Loss of sarcomeric proteins via upregulation of JAK/STAT signaling underlies interferon-γ-induced contractile deficit in engineered human myocardium.

The level of circulating interferon-γ (IFNγ) is elevated in various clinical conditions including autoimmune and inflammatory diseases, sepsis, acute coronary syndrome, and viral infections. As these conditions are associated with high risk of myocardial dysfunction, we investigated the effects of IFNγ on 3D fibrin-based engineered human cardiac tissues ("cardiobundles"). Cardiobundles were fabricated from human pluripotent stem cell-derived cardiomyocytes, exposed to 0-20 ng/ml of IFNγ on culture days 7-14, and assessed for changes in tissue structure, viability, contractile force and calcium transient generation, action potential propagation, cytokine secretion, and expression of select genes and proteins. We found that application of IFNγ induced a dose-dependent reduction in contractile force generation, deterioration of sarcomeric organization, and cardiomyocyte disarray, without significantly altering cell viability, action potential propagation, or calcium transient amplitude. At molecular level, the IFNγ-induced structural and functional deficits could be attributed to altered balance of pro- and anti-inflammatory cytokines, upregulation of JAK/STAT signaling pathway (JAK1, JAK2, and STAT1), and reduced expression of myosin heavy chain, myosin light chain-2v, and sarcomeric α-actinin. Application of clinically used JAK/STAT inhibitors, tofacitinib and baricitinib, fully prevented IFNγ-induced cardiomyopathy, confirming the critical roles of this signaling pathway in inflammatory cardiac disease. Taken together, our in vitro studies in engineered myocardial tissues reveal direct adverse effects of pro-inflammatory cytokine IFNγ on human cardiomyocytes and establish the foundation for a potential use of cardiobundle platform in modeling of inflammatory myocardial disease and therapy. STATEMENT OF SIGNIFICANCE: Various inflammatory and autoimmune diseases including rheumatoid arthritis, sepsis, lupus erythematosus, Chagas disease, and others, as well as viral infections including H1N1 influenza and COVID-19 show increased systemic levels of a pro-inflammatory cytokine interferon-γ (IFNγ) and are associated with high risk of heart disease. Here we explored for the first time if chronically elevated levels of IFNγ can negatively affect structure and function of engineered human heart tissues in vitro. Our studies revealed IFNγ-induced deterioration of myofibrillar organization and contractile force production in human cardiomyocytes, attributed to decreased expression of multiple sarcomeric proteins and upregulation of JAK/STAT signaling pathway. FDA-approved JAK inhibitors fully blocked the adverse effects of IFNγ, suggesting a potentially effective strategy against human inflammatory cardiomyopathy.

Exercise mimetics and JAK inhibition attenuate IFN-γ-induced wasting in engineered human skeletal muscle.

Chronic inflammatory diseases often lead to muscle wasting and contractile deficit. While exercise can have anti-inflammatory effects, the underlying mechanisms remain unclear. Here, we used an in vitro tissue-engineered model of human skeletal muscle ("myobundle") to study effects of exercise-mimetic electrical stimulation (E-stim) on interferon-γ (IFN-γ)-induced muscle weakness. Chronic IFN-γ treatment of myobundles derived from multiple donors induced myofiber atrophy and contractile loss. E-stim altered the myobundle secretome, induced myofiber hypertrophy, and attenuated the IFN-γ-induced myobundle wasting and weakness, in part by down-regulating JAK (Janus kinase)/STAT1 (signal transducer and activator of transcription 1) signaling pathway amplified by IFN-γ. JAK/STAT inhibitors fully prevented IFN-γ-induced myopathy, confirming the critical roles of STAT1 activation in proinflammatory action of IFN-γ. Our results reveal a previously unknown mechanism of the cell-autonomous anti-inflammatory effects of muscle exercise and establish the utility of human myobundle platform for studies of inflammatory muscle disease and therapy.

Engineered skeletal muscles for disease modeling and drug discovery.

