A fully-automated low-cost cardiac monolayer optical mapping robot.

Scalable and high-throughput electrophysiological measurement systems are necessary to accelerate the elucidation of cardiac diseases in drug development. Optical mapping is the primary method of simultaneously measuring several key electrophysiological parameters, such as action potentials, intracellular free calcium and conduction velocity, at high spatiotemporal resolution. This tool has been applied to isolated whole-hearts, whole-hearts in-vivo, tissue-slices and cardiac monolayers/tissue-constructs. Although optical mapping of all of these substrates have contributed to our understanding of ion-channels and fibrillation dynamics, cardiac monolayers/tissue-constructs are scalable macroscopic substrates that are particularly amenable to high-throughput interrogation. Here, we describe and validate a scalable and fully-automated monolayer optical mapping robot that requires no human intervention and with reasonable costs. As a proof-of-principle demonstration, we performed parallelized macroscopic optical mapping of calcium dynamics in the well-established neonatal-rat-ventricular-myocyte monolayer plated on standard 35 mm dishes. Given the advancements in regenerative and personalized medicine, we also performed parallelized macroscopic optical mapping of voltage dynamics in human pluripotent stem cell-derived cardiomyocyte monolayers using a genetically encoded voltage indictor and a commonly-used voltage sensitive dye to demonstrate the versatility of our system.

Sox6 as a new modulator of renin expression in the kidney.

Juxtaglomerular (JG) cells, major sources of renin, differentiate from metanephric mesenchymal cells that give rise to JG cells or a subset of smooth muscle cells of the renal afferent arteriole. During periods of dehydration and salt deprivation, renal mesenchymal stromal cells (MSCs) differentiate from JG cells. JG cells undergo expansion and smooth muscle cells redifferentiate to express renin along the afferent arteriole. Gene expression profiling comparing resident renal MSCs with JG cells indicates that the transcription factor Sox6 is highly expressed in JG cells in the adult kidney. In vitro, loss of Sox6 expression reduces differentiation of renal MSCs to renin-producing cells. In vivo, Sox6 expression is upregulated after a low-Na+ diet and furosemide. Importantly, knockout of Sox6 in Ren1d+ cells halts the increase in renin-expressing cells normally seen during a low-Na+ diet and furosemide as well as the typical increase in renin. Furthermore, Sox6 ablation in renin-expressing cells halts the recruitment of smooth muscle cells along the afferent arteriole, which normally express renin under these conditions. These results support a previously undefined role for Sox6 in renin expression.

Demethylation of H3K27 Is Essential for the Induction of Direct Cardiac Reprogramming by miR Combo.

RATIONALE: Direct reprogramming of cardiac fibroblasts to cardiomyocytes has recently emerged as a novel and promising approach to regenerate the injured myocardium. We have previously demonstrated the feasibility of this approach in vitro and in vivo using a combination of 4 microRNAs (miR-1, miR-133, miR-208, and miR-499) that we named miR combo. However, the mechanism of miR combo mediated direct cardiac reprogramming is currently unknown. OBJECTIVE: Here, we investigated the possibility that miR combo initiated direct cardiac reprogramming through an epigenetic mechanism. METHODS AND RESULTS: Using a quantitative polymerase chain reaction array, we found that histone methyltransferases and demethylases that regulate the trimethylation of H3K27 (H3K27me3), an epigenetic modification that marks transcriptional repression, were changed in miR combo-treated fibroblasts. Accordingly, global H3K27me3 levels were downregulated by miR combo treatment. In particular, the promoter region of cardiac transcription factors showed decreased H3K27me3 as revealed by chromatin immunoprecipitation coupled with quantitative polymerase chain reaction. Inhibition of H3K27 methyltransferases or of the PRC2 (Polycomb Repressive Complex 2) by pharmaceutical inhibition or siRNA reduced the levels of H3K27me3 and induced cardiogenic markers at the RNA and protein level, similarly to miR combo treatment. In contrast, knockdown of the H3K27 demethylases Kdm6A and Kdm6B restored the levels of H3K27me3 and blocked the induction of cardiac gene expression in miR combo-treated fibroblasts. CONCLUSIONS: In summary, we demonstrated that removal of the repressive mark H3K27me3 is essential for the induction of cardiac reprogramming by miR combo. Our data not only highlight the importance of regulating the epigenetic landscape during cell fate conversion but also provide a framework to improve this technique.

