells

Rationale: The mechanism by which endogenous progenitor cells contribute to functional and beneficial effects in stem cell therapy remains unknown. Objective: Utilizing a novel 31P magnetic resonance spectroscopy–2-dimensional chemical shift imaging method, this study examined the heterogeneity and bioenergetic consequences of postinfarction left ventricular (LV) remodeling and the mechanisms of endogenous progenitor cell contribution to the cellular therapy. Methods and Results: Human embryonic stem cell–derived vascular cells (hESC-VCs) that stably express green fluorescent protein and firefly luciferase (GFP+/Luc+) were used for the transplantation. hESC-VCs may release various cytokines to promote angiogenesis, prosurvival, and antiapoptotic effects. Both in vitro and in vivo experiments demonstrated that hESC-VCs effectively inhibit myocyte apoptosis. In the mouse model, a fibrin patch–based cell delivery resulted in a significantly better cell engraftment rate that was accompanied by a better ejection fraction. In the swine model of ischemia-reperfusion, the patch-enhanced delivery of hESC-VCs resulted in alleviation of abnormalities including border zone myocardial perfusion, contractile dysfunction, and LV wall stress. These results were also accompanied by a pronounced recruitment of endogenous c-kit+ cells to the injury site. These improvements were directly associated with a remarkable improvement in myocardial energetics, as measured by a novel in vivo 31P magnetic resonance spectroscopy–2-dimensional chemical shift imaging technology. Conclusions: The findings of this study demonstrate that a severely abnormal heterogeneity of myocardial bioenergetics in hearts with postinfarction LV remodeling can be alleviated by the hESC-VCs therapy. These findings suggest an important therapeutic target of peri-scar border zone and a promising therapeutic potential for using hESC-VCs together with the fibrin patch–based delivery system. # Novelty and Significance {#article-title-51}Rationale: The mechanism by which endogenous progenitor cells contribute to functional and beneficial effects in stem cell therapy remains unknown. Objective: Utilizing a novel 31P magnetic resonance spectroscopy–2-dimensional chemical shift imaging method, this study examined the heterogeneity and bioenergetic consequences of postinfarction left ventricular (LV) remodeling and the mechanisms of endogenous progenitor cell contribution to the cellular therapy. Methods and Results: Human embryonic stem cell–derived vascular cells (hESC-VCs) that stably express green fluorescent protein and firefly luciferase (GFP+/Luc+) were used for the transplantation. hESC-VCs may release various cytokines to promote angiogenesis, prosurvival, and antiapoptotic effects. Both in vitro and in vivo experiments demonstrated that hESC-VCs effectively inhibit myocyte apoptosis. In the mouse model, a fibrin patch–based cell delivery resulted in a significantly better cell engraftment rate that was accompanied by a better ejection fraction. In the swine model of ischemia-reperfusion, the patch-enhanced delivery of hESC-VCs resulted in alleviation of abnormalities including border zone myocardial perfusion, contractile dysfunction, and LV wall stress. These results were also accompanied by a pronounced recruitment of endogenous c-kit+ cells to the injury site. These improvements were directly associated with a remarkable improvement in myocardial energetics, as measured by a novel in vivo 31P magnetic resonance spectroscopy–2-dimensional chemical shift imaging technology. Conclusions: The findings of this study demonstrate that a severely abnormal heterogeneity of myocardial bioenergetics in hearts with postinfarction LV remodeling can be alleviated by the hESC-VCs therapy. These findings suggest an important therapeutic target of peri-scar border zone and a promising therapeutic potential for using hESC-VCs together with the fibrin patch–based delivery system.

ATP production rate via creatine kinase or ATP synthase in vivo: a novel superfast magnetization saturation transfer method.

