In the field of life sciences, cell therapy has always been a hot topic, but the controversy surrounding mesenchymal stromal cells and mesenchymal stem cells has lasted for more than two decades, which has become a major obstacle restricting the research and clinical application of MSCs. The core issue is that mesenchymal stem cells should not be approved for clinical use without comprehensive safety testing. However, all preclinical studies have shown that the currently used MSCs do not present stem-cell-related risks in terms of safety and should therefore be accurately defined as "mesenchymal stromal cells" rather than "stem cells."

In response to this problem, The Regenerative Medicine Research Center of West China Hospital and the Laboratory of Stem Cell Biology of Tasly jointly published the title "Unveiling Distinctions Between Mesenchymal Stromal Cells and Stem Cells by" at HELIYON The research article of Single-Cell Transcriptomic Analysis (revealing the difference between mesenchymal stromal cells and stem cells through single-cell transcriptomic analysis) has provided a breakthrough theoretical basis and completely solved this difficult problem that has long plagued the development of the industry.

Using advanced single-cell RNA sequencing (RNA-SEQ) and pseudo-time locus analysis, the research team found that stem cells exhibit strong self-renewal and differentiation capabilities, while mesenchymal stromal cells (MSCS) lack the expression of these key dry genes. These genes include SOX2, NANOG, POU5F1, SFRP2, DPPA4, SALL4, ZFP42, and MYCN. On the other hand, five key stromal cell functional genes, TMEM119, FBLN5, KCNK2, CLDN11 and DKK1, were expressed only in mesenchymal stromal cells and not in stem cells.

The study found that the MSCs from different tissues that are widely used at present are mesenchymal stromal cells. However, these cells (MSCs) have long been misused as stem cells in many cases. Although safety concerns have been ignored due to the nature of MSCs, the therapeutic efficacy of these cells has been a major drawback due to confusion over their mechanism of action. The mechanism of action of stem cells is to replace damaged functional cells through their ability to differentiate, while the mechanism of action of stromal cells is to lead to the restoration of the microenvironment through homing and excretion functions. Of course, these two different types of cells must be used in different ways because they perform different functions in the host body.

Specifically:

1. The single-cell transcriptome reveals the difference between MSCs and stem cells

The research team integrated all the RNA-SEQ data from the dataset and samples from this study, and found that pluripotent stem cells (PSCs), adult stem cells (ASCs), and mesenchymal stromal cells (MSCs) showed significant clustering through UMAP analysis. Further analysis showed that the highly expressed genes in PSCs were mainly involved in self-renewal and proliferation. The genes highly expressed in ASCs were more involved in differentiation and metabolism. However, the genes highly expressed in MSCs are mainly related to cell metabolism and interaction, and lack of genes involved in self-renewal or differentiation.

In order to define the developmental state and interconnections between different cell types, the research team conducted a quasi-temporal developmental trajectory analysis based on the expression of self-renewal/differentiation related genes. The results showed that different cell types showed obvious clustering distribution according to their developmental stages, and the trajectory was divided into early, middle and late stages, corresponding to PSCs, ASCs and MSCs respectively. The localization of the cells at the advanced stage indicates that the lack of self-renewal and differentiation related genes is a key feature that distinguishes MSCs from stem cells.

Functional gene analysis of MSCs at single cell level

2. MSCs do not express key genes for self-renewal and differentiation

Through single-cell transcripome analysis, eight key genes involved in self-renewal and differentiation were identified, including core genes SOX2, NANOG and POU5F1, which maintain self-renewal ability; SFRP2, which plays an important signaling role in self-renewal; DPPA4, SALL4 and ZFP4, which participate in downstream transcriptional regulation of self-renewal. And MYCN, which plays a key driving role in ectodermal differentiation.