Tissue-engineered 3-dimensional (3D) microenvironment enhances the direct reprogramming of fibroblasts into cardiomyocytes by microRNAs.

We have recently shown that a combination of microRNAs, miR combo, can directly reprogram cardiac fibroblasts into functional cardiomyocytes in vitro and in vivo. Reprogramming of cardiac fibroblasts by miR combo in vivo is associated with improved cardiac function following myocardial infarction. However, the efficiency of direct reprogramming in vitro is relatively modest and new strategies beyond the traditional two-dimensional (2D) culture should be identified to improve reprogramming process. Here, we report that a tissue-engineered three-dimensional (3D) hydrogel environment enhanced miR combo reprogramming of neonatal cardiac and tail-tip fibroblasts. This was associated with significantly increased MMPs expression in 3D vs. 2D cultured cells, while pharmacological inhibition of MMPs blocked the effect of the 3D culture on enhanced miR combo mediated reprogramming. We conclude that 3D tissue-engineered environment can enhance the direct reprogramming of fibroblasts to cardiomyocytes via a MMP-dependent mechanism.

Transcriptomic Analysis of Adult Renal Derived Mesenchymal Stem-Like Cells.

Mesenchymal stem cells (MSC) from bone marrow or adult tissues are widely studied to evaluate their potential for tissue repair. Differences in tissue of origin, donor variation, or in vitro handling exist and it is still unclear how they affect cell function and regenerative potential. Large-scale gene expression analysis of these cells not only allows researchers to compare and contrast the differences between each MSC subset but also allows for the development of better analytical tools for their characterization and utilization. Here, we describe a protocol for transcriptomics analysis of MSC-like cells derived from adult kidneys.

Tracking mesenchymal stromal cells using an ultra-bright TAT-functionalized plasmonic-active nanoplatform.

High-resolution tracking of stem cells remains a challenging task. An ultra-bright contrast agent with extended intracellular retention is suitable for in vivo high-resolution tracking of stem cells following the implantation. Here, a plasmonic-active nanoplatform was developed for tracking mesenchymal stromal cells (MSCs) in mice. The nanoplatform consisted of TAT peptide-functionalized gold nanostars (TAT-GNS) that emit ultra-bright two-photon photoluminescence capable of tracking MSCs under high-resolution optical imaging. In vitro experiment showed TAT-GNS-labeled MSCs retained a similar differentiability to that of non-labeled MSCs controls. Due to their star shape, TAT-GNS exhibited greater intracellular retention than that of commercial Q-Tracker. In vivo imaging of TAT-GNS-labeled MSCs five days following intra-arterial injections in mice kidneys showed possible MSCs implantation in juxta-glomerular (JG) regions, but non-specifically in glomeruli and afferent arterioles as well. With future design to optimize GNS labeling specificity and clearance, plasmonic-active nanoplatforms may be a useful intracellular tracking tool for stem cell research. An ultra-bright intracellular contrast agent is developed using TAT peptide-functionalized gold nanostars (TAT-GNS). It poses minimal influence on the stem cell differentiability. It exhibits stronger two-photon photoluminescence and superior labeling efficiency than commercial Q-Tracker. Following renal implantation, some TAT-GNS-labeled MSCs permeate blood vessels and migrate to the juxta-glomerular region.

Inhibition of Wnt6 by Sfrp2 regulates adult cardiac progenitor cell differentiation by differential modulation of Wnt pathways.

