Multipotent Human Mesenchymal Stem/Stromal Cells: An Updated Review on Historical Background, Recent Trends and Advances in their Clinical Applications

Early reports demonstrated the presence of cells with stem-like properties in bone marrow, with these cells having both hematopoietic and mesenchymal lineages. Over the years, various investigations have purified and characterized mesenchymal stromal/stem cells (MSCs) from different human tissues as cells with multi-lineage differentiation potential under the appropriate conditions. Due to their appealing characteristics and potential, MSCs are leveraged in many applications including medicine, oncology, bioprinting and as recent as treatment of COVID-19. To date, reports indicate mesenchymal stromal/stem cells have varied differentiation capabilities into different cell types and demonstrate immunomodulating and antiinflammatory properties. Reports indicate that different MSCs microenvironments or niche and the resulting heterogeneity may influence their behavior and differentiation capacity. The potential clinical applications of mesenchymal stromal/stem cells have led to an avalanche of research reports on their properties and hundreds of clinical trials being undertaken. The future looks bright and promising for mesenchymal stem cell research with many clinical trials under way to prove their utility in many applications and in the clinic. This report provides an update on the potential broader use of mesenchymal stromal/stem cells, review early observations of the presence of these cells in the bone marrow and their magnificent differentiation capabilities and immunomodulation.


Mesenchymal Stem/Stromal cells: What's in a Name?
Early reports indicated that the bone marrow contained both haematopoietic stem cells as well as mesenchymal stromal/stem cells (MSCs) [1,2]. Classic transplantation experiments by Friedensten and colleagues demonstrated that cells from the bone marrow can create a bone marrow microenvironment in the kidney as well as formation of bone tissue [2]. This indicated that beside haematopoietic stem cells, there was an additional stem cell type, later identified as mesenchymal stromal/stem cells, a term coined by Caplan in the 1990s (Figure 1) [1][2][3][4].
Mesenchymal stem cells (MSCs) have the ability to grow in vitro, display enhanced differentiation capabilities to form the connective tissues present in different organs compared to other cells and release large amounts of biomolecules such as growth factors and cytokines [4,5].
MSCs have been called by various names and experiments have shown that these cells demonstrate different degrees of stemness and differentiation capacities, capacities that appear to diminish with age [6][7][8]. In addition to mesenchymal stem cells, these cells can be referred to as mesenchymal stromal cells as well as multipotent stromal cells among many names [9][10][11]. Recent efforts have been made to name MSCs based on their mechanism of action, which is mainly secretion of various biological molecules including growth factors and cytokines [12][13][14]. In this regard, MSCs can be seen as cells involved in influencing cellular signaling [12,13]. It is important to note that MSCs differ in their origins, differentiation ability, functions and consequently their therapeutic uses. As early as 2006, the International Society for Cellular Therapy (ISCT) defined MSCs as multipotent mesenchymal stromal cells and published a set of conditions needed to be fulfilled for cells to be called such. These conditions included the expression of several surface antigens such as cluster of differentiation 90 (CD90), CD 105 as well as being able to grow in vitro as substrate-adhering cells [9,15,16]. As we demonstrated in our earlier report in addition to other reports, MSCs can differentiate into adipocytes, chondroblasts and osteoblasts as well as lacking CD14, CD34 and CD45 expression [9,16].
Additional markers also proved their utility in the isolation and identification of MSCs. For example, CD106, CD146 and stromal-1 antigen (STRO-1) are additional markers used to identify MSCs in vitro and are expressed by cells with differentiation abilities into multi-lineages [17][18][19].
The term 'mesenchymal' present in many of the names given to MSCs comes from the word 'mesenchyme' which is used to describe cells from the mesoderm of a developing embryo. These cells migrate and are distributed throughout the body of the developing embryo [4]. In adults, the mesenchymal cells forms the connective tissue including cartilage, muscle and bone marrow [20]. These cells are characterized based on their expression of markers such as fibronectin, laminin B1 and vimentin [21,22]. It is important to note that the origin of MSCs hasn't been proven since their discovery. Several reports have shown that MSCs can also be derived from cells of the ectoderm [23][24][25][26]. Different MSC cells have been identified based on expression of markers such as Nestin as well as cell proliferation in bone marrows of developing embryos, with some cells being derived from the mesoderm whilst others are derived from the neural crest [26].
Thus the origin of MSCs is broad-based and is linked to cells of different germ layers. In summary, the germ layers from which MSCs are derived determine their final function in the adult body. Figure 1 Historical background of Mesenchymal Stem cells. Cells isolated from the bone marrow were shown to form clonogenic colonies. Studies later showed that cells from the bone marrow can differentiate into the connective tissue.

