**Author details**

Nur Liyana Ismail\*, Sara Shahruddin and Jofry Othman PETRONAS Research Sdn. Bhd, Kajang, Selangor, Malaysia

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© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Perspective Chapter: Overview of Bio-Based Surfactant – Recent Development, Industrial… DOI: http://dx.doi.org/10.5772/intechopen.100542*


**1. Introduction**

The first derivation of human embryonic stem cells (hESCs) (Thomson et al., 1998) and the more recently development of human induced pluripotent stem cells (iPSCs) (Park et al., 2008; Takahashi et al., 2007; Takahashi & Yamanaka, 2006; Wernig et al., 2007; Yu et al., 2007) have marked the beginning of a new era in biomedical research. These two types of human pluripotent stem cells (hPSCs) are characterized by an unlimited capacity to selfrenew while retaining their potential to differentiate into almost all cell types of the body (Odorico et al., 2001; Reubinoff et al., 2000; Silva & Smith, 2008). These remarkable properties turn hPSCs into one of the most interesting cell types for toxicology and drug discovery, tissue engineering and regenerative medicine (Battey, 2007; Mountford, 2008). In fact, work with hPSCs has already provided new and exciting developments that may eventually lead to the creation of novel cell-based therapies for the treatment of a wide range of human diseases including Parkinson's and other neurodegenerative diseases, diabetes, cardiac and vascular diseases (Kiskinis & Eggan, 2010; Ronaghi et al., 2010). However, a major challenge for the widespread application of hPSCs is the development of efficient protocols for cryopreservation.

To date, two techniques are mainly applied for the cryopreservation of hPSCs: conventional slow freezing and vitrification. The conventional slow-freezing and rapid-thawing procedure using dimethylsulfoxide (DMSO) as a cryoprotectant is the most commonly used method (Grout et al., 1990; Meryman, 2007). While this established technique is effective for somatic cell lines and even murine embryonic stem cells (mESCs), hematopoietic and mesenchymal human stem cells, this is not the case for hPSCs, due to low recovery rates and high levels of differentiation (Berz et al., 2007; Reubinoff et al., 2001; Richards et al., 2004; Thirumala et al., 2010). In contrast, vitrification of hPSCs by the "open pulled straw" method

Cryopreservation of Human Pluripotent Stem Cells: Are We Going in the Right Direction? 141

An important revolution in the stem cell research field was accomplished when several groups in different studies demonstrated that using a cocktail of four factors, somatic cells could be reprogrammed into iPSCs (Maherali et al., 2007; Okita et al., 2007; Park et al., 2008; Takahashi et al., 2007; Takahashi & Yamanaka, 2006; Wernig et al., 2007; Yu et al., 2007). Consequently, it was shown that such cells could be generated from patient-specific cells for a wide variety of diseases (Kiskinis & Eggan, 2010; Raya et al., 2009; Raya et al., 2010) and from a wide variety of somatic cell types (Sun et al., 2010). These cells are morphologically similar to hESCs, express typical hESC-specific cell surface antigens and genes, differentiate into multiple lineages *in vitro*, and form teratomas containing differentiated derivatives of all three primary germ layers when injected into immunocompromised mice. Indeed, these new pluripotent cell lines satisfy all the original criteria proposed for hESCs (Thomson et al., 1998). Nevertheless, some differences have been observed between hESCs and iPSCs (Chin et al., 2009); but it remains unclear whether the small percentage of genes that are differentially expressed between these two types of hPSCs are shared among different lines

Developing iPSCs into therapeutic reagents faces a number of practical hurdles, including risks associated with cell processing, the difficulty of ensuring the purity and characteristics of the reprogrammed population and the safety and efficacy of reprogrammed cells *in vivo* (Condic & Rao, 2008; Rao & Condic, 2008; Rao & Condic, 2009). Moreover, a case of rejection has been recently described after iPSCs autologous transplantation (Apostolou & Hochedlinger, 2011). Nonetheless, there is cause for considerable optimism that patientspecific iPSC lines will both enhance the study of human diseases and advance these studies

