If stem cells are therapeutic, and they exert some of their actions by excreting EVs (extracellular vesicles) , then might EVs be therapeutic on their own, without stem cells?
The answer is “yes”. They are. And these are being studied to find what factors inside the EVs are exerting the positive effects seen.
Summary
EVs (extracellular vesicles) are small particles given off by stem cells, and perform much of the stem cell magic. We don’t know what all is inside them, but it would be useful to know that. If you aren’t a scientist, that may be the big take-away from this article. If you want to know the details, read the next section. If not, just skip it and go to the “Futures” section.
Details
One example of this is a study that concluded that the observed therapeutic effects of umbilical derived MSCs (Mesenchymal Stem Cells) on Pulmonary Fibrosis (in mice) is likely through their secreted EVs. These EVs contained several factors, including nucleic acids, proteins, and lipids, as you probably expected, but MSC derived EVs alleviated pulmonary fibrosis in mice, by inhibiting myofibroblast differentiation. MiR-21 and miR-23 RNAs carried by uMSC-EVs (EVs from Umbilical Mesenchymal Stem Cells) appear to downregulate the expression of TGF-β 2 and TGF-β R2 in the TGF-β signaling pathway. In other words, Extracellular vesicles derived from umbilical cord mesenchymal stromal cells alleviate pulmonary fibrosis by means of transforming growth factor-β signaling inhibition via RNA messengers.[1]
Another study found that EVs from umbilical MSCs ([CD44], CD73, CD90, CD105) had miRNA contents similar to those of their original MSC cells, targeting ion channel regulation (calcium channel activity), oxidative function, and inflammation regulation. [2] and these all have key roles in tissue injury repair. An attempt was made to predict what the effects would be of the16 miRNAs that had higher than normal levels. And they appeared to target over 100 genes in target cells and many biochemical pathways. For example, EV-enriched miRNAs participate in adrenaline and noradrenaline biosynthesis, dopamine receptor-mediated signaling pathways, including “enkephalin release, the oxidative stress response, Parkinson’s disease, the pyridoxal phosphate salvage pathway, pyridoxal-5-phosphate biosynthesis, synaptic vesicle trafficking, T cell activation, tricarboxylic acid cycle, toll-like receptor signaling, vitamin B6 metabolism, the vitamin D metabolic pathway, mRNA splicing, and the p38 mitogen-activated protein kinase pathway.” [2] This is a large number of potential effects from these small EVs. It appears that we are only starting to understand what is inside of EVs.
Another study on EVs from human umbilical cord mesenchymal stem cell-derived EVs (hUCMSC-EVs) “improved functional recovery and nerve regeneration in a rat model” of sciatica. This study found the EVs caused Schwann cells growth, by activating the PI3K/AKT signaling pathway via an RNA (miR-21).[3]
A 2002 study of EVs from human umbilical derived MSCs showed that the EVs can reduce immune inflammation, by reducing IgE-stimulated KU812 cell activation and reducing the level of NF-κB, thereby reducing inflammation factors like IL-1β, TNF-α and IL-6. The EVs also interfered with mast cell activation, and the IgE-induced signaling proteins p-P38, p-JNK and p-STAT5. This is apparently one of the mechanisms that MSCs modulation the target immune system, and if EVs are the mechanism, this lends support to the idea that EVs by themselves can be used as a therapeutic, an an immune-modulator, without the source MSCs. [4]
It is also worth mentioning that a 2011 study of umbilical cord derived stem cells found mRNA and proteins from Yamanaka Factors (Sox2, Oct4a, Nanog, ABCG2, c-Myc) that coincide with stem cell status). [6]
Futures?
There is an idea mentioned by several researchers - that EVs might be a better therapeutic than stem cells, as they can be stored easily at -80C [4] They have been used to treat heart, kidney, liver, brain, and skin issues. [4] Use of EVs instead of stem cells would avoid using living cells, and so avoid some tumor/immunity risks. EV use may also avoid infections from living cells. They can be tested for potency and safety more easily than living cell cultures. They can be stored without cytotoxic chemicals like Dimethyl Sulfoxide (DMSO) for long periods and still retain potency. And they could, in theory, be mass produced from ESC or other stem cell lines. ** [1]
I’d like to see more research where they tease out the functions of the hundreds of proteins, lipids, and RNAs in these EVs, to figure out what all they do. Also, different types of cells produce different EV contents, and I suspect this can also vary depending on the state of the source cell. (recall treatments like hypoxia on cells)
Right now, we are just treating stem cells like a “black box” where you do X and it causes Y, and that is a start, but not very scientific. Now we are beginning to talk about using EVs in the same way. We measure what they do, and we are just beginning to figure out Why.
