Stem cells are cells that have the ability to self-renew and differentiate into other types of cells. They have played many roles in development and maintaining the health of an organism. They consist of several levels of differentiation potential, namely, totipotent, pluripotent, multipotent, and unipotent. Besides differentiation potential, the fate of these stem cells is also influenced by the interplay of various external and internal factors, including their microenvironments, or niches which provides specific cues to the cells (Zakrzewski et al., 2019). These regenerative features of stem cells have attracted researchers to explore and utilize them in many ways, especially in treating incurable and degenerative diseases.

Although this field of research has been studied for a long time, there are still many challenges to overcome. Some examples of current limitations in stem cell differentiation include difficulties in controlling differentiation, where attenuation of SP7 gene responsible for undesired hypertrophic differentiation had compromised bone marrow derived stem cell (BMSC) chondrogenesis in cartilage defect repair study (Franco et al., 2023). The tendency of exogenous mesenchymal stem cells (MSCs) to go through hypertrophic differentiation is also a long-term clinical inferiority (Wang et al., 2019a). Unexpected outcomes of neuronal MSC differentiation also remained a complicated challenge, with the appearance of de-differentiation, poor differentiation products, limited functionality, and many more as reviewed in George et al. (2019). In addition to that, genomic instability and the risk of tumour formation were among the obstacles mentioned by Golchin et al. (2021) in the differentiation of human embryonic stem cells (hESC). Whereas (Hoang et al., 2022) highlighted immunogenicity and post-administration concerns of MSCs, as well as laborious and time-consuming protocols for human induced pluripotent stem cell (hiPSC)-derived retinal pigment epithelium (RPE) differentiation. Other limitations are inadequate spontaneous migration of endogenous MSC towards impaired bone site for cartilage regeneration (Wang et al., 2019a), and loss of implanted exogenous MSCs in bone tissue regeneration (Wang et al., 2023). However, there are other hurdles to guide stem cell differentiation that are not mentioned here, which, together, greatly forced scientists to look for solutions so that stem cell-based research and therapies continue to be the future promise.

To overcome these limitations, many methods and technologies were explored. This includes the use of design molecules such as aptamer, phage, biological vector, and 3-dimensional (3D) biomaterial. Design molecules are molecules that are designed to modulate desired functions. Aptamer, phage, biological vector, and 3D biomaterial are some examples of design molecules used in recent tissue engineering and therapeutic studies. They are designed to specifically recognise their target cells or molecules, and perform their intended functions such as recruiting cells, displaying ligands, delivering specific genes, mimicking niches, or providing cues to direct stem cell differentiation thus, promoting cell or tissue regeneration. These strategies have proven to be the most effective approaches for directing stem cell differentiation, depending on the application and non-specific response (Jang et al., 2020; Xing et al., 2019). Biochemical factors such as small molecules and growth factors generally have a short half-life, requiring greater dosage or repeated administrations, and may evoke undesired multiple lineage differentiation (Halim et al., 2020). Consequently, effective and specific designed delivery systems are needed to enable controlled and prolonged release of these biochemical factors, especially for in vivo studies (Oliveira et al., 2021). For these reasons, design molecules like aptamer, phage and biological vector as illustrated in Figure 1 are being developed.

Aptamer and phage can specifically target and recruit more endogenous MSCs at the site of injury, as well as regulating biochemical factors or pathways involved in promoting tissue regeneration (Wan et al., 2019; Wang et al., 2019a; Wang et al., 2023). In this way, issues like insufficient migration and unspecific differentiation of endogenous or exogenous MSCs can be resolved through their high specificity and binding ability toward the targeted cells or molecules. On the other hand, biological vectors that include both viral and non-viral vectors are excellent tools for gene delivery (Powell et al., 2015). In addition to allowing the delivery of specific genes to targeted cells, they can also be engineered to enhance target specificity and transgene expression for short-term or permanent long-term expression in targeted cells or tissues (Lundstrom, 2018; Ghosh et al., 2020). Therefore, non-specific reactions can be reduced and safe use in directing stem cell differentiation could be better ensured. In this review, we focus on the discussion of aptamers, phage, and biological vector as design molecules, as well as their roles in directing stem cell differentiation. Hence, the 3D biomaterial and other molecules are excluded from this review.

