Whether in vitro grown Mesenchymal stem cells represent any stage of natural in vivo MSCs, or similar cells observed during development, is a hot topic of discussion. It is important to keep in mind that the formation of mesenchymal tissues during embryology is complicated and involves both the trunk and head (produced from the neural crest) mesenchyme; these two cell sources are intertwined in some tissues, such as the heart. Cellular differentiation inside a developing organism would appear to be deterministic: we can anticipate the fate of similar cells in the following generation’s children, all subsequent generations’ offspring, or even in distant or unrelated species. However, numerous investigations in embryology have shown that cells from one presumed tissue can be implanted in different tissue and take on a different fate.

The new environment controls their destiny locally, and the implanted cells’ subsequent development is selective rather than directive. This is a quality of stem/progenitor cells like MSCs, not a defect. Early development may have something to teach us about using cultured Mesenchymal stem cells for repairing and regenerating adult tissues, but it is currently unclear what that will be. The early human developmental biology of mesenchymal tissues represents a very specific series of temporal events and is far removed from the tissue repair that occurs in the adult 15–80 years later.

Regrettable increase in unlicensed stem cell clinics

Although MSCs and other stem cells have incredible promise, our knowledge of their science and medicinal uses is not yet sufficient for unrestricted, widespread use. Given the complexity of tissue repair and cell replacement, it is obvious that the proliferation of dubious “stem cell clinics” and offshore medical tourism facilities advertising their autologous “stem cell treatments” with unknown and unproven efficacy will not significantly alleviate patient ills. According to Galipeau et al., the distance between reliable clinical trials and the early public marketing of stem cell treatments has grown, creating misunderstanding in the media. There are about 700 clinics that promote “stem cell” therapies directly to consumers. Any treatment using Mesenchymal stem cells that do not use defined cell products, keep accurate records, monitor intermediate parameters, use predetermined surrogate endpoints, track and report final patient results, and maintain accurate records is not something we can support or advocate (s). These procedures are standard in FDA-registered trials, but they are too onerous for clinics that are poorly or unregulated regulated. With the acronym DOSES (donor, origin tissue, separation method, exhibit characteristics, site of delivery), a recent study has provided a consensus report on the elements required to enhance the outcomes of cell treatment for both patients and practitioners.

Microheterogeneity, temporal stochasticity, and variety at the single cell level in Mesenchymal stem cells cell biology

A vertebrate stem cell is distinguished by its capacity for symmetrical or asymmetrical division, motility, differentiation into many lineages, and organization into multifunctional clusters. Stem cells need an environment that is both receptive and instructional to become functionally organized. Therefore, the cellular environment, the timing of the application of instructive agents, and their persistence are all necessary for the phenotypic reprogramming of stem cells. This characteristic is demonstrated in MSCs by their gradual acquisition of osteogenic, adipogenic, or chondrogenic characteristics over 1–3 weeks, as demonstrated by Terzic and colleagues. This process involves gradually altering the cultural conditions over 3–4 weeks. The results of Mesenchymal stem cell population differentiation are a composite and represent characteristics at the single-cell level, but the timing of events may differ slightly for each cell. It is well known that even clonally produced stem cell populations are not uniform; rather, the cells within them frequently act as individual cells. Stem/progenitor cells frequently exhibit this temporal stochasticity, which happens all through development. Although stem/progenitor cell stochastic events and processes are likely the hardest to model or approach experimentally, we can observe similar phenomena in vitro. A single cell in the case of Mesenchymal stem cells may undergo repeated cell division to give rise to a population with millions of cells, or it may succumb to apoptosis in response to food deprivation, DNA damage, membrane injury, etc. For instance, stochastic processes result in the loss of some clones and the proliferation of others when Mesenchymal stem cells in culture are labeled with lentivirus vectors encoding individual tags to track the fate of daughter cells. As a result, a cultured MSC population with an initial complexity of 70 is reduced to the complexity of two surviving clones, and these resulting clones do not represent the most abundant clones at the start. This reduction in MSC population complexity may not result in the loss of potential or utility while challenging our perception of culture-expanded MSCs as a homogeneous population provided all the remaining cell clones share the same stereotyped behavior or “abilities” as the initial cells. Our biology and clinical potential understanding depend on studies to determine how these events modify adult Mesenchymal stem cells and Mesenchymal stem cells’ population composition and function, concerning both their stem/progenitor and paracrine activities, in vitro and in vivo. The rise and fall of individual clones have long been observed in studies of HSC clonal activity, however, this may not affect their clinical or functional results. Strong experimental support exists for the clonal expansion/extinction process in vivo intestinal epithelial stem cells as well. Recent research suggests that MSCs may create growth factors, cytokines, and other bioactive substances that are encapsulated in exosomes and microvesicles that have paracrine functions. Exosome and microvesicle formation and composition are mainly unknown about stochastic processes, clonal proliferation, culture complexity, and MSC differentiation, even though their significance in normal MSC physiology and as therapeutic agents are emerging.

