Advanced Technologies to Target Cardiac Cell Fate Plasticity for Heart Regeneration

The adult human heart can only adapt to heart diseases by starting a myocardial remodeling process to compensate for the loss of functional cardiomyocytes, which ultimately develop into heart failure. In recent decades, the evolution of new strategies to regenerate the injured myocardium based on cellular reprogramming represents a revolutionary new paradigm for cardiac repair by targeting some key signaling molecules governing cardiac cell fate plasticity. While the indirect reprogramming routes require an in vitro engineered 3D tissue to be transplanted in vivo, the direct cardiac reprogramming would allow the administration of reprogramming factors directly in situ, thus holding great potential as in vivo treatment for clinical applications. In this framework, cellular reprogramming in partnership with nanotechnologies and bioengineering will offer new perspectives in the field of cardiovascular research for disease modeling, drug screening, and tissue engineering applications. In this review, we will summarize the recent progress in developing innovative therapeutic strategies based on manipulating cardiac cell fate plasticity in combination with bioengineering and nanotechnology-based approaches for targeting the failing heart.

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Constructing stem cell microenvironments using bioengineering approaches

Physiological Genomics ◽

10.1152/physiolgenomics.00099.2013 ◽

2013 ◽

Vol 45 (23) ◽

pp. 1123-1135 ◽

Cited By ~ 29

Author(s):

David A. Brafman

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Cell Fate ◽

Disease Modeling ◽

Mechanical Stimuli ◽

Culture Methods ◽

Stem Cell Fate ◽

And Function

Within the adult organism, stem cells reside in defined anatomical microenvironments called niches. These architecturally diverse microenvironments serve to balance stem cell self-renewal and differentiation. Proper regulation of this balance is instrumental to tissue repair and homeostasis, and any imbalance can potentially lead to diseases such as cancer. Within each of these microenvironments, a myriad of chemical and physical stimuli interact in a complex (synergistic or antagonistic) manner to tightly regulate stem cell fate. The in vitro replication of these in vivo microenvironments will be necessary for the application of stem cells for disease modeling, drug discovery, and regenerative medicine purposes. However, traditional reductionist approaches have only led to the generation of cell culture methods that poorly recapitulate the in vivo microenvironment. To that end, novel engineering and systems biology approaches have allowed for the investigation of the biological and mechanical stimuli that govern stem cell fate. In this review, the application of these technologies for the dissection of stem cell microenvironments will be analyzed. Moreover, the use of these engineering approaches to construct in vitro stem cell microenvironments that precisely control stem cell fate and function will be reviewed. Finally, the emerging trend of using high-throughput, combinatorial methods for the stepwise engineering of stem cell microenvironments will be explored.

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Yorkie and JNK revert syncytial muscles into myoblasts during Org-1 dependent lineage reprogramming

10.1101/607820 ◽

2019 ◽

Author(s):

Christoph Schaub ◽

Marcel Rose ◽

Manfred Frasch

Keyword(s):

Cell Fate ◽

Present Report ◽

Cellular Reprogramming ◽

List Type ◽

T Box ◽

Lineage Reprogramming ◽

Kinase Cascade ◽

Alary Muscles

SummaryLineage reprogramming has become a prominent focus in research since it was demonstrated that lineage restricted transcription factors can be used in vitro for direct reprogramming [1]. Recently, we reported that the ventral longitudinal musculature (VLM) of the adult Drosophila heart arises in vivo by direct lineage reprogramming from alary muscles (AM), a process which starts with dedifferentiation and fragmentation of syncytial alary muscles into mononucleate myoblasts. Central upstream activators of the genetic program regulating the development of VLMs from alary muscles are the T-box factor Org-1 (Drosophila Tbx1) and the LIM homeodomain factor Tup (Drosophila Islet1) [2]. However, the events downstream of Org-1 and Tup that exert dedifferentiation and fragmentation of alary muscles have been unknown. In the present report, we shed light on the initiation of this first step of transdifferentiation and show that AM lineage specific activation of Yorkie (Yki), the transcriptional co-activator of the transcription factor Scalloped (Sd), has a key role in initiating AM lineage reprogramming. An additional necessary input comes from active dJNK signaling, which contributes to the inactivation of the Hippo kinase cascade upstream of Yki and furthermore activates dJun. The synergistic activities of the Yki/Sd and dJun/dFos (AP-1) transcriptional activator complexes in the absence of Hippo activity initiate AM dedifferentiation and lead to the expression of Myc and piwi, which are crucial for different aspects of AM transdifferentiation. Our results provide new insights into the mechanisms that mediate muscle lineage plasticity during a cellular reprogramming process occurring in vivo.HighlightsDirect lineage reprogramming of alary muscles depends on Yorkie and JNKYorkie and JNK mediate reversal of syncytial muscle cell fateYki/Sd and AP-1 induce alary muscle dedifferentiation synergisticallyYki dependent Myc induces and Piwi mediates reprogramming of alary muscles