Wnt signaling has recently emerged as an important regulator of cardiac progenitor cell proliferation and differentiation, but the exact mechanisms by which Wnt signaling modulates these effects are not known. Understanding these mechanisms is essential for advancing our knowledge of cardiac progenitor cell biology and applying this knowledge to enhance cardiac therapy. Here, we explored the effects of Sfrp2, a canonical Wnt inhibitor, in adult cardiac progenitor cell (CPC) differentiation and investigated the molecular mechanisms involved. Our data show that Sfrp2 treatment can promote differentiation of CPCs after ischemia-reperfusion injury. Treatment of CPCs with Sfrp2 inhibited CPC proliferation and primed them for cardiac differentiation. Sfrp2 binding to Wnt6 and inhibition of Wnt6 canonical pathway was essential for the inhibition of CPC proliferation. This inhibition of Wnt6 canonical signaling by Sfrp2 was important for activation of the non-canonical Wnt/Planar Cell Polarity (PCP) pathway through JNK, which in turn induced expression of cardiac transcription factors and CPC differentiation. Taken together, these results demonstrate a novel role of Sfrp2 and Wnt6 in regulating the dynamic process of CPC proliferation and differentiation, as well as providing new insights into the mechanisms of Wnt signaling in cardiac differentiation.

MicroRNAs and Cardiac Regeneration.

The human heart has a limited capacity to regenerate lost or damaged cardiomyocytes after cardiac insult. Instead, myocardial injury is characterized by extensive cardiac remodeling by fibroblasts, resulting in the eventual deterioration of cardiac structure and function. Cardiac function would be improved if these fibroblasts could be converted into cardiomyocytes. MicroRNAs (miRNAs), small noncoding RNAs that promote mRNA degradation and inhibit mRNA translation, have been shown to be important in cardiac development. Using this information, various researchers have used miRNAs to promote the formation of cardiomyocytes through several approaches. Several miRNAs acting in combination promote the direct conversion of cardiac fibroblasts into cardiomyocytes. Moreover, several miRNAs have been identified that aid the formation of inducible pluripotent stem cells and miRNAs also induce these cells to adopt a cardiac fate. MiRNAs have also been implicated in resident cardiac progenitor cell differentiation. In this review, we discuss the current literature as it pertains to these processes, as well as discussing the therapeutic implications of these findings.

Salt restriction leads to activation of adult renal mesenchymal stromal cell-like cells via prostaglandin E2 and E-prostanoid receptor 4.

Despite the importance of juxtaglomerular cell recruitment in the pathophysiology of cardiovascular diseases, the mechanisms that underlie renin production under conditions of chronic stimulation remain elusive. We have previously shown that CD44+ mesenchymal-like cells (CD44+ cells) exist in the adult kidney. Under chronic sodium deprivation, these cells are recruited to the juxtaglomerular area and differentiate to new renin-expressing cells. Given the proximity of macula densa to the juxtaglomerular area and the importance of macula densa released prostanoids in renin synthesis and release, we hypothesized that chronic sodium deprivation induces macula densa release of prostanoids, stimulating renal CD44+ cell activation and differentiation. CD44+ cells were isolated from adult kidneys and cocultured with the macula densa cell line, MMDD1, in normal or low-sodium medium. Low sodium stimulated prostaglandin E2 production by MMDD1 and induced migration of CD44+ cells. These effects were inhibited by addition of a cyclooxygenase 2 inhibitor (NS398) or an E-prostanoid receptor 4 antagonist (AH23848) to MMDD1 or CD44+ cells, respectively. Addition of prostaglandin E2 to CD44+ cells increased cell migration and induced renin expression. In vivo activation of renal CD44+ cells during juxtaglomerular recruitment was attenuated in wild-type mice subjected to salt restriction in the presence of cyclooxygenase 2 inhibitor rofecoxib. Similar results were observed in E-prostanoid receptor 4 knockout mice subjected to salt restriction. These results show that the prostaglandin E2/E-prostanoid receptor 4 pathway plays a key role in the activation of renal CD44+ mesenchymal stromal cell-like cells during conditions of juxtaglomerular recruitment; highlighting the importance of this pathway as a key regulatory mechanism of juxtaglomerular recruitment.

MicroRNA induced cardiac reprogramming in vivo: evidence for mature cardiac myocytes and improved cardiac function.

