Mesenchymal Stromal Cells (MSCs): Definition, Characteristics, Sources, Functions and Uses

Mesenchymal stromal cells (MSCs) are heterogeneous populations of stromal cells which are widely studied in tissue biology and regenerative medicine. They can be isolated and culture-expanded from different tissues, including bone marrow, adipose tissue, umbilical cord and several other tissue sources. The properties of these cells are not exactly same between every tissue source, donor and culture condition.

Classically, human MSCs are characterized using three minimum criteria proposed by the International Society for Cellular Therapy (ISCT). The cells should show plastic adherence under standard culture conditions. They express CD105, CD73 and CD90, while lacking the major hematopoietic markers such as CD45, CD34, CD14/CD11b, CD79a/CD19 and HLA-DR. They also show osteogenic, adipogenic and chondrogenic differentiation under suitable in vitro conditions. These are minimum characterization criteria and by themselves do not prove that every cell of the population is a true stem cell.

The biological activities of MSCs are not based only on their differentiation capacity. These cells interact with stromal and immune cells and release different soluble factors and extracellular vesicles into the surrounding microenvironment. Through these activities, they can influence tissue repair, cell survival, inflammation and immune responses. Paracrine signaling and immune regulation are therefore important properties of MSC biology, together with their differentiation and stromal functions.

The terms mesenchymal stromal cell and mesenchymal stem cell should not be considered as completely equivalent terms. The term mesenchymal stromal cell is preferred for the heterogeneous culture-expanded populations unless sufficient evidence for stemness, including self-renewal and appropriate differentiation ability, has been demonstrated. The abbreviation MSC is still commonly used for both, which can sometimes produce confusion in the literature.

What Are Mesenchymal Stromal Cells?

Mesenchymal stromal cells (MSCs) are heterogeneous populations of non-hematopoietic stromal cells that can be isolated and expanded from different tissues. They are generally described as multipotent stromal cells because some cells within these populations have capacity to differentiate into more than one mesenchymal lineage. MSCs should not be considered as pluripotent cells.

The abbreviation MSC is commonly used for mesenchymal stromal cells. In culture, these cells are plastic-adherent and generally develop a spindle-shaped or fibroblast-like appearance. However, MSC cultures are not formed of a completely identical cell population. Differences are found according to tissue source, donor and culture condition.

MSCs are non-hematopoietic in nature and are different from the blood-forming hematopoietic cells of bone marrow. According to the classical ISCT criteria, culture-expanded human MSCs lack major hematopoietic markers such as CD45 and show a characteristic stromal surface-marker profile. They can form osteogenic, adipogenic and chondrogenic lineages under suitable in vitro differentiation conditions. This tri-lineage differentiation is the standard pattern used for their classical characterization.

The bone marrow was the first major tissue source studied for these cells. MSC or MSC-like populations have later been isolated from adipose tissue, umbilical cord, synovial tissue and several other connective tissues. The characteristics of cells obtained from different tissues are not always exactly same, therefore the tissue source is important while describing an MSC population.

MSCs have biological importance because they form part of stromal environments and interact with surrounding cells. Their activities also involve secretion of soluble factors, regulation of immune responses and changes in the local tissue environment. Thus, their biological effects are not only based on direct differentiation into new cells.

These properties have made MSCs an important subject in regenerative medicine and tissue engineering research. Their culture expansion, mesenchymal differentiation capacity and different paracrine and immunoregulatory activities are being studied for tissue repair and cell-based therapeutic approaches. The therapeutic effect, however, can vary with cell source, manufacturing method and biological condition.

Discovery and Historical Background

Early studies of MSCs started from observations of the bone marrow stromal compartment. During the late 1960s and 1970s, Alexander Friedenstein and his coworkers showed that bone marrow contains adherent fibroblast-like precursor cells. When these cells were cultured at low density, individual fibroblastic colonies could be formed. Some marrow-derived cells also showed bone-forming capacity in experimental systems.

These clonogenic stromal precursors became associated with the colony-forming unit-fibroblast (CFU-F) concept. CFU-F assays demonstrated that only a small fraction of marrow cells could produce such fibroblastic colonies. Later studies also showed that the CFU-F population itself was heterogeneous, with different degrees of self-renewal and differentiation capacity.

The term “mesenchymal stem cells” became widely established after the work of Arnold Caplan, particularly his 1991 publication describing mesenchymal stem cells and their differentiation toward skeletal tissue lineages. The name became commonly used in regenerative medicine and tissue engineering studies.

With increasing studies, it became clear that every plastic-adherent MSC culture could not automatically be demonstrated as a true stem-cell population. In 2005, the International Society for Cellular Therapy (ISCT) therefore recommended the term “multipotent mesenchymal stromal cells” for these heterogeneous culture-expanded populations, while retaining the abbreviation MSC. The term mesenchymal stem cell is more properly used when stemness has been demonstrated with suitable evidence. This stromal terminology was again supported in later ISCT recommendations.

Mesenchymal Stromal Cells vs Mesenchymal Stem Cells

The terms mesenchymal stromal cells and mesenchymal stem cells are both used for MSCs in different scientific studies. The term mesenchymal stem cell was used widely during the earlier development of this field. However, these two terms do not exactly have the same biological meaning.

The term mesenchymal stem cell became common after the work of Arnold Caplan in 1991. It was used for cells having the ability of self-renewal and differentiation into different mesenchymal tissues. With increasing studies, the term MSC became widely used in tissue engineering and regenerative medicine.

However, the cells obtained from bone marrow or other tissues and expanded in culture are generally a heterogeneous population. All cells present in such culture do not necessarily have same stem-cell properties. Some cells may show greater colony-forming and differentiation capacity while others show less.

The standard properties such as plastic adherence, surface markers and in vitro differentiation are used for characterization of MSCs. These characteristics mainly describe the cultured stromal population. They alone do not prove that every cell present in the culture is a stem cell.

For this reason, the International Society for Cellular Therapy (ISCT) proposed the term multipotent mesenchymal stromal cells in 2005. The abbreviation MSC was retained. The term mesenchymal stem cell can be used when stem-cell properties are demonstrated properly.

The main difference is therefore based on the meaning of stemness. A stromal cell population can fulfil the commonly used MSC characteristics without every cell behaving as a true stem cell. Stem-cell terminology gives a stronger meaning, because self-renewal and differentiation capacity should be demonstrated.

The ISCT again discussed this nomenclature problem in 2019. It recommended that the tissue source should be clearly given while describing MSCs. If the term mesenchymal stem cell is used, sufficient evidence for its stem-cell properties should also be present.

The expression mesenchymal stem/stromal cells is also found in many scientific articles. This form includes both the historically used stem-cell name and the stromal-cell terminology. The same abbreviation MSC is used, which is one of the major reasons for continuation of both names.

Mesenchymal Stromal Cells vs Mesenchymal Stem Cells

Medicinal Signaling Cell Proposal

The term medicinal signaling cells was proposed by Arnold Caplan in 2017, while keeping the abbreviation MSC. This name was suggested because many effects of administered MSCs are related to the release of different bioactive factors and interaction with surrounding cells.

According to this concept, MSCs may support tissue repair by signaling to other cells rather than becoming the replacement tissue themselves. Their secreted factors can affect immune cells, vascular cells and other cells present at the damaged region.