Aptamer is a short, 25–80 bases long single-stranded oligonucleotide sequence that resembles monoclonal antibody (Ni et al., 2021). It can fold into tertiary structures and bind with high specificity and affinity to the targets (Figure 1) (Ni et al., 2021). There are two main types of aptamers, nucleic acid aptamers and peptide aptamers. Nucleic acid aptamers consist of nucleotide sequences that form complex secondary or tertiary structures that bind to targets using their entire sequence. However, peptide aptamers are amino acid sequences with a short peptide region embedded within a scaffold protein that acts as the backbone (Söylemez et al., 2019). The peptide region is involved in binding with the target protein while the binding specificity and affinity are enhanced by the scaffold conformation (Söylemez et al., 2019).

When mentioning aptamers however, it usually refers to nucleic acid aptamers instead of peptide aptamers, unless specifically stated. Also, in terms of directing stem cell differentiation, nucleic acid aptamers are more widely used compared to peptide aptamers which are more applicable in other research fields. Leaving that aside, nucleic acid aptamers can be further classified into DNA and RNA aptamers, whereby DNA aptamers are preferred since they are chemically more stable and do not require reverse transcription steps when developing RNA aptamers (Söylemez et al., 2019; Acquah et al., 2020). Nevertheless, the interesting characteristics possessed by aptamers have attracted scientists to employ them for various research applications in many ways.

The aptamers are usually developed through a conventional method known as SELEX (Systematic Evolution of Ligands by EXponential Enrichment). It is an in vitro selection process that isolates aptamers with high affinity and specificity toward their targets (Söylemez et al., 2019; Ni et al., 2021). To simplify, SELEX involves the synthesis of a random oligonucleotide library and introduced with target molecules to obtain fragments that could specifically bind to the targets (Figure 2). This selection process is usually carried out in 8–15 rounds to obtain high affinity aptamers (Söylemez et al., 2019). However, due to time-consuming and laborious process, researchers have modified it with different methodologies such as Microfluidic SELEX, High Fidelity (Hi-Fi) SELEX, Atomic Force Microscopy (AFM) SELEX, as well as incorporating the Next-Generation Sequencing (NGS), High throughput-SELEX (HTS) which have reduced the selection cycles into just about two to six cycles, along with other types of SELEX offering different benefits, as reviewed in (Bayat et al., 2018; Komarova and Kuznetsov, 2019; Acquah et al., 2020).

However, since SELEX involves polymerase chain reaction (PCR) to amplify the target-bound aptamer sequences, it poses some disadvantages in determining aptamer candidates. The success rate remains low and the overall process still consumes a great amount of time and effort (Chen et al., 2021; Lee et al., 2023). The quality of aptamer products can also be affected by PCR amplification bias and accumulated flaws in the multistep SELEX procedure (Komarova and Kuznetsov, 2019). Hence, silico approaches in designing the aptamers have been introduced and incorporated into some SELEX methods. In silico methods are computer-based (Navien et al., 2021) and involve four main steps; 1) secondary structure prediction, 2) tertiary structure optimization, 3) structural docking simulation, 4) molecular dynamics simulation, and then followed by modifications for further quality improvements (Buglak et al., 2020; Chen et al., 2021; Lee et al., 2023). Some examples of programmes used in these steps are listed in Table 1 below (Ahmad et al., 2021; Buglak et al., 2020; Chen et al., 2021; Lee et al., 2023; Navien et al., 2021). It should be noted that the tools are not limited to those listed, as there are many others available for diverse applications.

