Direct Cardiac Reprogramming: Current Status and Future Prospects.

Advances in cellular reprogramming articulated the path for direct cardiac lineage conversion, bypassing the pluripotent state. Direct cardiac reprogramming attracts major attention because of the low or nil regenerative ability of cardiomyocytes, resulting in permanent cell loss in various heart diseases. In the field of cardiology, balancing this loss of cardiomyocytes was highly challenging, even in the modern medical world. Soon after the discovery of cell reprogramming, direct cardiac reprogramming also became a promising alternative for heart regeneration. This review mainly focused on the various direct cardiac reprogramming approaches (integrative and non-integrative) for the derivation of induced autologous cardiomyocytes. It also explains the advancements in cardiac reprogramming over the decade with the pros and cons of each approach. Further, the review highlights the importance of clinically relevant (non-integrative) approaches and their challenges for the prospective applications for personalized medicine. Apart from direct cardiac reprogramming, it also discusses the other strategies for generating cardiomyocytes from different sources. The understanding of these strategies could pave the way for the efficient generation of integration-free functional autologous cardiomyocytes through direct cardiac reprogramming for various biomedical applications.

Generation of a recombinant version of a biologically active cell-permeant human HAND2 transcription factor from E. coli.

Transcription factor HAND2 has a significant role in vascularization, angiogenesis, and cardiac neural crest development. It is one of the key cardiac factors crucial for the enhanced derivation of functional and mature myocytes from non-myocyte cells. Here, we report the generation of the recombinant human HAND2 fusion protein from the heterologous system. First, we cloned the full-length human HAND2 gene (only protein-coding sequence) after codon optimization along with the fusion tags (for cell penetration, nuclear translocation, and affinity purification) into the expression vector. We then transformed and expressed it in Escherichia coli strain, BL21(DE3). Next, the effect (in terms of expression) of tagging fusion tags with this recombinant protein at two different terminals was also investigated. Using affinity chromatography, we established the one-step homogeneous purification of recombinant human HAND2 fusion protein; and through circular dichroism spectroscopy, we established that this purified protein had retained its secondary structure. We then showed that this purified human protein could transduce the human cells and translocate to its nucleus. The generated recombinant HAND2 fusion protein showed angiogenic potential in the ex vivo chicken embryo model. Following transduction in MEF2C overexpressing cardiomyoblast cells, this purified recombinant protein synergistically activated the α-MHC promoter and induced GFP expression in the α-MHC-eGFP reporter assay. Prospectively, the purified bioactive recombinant HAND2 protein can potentially be a safe and effective molecular tool in the direct cardiac reprogramming process and other biological applications.

Soluble expression, purification, and secondary structure determination of human MESP1 transcription factor.

Transcription factor MESP1 is a crucial factor regulating cardiac, hematopoietic, and skeletal myogenic development. Besides, it also contributes to the generation of functional cardiomyocytes. Here, we report the soluble expression and purification of the full-length human MESP1 protein from the heterologous system, which can be delivered into the target mammalian cells. To generate this biological macromolecule, we cloned its codon-optimized gene sequence fused to a nuclear localization sequence, a cell-penetrating peptide, and a His-tag into the protein expression vector and expressed in the bacterial system (E. coli strain BL21(DE3)). Subsequently, we have screened and identified the optimal expression parameters to obtain this recombinant fusion protein in soluble form from E. coli and examined its expression concerning the placement of fusion tags at either terminal. Further, we have purified this recombinant fusion protein to homogeneity under native conditions. Notably, this purified fusion protein has maintained its secondary structure after purification, primarily comprising α-helices and random coils. This molecular tool can potentially replace its genetic and viral forms in the cardiac reprogramming of fibroblasts to induce a cardiac transcriptional profile in an integration-free manner and elucidating its role in various biological processes and diseases. KEY POINTS: • Screening of the suitable gene construct was performed and identified. • Screening of optimal expression conditions was performed and identified. • Native purification of recombinant human MESP1 protein from E. coli was performed. • Recombinant MESP1 protein has retained its secondary structure after purification.

Generation of cell-permeant recombinant human transcription factor GATA4 from E. coli.

Transcription factor GATA4 is expressed during early embryogenesis and is vital for proper development. In addition, it is a crucial reprogramming factor for deriving functional cardiomyocytes and was recently identified as a tumor suppressor protein in various cancers. To generate a safe and effective molecular tool that can potentially be used in a cell reprogramming process and as an anti-cancer agent, we have identified optimal expression parameters to obtain soluble expression of human GATA4 in E. coli and purified the same to homogeneity under native conditions using immobilized metal ion affinity chromatography. The identity of GATA4 protein was confirmed using western blotting and mass spectrometry. Using circular dichroism spectroscopy, it was demonstrated that the purified recombinant protein has maintained its secondary structure, primarily comprising of random coils and α-helices. Subsequently, this purified recombinant protein was applied to human cells and was found that it was non-toxic and able to enter the cells as well as translocate to the nucleus. Prospectively, this cell- and nuclear-permeant molecular tool is suitable for cell reprogramming experiments and can be a safe and effective therapeutic agent for cancer therapy.

Codon Optimization, Cloning, Expression, Purification, and Secondary Structure Determination of Human ETS2 Transcription Factor.