However, medicinal signaling cell is a proposed name. It has not replaced mesenchymal stromal cell as the general ISCT terminology. Thus, mesenchymal stromal cell remains the more suitable term for many heterogeneous culture-expanded MSC preparations unless their stem-cell properties are specifically demonstrated.

Characteristics of Mesenchymal Stromal Cells (MSCs)

Some of the important characteristics of Mesenchymal Stromal Cells (MSCs) are as follows-

• Non-hematopoietic nature- MSCs are non-hematopoietic stromal cell populations. They are different from the hematopoietic cells which form different blood cells.
• Plastic adherence- Human MSCs adhere to plastic surface when maintained under standard culture conditions. This is one of the classical minimum characteristics used for identification of cultured MSCs.
• Fibroblast-like morphology- During culture, MSCs generally show elongated spindle-shaped or fibroblast-like appearance. The appearance can change with cell density, passage and culture condition.
• Surface markers- MSCs generally express CD105, CD73 and CD90. According to the classical ISCT criteria, they lack expression of CD45, CD34, CD14 or CD11b, CD79α or CD19 and HLA-DR. These markers are used together and not as a single specific MSC marker.
• Multipotent differentiation- MSCs can show differentiation into osteogenic, adipogenic and chondrogenic lineages under suitable in vitro conditions. This tri-lineage differentiation forms one of the minimum ISCT criteria. It should not be taken as evidence of pluripotency.
• Heterogeneous population- Culture-expanded MSCs are not completely identical cells. Differences occur between individual cells and also between MSC preparations. The heterogeneity can be related to tissue source, donor, isolation method and culture conditions.
• Different tissue sources- MSC populations can be obtained from bone marrow, adipose tissue, umbilical tissues, placenta and several other tissues. Their properties are not exactly same from every source. For this reason, the tissue origin should be mentioned while describing an MSC preparation.
• Culture expansion- MSCs can proliferate and their number can be increased during in vitro culture. But the proliferation capacity is affected by donor, source, passage number and culture medium. Different culture conditions can also change their colony-forming and functional properties.
• Paracrine activity- MSCs release different cytokines, growth factors and other bioactive molecules. Extracellular vesicles are also released by these cells. These secreted products can influence nearby cells and the tissue environment.
• Immunoregulatory properties- MSCs can interact with different cells of immune system and modify immune responses. The activity is not fixed under every condition and can be influenced by the surrounding inflammatory environment.
• Stromal interaction- MSCs are associated with stromal environments and can interact with surrounding cells and extracellular matrix. Their biological activity is therefore not limited only to differentiation into other cell types.
• No single defining property- A single marker, fibroblast-like shape or plastic adherence alone cannot define an MSC population. The classical criteria use adherence, immunophenotype and tri-lineage differentiation together. Functional characterization may also be required according to their intended use.

Morphology of Mesenchymal Stromal Cells (MSCs)

The morphology of Mesenchymal Stromal Cells (MSCs) can differ according to tissue source, culture condition and stage of culture. Some of the common morphological features of MSCs are as follows-

• Spindle-shaped cells- MSCs grown under standard culture conditions generally have elongated spindle-shaped cells. They show a fibroblast-like appearance, especially during active proliferation.
• Plastic-adherent appearance- MSCs attach to plastic culture surfaces and spread over the surface during in vitro culture. Plastic adherence is one of the minimum characteristics given by the International Society for Cellular Therapy (ISCT) for cultured human MSCs.
• Cell shape is not completely uniform- All cells in an MSC culture do not have exactly same morphology. Small spindle-shaped cells, larger flattened cells and other intermediate forms can be present. In human bone marrow stromal cell colonies, morphologically different cell populations have been observed.
• Cell size- MSCs also show variation in their cell size. Rapidly growing cells can be smaller, whereas large and flat cells are commonly associated with lower proliferative activity. Thus, size and shape of the cells may change even within the same culture.
• Cellular processes- Cultured MSCs can possess different cytoplasmic extensions. Thick processes, slender processes and filopodia have been observed during ultrastructural studies of adipose-derived MSCs. These processes contain cytoskeletal elements and sometimes vesicular structures.
• Nucleus and cytoplasm- The nucleus is generally round to oval in cultured MSCs, but its appearance can differ with the functional state of cell. Ultrastructural studies of umbilical cord-derived MSCs have shown cells with a prominent nucleus and variable amount of cytoplasmic organelles.
• Internal cell structure- MSCs contain mitochondria, rough endoplasmic reticulum, microtubules and intermediate filaments. The amount and arrangement of these structures are not fixed and can change according to culture medium and activity of the cells.
• Colony formation- When marrow-derived stromal cells are cultured at low density, individual cells can give rise to fibroblast-like colonies. These colonies are associated with colony-forming unit-fibroblast (CFU-F) activity, although all cells present in a colony are not morphologically or functionally identical.
• Changes during prolonged culture- The morphology of MSCs changes with continued culture expansion. Cells with reduced proliferative activity tend to become larger and flatter, and culture aging is associated with development of cellular senescence.
• Source-dependent morphology- The common spindle-shaped morphology is found in MSCs from different sources, but their size and exact appearance can vary. Therefore morphology from one tissue-derived MSC population should not be considered exactly same for all MSC preparations.
• Morphology is not sufficient for identification- A fibroblast-like or spindle-shaped appearance alone cannot confirm that cultured cells are MSCs. Morphology is considered together with plastic adherence, surface-marker profile and differentiation characteristics during MSC characterization.

ISCT Minimal Criteria for Mesenchymal Stromal Cells (MSCs)

The International Society for Cellular Therapy (ISCT) proposed minimal criteria for identification of human Mesenchymal Stromal Cells (MSCs) in 2006. These criteria were given because different methods were being used for isolation, culture and characterization of MSCs. Three minimum characteristics were proposed.

The following are the ISCT minimal criteria for MSCs–

1. Plastic Adherence

MSCs must be plastic-adherent when maintained under standard culture conditions. The cells attach to the surface of tissue culture flasks and grow as adherent cells. Plastic adherence is therefore the first minimum criteria used for identification of cultured human MSCs.

Fibroblast-like morphology is commonly seen during culture, but fibroblast-like shape itself is not a separate ISCT minimum criterion.

2. Surface Antigen Expression

The second criteria is based on the expression and absence of specific cell surface markers. These are generally determined by flow cytometry.

At least 95% of the MSC population should express-

• CD105
• CD73
• CD90

The cells should show 2% or less positive expression for-

• CD45
• CD34
• CD14 or CD11b
• CD79a or CD19
• HLA-DR

Thus, MSCs show positive expression of CD105, CD73 and CD90 while the listed hematopoietic and immune-associated markers are largely absent. There is no single surface marker which alone defines an MSC population in these criteria.

3. Tri-lineage Differentiation

MSCs must have the ability to differentiate into osteogenic, adipogenic and chondrogenic lineages under standard in vitro differentiation conditions. This is the third minimum criteria.

The differentiation pattern includes-

• Osteoblasts – osteogenic differentiation.
• Adipocytes – adipogenic differentiation.
• Chondroblasts – chondrogenic differentiation.

This tri-lineage differentiation demonstrates the commonly used multipotent differentiation property of MSC cultures. It does not mean that the cells are pluripotent.