RATIONALE: A major goal for the treatment of heart tissue damaged by cardiac injury is to develop strategies for restoring healthy heart muscle through the regeneration and repair of damaged myocardium. We recently demonstrated that administration of a specific combination of microRNAs (miR combo) into the infarcted myocardium leads to direct in vivo reprogramming of noncardiac myocytes to cardiac myocytes. However, the biological and functional consequences of such reprogramming are not yet known. OBJECTIVE: The aim of this study was to determine whether noncardiac myocytes directly reprogrammed using miRNAs in vivo develop into mature functional cardiac myocytes in situ, and whether reprogramming leads to improvement of cardiac function. METHODS AND RESULTS: We subjected fibroblast-specific protein 1-Cre mice/tandem dimer Tomato (tdTomato) mice to cardiac injury by permanent ligation of the left anterior descending coronary artery and injected lentiviruses encoding miR combo or a control nontargeting miRNA. miR combo significantly increased the number of reprogramming events in vivo. Five to 6 weeks after injury, morphological and physiological properties of tdTomato(-) and tdTomato(+) cardiac myocyte-like cells were analyzed ex vivo. tdTomato(+) cells expressed cardiac myocyte markers, sarcomeric organization, excitation-contraction coupling, and action potentials characteristic of mature ventricular cardiac myocytes (tdTomato(-) cells). Reprogramming was associated with improvement of cardiac function, as analyzed by serial echocardiography. There was a time delayed and progressive improvement in fractional shortening and other measures of ventricular function, indicating that miR combo promotes functional recovery of damaged myocardium. CONCLUSIONS: The findings from this study further validate the potential use of miRNA-mediated reprogramming as a therapeutic approach to promote cardiac regeneration after myocardial injury.

Reprogramming approaches in cardiovascular regeneration.

OPINION STATEMENT: Reconstitution of cardiac muscle as well as blood vessels to provide sufficient oxygenation and nutrients to the myocardium is an important component of any therapeutic approach for cardiac repair after injury. Recent reports of reprogramming somatic cells directly to cells of another lineage raised the possibility of using cell reprogramming for cardiac regenerative therapy. Here, we provide an overview of the current reprogramming strategies to generate cardiomyocytes (CMs), endothelial cells (ECs) and smooth muscle cells (SMCs), and the implications of these methods for cardiac regeneration. We also discuss the challenges and limitations that need to be addressed for the development of future therapies.

Classical Renin-Angiotensin system in kidney physiology.

The renin-angiotensin system has powerful effects in control of the blood pressure and sodium homeostasis. These actions are coordinated through integrated actions in the kidney, cardiovascular system and the central nervous system. Along with its impact on blood pressure, the renin-angiotensin system also influences a range of processes from inflammation and immune responses to longevity. Here, we review the actions of the "classical" renin-angiotensin system, whereby the substrate protein angiotensinogen is processed in a two-step reaction by renin and angiotensin converting enzyme, resulting in the sequential generation of angiotensin I and angiotensin II, the major biologically active renin-angiotensin system peptide, which exerts its actions via type 1 and type 2 angiotensin receptors. In recent years, several new enzymes, peptides, and receptors related to the renin-angiotensin system have been identified, manifesting a complexity that was previously unappreciated. While the functions of these alternative pathways will be reviewed elsewhere in this journal, our focus here is on the physiological role of components of the "classical" renin-angiotensin system, with an emphasis on new developments and modern concepts.

Direct reprogramming of cardiac fibroblasts to cardiomyocytes using microRNAs.

The therapeutic administration of microRNAs represents an innovative reprogramming strategy with which to advance cardiac regeneration and personalized medicine. Recently, a distinct set of microRNAs was found capable of converting murine fibroblasts to cardiomyocyte-like cells in vitro. Further treatment with JAK inhibitor I significantly enhanced the efficiency of the microRNA-mediated reprogramming (Jayawardena et al., Circ Res 110(11):1465-1473, 2012). This novel technique serves as an initial tool for switching the cell fate of cardiac fibroblasts toward the cardiomyocyte lineage using microRNAs. As the budding field of reprogramming biology develops, we hope that a thorough examination of the chemical, physical, and temporal parameters determining reprogramming efficiency and maturation will enable a better understanding of the mechanisms governing cardiac cell fate and provide new approaches for drug discovery and therapy for cardiovascular diseases.

Stem cells: potential and challenges for kidney repair.