Transcription factor ETS2 regulates genes involved in development, differentiation, angiogenesis, proliferation, and apoptosis. In addition, it is one of the core reprogramming factors responsible for the generation of human cardiomyocytes from adult somatic cells. In this study, we report the heterologous expression of human ETS2 in E. coli to produce a highly pure recombinant protein. To accomplish this, the codon-optimized 1507 bp coding sequence of the human ETS2 gene in fusion with a His-tag, a cell-penetrating peptide, and a nuclear localization sequence was cloned in the protein expression vector and transformed into E. coli strain BL21(DE3) for expression. The recombinant protein was purified to homogeneity under native conditions using immobilized metal ion affinity chromatography, and its identity was confirmed by Western blotting with an ETS2 antibody. Using far-UV circular dichroism spectroscopy, we have demonstrated that the recombinant protein has retained its secondary structure, predominantly comprising of random coils and ß-sheets. Prospectively, this biological recombinant ETS2 protein can substitute viral and genetic forms of ETS2 in a cell reprogramming process to facilitate the generation of clinical-grade cells. It can also be used to investigate its molecular role in various biological processes and diseases and for biochemical and structural studies.

An Insight into Reprogramming Barriers to iPSC Generation.

Derivation of induced Pluripotent Stem Cells (iPSCs) by reprogramming somatic cells to a pluripotent state has revolutionized stem cell research. Ensuing this, various groups have used genetic and non-genetic approaches to generate iPSCs from numerous cell types. However, achieving a pluripotent state in most of the reprogramming studies is marred by serious limitations such as low reprogramming efficiency and slow kinetics. These limitations are mainly due to the presence of potent barriers that exist during reprogramming when a mature cell is coaxed to achieve a pluripotent state. Several studies have revealed that intrinsic factors such as non-optimal stoichiometry of reprogramming factors, specific signaling pathways, cellular senescence, pluripotency-inhibiting transcription factors and microRNAs act as a roadblock. In addition, the epigenetic state of somatic cells and specific epigenetic modifications that occur during reprogramming also remarkably impede the generation of iPSCs. In this review, we present a comprehensive overview of the barriers that inhibit reprogramming and the understanding of which will pave the way to develop safe strategies for efficient reprogramming.

An insight into non-integrative gene delivery approaches to generate transgene-free induced pluripotent stem cells.

Over a decade ago, a landmark study that reported derivation of induced Pluripotent Stem Cells (iPSCs) by reprogramming fibroblasts has transformed stem cell research attracting the interest of the scientific community worldwide. These cells circumvent the ethical and immunological concerns associated with embryonic stem cells, and the limited self-renewal ability and restricted differentiation potential linked to adult stem cells. iPSCs hold great potential for understanding basic human biology, in vitro disease modeling, high-throughput drug testing and discovery, and personalized regenerative medicine. The conventional reprogramming methods involving retro- and lenti-viral vectors to deliver reprogramming factors in somatic cells to generate iPSCs nullify the clinical applicability of these cells. Although these gene delivery systems are efficient and robust, they carry an enormous risk of permanent genetic modifications and are potentially tumorigenic. To evade these safety concerns and derive iPSCs for human therapy, tremendous technological advancements have resulted in the development of non-integrating viral- and non-viral approaches. These gene delivery techniques curtail or eliminate the risk of any genomic alteration and enhance the prospects of iPSCs from bench-to-bedside. The present review provides a comprehensive overview of non-integrating viral (adenoviral vectors, adeno-associated viral vectors, and Sendai virus vectors) and DNA-based, non-viral (plasmid transfection, minicircle vectors, transposon vectors, episomal vectors, and liposomal magnetofection) approaches that have the potential to generate transgene-free iPSCs. The understanding of these techniques could pave the way for the use of iPSCs for various biomedical applications.

An Insight into DNA-free Reprogramming Approaches to Generate Integration-free Induced Pluripotent Stem Cells for Prospective Biomedical Applications.

More than a decade ago, a pioneering study reported generation of induced Pluripotent Stem Cells (iPSCs) by ectopic expression of a cocktail of reprogramming factors in fibroblasts. This study has revolutionized stem cell research and has garnered immense interest from the scientific community globally. iPSCs hold tremendous potential for understanding human developmental biology, disease modeling, drug screening and discovery, and personalized cell-based therapeutic applications. The seminal study identified Oct4, Sox2, Klf4 and c-Myc as a potent combination of genes to induce reprogramming. Subsequently, various reprogramming factors were identified by numerous groups. Most of these studies have used integrating viral vectors to overexpress reprogramming factors in somatic cells to derive iPSCs. However, these techniques restrict the clinical applicability of these cells as they may alter the genome due to random viral integration resulting in insertional mutagenesis and tumorigenicity. To circumvent this issue, alternative integration-free reprogramming approaches are continuously developed that eliminate the risk of genomic modifications and improve the prospects of iPSCs from lab to clinic. These methods establish that integration of transgenes into the genome is not essential to induce pluripotency in somatic cells. This review provides a comprehensive overview of the most promising DNA-free reprogramming techniques that have the potential to derive integration-free iPSCs without genomic manipulation, such as sendai virus, recombinant proteins, microRNAs, synthetic messenger RNA and small molecules. The understanding of these approaches shall pave a way for the generation of clinical-grade iPSCs. Subsequently, these iPSCs can be differentiated into desired cell type(s) for various biomedical applications.