The three criteria are therefore plastic adherence, specific surface antigen expression and in vitro tri-lineage differentiation. They were proposed as minimum identifying criteria for human MSCs used in research and pre-clinical studies. They should not be considered as clinical product release specifications.

The classical ISCT criteria also do not prove that every cell in the culture is a true stem cell or that an MSC preparation has a particular therapeutic activity. Later ISCT recommendations stated that the tissue source should be given and suitable functional assays should be used according to the proposed biological or therapeutic function of MSCs. Rigorous evidence is required when the cells are specifically called mesenchymal stem cells.

Surface Markers of Mesenchymal Stromal Cells (MSCs)

The surface marker profile is one of the important criteria used for characterization of human Mesenchymal Stromal Cells (MSCs). According to the classical ISCT criteria, MSCs show positive expression of CD73, CD90 and CD105 and lack or show very low expression of selected hematopoietic and immune cell markers.

MSC Surface Markers

For the positive markers, 95% or more of the cultured cell population should express CD73, CD90 and CD105. For the negative marker panel, 2% or less of cells should express CD45, CD34, CD14 or CD11b, CD79α or CD19 and HLA-DR according to the original ISCT proposal.

Positive Markers

CD73, CD90 and CD105 are the three positive surface markers included in the classical ISCT minimum criteria.

• CD73- It is also known as ecto-5′-nucleotidase. CD73 is commonly expressed on culture-expanded MSCs.
• CD90- It is also called Thy-1. High expression of CD90 is generally found in cultured MSC preparations.
• CD105- It is also known as endoglin. CD105 is another commonly used positive marker in the classical MSC immunophenotype.

These markers are generally examined together. Expression of only one of these markers cannot identify a cell as an MSC.

Negative/Low Markers

The classical ISCT panel also includes markers that should be absent or present only at very low levels.

• CD45- It is a common leukocyte marker and is used to exclude hematopoietic cells.
• CD34- It is included as a negative marker in the classical ISCT criteria for culture-expanded human MSCs.
• CD14 or CD11b- These markers are associated mainly with monocyte and myeloid cell populations. Their low expression helps to exclude these contaminating cells.
• CD79α or CD19- These are B-cell-associated markers. MSC preparations should show very low or absent expression.
• HLA-DR- It is an MHC class II molecule. Classical culture-expanded MSC preparations should show low or absent HLA-DR expression under the minimum ISCT framework.

The surface marker pattern should not be considered as a single unique molecular barcode of MSCs. Many of these markers can also be expressed by other stromal or connective-tissue cells. Similarly, absence of the negative markers alone cannot prove MSC identity.

Types of Mesenchymal Stromal Cells (MSCs)

Mesenchymal Stromal Cells (MSCs) are commonly named according to the tissue from which they are obtained. The ISCT also recommends mentioning the tissue-source origin while describing MSCs, because cells obtained from different sources can have different properties.

Some of the common types of MSCs are as follows-

1. Bone Marrow-Derived MSCs (BM-MSCs)- These are obtained from the stromal compartment of bone marrow. BM-MSCs are the classical and one of the most extensively studied MSC populations. They show osteogenic, adipogenic and chondrogenic differentiation under suitable in vitro conditions.
2. Adipose-Derived MSCs (AD-MSCs)- These cells are obtained from adipose tissue. Adipose tissue contains a heterogeneous stromal vascular fraction (SVF) from which adipose-derived stromal cells can be isolated and culture-expanded. SVF itself should not be considered the same as culture-expanded AD-MSCs.
3. Umbilical Cord-Derived MSCs (UC-MSCs)- These are obtained from different regions of umbilical cord tissue. Wharton’s jelly is one of the commonly used regions and the cells are also called WJ-MSCs. They generally show high proliferative activity during culture.
4. Placenta-Derived MSCs (P-MSCs)- MSC populations can be isolated from different regions of the placenta. Placenta is a perinatal tissue and contains several different cell populations, including mesenchymal stromal populations. The exact placental region should be mentioned because placenta is not a single uniform tissue.
5. Dental Tissue-Derived MSCs- Different mesenchymal stromal/stem cell populations are obtained from dental and periodontal tissues. Dental pulp stem/stromal cells (DPSCs) are isolated from dental pulp. Other oral sources include periodontal tissues and related dental structures. These cells are mainly studied in dental and craniofacial tissue research.
6. Synovium-Derived MSCs (S-MSCs)- These MSC populations are obtained from synovial membrane and related synovial tissues of joints. MSC-like populations can also be recovered from synovial fluid. Synovial MSCs are particularly studied for their chondrogenic properties and cartilage-related research.
7. Other Tissue-Derived MSCs- MSC-like stromal populations have also been isolated from different adult and perinatal tissues. These include skeletal muscle and several connective or perivascular tissues. The properties are not exactly same from every tissue source, and culture conditions also affect the obtained population.

Where Are Mesenchymal Stromal Cells (MSCs) Found?

Mesenchymal Stromal Cells (MSCs) can be isolated and culture-expanded from different adult and perinatal tissues. Bone marrow is the classical and one of the most studied sources. Later, MSC-like stromal populations have been obtained from adipose tissue, umbilical tissues, placenta and several other tissues.

The following are some of the important tissue sources of MSCs-

• Bone marrow- It is the classical source of MSCs. Bone marrow contains non-hematopoietic stromal progenitor cells which can form fibroblast-like colonies after culture. Bone marrow-derived MSCs are generally called BM-MSCs.
• Adipose tissue- Adipose tissue is another major source. MSC-like stromal cells can be isolated from the stromal vascular fraction (SVF) of adipose tissue and then expanded in culture. These are commonly referred to as adipose-derived MSCs (AD-MSCs).
• Umbilical cord- MSCs can be obtained from different regions of umbilical cord, particularly Wharton’s jelly. Perivascular, intervascular and subamnion regions of the cord have also been used for isolation of MSC-like populations. Umbilical cord tissue should not be considered exactly same as umbilical cord blood as an MSC source.
• Placenta- Placental tissues are also a source of MSC populations. Placenta together with umbilical cord is generally included among the birth-associated or perinatal tissues used for MSC isolation.
• Dental tissues- Mesenchymal stromal/stem cell populations have been isolated from dental pulp and other dental tissues. Dental-derived populations are particularly studied in dental and craniofacial tissue research.
• Synovial tissue- MSC populations can also be isolated from synovial tissue. Synovium-derived MSCs show mesenchymal characteristics after culture and are studied particularly for cartilage and joint-related research.
• Skeletal muscle and other connective tissues- MSC-like progenitor populations have been obtained from skeletal muscle and several other connective tissues. Their characteristics and differentiation capacities are not exactly same between every tissue source.

Location of MSCs Within Tissues

The exact identity of MSCs inside the living tissue is more difficult to define than culture-expanded MSCs. Studies from different human organs have shown that some MSC-like progenitor cells are associated with blood vessel walls and perivascular regions. Pericytes around small vessels and adventitial cells around larger vessels can develop MSC-like characteristics after culture.

This perivascular association does not mean that every MSC from every tissue is simply a pericyte. The in vivo identity of MSC populations is more complex and tissue-dependent. Many commonly used MSC definitions are based on cells after isolation and culture expansion rather than one single cell type identified directly inside all tissues.

Comparison of MSCs From Different Tissue Sources

Mesenchymal Stromal Cells (MSCs) can be obtained from different tissues. Bone marrow, adipose tissue and umbilical cord are among the most commonly studied sources. The cells show many common MSC characteristics, but they are not exactly same in their biological properties.