Phenomics and the Mesenchymal stem cell transcriptome

MSCs are still primarily identified by their in vitro expression of a small number of cell surface proteins and their ability to differentiate into three different cell types in response to stimuli. The field should gain from the vast collection of gene expression-based data (GEO Datasets) that examines the MSC transcriptome as well as the significant expression changes that result from culture expansion, hypoxia preconditioning, stimulus-directed differentiation, trans-differentiation, exposure to biologics, and coculture with other cell types. While this simple definition has been sufficient for almost two decades and is still widely used today. The biological makeup of MSCs, their anticipated physiological function, their part in disease pathophysiology, and their likely therapeutic mechanism of action can all be understood with the help of genome-wide gene expression investigations. Understanding MSC gene expression data holds promise for improving the operational definition of MSCs, elucidating their native physiological function, and guiding the most effective culture conditions and clinical manufacturing protocols for characterizing their composition and function prior to patient administration.

The main goal of MSC gene expression investigations was to provide a standardized identification for in vitro bone marrow-derived MSCs (BM-MSCs) that could be used by different labs. In order to achieve this, serial analysis of gene expression (SAGE) was used to catalog the transcriptome of human and mouse BM-MSCs, and it was discovered that the characteristics of the cataloged transcripts reflected their stem/progenitor properties and paracrine activities related to skeletal homeostasis and hematopoiesis support. The gene expression data support the skeletogenic, angiogenic, anti-inflammatory, and immunomodulatory actions of MSC populations that are commonly used for therapeutic therapy today, along with relevant cellular investigations. A physiological explanation for the widespread anatomical distribution of MSCs or MSC-like cells in vivo can be found in studies that demonstrate that MSCs isolated and cultured from various tissues and organs are more closely related to one another than other mesodermal lineages and share perivascular cells’ phenotypic characteristics (see below.) For MSCs to be useful in clinical settings, it is still critical to understand how much their cultural conditions can affect and regulate their gene expression.

Our knowledge of how MSCs react to stimuli that cause differentiation at the cellular level has improved as a result of recent RNA-seq investigations. The epigenome of MSC-derived osteoblasts, but not adipocytes, more closely mirrored that of naive cultured MSCs, according to ChIP-Seq studies. For instance, one such study detected significant alterations in the MSC transcriptome following differentiation to the adipogenic vs. osteogenic lineage. Additionally, it was discovered that the master transcriptional regulators RUNX2 and C/EBP, whose binding sites are epigenetically shrunk after differentiation, have a high degree of overlap in the MSC genome. These promoter regions also exhibited high plasticity, allowing MSCs to trans-differentiate from adipocytes to osteoblasts and vice versa. These transcriptional pathways might be important for how MSCs differentiate in vivo. Recent research has revealed that the osteo- and adipogenic lineages are modulated by the Wnt intracellular signaling molecule (WISP-1 or CCN4). These discoveries expand on the idea of in vitro lineage priming, which was initially used to reveal the molecular underpinnings for MSC multi-potency and may lead to more effective treatments.

