The reprogramming of human somatic cells into a pluripotent state has been accomplished by the ectopic expression of well-defined transcriptional factors such as OCT4 (also known as POU5F1), SOX2, KLF4, MYC (also known as c-MYC); these factors are collectively referred to as the OSKM factors (Yamanaka, 2012; Swaidan et al., 2020). Human induced pluripotent stem cells (hiPSCs), which possess embryonic stem cell (ESC)-like characteristics, have increased the possibility of generating patient-specific cells (Dimos et al., 2008; Soldner et al., 2009). These hiPSCs are distinguished by their ability to self-renew and differentiate into any cell type of the body. Consequently, these cells can serve purposes such as comprehending disease progression, developing and testing novel drugs, and conducting autologous replacement therapies for degenerated cells (Dimos et al., 2008). This is particularly relevant to degenerative diseases of the central nervous system (CNS) such as Parkinson’s disease (PD), in which dopaminergic neurons are lost (Soldner et al., 2009).

PD is the second most common chronic progressive neurodegenerative disease after Alzheimer’s disease. It is characterized by the loss of the nigrostriatal dopamine-releasing neurons. Despite the fact that genetic factors are implicated in the pathogenesis of PD, the majority of PD cases are sporadic, not associated with a known genetic mutation (Soldner et al., 2009). The pathological hallmark of PD is the aggregation and deposition of abnormal α-synuclein protein within the neuronal cell bodies (Stefanis, 2012). Numerous mutations have been reported in the gene responsible for encoding this protein. The α-synuclein point mutation (A53T) is believed to be the most frequent and associated with the familial type of PD (Swaidan et al., 2020).

While OSKM factors have demonstrated a robust method for reprogramming somatic cells that has potential for regenerative medicine, the process is still inefficient since only a small fraction of cells undergoes a complete and successful reprogramming (Swaidan et al., 2020). To overcome low reprogramming efficiency, several studies have examined factors that could enhance the activation of the pluripotency pathway and prevent the differentiation of stem cells (M. Wang et al., 2017; Bogliotti et al., 2018; Xu et al., 2016; Theka et al., 2017). In our earlier research, we pinpointed five genes (GBX2, NANONGP8, SP8, PEG3, and ZIC1) exhibiting differential expression in iPSCs generated from fibroblast cells. These genes maintained their differential expression regardless of the presence or absence of the PD A53T mutation, while also showcasing interactions with OSKM factors (Swaidan et al., 2020). Building upon our prior research, this paper focuses on a distinct sample (named A53T-PD1) that displayed enhanced colony integrity and stability in comparison to other samples (A53T-PD2, ID-PD and HC). This observation holds true despite the fact that a smaller number of colonies were formed during the reprogramming process of this sample.

Hence, with the aim of enhancing the regeneration process’s efficiency, our focus lies in investigating genes related to genomic integrity/stability and cellular survival. These genes exhibit differential expression in A53T-PD1 cells and could potentially be genetically modified or suppressed to bolster and sustain iPSC pluripotency.

ESCs and iPSCs offer immense potential, not only in the field of regenerative medicine but also as valuable tools for investigating developmental processes, pathologies, and the assessment of novel drug interventions. However, the maintenance of genomic integrity and cellular stability presents a formidable challenge to sustaining the self-renewal and pluripotency of PSCs. Genomic instability can impair self-renewal pathways, activate the p53 apoptotic pathway, and promote PSC differentiation (Su et al., 2019). It is therefore imperative to safeguard the genomic integrity of PSCs and explore the genes that may be involved in this process, all while promoting robust growth to preserve their vital functions and self-renewal capability. Several factors, including culture conditions, can contribute to genomic instability in ESCs, and exposure to endogenous or exogenous genotoxic stresses can lead to cell apoptosis due to DNA damage (Martins-Taylor and Xu, 2012). It has been noted that ESCs have shown distinct mechanisms to shield themselves from DNA double-strand breaks, setting them apart from somatic cells (Nagaria et al., 2013).

In our previous study (Swaidan et al., 2020), iPS cell lines were successfully generated from a PD patient carrying the A53T mutation (A53T-PD1). Although the generated colonies number was limited, these colonies exhibited remarkable genomic resilience and prolonged viability compared to other iPS cell lines that were generated in parallel. This intriguing finding prompted us to consider additional factors influencing genomic integrity beyond age and disease mutation. Our hypothesis regarding the potential roles of certain molecules specific to A53T-PD1 cell lines, at both nuclear and cellular levels, in maintaining genomic and cellular stability were substantiated by transcriptomic analysis. This analysis revealed three differentially expressed genes in the A53T-PD1 cell line (CCN3, POSTN, and PTHLH), all of which are implicated in cellular stability, DNA repair mechanisms, and apoptosis inhibition.