Bone Marrow vs Adipose vs Umbilical Cord MSCs

The differences given in the table are general tendencies. They are not fixed properties of every MSC preparation. Even cells from the same tissue can differ with donor, age, isolation method, culture medium, passage number and experimental condition.

Differentiation Potential of Mesenchymal Stromal Cells (MSCs)

Mesenchymal Stromal Cells (MSCs) have the capacity to differentiate into different mesenchymal cell lineages under suitable in vitro conditions. The classical differentiation includes osteogenic, adipogenic and chondrogenic lineages. These three differentiation properties are also included in the minimum criteria given by ISCT for characterization of human MSCs.

Osteogenic Differentiation

During osteogenic differentiation, MSCs are induced toward osteogenic lineage and develop osteoblast-like characteristics. The cells gradually produce an extracellular matrix which can undergo mineral deposition.

Some of the common changes and assays are-

• Mineral deposition- Calcium-rich mineralized deposits are formed during osteogenic differentiation.
• Alkaline phosphatase (ALP)- Increased ALP activity is commonly used during evaluation of osteogenic differentiation.
• Alizarin Red S staining- It is used to detect calcium-containing mineralized deposits formed by differentiated cells.

Osteogenic differentiation is one of the three in vitro differentiation properties used in the classical identification of MSCs. It is also widely studied for bone formation and osteogenic research.

Adipogenic Differentiation

MSCs can differentiate into adipocyte-like cells when maintained in a suitable adipogenic induction medium. During this process, intracellular lipid droplets start to accumulate within the differentiated cells.

The following are commonly used for its laboratory confirmation-

• Lipid droplet formation- Accumulation of lipid droplets is an important morphological change during adipogenic differentiation.
• Oil Red O staining- It is commonly used to stain the intracellular lipid droplets and demonstrate adipogenic differentiation.

Adipogenic differentiation forms the second component of the classical tri-lineage differentiation used for characterization of MSC populations.

Chondrogenic Differentiation

During chondrogenic differentiation, MSCs are induced toward chondroblast or chondrocyte-like lineage. Chondrogenic culture is often carried out using a high-density or pellet culture system with suitable induction factors.

The differentiated cells produce a cartilage-like extracellular matrix. This matrix contains molecules associated with cartilage, including type II collagen, aggrecan and proteoglycans.

Some of the common observations are-

• Cartilage matrix formation- Extracellular matrix rich in proteoglycans is formed during chondrogenesis.
• Alcian Blue staining- It can be used to demonstrate proteoglycan or glycosaminoglycan-rich matrix.
• Type II collagen and aggrecan- These are commonly examined as cartilage-associated components during chondrogenic studies.

Chondrogenic differentiation is the third lineage included in the classical ISCT minimum differentiation criteria.

Can MSCs Form Other Cell Types?

Different studies have reported broader differentiation properties of MSCs beyond bone, fat and cartilage lineages. Earlier studies have also discussed differentiation toward other mesenchymal tissues and different experimental studies use various induction conditions.

The obtained differentiation response is not fixed. Culture medium, induction factors and other experimental conditions can affect the differentiation capacity of cultured MSCs. Even MSC preparations maintained under different culture conditions can show differences in their differentiation potential.

The standard minimum defining framework, however, remains in vitro differentiation into osteoblasts, adipocytes and chondroblasts. Broader lineage observations are not part of the classical ISCT minimal criteria.

MSCs should not be described as pluripotent cells from this tri-lineage property. They are generally described as multipotent stromal populations, and stronger stem-cell claims require suitable evidence rather than only routine in vitro differentiation.

How Are Mesenchymal Stromal Cells Isolated and Cultured?

Mesenchymal Stromal Cells (MSCs) can be isolated from bone marrow, adipose tissue, umbilical cord and other tissues. The exact isolation method is different for different sources. In many methods, the tissue cells are first collected or released and then the plastic-adherent stromal cells are expanded in in vitro culture.

The following are the general steps involved in isolation and culture of MSCs-

1. Collection of Tissue

The first step is collection of the required tissue sample. Bone marrow aspirate, adipose tissue or lipoaspirate and umbilical cord tissue are commonly used sources.

The starting sample is not a pure population of MSCs. It contains different cells according to the tissue from which it is obtained.

2. Processing of Tissue

The tissue is processed to obtain the cell-containing fraction.

• Bone marrow- Bone marrow aspirate can be processed to obtain a low-density mononuclear cell fraction. These cells are then placed in culture. Earlier human marrow studies used low-density mononuclear cells followed by selection of the adherent stromal population.
• Adipose tissue- The adipose tissue is generally washed and digested with an enzyme such as collagenase. After digestion and centrifugation, a cell-rich stromal vascular fraction (SVF) is obtained. The SVF itself contains different cell types and is not the same as an expanded MSC culture.
• Umbilical cord- Different methods can be used. Small pieces of cord tissue can be placed directly for explant culture, or the tissue can be enzymatically digested to release the cells. Both methods have been used for Wharton’s jelly-derived MSCs. The isolation method can also affect cell yield and proliferation.

3. Primary Plating

The obtained cells are placed in a suitable cell culture vessel containing growth medium. MSCs attach to the plastic surface during culture.

In bone marrow cultures, many non-adherent hematopoietic cells remain suspended. These cells are removed with medium replacement, while the adherent stromal cells remain attached and continue their growth.

4. Growth of Adherent Cells

After attachment, the MSC population starts to proliferate over the culture surface. The growing cells generally show elongated and fibroblast-like morphology and form an adherent monolayer.

Different basal media and supplements are used for MSC culture. The culture medium is not exactly same in every laboratory. Changes in medium composition can affect cell growth, differentiation and surface-marker expression.

5. Removal of Non-adherent Cells

During early culture, the medium is changed and unattached cells are removed. The attached cells are allowed to remain on the culture surface.

Plastic adherence helps in obtaining the stromal population, but it is not a specific purification marker for MSCs. Other fibroblast-like stromal cells can also adhere to plastic. The cultured cells still require proper characterization.

6. Expansion of MSCs

The adherent cells continue to multiply and increase their number. This process is referred to as culture expansion.

MSCs obtained initially from tissue are generally limited in number. Culture expansion is therefore used to produce a larger population for laboratory studies. The cells can pass through different growth phases during expansion and their biological state also changes with culture density.

7. Passaging

When the adherent culture reaches a suitable cell density, the cells are detached from the culture surface and transferred into new culture vessels. This is referred to as passaging or subculture.

Trypsinization has traditionally been used for detachment of cultured MSCs. The collected cells are counted and again seeded into fresh culture vessels for further expansion. Repeated and prolonged culture can change their proliferation and some biological characteristics, so passage history is an important culture information.

8. Characterization of Cultured MSCs

The obtained adherent cells are not identified as MSCs only from their isolation method or fibroblast-like appearance. The cultured population is further characterized.

According to the classical ISCT minimal criteria, human MSCs should-

• Remain plastic-adherent under standard culture conditions.
• Express CD73, CD90 and CD105.
• Show very low or absent expression of CD45, CD34, CD14 or CD11b, CD79α or CD19 and HLA-DR.
• Differentiate into osteogenic, adipogenic and chondrogenic lineages under suitable in vitro conditions.

