Unveiling the Characteristics of Mid-Old Cells: A Key to Understanding Aging

24 December 2023

Exploring the Markers and Functions of Mid-Old Cells in Vitro and In Vivo

As the population ages, understanding the process of aging and its impact on human health has become increasingly important. One area of interest is the study of mid-old cells, a distinct group of cells that exhibit characteristics between young and old cells. By identifying the markers and functions of mid-old cells, researchers hope to gain valuable insights into the aging process and potentially develop strategies to delay or reverse age-related diseases. In this article, we delve into the latest research on mid-old cells, exploring their unique characteristics and their implications for aging.

Identifying Mid-Old Cells in Vitro

Researchers have developed an in vitro culture model to identify and study mid-old cells. By sub-culturing primary human fibroblasts in increasing passages, they observed distinct changes in cell morphology, proliferation, and gene expression. These changes allowed them to categorize the cells into three groups: young, mid-old, and old. Mid-old cells exhibited a doubling time of 5-7 days and a low percentage of senescence-associated beta-galactosidase (SA-β-Gal) positivity. Importantly, mid-old cells displayed a unique gene expression pattern, distinct from both young and old cells. They showed a p53-p21Waf1-dependent cell cycle inhibition, suggesting a different mechanism of senescence compared to old cells. RNA-sequencing further revealed that mid-old cells were more closely related to young cells than old cells at the gene expression level.

Functional Changes in Mid-Old Cells

To understand the functional changes in mid-old cells, researchers conducted gene set enrichment analysis (GSEA) using hallmark gene sets. They found that mid-old cells exhibited a clear senescence phenotype only in old cells, as indicated by the upregulation of senescence-related gene sets. However, mid-old cells did not show significant changes in these gene sets compared to young cells. Instead, they displayed upregulation of genes related to peptide metabolism, suggesting an increase in protein metabolism as a characteristic of mid-old cells. Additionally, GSEA revealed a decrease in gene expression related to DNA and mRNA metabolism in old cells compared to young and mid-old cells. These findings suggest that mid-old cells maintain some functional characteristics of young cells, while also exhibiting distinct changes associated with aging.

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Presence of Mid-Old Cells in Elderly Tissues

To investigate the presence of mid-old cells in vivo, researchers analyzed elderly tissues using immunohistochemistry (IHC). They found marked upregulation of p21Waf1 in stromal cells of elderly tissues, indicating the presence of mid-old cells. Furthermore, mid-old-specific inflammatory genes, such as IL1β and SAA1, were significantly upregulated in fibroblast-rich organs of elderly subjects. Interestingly, SAA1 expression was also elevated in smooth muscle cells of muscular mucosa, arteries, and bronchioles, suggesting a broader role of mid-old cells in tissue inflammation. Conversely, anti-inflammatory factors, such as SLIT2 and CXCL12, were downregulated in elderly tissues. These findings suggest that elderly tissues undergo a shift towards a chronically inflammatory state, characterized by the upregulation of specific inflammatory genes and the downregulation of anti-inflammatory factors.

Functional Role of SAA1 in Elderly Tissues

Further investigation revealed that SAA1, a pro-inflammatory cytokine, played a significant role in the functional changes observed in mid-old cells. Treatment with recombinant human SAA1 (rhSAA1) increased the expression of matrix metalloproteinases (MMPs) in fibroblasts and smooth muscle cells, potentially contributing to the degradation of extracellular matrix (ECM) components. In tissues from elderly subjects, increased expression of SAA1 was accompanied by enhanced MMP9 protein expression. This suggests that SAA1 may contribute to the development of aging-related microenvironments by promoting ECM degradation. Additionally, SAA1 was found to induce muscle atrophy-related genes in smooth muscle cells, highlighting its role in age-related muscle loss. The downregulation of type IV collagen, a major component of the basement membrane, in elderly tissues further supports the involvement of ECM degradation in aging-related functional decline.

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Reversal of Aging Phenotype by Young Cells

Researchers hypothesized that young cells may prevent the onset of senescence in adjacent mid-old cells. Co-culturing mid-old cells with young cells resulted in a significant increase in the proliferative capacity of mid-old cells, suggesting the presence of factors released by young cells that reverse the aging phenotype. RNA-sequencing analysis revealed that co-cultured mid-old cells exhibited functional recovery in various criteria, including self-replicative ability, ECM production, tissue regeneration, and inflammation regulation. These findings suggest the presence of “Juvenile-Associated Secretory Phenotypes (JASPs)” released by young cells that reverse the aging phenotype of mid-old cells. Further analysis identified specific factors, such as SLIT2 and Lnc-RNA-SBLC, which were abundantly expressed in young cells and showed anti-inflammatory effects on mid-old cells.

The Role of SLIT2 in Reversing Aging Phenotype

SLIT2, a known anti-inflammatory protein, was found to have a significant impact on mid-old cells. Treatment with recombinant human SLIT2 (rhSLIT2) decreased the expression of inflammatory genes and proteins in mid-old cells, including SAA1 and IL1β. Moreover, long-term treatment with rhSLIT2 restored the morphology and proliferative capacity of mid-old cells, accompanied by decreased levels of p21Waf1. Knockdown of SLIT2 in young cells induced the expression of inflammation-related genes and proteins, suggesting that decreased SLIT2 expression could promote the senescence process. Importantly, the reversal of aging phenotype in mid-old cells was abolished when SLIT2 was downregulated in co-culture experiments. These findings highlight the crucial role of SLIT2 in reversing the aging phenotype of mid-old cells.

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Anti-Aging Effects of SLIT2 in Aged Mice

To further investigate the anti-aging effects of SLIT2, aged mice were treated with recombinant mouse SLIT2 protein (rmSLIT2). Treatment with rmSLIT2 resulted in increased activity in the cage and decreased levels of the inflammatory marker SAA1 in the blood. Necropsy results demonstrated inhibition of muscle mass reduction and a decrease in the number of p21Waf1-positive mid-old cells in various organs of rmSLIT2-treated aged mice. These findings suggest that SLIT2 functions as an anti-aging molecule in aged mice, improving aging-related characteristics and promoting tissue rejuvenation.


Mid-old cells, a distinct group of cells between young and old cells, exhibit unique characteristics and functions that provide valuable insights into the aging process. By identifying the markers and functions of mid-old cells, researchers have shed light on the complex mechanisms underlying aging and age-related diseases. The discovery of factors released by young cells, such as SLIT2, that can reverse the aging phenotype of mid-old cells opens up new possibilities for developing interventions to delay or reverse age-related diseases. Further research is needed to fully understand the mechanisms involved and translate these findings into potential therapeutic strategies for promoting healthy aging.

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