Other therapies have been found that modify the biological activity of MSCs by changing gene expression in addition to differentiation-inducing stimuli. For instance, it has long been known that rodent MSCs have improved development and osteogenic potential when exposed to settings that mirror the bone marrow niche, such as low oxygen levels (5 percent). Afterward, it was established that brief exposure of MSCs to hypoxic conditions (2% oxygen saturation) increases their proangiogenic activity in vitro and in vivo and favorably affects growth and survival. Profiling studies have shown that a small group of genes associated with cell proliferation and survival, glycolysis, and vasculogenesis/angiogenesis in MSCs is dramatically altered by hypoxia preconditioning, the majority of which were elevated. To learn more about MSCs’ anti-inflammatory and immunomodulatory properties, a similar method is being employed to examine how they react to inflammatory stimuli. Using lipopolysaccharides as an example, which is a ligand for TRL4, human MSCs were stimulated in vitro. This led to the expression of transcripts involved in chemotaxis and inflammatory responses, which were primarily controlled by interferon regulatory factor (IRF1) and nuclear factor kappa B (NF-B).  As an alternative, stimulation by TNF increases the expression of the anti-inflammatory protein TSG-6, and exposure to interferon (IFN)-gamma licenses the immunosuppressive activity of MSCs by inducing the expression of indoleamine 2,3-dioxygenase (IDO1), an enzyme in the kynurenine pathway that consumes tryptophan and reduces inflammation. The immunosuppressive proteins IL-4, IL-10, CD274/PD-L1, and IDO were found to work in concert with TNF and IFN gamma to universally polarize MSCs toward a Th1 phenotype, although these proteins induced MSCs to produce different sets of proinflammatory factors. This discovery is also relevant since hierarchical clustering of gene expression data from MSC donors at the population level showed that nonstimulated populations displayed a considerably higher amount of inter-donor heterogeneity. Therefore, “cytokine priming” should be investigated in both animal and human studies. It may greatly reduce interdonor disparities in MSC function and has a normalizing effect on the population.

It is crucial to note that exposure to inflammatory stimuli like TNF and IFN-gamma generates other noticeable effects on MSCs in addition to boosting paracrine signaling. For instance, IFN-gamma has a profoundly detrimental effect on MSC growth and survival because it upregulates the expression of genes linked to cellular apoptosis and programmed cell death. A change in gross morphology is also present in these gene expression responses of MSCs to IFN-gamma therapy. IFN-gamma-induced genes have been demonstrated to be upregulated in MSCs driven toward the osteogenic lineage, and this is accompanied by a reduction in angiogenic activity. Analyzing MSCs from TSG-6 mutant mice also uncovered significant changes in cell shape, poor growth, and lack of tri-lineage differentiation capacity. 65 As compared to wild-type cells, RNA-seq analysis in this study found that 1537 genes were downregulated and 1487 were upregulated in TSG-6 null MSCs. These genes were mapped to biological processes like cell cycle, cell death and survival, cell morphology, cellular movement, DNA replication, and repair. Notably, TSG-6 deletion was found to suppress the production of numerous transcription factors that control cell division, stem cell differentiation, and Wnt signaling. These findings are in line with recent research that demonstrates the mechanistic interdependence of the pathways governing cell proliferation, differentiation, and paracrine signaling as well as their potential mutual exclusion. For instance, a recent study has shown that interleukin-1 and/or TNF treatment of horse adipose-derived stromal cells damaged their tenogenic capabilities by lowering the expression of the tenogenic transcription factor scleraxis. To forecast which culture conditions are most appropriate for a certain cellular response or a specific therapeutic indication, more research is required.

