Biodistribution and Clearance of Human Mesenchymal Stem Cells by Quantitative Three-Dimensional Cryo-Imaging After Intravenous Infusion in a Rat Lung Injury Model

Stem Cells Translational Medicine · 2016-07-26 · 62 citations

: Cell tracking is a critical component of the safety and efficacy evaluation of therapeutic cell products. To date, cell-tracking modalities have been hampered by poor resolution, low sensitivity, and inability to track cells beyond the shortterm. Three-dimensional (3D) cryo-imaging coregisters fluorescent and bright-field microcopy images and allows for single-cell quantification within a 3D organ volume. We hypothesized that 3D cryo-imaging could be used to measure cell biodistribution and clearance after intravenous infusion in a rat lung injury model compared with normal rats. A bleomycin lung injury model was established in Sprague-Dawley rats (n = 12). Human mesenchymal stem cells (hMSCs) labeled with QTracker655 were infused via jugular vein. After 2, 4, or 8 days, a second dose of hMSCs labeled with QTracker605 was infused, and animals were euthanized after 60, 120, or 240 minutes. Lungs, liver, spleen, heart, kidney, testis, and intestine were cryopreserved, followed by 3D cryo-imaging of each organ. At 60 minutes, 82% ± 9.7% of cells were detected; detection decreased to 60% ± 17% and 66% ± 22% at 120 and 240 minutes, respectively. At day 2, 0.06% of cells were detected, and this level remained constant at days 4 and 8 postinfusion. At 60, 120, and 240 minutes, 99.7% of detected cells were found in the liver, lungs, and spleen, with cells primarily retained in the liver. This is the first study using 3D cryo-imaging to track hMSCs in a rat lung injury model. hMSCs were retained primarily in the liver, with fewer detected in lungs and spleen. SIGNIFICANCE: Effective bench-to-bedside clinical translation of cellular therapies requires careful understanding of cell fate through tracking. Tracking cells is important to measure cell retention so that delivery methods and cell dose can be optimized and so that biodistribution and clearance can be defined to better understand potential off-target toxicity and redosing strategies. This article demonstrates, for the first time, the use of three-dimensional cryo-imaging for single-cell quantitative tracking of intravenous infused clinical-grade mesenchymal stem cells in a clinically relevant model of lung injury. The important information learned in this study will help guide future clinical and translational stem cell therapies for lung injuries.

Cardiopulmonary and histological characterization of an acute rat lung injury model demonstrating safety of mesenchymal stromal cell infusion

Cytotherapy · 2016-03-10 · 9 citations

Feasibility of Atrial Delivery and Tracking of Stem Cells in a Porcine Model

Journal of patient-centered research and reviews · 2015-11-20

Background: Many patients undergoing open heart surgery have sinus node dysfunction and atrial fibrillation, leading to adverse outcomes. Mesenchymal stem cells (MSC) delivered at the time of surgery may have a reparative effect on atrial tissue, thereby improving sinus node function and reducing or preventing atrial fibrillation. Stem cell delivery to the atrium is entirely unstudied. This is a significant gap in medical research, as atrial disease contributes significantly to health care costs. Purpose: The purpose of this pilot study is to establish a technique to deliver MSC to the atria through an open-chest model, to assess the safety of this technique, and to evaluate the acute retention of the delivered cells. Methods: All in vivo animal experimentation was approved by the University of Wisconsin Animal Care and Use Committee and took place in the Cardiovascular Physiology Core Facility at UW-Madison. MSC (3-5×106 in 50 μl per site) were injected intramyocardially during an open-chest procedure in anesthetized pigs. To track the cells in vivo, MSC were labeled with 18FDG then visualized at 1 and 6 hours postinjection by PET/CT. Pigs were monitored for intraoperative arrhythmia, bleeding and hypotension. Results: By gently repositioning the heart, both atria were accessible for the injections. The thickest part of each atrium was isolated and stabilized briefly for the injection using a hemostat. The injected cells were visible by PET/CT 1 and 6 hours postinjection. However, when the MSC were labeled with 10mCi 18FDG, the signal was too high, causing a bloom around the areas of injection. So the dose was lowered to 5mCi 18FDG, which resulted in a clear signal at 1 hour in both atria. At 6 hours, the right atrial injection was still easy to read, but the left injection was difficult to resolve from background signal. All injections resulted in cell leakage from the injection site and uptake of the signal into the lungs. However, pulmonary function as measured by SpO2 and EtCO2 was unchanged. Intraoperative arrhythmias detected during the injections were caused by manipulation of the heart. No additional arrhythmias were detected. No bleeding or hypotension was observed as a result of the injections. Conclusion: This pilot study demonstrated that atrial delivery of MSC is feasible and safe in an open-chest porcine model and that MSC are retained for at least 6 hours postinjection. Subsequent studies will determine the ability of MSC to downregulate inflammation, decrease scarring and prevent sinus node dysfunction.