Skeletal muscle is the largest organ of human body with several important roles in everyday movement and metabolic homeostasis. The limited ability of small animal models of muscle disease to accurately predict drug efficacy and toxicity in humans has prompted the development in vitro models of human skeletal muscle that fatefully recapitulate cell and tissue level functions and drug responses. We first review methods for development of three-dimensional engineered muscle tissues and organ-on-a-chip microphysiological systems and discuss their potential utility in drug discovery research and development of new regenerative therapies. Furthermore, we describe strategies to increase the functional maturation of engineered muscle, and motivate the importance of incorporating multiple tissue types on the same chip to model organ cross-talk and generate more predictive drug development platforms. Finally, we review the ability of available in vitro systems to model diseases such as type II diabetes, Duchenne muscular dystrophy, Pompe disease, and dysferlinopathy.

Engineering human pluripotent stem cells into a functional skeletal muscle tissue.

The generation of functional skeletal muscle tissues from human pluripotent stem cells (hPSCs) has not been reported. Here, we derive induced myogenic progenitor cells (iMPCs) via transient overexpression of Pax7 in paraxial mesoderm cells differentiated from hPSCs. In 2D culture, iMPCs readily differentiate into spontaneously contracting multinucleated myotubes and a pool of satellite-like cells endogenously expressing Pax7. Under optimized 3D culture conditions, iMPCs derived from multiple hPSC lines reproducibly form functional skeletal muscle tissues (iSKM bundles) containing aligned multi-nucleated myotubes that exhibit positive force-frequency relationship and robust calcium transients in response to electrical or acetylcholine stimulation. During 1-month culture, the iSKM bundles undergo increased structural and molecular maturation, hypertrophy, and force generation. When implanted into dorsal window chamber or hindlimb muscle in immunocompromised mice, the iSKM bundles survive, progressively vascularize, and maintain functionality. iSKM bundles hold promise as a microphysiological platform for human muscle disease modeling and drug development.

p53 isoform Δ133p53 promotes efficiency of induced pluripotent stem cells and ensures genomic integrity during reprogramming.

Human induced pluripotent stem (iPS) cells have great potential in regenerative medicine, but this depends on the integrity of their genomes. iPS cells have been found to contain a large number of de novo genetic alterations due to DNA damage response during reprogramming. Thus, to maintain the genetic stability of iPS cells is an important goal in iPS cell technology. DNA damage response can trigger tumor suppressor p53 activation, which ensures genome integrity of reprogramming cells by inducing apoptosis and senescence. p53 isoform Δ133p53 is a p53 target gene and functions to not only antagonize p53 mediated apoptosis, but also promote DNA double-strand break (DSB) repair. Here we report that Δ133p53 is induced in reprogramming. Knockdown of Δ133p53 results 2-fold decrease in reprogramming efficiency, 4-fold increase in chromosomal aberrations, whereas overexpression of Δ133p53 with 4 Yamanaka factors showes 4-fold increase in reprogamming efficiency and 2-fold decrease in chromosomal aberrations, compared to those in iPS cells induced only with 4 Yamanaka factors. Overexpression of Δ133p53 can inhibit cell apoptosis and promote DNA DSB repair foci formation during reprogramming. Our finding demonstrates that the overexpression of Δ133p53 not only enhances reprogramming efficiency, but also results better genetic quality in iPS cells.

Generating hESCs with reduced immunogenicity by disrupting TAP1 or TAPBP.

Human embryonic stem cells (hESCs) are thought to be a promising resource for cell therapy, while it has to face the major problem of graft immunological rejection. Major histocompatibility complex (MHC) class I expressed on the cell surface is the major cause of graft rejection. Transporter associated with antigen presentation 1 (TAP1) and TAP-associated glycoprotein (TAPBP) play important roles in regulating MHC class I expression. In this study, we generated TAP1- and TAPBP-deficient hESC lines, respectively, using transcription activator-like effector nucleases technique. These cells showed deficient expression of MHC class I on the cell surface and reduced immunogenicity compared with wild types, but maintained normal pluripotency, karyotypes, and differentiation ability. Thus, our findings are instrumental in developing a universal cell resource with both pluripotency and hypo-immunogenicity for transplantation therapy in the future.

