Source: Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) were first identified in bone marrow and are widely distributed in the human body, such as bone marrow, umbilical cord, fat, umbilical cord blood, amniotic membrane, (placental) chorionic membrane, dental pulp, thymus, synovial membrane, fetal blood and liver. MSCs are currently thought to exist in the body around large blood vessels. Although MSCs from different tissues all meet the minimum standards (definitions) set by the International Society for Cell Therapy (ISCT) in 2006, there are still many research results suggesting that MSCs from different tissues have some differences, mainly reflected in the proliferation rate, secretion cytokine profile and immune regulation ability of MSCs. MSCs repair damaged tissues not by differentiating into cells of tissues and organs (autologous MSCs treatment may involve differentiation mechanism), but by secreting cytokines, reducing inflammation, reducing apoptosis of tissue cells, eliminating fibrosis, and promoting the proliferation of endogenous stem/progenitor cells of tissues and organs, so as to achieve the effect of repairing tissues and organs. This paper focuses on the differences of MSCs from different sources in content, proliferation capacity, immunomodulatory capacity and secretion cytokine profile.

First, the content of MSCs in different tissues is different

According to the fibroblast clonoforming unit (CFU-F) test, the content of MSCs in bone marrow accounted for about 0.001% to 0.01% of the mononuclear cells (MNC). Placental amniotic membrane and umbilical cord MSCs account for about 0.2% to 1.8% of MNC. Other studies have shown that the content of MSCs in MNC isolated from amniotic membrane and umbilical cord is as high as 80% to 100%. But only 8% of the MNC isolated from cord blood were MSCs.

The number of MSCs in cord blood is small, and the probability of successful isolation and cultivation from cord blood is only 5.7% to 10%. A research team isolated 118 MNC from cord blood, and only 11 MSCs could be cultured. Some studies have even suggested that these quantities are undetectable and cannot be amplified in vitro.

At present, it is not clear what proportion of MSCs is in single-cell fat digestive fluid, but relevant studies suggest that the content is not more than 50%, and this 50% also contains endothelial cells and adipose stromal cells.

Interestingly, MSCs have been detected in the amniotic fluid in some laboratories, and MSCs are also present in the amniotic fluid of 3 to 6 months of age. With the increase of pregnancy, the number of MSCs in the amniotic fluid continues to decline, until after the fetal development and maturity, MSCs cannot be detected in the amniotic fluid. The MSCs in amniotic fluid accounted for 0.9% to 1.5% of MNC, which was slightly higher than that in cord blood. However, this source (early fetal amniotic fluid) can easily raise ethical issues and is not suitable for routine access.

Even from the source of umbilical cord, MSCs in different parts of umbilical cord (umbilical cord membrane, umbilical cord submembrane, perivascular cord, Watton's gum) were different to some extent. The MSCs content of Watton's gum was the highest and the proliferation ability was the strongest.

Second, the proliferation ability of MSCs in different tissues is different

Bone marrow derived MSCs are the most widely studied, so bone marrow MSCs are often the reference objects for comparative studies of MSCs from other tissues.

After the separation of fat MSCs and bone marrow MSCs, it takes about 15 to 22 days for P1 generation cells to become full, but it takes 30 days for cord blood MSCs to become full. After P1 generation, the doubling time of MSCs in amniotic fluid (the time required for cell number doubling) was 1.6 days, while that of MSCs in bone marrow was 3.75 days. The proliferation capacity of adipose MSCs was close to that of amniotic fluid MSCs and was still better than the doubling time of bone marrow MSCs (about 44 hours Vs 76 hours). Other studies have shown that there is no significant difference in the proliferation ability of human umbilical cord perivascular MSCs and human bone marrow MSCs before 3 generations of cell culture. However, after 7 to 14 days (after 3 generations), the doubling time of umbilical perivascular MSCs was significantly shorter than that of bone marrow MSCs. The multiplication time of umbilical MSCs was shorter than that of placental MSCs, indicating that umbilical MSCs had better proliferation ability than placental MSCs.

Compared with umbilical cord, umbilical cord blood and amniotic membrane, bone marrow derived MSCs with the same algebra had the weakest cloning ability. The clonogenesis ability can be used as an important index to evaluate the quality of MSCs.

Under the same laboratory culture conditions, MSCs derived from bone marrow can only be expanded to the 10th generation, and MSCs derived from umbilical cord, cord blood, and amniotic membrane can only be expanded to 12-14 generations. There are even laboratories that can effectively expand the cultivation of umbilical cord MSCs to 40 generations, and still have multi-directional differentiation potential.

Karyotype analysis showed that the bone marrow MSCs had chromosome abnormalities and telomerase shortening at the 18th generation, while the umbilical cord MSCs did not have chromosome abnormalities until the 30th generation. No matter what kind of tissue MSCs are derived from, no genetic mutations have been found to possess the characteristics of tumor cells after expansion in vitro.

With the increase of age, the number and proliferation capacity of MSCs in bone marrow decreased significantly. Therefore, it is easy to understand that the proliferation ability of MSCs derived from fetal tissues (including umbilical cord, umbilical cord blood, amniotic membrane, amniotic fluid, placenta, etc.) is stronger than that of adult tissues (including adult bone marrow, adult fat, etc.).

Interestingly, sex also affects the size and proliferation capacity of MSCs. The cell diameter of female bone marrow MSCs is 20.9±0.8μm, and the doubling time is about 3.3±1.9 days, while the cell diameter of male bone marrow MSCs is 22±1.1μm, and the doubling time is about 5.0±3.7 days.

