Sequential paracrine mechanisms are necessary for the therapeutic benefits of stem cell therapy.

Stem cell injections are an attractive therapeutic tool. It has been demonstrated that injected stem cells promote tissue repair and regeneration via paracrine mechanisms. However, the effects of injected stem cells continue for far longer than they are present. We hypothesized that the effects of injected stem cells are prolonged because of a sequential paracrine relay mechanism. Conditioned media was collected from mesenchymal stem cells (MSCs) after 24 h. This media was then added to RAW264.7. Media was collected from the macrophages after 24 h and was then added to endothelial cells (ECs). This conditioned macrophage media, but not control media, promoted wound healing and induced EC differentiation. Similar results were observed with primary macrophages. To identify the active paracrine factors released by macrophages in response to stimulation by MSC conditioned media we used an antibody array, identifying increased expression of the angiogenesis-related proteins stromal cell-derived factor 1 (SDF1) and plasminogen activator inhibitor-1 (PAI-1). Knockdown of either protein inhibited the ability of conditioned media derived from MSC paracrine factor-stimulated macrophages to induce EC differentiation both in vitro and in vivo. Conditioned media derived from postnatal day 7 (P7) mouse macrophages induced EC differentiation. Moreover, SDF1 and PAI-1 levels were >120 higher in P7 macrophages compared with adult macrophages, suggesting that MSC paracrine factors promote adult macrophages to adopt a juvenile phenotype. These results indicate that MSC paracrine factors induce macrophages to secrete SDF1 and PAI-1, in-turn inducing endothelial cells to differentiate. Identification of a sequential paracrine mechanism opens new therapeutic avenues for stem cell therapy.

Optimizing delivery for efficient cardiac reprogramming.

Following heart injury, cardiomyocytes, are lost and are not regenerated. In their place, fibroblasts invade the dead tissue where they generate a scar, which reduces cardiac function. We and others have demonstrated that combinations of specific miRNAs (miR combo) or transcription factors (GMT), delivered by individual lenti-/retro-viruses in vivo, can convert fibroblasts into cardiomyocytes and improve cardiac function. However, the effects are relatively modest due to the low efficiency of delivery of miR combo or GMT. We hypothesized that efficiency would be improved by optimizing delivery. In the first instance, we developed a multicistronic system to express all four miRNAs of miR combo from a single construct. The order of each miRNA in the multicistronic construct gave rise to different levels of miRNA expression. A combination that resulted in equivalent expression levels of each of the four miRNAs of miR combo showed the highest reprogramming efficiency. Further efficiency can be achieved by directly targeting fibroblasts. Screening of several AAV serotypes indicated that AAV1 displayed tropism towards cardiac fibroblasts. Combining multicistronic expression with AAV1 delivery robustly reprogrammed cardiac fibroblasts into cardiomyocytes in vivo.

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.

Insights from molecular signature of in vivo cardiac c-Kit(+) cells following cardiac injury and ß-catenin inhibition.

There is much interest over resident c-Kit(+) cells in tissue regeneration. Their role in cardiac regeneration has been controversial. In this study we aim to understand the in vivo behavior of cardiac c-Kit(+) cells at baseline and after myocardial infarction and in response to Sfrp2. This approach can accurately study the in vivo transcript expressions of these cells in temporal response to injury and overcomes the limitations of the in vitro approach. RNA-seq was performed with c-Kit(+) cells and cardiomyocytes from healthy non-injured mice as well as c-Kit(+) cells from 1 day post-MI and 12 days post-MI mice. When compared to in vivo c-Kit(+) cells isolated from a healthy non-injured mouse heart, cardiomyocytes were enriched in transcripts that express anion channels, cation channels, developmental/differentiation pathway components, as well as proteins that inhibit canonical Wnt/ß-catenin signaling. Myocardial infarction (MI) induced in vivo c-Kit(+) cells to transiently adopt the cardiomyocyte-specific signature: expression of a number of cardiomyocyte-specific transcripts was maximal 1 day post-MI and declined by 12 days post-MI. We next studied the effect of ß-catenin inhibition on in vivo c-Kit(+) cells by administering the Wnt inhibitor Sfrp2 into the infarct border zone. Sfrp2 both enhanced and sustained cardiomyocyte-specific gene expression in the in vivo c-Kit(+) cells: expression of cardiomyocyte-specific transcripts was higher and there was no decline in expression by 12 days post-MI. Further analysis of the biology of c-Kit(+) cells identified that culture induced a significant and irreversible change in their molecular signature raising questions about reliability of in vitro studies. Our findings provide evidence that MI induces in vivo c-Kit(+) cells to adopt transiently a cardiomyocyte-specific pattern of gene expression, and Sfrp2 further enhances and induces sustained gene expression. Our approach is important for understanding c-Kit(+) cells in cardiac regeneration and also has broad implications in the investigation of in vivo resident stem cells in other areas of tissue regeneration.