Among these genes, CCN3, also known as NOV, exhibited significant upregulation in the A53T-PD1 iPS cell line compared to other cell lines (34.9-fold, FDR <0.001). CCN3 belongs to the CCN family of matricellular proteins, known for its pivotal role in extracellular signaling and the maintenance of cellular viability (Kubota and Takigawa, 2013). Previous studies have highlighted the importance of CCN3, particularly its role in cellular adhesion and the regulation of integrin expression (Lafont et al., 2005; Lin et al., 2005; Vallacchi et al., 2008). These findings support our hypothesis that CCN3 plays a crucial role in pluripotency circuits and their maintenance, particularly in cell adhesion receptor systems and integrin expression (Vitillo and Kimber, 2017; Yu et al., 2018). Moreover, the involvement of CCN3 in regulating integrin expression not only underscores its significance in initiating and sustaining pluripotency but also implies its role in maintaining the compact morphology of pluripotent colonies, dependent on integrin activity (Yu et al., 2018). In addition, increased cellular proliferation, a hallmark of cells undergoing reprogramming, can be achieved by inhibiting p53, which is responsible for programmed cell death, and promoting LIN28 expression, which regulates cell cycle genes like CDK genes. This mechanism contributes to the efficiency of reprogramming processes and the stability of hiPSCs (Hong et al., 2009; Kawamura et al., 2009; Hawkins et al., 2014).

Through STRING analysis, we observed that CCN3 is involved in promoting cell survival. Notably, it directly interacts with key players in the p53 pathway, including TP53 and CDKN1A, which regulate cell apoptosis and survival. Reactome analysis further elucidated these interactions, revealing CCN3’s direct interaction with POU domain transcription factors that modulate p53 and regulate the transcription of pro-apoptotic genes (Figure 6A). When the P53 pathway is activated in stem cells, it initiates a cascade of events leading to cell cycle arrest, commitment to a differentiation pathway, and the formation of progenitor cells. P53 serves as a guardian of genomic stability, and its activation is pivotal for maintaining cellular integrity (Levine et al., 2016). Furthermore, CCN3’s interaction with FGF2, a key factor in the FGF2 pathway governing reprogramming initiation and stabilization, provides additional evidence of its role in improving reprogramming efficiency. FGF2 accelerates cell proliferation during the initial phases of reprogramming, and it has been noted that hiPSCs generated in the presence of Activin/Nodal and FGF2 ligands tend to stabilize in the primed state (Jiao et al., 2013; Hawkins et al., 2014). In addition, FGF2 is known to influence the self-renewal and clonogenic capacity of pluripotent stem cells (Aprile et al., 2023). These findings highlight CCN3’s significance in orchestrating key signaling pathways that govern cellular fate and function. Its active participation in both the P53 and FGF2 pathways positions CCN3 as a central player in the intricate network of molecular events that determine cell behavior, stability, and responsiveness to environmental cues. Our results were further substantiated by pluripotency pathways extracted from the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, which highlighted the significance of the FGF2 pathway in governing reprogramming process in primed stem cells (Figure 8). Interestingly, the CCN3 gene has been documented to collaborate with FGF2 as a substrate, leading to enhanced cell proliferation and improved cell survival when they are combined (Lafont et al., 2005; van Roeyen et al., 2008). Reactome analysis also revealed CCN3’s direct interaction with FSR3, an adapter protein linking FGF receptor to downstream signaling pathways (Figure 6B).

Moreover, CCN3 was found to enhance the expression of BMP-4, a protein critical for pluripotency initiation and maintenance through various mechanisms (Qi et al., 2004; Tan et al., 2012; Hawkins et al., 2014; Yu et al., 2020). KEGG database results highlighted the BMP-4 pathway’s involvement in governing reprogramming process in primed stem cells (Figure 8). Collectively, these findings strengthen our hypothesis regarding CCN3’s role in initiating and maintaining the reprogramming process, as well as its contribution to stabilizing and preserving reprogrammed cells from apoptosis.

