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

PurposeThe 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).MethodsMagnetic 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.ResultsWe 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.ConclusionThe 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

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 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

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

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 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

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

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-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. 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

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

BackgroundThe 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.ObjectiveThe purpose of this study was to test the electrotactic behaviors of several types of cardiac cells.MethodsCardiac progenitor cells (CPCs), cardiac fibroblasts (CFs), and human induced pluripotent stem cell-derived cardiac progenitor cells (hiPSC-CPCs) were used.ResultsCPCs 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.ConclusionThe electrotactic behaviors of these therapeutic cardiac cells may be used to improve cell-based therapy for recovering function in damaged myocardium

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