Rationale Dual cell transplantation of cardiac progenitor cells (CPCs) and mesenchymal stem cells (MSCs) after infarction improves myocardial repair and performance in large animal models relative to delivery of either cell population. Objective To demonstrate that CardioChimeras (CCs) formed by fusion between CPCs and MSCs have enhanced reparative potential in a mouse model of myocardial infarction relative to individual stem cells or combined cell delivery. Methods and Results Two distinct and clonally derived CCs, CC1 and CC2 were utilized for this study. CCs improved left ventricular anterior wall thickness (AWT) at 4 weeks post injury, but only CC1 treatment preserved AWT at 18 weeks. Ejection fraction was enhanced at 6 weeks in CCs, and functional improvements were maintained in CCs and CPC + MSC groups at 18 weeks. Infarct size was decreased in CCs, whereas CPC + MSC and CPC parent groups remained unchanged at 12 weeks. CCs exhibited increased persistence, engraftment, and expression of early commitment markers within the border zone relative to combinatorial and individual cell population-injected groups. CCs increased capillary density and preserved cardiomyocyte size in the infarcted regions suggesting CCs role in protective paracrine secretion. Conclusions CCs merge the application of distinct cells into a single entity for cellular therapeutic intervention in the progression of heart failure. CCs are a novel cell therapy that improves upon combinatorial cell approaches to support myocardial regeneration.

Treg1 cell therapy is a promising approach for transplant rejection and severe autoimmunity. Unfortunately, clinically meaningful Treg numbers can be obtained only upon in vitro culture. Functional stability of human expanded (e)Tregs2 and induced (i)Tregs3 has not been thoroughly addressed for all proposed protocols, hindering clinical translation. We undertook a systematic comparison of eTregs and iTregs to recommend the most suitable for clinical implementation, and then tested their effectiveness and feasibility in rheumatoid arthritis (RA)4. Regardless of the treatment, iTregs acquired suppressive function and FOXP3 expression, but lost them upon secondary restimulation in the absence of differentiation factors, which mimics in vivo reactivation. In contrast, eTregs expanded in the presence of Rapamycin (rapa)5 retained their regulatory properties and FOXP3 demethylation upon restimulation with no stabilizing agent. FOXP3 demethylation predicted Treg functional stability upon secondary TCR engagement. Rapa eTregs suppressed conventional T cell (Tconv)6 proliferation via both surface (CTLA-4) and secreted (IL-10, TGF-β and IL-35) mediators, similarly to ex vivo Tregs. Importantly, Treg expansion with rapa from RA patients produced functionally stable Tregs with yields comparable to healthy donors (HD)7. Moreover, rapa eTregs from RA patients were resistant to suppression reversal by the pro-inflammatory cytokine TNF-α, and were more efficient in suppressing synovial Tconv proliferation compared to their ex vivo counterparts, suggesting that rapa improves both Treg function and stability. In conclusion, our data indicate Treg expansion with rapa as the protocol of choice for clinical application in rheumatological settings, with assessment of FOXP3 demethylation as a necessary quality control step.

Rheumatoid arthritis (RA) is an autoimmune disease hallmarked by aberrant cellular homeostasis, resulting in hyperactive CD4+ T cells that are more resistant to apoptosis. Both hyperactivation and resistance to apoptosis may contribute to the pathogenicity of these T cells in the autoimmune process. A better knowledge of the mechanisms determining such impaired homeostasis could contribute significantly to both the understanding and the treatment of the disease. Here we investigated whether autophagy, is dysregulated in CD4+ T cells of RA patients, resulting in disturbed T cell homeostasis. We demonstrate that the rate of autophagy is significantly increased in CD4+ T cells from RA patients, and that increased autophagy is also a feature of in vitro activated CD4+ T cells. The increased apoptosis resistance observed in CD4+ T cells from RA patients was significantly reversed upon autophagy inhibition. These mechanisms may contribute to RA pathogenesis, as autophagy inhibition reduced both arthritis incidence and disease severity in a mouse collagen induced arthritis mouse model. Conversely, in Atg5flox/flox-CD4-Cre+ mice, in which all T cells are autophagy-deficient, T cells showed impaired activation and proliferation. These data provide novel insight into the pathogenesis of RA and underscore the relevance of autophagy as a promising therapeutic target.

