A Comprehensive Review on the Role of ZSCAN4 in Embryonic Development, Stem Cells, and Cancer.

ZSCAN4 is a transcription factor that plays a pivotal role during early embryonic development. It is a unique gene expressed specifically during the first tide of de novo transcription during the zygotic genome activation. Moreover, it is reported to regulate telomere length in embryonic stem cells and induced pluripotent stem cells. Interestingly, ZSCAN4 is expressed in approximately 5% of the embryonic stem cells in culture at any given time, which points to the fact that it has a tight regulatory system. Furthermore, ZSCAN4, if included in the reprogramming cocktail along with core reprogramming factors, increases the reprogramming efficiency and results in better quality, genetically stable induced pluripotent stem cells. Also, it is reported to have a role in promoting cancer stem cell phenotype and can prospectively be used as a marker for the same. In this review, the multifaceted role of ZSCAN4 in embryonic development, embryonic stem cells, induced pluripotent stem cells, cancer, and germ cells are discussed comprehensively.

Tissue-Restricted Stem Cells as Starting Cell Source for Efficient Generation of Pluripotent Stem Cells: An Overview.

Induced pluripotent stem cells (iPSCs) have vast biomedical potential concerning disease modeling, drug screening and discovery, cell therapy, tissue engineering, and understanding organismal development. In the year 2006, a groundbreaking study reported the generation of iPSCs from mouse embryonic fibroblasts by viral transduction of four transcription factors, namely, Oct4, Sox2, Klf4, and c-Myc. Subsequently, human iPSCs were generated by reprogramming fibroblasts as a starting cell source using two reprogramming factor cocktails [(i) OCT4, SOX2, KLF4, and c-MYC, and (ii) OCT4, SOX2, NANOG, and LIN28]. The wide range of applications of these human iPSCs in research, therapeutics, and personalized medicine has driven the scientific community to optimize and understand this reprogramming process to achieve quality iPSCs with higher efficiency and faster kinetics. One of the essential criteria to address this is by identifying an ideal cell source in which pluripotency can be induced efficiently to give rise to high-quality iPSCs. Therefore, various cell types have been studied for their ability to generate iPSCs efficiently. Cell sources that can be easily reverted to a pluripotent state are tissue-restricted stem cells present in the fetus and adult tissues. Tissue-restricted stem cells can be isolated from fetal, cord blood, bone marrow, and other adult tissues or can be obtained by differentiation of embryonic stem cells or trans-differentiation of other tissue-restricted stem cells. Since these cells are undifferentiated cells with self-renewal potential, they are much easier to reprogram due to the inherent characteristic of having an endogenous expression of few pluripotency-inducing factors. This review presents an overview of promising tissue-restricted stem cells that can be isolated from different sources, namely, neural stem cells, hematopoietic stem cells, mesenchymal stem cells, limbal epithelial stem cells, and spermatogonial stem cells, and their reprogramming efficacy. This insight will pave the way for developing safe and efficient reprogramming strategies and generating patient-specific iPSCs from tissue-restricted stem cells derived from various fetal and adult tissues.

Generation of biologically active recombinant human OCT4 protein from E. coli.

Octamer-binding transcription factor 4 (OCT4) is vital for early embryonic development and is a master regulator of pluripotency in embryonic stem cells. Notably, OCT4 is a key reprogramming factor to derive induced pluripotent stem cells, which have tremendous prospects in regenerative medicine. In the current study, we report heterologous expression and purification of human OCT4 in E. coli to produce pure recombinant protein under native conditions. To achieve this, the 1083 bp coding sequence of the human OCT4 gene was codon-optimized for heterologous expression in E. coli. The codon-optimized sequence was fused with fusion tags, namely a cell-penetrating peptide sequence for intracellular delivery, a nuclear localization sequence for intranuclear delivery, and a His-tag for affinity purification. Subsequently, the codon-optimized sequence and the fusion tags were cloned in the protein expression vector, pET28a(+), and transformed into E. coli strain BL21(DE3) for expression. The recombinant OCT4 protein was purified from the soluble fraction under native conditions using immobilized metal ion affinity chromatography in a facile manner, and its identity was confirmed by Western blotting and mass spectrometry. Furthermore, the secondary structure of the recombinant protein was analyzed using far ultraviolet circular dichroism spectroscopy, which confirmed that the purified fusion protein maintained a secondary structure conformation, and it predominantly composed of α-helices. Next, the recombinant OCT4 protein was applied to human cells, and was found that it was able to enter the cells and translocate to the nucleus. Furthermore, the biological activity of the transduced OCT4 protein was also demonstrated on human cells. This recombinant tool can substitute for genetic and viral forms of OCT4 to enable the derivation of integration-free pluripotent cells. It can also be used to elucidate its biological role in various cellular processes and diseases and for structural and biochemical studies. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02758-z.

Generation of a Recombinant Stem Cell-Specific Human SOX2 Protein from Escherichia coli Under Native Conditions.

The stem cell-specific SOX2 transcription factor is critical for early embryonic development and the maintenance of embryonic and neural stem cell identity. It is also crucial for the generation of induced pluripotent and neural stem cells, thus providing immense prospect in patient-specific therapies. Here, we report soluble expression and purification of human SOX2 protein under native conditions from a bacterial system. To generate this macromolecule, we codon-optimized the protein-coding sequence and fused it to a nuclear localization signal, a protein transduction domain, and a His-tag. This was then cloned into a protein expression vector and was expressed in Escherichia coli. Subsequently, we have screened and identified the optimal expression conditions to obtain recombinant fusion protein in a soluble form and studied its expression concerning the position of fusion tags at either terminal. Furthermore, we purified two versions of recombinant SOX2 fusion proteins to homogeneity under native conditions and demonstrated that they maintained their secondary structure. This molecular tool can substitute genetic and viral forms of SOX2 to facilitate the derivation of integration-free induced pluripotent and neural stem cells. Furthermore, it can be used in elucidating its role in stem cells, various cellular processes and diseases, and for structural and biochemical studies.

An Insight into the Role of UTF1 in Development, Stem Cells, and Cancer.

The curiosity to understand the mechanisms regulating transcription in pluripotent cells resulted in identifying a unique transcription factor named Undifferentiated embryonic cell transcription factor 1 (UTF1). This proline-rich, nuclear protein is highly conserved among placental mammals with prominent expression observed in pluripotent, germ, and cancer cells. In pluripotent and germ cells, its role has been implicated primarily in proper cell differentiation, whereas in cancer, it shows tissue-specific function, either as an oncogene or a tumor suppressor gene. Furthermore, UTF1 is crucial for germ cell development, spermatogenesis, and maintaining male fertility in mice. In addition, recent studies have demonstrated the importance of UTF1 in the generation of high quality induced Pluripotent Stem Cells (iPSCs) and as an excellent biomarker to identify bona fide iPSCs. Functionally, UTF1 aids in establishing a favorable chromatin state in embryonic stem cells, reducing "transcriptional noise" and possibly functions similarly in re-establishing this state in differentiated cells upon their reprogramming to generate mature iPSCs. This review highlights the multifaceted roles of UTF1 and its implication in development, spermatogenesis, stem, and cancer cells.