Sources and Niche of Adult Mesenchymal Stem/Stromal Cells
Well-known sources of mesenchymal stromal/stem cells include the bone marrow, umbilical cord tissue, adipose tissue as well the placenta ( Figure 2) [27, 28]. In most cases, some of these tissues do not raise ethical issues regarding harvesting of cells as they can be considered medical waste for example during liposuction and after child-birth [29][30][31][32]. Several factors such as the final use of the isolated MSCs, the differentiation potential of the MSCs and the total number of cells needed for the application can influence the source of the cells [33][34][35].   [57][58][59][60][61]. Overall, the interconnectivity between MSCs and pericytes require further investigations.
Cell-cell interactions are important within MSCs' in vivo niche for the maintenance of stem cell properties and cadherins such as N-cadherin have been shown to play key roles [62,63].
Migration of MSCs from their niche result in new environments characterized by increased ECM molecules including collagens and proteoglycans [64][65][66]. To be able to interact with ECM molecules, MSCs express integrins and are able to form focal adhesions [67,68]. Culture of MSCs in vitro induce cellular changes and influence differentiation into specific lineages. For example, culture on rigid surfaces can induce osteogenic and adipogenic differentiation [9,69,70]. Soft tissues and surfaces induce MSCs towards the myogenic and neurogenic lineages [71][72][73][74][75]. Thus the stiffness of a surface or microenvironment can influence the expression of markers and shape of MSCs both in vivo and in vitro ( Figure 3) [76][77][78]. Generally, a stiff surface or environment tends to favour MSC differentiation into lineages associated with stiff tissues such as bones [69,78]

Heterogeneity of MSCs
MSCs display great heterogeneity in terms of their functionality and consequently application despite their sharing of several characteristics. Thus, whilst MSCs from different tissues meet the minimum criteria needed for classification as MSCs, those isolated from different tissues and developmental stages, display differences that impact on their use [79][80][81]. Initially isolated from the bone marrow, MSCs can now be obtained from a variety of sources such as Wharton's Jelly, adipose, blood, placenta and amniotic fluid ( Figure 2) [82][83][84][85][86]. MSCs from fetal tissues tend to proliferate at a higher rate and have longer telomeres than those from adult tissues [87,88]. In addition, several reports also indicate that fetal MSCs have a higher differentiation potential than adult tissue-derived MSCs [89][90][91]. Furthermore, MSCs from fetal tissues can proliferate in vitro for a longer period of time before displaying characteristics of senescence [91]. In contrast, MSCs from the bone marrow and adult adipose tissue display a higher level of stemness as demonstrated by the formation of more colony-forming units compared to fetal tissue-derived MSCs [92][93][94][95]. In a detailed analysis of MSCs from different sources, Heo and colleagues demonstrated that MSCs from bone marrow express DLX5 and Sox2 needed for brain neuron development [96]. In addition, the authors showed that bone marrow-and adipose tissue-derived MSCs displayed similar differentiation capacities as well as gene expression. MSCs from different sources also display multi-lineage differentiation as well as immunomodulatory behaviours. Kern and colleagues demonstrated that MSCs from the bone marrow and adipose tissue can easily be isolated compared to MSCs from the umbilical cord blood [97]. In addition, the authors also demonstrated that umbilical cord blood MSCs showed no adipogenic differentiation abilities compared to those from the bone marrow and adipose tissue [97]. Several studies including our own have demonstrated that indeed, adipose-derived MSCs can undergo multi-lineage differentiation under different conditions [9,[98][99][100].
MSCs from different donors and of different ages also display great heterogeneity. Phinney and colleagues demonstrated that bone marrow-derived MSCs from different donors show differences in differentiation capabilities as well as osteogenic potentials [101]. The authors showed that MSCs from different donors show different growth rates and alkaline phosphatase activity in culture [101]. In addition, the authors postulated that MSCs heterogeneity and differentiation potential were also influenced by method of harvest used. For example, Pezzi and colleagues demonstrated that oxygen levels impact on MSC heterogeneity in vitro and this has huge influence on long term culture of MSCs for therapy [102]. Other reports have postulated 7 that MSCs maybe a product of long term in vitro culture [102][103][104]. Furthermore, other reports demonstrate bone marrow derived-MSC heterogeneity due to age of donor, with more MSCs from older donors undergoing apoptosis and having a low proliferation rate than those from younger donors [105][106][107]. Naïve MSCs have been shown to exhibit heterogeneity based on transcription data [108]. If MSCs are to be used for therapy there may be need to synchronise the cells via stimulation or growth in media with no growth factors [103,109,110]. 'Priming' of MSCs through the use of a stimulus must be done with caution as some factors can induce undesirable effects such as apoptosis and senescence [111][112][113]. Current data show that priming of MSCs can be done via the use of cytokines, drugs, growth factors, biomaterials and the extracellular matrix as well as hypoxia. When cultured in vitro, MSCs display three main morphologies. Colter and colleagues as well as others demonstrated that beside the fibroblastic spindle shaped cells and the large flat cells, a third type of cells characterized as small, round and having enhanced self-renewal abilities also exists in vitro [114][115][116][117]. Studies now show that cells from a single colony are not all the same and can demonstrate multipotence, dipotence or unipotence [104]. Thus, a single MSC colony can give rise to different cell types with varied differentiation potentials. With publication of different data sets, it has been theorized that even within MSC populations, cells exist in a hierarchical structure, with some remaining as unipotential or dipotential whilst others become multipotential, a characteristic which they can eventually loose [118][119][120][121]. Several pieces of evidence suggest that the transformation of MSCs may be linked to a lower proliferation rate and decreased expression of markers such as CD146 [122][123][124].