Renal damage resulting from acute and chronic kidney injury poses an important problem to public health. Currently, patients with end-stage renal disease rely solely on kidney transplantation or dialysis for survival. Emerging therapies aiming to prevent and reverse kidney damage are thus in urgent need. Although the kidney was initially thought to lack the capacity for self-repair, several studies have indicated that this might not be the case; progenitor and stem cells appear to play important roles in kidney repair under various pathological conditions. In this review, we summarize recent findings on the role of progenitor/stem cells on kidney repair as well as discuss their potential as a therapeutic approach for kidney diseases.

HASF is a stem cell paracrine factor that activates PKC epsilon mediated cytoprotection.

Despite advances in the treatment of acute tissue ischemia significant challenges remain in effective cytoprotection from ischemic cell death. It has been documented that injected stem cells, such as mesenchymal stem cells (MSCs), can confer protection to ischemic tissue through the release of paracrine factors. The study of these factors is essential for understanding tissue repair and the development of new therapeutic approaches for regenerative medicine. We have recently shown that a novel factor secreted by MSCs, which we called HASF (Hypoxia and Akt induced Stem cell Factor), promotes cardiomyocyte proliferation. In this study we show that HASF has a cytoprotective effect on ischemia induced cardiomyocyte death. We assessed whether HASF could potentially be used as a therapeutic agent to prevent the damage associated with myocardial infarction. In vitro treatment of cardiomyocytes with HASF protein resulted in decreased apoptosis; TUNEL positive nuclei were fewer in number, and caspase activation and mitochondrial pore opening were inhibited. Purified HASF protein was injected into the heart immediately following myocardial infarction. Heart function was found to be comparable to sham operated animals one month following injury and fibrosis was significantly reduced. In vivo and in vitro HASF activated protein kinase C ε (PKCε). Inhibition of PKCε blocked the HASF effect on apoptosis. Furthermore, the beneficial effects of HASF were lost in mice lacking PKCε. Collectively these results identify HASF as a protein of significant therapeutic potential, acting in part through PKCε.

C3orf58, a novel paracrine protein, stimulates cardiomyocyte cell-cycle progression through the PI3K-AKT-CDK7 pathway.

RATIONALE: The regenerative capacity of the heart is markedly diminished shortly after birth, coinciding with overall withdrawal of cardiomyocytes from cell cycle. Consequently, the adult mammalian heart has limited capacity to regenerate after injury. The discovery of factors that can induce cardiomyocyte proliferation is, therefore, of high interest and has been the focus of extensive investigation throughout the past years. OBJECTIVE: We have recently identified C3orf58 as a novel hypoxia and Akt induced stem cell factor (HASF) secreted from mesenchymal stem cells, which can promote cardiac repair through cytoprotective mechanisms. Here, we tested the hypothesis that HASF can also contribute to cardiac regeneration by stimulating cardiomyocyte division and proliferation. METHODS AND RESULTS: Neonatal ventricular cardiomyocytes were stimulated in culture for 7 days with purified recombinant HASF protein. Compared with control untreated cells, HASF-treated neonatal cardiomyocytes exhibited 60% increase in DNA synthesis as measured by bromodeoxyuridine incorporation. These results were confirmed by immunofluorescence confocal microscopy showing a 50% to 100% increase in the number of cardiomyocytes in the mitotic and cytokinesis phases. Importantly, in vivo cardiac overexpression of HASF in a transgenic mouse model resulted in enhanced level of DNA synthesis and cytokinesis in neonatal and adult cardiomyocytes. These proliferative effects were modulated by a phosphoinositide 3-kinase-protein kinase B-cycle-dependent kinase 7 pathway as revealed by the use of phosphoinositide 3-kinase -pathway-specific inhibitors and silencing of the Cdk7 gene. CONCLUSIONS: Our studies support the hypothesis that HASF induces cardiomyocyte proliferation via a phosphoinositide 3-kinase-protein kinase B-cycle-dependent kinase 7 pathway. The implications of this finding may be significant for cardiac regeneration biology and therapeutics.

Adult renal mesenchymal stem cell-like cells contribute to juxtaglomerular cell recruitment.