The isolation and culture conditions are not fixed for all MSC preparations. Tissue processing method, culture medium, cell density and duration of expansion can change the properties of the obtained cells.

How Do Mesenchymal Stromal Cells (MSCs) Work?

The biological effects of Mesenchymal Stromal Cells (MSCs) can occur through different mechanisms. Earlier, much attention was given to their differentiation into replacement cells. Now many of their effects are associated with paracrine signaling, interaction with immune cells and release of extracellular vesicles. Direct differentiation is still a property of MSCs, but it cannot explain all of their reported effects.

The following are the important mechanisms of MSCs-

• Paracrine signaling- MSCs release different cytokines, chemokines and growth factors into the surrounding environment. These soluble molecules act on nearby cells. This is one of the major ways by which MSCs can affect tissue responses.
• Immunomodulation- MSCs interact with different cells of immune system. They can change the activity of T cells, B cells, natural killer cells, dendritic cells and macrophages under different experimental conditions. The immune effect is influenced by inflammatory signals present around the MSCs and is not exactly same under all conditions.
• Regulation of T cells– MSCs can inhibit proliferation and activation of inflammatory T cells in different experimental systems. They can also favour development or activity of regulatory T cells (Tregs) under some conditions.
• Extracellular vesicle release- MSCs release extracellular vesicles (EVs) containing proteins, lipids and different RNA molecules. These vesicles can transfer biological signals to recipient cells. MSC-derived EVs are now studied as one component of the paracrine action of MSCs. Most therapeutic evidence for isolated MSC-EVs is still largely from preclinical studies.
• Support of tissue repair- The secreted products of MSCs can affect survival, proliferation and activity of tissue-resident cells. Some MSC-derived factors can reduce apoptosis and support repair responses in damaged tissues. These actions are sometimes referred to as trophic effects.
• Angiogenic activity- MSCs can secrete molecules that affect endothelial cells and blood-vessel formation. Factors associated with their secretome can promote angiogenesis in different experimental models, especially under ischemic or damaged tissue conditions.
• Homing and migration- MSCs can respond to chemokines and other signals associated with injured or inflamed tissues. Cell adhesion, chemokine signaling and migration through extracellular matrix are involved in this process. However, homing after systemic administration is generally inefficient and only a small proportion of administered cells may reach the intended tissue.
• Limited long-term engraftment- Therapeutic effects have been observed even when long-term survival or permanent engraftment of administered MSCs is low. This finding is one reason why secreted factors and interaction with host cells have received greater attention than permanent replacement of damaged tissue by MSCs.
• Differentiation- MSCs can differentiate into osteogenic, adipogenic and chondrogenic lineages under suitable in vitro conditions. Differentiation can have importance in tissue biology and tissue-engineering studies. It should not be considered as the only mechanism by which administered MSCs may produce biological effects.
• Cell-to-cell interaction- Some MSC effects require direct or close interaction with surrounding cells. Surface molecules and local signaling can take part in immune regulation together with the soluble factors released by MSCs.
• Apoptosis and efferocytosis- Some administered MSCs can undergo apoptosis after entering the recipient. These apoptotic MSCs can be engulfed by host phagocytic cells, a process called efferocytosis. This interaction has been linked with immunomodulatory effects and is another mechanism being studied for MSC therapy.
• Mitochondrial transfer- Experimental studies also show that MSCs can transfer mitochondria or mitochondrial components to other cells. Transfer can occur through structures such as tunnelling nanotubes and through other intercellular routes. This mechanism is mainly studied in experimental tissue injury and regenerative models.

Functions of Mesenchymal Stromal Cells (MSCs)

Mesenchymal Stromal Cells (MSCs) perform different stromal and regulatory functions. Their activity can change according to tissue source and surrounding conditions. Some of the important functions of MSCs are as follows-

• Support of stromal microenvironment- MSCs take part in formation and maintenance of stromal environment of tissues. In bone marrow, mesenchymal stromal populations form an important component of the niche associated with hematopoietic stem cells.
• Support of hematopoiesis– Bone marrow stromal populations produce signals required for maintenance of hematopoietic stem cells (HSCs). CXCL12 produced by mesenchymal progenitor populations is one of the important factors involved in this function.
• Differentiation- MSCs have capacity to differentiate into osteogenic, adipogenic and chondrogenic lineages under suitable in vitro conditions. This property is widely used in MSC characterization and mesenchymal tissue research.
• Paracrine function- MSCs release different cytokines, chemokines and growth factors. These secreted molecules can act on surrounding cells and alter their survival, proliferation and activity.
• Immunomodulation- MSCs can regulate the activity of immune cells including T cells, B cells, macrophages, dendritic cells and natural killer cells. The type and degree of immune regulation depends upon inflammatory signals and other conditions around the cells.
• Tissue repair support- MSC-derived bioactive factors can support repair activity of tissue-resident cells. They can stimulate proliferation and activity of local reparative cells and also provide trophic support at damaged tissue regions.
• Cell survival- Some factors secreted by MSCs can reduce apoptosis and support survival of stressed or damaged cells in experimental systems. This is included among the trophic activities of MSCs.
• Angiogenic function- MSCs release factors which can act on endothelial cells and support formation of new blood vessels. Their secretome has shown pro-angiogenic activity in different in vitro and in vivo experimental models.
• Extracellular vesicle secretion- MSCs release extracellular vesicles (EVs) containing proteins, lipids and RNA molecules. These vesicles take part in transfer of biological signals between MSCs and other cells. Their regenerative and immune-regulatory activities are mainly being studied in preclinical systems.
• Regulation of local inflammation- MSCs can change inflammatory responses through soluble factors and interaction with immune cells. This activity is not always fixed as suppressive, because the response of MSCs changes according to the local inflammatory environment.
• Support of endogenous reparative cells- Factors released from MSCs can stimulate tissue-intrinsic progenitor or reparative cells. This function does not require the MSC itself to become the replacement cell of the damaged tissue.

Clinical Applications of Mesenchymal Stromal Cells (MSCs)

Mesenchymal Stromal Cells (MSCs) are studied for different clinical applications because of their immunomodulatory, stromal and paracrine properties. Clinical evidence is not same for every disease. Many MSC applications are still under clinical investigation.