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Induced Neurons for Disease Modeling and Repair: A Focus on Non-fibroblastic Cell Sources in Direct Reprogramming

Frontiers in Bioengineering and Biotechnology ◽

10.3389/fbioe.2021.658498 ◽

2021 ◽

Vol 9 ◽

Author(s):

Kathryn M. Kim ◽

Mentor Thaqi ◽

Daniel A. Peterson ◽

Robert A. Marr

Keyword(s):

Disease Modeling ◽

Cellular Reprogramming ◽

Cerebral Injury ◽

Direct Reprogramming ◽

Fibroblastic Cell ◽

Donor Cells ◽

Cell Sources ◽

Induced Neurons

Direct cellular reprogramming exhibits distinct advantages over reprogramming from an induced pluripotent stem cell intermediate. These include a reduced risk of tumorigenesis and the likely preservation of epigenetic data. In vitro direct reprogramming approaches primarily aim to model the pathophysiological development of neurological disease and identify therapeutic targets, while in vivo direct reprogramming aims to develop treatments for various neurological disorders, including cerebral injury and cancer. In both approaches, there is progress toward developing increased control of subtype-specific production of induced neurons. A majority of research primarily utilizes fibroblasts as the donor cells. However, there are a variety of other somatic cell types that have demonstrated the potential for reprogramming into induced neurons. This review highlights studies that utilize non-fibroblastic cell sources for reprogramming, such as astrocytes, olfactory ensheathing cells, peripheral blood cells, Müller glia, and more. We will examine benefits and obstructions for translation into therapeutics or disease modeling, as well as efficiency of the conversion. A summary of donor cells, induced neuron types, and methods of induction is also provided.

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De Novo Human Cardiac Myocytes for Medical Research: Promises and Challenges

Stem Cells International ◽

10.1155/2017/4528941 ◽

2017 ◽

Vol 2017 ◽

pp. 1-7 ◽

Cited By ~ 9

Author(s):

Veronique Hamel ◽

Kang Cheng ◽

Shudan Liao ◽

Aizhu Lu ◽

Yong Zheng ◽

...

Keyword(s):

Stem Cells ◽

Cardiac Myocytes ◽

Disease Modeling ◽

De Novo ◽

Somatic Cells ◽

Embryonic Stem ◽

Cellular Reprogramming ◽

Great Promise

The advent of cellular reprogramming technology has revolutionized biomedical research. De novo human cardiac myocytes can now be obtained from direct reprogramming of somatic cells (such as fibroblasts), from induced pluripotent stem cells (iPSCs, which are reprogrammed from somatic cells), and from human embryonic stem cells (hESCs). Such de novo human cardiac myocytes hold great promise for in vitro disease modeling and drug screening and in vivo cell therapy of heart disease. Here, we review the technique advancements for generating de novo human cardiac myocytes. We also discuss several challenges for the use of such cells in research and regenerative medicine, such as the immature phenotype and heterogeneity of de novo cardiac myocytes obtained with existing protocols. We focus on the recent advancements in addressing such challenges.