Cryopreservation is the process of cooling and storing cells, tissues or organs at sub-freezing temperatures, below −80°C and typically below −140°C, to maintain viability (Baust et al., 2009). The freezing process involves complex phenomena of water crystallization and changes in solute concentration both outside and inside the cell that can be detrimental to cell survival. In addition, exposure to low temperatures has been reported to induce a stress response resulting in biomolecular-based cell death for different cell types (Baust et al., 2001;

In general, the major steps used in cryopreservation of most cell types can be summarized as follows (figure 1): i) harvesting the cells, ii) addition of cryoprotective agents within a carrier media to the cell suspension, iii) ice crystal induction in cell suspension following a determined cooling rate (ranging from -1 to -10ºC/min), iv) long-term storage at cryogenic temperatures (normally in liquid nitrogen), v) rapid thawing by warming the cell suspension in a 37-40ºC water bath, vi) removal of cryoprotective agent by centrifugation and vii) seeding down the cells to allow culture growth (Gao et al., 1998; Hubel, 1997).

Cryoinjury can be due to one or a combination of the following processes: 1) cytotoxicity of cryoprotective agents (Muldrew & McGann, 1994; Schneider & Maurer, 1983); 2) osmotic injury due to excursion of cryoprotective agents upon freeze-thawing (Gao et al., 1995; Mazur & Schneider, 1986); 3) intracellular ice formation in the cooling process (Fujikawa,

**1.1.2 Human induced pluripotent stem cells**

and whether these differences are biologically significant.

Fu et al., 2001; Paasch et al., 2004; Xiao & Dooley, 2003).

toward clinical applications.

**1.2 Cryopreservation of hPSCs**

using high cryoprotectant concentrations together with flash-freezing in liquid nitrogen has reported higher cell survival rates (Li et al., 2010b; Reubinoff et al., 2001; Richards et al., 2004). However, there are several disadvantages preventing the widespread use of this technique. First, high concentrations of cryoprotectors, which are cytotoxic above 4ºC, are needed. Second, these procedures are tedious to perform manually. Additionally, as vitrification is mostly performed in open pulled straws, contact between the liquid nitrogen and the cells is unavoidable, which carries the risk of contamination. Finally, and one of the most limiting disadvantages of this technique is that it is clearly unsuited for freezing bulk quantities of hPSCs.

During the last decade, several groups have been studying different approaches to improve the above described cryopreservation protocols. In the present work we will review the recent advances in the cryopreservation field trying to point out how a better understanding of the sensitivity of hPSCs to the cryopreservation process will help to develop more efficient protocols.


*5.3. Third Tier: Utilization*

The third tier is governmental utilization of religion to achieve national security goals. This utilization of religion for security objectives is common, multifaceted and present in the four classic instruments of national power. Israel's use of Islamic messaging to delegitimize Hamas is an example of utilization in the informational instrument. The utilization, to be discussed below, of Hamas's religious objection to the idea of a Jewish state in Palestine by Olmert is an example of religion in the diplomatic instrument. Examples of utilization of religion in the military instrument include Ben-Gurion's use of the Bible to enhance the fighting spirit of immigrants soldiers, and the IDF's tactic of deliberately launching an aerial bombing operation on the Sabbath, in order to surprise the enemy, who knows that, except for emergencies, the IDF refrains from acting on the Sabbath, a holy day for the Jewish public and orthodox Jewish soldiers (Hassner 2016, pp. 35, 173 note 8; Shelah 2015, p. 96). As for the economic instrument, Israel does not seem to utilize religion there (nor does France), but other countries do. For example, the US conditioned foreign aid or trade agreements on increasing religious freedom and, since 1984, every Republican president has withheld USAID funding from any overseas family planning organization o ffering or providing information about abortions, including the UN Population Fund (Lucius 2013, pp. 63–66; Rankin 2017). Along with religion's utilization in the four instruments, there is also a fifth type of utilization—the reorganization of state–religion relations for national security purposes. This utilization is absent in the Israeli case, but exists in the French case.