The following are some of the important clinical applications of MSCs-

1. Graft-versus-host disease (GVHD)- MSCs are used and studied mainly for steroid-refractory acute graft-versus-host disease (SR-aGVHD) after allogeneic hematopoietic stem cell transplantation. In the United States, bone marrow-derived remestemcel-L-rknd (Ryoncil) is approved for SR-aGVHD in pediatric patients of 2 months of age and older. Clinical responses are reported, although survival benefit has not been consistently demonstrated in randomized trials.
2. Perianal fistulas in Crohn’s disease- Adipose and bone marrow-derived MSCs have been studied for treatment of complex perianal fistulas. Local administration is generally used around the fistula tract. Results between trials are variable. Darvadstrocel was previously authorized in the European Union, but its EU authorization was withdrawn in December 2024 after a confirmatory study failed to demonstrate sufficient clinical benefit.
3. Osteoarthritis and cartilage disorders- Intra-articular MSC administration is studied mainly in knee osteoarthritis and cartilage lesions. Some clinical trials report reduction in pain and changes in joint function. The certainty of evidence is still limited and the results are different between products and studies.
4. Bone regeneration- MSCs are used experimentally with scaffolds and biomaterials for bone defects and bone tissue engineering. Human clinical studies have reported bone formation and healing, but the number of patients in many studies is small and procedures are not uniform.
5. Heart diseases- MSCs have been studied in myocardial infarction and heart failure. Some trials show modest improvement in left ventricular function after cell administration. Reduction of major cardiovascular events has not been consistently demonstrated.
6. Ischemic stroke- MSC administration has been tested in patients with ischemic stroke. Different studies report possible changes in neurological function, disability and motor recovery. Cell source, dose, administration route and timing are different between studies and routine clinical use is not established.
7. Acute respiratory distress syndrome (ARDS)- MSCs have been investigated because of their immunomodulatory and tissue-supporting activities in injured lung. Recent randomized-trial analysis did not show significant improvement in major outcomes such as 28-day mortality, and the certainty of evidence was low to very low.
8. Liver diseases- Bone marrow and umbilical cord-derived MSCs have been tested in liver cirrhosis, liver failure and other chronic liver diseases. Different clinical studies examine changes in liver function, fibrosis and inflammatory responses. These therapies remain under clinical investigation and are not a replacement for liver transplantation in end-stage disease.
9. Complications after hematopoietic stem cell transplantation- Apart from GVHD, MSCs are being studied for poor graft function and support of the damaged bone marrow stromal environment after transplantation. Clinical information for these uses is still limited compared with SR-aGVHD.
10. Regenerative medicine and tissue engineering- MSCs are combined with biomaterials, scaffolds and different culture systems for bone, cartilage and other tissue-repair studies. Their differentiation capacity is used in some approaches, while many effects are associated with release of bioactive factors rather than permanent replacement of damaged tissue.

MSCs and Fibroblasts: Are They the Same?

Mesenchymal Stromal Cells (MSCs) and fibroblasts are closely related stromal cell populations, but they should not simply be considered as the same cells. A major problem is that both become very similar after in vitro culture. They have spindle-shaped morphology and grow as plastic-adherent fibroblast-like cells.

Fibroblasts are stromal cells present in connective tissues. They are strongly involved in production and organization of extracellular matrix (ECM). MSCs are also stromal cells and they produce extracellular matrix components. Because of these common properties, distinction between cultured fibroblasts and MSCs is sometimes difficult.

The common surface markers also show considerable overlap. Fibroblasts can express markers such as CD73, CD90 and CD105, which are included in the classical MSC marker panel. Many other markers commonly associated with cultured MSCs are also expressed by fibroblasts. Therefore these markers are not specific molecular markers only for MSCs.

The difference cannot always be made from differentiation assays also. Some human fibroblast preparations have shown osteogenic, adipogenic and chondrogenic differentiation under experimental induction conditions. One comparative study found dermal fibroblasts and adipose-derived MSCs similar in morphology, surface phenotype and differentiation ability.

Some functional differences can still occur. In the same comparative study, adipose-derived MSCs showed stronger angiogenic and anti-inflammatory activities than the tested dermal fibroblasts. Other studies have also found differences in gene expression and protein profiles between fibroblasts and MSC populations. These differences are affected by tissue source and culture condition.

A single marker which can reliably separate every MSC from every fibroblast has not been established. Markers such as CD146, CD106 and integrin α11 have shown differences in particular studies, but their expression can change during culture and passage. They are not universal identifying markers.

The classical ISCT minimal criteria identify culture-expanded MSCs by plastic adherence, surface-marker profile and tri-lineage differentiation. These criteria were developed as a minimum framework for MSC characterization. They were not designed as a specific test to distinguish MSCs from fibroblasts.

More recent molecular studies also show that fibroblasts and MSC populations can share many phenotypic characteristics while still having different molecular signatures. Proteomic analysis has identified differences between human dermal fibroblasts and MSC populations from adipose and dental tissues, even when several conventional characteristics were similar.

For MSCs, tissue origin and functional properties need to be given together with the common phenotype. The ISCT also recommends tissue-source designation and suitable functional assays rather than considering the basic MSC marker pattern alone as sufficient biological identity.

Limitations and Challenges of Mesenchymal Stromal Cells (MSCs)

Mesenchymal Stromal Cells (MSCs) have several limitations during isolation, culture expansion and clinical use. The properties of MSCs are not fixed between every preparation. Some of the important limitations and challenges are as follows-

• Heterogeneity- MSC cultures are heterogeneous cell populations. Cells obtained from different tissue sources do not show exactly same molecular and functional properties. Variations are also found between donors and between different cell populations from the same tissue.
• Donor-to-donor variation- Age, health condition and other donor-related factors can affect MSC growth and biological properties. The cells from one donor may not show the same proliferation, differentiation or functional activity as cells obtained from another donor.
• No single specific marker- A unique surface marker which identifies all MSCs has not been established. The commonly used CD73, CD90 and CD105 profile is a minimum characterization framework. It does not give complete functional identity of the cells. The ISCT also recommends tissue-source and functional characterization of MSC preparations.
• Variation in isolation and culture methods- Different laboratories use different isolation methods, culture media, supplements, cell densities and expansion methods. These differences can change the obtained MSC population and its properties. This makes comparison between different studies difficult.
• Changes during culture expansion- MSCs need culture expansion when large numbers of cells are required. Prolonged expansion can result in changes in morphology, gene expression and cell function. Culture-aged cells are not always equivalent to cells present during earlier passages.
• Cellular senescence- MSCs have a limited proliferative life during in vitro culture. With repeated cell divisions, some cells enter cellular senescence and their proliferation decreases. Other biological properties and secretory activity can also change during this process.
• Difficulty in measuring potency- The biological activity of an MSC product cannot always be predicted from cell viability and surface markers. Different therapeutic uses may depend on different mechanisms such as immunomodulation or paracrine activity. Development of a suitable potency assay for each intended function is therefore a major challenge.
• Effect of cryopreservation- MSCs are commonly cryopreserved for cell banking and later use. Some studies have shown reduced immunosuppressive activity immediately after thawing, which can recover after a period of culture. The effect is affected by freezing, thawing and post-thaw handling conditions and is not exactly same with every MSC product.
• Poor homing and retention- Systemically administered MSCs do not efficiently reach and remain in every target tissue. After intravenous administration, a large proportion of cells can initially become retained in the lungs, known as the pulmonary first-pass effect. Long-term persistence of administered cells is also generally limited.
• Not completely immune privileged- MSCs were earlier frequently described as immune-privileged cells. However, allogeneic MSCs can be recognized by the recipient immune system and immune responses against donor MSCs have been reported. They are more appropriately considered relatively immune-evasive rather than completely immune privileged.
• Coagulation-related effects- Some MSC preparations express tissue factor (TF/CD142) which can activate coagulation after contact with blood. The level differs according to MSC source and product characteristics. Hemocompatibility becomes an important concern particularly for intravascular administration.
• Batch-to-batch variation- MSC products prepared at different times can have differences in cell composition and functional potency. Donor source, manufacturing process, passage number, storage and post-thaw procedures can contribute to this variation.
• Dose and administration route- The number of MSCs administered, route of delivery and timing of administration are not standardized for all diseases. These factors also affect cell distribution and activity after administration.
• Clinical response is variable- Strong effects obtained in experimental models are not always reproduced in human clinical studies. MSC products used between trials can differ in tissue source, manufacturing method, potency and administration procedure. Variable clinical responses remain an important problem in development of MSC-based therapies.
• Large-scale manufacturing- Clinical use can require large numbers of cells. Expansion of MSCs to large numbers while keeping their identity, viability and functional activity consistent is difficult. Manufacturing also requires controlled processing and appropriate quality testing of the final cell product.