The role of CCN3 in cellular stability and integrity emerges as a complex and multifaceted process, as evidenced by the findings from various studies employing overexpression and knockdown approaches. A study focused on CCN3 overexpression in bone marrow-derived stroma cell line (ST-2) cells revealed its inhibitory influence on BMP signaling, particularly preventing BMP-2 from phosphorylating Smad1/5/8, ultimately leading to the inhibition of osteoblastogenesis. This implies that CCN3 may play a crucial role in regulating bone homeostasis by modulating BMP signaling, a pathway fundamental to osteoblastic differentiation (Zuo et al., 2010), which in turn indicates the stability of ST-2 cells in its undifferentiated form. Moreover, CCN3’s involvement in the regulation of primitive hematopoietic stem cells adds another layer to its significance in cellular homeostasis. Studies employing CCN3 knockdown or forced expression in CD34⁺ cells have shown the necessity of CCN3 for the functional self-renewal of hematopoietic stem cells (Zuo et al., 2010).In a different investigation, CCN3 levels were modified by employing adenoviruses that either overexpressed CCN3 (Ad-CCN3) or interfered with CCN3 (Ad-siCCN3) in mouse embryonic fibroblasts (MEFs). This study aimed to examine the impacts and mechanisms of CCN3 on the process of osteoblastic differentiation. The findings indicated a notable hindrance of osteogenic differentiation in MEFs with higher CCN3 expression, primarily attributed to the suppression of BMP signaling and the reciprocal inhibition with DLL1(Su et al., 2018). Mechanistic analyses further uncovered CCN3’s suppressive effects on BMP/Smad and BMP/MAPK signaling pathways, along with its mutual inhibition with DLL1, a prominent membrane protein ligand associated with the Notch signaling pathway. Significantly, Hey1, a target gene shared by both BMP and Notch signaling pathways, emerged as a pivotal factor, partially counteracting the inhibitory impact of CCN3 on osteoblastic differentiation (Su et al., 2018). These regulatory roles of CCN3 in both bone formation and hematopoiesis underscores its vitality in maintaining cellular stability within distinct cellular contexts.

Another gene exclusively upregulated in the A53T-PD1 iPS cell line compared to other cell lines was PTHLH (59-fold, FDR <0.001). STRING analysis unveiled an unknown interaction between PTHLH and CDK2, which plays a role in the cell cycle and TP53 inhibition. Previous research has highlighted PTHLH as a crucial molecular regulator of cellular development and survival (Guo et al., 2012). Additionally, in Pthlh-depleted embryos at the morula stage, the expression of pluripotency-related genes NANOG and POU5F1 was markedly diminished (Min et al., 2022). While there is no direct evidence suggesting PTHLH’s involvement in histone acetylation during early embryonic development before implantation, previous research has indicated its direct influence on histone deacetylase 4 (HDAC4) (Kozhemyakina et al., 2009; Correa et al., 2010). Moreover, PTHLH exerts its influence through a complex network of signaling pathways involving ERK/MAPK pathways (Datta et al., 2005). ERK depletion has been associated with dysregulated pluripotency gene expression, decreased proliferation rates, G1 cell cycle arrest, increased apoptosis, faster telomere shortening, and impaired genomic stability (Chen et al., 2015). This data correlates well our data that PTHLH may play a role in increasing the cell survival and stability.

Reactome analysis confirmed PTHLH’s role in intracellular signaling, through its binding to GPCR leading to the activation of GNAS (Figure 7). G-proteins are essential regulators of cellular apoptotic cascades, with GNAS being linked to both proapoptotic and anti-apoptotic pathways (Melien, 2007; Yanamadala et al., 2009). GNAS overexpression has been shown to inhibit hydrogen peroxide-induced apoptosis by downregulating Bcl-xl (Zhao et al., 2006). Furthermore, endogenous GNAS activation through agonists has inhibited apoptosis mediated by cAMP production and PKA activation, leading to the inhibition of pro-apoptotic transcription factors such as AP-1, NF-κB, and NFAT (Choi et al., 2009). Interestingly, the Gs-alpha pathway has been proposed to regulate pluripotency in ESCs. Activation of Gs-alpha and amplification of cAMP levels have been shown to promote Oct4 expression, stimulating ESC self-renewal and pluripotency (Faherty et al., 2007; Layden et al., 2010). Therefore, we hypothesize that PTHLH may exert indirect influences on the expression of pluripotency-related genes, leading to more cellular stability, although the precise underlying mechanism warrants further investigation.