**4. Discussion**

Using molecular markers, genetic differences can be quickly and easily identified at the DNA level without phenotyping. RAPD markers can easily identify polymorphisms by using short random primers, commonly 10 bp in length. In addition, it does not rely on knowing the target DNA sequence information, and RAPD is inexpensive, simple, quick, and easy to use [25–27]. Based on the RAPD results, small genetic differences between large genomes can be identified. Genetic relationships between the onion lines can also be confirmed through the unweighted pair group method with an arithmetic mean cluster dendrogram [15,16].

In this study, a phylogenetic analysis of gray mold disease-resistant and -susceptible lines was conducted using RAPD. The phylogenic analysis obtained from the RAPD showed that the resistant lines were closely related to other resistant lines, and the susceptible lines were closely related to other susceptible lines. S&P 7522, S&P 7521, S&P 7129, and S&P 7168 belong to the gray mold disease-resistant line group, and S&P 7483, S&P 7130, and S&P 7175 belong to the gray mold disease-susceptible line group. Additionally, a close genetic relationship appeared between the resistant S&P 7522 and S&P 7521 lines and between the resistant S&P 7129 and S&P 7168 lines. Similarly, susceptible lines S&P 7483, S&P 7130, and S&P 7175 showed close genetic relationships in the phylogenic analysis. These results were similar to the pedigree of the 'Seeds & People' Co. onion lines. According to the pedigrees, two gray mold disease-resistant lines, S&P 7129 and S&P 7168, were bred by crossing the resistant lines S&P 7522 and S&P 7521. The gray mold disease-susceptible lines S&P 7130 and S&P 7175 were bred through the crossing of the susceptible lines S&P 7406 and S&P 7405. A comparison of the results of the phylogenetic analysis obtained from the RAPD with the pedigree of the breeding lines revealed similar genetic relationships, thereby demonstrating the reliability of the RAPD results.

The SCAR-OPAN1 marker was developed using the polymorphic fragment in the RAPD analysis. The SCAR-OPAN1 marker sequence was designed to extend longer, including the 5 -ACT CCA CGT C-3 sequence of the OPAN-1 random primer. The primer set for the SCAR- OPAN1 marker was designed considering the annealing temperature, GC content, the possibility of the dimer formation, and the hairpin loops. A BLAST analysis was performed to analyze the products amplified by the SCAR marker. In the BLAST

analysis of the DNA sequence of the fragment amplified from the SCAR marker, there was no significant percentage of sequences matched to the other sequences from onions and other species; therefore, additional analysis is required. SCAR markers have been used to distinguish between various cultivars and species [28,29]. SCAR markers can be used to select individuals with specific traits and to identify disease-resistant individuals. Using SCAR markers, the selection of male infertility-dominant *Ms* and -recessive *ms* in onions [30], anthrax resistance in grapes [31], and Acokita blight resistance in lentils [32] have been reported. It was confirmed that the selection of gray mold disease-resistant onions was possible using this SCAR- OPAN1 marker in the onion breeding lines of 'Seeds & People' Co.

In addition, to validate the developed SCAR-OPAN1 marker, additional gray mold disease-resistant and -susceptible lines from the 'Asia Seed' Co. were analyzed. In the analysis of the gray mold disease-resistant and -susceptible 'Asia Seed' Co. lines, it was confirmed that a product of 2Kb was amplified only in the resistant lines. Therefore, it was confirmed that the SCAR-OPAN1 marker developed in this study not only facilitates the selection of gray mold-resistant onions but also facilitates easy selection in a considerably short time.

The HRM marker was developed to select gray mold disease-resistant onion lines by using the selected transcripts through a DEG analysis from RNA sequencing. After analyzing the RNA-seq, the selected 14 transcripts were analyzed using the Arabidopsis Information Resource (TAIR) ID derived from Arabidopsis. The matched transcripts were analyzed using GO annotation and were largely divided into cellular components, molecular functions, and biological processes. Among them, the transcripts related to disease resistance, classified as 'response to biotic stimulus' and 'response to stress', were selected.

Plant cells synthesized reactive oxygen species (ROS) and pathogenesis-related proteins (PR proteins) after the detection of the presence of pathogens, along with a hypersensitive response (HR) to prevent the growth of pathogens. These resistance responses can induce a systemic acquired resistance (SAR) response, which is a resistance response of the whole plant. When SAR is activated by pathogens such as fungi, bacteria, and viruses, salicylic acid (SA) accumulates, and the accumulation of SA is essential for SAR expression [33,34].