Safety and Regulatory Considerations of Mesenchymal Stromal Cells (MSCs)

Mesenchymal Stromal Cells (MSCs) used for clinical treatment require proper safety testing and controlled manufacturing. The safety is affected by tissue source, donor, culture expansion, cell dose, route of administration and final cell product. Clinical-grade MSCs are therefore regulated as cell-based medicinal products in many settings.

Some of the important safety and regulatory considerations are as follows-

1. Infusion-related reactions- Fever is one of the more consistently reported adverse events after intravascular MSC administration. A 2026 meta-analysis of umbilical cord-derived MSC trials found increased risk of fever, while no significant safety signal was detected for the other prespecified serious events.
2. Thrombosis and coagulation- MSCs can express tissue factor (TF/CD142) and interact with the coagulation system after entering blood. This can produce an instant blood-mediated inflammatory reaction (IBMIR). The activity can vary with donor, cell dose, tissue source and culture passage.
3. Infection and microbial contamination- MSC products are living cell preparations and contamination can occur during collection, processing or culture. Testing for bacteria, fungi, mycoplasma and other adventitious agents is required during development of clinical cell products.
4. Tumorigenicity- Tumour formation is an important theoretical safety concern for expanded cell products. Cell proliferation, genomic changes and transformation potential need to be evaluated during product development. Available randomized clinical data have not shown a clear increased malignancy signal with MSC administration, but rare or late events require longer follow-up.
5. Ectopic tissue formation- MSCs have mesenchymal differentiation capacity. Formation of unwanted tissue at an unintended location is therefore considered during safety assessment, although clinical trials have not shown a clear signal of ectopic tissue formation in systematic safety analyses.
6. Immune reactions- Allogeneic MSCs should not be considered completely immune privileged. The recipient can respond to donor-derived cells, and immunological properties may also change with inflammatory stimulation and culture conditions. Immune-related safety therefore forms part of clinical evaluation of an MSC product.
7. Culture-related changes- MSCs are usually expanded in vitro to obtain sufficient cell numbers. Long culture can change morphology, physiology and function of the cells. Culture-induced heterogeneity is one of the problems during production of a consistent MSC preparation.
8. Product identity and purity- A clinical MSC preparation needs proper characterization. Identity, purity, viability and biological activity are important quality parameters. The commonly used MSC surface markers alone do not describe all functional properties of the final product.
9. Potency testing- The biological activity of the manufactured cells needs to be linked with their proposed mechanism or clinical use. A simple viable cell count cannot always show the functional potency of an MSC product. Potency testing remains an important part of cellular therapy development.
10. Good Manufacturing Practice (GMP)- MSCs prepared for clinical use require controlled manufacturing conditions. GMP production involves controlled starting materials, culture process, storage, quality testing and documentation. Large-scale expansion while maintaining consistent cell quality is one of the major manufacturing problems.
11. Donor and starting-material safety- The tissue donor and starting biological material need suitable screening and control. Materials used during culture can also introduce contamination or other safety problems. Animal-derived serum and other biological reagents require particular quality control when they are used.
12. Cryopreservation and storage- Many MSC products are frozen before clinical administration. Freezing, thawing and post-thaw handling can alter cell viability and functional state. These conditions need to remain controlled during manufacturing and product release.
13. Route and dose of administration- Safety can differ according to cell number and route of delivery. Intravascular administration exposes MSCs directly to blood and coagulation components. Higher cell dose, cell size and procoagulant activity can become important during this type of administration.
14. Regulation in the United States- Human cell and tissue products are regulated by the U.S. Food and Drug Administration (FDA). Products that do not fulfil all requirements for regulation solely under section 361 of the Public Health Service Act can be regulated as drugs and biological products and require additional regulatory review.
15. FDA-approved MSC therapy- Ryoncil (remestemcel-L-rknd) became the first FDA-approved MSC therapy in December 2024. It is an allogeneic bone marrow-derived MSC product approved for steroid-refractory acute graft-versus-host disease (SR-aGVHD) in pediatric patients 2 months of age and older. This approval is for this defined product and indication, not for MSC treatment of diseases in general.
16. Unapproved MSC products- Many cell products marketed as stem-cell or regenerative therapies do not have FDA approval. In May 2026, FDA again warned that unapproved human cell or tissue products marketed for treatment of different diseases have not been reviewed for their quality, safety, purity or potency.
17. Regulation in the European Union- Culture-expanded MSC products can fall under Advanced Therapy Medicinal Product (ATMP) regulation in the European Union depending on their manipulation and intended use. EMA has classified ex vivo-expanded bone marrow MSC products as somatic cell therapy medicinal products or tissue-engineered products in specific cases.
18. Long-term safety monitoring- Some safety problems such as malignancy or other uncommon adverse events may require longer observation before they can be properly detected. Continued adverse-event reporting and follow-up are important during clinical development of MSC products.

Current Research and Future Directions of Mesenchymal Stromal Cells (MSCs)

Research on Mesenchymal Stromal Cells (MSCs) is now based on their heterogeneity, functional activity and development of more defined cell products. Different MSC preparations do not give exactly same biological response. Current studies include the following areas-