Abstract 45: Systemic Illness in Ece1 Ablated Adult Mice

Circulation Research · 2015-07-17

Endothelin converting enzyme-1 (ECE1) catalyzes the conversion of inactive big endothelin 1 (ET1) to active ET1. Homozygous Ece1 knock out (KO) mice die in utero or at birth, displaying multiple abnormalities including mandibular hypoplasia and cardiac outflow tract malformations, in spite of the presence of ample tissue ET1. However, increased ECE1 activity and circulating and/or tissue ET1 are associated with many adult cardiovascular diseases, including idiopathic pulmonary fibrosis (IPF), a chronic and fatal lung disease. There is an apparent paradox between the need for ET1 in development and its harmful effects in adult disease. Therefore, our lab developed a conditional Ece1 KO mouse, in which Ece1 is ablated following tamoxifen (tam) treatment. We hypothesized that ECE1 serves to localize ET1 signals to specific cell populations and is essential in normal adult physiology. We studied the following groups: mice given vehicle rather than tam, mice lacking tam-inducible Cre recombinase, mice harboring a normal Ece1 allele ( Ece1 +/flox ), and the experimental animals (Cre Ece1 -/flox ). Mice were treated with vehicle or tam at 8-9 weeks of age. Cre Ece1 -/flox mice showed 85-100% mRNA knock-down efficiency 8 weeks after tam treatment. By 17 weeks of age, Cre Ece1 -/flox mice have tachypnea, decreased activity, and weight loss, requiring euthanasia for humane considerations. They display depleted adipose tissue mass compared to controls. By two weeks after treatment, Cre Ece1 -/flox mice had lower blood pressure relative to controls, which persisted until euthanasia at 17-20 weeks old (p=0.004). Between 17-20 weeks of age, most of Cre Ece1 -/flox mice develop pectus excavatum, enlarged right hearts and have reduced stroke volume and cardiac output as analyzed by echocardiography. Histological examination revealed eosinophilic crystalline pneumonia and increased collagen in the lung and heart. These findings are consistent with development of IPF in the experimental mice. Our findings show that Ece1 ablation in post-natal animal results in a severe cardiorespiratory disease, suggesting that ectopic activation of ET1 by other tissue proteases is the primary mechanism underlying the association of increased ET1 signaling in disease states.

Mesenchymoangioblast-derived mesenchymal stromal cells inhibit cell damage, tissue damage and improve peripheral blood flow following hindlimb ischemic injury in mice

Cytotherapy · 2015-12-28 · 18 citations

Intravenous Followed by X-ray Fused with MRI-Guided Transendocardial Mesenchymal Stem Cell Injection Improves Contractility Reserve in a Swine Model of Myocardial Infarction

Journal of Cardiovascular Translational Research · 2015-09-15 · 17 citations

Bilateral administration of autologous CD133+ cells in ambulatory patients with refractory critical limb ischemia: lessons learned from a pilot randomized, double-blind, placebo-controlled trial

Cytotherapy · 2014-09-18 · 44 citations

Local Effects of Pregnancy on Connexin Proteins That Mediate Ca ²⁺ -Associated Uterine Endothelial NO Synthesis

Hypertension · 2013-12-24 · 20 citations

UNLABELLED: Uterine artery adaptations during gestation facilitate increases in uterine blood flow and fetal growth. HYPOTHESIS: local expression and distribution of uterine artery connexins play roles in mediating in vivo gestational eNOS activation and NO production. We established an ovine model restricting pregnancy to a single uterine horn and measured uterine blood flow, uterine artery shear stress, connexins 37/43, and P(635)eNOS protein levels in uterine artery and systemic artery (omental and renal) endothelium and connexins in vascular smooth muscle. Uterine blood flow and shear stress were locally (unilaterally) and substantially elevated by gestation. During pregnancy, uterine artery endothelial gap junction proteins connexins 37/43 were locally regulated in the gravid horn and elevated 10.3- and 25.6-fold; uterine artery endothelial P(635)eNOS and total eNOS were elevated 3.3- and 2.9-fold; whereas uterine artery vascular smooth muscle connexins 37/43 were locally elevated 12.5- and 5.9-fold, respectively. Less pronounced changes were observed in systemic vasculature except for significant pregnancy-associated increases in omental artery vascular smooth muscle connexin 43 and omental artery endothelial P(635)eNOS and total eNOS. Gap junction blockade using connexin 43, but not connexin 37-specific Gap peptides, abrogated uterine artery endothelial ATP-induced Ca(2+)-mediated NO production. Thus, uterine artery endothelial connexin 43, but not connexin 37, regulates Ca(2+)-mediated NO production required for the vasodilation to accommodate increases in uterine blood flow and shear stress during healthy pregnancies.

