Comprehensive UHPLC- and CE-based methods for engineered Cas9 characterization.

CRISPR (clustered regularly interspaced short palindromic repeats)-associated proteins (Cas) are powerful gene-editing tools used in therapeutic applications. Efforts to minimize off-target cleavage by CRISPR-Cas9 have motivated the development of engineered Cas9 variants. The wild-type (WT) Streptococcus pyogenes (SpCas9) has been engineered into a high-fidelity Cas9 (SpyFi Cas9) that shows promising results in providing high on-target activity (targeting efficiency) while reducing off-target editing (unwanted mutations). This work describes for the first time the development of ultra-high-performance liquid chromatography (UHPLC) and capillary electrophoresis (CE)-based methods for a full characterization of different engineered Cas9 variants, including determination of purity, size variants, isoelectric points (pI), post-translational modifications (PTMs), and functional activities. The purity and size variant characterization were first determined by CE-sodium dodecyl sulfate (SDS). An in vitro DNA cleavage assay using an automated electrophoresis tool was employed to investigate the functional activity of ribonucleoprotein (RNP) complexes derived from Cas9 variants. The pIs of the engineered Cas9 proteins were determined by imaged capillary isoelectric focusing (icIEF), while intact mass measurements were performed by reversed-phase (RP)-UHPLC coupled with high-resolution mass spectrometry (HRMS). A peptide mapping assay based on LC-UV-MS/MS using endoproteinase Lys-C under non-reducing conditions was developed to confirm amino acid sequences, allowing differentiation of SpyFi Cas9 from WT SpCas9. The potential of using a low-resolution MS detector, especially for a GMP environment, as a low-cost and simple method to identify SpyFi Cas9 is discussed.

Physicochemical and Functional Characterization of Differential CRISPR-Cas9 Ribonucleoprotein Complexes.

Advances in gene-editing technology enable efficient, targeted ex vivo engineering of different cell types, which offer a potential therapeutic platform for most challenging disease areas. CRISPR-Cas9 is a widely used gene-editing tool in therapeutic applications. The quality of gene-editing reagents (i.e., Cas9 nuclease, single guide (sg)RNA) is associated with the final cellular product quality as they can impact the gene-editing accuracy and efficiency. To assess the impact of the quality of Cas9 protein and sgRNA in the formation of a Cas9 ribonucleoprotein (RNP) complex, stability, and functional activities, we developed a size exclusion chromatography method that utilizes multiple detectors and an in vitro DNA cleavage assay using anion-exchange chromatography. Using these methods, we characterized the formation and stability of Cas9 RNP complexes associated with Cas9 and sgRNA characteristics as well as their functional activities. Multi-angle light scattering characterization showed different types and levels of aggregates in different source sgRNA materials, which contribute to form different Cas9 RNP complexes. The aggregations irreversibly dissociated at high temperatures. When the Cas9 RNP complexes derived from non-heated and heated sgRNAs were characterized, the data showed that specific RNP peaks were impacted. The Cas9 RNP complexes derived from the heated sgRNA retained their biological function and cleaved the double-strand target DNA at a higher rate. This work provides new tools to characterize the Cas9 RNP complex formation, stability, and functional activity and provides insights into sgRNA properties and handling procedures to better control the Cas9 RNP complex formation.

Panobinostat Effectively Increases Histone Acetylation and Alters Chromatin Accessibility Landscape in Canine Embryonic Fibroblasts but Does Not Enhance Cellular Reprogramming.

Robust and reproducible protocols to efficiently reprogram adult canine cells to induced pluripotent stem cells are still elusive. Somatic cell reprogramming requires global chromatin remodeling that is finely orchestrated spatially and temporally. Histone acetylation and deacetylation are key regulators of chromatin condensation, mediated by histone acetyltransferases and histone deacetylases (HDACs), respectively. HDAC inhibitors have been used to increase histone acetylation, chromatin accessibility, and somatic cell reprogramming in human and mice cells. We hypothesized that inhibition of HDACs in canine fibroblasts would increase their reprogramming efficiency by altering the epigenomic landscape and enabling greater chromatin accessibility. We report that a combined treatment of panobinostat (LBH589) and vitamin C effectively inhibits HDAC function and increases histone acetylation in canine embryonic fibroblasts in vitro, with no significant cytotoxic effects. We further determined the effect of this treatment on global chromatin accessibility via Assay for Transposase-Accessible Chromatin using sequencing. Finally, the treatment did not induce any significant increase in cellular reprogramming efficiency. Although our data demonstrate that the unique epigenetic landscape of canine cells does not make them amenable to cellular reprogramming through the proposed treatment, it provides a rationale for a targeted, canine-specific, reprogramming approach by enhancing the expression of transcription factors such as CEBP.