Besides, some studies have also used Quantitative Structure-Activity Relationship (QSAR) method for aptamer design and prediction of binding ability (Kumar and Kumar, 2020; Yu et al., 2019). Other techniques in developing aptamer are particle display and multiparametric particle display that involve fluorescence-activating cell sorting (FACS) aptamer screening, alkyne-azide chemistry-based particle display, and non-natural aptamer array (N2A2) platform for base-modified aptamers (Wu et al., 2022). On the other side, non-equilibrium capillary electrophoresis of equilibrium mixtures (NECEEM)-based non-SELEX was also developed to eliminate the repetitive PCR amplification steps in SELEX-based procedures thus, reducing the errors they posed (Bayat et al., 2018). Whereas, to increase the quality of SELEX products and aptamers efficacy, chemical modifications have been implemented and can be applied pre- or after SELEX (Bayat et al., 2018). Modifications to nucleotide bases, sugar rings, and phosphates have improved the binding affinity and total number of aptamer candidates obtained in SELEX (Bayat et al., 2018). Chemical modification is one of the most effective approaches to improve delivery of oligonucleotides. Nucleic acid backbone modification, ribosomal sugar moiety modification and nucleobase modification have all extensively used to improve the drug delivery (Roberts et al., 2020). Modification is used to improve the oligonucleoside pharmacokinetics, pharmacodynamics and biodistribution. Most of the chemical modification method have a higher binding affinity and decrease the immune stimulation (Dhuri et al., 2020). Various base modifications and the creation of mirror image aptamers, or Spiegelmers, have facilitated the avoidance of nuclease degradation, while the composition of aptamers with bulky moieties helped to minimize the quick elimination of renal filtration (Bayat et al., 2018; Ni et al., 2021). Spiegelmers also known as synthetically prepared oligonucleotide with L-configuration, are not only highly selective and ligand-like, but also bio structural due to their resistance to enzyme in biological fluids (Rana et al., 2021). Although seems favorable, there are issues arise when dealing with chemical modifications, as unnatural nucleotides, and addition of bulky moieties like PEG (polyethylene glycol) may result in chemical toxicity and immune reactions risks respectively (Ni et al., 2021).

Whilst all these strategies are available, producing good quality aptamers are largely influenced by the aptamer sequence design since their binding specificity and affinity are target-dependent. So, knowing the targets (cells or molecules) and the way they work well will assist in good aptamer sequence creations. Furthermore, as aptamer synthesis generally consist of many steps, conducting each step, especially the initial oligonucleotide library design (Bayat et al., 2018) with great care and precision would certainly reduce the accumulation of faults thus, generating more and better aptamer products. The incorporation of suitable modifications as well would guide in the quality enhancement.

Bacteriophage, or phage, is a human-safe bacteria-infecting virus made up of protein capsid as the outer structure, while containing its genetic materials (Figure 1) (Cao et al., 2019). There are two lifecycles, the lytic and lysogenic lifecycles, which the phage could undergo. For lytic lifecycle, the host cell’s machinery system will be taken over upon the injection of the phage’s genome to produce more of itself. The lysogenic lifecycle only involves the integration of the phage genome into the host genome and will remain dormant and replicated along with the host during cell division (Monteiro et al., 2019). These distinctive lifecycle modes consequently classify phage into different types, the lytic, temperate, and non-lytic phage (Cao et al., 2019; Monteiro et al., 2019). Basically, lytic and nonlytic phages strictly undergo lytic life cycle and lysogenic lifecycle respectively, while temperate phages carry out lysogenic lifecycle and could switch into lytic lifecycle when the host cell is put under environmental stress (Cao et al., 2019; Monteiro et al., 2019).

Exploiting the intriguing nature of phage, scientists have been experimenting with phage for various research purposes such as delivering or expressing desired genes, displaying desired coat protein, targeting specific bacteria or cells, and many others, which mostly were made possible through advancements of genetic engineering. In general, different types of phages are utilized according to their natural behaviours. Instinctively, a lytic phage will be used when its lytic activity is needed. Otherwise, non-lytic phage will be preferred. Temperate phages have been previously less used since they pose the risk of unexpected lifecycle switching. But advances in technologies have now made researchers explore ways to utilize them, by eliminating lysogenic-contributing genes that convert them into lytic phage and omit concerns of possible horizontal gene transfer that can cause bacterial resistance or superinfection immunity (Monteiro et al., 2019; Peng and Chen, 2021). Likewise, when bactericidal effect and its accompanied release of endotoxin are undesired, lytic genes can be preferably inhibited (Monteiro et al., 2019; Peng and Chen, 2021). Therefore, scientists have the opportunity to investigate the potential use of all types of phages, allowing them to be applied in various studies.