The renin-angiotensin-aldosterone system (RAAS) regulates BP and salt-volume homeostasis. Juxtaglomerular (JG) cells synthesize and release renin, which is the first and rate-limiting step in the RAAS. Intense pathologic stresses cause a dramatic increase in the number of renin-producing cells in the kidney, termed JG cell recruitment, but how this occurs is not fully understood. Here, we isolated renal CD44(+) mesenchymal stem cell (MSC)-like cells and found that they differentiated into JG-like renin-expressing cells both in vitro and in vivo. Sodium depletion and captopril led to activation and differentiation of these cells into renin-expressing cells in the adult kidney. In summary, CD44(+) MSC-like cells exist in the adult kidney and can differentiate into JG-like renin-producing cells under conditions that promote JG cell recruitment.

Abi3bp is a multifunctional autocrine/paracrine factor that regulates mesenchymal stem cell biology.

Mesenchymal stem cells (MSCs) transplanted into injured myocardium promote repair through paracrine mechanisms. We have previously shown that MSCs over-expressing AKT1 (Akt-MSCs) exhibit enhanced properties for cardiac repair. In this study, we investigated the relevance of Abi3bp toward MSC biology. Abi3bp formed extracellular deposits with expression controlled by Akt1 and ubiquitin-mediated degradation. Abi3bp knockdown/knockout stabilized focal adhesions and promoted stress-fiber formation. Furthermore, MSCs from Abi3bp knockout mice displayed severe deficiencies in osteogenic and adipogenic differentiation. Knockout or stable knockdown of Abi3bp increased MSC and Akt-MSC proliferation, promoting S-phase entry via cyclin-d1, ERK1/2, and Src. Upon Abi3bp binding to integrin-ß1 Src associated with paxillin which inhibited proliferation. In vivo, Abi3bp knockout increased MSC number and proliferation in bone marrow, lung, and liver. In summary, we have identified a novel extracellular matrix protein necessary for the switch from proliferation to differentiation in MSCs.

Lung stem and progenitor cells.

Over the past few years, new insights have been added to the study of stem cells in the adult lung. The exploration of endogenous lung progenitors as well as the study of exogenously delivered stem cell populations holds promise for advancing our understanding of the biology of lung repair mechanisms. Moreover, it opens new possibilities for the use of stem cell therapy for the development of regenerative medicine approaches for the treatment of lung disease. Here, we discuss the main types of lung epithelial progenitor populations; the potential of endothelial progenitors, mesenchymal stem cells and embryonic stem cells for lung therapy, as well as summarize the cellular mechanisms involved.

MicroRNA-mediated in vitro and in vivo direct reprogramming of cardiac fibroblasts to cardiomyocytes.

RATIONALE: Repopulation of the injured heart with new, functional cardiomyocytes remains a daunting challenge for cardiac regenerative medicine. An ideal therapeutic approach would involve an effective method at achieving direct conversion of injured areas to functional tissue in situ. OBJECTIVE: The aim of this study was to develop a strategy that identified and evaluated the potential of specific micro (mi)RNAs capable of inducing reprogramming of cardiac fibroblasts directly to cardiomyocytes in vitro and in vivo. METHODS AND RESULTS: Using a combinatorial strategy, we identified a combination of miRNAs 1, 133, 208, and 499 capable of inducing direct cellular reprogramming of fibroblasts to cardiomyocyte-like cells in vitro. Detailed studies of the reprogrammed cells demonstrated that a single transient transfection of the miRNAs can direct a switch in cell fate as documented by expression of mature cardiomyocyte markers, sarcomeric organization, and exhibition of spontaneous calcium flux characteristic of a cardiomyocyte-like phenotype. Interestingly, we also found that miRNA-mediated reprogramming was enhanced 10-fold on JAK inhibitor I treatment. Importantly, administration of miRNAs into ischemic mouse myocardium resulted in evidence of direct conversion of cardiac fibroblasts to cardiomyocytes in situ. Genetic tracing analysis using Fsp1Cre-traced fibroblasts from both cardiac and noncardiac cell sources strongly suggests that induced cells are most likely of fibroblastic origin. CONCLUSIONS: The findings from this study provide proof-of-concept that miRNAs have the capability of directly converting fibroblasts to a cardiomyocyte-like phenotype in vitro. Also of significance is that this is the first report of direct cardiac reprogramming in vivo. Our approach may have broad and important implications for therapeutic tissue regeneration in general.