Pim1 maintains telomere length in mouse cardiomyocytes by inhibiting TGFß signalling.

AIMS: Telomere attrition in cardiomyocytes is associated with decreased contractility, cellular senescence, and up-regulation of proapoptotic transcription factors. Pim1 is a cardioprotective kinase that antagonizes the aging phenotype of cardiomyocytes and delays cellular senescence by maintaining telomere length, but the mechanism remains unknown. Another pathway responsible for regulating telomere length is the transforming growth factor beta (TGFß) signalling pathway where inhibiting TGFß signalling maintains telomere length. The relationship between Pim1 and TGFß has not been explored. This study delineates the mechanism of telomere length regulation by the interplay between Pim1 and components of TGFß signalling pathways in proliferating A549 cells and post-mitotic cardiomyocytes. METHODS AND RESULTS: Telomere length was maintained by lentiviral-mediated overexpression of PIM1 and inhibition of TGFß signalling in A549 cells. Telomere length maintenance was further demonstrated in isolated cardiomyocytes from mice with cardiac-specific overexpression of PIM1 and by pharmacological inhibition of TGFß signalling. Mechanistically, Pim1 inhibited phosphorylation of Smad2, preventing its translocation into the nucleus and repressing expression of TGFß pathway genes. CONCLUSION: Pim1 maintains telomere lengths in cardiomyocytes by inhibiting phosphorylation of the TGFß pathway downstream effectors Smad2 and Smad3, which prevents repression of telomerase reverse transcriptase. Findings from this study demonstrate a novel mechanism of telomere length maintenance and provide a potential target for preserving cardiac function.

Chromatin accessibility in canine stromal cells and its implications for canine somatic cell reprogramming.

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.

Concise Review: Canine Diabetes Mellitus as a Translational Model for Innovative Regenerative Medicine Approaches.

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 ß-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 ß-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.

Cardiomyocyte cell cycle dynamics and proliferation revealed through cardiac-specific transgenesis of fluorescent ubiquitinated cell cycle indicator (FUCCI).

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 α-myosin 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 cell cycle without concurrent mitotic activity, indicating lack of cardiomyogenesis. In comparison, cardiomyogenic activity during early postnatal development correlated with coincidence of FUCCI and cKit+ cells that were undetectable in the adult myocardium. CONCLUSIONS: Cardiomyocyte-specific expression of Fluorescence Ubiquitination-based Cell Cycle Indicators (FUCCI) reveals previously unappreciated aspects of cardiomyocyte cell cycle arrest and biological activity in postnatal development and in response to pathologic damage. Compared to many other methods and model systems, the FUCCI transgenic (FUCCI-Tg) mouse represents a valuable tool to unambiguously track cell cycle and proliferation of the entire cardiomyocyte population in the adult murine heart. FUCCI-Tg provides a desperately needed novel approach in the armamentarium of tools to validate cardiomyocyte proliferative activity that will reveal cell cycle progression, discriminate between cycle progression, DNA replication, and proliferation, and provide important insight for enhancing cardiomyocyte proliferation in the context of adult myocardial tissue.

Highly efficient transfection of human induced pluripotent stem cells using magnetic nanoparticles.

PURPOSE: The delivery of transgenes into human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (hiPSC-CMs) represents an important tool in cardiac regeneration with potential for clinical applications. Gene transfection is more difficult, however, for hiPSCs and hiPSC-CMs than for somatic cells. Despite improvements in transfection and transduction, the efficiency, cytotoxicity, safety, and cost of these methods remain unsatisfactory. The objective of this study is to examine gene transfection in hiPSCs and hiPSC-CMs using magnetic nanoparticles (NPs). METHODS: Magnetic NPs are unique transfection reagents that form complexes with nucleic acids by ionic interaction. The particles, loaded with nucleic acids, can be guided by a magnetic field to allow their concentration onto the surface of the cell membrane. Subsequent uptake of the loaded particles by the cells allows for high efficiency transfection of the cells with nucleic acids. We developed a new method using magnetic NPs to transfect hiPSCs and hiPSC-CMs. HiPSCs and hiPSC-CMs were cultured and analyzed using confocal microscopy, flow cytometry, and patch clamp recordings to quantify the transfection efficiency and cellular function. RESULTS: We compared the transfection efficiency of hiPSCs with that of human embryonic kidney (HEK 293) cells. We observed that the average efficiency in hiPSCs was 43%±2% compared to 62%±4% in HEK 293 cells. Further analysis of the transfected hiPSCs showed that the differentiation of hiPSCs to hiPSC-CMs was not altered by NPs. Finally, robust transfection of hiPSC-CMs with an efficiency of 18%±2% was obtained. CONCLUSION: The difficult-to-transfect hiPSCs and hiPSC-CMs were efficiently transfected using magnetic NPs. Our study offers a novel approach for transfection of hiPSCs and hiPSC-CMs without the need for viral vector generation.