MSCs and the Immune System.
Reports over the years have shown that MSCs have immunomodulatory properties. MSCs were shown to influence the behaviour of cells involved in immune responses and to be able to impact a lot of cellular processes ( Figure 4) [125]. MSCs achieve their immunomodulating effects via prevention of apoptosis in native and activated neutrophils, preventing the action of peripheral blood mononuclear cell proliferation, preventing the recruitment of cells at wound sites as well as preventing the interaction between neutrophils and vascular endothelial cells (Table 1) [126][127][128][129]. Several reports have shown that native or transformed MSCs release several cytokines including TGF-B, which can impact other cells such as tumor cells and neutrophils [5,130,131]. Furthermore, MSCs have been shown to release various chemokines involved in recruiting macrophages and neutrophils to wound areas [132][133][134][135]. Brown and colleagues demonstrated that MSCs prevent mast cell degranulation and limit cytokines secretion by these cells [136]. In addition, MSCs can inhibit the proliferation of natural killer cells and have been shown to suppress natural killer cell cytotoxicity [137,138].
Several MSC-secreted factors have been shown to prevent the differentiation and maturation of blood monocytes into dendritic cells as well as preventing dendritic cells from migrating to tissues [139][140][141]. MSCs have been shown to induce transformation of M1 macrophages into M2 macrophages, via nuclear factor-kB (NF-kB) and signal transducer and activator of transcription 3 (STAT3) pathways, with anti-inflammatory properties [142,143]. The resulting M2 macrophages are anti-inflammatory and release cytokines such as interleukin 10 (IL-10) that can prevent T cell proliferation [142,144,145]. Glennie and colleagues demonstrated that bone marrow-derived MSCs can inhibit growth of activated T cells and lymphocytes [146]. Overall, MSCs induce a reduction in synthesis and release of cytokines that promote inflammation and increase the synthesis of anti-inflammatory cytokines by T-lymphocytes [147,148]. MSCs have also been shown to prevent the differentiation of CD4-positive T lymphocytes into T helper cells, whilst inducing differentiation of T cells into CD4-positive regulatory T cells [149,150]. It has been shown that MSCs suppress chemokines expression by B lymphocytes and this impact the ability of the B lymphocytes to migrate [151,152]. Finally, MSCs have been shown to inhibit the synthesis of several immunoglobulins by activated B cells and in the process prevents formation of plasma cells [153][154][155].  Downregulate the synthesis of inflammatory cytokines by lymphocytes whilst inducing anti-inflammatory cytokine synthesis.
Prevents naïve lymphocyte differentiation into T helper cells.
Inhibit the synthesis of various immunoglobulins. [146,147,149,150] MSCs Macrophages Conversion of M1 macrophages into M2 macrophages that synthesize IL-10, which inhibit proliferation of T-cells.
Induce migration of macrophages and recruitment to injury sites.
Suppress natural killer cell cytotoxicity activity. [137,138] MSCs Neutrophils Reduce binding between neutrophils and vascular endothelial cells.
Prevents neutrophils from mobilizing at injury sites.

