Introduction to the Concept of Stroma
The actual multipotent differentiation ability of mesenchymal stem cells remains a subject of debate. Although they have the potential for multidirectional differentiation within the body, whether they can be differentiated in a desired direction to produce various types of cells is still questionable. What can be differentiated in the laboratory and what succeeds clinically inside the body are completely different matters. If it were possible to replicate or regenerate cartilage or bone using mesenchymal stem cells, it would perhaps fulfill the dream of regenerative medicine. However, this area does not yet seem to have achieved significant success. Because of this, calling them “stem cells” may not be entirely accurate, and since this can sometimes raise excessive expectations and misunderstandings, there are opinions that we should call them mesenchymal stromal cells rather than mesenchymal stem cells. [1]
I, too, find it ambiguous whether to call them mesenchymal stem cells or stromal cells, but ironically, both have the same English abbreviation—MSC. Therefore, I would like to briefly examine the background to explain this controversy. To understand what stromal cells are, let us first briefly review the concept of “parenchyma” and “stroma,” and the connective tissues closely related to “stroma.”
Parenchyma vs. Stroma
What are stromal cells? In any part of the world, if there are protagonists, there are always supporting groups surrounding them—those who provide firm support, assistance, and protection so that the protagonists can function properly. If cooperation among them is well established, the community will successfully achieve its intended goals. The same applies to the world of cells. For a specific tissue, or further, an organ to perform a particular function or task, there are the essential parenchymal cells that carry out that function, and there are surrounding supporting cells that provide nutrition and structural support.
Let us take the heart as a simple example. For the heart to beat properly, there are central cells such as cardiomyocytes, sinoatrial node cells, and atrioventricular node cells, and surrounding them are vascular endothelial cells, vascular smooth muscle cells, fibroblasts (which produce collagen and elastin), immune cells such as macrophages, nerve cells, and perivascular cells (which stabilize capillaries). These work together so that cardiomyocytes can contract and relax effectively. The main cells responsible for the pumping and rhythm of the heart are called parenchymal cells, while the surrounding supporting cells are called stromal cells.
It is also noteworthy that the cells differentiated from mesenchymal stem cells mostly form connective tissues and act as stromal cells, and that mesenchymal stem cells regulate and support the environment that allows hematopoietic stem cells to differentiate in specific directions. The relationship between these two—both derived from the mesoderm and both possessing stem cell properties—is quite intriguing. Observing their interaction within the same bone marrow, interestingly, mesenchymal stem cells create a microenvironment where hematopoietic stem cells can reside safely, regulate their activity, and protect them. In other words, even between these two, we can observe the characteristics of parenchymal cells and stromal cells.
Connective Tissue
Let us understand that connective tissue itself essentially possesses the nature of stroma. That is, the cells that make up connective tissue mostly function as stromal cells. This could serve as the basis for the argument that mesenchymal stem cells, which differentiate into various types of connective tissues, should be regarded as stromal cells.
Among the four basic tissue types that make up the human body, the main functions of connective tissue are to support and connect cells and tissues and to protect delicate organs and the skeletal system. Bone, cartilage, tendons that maintain skeletal structure, immune cells that protect the body from invading microorganisms, blood and lymph (fluid connective tissues that transport nutrients, waste, and signaling molecules), and adipose tissues that protect organs—all belong to connective tissue.
Cells that make up connective tissue, unlike epithelial cells in epithelial tissues, are not closely packed together but are rather sparsely distributed, because the abundant extracellular matrix (ECM) secreted and released by these cells fills the spaces between them. The role of this extracellular matrix in connective tissue is extremely important. For instance, in bone, this extracellular matrix becomes mineralized, forming the hard bony structure we know. Within connective tissue exist fibroblasts, adipocytes, and mesenchymal stem cells. Fibroblasts, which can be considered the most abundant cells in connective tissue, secrete polysaccharides and various forms of fibrous proteins (collagen fibers, elastic fibers, reticular fibers). These combine with extracellular fluid to form the fibrous extracellular matrix. Adipocytes store fat in the cytoplasm in the form of lipid droplets, and depending on the type and location of the tissue, the number and type of fat (white adipose tissue, brown adipose tissue) appear differently. Fat acts as an insulator against cold and mechanical damage, absorbs shock, and protects organs.