Exists a gene expression profile for MSCs that can be utilized to identify them and forecast how well they would respond to treatment? An “MSC classifier” was created using a sizable integrative study of the genomic datasets now available for MSCs, and it successfully identifies MSC samples from non-MSC samples with over 97 percent accuracy. The MSC classifier’s gene expression and protein data can make the existing definition of MSCs more precise. Similar to this, a “Clinical Indications Prediction” (CLIP) scale based on in vitro TWIST1 expression levels was recently developed using a comparative genomics approach to highlight differences in the biological activity of various donor MSCs.The CLIP scale may be useful for matching the biological activity of MSC donor populations to particular disease indications, which could improve the results of clinical research that use MSC-based preclinical illness models. Additionally, this method might examine in real time how preconditioning routines, cytokine priming, and manufacturing procedures might increase the predictability of MSC biological activity and clinical results. The analysis of repeated bone marrow isolations and comparison of the outcomes to HSCs, NSCs, and ESCs provides additional evidence for the repeatability of MSC isolation and gene expression. It was discovered that the MSCs grouped more densely than the other stem cell types examined. Another crucial differentiating factor of phenotype is believed to be the function of circular and microRNAs in maintaining MSC identity. Both BM-MSCs and ESC-derived MSCs had a wide range of expressed genes that were analyzed using RNA deep sequencing and translated proteins that were analyzed using nano-liquid chromatography MS/MS. This analysis revealed many similarities and proposed novel membrane surface proteins that may be helpful for phenotypic identification in future studies.

Remember that the FDA requests a release test relevant to the clinically proposed function of the MSCs in vivo at phase 1 and that it is necessary by phase 3 for therapeutic MSC products. Because of this, these RNA-Seq and ChIP databases and associated resources offer a toolkit that has to be more fully examined to match suitable donor MSCs, manufacturing procedures, and patients to increase response rates. This indicates that MSCs will be created as a concentrated product for each therapeutic use and will need more precise release criteria, which are likely to incorporate both an individual cell measure like flow cytometry and a population gene expression measure like the TWIST-based CLIP assay. An affordable and widely available flow cytometry assay, like that of Ribeiro et al.72, that can quickly test >10,000 individual MSCs in minutes gives a readout of both the single-cell analysis and a population (product) assay, should reflect the anti-inflammatory activity of the MSC product for MSCs for the treatment of graft vs. host disease (GVHD). But if it turns out that the therapeutic mechanism is not a cell but rather a secreted cytokine, miRNA, or factor, then more precise assays will be needed. Recently, Kaushal and colleagues were able to show that enlarged cardiac-derived cell populations (cardiac progenitor cells, or CPCs), which include an MSC-like population, adjust their advantageous exosome expression in the in vivo situation after injection into heart tissues. For the development of cellular treatments, more knowledge of the localized response(s) of ex vivo expanded progenitor cells introduced into the in vivo injured tissue context is required.