Paracrine Properties of MSCs
Recent reports show that MSCs secrete several factors that act in both autocrine and paracrine fashion. Thus, one area of MSCs being investigated thoroughly is that of provision of factors rather than the differentiation of MSCs into different lineages. If MSCs can release factors necessary for immune modulation, tissue repair and wound healing, then MSCs can be useful in various ways. One primary function of secreted factors is the regeneration of damaged or diseased tissues [11,[160][161][162][163]. Factors secreted by MSCs include growth factors, enzymes, cytokines, chemokines as well as ECM proteins [133,161]. MSC-secreted factors act in a context-dependent manner. For example, secreted factors such as VEGF and TIMP-1 and TIMP-2 are known to regulate angiogenesis in opposing ways, with VEGF promoting angiogenesis whilst TIMP-1 and TIMP-2 inhibit angiogenesis [164,165]. Additionally, VEGF secreted by MSCs is known to influence other processes such as fibrosis and apoptosis [166][167][168]. MSC-derived VEGF, IGF-1, IGF-2 and HGF have anti-apoptotic effects [169][170][171][172][173]. Thus, an increase in the expression of one factor can have multiple effects on the tissue into which it is released. Cantinieaux and colleagues demonstrated that conditioned media from bone marrowderived MSCs can protect neurons from apoptosis as well as activates macrophages [164].
Menezes and colleagues demonstrated that MSCs can recruit pericytes and induce angiogenesis via release of factors such as VEGF during the repair of spinal cord injury in rats [174].
In classic co-culture experiments of both esophageal and breast cancer cells with Wharton Jellyderived MSCs, we demonstrated that MSCs reduced the effects of paclitaxel and cisplatin on cancer cells [5]. One factor that was released in large quantities by both cancer cells and MSCs was TGF-B and was involved in transformation of MSCs into cancer-associated fibroblasts (CAFs) [5]. Thus MSCs protected cancer cells from drug-induced apoptosis. Several other pieces of evidence substantiated our findings [175]. For example, Eliopoulos and colleagues demonstrated that bone marrow-derived MSCs also reduce the renotoxicity of cisplatin in mice [176]. Bergfeld [179]. In yet another study, Ohlsson and colleagues showed that mesenchymal progenitor cells can inhibit tumor growth when grown in a gelatin matrix [180]. Furthermore, Maestroni and colleagues demonstrated that factors secreted by bone marrow-derived stromal cells can inhibit the growth of Lewis lung carcinoma and B16 melanoma cells in mice [181]. The contrasting data as presented above demonstrate the importance of accurate reporting and understanding the effects of MSCs and factors they release on cancer cells [182,183]. In addition, one of our studies showed that Wharton's Jelly-derived MSCs when cultured on an ECM activate apoptosis in in a p21dependent mechanism in esophageal and breast cancer cells [10]. It appears the effect of MSCs on cancer cells is context-dependent, may depend on the paracrine factors released and requires further investigations.

Mesenchymal Stem Cell-derived Extracellular Vesicles
MSCs have also been suggested to secrete extracellular vesicles (EVs), through which they can relay signals and cues to other cells. These extracellular vesicles may include apoptotic bodies, microvesicles and exosomes. The biomolecules and other factors that are carried by EVs also called cargo are largely dependent on the cell type from which they originate, although some reports indicate some processing can take place during transportation. EV cargo is composed mainly of lipids, nucleic acids, and proteins and thus mainly functions to regulate cellular processes, cellular functions, immune response and also the maintenance of homeostasis [201,202].  [215]. Furthermore, Guo and colleagues showed that MSC-derived EVs containing miR-130b-3p promotes the progression of lung cancer [216].