And that works. We can use these black boxes and get some meaningful results. But once the EV contents are analyzed, then we might know Why stem cells and EVs do the functions that they do. And that is analogous to analyzing herbs to create drugs from them. In theory, we could create artificial EVs with the right contents for disease X or condition Y, and no longer depend on black boxes. We could even begin constructing our own black boxes.
References
[1] Shi, L., Ren, J., Li, J. et al. Extracellular vesicles derived from umbilical cord mesenchymal stromal cells alleviate pulmonary fibrosis by means of transforming growth factor-β signaling inhibition. Stem Cell Res Ther 12, 230 (2021). https://doi.org/10.1186/s13287-021-02296-8
[2] Xiang-yu Zou, Yongjiang Yu, Sihao Lin, Liang Zhong, Jie Sun, Guangyuan Zhang, Yingjian Zhu; Comprehensive miRNA Analysis of Human Umbilical Cord-Derived Mesenchymal Stromal Cells and Extracellular Vesicles. Kidney Blood Press Res 27 March 2018; 43 (1): 152–161. https://doi.org/10.1159/000487369
[3] Ma, Yongbin, Zhou, Dan, Zhang, Huanyan, Tang, Liming, Qian, Fen, Su, Jianhua, Human Umbilical Cord Mesenchymal Stem Cell-Derived Extracellular Vesicles Promote the Proliferation of Schwann Cells by Regulating the PI3K/AKT Signaling Pathway via Transferring miR-21, Stem Cells International, 2021, 1496101, 11 pages, 2021. https://doi.org/10.1155/2021/1496101
[4] Lin, T.-Y., Chang, T.-M., & Huang, H.-C. (2022). Extracellular Vesicles Derived from Human Umbilical Cord Mesenchymal Stem Cells Attenuate Mast Cell Activation. Antioxidants, 11(11), 2279. https://doi.org/10.3390/antiox11112279
** The FDA could have issues with use of totipotent ESC type cell lines, although these culture well, but with MSC stem cells, which are harder to grow, many safety issues are better IMHO.
[5] https://www.cell.com/molecular-therapy-family/molecular-therapy/fulltext/S1525-0016(18)30383-6
[6] “Umbilical Cord Wharton Jelly” TISSUE ENGINEERING: Part A. Volume 17, Numbers 21 and 22, 2011 Mary Ann Liebert, Inc. DOI: 10.1089/ten.tea.2010.0587
Methods of EV isolation
Method1
“uMSCs were grown to 80% density in DMEM with FBS and then replaced with serum-free medium (BI) and cultured for 48 h. The culture supernatant was collected, filtered through a 0.1-μm filter device, and then ultracentrifuged at 100,000g for 3 h. The precipitates (EVs) were washed thrice using PBS. uMSC-EVs were confirmed using transmission electron microscopy and the exosomal markers CD9, CD63, and TSG101 (Beyotime, China) through western blot. The EVs were stored at − 80 °C until use.“ [1] My note: 100000g means an Ultra centrifuge, which can be expensive.
Method 2
“Condition medium was collected and centrifuged at 2000 x g to remove the cell debris, and then centrifuged again at a speed of 100, 000 x g for 1 h at 4°C to acquire the EVs” [2]
Method 3
“Isolation of Exosomes from Cell Culture Media
Conditioned media were collected from cells grown in serum-free media for 48 hr. Dead cells and contaminating cell debris were removed by centrifugation at 300 × g for 10 min and then at 2,000 × g for 10 min at 4°C. Media were subjected to ultracentrifugation (SW32Ti, Ultra-Clear tube; Beckman Coulter, Brea, CA, USA) at 175,000 × g for 120 min at 4°C; ultracentrifugation of the resulting pellet was repeated following washing with PBS to produce a pellet containing extracellular vesicles including exosomes.”
Definitions
PF or Pulmonary fibrosis is a final stage of some lung diseases. It is the accumulation of collagen and other extracellular matrix molecules.
MSC mesenchymal stem cell. An adult stem cell that is know to have immune modulation and healing effects.
Umbilical. Umbilical cords from child birth are full of stem cells, as well as EVs, proteins, etc, and these have been found to have therapeutic effects.
DMEM with FBS. Growth media used to grow cells in the lab.
Markers. detectable proteins found on the surface of cells, which can be used to identify the cell type for clinical or research purposes.
DMSO Dimethyl sulfoxide. A cryoprotectant chemical that allows freezing cells without ice crystals forming and damaging the cells.
Indeed, Electric Vehicles are the medicine of the future,
but this is a weird statement? "Another study found that EVs from umbilical MSCs ([CD44], CD73, CD90, CD105) had miRNA contents similar to those of their original MSC cells"
The MV,MVB, ILV feel left out, all this EV acronym hype.