Cardiomyocyte Maturation Requires TLR3 Activated Nuclear Factor Kappa B.

The process by which committed precursors mature into cardiomyocytes is poorly understood. We found that TLR3 inhibition blocked cardiomyocyte maturation; precursor cells committed to the cardiomyocyte lineage failed to express maturation genes and sarcomeres did not develop. Using various approaches, we found that the effects of TLR3 upon cardiomyocyte maturation were dependent upon the RelA subunit of nuclear factor kappa B (NFκB). Importantly, under conditions that promote the development of mature cardiomyocytes NFκB became significantly enriched at the promoters of cardiomyocyte maturation genes. Furthermore, activation of the TLR3-NFκB pathway enhanced cardiomyocyte maturation. This study, therefore, demonstrates that the TLR3-NFκB pathway is necessary for the maturation of committed precursors into mature cardiomyocytes. Stem Cells 2018;36:1198-1209.

The proximity-labeling technique BioID identifies sorting nexin 6 as a member of the insulin-like growth factor 1 (IGF1)-IGF1 receptor pathway.

The insulin-like growth factor 1 receptor (IGF1R) is a receptor tyrosine kinase with critical roles in various biological processes. Recent results from clinical trials targeting IGF1R indicate that IGF1R signaling pathways are more complex than previously thought. Moreover, it has become increasingly clear that the function of many proteins can be understood only in the context of a network of interactions. To that end, we sought to profile IGF1R-protein interactions with the proximity-labeling technique BioID. We applied BioID by generating a HEK293A cell line that stably expressed the BirA* biotin ligase fused to the IGF1R. Following stimulation by IGF1, biotinylated proteins were analyzed by MS. This screen identified both known and previously unknown interactors of IGF1R. One of the novel interactors was sorting nexin 6 (SNX6), a protein that forms part of the retromer complex, which is involved in intracellular protein sorting. Using co-immunoprecipitation, we confirmed that IGF1R and SNX6 physically interact. SNX6 knockdown resulted in a dramatic diminution of IGF1-mediated ERK1/2 phosphorylation, but did not affect IGF1R internalization. Bioluminescence resonance energy transfer experiments indicated that the SNX6 knockdown perturbed the association between IGF1R and the key adaptor proteins insulin receptor substrate 1 (IRS1) and SHC adaptor protein 1 (SHC1). Intriguingly, even in the absence of stimuli, SNX6 overexpression significantly increased Akt phosphorylation. Our study confirms the utility of proximity-labeling methods, such as BioID, to screen for interactors of cell-surface receptors and has uncovered a role of one of these interactors, SNX6, in the IGF1R signaling cascade.

Mesenchymal stem cells in obesity: insights for translational applications.