Rationale: 31P magnetization saturation transfer (MST) experiment is the most widely used method to study ATP metabolism kinetics. However, its lengthy data acquisition time greatly limits the wide biomedical applications in vivo, especially for studies requiring high spatial and temporal resolutions. Objective: We aimed to develop a novel superfast MST method that can accurately quantify ATP production rate constants (kf) through creatine kinase (CK) or ATP synthase (ATPase) with 2 spectra. Methods and Results: The T1nom (T1 nominal) method uses a correction factor to compensate the partially relaxed MST experiments, thus allowing measurement of enzyme kinetics with an arbitrary repetition time and flip angle, which consequently reduces the data acquisition time of a transmurally differentiated CK kf measurement by 91% as compared with the conventional method with spatial localization. The novel T1nom method is validated theoretically with numeric simulation, and further verified with in vivo swine hearts, as well as CK and ATPase activities in rat brain at 9.4 Tesla. Importantly, the in vivo data from swine hearts demonstrate, for the first time, that within an observation window of 30 minutes, the inhibition of CK activity by iodoacetamide does not limit left ventricular chamber contractile function. Conclusions: A novel MST method for superfast examination of enzyme kinetics in vivo has been developed and verified theoretically and experimentally. In the in vivo normal heart, redundant multiple supporting systems of myocardial ATP production, transportation, and utilization exist, such that inhibition of one mechanism does not impair the normal left ventricular contractile performance.Rationale: 31P magnetization saturation transfer (MST) experiment is the most widely used method to study ATP metabolism kinetics. However, its lengthy data acquisition time greatly limits the wide biomedical applications in vivo, especially for studies requiring high spatial and temporal resolutions. Objective: We aimed to develop a novel superfast MST method that can accurately quantify ATP production rate constants ( k f) through creatine kinase (CK) or ATP synthase (ATPase) with 2 spectra. Methods and Results: The T1nom (T1 nominal) method uses a correction factor to compensate the partially relaxed MST experiments, thus allowing measurement of enzyme kinetics with an arbitrary repetition time and flip angle, which consequently reduces the data acquisition time of a transmurally differentiated CK k f measurement by 91% as compared with the conventional method with spatial localization. The novel T1nom method is validated theoretically with numeric simulation, and further verified with in vivo swine hearts, as well as CK and ATPase activities in rat brain at 9.4 Tesla. Importantly, the in vivo data from swine hearts demonstrate, for the first time, that within an observation window of 30 minutes, the inhibition of CK activity by iodoacetamide does not limit left ventricular chamber contractile function. Conclusions: A novel MST method for superfast examination of enzyme kinetics in vivo has been developed and verified theoretically and experimentally. In the in vivo normal heart, redundant multiple supporting systems of myocardial ATP production, transportation, and utilization exist, such that inhibition of one mechanism does not impair the normal left ventricular contractile performance. # Novelty and Significance {#article-title-33}Rationale and Objective— 31 P magnetization saturation transfer (MST) experiment is the most widely used method to study ATP metabolism kinetics. However, its lengthy data acquisition time greatly limits the wide biomedical applications in vivo, especially for studies requiring high spatial and temporal resolutions. We aim to develop a novel superfast MST method that can accurately quantify ATP production rate constants (kf) through creatine kinase (CK) or ATP synthase (ATPase) with two spectra. Methods and Results—The T1 nom (T1 nominal) method utilizes a correction factor to compensate the partially relaxed MST experiments, thus allowing measurement of enzyme kinetics with an arbitrary repetition time and flip angle, which consequently reduces the data acquisition time of a transmurally differentiated CK kf measurement by 91% as compared to the conventional method with spatial localization. The novel T1 nom method is validated theoretically with numerical simulation, and further verified with in vivo swine hearts, as well as CK and ATPase activities in rat brain at 9.4 Tesla. Importantly, the in vivo data from swine hearts demonstrate for the first time, that within an observation window of 30 minutes, the inhibition of CK activity by iodoacetamide does not limit LV chamber contractile function. Conclusions—A novel MST method for superfast examination of enzyme kinetics in vivo has been developed and verified theoretically and experimentally. In the in vivo normal heart, redundant multiple supporting systems of myocardial ATP production, transportation, and utilization exist, such that inhibition of one mechanism, does not impair the normal LV contractile performance.

Derivation and High Engraftment of Patient-Specific Cardiomyocyte-Sheet Using Induced Pluripotent Stem Cells Generated From Adult Cardiac Fibroblast

Background—Induced pluripotent stem cells (iPSCs) can be differentiated into potentially unlimited lineages of cell types for use in autologous cell therapy. However, the efficiency of the differentiation procedure and subsequent function of the iPSC-derived cells may be influenced by epigenetic factors that the iPSCs retain from their tissues of origin; thus, iPSC-derived cells may be more effective for treatment of myocardial injury if the iPSCs were engineered from cardiac-lineage cells, rather than dermal fibroblasts. Methods and Results—We show that human cardiac iPSCs (hciPSCs) can be generated from cardiac fibroblasts and subsequently differentiated into exceptionally pure (>92%) sheets of cardiomyocytes (CMs). The hciPSCs passed through all the normal stages of differentiation before assuming a CM identity. When using the fibrin gel–enhanced delivery of hciPSC-CM sheets at the site of injury in infarcted mouse hearts, the engraftment rate was 31.91%±5.75% at Day 28 post transplantation. The hciPSC-CM in the sheet also appeared to develop a more mature, structurally aligned phenotype 28 days after transplantation and was associated with significant improvements in cardiac function, vascularity, and reduction in apoptosis. Conclusions—These data strongly support the potential of hciPSC-CM sheet transplantation for the treatment of heart with acute myocardial infarction.