1. Identification of MSC subpopulations- MSC cultures contain different cell populations. Single-cell RNA sequencing (scRNA-seq) is being used to identify these differences at individual cell level. Tissue-specific and donor-related MSC subpopulations have been detected using this approach. (pubmed.ncbi.nlm.nih.gov)
2. Study of MSC heterogeneity- Tissue source, donor and manufacturing method can change MSC phenotype and function. Current studies are trying to define more uniform populations and determine which cellular properties are associated with the required activity.
3. Development of better potency assays- Cell viability and common surface markers do not completely show the functional activity of an MSC product. Research is directed toward potency assays related to the proposed mechanism, such as immunomodulation or secretory activity. A recent precision-product concept also proposes matching MSC functional state with the particular disease environment. This remains a developing framework.
4. MSC preconditioning or priming- MSCs can be exposed to selected conditions before administration to change their functional activity. Hypoxia, inflammatory cytokines, mechanical signals and different small molecules are being investigated for this purpose. These methods are mostly aimed at improving survival, secretory activity or immunomodulatory function of MSCs.
5. Genetically modified MSCs- Gene modification is also being studied to alter selected functions of MSCs. CRISPR/Cas systems, microRNA/siRNA modification and viral or non-viral gene transfer are among the methods under investigation. Much of this work is still experimental and safety of modified cells requires proper evaluation.
6. MSC-derived extracellular vesicles- Extracellular vesicles (EVs) released by MSCs contain proteins, lipids and nucleic acids. MSC-EVs are studied as cell-free products and also as carriers for delivery of therapeutic molecules. Their isolation, dose, purity, cargo and large-scale production are still important problems before wider clinical use.
7. Three-dimensional MSC culture- Conventional MSCs are mostly expanded as two-dimensional monolayers. 3D spheroids and cell aggregates are now studied because cell-cell and cell-matrix interactions are different in these conditions. Recent studies have also shown source-dependent changes in immunomodulatory activity during 3D culture.
8. Biomaterials and tissue engineering- MSCs are being combined with hydrogels, scaffolds and other biomaterials for bone, cartilage and other tissue-regeneration studies. These materials can provide biochemical and mechanical signals to the cultured cells. Donor-to-donor differences are still found even when MSCs are grown in 3D biomaterial systems.
9. Large-scale MSC manufacturing- Clinical application can require large numbers of cells. Automated expansion systems and bioreactors are being developed for production of clinical-grade MSCs. Serum-free or humanized culture systems, controlled cryopreservation and automated processing are also major areas of manufacturing research.
10. Standardization of MSC products- Different tissue sources, culture media, passage numbers and storage methods can produce different MSC preparations. Standardization of manufacturing and reporting is being studied to make clinical products and results more comparable.
11. Omics-based characterization- Transcriptomics, proteomics and metabolomics are being used to study the biological state of MSCs and their extracellular vesicles. Metabolic profiles are also being investigated as possible biomarkers of MSC potency, although such assays still need larger validation studies.
12. Patient and disease-specific MSC therapy- The same MSC preparation may not act equally in every inflammatory or regenerative condition. Current work is examining biomarkers, disease environment and timing of treatment for selection of suitable MSC products and patients. This type of approach is still developing.
13. Optimization of dose and administration- Cell dose, route of administration, timing and repeated dosing can affect MSC distribution and activity. These variables are being examined separately for different disease conditions instead of using one administration method for every MSC treatment.
14. Controlled clinical studies- Many experimental MSC applications still require larger and properly controlled clinical trials. Future studies need defined cell products, suitable potency measurements and disease-specific clinical endpoints. MSC-EVs and genetically modified MSC products also require controlled clinical evaluation before their wider therapeutic use.

Mesenchymal Stromal Cells (MSCs) vs Hematopoietic Stem Cells (HSCs)

Mesenchymal Stromal Cells (MSCs) and Hematopoietic Stem Cells (HSCs) are different cell populations. Both are present in bone marrow, but their nature, markers and functions are different.

Autologous vs Allogeneic Mesenchymal Stromal Cells (MSCs)

Autologous MSCs are obtained from the same individual who will receive the cells. Allogeneic MSCs are obtained from another donor. Both types are used in MSC research and clinical studies.

Frequently Asked Questions (FAQs) About Mesenchymal Stromal Cells

1. What are mesenchymal stromal cells?

Mesenchymal Stromal Cells (MSCs) are heterogeneous non-hematopoietic stromal cell populations. They can be isolated and culture-expanded from different tissues. Cultured MSCs show plastic adherence and under suitable in vitro conditions can differentiate into osteogenic, adipogenic and chondrogenic lineages.

2. Are mesenchymal stromal cells the same as mesenchymal stem cells?

No, not exactly. Both terms use the abbreviation MSC, but their biological meaning is different. Mesenchymal stromal cell is used for the heterogeneous culture-expanded stromal population, while the term mesenchymal stem cell needs proper evidence of stemness.

3. Why is the term “mesenchymal stromal cell” preferred?

The term mesenchymal stem cell was widely used earlier. However, all cells present in an MSC culture do not necessarily fulfil the accepted properties of a true stem cell. The ISCT therefore recommended the term multipotent mesenchymal stromal cells for these fibroblast-like plastic-adherent populations unless stemness is properly demonstrated.

4. Where are mesenchymal stromal cells found?

MSC-like stromal populations have been isolated from many tissues. They are found in stromal and often perivascular regions of different tissues, although their exact in vivo identity is not same in every tissue. Bone marrow is the classical source.

5. What are the main sources of MSCs?

The commonly studied sources are bone marrow, adipose tissue and umbilical cord tissue. MSC populations are also obtained from placenta, synovial tissue, dental tissues and several other connective tissues. The properties of MSCs obtained from different sources can differ.

6. What are the ISCT criteria for identifying MSCs?

The classical ISCT minimal criteria include three characteristics. MSCs should be plastic-adherent during standard culture, show a defined surface-marker profile and differentiate into osteoblasts, adipocytes and chondroblasts in vitro. These are minimum characterization criteria.

7. Which surface markers are expressed by mesenchymal stromal cells?

According to the classical ISCT panel, 95% or more cells should express CD73, CD90 and CD105. CD45, CD34, CD14 or CD11b, CD79α or CD19 and HLA-DR should be present in 2% or less of the population. This marker profile is not a unique molecular barcode for MSCs.

8. What can mesenchymal stromal cells differentiate into?

The standard defining differentiation pattern includes osteogenic, adipogenic and chondrogenic lineages. These form osteoblast-like, adipocyte-like and chondroblast/chondrocyte-like cells under suitable in vitro conditions. MSCs should not be described as pluripotent cells from this property.

9. How do mesenchymal stromal cells work?

MSCs can work through several mechanisms. A major activity is release of cytokines, growth factors and other paracrine factors. Cell-to-cell interaction, extracellular vesicle release, immune regulation and differentiation can also take part according to the biological condition.

10. How do MSCs regulate the immune system?

MSCs can interact with T cells, B cells, natural killer cells, dendritic cells and macrophages. They release different soluble factors and can change immune-cell activation and inflammatory responses. The activity depends on the inflammatory environment and is not fixed under every condition.

11. What is the MSC secretome?

The MSC secretome refers to substances released by MSCs into their surrounding environment. It contains soluble factors such as cytokines and growth factors together with extracellular vesicles carrying different biological molecules. Its composition can change according to MSC source and culture condition.

12. Are MSC-derived exosomes the same as mesenchymal stromal cells?

No. MSCs are living cells, while MSC-derived exosomes or other extracellular vesicles are membrane-bound particles released from these cells. They can contain proteins, lipids and nucleic acids and can carry signals to other cells. They do not contain the complete MSC and are being studied mainly as cell-free products.

13. What is the difference between MSCs and hematopoietic stem cells?

MSCs are non-hematopoietic stromal populations. Hematopoietic Stem Cells (HSCs) are true blood-forming stem cells having long-term self-renewal and multilineage hematopoietic differentiation. HSCs produce the different blood-cell lineages, while MSCs form and regulate parts of the stromal environment and show mesenchymal differentiation properties.

14. What is the difference between autologous and allogeneic MSCs?

Autologous MSCs are obtained from the same individual who receives the cells. Allogeneic MSCs are obtained from another donor and can be expanded and banked for use in different recipients. Allogeneic MSCs have immune-evasive properties but they should not be considered completely immune privileged.

15. Are any mesenchymal stromal cell therapies FDA approved?

Yes. On December 18, 2024, the U.S. FDA approved Ryoncil (remestemcel-L-rknd). It is an allogeneic bone marrow-derived MSC therapy for steroid-refractory acute graft-versus-host disease (SR-aGVHD) in pediatric patients 2 months of age and older. Ryoncil became the first FDA-approved MSC therapy. The approval is for this specific product and indication.

16. What are the major limitations of MSC therapy?

MSC preparations show considerable heterogeneity. Tissue source, donor characteristics, isolation method, culture medium, passage, storage and administration method can change their properties. Other problems include development of suitable potency assays, limited persistence after administration and variable clinical responses between different MSC products and studies.

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