Mesenchymal stem cells are multipotent adult stem cells that have the potential to differentiate into various types of connective tissue cells necessary for repairing and healing damaged tissues. In addition, along with these cells present within the connective tissue, immune cells such as macrophages, mast cells, and lymphocytes enter or leave the connective tissue in response to chemical signals.
Connective tissue can be classified in various ways. First, according to the extracellular matrix (ECM) composition: Connective tissue proper, which contains abundant fibrous proteins and forms a soft yet firm structure that surrounds organs and connects them flexibly; Supportive connective tissue, such as bone and cartilage, where the ECM is mineralized or highly rigid to withstand compression and mechanical load; And fluid connective tissue, which includes blood and lymph.
Connective tissue proper can be divided into loose connective tissue and dense connective tissue based on the density of fibers. Loose connective tissue fills the spaces between organs and surrounds blood vessels and includes areolar tissue, adipose tissue, and reticular tissue. Reticular tissue connects reticular fibers in a network-like fashion, allowing cells to attach to this mesh, forming a scaffolding that supports soft tissues such as lymphatic tissues, the spleen, and the liver. Dense connective tissue, which contains a large amount of collagen fibers, is found mainly in the skin, ligaments, and tendons—where high tensile strength is required.
When I first learned that blood and lymph, classified as fluid connective tissues, are also considered types of connective tissue, I found it a bit puzzling. However, considering that blood carries oxygen and nutrients to other cells, collects waste products that need to be excreted, and transports immune cells that fight invading pathogens, it makes sense that these functions align well with the connective tissue’s role of supporting and protecting other tissues. The only difference is that while other connective tissues contain fibrous elements and have solid or elastic extracellular matrices, blood and lymph each possess liquid extracellular matrices—plasma and lymphatic fluid—thus they are categorized as fluid connective tissues. However, since the functions of blood and lymph are more directly practical and functional for other cells rather than merely providing structural support, many argue that they should be regarded as parenchymal tissues rather than stromal tissues.
Now that we have reviewed stromal cells and connective tissue, let us return to examine how research on mesenchymal stem/stromal cells (MSC) has progressed historically.
History of Mesenchymal Stem/Stromal Cell Research
In 1965, Dr. Marshall R. Urist discovered that when the mineral components such as calcium were removed from the bone matrix and the demineralized bone fragment was implanted into the muscle or subcutaneous tissue of an animal (rabbit), new bone formation occurred there. [2] He identified powerful growth factors naturally present within the bone matrix that send signals to promote bone regeneration and repair and named them Bone Morphogenetic Proteins (BMPs). Later, BMPs were found to belong to the Transforming Growth Factor-β (TGF-β) superfamily. That is, when minerals were removed, the exposed BMPs emitted strong signals that stimulated nearby mesenchymal stem cells to differentiate into bone or cartilage cells. As we saw earlier in the relationship between osteoclasts and osteoblasts, BMPs exposed during bone resorption promote osteoblast differentiation. Strictly speaking, Dr. Urist’s experiment did not show that stem cells capable of forming bone could be transplanted; rather, it showed that the bone fragments provided substances that send signals to stimulate stem cells already existing in the host to form new bone.
In the early 1970s, A. Friedenstein isolated and cultured various cells from bone marrow. Most of the cells died, but some adhered to plastic surfaces and proliferated. These cells, resembling fibroblasts in appearance, began to proliferate at certain points, forming colonies. The fact that they formed colonies meant that a single cell could self-replicate to produce a cluster of cells—an important feature of stem cells. These fibroblast-like cell clusters were literally named Colony-Forming Unit–Fibroblast (CFU-F), and from these, bone cells, cartilage cells, and adipocytes were actually differentiated.