Generating hypoimmunogenic human embryonic stem cells by the disruption of beta 2-microglobulin.

Immune rejection hinders the application of human embryonic stem cells (hESCs) in transplantation therapy. Human leukocyte antigens (HLAs) on the cell surface are the major cause of graft rejection. In this study, we generated HLA class I-deficient hESCs via disruption of beta 2-microglobulin (ß2m), the light chain of HLA Class I. We found that HLA class I proteins were not present on the cell surface of ß2m-null hESCs. These cells showed the same pluripotency as wildtype hESCs and demonstrated hypoimmunogenicity. Thus, HLA class I-deficient hESCs might serve as an unlimited cell source for the generation of universally compatible "off-the-shelf" cell grafts, tissues or organs in the future.

Highly efficient derivation of skeletal myotubes from human embryonic stem cells.

Human embryonic stem cells (hESCs) are a promising model for the research of embryonic development and regenerative medicine. Since the first hESC line was established, many researchers have shown that pluripotent hESCs can be directed into many types of functional adult cells in culture. However, most of the reported methods have induced differentiation through the alteration of growth factors in the culture medium. These methods are time consuming; moreover, it is difficult to obtain a pure population of the desired cells because of the low efficiency of induction. In this study, we used a lentiviral-based inducible gene-expression system in hESCs to control the ectopic expression of MyoD, which is an essential transcription factor in skeletal muscle development. The induction of MyoD can efficiently direct the pluripotent hESCs into mesoderm in 24 h. The cells then become proliferated myoblasts and finally form multinucleated myotubes in vitro. The whole procedure took about 10 days, with an induction efficiency of over 90%. To our knowledge, this is the first time that hESCs have been induced into terminally differentiated cells with only one factor. In the future, these results could be a potential resource for cell therapy for diseases of muscle dysfunction.

Derivation and characterization of human embryonic stem cell lines from the Chinese population.

Human embryonic stem cells (hESCs) can self-renew indefinitely and differentiate into all cell types in the human body. Therefore, they are valuable in regenerative medicine, human developmental biology and drug discovery. A number of hESC lines have been derived from the Chinese population, but limited of them are available for research purposes. Here we report the derivation and characterization of two hESC lines derived from human blastocysts of Chinese origin. These hESCs express alkaline phosphatase and hESC-specific markers, including Oct4, Nanog, SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81. They also have high levels of telomerase activity and normal karyotypes. These cells can form embryoid body in vitro and can be differentiated into all three germ layers in vivo by teratoma formation. The newly established hESCs will be distributed for research purposes. The availability of hESC lines from the Chinese population will facilitate studies on the differences in hESCs from different ethnic groups.

Reprogramming of ovine adult fibroblasts to pluripotency via drug-inducible expression of defined factors.

Reprogramming of somatic cells in the enucleated egg made Dolly, the sheep, the first successfully cloned mammal in 1996. However, the mechanism of sheep somatic cell reprogramming has not yet been addressed. Moreover, sheep embryonic stem (ES) cells are still not available, which limits the generation of precise gene-modified sheep. In this study, we report that sheep somatic cells can be directly reprogrammed to induced pluripotent stem (iPS) cells using defined factors (Oct4, Sox2, c-Myc, Klf4, Nanog, Lin28, SV40 large T and hTERT). Our observations indicated that somatic cells from sheep are more difficult to reprogram than somatic cells from other species, in which iPS cells have been reported. We demonstrated that sheep iPS cells express ES cell markers, including alkaline phosphatase, Oct4, Nanog, Sox2, Rex1, stage-specific embryonic antigen-1, TRA-1-60, TRA-1-81 and E-cadherin. Sheep iPS cells exhibited normal karyotypes and were able to differentiate into all three germ layers both in vitro and in teratomas. Our study may help to reveal the mechanism of somatic cell reprogramming in sheep and provide a platform to explore the culture conditions for sheep ES cells. Moreover, sheep iPS cells may be directly used to generate precise gene-modified sheep.