Short Telomeres Induce p53 and Autophagy and Modulate Age-Associated Changes in Cardiac Progenitor Cell Fate.

Aging severely limits myocardial repair and regeneration. Delineating the impact of age-associated factors such as short telomeres is critical to enhance the regenerative potential of cardiac progenitor cells (CPCs). We hypothesized that short telomeres activate p53 and induce autophagy to elicit the age-associated change in CPC fate. We isolated CPCs and compared mouse strains with different telomere lengths for phenotypic characteristics of aging. Wild mouse strain Mus musculus castaneus (CAST) possessing short telomeres exhibits early cardiac aging with cardiac dysfunction, hypertrophy, fibrosis, and senescence, as compared with common lab strains FVB and C57 bearing longer telomeres. CAST CPCs with short telomeres demonstrate altered cell fate as characterized by cell cycle arrest, senescence, basal commitment, and loss of quiescence. Elongation of telomeres using a modified mRNA for telomerase restores youthful properties to CAST CPCs. Short telomeres induce autophagy in CPCs, a catabolic protein degradation process, as evidenced by reduced p62 and increased accumulation of autophagic puncta. Pharmacological inhibition of autophagosome formation reverses the cell fate to a more youthful phenotype. Mechanistically, cell fate changes induced by short telomeres are partially p53 dependent, as p53 inhibition rescues senescence and commitment observed in CAST CPCs, coincident with attenuation of autophagy. In conclusion, short telomeres activate p53 and autophagy to tip the equilibrium away from quiescence and proliferation toward differentiation and senescence, leading to exhaustion of CPCs. This study provides the mechanistic basis underlying age-associated cell fate changes that will enable identification of molecular strategies to prevent senescence of CPCs. Stem Cells 2018;36:868-880.

Action Potential Shortening and Impairment of Cardiac Function by Ablation of Slc26a6.

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 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.

Electrotaxis of cardiac progenitor cells, cardiac fibroblasts, and induced pluripotent stem cell-derived cardiac progenitor cells requires serum and is directed via PI3'K pathways.

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 used to improve recovery in a number of tissues but has not been investigated for treating myocardial damage. OBJECTIVE: The purpose of this study was 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 toward the anode of a direct current electric field, whereas 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 to serum-free media restores directed migration. We provide evidence that CPC and CF electrotaxis is mediated through phosphatidylinositide 3-kinase signaling. In addition, very late antigen-4, an integrin and growth factor receptor, is required for electrotaxis and localizes to the anodal edge of CPCs in response to direct current electric field. The hiPSC-derived CPCs do not express very late antigen-4, migrate toward the cathode in a voltage-dependent manner, and, similar to CPCs and CFs, require media serum and phosphatidylinositide 3-kinase activity for electrotaxis. CONCLUSION: The electrotactic behaviors of these therapeutic cardiac cells may be used to improve cell-based therapy for recovering function in damaged myocardium.

Increased autophagy in CD4+ T cells of rheumatoid arthritis patients results in T-cell hyperactivation and apoptosis resistance.

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 CD4+ 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.

Cardiac Stem Cell Hybrids Enhance Myocardial Repair.

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 used for this study. CCs improved left ventricular anterior wall thickness at 4 weeks post injury, but only CC1 treatment preserved anterior wall thickness 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 on combinatorial cell approaches to support myocardial regeneration.

Ex vivo-expanded but not in vitro-induced human regulatory T cells are candidates for cell therapy in autoimmune diseases thanks to stable demethylation of the FOXP3 regulatory T cell-specific demethylated region.

Regulatory T cell (Treg) 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)Tregs and induced (i)Tregs 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). 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) 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 proliferation via both surface (CTLA-4) and secreted (IL-10, TGF-ß, 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. Moreover, rapa eTregs from RA patients were resistant to suppression reversal by the proinflammatory cytokine TNF-α, and were more efficient in suppressing synovial conventional T cell proliferation compared with 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.