By analyzing the expression of these genes, the study found that these genes are widely expressed in stem cells, but in four different tissue-derived mesenchymal stromal cells (MSCs), Almost no expression was found in adipose derived MSCs (AD-MSCs), umbilical cord derived MSCs (UC-MSCs), placenta derived MSCs (PM-MSCs) and bone marrow derived MSCs (BM-MSCs). Further analysis revealed that the proportion of cells expressing these genes in MSCs was extremely low, indicating that MSCs differ significantly from stem cells in their ability to self-renew and differentiate.

Single cell transcriptome analysis of different mesenchymal stem cells

3. Stem cells do not express functional characteristic genes of MSCs

The above single-cell transcriptome and locus analysis revealed that MSCs were missing eight key genes for self-renewal and differentiation, but had high expression of specific functional genes. Cluster analysis of gene expression profiles of MSCs and stem cells from different sources showed that five key functional genes (TMEM119, FBLN5, KCNK2, CLDN11 and DKK1) were expressed in all MSCs, but not in stem cells. At the same time, a large number of genes are expressed in common in all MSCs, and some genes are only expressed in specific types of MSCs, especially in AD-MSCs, which indicates that there are functional differences among different MSCs.

To verify these genetic markers, the research team analyzed FBLN5 protein expression levels in AD-MSCs samples by flow cytometry, and the results showed that 90.32% of AD-MSCs expressed FBLN5 protein, which was consistent with single-cell transcriptomic analysis.

4. MSCs of different tissue origin have unique single-cell transcriptome

The research team performed cluster analysis on MSCs from four different tissue sources and found that their clustering was different, suggesting that they may have different functions. Functional analysis of single cell transcriptomes showed that there were differences in the expression of functional genes of MSCs from different sources. For example, the high-expression genes of AD-MSCs were related to "chemical signal reception", and the high-expression genes of UC-MSCs and PM-MSCs were enriched in "chemokine perception". The trend of BM-MSCs gene expression is related to PDGF-induced cell motility. To further explore the differences between MSCs of different origin, the research team performed cell fate locus analysis of different MSCs relative to embryonic stem cells (ESCs). The results showed that PM-MSCs and UC-MSCs were located in the same fate cluster, BM-MSCs were close to it but located in different clusters, AD-MSCs were furthest away from ESC, and all MSCs and ESCs had obvious developmental boundaries.

The therapeutic effect of MSCs is believed to be closely related to the bioactive factors secreted by MSCS. The research team analyzed the expression differences of secretion-related genes, and the results showed that MSCs from different tissues expressed 455 secretory factors, which could interact with 217 cell membrane receptors and play common functions, and their expressions were different among different samples. AD-MSCs were actively expressed in vascularization and other aspects. BM-MSCs are moderately active but do not express anti-apoptotic genes, PM-MSCs are expressed with low intensity, and AD-MSCs are weakly expressed and do not express ECM remodeling genes.

5. Application of mesenchymal stromal cell identification criteria in the analysis of mixed cell samples

The research team attempted to apply the criteria established above to the analysis of mixed cell samples, focusing on the content of intermediate mesenchymal stromal cells (MSCs) and stem cells (n=11). The results showed that the expression of stem cell marker genes was high in the amniotic fluid samples, while the expression of MSCs functional genes was low. Further analysis showed that stem cells accounted for 13.66% (range 10.45%-21.82%) and MSCs accounted for only 1.52% (range 0.06%-4.49%) in the amniotic fluid samples.

Mesenchymal stem cells and stem cell content and marker gene expression

This research result not only ends the long-term controversy over the identity of MSCs, but also provides a solid scientific basis for the re-planning of the clinical application path of MSCs. The study clearly distinguishes the mechanisms of action of two types of cells: stem cells rely on differentiation to replace damaged cells, while stromal cells reshape the microenvironment through homing and secretion. This breakthrough completely corrects long-standing misconceptions about the safety and efficacy of MSCs, establishes criteria for the identification of mesenchymal stromal cells, and opens up new avenues for personalized therapy. With the promotion of this discovery, MSCs therapy will be more widely and effectively used in clinical practice, undoubtedly driving its revolutionary breakthrough in disease treatment.