MSCs Differentiation Potential
One of the criteria used to characterize MSCs is the ability to differentiate into multi-lineages ( Figure 5) [9,11,21,54,210]. Several reports including one by Dominici and colleagues spelt out the minimum criteria required for cells to be defined as MSCs as stipulated by The International Society for Cellular Therapy [16]. As we showed in our early publication, this can be achieved through culturing the MSCs in differentiation media with specific supplements and then evaluating adipogenesis markers, osteogenesis markers and chondrogenesis markers [9]. and Sox9 as well as staining with Toluidine Blue O for proteoglycans that are visible as purple [9]. These conditions have been utilized by several laboratories worldwide and are the stipulated conditions for such analysis [28, [217][218][219]. Beside these differentiating conditions, MSCs are also characterized based on expression of several surface markers as we showed in our study.
Specifically, our data show that our adipose-derived MSCs expressed markers such as CD73, CD90, and CD 105, with no expression of CD34 and CD45 [9]. In addition, we also utilized and characterized Wharton's Jelly derived MSCs [10]. Flow cytometric analysis of Wharton's Jelly-derived MSCs using antibodies against CD44, CD45, CD73, CD90 and CD105, showed that our MSCs fulfilled the minimum requirements for MSCs as stipulated by the ISCT [10]. Cells characterized in our laboratory demonstrated the classical MSC phenotype of CD44+CD73+CD90+CD105+CD45-cell population [10]. Furthermore, lineage specific differentiation capacity of Wharton's jelly-derived MSCs fulfilled stipulated criteria by the ISCT. Figure 5 The differentiation potential of mesenchymal stem cells.
In addition to having multi-lineage differentiation potential, MSCs have been shown to transform to other cell types, given the right cues or signal. in markers such as Runx2, osteopontin, p-TGFB-RII [9]. Furthermore, our study showed that the use of siRNA and a mutant Notch1 construct showed that the mechanism through which MSCs differentiated towards chondrogenic phenotype involved Notch1 and β-catenin signaling [9]. Overall, these and other studies indicate that Cs, given the right cues and signaling molecules, can differentiate into multi-lineage cells and form several tissues of the human body.

Challenges, Prospects and Conclusion
The discovery that the bone marrow contained more than just hematopoietic stem cells, initiated a frantic study of these cells, resulting in the realisation that some cells within the bone marrow can form the connective tissue of the body. Mesenchymal stem cell research entered an exciting period mainly due to their appealing properties including the easy accessibility and multi-lineage differentiation. Further studies revealed that MSCs have immunomodulatory properties and can be a source of many difficult-to-repair cells and tissues, astounding many scientists in the process. Ever since the discovery of MSCs, these cells have been receiving great attention in different fields of biology from regenerative medicine, cancer research and even the treatment of infectious diseases [10,228]. It is important that MSCs prepared for therapy are properly 'synchronized' or 'tuned' in order to provide optimum effect. Factors such as MSC isolation method, culture method, metabolic state and doses used must be carefully considered during treatment. Thus, MSCs intended for different applications must be prepared differently to increase 'therapeutic effect'. Knowledge of the 'microenvironments' that MSCs are likely to encounter when in vivo, means that the MSCs can be subjected to the same treatments or conditions during preparation time. To overcome challenges such as donor differences in MSCs characteristics, longitudinal culture analysis can be done together with genetic tagging. In addition, MSCs intended for tissue repair may benefit from 'priming' for certain environments, can be grown on specific surfaces such as the ECM as we have done previously [9]. MSCs can be 'tuned' for specific therapeutic use or they can be used to provide extracellular vesicles with a specific biological factor(s) 'package'. Furthermore, cells do not perform tissue repair in isolation.
If MSCs are intended for tissue repair or regenerative purposes, co-culture or co-transplantation with other cells may increase 'therapeutic effect' or engraftment and differentiation. Based on our studies and those by others, the use of decellularized extracellular matrices and other scaffolds may increase both MSCs differentiation and engraftment [9,10,51,176,184,229].
Although MSCs can be isolated and purified effectively, they comprise a very small fraction of cells in tissues. Once isolated, MSCs will therefore require in vitro expansion, a process called Biobanking. In most cases Good Manufacturing Practises are adopted and the isolated and In order to curtail the mushrooming of unapproved MSC treatment, new and strict regulatory practices are now in place. New rules require that any medical product preparation with living cells must first seek 'investigational new drug' status before being used in clinical trials for example. The EMA has the Centralized Marketing Authorization, a series of steps involving quality checks, safety checks and final approval before any MSC-containing product can be released to the market. Based on current trajectory, the MSC products market is set for a boom.
Soon it appears many MSC products will be available in the market for the treatment of various conditions. Many global health challenges including disease like cancer, HIV/AIDS, TB and degenerative diseases still remain with modern medicine and drugs unable to provide cure for [230][231][232]. Hence the search for solutions continues and MSCs may offer a new and untried way to treat these conditions. It is our belief that advances in the MSC industry and products will transform medicine and provide innovative strategies for different conditions.