An overview of reprogramming approaches to derive integration-free induced pluripotent stem cells for prospective biomedical applications

Dey, Chandrima; Raina, Khyati; Haridhasapavalan, Krishna Kumar; Thool, Madhuri; Sundaravadivelu, Pradeep Kumar; Adhikari, Poulomi; Gogoi, Ranadeep; Thummer, Rajkumar P.

doi:10.1016/b978-0-12-822231-7.00011-4

Cited by 24 publications

(17 citation statements)

References 205 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Most importantly, in all these studies, the presence of the fusion tags (TAT and NLS) did not hinder their functionality. Hence, this purified HAND2 recombinant protein can be utilized to generate transgene-free cells as well as for other biological applications; thus, eliminating the limitations that accrue due to the plasmid or viral-based approaches [69,70,71].…”

Section: Discussionmentioning

confidence: 99%

Generation of transducible version of a recombinant human HAND2 transcription factor from Escherichia coli

Haridhasapavalan

Sundaravadivelu

Mohapatra

et al. 2021

Preprint

Self Cite

View full text Add to dashboard Cite

Transcription factor HAND2 has a significant role in vascularization, angiogenesis, and cardiac neural crest development. Also, 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 (E. coli) strain, BL21(DE3). Next, the effect (in terms of expression) of tagging of fusion tags with this recombinant protein at two different terminals was also investigated. Notably, using affinity chromatography, we established the one-step homogeneous purification of human recombinant HAND2 protein; and through circular dichroism spectroscopy, we established that this purified protein had retained its secondary structure. Furthermore, we show that this purified human protein could transduce the human cells and translocate to its nucleus. Prospectively, the purified recombinant HAND2 protein can potentially be a safe and effective molecular tool in the direct cardiac reprogramming process and other biological applications.

show abstract

Section: Discussionmentioning

confidence: 99%

Generation of transducible version of a recombinant human HAND2 transcription factor from Escherichia coli

Haridhasapavalan

Sundaravadivelu

Mohapatra

et al. 2021

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

“…Since then, many genes (Dey et al 2022 ) and cell types (Ray et al 2021 ; Sundaravadivelu et al 2021 ) have been identified that play a crucial role in generating iPSCs efficiently. Various technological advancements have been made with an aim to prospectively generate clinical-grade iPSCs for future biomedical applications (Dey et al 2017 , 2021 ; Saha et al 2018a ; Haridhasapavalan et al 2019 , 2020 ; Borgohain et al 2019 ). Over the years since the discovery of iPSCs, the use of these cells in disease modelling applications has increased substantially (Young 2012 ; Singh et al 2015 ; Tang et al 2016 ; Saha et al 2018b ; Agrawal et al 2021 ).…”

Section: A Brief Summary Of Crispr and Ipscsmentioning

confidence: 99%

“…This is significant as brain tissue is among the most inaccessible tissue systems in the body and brain biopsies are inherently risky. Cell lines derived from any easily accessible region of a patient’s body can be converted into human iPSCs (Dey et al 2017 , 2021 ; Haridhasapavalan et al 2019 ; Borgohain et al 2019 ; Ray et al 2021 ; Sundaravadivelu et al 2021 ). The iPSCs can subsequently be differentiated into the desired cell type by treating it with an optimized cocktail of growth factors and culture conditions (Young 2012 ; Hargus et al 2014 ; Singh et al 2015 ; Tang et al 2016 ; Saha et al 2018b ; Agrawal et al 2021 ).…”

Section: Crispr and Ipscs In Disease Modellingmentioning

confidence: 99%

CRISPR and iPSCs: Recent Developments and Future Perspectives in Neurodegenerative Disease Modelling, Research, and Therapeutics

Sen

Thummer

2022

Neurotox Res

Self Cite

View full text Add to dashboard Cite

Neurodegenerative diseases are prominent causes of pain, suffering, and death worldwide. Traditional approaches modelling neurodegenerative diseases are deficient, and therefore, improved strategies that effectively recapitulate the pathophysiological conditions of neurodegenerative diseases are the need of the hour. The generation of human-induced pluripotent stem cells (iPSCs) has transformed our ability to model neurodegenerative diseases in vitro and provide an unlimited source of cells (including desired neuronal cell types) for cell replacement therapy. Recently, CRISPR/Cas9-based genome editing has also been gaining popularity because of the flexibility they provide to generate and ablate disease phenotypes. In addition, the recent advancements in CRISPR/Cas9 technology enables researchers to seamlessly target and introduce precise modifications in the genomic DNA of different human cell lines, including iPSCs. CRISPR-iPSC-based disease modelling, therefore, allows scientists to recapitulate the pathological aspects of most neurodegenerative processes and investigate the role of pathological gene variants in healthy non-patient cell lines. This review outlines how iPSCs, CRISPR/Cas9, and CRISPR-iPSC-based approaches accelerate research on neurodegenerative diseases and take us closer to a cure for neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, Amyotrophic Lateral Sclerosis, and so forth.

show abstract

“…The poly-histidine tag is the most widely used tag for purification since it is inexpensive and does not alter the characteristics of the proteins 11 , 12 . The introduction of recombinant proteins into mammalian cells has been proven to be an effective alternative since it does not integrate and alter the genome, and also manipulation of cell fate can be done in a time- and dosage-dependent manner 13 – 17 . Thus, recombinant proteins contribute to a major and vital part in therapeutics and in safer and non-integrative cell reprogramming processes.…”

Section: Introductionmentioning

confidence: 99%

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

Haridhasapavalan

Sundaravadivelu

Joshi

et al. 2022

Sci Rep

Self Cite

View full text Add to dashboard Cite

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.

show abstract

An overview of reprogramming approaches to derive integration-free induced pluripotent stem cells for prospective biomedical applications

Cited by 24 publications

References 205 publications

Generation of transducible version of a recombinant human HAND2 transcription factor from Escherichia coli

Generation of transducible version of a recombinant human HAND2 transcription factor from Escherichia coli

CRISPR and iPSCs: Recent Developments and Future Perspectives in Neurodegenerative Disease Modelling, Research, and Therapeutics

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

Contact Info

Product

Resources

About