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Role of SHH in Patterning Human Pluripotent Cells towards Ventral Forebrain Fates

Cells ◽

10.3390/cells10040914 ◽

2021 ◽

Vol 10 (4) ◽

pp. 914

Author(s):

Melanie V. Brady ◽

Flora M. Vaccarino

Keyword(s):

Stem Cell ◽

Human Brain ◽

Human Development ◽

Disease Modeling ◽

Model Systems ◽

Advantages And Disadvantages ◽

Technical Ability ◽

Cellular Phenotypes

The complexities of human neurodevelopment have historically been challenging to decipher but continue to be of great interest in the contexts of healthy neurobiology and disease. The classic animal models and monolayer in vitro systems have limited the types of questions scientists can strive to answer in addition to the technical ability to answer them. However, the tridimensional human stem cell-derived organoid system provides the unique opportunity to model human development and mimic the diverse cellular composition of human organs. This strategy is adaptable and malleable, and these neural organoids possess the morphogenic sensitivity to be patterned in various ways to generate the different regions of the human brain. Furthermore, recapitulating human development provides a platform for disease modeling. One master regulator of human neurodevelopment in many regions of the human brain is sonic hedgehog (SHH), whose expression gradient and pathway activation are responsible for conferring ventral identity and shaping cellular phenotypes throughout the neural axis. This review first discusses the benefits, challenges, and limitations of using organoids for studying human neurodevelopment and disease, comparing advantages and disadvantages with other in vivo and in vitro model systems. Next, we explore the range of control that SHH exhibits on human neurodevelopment, and the application of SHH to various stem cell methodologies, including organoids, to expand our understanding of human development and disease. We outline how this strategy will eventually bring us much closer to uncovering the intricacies of human neurodevelopment and biology.

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Up-Regulating Relaxin Expression by G-Quadruplex Interactive Ligand to Achieve Antifibrotic Action

Endocrinology ◽

10.1210/en.2012-1114 ◽

2012 ◽

Vol 153 (8) ◽

pp. 3692-3700 ◽

Cited By ~ 28

Author(s):

Hui-Ping Gu ◽

Sen Lin ◽

Ming Xu ◽

Hai-Yi Yu ◽

Xiao-Jun Du ◽

...

Keyword(s):

Rna Polymerase Ii ◽

Cardiac Fibrosis ◽

Cardiac Fibroblasts ◽

Heart Diseases ◽

Gene Promoter ◽

Binding Motif ◽

Endogenous Expression ◽

G Quadruplex

Myocardial fibrosis is a key pathological change in a variety of heart diseases contributing to the development of heart failure, arrhythmias, and sudden death. Recent studies have shown that relaxin prevents and reverses cardiac fibrosis. Endogenous expression of relaxin was elevated in the setting of heart disease; the extent of such up-regulation, however, is insufficient to exert compensatory actions, and the mechanism regulating relaxin expression is poorly defined. In the rat relaxin-1 (RLN1, Chr1) gene promoter region we found presence of repeated guanine (G)-rich sequences, which allowed formation and stabilization of G-quadruplexes with the addition of a G-quadruplex interactive ligand berberine. The G-rich sequences and the G-quadruplexes were localized adjacent to the binding motif of signal transducer and activator of transcription (STAT)3, which negatively regulates relaxin expression. Thus, we hypothesized that the formation and stabilization of G-quadruplexes by berberine could influence relaxin expression. We found that berberine-induced formation of G-quadruplexes did increase relaxin gene expression measured at mRNA and protein levels. Formation of G-quadruplexes significantly reduced STAT3 binding to the promoter of relaxin gene. This was associated with consequent increase in the binding of RNA polymerase II and STAT5a to relaxin gene promoter. In cardiac fibroblasts and rats treated with angiotensin II, berberine was found to suppress fibroblast activation, collagen synthesis, and extent of cardiac fibrosis through up-regulating relaxin. The antifibrotic action of berberine in vitro and in vivo was similar to that by exogenous relaxin. Our findings document a novel therapeutic strategy for fibrosis through up-regulating expression of endogenous relaxin.

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Abstract 18845: Induced Cardiac Progenitor Cells Differentiate Into Cardiac Lineage Cells in vivo and Improve Survival in Mice post Myocardial Infarction

Circulation ◽

10.1161/circ.132.suppl_3.18845 ◽

2015 ◽

Vol 132 (suppl_3) ◽

Author(s):

Pratik A Lalit ◽

Max R Salick ◽

Daryl O Nelson ◽

Jayne M Squirrell ◽

Christina M Shafer ◽

...