Rationale: Understanding and manipulating the cardiomyocyte cell cycle has been the focus of decades of research, however the ultimate goal of activating mitotic activity in adult mammalian cardiomyocytes remains elusive and controversial. The relentless pursuit of controlling cardiomyocyte mitosis has been complicated and obfuscated by a multitude of indices used as evidence of cardiomyocyte cell cycle activity that lack clear identification of cardiomyocyte "proliferation" versus cell cycle progression, endoreplication, endomitosis, and even DNA damage. Unambiguous appreciation of the complexity of cardiomyocyte replication that avoids oversimplification and misinterpretation is desperately needed.Objective: Track cardiomyocyte cell cycle activity and authenticate fidelity of proliferation markers as indicators of de novo cardiomyogenesis in post-mitotic cardiomyocytes. Methods and Results:Cardiomyocytes expressing the FUCCI construct driven by the αmyosin heavy chain promoter were readily and uniformly detected through the myocardium of transgenic mice. Cardiomyocyte cell cycle activity peaks at postnatal day 2 and rapidly declines thereafter with almost all cardiomyocytes arrested at the G1/S cell cycle transition. Myocardial infarction injury in adult hearts prompts transient small increases in myocytes progressing through

Naturally occurring disease in pet dogs is an untapped and unique resource for stem cell‐based regenerative medicine translational research, given the many similarities and complexity such disease shares with their human counterparts. Canine‐specific regulators of somatic cell reprogramming and pluripotency maintenance are poorly understood. While retroviral delivery of the four Yamanaka factors successfully reprogrammed canine embryonic fibroblasts, adult stromal cells remained resistant to reprogramming in spite of effective viral transduction and transgene expression. We hypothesized that adult stromal cells fail to reprogram due to an epigenetic barrier. Here, we performed assay for transposase‐accessible chromatin using sequencing (ATAC‐seq) on canine stromal and pluripotent stem cells, analyzing 51 samples in total, and establishing the global landscape of chromatin accessibility before and after reprogramming to induced pluripotent stem cells (iPSC). We also studied adult stromal cells that do not yield iPSC colonies to identify potential reprogramming barriers. ATAC‐seq analysis identified distinct cell type clustering patterns and chromatin remodeling during embryonic fibroblast reprogramming. Compared with embryonic fibroblasts, adult stromal cells had a chromatin accessibility landscape that reflects phenotypic differentiation and somatic cell‐fate stability. We ultimately identified 76 candidate genes and several transcription factor binding motifs that may be impeding somatic cell reprogramming to iPSC, and could be targeted for inhibition or activation, in order to improve the process in canines. These results provide a vast resource for better understanding of pluripotency regulators in dogs and provide an unbiased rationale for novel canine‐specific reprogramming approaches.