Vascular cell types that are related to MSCs

Despite intensive efforts to describe MSCs, these nevertheless stem from the prolonged cell culture conditions under which they were created. They come from tissue, but in vivo, are they “genuine” stem cells? Do tissue dissociation, adhesion to tissue culture plastic, and growth in serum-supplemented medium isolate and drive the sustained proliferation of a rare, elusive tissue-residing progenitor cell(s), or has our expertise in tissue culture led to the creation of a valuable “artifact” of the process? Although MSCs are now widely used in tissue engineering and regenerative medicine, some studies may have equated the MSC to a previously histologically identified cell in bone marrow such as the reticulocyte, Weston-Bainton cell, stromal cell, fibroblast, etc. The rare nature of MSCs makes this unlikely, and the in vivo identity(s) of MSCs remains obscure. Aside from that, MSC-like cells have been isolated from a variety of tissue sources, including harvested adipose tissue, umbilical Wharton’s jelly, placenta, skin, and the roots of shed teeth, even though bone marrow was the first tissue used for MSC isolation and was thought to be a renewable source. Studies on microvascular pericytes quickly converged with efforts to understand the innate identity of in vivo MSCs and revealed phenotypic overlaps between the two. Crisan et al. demonstrated that pericytes purified by flow cytometry from various human organs and cultured for several passages are identical to conventional, bone marrow-derived MSCs in terms of morphology, proliferation kinetics, surface antigen expression, and differentiation potential, in v Additionally, pericytes can be detected in situ with the classic MSC surface marker combination of CD44+/CD73+/CD90+/CD105+. This paradigm was later applied to the perivascular regions surrounding bigger arteries and veins, where a population of fibroblast-like presumed MSCs can be found in the tunica adventitia’s outermost layer.  The existence of comparable cells near the microvasculature is now being researched. MSCs are in a position to react swiftly to tissue injury because of their association with the vasculature. The transcriptome of single cells sorted from human adipose tissue was determined to better understand the potential of pericytes and adventitial progenitors. Differential gene expression, principal component, and clustering analysis, as well as the creation of gene coregulation networks, revealed that adventitial cells are a more “primitive” population that expresses genes linked to “stemness,” such as Nanog, c-myc, klf2, -4, -6, and osteog (runx2, nox4, notch2).

In contrast, the pericytes displayed a more differentiated appearance overall and expressed genes (angpt2, acta2) related to angiogenesis and smooth muscle cell activity, which was consistent with the in vivo function of these cells.

Accordingly, recent research has shown that adventitial cells are more directly involved in bone production during osseous regeneration in vivo, whereas pericytes primarily induce neoangiogenesis. Both perivascular cell types generate an MSC population when cultured in vitro, however, it is still unclear how pericytes and adventitial cells contribute to the multi-passage cultured MSC.

Whether these cells serve the same progenitor function in their in vivo environment is a significant unresolved topic posed by the potential discovery of perivascular cells as innate MSC forerunners. Unsurprisingly, RNA-Seq analyses of human adventitial perivascular cells and pericytes before and after culture revealed striking differences in gene expression linked to the establishment of these cells in culture and the transition to the in vitro MSC phenotype, with up to one- of all expressed genes being significantly up- or downregulated. (Hardy et al., manuscript under development). However, cell lineage tracking in reporter transgenic mice has revealed roles for pericytes as mesenchymal progenitors, in the adult, for white adipocytes, myoblasts, follicular dendritic cells, and profibrotic myofibroblasts, and both pericytes and adventitial progenitor cells are involved in the turnover and repair of dental tissues. This may imply that perivascular cell-derived MSCs are profoundly modified, or even entirely. A recent study that verified the MSC potential of mouse perivascular adventitial cells also revealed a pathologic correlation and showed that adventitial cells directly contribute to the development of atheroma and calcification in the remodeling of large vessels, by differentiating into smooth muscle cells and osteoblasts, respectively. As a result, the contemporary method of embryological tissue transplantation implies that MSCs are multipotent both in vivo and in vitro.

The effectiveness of the system to repair/regenerate tissues in the living adult organism would seem to be surprisingly low, however, given the hundreds of billions of pericytes associated with the 50,000 miles of capillaries present in the human body—plus the other blood vessels—and even though no more than one in ten perivascular cells yields MSCs in culture. To better understand and possibly facilitate the molecular control of MSCs’ in situ reprogramming into stem-like reparative cells, investigations regarding the clonal selection and gene expression alterations that accompany their establishment in vitro are warranted given the observed discrepancy between the robust potential displayed by in vitro cultured MSCs from perivascular tissue and the modest endogenous role evidenced in vivo. It’s also possible that the perivascular MSCs are performing a crucial tissue role and can’t be mobilized. The vascular-derived MSCs appear to be free to explore alternative tasks after being released from this commitment through tissue collection and in vitro culture. Pericytes have a variety of functions in the kidney, including mesangial cells in the glomeruli and renin-secreting cells in afferent arterioles. Yet when purified, grown, and tested, these particular pericytes produce “MSCs.”