Keyword(s):

Progenitor Cells ◽

Disease Modeling ◽

Ex Vivo ◽

Cardiac Progenitor Cells ◽

Heart Tube ◽

Whole Embryo Culture ◽

Cardiac Actin ◽

Cardiac Lineage

Several studies have reported reprogramming of fibroblasts (Fibs) to induced cardiomyocytes, and we have recently reprogrammed mouse Fibs to induced cardiac progenitor cells (iCPCs), which may be more favorable for cardiac repair because of their expandability and multipotency. Adult cardiac (AC), lung and tail-tip Fibs from an Nkx2.5-EYFP reporter mouse were reprogrammed using a combination of five defined factors into iCPCs. Transcriptome and immunocytochemistry analysis revealed that iCPCs were cardiac mesoderm-restricted progenitors that expressed CPC markers including Nkx2.5, Gata4, Irx4, Tbx5, Cxcr4, Flk1 etc. iCPCs could be extensively expanded (over 30 passages) while maintaining multipotency to differentiate in vitro into cardiac lineage cells including cardiomyocytes (CMs), smooth muscle cells and endothelial cells. iCPC derived CMs upon co-culture with mESC-derived CMs formed intercellular gap junctions, exhibited calcium transients, and contractions. The purpose of this study was to determine the in vivo potency of iCPCs. Given that the Nkx2.5-EYFP reporter identifies embryonic CPCs, we first tested the embryonic potency of iCPCs using an ex vivo whole embryo culture model injecting cells into the cardiac crescent (CC) of E8.5 mouse embryos and culturing for 24 to 48 hours. GFP labeled AC Fibs were first tested and live imaging revealed that after 24 hours these cells were rejected from the embryo proper and localized to the ecto-placental cone. In contrast, iCPCs reprogrammed from AC Fibs when injected into the CC localized to the developing heart tube and differentiated into MLC2v, αMHC and cardiac actin expressing CMs. Further we injected iCPCs into infarcted adult mouse hearts and determined their regenerative potential after 1-4 wks. The iCPCs significantly improved survival (p<0.01 Mantel-Cox test) in treated animals (75%) as compared to control (11%). Immunohistochemistry revealed that injected iCPCs localized to the scar area and differentiated into cardiac lineage cells including CMs (cardiac actin). These results indicate that lineage reprogramming of adult somatic cells into iCPCs provides a scalable cell source for cardiac regenerative therapy as well as drug discovery and disease modeling.

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Abstract 518: In Vitro and in vivo Disease Modeling Using Patient Derived iPSCs to Characterize the Calcification Phenotype in Arterial Calcification Due to Deficiency of CD73

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.36.suppl_1.518 ◽

2016 ◽

Vol 36 (suppl_1) ◽

Author(s):

Cynthia St. Hilaire ◽

Hui Jin ◽

Yuting Huang ◽

Dan Yang ◽

Alejandra Negro ◽

...

Keyword(s):

Vascular Calcification ◽

Disease Modeling ◽

Induced Pluripotent Stem Cell ◽

Dermal Fibroblasts ◽

In Vivo Studies ◽

Arterial Calcification ◽

Patient Specific ◽

And Control

Objective: The objective of this study was to develop a patient-specific induced pluripotent stem cell (iPSC)-based disease model to understand the process by which CD73-deficiency leads to vascular calcification in the disease, Arterial Calcification due to Deficiency of CD73 (ACDC). Approach & Results: ACDC is an autosomal recessive disease resulting from mutations in the gene encoding for CD73, which converts extracellular AMP to adenosine. CD73-deficiency manifests with tortuosity and vascular calcification of the medial layer of lower-extremity arteries, a pathology associated with diabetes and chronic kidney disease. We previously identified that dermal fibroblasts isolated from ACDC patients calcify in vitro, however in vivo studies of the vasculature are limited, as murine models of CD73 deficiency do not recapitulate the human disease phenotype. Thus, we created iPSCs from ACDC patients and control fibroblasts. ACDC and Control iPSCs form teratomas when injected in immune-compromised mice, however ACDC iPSC teratomas exhibit extensive calcifications. Control and ACDC iPSCs were differentiated down the mesenchymal lineage (MSC) and while there was no difference in chondrogenesis and adipogenesis, ACDC iMSCs underwent osteogenesis sooner than control iPSC, have higher activity of tissue-nonspecific alkaline phosphatase (TNAP), and lower levels of extracellular adenosine. During osteogenic simulation, TNAP activity in ACDC cells significantly increased adenosine levels, however, not to levels needed for functional compensatory stimulation of the adenosine receptors. Inhibition of TNAP with levimisole ablates this increase in adenosine. Treatment with an A2b adenosine receptor (AR) agonist drastically reduced TNAP activity in vitro, and calcification in ACDC teratomas, as did treatment with etidronate, which is currently being tested in a clinical trial on ACDC patients. Conclusions: These results illustrate a pro-osteogenic phenotype in CD73-deficient cells whereby TNAP activity attempts to compensate for CD73 deficiency, but subsequently induces calcification that can be reversed by activation of the A2bAR. The iPSC teratoma model may be used to screen other potential therapeutics for calcification disorders.

