Research Projects

Embryonic stem cells (ESCs) are pluripotent and hold promise for regenerative medicine, drug screening, and disease modeling. However, ethical concerns and the risk of immune rejection limit their clinical utility, prompting the development of alternative pluripotent cell sources. This led to the generation of induced pluripotent stem cells (iPSCs), where somatic cells were reprogrammed to an ESC-like state using defined transcription factors. Despite mimicking ESCs, iPSC generation often suffers from genomic instability. iPSCs may acquire genetic mutations, copy number variations, or epigenetic abnormalities during reprogramming or prolonged culture. These alterations can affect cell function, differentiation capacity, and increase tumorigenic risk. Zinc finger and SCAN domain-containing protein 4 (ZSCAN4) has emerged as a promising factor in this context. In mouse ESCs and iPSCs, ZSCAN4 plays a vital role in telomere elongation and genomic stability. When included in the reprogramming cocktail, mouse ZSCAN4 improves reprogramming efficiency and yields iPSCs with fewer single nucleotide polymorphisms, reduced DNA damage, and better genomic integrity (Zalzman et al., 2010; Hirata et al., 2012; Jiang et al., 2013; Su et al., 2013). However, the role of human ZSCAN4 in the reprogramming and maintenance of iPSCs remains unexplored. Furthermore, to date, no studies have reported the knockout of ZSCAN4 in mouse cells specifically in the context of cellular reprogramming. This study aims to elucidate the role of human ZSCAN4 in the induction and maintenance of pluripotency. Specifically, we aim to address key questions: (i) Is human ZSCAN4, like mouse ZSCAN4, essential for the successful generation of iPSCs? (ii) Do ZSCAN4-deficient iPSCs retain key features of stemness and differentiation potential? (iii) Are iPSCs generated from ZSCAN4-deficient fibroblasts genomically stable and suitable for potential therapeutic applications? (iv) If ZSCAN4 is essential, what is its functional role during the reprogramming process, and at which phase (initiation, maturation, or stabilization) is it most critical? Additionally, we aim to identify key molecular partners that cooperate with ZSCAN4 to regulate pluripotency. To address these objectives, we have constructed a non-integrating episomal expression plasmid encoding the human ZSCAN4 gene by cloning its coding sequence into the pCXLE-EGFP backbone. Homozygous ZSCAN4-knockout (ZSCAN4-KO) fibroblasts will be generated using CRISPR-Cas9 genome editing to ensure complete loss of ZSCAN4 function. These wild-type and ZSCAN4-KO fibroblasts will then be subjected to reprogramming using the Yamanaka Y4 episomal plasmids (Okita et al., 2011), with or without ZSCAN4 episomal plasmid. We will assess whether ectopic expression of ZSCAN4 enhances reprogramming in wild-type cells and whether it can rescue iPSC generation in ZSCAN4-KO cells. This will determine whether exogenous ZSCAN4 can functionally compensate for its genetic loss. In parallel, ZSCAN4 will be deleted from multiple in-house, well-characterized human iPSC lines to investigate its role in maintaining established pluripotency. To gain mechanistic insights, RNAseq will be performed on wild-type and ZSCAN4-KO fibroblasts, as well as the resulting iPSC lines: wild-type, ZSCAN4-KO, ZSCAN4-rescued, and ZSCAN4-overexpressing. Comparative transcriptomic analysis will help identify pathways regulated by ZSCAN4 during reprogramming and pluripotency maintenance. This study will determine whether human ZSCAN4 enhances the efficiency and genomic integrity of iPSC generation. Evidence of improved telomere maintenance, reduced DNA damage, and lower mutational burden in ZSCAN4-expressing iPSCs would support its role as a key factor for producing high-quality, clinically relevant iPSCs. These findings have the potential to establish ZSCAN4 as a key component of next-generation reprogramming strategies for regenerative medicine.