Obesity is now a major public health problem worldwide. Lifestyle modification to reduce the characteristic excess body adiposity is important in the treatment of obesity, but effective therapeutic intervention is still needed to control what has become an obesity epidemic. Unfortunately, many anti-obesity drugs have been withdrawn from market due to adverse side effects. Bariatric surgery therefore remains the most effective therapy for severe cases, although such surgery is invasive and researchers continue to seek new control strategies for obesity. Mesenchymal stem cells (MSCs) are a major source of adipocyte generation, and studies have been conducted into the potential roles of MSCs in treating obesity. However, despite significant progress in stem cell research and its potential applications for obesity, adipogenesis is a highly complex process and the molecular mechanisms governing MSC adipogenesis remain ill defined. In particular, successful clinical application of MSCs will require extensive identification and characterization of the transcriptional regulators controlling MSC adipogenesis. Since obesity is associated with the incidence of multiple important comorbidities, an in-depth understanding of the relationship between MSC adipogenesis and the comorbidities of obesity is also necessary to evaluate the potential of effective and safe MSC-based therapies for obesity. In addition, brown adipogenesis is an attractive topic from the viewpoint of therapeutic innovation and future research into MSC-based brown adipogenesis could lead to a novel breakthrough. Ongoing stem cell studies and emerging research fields such as epigenetics are expected to elucidate the complicated mechanisms at play in MSC adipogenesis and develop novel MSC-based therapeutic options for obesity. This review discusses the current understanding of MSCs in adipogenesis and their potential clinical applications for obesity.

Deletion of angiotensin II type 2 receptor accelerates adipogenesis in murine mesenchymal stem cells via Wnt10b/beta-catenin signaling.

Recent evidence suggests that the renin-angiotensin system (RAS) has a vital role in adipocyte biology and the pathophysiology of metabolic syndrome. Obesity is the main culprit of metabolic syndrome; and mesenchymal stem cells (MSCs) have been forwarded as a major source of adipocyte generation. Previously, we reported that MSCs have a local RAS and that pharmacological blockade of angiotensin II type 2 receptor (AT2R) promotes adipogenesis in human MSCs. However, the definitive roles of AT2R and how AT2R functions in adipogenesis remains unknown. To this end, we employed AT2R-null murine MSCs to characterize how AT2R affects the differentiation of MSCs into adipocytes. Murine MSCs were isolated from AT2R-null mice and wild-type littermates, grown to confluency, and then differentiated into adipocytes. Adipogenesis was quantitated by assessing the lipid droplet accumulation. Using the lipophilic fluorescent dye, the AT2R-null cells showed significantly increased total fluorescence (261.6±49.6% vs littermate) on day 7. Oil red O staining followed by extraction of the absorbed dye and measurement of the absorbance on day 14 also exhibited significantly increased lipid droplet accumulation in the AT2R-null cells (202.7±14.1% vs littermate). We also examined the expression of adipogenic marker genes by quantitative RT-PCR. The AT2R-null group exhibited significantly increased expression of PPAR-gamma, fatty acid synthase, and adiponectin (vs littermate). We further examined the role of Wnt10b/beta-catenin signaling, which reportedly has an important inhibitory role in adipogenesis. The AT2R-null group exhibited significantly decreased Wnt10b expression accompanied by decreased beta-catenin (vs littermate). Our results thus revealed that the AT2R inhibits adipogenic differentiation in murine MSCs. Moreover, this inhibitory effect is associated with Wnt10b/beta-catenin signaling. These results provide important insights into the pathophysiology of obesity and obesity-related consequences such as metabolic syndrome, hinting at possible future therapies.

Toward a Common Secure Future: Four Global Commissions in the Wake of Ebola.

Lawrence Gostin and colleagues offer a set of priorities for global health preparedness and response for future infectious disease threats.

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.

Emerging Concepts in Paracrine Mechanisms in Regenerative Cardiovascular Medicine and Biology.

In the past decade, substantial evidence supports the paradigm that stem cells exert their reparative and regenerative effects, in large part, through the release of biologically active molecules acting in a paracrine fashion on resident cells. The data suggest the existence of a tissue microenvironment where stem cell factors influence cell survival, inflammation, angiogenesis, repair, and regeneration in a temporal and spatial manner.

Nuclear hormone receptor LXRα inhibits adipocyte differentiation of mesenchymal stem cells with Wnt/beta-catenin signaling.