Many methods have been used to develop and designing phage with desirable features. The phage genome can be modified through genetic engineering methods (Figure 3) such as homologous recombination between the phage and donor DNA, producing new recombinant phages (Monteiro et al., 2019; Lenneman et al., 2021; Mahler et al., 2023). Also, bacteriophage recombineering of electroporated DNA (BRED) method has improved homologous recombination through electroporation of phage DNA and dsDNA containing desired modification into host cell carrying recombinase protein-encoding plasmid hence, increasing the rate of recombination (Monteiro et al., 2019; Mahler et al., 2023). Another variant called bacteriophage recombineering with infectious particles (BRIP) instead, involves only the electroporation of the dsDNA, while the phage genome is delivered through usual phage infection (Mahler et al., 2023). Furthermore, fragments of synthetically produced phage genomes can also be assembled and subjected to genome rebooting to produce functional phages. The synthetic fragments can be assembled through yeast-based platform and Gibson assembly (Lenneman et al., 2021; Mahler et al., 2023). The yeast-based platform involves homologous recombination between fragments and the yeast artificial chromosome (YAC), creating YAC-phage DNA, while Gibson assembly implies an in vitro enzymatic assembly of synthetic genomes (Monteiro et al., 2019; Lenneman et al., 2021; Mahler et al., 2023). The resulting fully assembled genomes from either method will then be rebooted through transformation into host or L-form bacterial cells for recovery of functional phages, or using cell-free transcription-translation (TXTL) systems (Monteiro et al., 2019; Lenneman et al., 2021; Mahler et al., 2023).

Apart from that, clustered regularly interspaced short palindromic repeats-CRISPR associated protein (CRISPR-Cas) genome editing has also been employed in phage engineering and there are many CRISPR-Cas systems that can be used (Mahler et al., 2023). For example, CRISPR-Cas9 nuclease is used to cleave the injected phage genome to allow recombination of the phage genome and donor DNA, and the recovery of engineered phages (Monteiro et al., 2019). It should be brought to attention though that the use of CRISPR-Cas systems is, however, restricted to CRISPR-Cas encoding bacteria (Mahler et al., 2023). In addition to phage engineering, many researchers have also modified the capsid of the phage protein to display functional proteins (phage display) by adding amino groups, decoration proteins, and chemical modifications by bio conjugating functional groups (Carmody et al., 2021; Peng and Chen, 2021). Chemical modifications are generally less complicated than genetic engineering, yet there might be chemical changes induced that could interfere with the phage’s functions (Peng and Chen, 2021).

Despite the various methods used to develop and design phages with favorable features, achieving phages with desired functionality essentially depends on the type of phage used and the molecules or cells to be displayed or targeted by the modified phage. Hence, the modification of the phage sequences or genes needs to be well considered and accurate in ensuring the required specificity of phages for their targets. As mentioned before, different phages work differently and so the suitable phage must be carefully chosen to meet the targeted purpose in studies. Ergo, understanding the phage itself as well as the target molecules or cells are detrimental in achieving high quality phage with expected functionality and reliability.

During the past 20 years, gene transfer to stem cells has led to optimism that gene therapy approaches can provide long-lasting therapeutic benefits. Numerous research papers have advanced our knowledge of individual stem cell behaviour in various tissue environments. Genetic modification has played an important role in cellular biology studies that seek to elucidate cellular mechanisms and pathological processes (Reiser et al., 2005). The increased understanding of biological signaling pathways and networks has led to the emergence of a completely new area of synthetic biology where single or multiple gene transfer to cells, assigning new functions, and potentially affecting the entire metabolism of multicellular organisms (Vargas et al., 2016). Biological vectors consist of viral vectors and non-viral vectors that are used to deliver genetic materials by infecting the cells (Karami et al., 2023).

Viruses have been developed to be very efficient in providing nucleic acids to certain types of cells while eliminating immunosuppression by the infected host. These characteristics make viruses an ideal vector for gene therapy (Robbins and Ghivizzani, 1998). Gene therapy involves the delivery of specific genetic materials (DNA or RNA) via a carrier called a “transmission vector” that allows foreign genetic material to enter target cells. Viral vectors are composed of retroviruses, adenoviruses, lentiviruses, and adeno-associated viruses. Viral vectors cover a wide range of applications, including short-term expression delivery vehicles and long-term expression delivery vehicles. Both RNA and DNA viruses are represented, with a single- or double-stranded genome (Lundstrom, 2018). Nonviral vectors are composed of exosomes, piggyBac transposons, mRNAs, and plasmid DNAs.

Despite all the advancements in stem cell differentiation, there are concerns about the safety and efficacy of design molecules. For aptamer, the constructs has slightly inferior structural and functional properties compared to native meniscus (Li et al., 2022). Apt19S alone is not sufficient for osteogenic differentiation, as osteoinductive growth factor BMP2 is needed (Sun et al., 2021). Besides, rapid clearance of aptamer may lead to loss of targeting efficacy in the human body (Byun, 2021), and RBM-007 aptamers were also highlighted to have uncertain efficacy which needs further evaluation in human studies (Kimura et al., 2021). Also, the use of unnatural nucleotides or bulky moieties for chemical modifications can evoke safety risks, so should be avoided when necessary (Ni et al., 2021).