Friedenstein referred to cells forming bone, cartilage, fat, and connective tissues that perform structural and mechanical functions as mechanocytes, and he defined the bone marrow cells forming colonies—though fibroblast-like in shape—not as true fibroblasts secreting collagen, but as progenitor cells that generate mechanocytes (connective tissue cells). [3] He also emphasized that these progenitor cells not only differentiate into mechanical cells but also create a special environment for hematopoietic stem cells. In his various experiments, when hematopoietic stem cells were separated from these progenitor cells, the hematopoietic stem cells could not be maintained for long periods. This demonstrated that CFU-F cells are essential stromal components required for the maintenance of hematopoietic stem cells. At that time, he already suggested that although hematopoietic stem cells and CFU-F (later called mesenchymal stem cells) are of different lineages, they have a mutually dependent and intimate relationship, thus laying the groundwork for today’s concept of the stem cell niche—the unique microenvironment for hematopoietic stem cells.
Stem Cells? Stromal Cells?: The Dual Identity of MSCs
In the early 1990s, the self-renewal capacity of colony-forming cells demonstrated by Friedenstein became the foundation for what came to be known as mesenchymal stem cells (MSCs), and these became the subject of modern research. However, unlike the in vitro experiments on animals in which these cells proliferated and successfully formed colonies, it remains unclear whether such self-renewal occurs as desired in vivo within the human body. Moreover, it has also been revealed that not all colony-forming cells differentiate into multiple lineages, and thus, there have been ongoing questions as to whether they can truly be called stem cells in the strict sense.
In 1965, Urist’s experiment showed that the formation of new bone was not due to the bone fragment itself creating new bone, but rather due to a paracrine interaction—a cell-to-cell signaling mechanism—where growth factors within the bone fragment stimulated mesenchymal stem cells in the muscle to differentiate into bone and cartilage. Friedenstein isolated cells capable of self-renewal and colony formation, and showed that they secreted various factors that activated other surrounding stem cells (such as resident stem cells or hematopoietic stem cells). As research continued, the role of mesenchymal stem cells in maintaining the microenvironment (niche) of hematopoietic stem cells within the body, regulating the immune system, and mediating intercellular signaling became increasingly emphasized. Furthermore, as functions of pericytes—cells located around blood vessels throughout the body—that share morphological and molecular similarities with mesenchymal stem cells were identified, it became clear that the scope and function of MSCs were much broader and more complex. Thus, the prevailing view is that labeling them simply as either stromal cells or stem cells is overly simplistic and generalized. For reference, since they in fact possess the characteristics of both, in this text, for convenience, they will collectively be referred to as MSCs, as both “stem cell” and “stromal cell” begin with the letter S.
Immunomodulatory Function of MSCs
Alongside research on the differentiation capacity of MSCs, another major area of study has focused on how MSCs create an environment for healing and recovery, playing a key role in the repair of damaged tissue. It has been discovered that MSCs reduce or suppress immune responses across both innate and adaptive immunity, thereby diminishing inflammation and creating an environment conducive to faster healing. When tissue damage occurs, inflammatory signaling begins, and MSCs that receive this signal immediately migrate to the site of injury (homing) and express various genes that help repair the damage, forming a restorative environment.