Pravastatin stimulates angiogenesis in a murine hindlimb ischemia model: a positron emission tomography imaging study with (64)Cu-NOTA-TRC105.

PubMed · 2013-01-01 · 19 citations

In this study, (64)Cu-NOTA-TRC105 (TRC105 is an anti-CD105 monoclonal antibody that binds to both human and murine CD105) positron emission tomography (PET) was used to assess the response to pravastatin treatment in a murine model of peripheral artery disease (PAD). Hindlimb ischemia was induced by ligation of the right femoral arteries in BALB/c mice under anesthesia, and the left hindlimb served as an internal control. Mice in the treatment group were given intraperitoneal pravastatin daily until the end of the study, whereas the animals in the control group were injected with 0.9% sodium chloride solution. Laser Doppler imaging showed that blood flow in the ischemic hindlimb plummeted to ~20% of the normal level after surgery, and gradually recovered to near normal level on day 10 in the treatment group and on day 20 in the control group. Angiogenesis was non-invasively monitored and quantified with (64)Cu-NOTA-TRC105 PET on postoperative days 3, 10, 17, and 24. Tracer uptake at 48 h post-injection in the ischemic hindlimb in the treatment group was significantly higher than that of the control group on day 10 (20.5 ± 1.9 %ID/g vs 11.4 ± 1.5 %ID/g), suggesting increased CD105 expression and higher level of angiogenesis upon pravastatin treatment, and gradually decreased to background levels in both groups (4.9 ± 0.8 %ID/g vs 3.4 ± 1.9 %ID/g on day 24). The in vivo PET data correlated well with ex vivo biodistribution studies performed on day 24. Increased CD105 expression on days 3 and 10 following ischemia was further confirmed by immunofluorescence staining. Taken together, our results indicated that (64)Cu-NOTA-TRC105 PET is a suitable and non-invasive method to monitor the angiogenesis and therapeutic response in PAD, which can also be utilized for non-invasive evaluation of other pro-angiogenic/anti-angiogenic drugs in other cardiovascular diseases and cancer.

Periconceptional growth hormone treatment alters early uterine environment

Open Journal of Animal Sciences · 2013-01-01

We have shown that an injection of sustained release growth hormone (GH), given just prior to breeding, results in lambs that are 25% heavier at birth, with an altered body composition as evidenced by an increased abdominal girth, but no difference in crown rump length. The mechanisms by which these differences occur from a single periconceptional injection are not yet known. Therefore, the objective of this experiment was to determine the effect of an injection of GH given prior to breeding on the composition of the uterine luminal environment and the embryo at the time of blastocyst. Ewes were synchronized with two injections of prostaglandin F2α given eight days apart. On the day of the second injection, ewes were randomly assigned to be given an injection of sustained release GH or remain as controls and penned with a ram. On day 6.5 following breeding embryos were collected. Prior to surgery a jugular blood sample was taken to determine plasma progesterone and urea concentration. The uterine content of urea, prostaglandin F2α, prostaglandin E2, transforming growth factor β-1 (TGF β), and nitric oxide metabolites were measured. Collected embryos were differentially stained to calculate a trophectoderm to inner cell mass ratio. The concentration of progesterone in maternal plasma was greater in the treated group compare to controls, but blood urea nitrogen was not different between groups. The uterine content of urea in the GH group was lower than that of the control group. There was a trend for TGF β to be increased in the GH treated group compared to control (P = 0.07). There was no difference (P > 0.05) in the total uterine content prostaglandin F2α, prostaglandin E2 or nitric oxide metabolites. The trophectoderm to inner cell mass ratio was not different between treatment groups. Thus, we suggest that the observed difference in fetal development following periconceptional growth hormone administration just prior to breeding may be the result of an alteration in the uterine luminal environment, which may alter the cellular program of the conceptus and later development beyond the embryonic stage.