For phage, short clearance rate poses challenges in research studies since the quick elimination of phage from the system reduces the phage efficacy (Caflisch et al., 2019). Although the use of M13 phage displaying functional peptides seems promising in improving engraftment and angiogenesis (Jang et al., 2020), had also noted some unexpected in vitro results of the pre-treated hCPCs. Plus, there are concerns of side effects and development of phage-resistant bacteria due to genetic mutation (Caflisch et al., 2019). Most phage genes are also still of unknown function which may cause undesirable events (Monteiro et al., 2019), and the methods of administration and dose of bacteriophage used are in need to be deliberately considered (Podlacha et al., 2021). Additionally (Peng and Chen, 2021), had highlighted the possibility of inducing chemical changes towards chemically modified phage which could interfere in the phage functionality. Hence, these issues highlight the importance of more evaluations and studies to be carried out to secure its safety and efficacy.

Viral vectors which have their origin in wild type viruses, can be used as treatments or prophylactics (Shirley et al., 2020). Effective prophylaxis may be crucial in controlling environmental risk assessment. It is important to assess if the alteration will lead to a lower sensitivity of viral vector to prophylaxis that is effective against the wild type (Reuter et al., 2012; Baldo et al., 2013). Characterizing the genetically modified virus (GMV) is crucial in identifying hazards. If the virus is attenuated through modification, the risk group of the viral vector maybe lower than the wild-type virus from which it is derived (Baldo et al., 2013). Transgene characteristics must also be considered when assigning recombinant vectors to risk groups. One method of effectively attenuating the viral vector is minimizing self-propagation in target cells while maintaining the ability to introduce the gene of interest (Rakowska et al., 2021).

Non-viral vector are used to prevent endonuclease destruction of the genetic material, which allowing nuclear absorption and vector unpacking (Zu and Gao, 2021). However, their larger particles can hinder their effectiveness, as DNA must be transported into the nucleus for therapeutic impact (Torres-Vanegas et al., 2021). Understanding the physiochemical and biological characteristics of genetic material and the carrier, and the molecular mechanistic understanding of vector-induced transfection, is crucial for creating and effective and safe non-viral vectors (Jones et al., 2013). Toxicology should be considered, as nanoparticles may cause an immunological response, despite being less immunotoxic than viral vectors. Additionally, the pH and ionic strength of the formulation buffer can affect vector transfection efficiencies. Gene therapy formulations are often stored by freezing or refrigeration which can increase temperature before delivery and reduce efficacy (Kafetzis et al., 2023).

Regenerative medicine is a field that uses stem cell technologies, and physical and chemical interventions to treat diseases. However, challenges remain, such as obtaining stem cells and controlling cell biological behavior. Techniques and technologies have been developed to direct stem cell differentiation, including designing molecules like aptamers, phages, and biological vectors.

Aptamers demonstrate robust plasticity and broad transformation potential for applied regenerative medicine research (Luo et al., 2022). However, challenges remain, such as cost control, developing new types of aptamers, increasing screening efficiencies, and optimizing connections with other biomedical materials. Future research should focus on solving the above issues to advance the clinical application of targeted aptamer-based therapeutic strategies. Aptamer-based targeted therapy is an approach that may overcome the limitations of current therapeutic strategies, so this area deserves further research attention (Liu et al., 2023).

In the era of synthetic biology, engineered phages have improved therapeutic properties, with the ability to carry long chain DNA or display protein, making them ideal for vaccine development and gene therapy (Tao et al., 2019). The TXTL cell-free synthesis method has made significant strides in recent years, and the CRISPR-Cas system has demonstrated its usefulness for various application due to its non-propagative and genome independent properties (Azam et al., 2021).

Advances in vector design have enabled vectors with better systemic distribution and spatial control for gene expression, driving gene therapy into clinical trials for a wide range of diseases (Nelson et al., 2020). A viral vector should meet safety, easy manufacturing, efficient reach to target cells, and reproducibility in animal model before being used in human clinical trials (Nance and Duan, 2018). Appropriate laboratory and manufacturing practices are necessary for bringing viral therapeutics into clinics with quality assurance required at every stage of production, validation, and storage.