Thymosin β4 increases the potency of transplanted mesenchymal stem cells for myocardial repair

Background— Thymosin &bgr;4 (T&bgr;4) has been shown to enhance the survival of cultured cardiomyocytes. Here, we investigated whether the cytoprotective effects of T&bgr;4 can increase the effectiveness of transplanted swine mesenchymal stem cells (sMSCs) for cardiac repair in a rat model of myocardial infarction (MI). Methods and Results— Under hypoxic conditions, cellular damage (lactate dehydrogenase leakage), apoptosis (terminal deoxynucleotidyl transferase dUTP nick end labelingc cells), and caspase-8 activity were significantly lower, whereas B-cell lymphoma-extra large protein expression was significantly higher, in sMSCs cultured with T&bgr;4 (1 &mgr;g/mL) than in sMSCs cultured without T&bgr;4, and T&bgr;4 also increased sMSC proliferation. For in vivo experiments, animals were treated with basal medium (MI: n=6), a fibrin patch (Patch: n=6), a patch containing sMSCs (sMSC: n=9), or a patch containing sMSCs and T&bgr;4 (sMSC/T&bgr;4: n=11); T&bgr;4 was encapsulated in gelatin microspheres to extend T&bgr;4 delivery. Four weeks after treatment, echocardiographic assessments of left-ventricular ejection fraction and fractional shortening were significantly better (P<0.05) in sMSC/T&bgr;4 animals (left-ventricular ejection fraction=51.7±1.1%; fractional shortening=26.7±0.7%) than in animals from MI (39±3%; 19.5±1.7%) and Patch (43±1.4%; 21.6±0.9%) groups. Histological assessment of infarct wall thickness was significantly higher (P<0.05) in sMSC/T&bgr;4 animals (50%, [45%, 80%]) than in animals from MI (25%, [20%, 25%]) group. Measurements in sMSC (left-ventricular ejection fraction=45±2.6%; fractional shortening=22.9±1.6%; TH=43% [25%, 45%]), Patch, and MI animals were similar. T&bgr;4 administration also significantly increased vascular growth, the retention/survival of the transplanted sMSCs, and the recruitment of endogenous c-Kit+ progenitor cells to the infarcted region. Conclusions— Extended-release T&bgr;4 administration improves the retention, survival, and regenerative potency of transplanted sMSCs after myocardial injury.

Novel strategy for measuring creatine kinase reaction rate in the in vivo heart

In the heart, the creatine kinase (CK) system plays an important role in the cascade of ATP production, transportation, and utilization. The forward pseudo-first-order rate constant for the CK reaction can be measured noninvasively by the (31)P-magnetic resonance (MR) spectroscopy magnetization saturation transfer (MST) techniques. However, the measurement of MST in the in vivo heart is limited by the lengthy data acquisition time, especially for studies requiring spatial localization. This technical report presents a new method for measuring ATP production rate via CK that can reduce the MST data acquisition time by 82%. This method is validated using an in vivo pig model to evaluate the forward pseudo-first-order rate constant of myocardial CK reaction noninvasively.

Aging Kit Mutant Mice Develop Cardiomyopathy

Both bone marrow (BM) and myocardium contain progenitor cells expressing the c-Kit tyrosine kinase. The aims of this study were to determine the effects of c-Kit mutations on: i. myocardial c-Kit+ cells counts and ii. the stability of left ventricular (LV) contractile function and structure during aging. LV structure and contractile function were evaluated (echocardiography) in two groups of Kit mutant (W/Wv and W41/W42) and in wild type (WT) mice at 4 and 12 months of age and the effects of the mutations on LV mass, vascular density and the numbers of proliferating cells were also determined. In 4 month old Kit mutant and WT mice, LV ejection fractions (EF) and LV fractional shortening rates (FS) were comparable. At 12 months of age EF and FS were significantly decreased and LV mass was significantly increased only in W41/W42 mice. Myocardial vascular densities and c-Kit+ cell numbers were significantly reduced in both mutant groups when compared to WT hearts. Replacement of mutant BM with WT BM at 4 months of age did not prevent these abnormalities in either mutant group although they were somewhat attenuated in the W/Wv group. Notably BM transplantation did not prevent the development of cardiomyopathy in 12 month W41/W42 mice. The data suggest that decreased numbers and functional capacities of c-Kit+ cardiac resident progenitor cells may be the basis of the cardiomyopathy in W41/W42 mice and although defects in mutant BM progenitor cells may prove to be contributory, they are not causal.