However, in order to understand the immunomodulatory function of MSCs, one must first note that MSCs are versatile cells that can either promote or suppress inflammation depending on the situation. The tendency of a cell’s function to lean in a specific direction depending on the external stimuli it receives is called polarization. A well-known example of this is the macrophage: when it encounters inflammatory cytokines or pathogens, it switches to an aggressive pro-inflammatory mode, and when the initial inflammation subsides and the environment requires tissue regeneration or wound healing, the macrophage transitions to an anti-inflammatory mode that suppresses immune responses to facilitate healing. These two states are distinguished as M1(pro-inflammatory) and M2(anti-inflammatory). Similarly, MSCs, like macrophages, can shift between a pro-inflammatory mode and an anti-inflammatory mode depending on their surroundings. The pro-inflammatory mode is called MSC1, and the anti-inflammatory mode is called MSC2. Depending on which direction the cell polarizes, it secretes different substances, expresses different marker proteins on its surface, and even alters its metabolic processes. These outwardly observable characteristics are called the phenotype. These phenotypes are not fixed; they can change again if the environment changes, and this ability to change is called plasticity—a term derived from the fact that plastic can change its shape.
Plasticity of MSCs and Polarization Toward the Anti-inflammatory Phenotype
Environmental conditions that induce polarization of MSCs into the anti-inflammatory phenotype MSC2 include high concentrations of inflammatory cytokines such as TNF-α, IFN-γ, activation of TLR-3, and hypoxic (low-oxygen) conditions. TLRs (Toll-like receptors) are important receptors in the innate immune system. They detect molecules that are found only in pathogens—so-called Pathogen-Associated Molecular Patterns (PAMPs)—or materials released from the cytoplasm of cells that are dying or damaged due to infection, known as Damage-Associated Molecular Patterns (DAMPs). When these receptors bind to such molecules, they activate intracellular signaling pathways that lead to cytokine secretion and the initiation of an inflammatory response. They act like the “Sentinels” in The Matrix movie, which detect signs of intrusion or danger. Humans possess a variety of TLRs, numbered TLR1–TLR10, some located on the cell membrane and others internally within endosomes. In MSCs, the activation of different TLRs tends to induce different phenotypes: activation of TLR-3 (located in endosomal structures within the cytoplasm) induces anti-inflammatory responses, while activation of TLR-4 (located on the cell membrane) induces polarization toward an inflammatory response mode. Activation of TLR-3 increases the secretion of anti-inflammatory cytokines such as IDO, IL-10, PGE₂, and IL-4, and can suppress T-cell activation, whereas activation of TLR-4 leads to the secretion of many inflammatory cytokines such as IL-6 and IL-8, which promote T-cell activation. [4] For reference, substances that activate the TLR-4 receptor include components of the outer membrane of pathogens (LPS, lipopolysaccharide), while ligands of TLR-3 include double-stranded RNA (dsRNA) generated during viral replication. Most RNA in human cells is single-stranded RNA (ssRNA).
Hypoxic environments also stimulate MSCs. When tissue damage occurs, numerous immune cells rush to the site to initiate an inflammatory response, consuming oxygen to fuel their activity. At the same time, blood flow may be temporarily blocked, reducing oxygen supply, thus creating a hypoxic environment. This leads to the release of hypoxia-inducible signals (HIF-1α, HIF-2α), which stimulate MSCs and polarize them into the anti-inflammatory mode.
However, when considering both sides of inflammation, MSCs are generally more inclined toward suppressing immune responses and performing anti-inflammatory functions rather than promoting inflammation. Given that MSCs support other stem cells and help maintain the niche environment of hematopoietic stem cells, it seems that MSCs are optimized not for “starting the fire” (inflammation), but rather for extinguishing it quickly and promoting healing and regeneration.
[References]
[1] Mesenchymal Stem Cells: Time to Change the Name!
https://doi.org/10.1002/sctm.17-0051
[2] Marshall R. Urist and the discovery of bone morphogenetic proteins
https://doi.org/10.1007/s00264-017-3402-9
[3] Alexander Friedenstein, Mesenchymal Stem Cells, Shifting Paradigms and Euphemisms
doi: 10.3390/bioengineering11060534
[4] Strategies for the induction of anti-inflammatory mesenchymal stem cells and their application in the treatment of immune-related nephropathy
doi: 10.3389/fmed.2022.891065
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