1.Introduction of MSCs

MSCs is a common acronym used to describe mesenchymal stemcell, Mesenchymal Stromal Cell, orMedicinalSignalingCell. However, the debate is still ongoing over which of these long names best describes MSCs [1]. They are an example of “adult” stem cells that could be derived from various tissue types.

MSCs have been isolated from almost all tissues [1] and have been reported to play critical roles in many physiological processes, such as tissue homeostasis, immunomodulation, and tissue regeneration [2].

Since the famous publications by Alexander Friedenstein et al., on MSCs, half a century ago, mounting evidence has been accumulating that bone marrow (BM)-derived MSCs are capable of differentiating into other cells of mesenchymal lineage (e.g., adipocytes, osteoblasts, chondroblasts, myocytes, and tenocytes, etc.,) ^[3][4][3,4]. The authors were able to isolate the plastic-adherent spindle-shaped cells that were capable of self-renewal and showed a multi-differentiation potential.

Later on, more reports unveiled potential pluripotency where these cells can transdifferentiate into cells of other lineages, endodermal (e.g., muscle, lung, and gut cells, etc.), and ectodermal (e.g., epithelial, and neural cells) Another interesting feature of MSCs is their homing ability, meaning that they can migrate into injured tissues where they can contribute to the physiological processes in ways more than one. They can differentiate into various local cell types at the injured sites, (ii) they can secrete chemokines, cytokines, and growth factors that help in tissue regeneration, (iii)

In addition to BM, MSCs can be obtained from various sources such as, adipose connective tissue, synovial fluid, hair follicles, dental pulp, salivary glands, amniotic fluid and membranes, endometrial lining, peripheral and menstrual blood, placenta and fetal membranes, umbilical cord blood, and Wharton’s jelly ^[5][6]. Therefore, due to the above-mentioned appealing features, MSCs have quickly made the transition from benchtop to bedside ^[6][7].

To clearly define MSCs, and develop universal criteria for such cell population, the Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy (ISCT) proposed a set of standards for pre-clinical research studies ^[7][8].

The minimal criteria of MSCs as determined by the ISCT are the following ones:

The MSCs population must be plastic-adherent when maintained in tissue culture vessels under standard culture conditions.

Nevertheless, such historical criteria have not been always correlated with the applicability of these cells in various biomedical purposes. For instance, while CD markers might stay consistent over successive passages, MSCs tend to lose their differentiation or immunomodulatory capabilities ^[8][9][9,10].

Despite the aforementioned criteria, ISCT now suggests considerable flexibility, particularly when it comes to MSCs the lack of expression of the HLA Class II marker is conditionally expressed once stimulated by specific cytokines.

Therefore, it is crucial to have the process of MSCs characterization well-standardized to enable accurate comparison of study outcomes and to guarantee safety and efficacy in the field. Unfortunately, to date, no single marker has been identified as being exclusively expressed by MSCs ^[10][11]. Yet, the number of MSCs markers (positive and negative) is expanding over time to help researchers verifying the MSCs features, thus increasing the confidence in the obtained/transplanted cells.

In addition, various research teams have developed and expanded innovative molecular markers (e.g., proteomic and epigenetic markers, transcriptome analysis, gene signature, etc.,). Despite all these trials to address the thorny question about MSCs identity, there is still little consensus on these characterization methods. Therefore, Arnold I. Caplan ^[11][12] has recently suggested the insignificance of characterizing every cell in every MSCs population in vitro. The author believes that most of the propagated MSCs populations have become culture-adapted and can no longer display their innate (in vivo) features, nor their therapeutic behavior, once transplanted.

2. MSCs as a Gene Delivery

The Food and Drug Administration (FDA) has defined gene therapy as “the administration of genetic material to modify or manipulate the expression of a gene product or to alter the biological properties of living cells for therapeutic use.” An essential aspect of gene therapy depends on designing a suitable gene delivery system to convey the cargo gene into the target cells. More than half a century after their introduction as a novel therapeutic approach, and despite some adverse effects seen in clinical trials, the concept of gene therapy remains to be acknowledged as a promising therapeutic alternative for various clinical disorders. However, the obstacles encountered have fueled research efforts that led to the improvement of gene carriers in terms of their efficacy and safety profiles.

Over the past decades, genetically engineered stem cells were feasibly used in cell-based gene delivery, providing long-term therapeutic effects. Furthermore, continuous research efforts have been directed toward understanding the behavior of individual stem cells in different tissue microenvironments, in vivo ^[12][17]. In parallel, the implementation of more accurate assays for MSCs and enhancement in gene vehicles have increased gene transfer efficiency. Nevertheless, quality control of the protocols applied in human gene therapy remains crucial, especially when cells are used as a gene carrier for the treatment of hereditary and acquired diseases.

For successful gene delivery to MSCs, the proper choice of the deliverable nucleic acid, as well as the delivery carrier/method, will determine the transfection outcome. Therefore, in the following section, we will review different types of exogenous nucleic acid cargo along with various non-viral nanocarriers used with MSCs.

Nucleic acids act as drugs that aim to treat and/or prevent countless intractable diseases, such as cancer, cardiovascular, neurodegenerative diseases by adding, replacing, editing, or even inhibiting specific target genes or their products ^[13][20]. Currently, therapeutic nucleic acids could be roughly classified according to their different structures into DNA and RNA drugs. Therefore, various therapeutics were developed and are now commercially available for various diseases (summarized in table 1).

Note: Antisense oligonucleotides (ASOs), small interfering RNAs (siRNAs).

Despite such achievements, myriad challenges remain to be overcome before their impact on patient’s care is fully understood. In this section, we have discussed some of the most popular nucleic acids used to transfect MSCs, highlighting their advantages and disadvantages (Summarized inTable 2)

Table 2. A summary of nucleic acids used to transfect MSCs: The advantages and disadvantages.

Plasmid-based gene therapy was attempted to correct single-gene disorders. On a molecular level, plasmids are circular, double-stranded DNA constructs varying in size from <1000 to >200 000 bp containing transgenes. Therefore, plasmid design can dramatically influence transgene expression ^[34][23]. ^[16][35][25,26] genes.

The decreased backbone size was shown to be directly correlated with the levels and extent of transgene expression in mammalian cells ^[18][29]. When compared to pDNA, Maria Florian et al., demonstrated that angiopoietin 1 (ANGPT1) encoded in mcDNAs -transfected MSCs could attain notably higher and prolonged secretion levels of ANGPT1 protein, resulting in superior therapeutic effects animals with acute lung injury ^[20][31]. On the other side, Serra J and team reported insignificant differences in transfection results in BM-MSCs with mcDNAs Efficient nuclear transport is still required to achieve notable transfection efficiency ^[18][29].

Nevertheless, the protein expression takes place for a shorter duration, which demands repeated transfection. To this end, BM-MSCs were transfected with mRNAs encoding several reprogramming factors (e.g., Oct4, Klf4, Sox2, cMyc, and Lin28) resulting in the formation of iPSC colonies ^[36][34]. Moreover, mRNA transfection is being used to simultaneously express multiple proteins such as in the study of Wenbin Liao et al. Such breakthrough would not have been possible without critical recent innovations in the production of high-quality mRNA as well as the development of safe and efficient materials for in vivo delivery.

3. Applications of Engineered MSCs