PTHLH has shown to be an upstream activator of GNAS which directly interacts with SRC, leading to SRC autophosphorylation and subsequent activation. SRC plays a diverse role in iPSCs, influencing key cellular processes such as survival, proliferation, and cell fate decisions. Its signaling pathways support iPSC self-renewal, prevent apoptosis, maintain stability, and impact commitment to specific lineages (Lu et al., 2007; Shoni et al., 2014; Zhang et al., 2014; Chaudhari et al., 2019). SRC also modulates pluripotency-associated pathways, including Wnt/β-catenin (Min et al., 2022), which is crucial for maintaining stem cell identity. During cellular reprogramming, SRC activation facilitates the transition to a pluripotent state, contributing to the erasure of cell identity (Ma et al., 2013; González and Huangfu, 2016). Additionally, SRC is associated with cellular senescence, potentially regulating senescence during reprogramming (Anerillas et al., 2022). The context-dependent nature of SRC in iPSCs, influenced by cellular context, reprogramming stage, and interactions with other pathways, underscores the dynamic complexity of its functions. Altogether, emphasize the important role of PTHLH in maintaining iPSC stability and integrity. Indeed, the downregulation of PTHLH emerged as a critical factor influencing cellular stability and integrity, particularly evident in a MYCN-amplified, TP53-mutated neuroblastoma cell line. The reduction of PTHLH levels led to a consequential decrease in MYCN expression. This event triggered a cascade of effects, including cell cycle arrest, induction of senescence, and impairment of migration and invasion capabilities within the neuroblastoma cell line (García et al., 2019). These findings highlight the intricate role of PTHLH in orchestrating key cellular processes and maintaining the equilibrium necessary for stability and integrity.

POSTN exhibited the highest upregulation, increasing by 70-fold (FDR <0.001), in A53T-PD1 iPS cell line compared to other iPS cell lines (Figure 3B). STRING analysis predicted that POSTN interacts with extracellular matrix components, including collagen-related proteins, Decorin and Tenascin C. Our findings align with previous research indicating that POSTN is highly expressed in a mouse ES cell line treated with Mek inhibitor PD0325901, in contrast to other untreated cell lines (Chen et al., 2015). PD is a pharmacological agent that inhibits the Mek/Erk signaling pathway, promoting self-renewal and pluripotency in mESCs (Chen et al., 2015). Additionally, POSTN has been associated with the augmentation of the Wnt signaling pathway in mouse breast cancer stem cells (Wang et al., 2013). This suggests a potential role for POSTN in maintaining self-renewal and pluripotency through the activation of the Wnt signaling pathway, which is essential for preserving naïve pluripotency and epigenetic stability in mESCs (de Jaime-Soguero et al., 2018; Theka et al., 2019).

Previous studies elucidate the intricate and context-dependent role of POSTN in cellular stability and integrity across various biological systems. In renal cell carcinoma (RCC), POSTN emerges as a key regulator, where its knockdown significantly suppresses epithelial-mesenchymal transition (EMT) through the IKL/AKT/mTOR pathway. Conversely, overexpression of POSTN facilitates EMT (Jia et al., 2021), indicating the crucial role of POSTN in cell stability and integrity. Moreover, overexpression of POSTN in the context of osteoblasts acts as a protective factor by impeding melatonin-induced apoptosis; hence, increase cell stability and integrity. This protective effect is achieved by inhibiting the eIF2α-ATF4 pathway through the suppression of the protein kinase R-like endoplasmic reticulum kinase (PERK) pathway (Zhu et al., 2021). These roles of POSTN highlights its versatile functions in maintaining cellular homeostasis. Furthermore, using mesenchymal stem cells (MSCs), POSTN plays a crucial role in the mineralization of the extracellular matrix (ECM) and supports tendon formation when overexpressed. This emphasizes POSTN’s involvement in ECM dynamics, which is essential for tissue development and integrity (Zhu et al., 2021).

In conclusion, our study sheds light on the potential roles of CCN3, PTHLH, and POSTN in maintaining genomic and cellular stability while preserving pluripotency in iPSCs. These findings warrant further investigations to unravel the precise mechanisms through which these genes contribute to these processes.

In summary, we identified three genes that were highly expressed in A53T-PD1 iPS cell line, from which robust and stable colonies were generated. The implications of these identified genes extend to potential selective interactions, which are vital for upholding the genomic integrity and maintaining pluripotency in both ESCs and iPSCs. Their functions may encompass activating DNA repair mechanisms, evading apoptosis through TP53 phosphorylation, and triggering signaling pathways that are crucial for preserving the pluripotent state of ESCs. To substantiate this conceptual framework and gain a deeper understanding of the underlying mechanisms, it becomes imperative to conduct thorough functional investigations. These endeavors hold the promise of unraveling crucial insights into the precise roles of these genes in ensuring genomic stability while upholding the self-renewal and pluripotency of the generated iPSCs.