In this study, the SAR gene was targeted by the SNP-10 transcript and showed DEGs and variants between the gray mold disease-resistant and -susceptible line groups (Table 2). After analyzing the relationship between SA accumulation and SAR in the early 1990s, SARs have been identified in *Arabidopsis thaliana*, tobacco, and cucumbers. The immune response by SAR is induced by PR proteins and has been studied to recognize SA as a signal [35–37]. When tobacco began to show resistance to *B. cinerea* and *Psequdomonas syringae*, it was confirmed that the resistance mechanism was initiated by the involvement of SAR, PR genes (PR-1 and PR-5), and SA before preparation in whole plants [38].

In contrast to SAR, the induced systemic resistance (ISR) with other signaling pathways induced by the plant hormones jasmonic acid (JA) and ethylene is also one of the resistance responses of plants. JA is a plant hormone that is related to disease resistance. The transcript targeted by SNP-1 was found to be the lipoxygenase (LOX) 3 gene (Table 2). The LOX gene is involved in JA biosynthesis. In particular, LOX3 was rapidly upregulated when the pathogen *B. cinerea* was inoculated in *Arabidopsis thaliana*. This LOX-upregulated response was shown in LOX3 and LOX4, which was presumably related to the early JA response of oligogalacturonides acting as damage-associated molecular patterns (DAMPs) [39].

In addition, the glutathione S-transferase (GST) gene, the transcript targeted by SNP-6, has been identified in many studies on disease resistance in plants (Table 2) [40–42]. GST plays a major role as an antioxidant and is resistant to plants in relation to a hypersensitive response to cell death. The transformed *Nicotiana benthamiana* was resistant to *Colletotrichum destructivum* and *C. orbiculare* by the GST gene [43].

In other selected transcripts, it was also confirmed that target genes have various functions related to plant resistance. Among them, the SNP-3 transcript developed as an HRM marker was confirmed to be an aldo-keto reductase gene (AKR) (Table 2). It was confirmed that the SNP-3 HRM marker related to the AKR gene can also select gray mold disease-resistant lines from 'Seeds & People' Co. and 'Asia Seed' Co. AKR gene expression was higher in the gray mold disease-resistant line group than in the -susceptible line group. AKR has been mentioned in previous studies and is known to increase the resistance at high expression levels [44,45].

Since the SNP-3 HRM marker was a gene-based marker, a qPCR analysis was conducted to confirm the expression level of the AKR gene. The expression level of AKR was higher in the resistant group than in the susceptible group. The qPCR results were compared with the in silico data. In the RNA-seq results, the expression level of AKR was higher in the resistant group than in the susceptible group, and S&P7522 showed the highest expression level. The similarity of the AKR gene expression levels between the qPCR analysis and in silico RNA-seq results showed that the gene was associated with resistance to gray mold disease, increasing the reliability of the in silico data used to develop the molecular markers.

It has also been identified that the AKR gene provides multiple stress tolerances in plants. In particular, there have been studies related to abiotic stresses such as herbicide resistance, heat stress tolerance, and biotic stress, such as mildew [41,46,47]. For biotic stress from pathogens such as microorganisms, the AKR gene group is mainly involved in plant secondary metabolic pathways, including flavonoid biosynthesis in plant-microbial interactions. Therefore, these AKRs primarily function as plant defense systems against biological stresses, such as a pathogen attack. However, despite reports identifying plant AKR as a potential target for developing abiotic and biotic stress-tolerant plant species, the importance of plant AKR has not yet been emphasized [46,48].

Various studies have revealed that AKR plays a stress-related role. This study also showed differences in the resistance and gene expression levels according to SNPs between the gray mold-resistant and -susceptible line groups. It was considered that the AKR gene affected the resistant group to show a resistance to gray mold disease. Therefore, further studies of AKR will be needed, and the SNP-3 HRM marker developed in this study could be used to select gray mold disease-resistant onion lines.