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The Drosophila extramacrochaetae protein antagonizes sequence-specific DNA binding by daughterless/achaete-scute protein complexes

Development ◽

10.1242/dev.113.1.245 ◽

1991 ◽

Vol 113 (1) ◽

pp. 245-255 ◽

Cited By ~ 32

Author(s):

M. Van Doren ◽

H.M. Ellis ◽

J.W. Posakony

Keyword(s):

Dna Binding ◽

Cell Fate ◽

Protein Complexes ◽

Competitive Inhibitor ◽

Binding Activity ◽

Regulatory Function ◽

Biochemical Basis ◽

Dna Binding Activity

In Drosophila, a group of regulatory proteins of the helix-loop-helix (HLH) class play an essential role in conferring upon cells in the developing adult epidermis the competence to give rise to sensory organs. Proteins encoded by the daughterless (da) gene and three genes of the achaete-scute complex (AS-C) act positively in the determination of the sensory organ precursor cell fate, while the extramacrochaetae (emc) and hairy (h) gene products act as negative regulators. In the region upstream of the achaete gene of the AS-C, we have identified three ‘E box’ consensus sequences that are bound specifically in vitro by hetero-oligomeric complexes consisting of the da protein and an AS-C protein. We have used this DNA-binding activity to investigate the biochemical basis of the negative regulatory function of emc. Under the conditions of our experiments, the emc protein, but not the h protein, is able to antagonize specifically the in vitro DNA-binding activity of da/AS-C and putative da/da protein complexes. We interpret these results as follows: the heterodimerization capacity of the emc protein (conferred by its HLH domain) allows it to act in vivo as a competitive inhibitor of the formation of functional DNA-binding protein complexes by the da and AS-C proteins, thereby reducing the effective level of their transcriptional regulatory activity within the cell.

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MicroRNAs in Valvular Heart Diseases: Biological Regulators, Prognostic Markers and Therapeutical Targets

International Journal of Molecular Sciences ◽

10.3390/ijms222212132 ◽

2021 ◽

Vol 22 (22) ◽

pp. 12132

Author(s):

Francesco Nappi ◽

Adelaide Iervolino ◽

Sanjeet Singh Avtaar Singh ◽

Massimo Chello

Keyword(s):

Mitral Valve ◽

Mitral Valve Prolapse ◽

Heart Diseases ◽

Osteoblastic Differentiation ◽

New Drugs ◽

Expression Vectors ◽

Chordae Tendineae ◽

Valvular Heart Diseases

miRNAs have recently attracted investigators’ interest as regulators of valvular diseases pathogenesis, diagnostic biomarkers, and therapeutical targets. Evidence from in-vivo and in-vitro studies demonstrated stimulatory or inhibitory roles in mitral valve prolapse development, aortic leaflet fusion, and calcification pathways, specifically osteoblastic differentiation and transcription factors modulation. Tissue expression assessment and comparison between physiological and pathological phenotypes of different disease entities, including mitral valve prolapse and mitral chordae tendineae rupture, emerged as the best strategies to address miRNAs over or under-representation and thus, their impact on pathogeneses. In this review, we discuss the fundamental intra- and intercellular signals regulated by miRNAs leading to defects in mitral and aortic valves, congenital heart diseases, and the possible therapeutic strategies targeting them. These miRNAs inhibitors are comprised of antisense oligonucleotides and sponge vectors. The miRNA mimics, miRNA expression vectors, and small molecules are instead possible practical strategies to increase specific miRNA activity. Advantages and technical limitations of these new drugs, including instability and complex pharmacokinetics, are also presented. Novel delivery strategies, such as nanoparticles and liposomes, are described to improve knowledge on future personalized treatment directions.

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