MSC responses: outside-in signaling on hard vs. soft substrates

The N-cadherins are assumed to be necessary for maintaining the stem cell state with the crucial interacting domains involving the peptide His-Ala-Val-Asp in the bone marrow MSCs’ in vivo habitats. There may be fewer cell-cell connections and more extracellular matrix interactions as MSCs leave their nourishing niche environment. On rigid plastic or glass surfaces but not on flexible substrates, the majority of types of in vitro cultured adherent cells, including MSCs, assemble integrin-based focal adhesions that engage extracellular matrix molecules (fibronectin, laminins, and collagens, initially supplied in vitro from serum). Negatively charged polystyrene dishes or flasks are frequently used in in vitro cell culture to facilitate the adhesion of extracellular matrix proteins to cells. However, these hard polymers might not be the best for interpreting cell lineage potentials in multipotent cells like MSCs. Only a small amount of myogenic differentiation occurs in MSCs cultured on the hard polystyrene culture surface, with only 1-2 percent of these cells expressing desmin and myosin heavy chain proteins. However, after spending several days in culture on flexible substrates, MSCs began to display additional traits that are typical of the soft-tissue Myo- and neuro-lineages. As determined by the elastic modulus (Young’s modulus) evaluated over a range of 0.1-100 kPa, tissues exhibit a variety of stiffness in vivo. Compared to tissue culture plastic or glass, which has a stiffness of »1000 kPa, the stiffness of brain and marrow tissue is about 0.3 kPa, fat is 3 kPa, muscle is 10 kPa, and calcified bone is 100 kPa. A small percentage of MSCs cultivated on soft gels that replicate the softness of marrow or neural tissue promote expression of nestin, 3 tubulin, and other neuro-typical features. MSCs in bone marrow can have dendritic forms and express some neuro-typical markers like CD271, TRK A, B, and C mRNAs. But as compared to differentiation on stiff substrates, soft gels have frequently been found to be more favorable for adipogenesis. Conversely, stiff 2D substrates substantially favor osteogenesis, which is relevant to the epitaxial growth of bone, and comparable findings have been observed for 3D gels. Longer-term cultivation of MSCs on either a soft or a stiff substrate gradually binds the cells to the appropriate lineages, rendering them resistant to fast lineage switching induced by both soluble factors and substrate modifications. It is yet unknown what biological mechanism underlies this commitment, whether it be DNA methylation, microRNA, soluble substances, or something else. It was recently shown that purified human pericytes can also be induced to differentiate in culture into either osteocyte, chondrocytes, or even neuron-like cells by modifying the stiffness of a hydrogel substrate. This is relevant to the perivascular localization of in vivo MSCs that was previously discussed.

The molecular processes that MSCs use to interpret a matrix’s receptivity to stimuli indicating lineage differentiation are still being investigated, and certain repeatable routes are beginning to emerge. On stiffer substrates for MSCs, actomyosin motility is more pronounced when substrate adherence is not a barrier (too little or too much). The larger forces applied to harder substrates promote a stiff tissue (bone) lineage, demonstrating the evident contribution of cytoskeletal contraction forces to differentiation. Although SMA expression can vary quite a bit between MSCs even on a uniformly stiff substrate, SMA does contribute to MSC osteogenesis. Smooth muscle actin (SMA) assembles into high tension stress fibers and is naturally increased in MSCs on stiff substrates. High levels of lamin-A in MSCs favor osteogenesis while low levels favor adipogenesis, which is consistent with observations that lamin-A is high in stiff tissues but relatively low in soft tissues. The structural protein lamin-A engages cytoskeletal stress fibers at the nuclear membrane via linkage proteins that span the nuclear envelope. In cells on stiff substrates, the transcriptional co-activators YAP and TAZ predominantly translocate into the nucleus to encourage the expression of differentiation genes for “stiff lineages.”