Third, the immunomodulatory function of MSCs in different tissues is different

Umbilical MSCs have lower immunogenicity and stronger immunomodulatory effects than bone marrow MSCs, and the inflammatory environment can increase the expression of HLA-DR in bone marrow MSCs (about 12%). Given TNF-α and IFN-γ stimulation, bone marrow MSCs upregulated HLA-DR expression, while placenta, umbilical cord and amniotic MSCs were not affected. However, other studies have shown that IFN-γ has no difference in the effects on fat MSCs and bone marrow MSCs. Low concentration (25-50 ng/ml) IFN-γ promotes the expression of HLA-DR in MSCs, while high concentration (100-500 ng/ml) IFN-γ down-regulates the expression of HLA-DR and promotes the secretion of IDO. However, IDO was not expressed in conventional MSCs (IDO plays an immunosuppressive role).

The improvement of HLA-DR means that the immunogenicity of MSCs is improved, and the antigen-presenting cells of the body's immune system recognize MSCs with high expression of HLA-DR and present them to T cells for killing and clearing. The high expression of HLA-DR leads to the acceleration of the body's clearance of MSCs and the shortening of the time for MSCs to play a role in the body, which directly affects the therapeutic effect. However, inflammatory environment could not improve the expression of HLA-DR in umbilical cord, cord blood, and amniotic source MSCs. According to the International Society of Cell Therapy criteria for the identification of MSCs, the expression of HLA-DR cannot be higher than 2%.

The immunosuppressive ability of MSCs from umbilical cord and amniotic membrane (80.6%± 4.2% and 85.6%±1.2%, respectively) was significantly better than that from bone marrow (69.3%±4.7%) and umbilical cord blood (44.8%±4%), and the immunosuppressive ability of MSCs from umbilical cord blood was the worst. The immunomodulatory capacity of adipose MSCs was better than that of bone marrow MSCs. Compared with bone marrow MSCs, adipose MSCs have more powerful regulatory capabilities on dendritic cells (DC cells), including promoting the proliferation of DC cells and the secretion of IL-10, down-regulating the expression of co-stimulatory molecules CD80, CD86 and CD83 on the surface of DC cells, and inhibiting the differentiation and maturation of DC cells.

In the 1:1 co-culture experiment of MSCs (P2 generation) and CD3+T cells, compared with the control group (no MSCs group), the bone marrow MSCs and cord blood MSCs inhibited the proliferation of CD3+T cells significantly, 48.2% and 42.9% respectively, while the placental MSCs only slightly inhibited the proliferation of CD3+T cells (77.0%).

4. MSCs secretion factor spectra are different in different tissues

Cord blood MSCs secrete more hematopoietic cytokines, so the effect of supporting hematopoietic stem cell cloning is better than that of bone marrow MSCs. However, some studies have shown that the ability of bone marrow MSCs to promote the formation of hematopoietic stem cell cloning is better than that of cord blood and cord sources. The cytokines secreted by umbilical MSCs (G-CSF, GM-CSF, HGF, IL-6, IL-8, IL-11) were much higher than those of bone marrow MSCs, but the amount of VEGF secreted by bone marrow MSCs was higher than that of umbilical MSCs. The phenomenon of high expression of HGF and low expression of VEGF in umbilical cord MSCs was further verified by another independent laboratory. This suggests that bone marrow MSCs have certain advantages over umbilical cord MSCs in promoting angiogenesis.

At the genetic level, fat MSCs expressed more BDNF than bone marrow MSCs, but bone marrow MSCs expressed higher NGF. Commercial bone marrow MSCs and fat MSCs showed similar proliferative and chemotactic abilities, but the proliferative and chemotactic abilities of different individual fat MSCs cultured in the laboratory showed significant differences.

Fifth, summary

Currently, MSCs are commonly found in four major tissue sources: bone marrow, umbilical cord, fat, and amniotic membrane. According to the current research results, some conclusions can be drawn: (1) The content of MSCs in tissues (cell abundance) is the highest in umbilical cord, followed by amniotic membrane and fat, and very little in bone marrow; (2) MSCs proliferation ability: Due to the age characteristics of MSCs, MSCs derived from umbilical cord and amniotic membrane have obvious advantages, followed by fat and bone marrow. (3) Immunomodulatory capacity: umbilical, amniotic and adipose MSCs were superior to bone marrow MSCs, and placental MSCs had the worst immunomodulatory capacity. (4) Secreted cytokine profiles: The total amount of growth factors secreted by umbilical MSCs was significantly higher than that of bone marrow MSCs, but the secreted cytokine profiles from different sources had obvious characteristics. Due to the characteristics of easy source acquisition and high proliferation ability, a large number of MSCs can be cultivated to meet the needs of clinical treatment, making umbilical cord, amniotic membrane and fat the most suitable tissue sources for MSCs cell therapy.

However, different laboratories, different separation methods and different culture systems may lead to inconsistent results of experimental studies on MSCs. Even from the same source, MSCs from different individuals may show functional heterogeneity. Therefore, it is necessary to be cautious about the findings and conclusions of the current study. MSCs have age properties, and the quality of MSCs cells decreases with the increase of donor age. In addition, improper culture methods can also accelerate the aging of MSCs (replicative aging).

In conclusion, (1) the quality of MSCs derived from umbilical cord and placental amniotic membrane was the highest, while that of bone marrow MSCs was the lowest; (2) The cell quality of MSCs depends on the cell culture system, and the core is the medium.