Nuclear hormone receptor liver X receptor-alpha (LXRα) has a vital role in cholesterol homeostasis and is reported to have a role in adipose function and obesity although this is controversial. Conversely, mesenchymal stem cells (MSCs) are suggested to be a major source of adipocyte generation. Accordingly, we examined the role of LXRα in adipogenesis of MSCs. Adult murine MSCs (mMSCs) were isolated from wild-type (WT) and LXR-null mice. Using WT mMSCs, we further generated cell lines stably overexpressing GFP-LXRα (mMSC/LXRα/GFP) or GFP alone (mMSC/GFP) by retroviral infection. Confluent mMSCs were differentiated into adipocytes by the established protocol. Compared with MSCs isolated from WT mice, MSCs from LXR-null mice showed significantly increased adipogenesis, as determined by lipid droplet accumulation and adipogenesis-related gene expression. Moreover, mMSCs stably overexpressing GFP-LXRα (mMSC/LXRα/GFP) exhibited significantly decreased adipogenesis compared with mMSCs overexpressing GFP alone (mMSC/GFP). Since Wnt/beta-catenin signaling is reported to inhibit adipogenesis, we further examined it. The LXR-null group showed significantly decreased Wnt expression accompanied by a decrease of cellular beta-catenin (vs WT). The mMSC/LXRα/GFP group exhibited significantly increased Wnt expression accompanied by an increase of cellular beta-catenin (vs mMSC/GFP). These data demonstrate that LXRα has an inhibitory effect on adipogenic differentiation in mMSCs with Wnt/beta-catenin signaling. These results provide important insights into the pathophysiology of obesity and obesity-related consequences such as metabolic syndrome and may identify potential therapeutic targets.

Blockade of angiotensin II type 2 receptor by PD123319 inhibits osteogenic differentiation of human mesenchymal stem cells via inhibition of extracellular signal-regulated kinase signaling.

Recent evidence indicates that the vasculature contains mesenchymal stem cells (MSCs). We hypothesized that angiotensin II (Ang II) type 2 receptors (AT2Rs) play a role in the osteogenesis of MSCs and may have a role in vascular calcification. Human MSCs were differentiated into osteoblasts. Expression of AT2R was significantly increased during osteogenesis, whereas the expression of Ang II type 1 receptors was not significantly changed. Incubation with the AT2R blocker PD123319 with or without Ang II significantly inhibited calcium deposition, whereas type 1 receptor blocker valsartan had no significant effect. PD123319 inhibited extracellular signal-regulated kinase (ERK) phosphorylation in the osteogenic process, whereas valsartan had no effect. Furthermore, PD123319 combined with Ang II also inhibited acute ERK phosphorylation in MSCs induced by insulin. In conclusion, AT2R is upregulated during osteogenesis. Blockade of AT2R inhibits osteogenesis and ERK phosphorylation of human MSCs. These results provide a novel insight into the pathophysiology of calcific vascular disease.

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.

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.

Abi3bp regulates cardiac progenitor cell proliferation and differentiation.

RATIONALE: Cardiac progenitor cells (CPCs) are thought to differentiate into the major cell types of the heart: cardiomyocytes, smooth muscle cells, and endothelial cells. We have recently identified ABI family, member 3 (NESH) binding protein (Abi3bp) as a protein important for mesenchymal stem cell biology. Because CPCs share several characteristics with mesenchymal stem cells, we hypothesized that Abi3bp would similarly affect CPC differentiation and proliferation. OBJECTIVE: To determine whether Abi3bp regulates CPC proliferation and differentiation. METHODS AND RESULTS: In vivo, genetic ablation of the Abi3bp gene inhibited CPC differentiation, whereas CPC number and proliferative capacity were increased. This correlated with adverse recovery after myocardial infarction. In vitro, CPCs, either isolated from Abi3bp knockout mice or expressing an Abi3bp shRNA construct, displayed a higher proliferative capacity and, under differentiating conditions, reduced expression of both early and late cardiomyocyte markers. Abi3bp controlled CPC differentiation via integrin-ß1, protein kinase C-ζ, and v-akt murine thymoma viral oncogene homolog. CONCLUSIONS: We have identified Abi3bp as a protein important for CPC differentiation and proliferation.

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.