Background The limited regenerative capacity of cardiac tissue has long been an obstacle to treating damaged myocardium. Cell-based therapy offers an enormous potential to the current treatment paradigms. However, the efficacy of regenerative therapies remains limited by inefficient delivery and engraftment. Electrotaxis, electrically guided cell movement, has been clinically utilized to improve recovery in a number of tissues, but has not been investigated for treating myocardial damage. Objectives The goal of the study is to test the electrotactic behaviors of several types of cardiac cells. Methods Cardiac Progenitor Cells (CPCs), Cardiac Fibroblasts (CFs), and human induced Pluripotent Stem cell-derived Cardiac Progenitor Cells (hiPSC-CPCs) were used. Results CPCs and CFs electrotax towards the anode of a direct current electric field (EF), while hiPSC-CPCs electrotax toward the cathode. The voltage-dependent electrotaxis of CPCs and CFs requires the presence of serum in the media. Addition of soluble vascular cell adhesion molecule (sVCAM) to serum-free media restores directed migration. We provide evidence that CPC and CF electrotaxis is mediated through phosphatidylinositide 3-kinases (PI3′K) signaling. In addition, Very Late Antigen-4 (VLA4), an integrin and growth factor receptor, is required for electrotaxis and localizes to the anodal edge of CPCs in response to direct current EF. HiPSC-derived CPCs do not express VLA4, migrate toward the cathode in a voltage-dependent manner, and similar to CPCs and CFs require media serum and PI3′K activity for electrotaxis. Conclusion The electrotactic behaviors of these therapeutic cardiac cells may be utilized for improving cell-based therapy for recovering function in damaged myocardium.

Diabetes mellitus (DM) is a common spontaneous endocrine disorder in dogs, which is defined by persistent hyperglycemia and insulin deficiency. Like type 1 diabetes (T1D) in people, canine DM is a complex and multifactorial disease in which genomic and epigenomic factors interact with environmental cues to induce pancreatic β‐cell loss and insulin deficiency, although the pathogenesis of canine DM is poorly defined and the role of autoimmunity is further controversial. Both diseases are incurable and require life‐long exogenous insulin therapy to maintain glucose homeostasis. Human pancreatic islet physiology, size, and cellular composition is further mirrored by canine islets. Although pancreatic or isolated islets transplantation are the only clinically validated methods to achieve long‐term normoglycemia and insulin independence, their availability does not meet the clinical need; they target a small portion of patients and have significant potential adverse effects. Therefore, providing a new source for β‐cell replacement is an unmet need. Naturally occurring DM in pet dogs, as a translational platform, is an untapped resource for various regenerative medicine applications that may offer some unique advantages given dogs' large size, longevity, heterogenic genetic background, similarity to human physiology and pathology, and long‐term clinical management. In this review, we outline different strategies for curative approaches, animal models used, and consider the value of canine DM as a translational animal/disease model for T1D in people. stem cells translational medicine 2019;8:450–455

Background Intracellular pH (pHi) is critical to cardiac excitation and contraction; uncompensated changes in pHi impair cardiac function and trigger arrhythmia. Several ion transporters participate in cardiac pHi regulation. Our previous studies identified several isoforms of a solute carrier Slc26a6 to be highly expressed in cardiomyocytes. We show that Slc26a6 mediates electrogenic Cl−/HCO3− exchange activities in cardiomyocytes, suggesting the potential role of Slc26a6 in regulation of not only pHi, but also cardiac excitability. Methods and Results To test the mechanistic role of Slc26a6 in the heart, we took advantage of Slc26a6 knockout (Slc26a6−/−) mice using both in vivo and in vitro analyses. Consistent with our prediction of its electrogenic activities, ablation of Slc26a6 results in action potential (AP) shortening. There are reduced Ca2+ transient and sarcoplasmic reticulum Ca2+ load, together with decreased sarcomere shortening in Slc26a6−/− cardiomyocytes. These abnormalities translate into reduced fractional shortening and cardiac contractility at the in vivo level. Additionally, pHi is elevated in Slc26a6−/− cardiomyocytes with slower recovery kinetics from intracellular alkalization, consistent with the Cl−/HCO3− exchange activities of Slc26a6. Moreover, Slc26a6−/− mice show evidence of sinus bradycardia and fragmented QRS complex, supporting the critical role of Slc26a6 in cardiac conduction system. Conclusions Our study provides mechanistic insights into Slc26a6, a unique cardiac electrogenic Cl−/HCO3− transporter in ventricular myocytes, linking the critical roles of Slc26a6 in regulation of pHi, excitability, and contractility. pHi is a critical regulator of other membrane and contractile proteins. Future studies are needed to investigate possible changes in these proteins in Slc26a6−/− mice.