Unveiling the Characteristics of Mid-Old Cells: Insights from In Vitro Studies and Aged Tissues

22 December 2023

Exploring the Markers and Functional Changes of Mid-Old Cells in Aging

As the human population continues to age, understanding the cellular changes associated with aging becomes increasingly important. In an effort to shed light on the characteristics of mid-old cells, researchers have turned to in vitro studies and analysis of aged tissues. By examining the markers and functional changes of mid-old cells, scientists hope to gain a deeper understanding of the aging process and potentially identify ways to mitigate its effects.

Exploring Mid-Old Cells in In Vitro Studies:

In order to identify mid-old cells in elderly tissue, researchers conducted in vitro studies using primary human fibroblasts. These fibroblasts were sub-cultured in increasing passages, allowing researchers to observe the changes in morphology, proliferation, and gene expression. Through this process, the fibroblasts were categorized into three groups: young, mid-old, and old cells. The mid-old cells exhibited distinct expression patterns of senescence markers and gene expression patterns that were more closely related to young cells than old cells.

Characteristics of Mid-Old Cells:

Further analysis of the mid-old cells revealed several characteristics. These cells displayed a unique gene expression pattern, including an upregulation of genes related to peptide metabolism. They also exhibited a decrease in gene expression related to DNA and mRNA metabolism, suggesting a shift in metabolic processes during aging. Additionally, mid-old cells showed a distinct inflammatory response, with an upregulation of the IL1β pathway. This inflammatory response was different from that observed in old cells, which exhibited upregulation of TNFα and IL6 signaling pathways.

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Presence of Mid-Old Cells in Aged Tissues:

To validate the findings from in vitro studies, researchers analyzed aged tissues for the presence of mid-old cells. Immunohistochemistry studies revealed an increase in markers specific to mid-old cells, such as p21Waf1 and IL1β, in stromal cells of elderly tissues. The expression of these markers was particularly prominent in fibroblast-rich organs, such as the colon and lung. Additionally, the expression of SAA1, an acute response protein, was elevated in smooth muscle cells of these tissues. These findings suggest that mid-old cells are present in aged tissues and contribute to the aging process.

Functional Role of Mid-Old Cell Markers:

Further investigation into the functional role of mid-old cell markers revealed their impact on tissue function. SAA1, which was highly upregulated in mid-old cells, was found to promote the degradation of extracellular matrix (ECM) components. This degradation of the ECM could contribute to the aging-related symptoms observed in the colon and lungs, such as distorted absorption and peristalsis. Additionally, SAA1 was found to induce muscle atrophy-related genes in smooth muscle cells. These findings suggest that mid-old cell markers play a role in tissue dysfunction associated with aging.

Reversal of Aging Phenotype by Young Cells:

To explore the potential for reversing the aging phenotype, researchers investigated the effects of young cells on mid-old cells. Co-culturing mid-old cells with young cells or treating mid-old cells with conditioned media from young cells resulted in a significant increase in the proliferative ability of mid-old cells. RNA-sequencing analysis revealed that functional criteria of mid-old cells, such as self-replication, extracellular matrix formation, tissue organization/regeneration, and immune response regulation, were restored. This suggests that factors secreted from young cells have the ability to reverse the aging phenotype of mid-old cells.

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Identification of Anti-Aging Factors:

To identify the factors responsible for the restoration of mid-old cell function, researchers analyzed the composition of conditioned media from young cells. Long non-coding RNAs (Lnc-RNAs), including SBLC and SLIT2, were found to be abundantly expressed in young fibroblasts. Overexpression of SBLC in mid-old cells decreased the expression of p21Waf1, a marker of senescence. However, SLIT2 was found to have a more significant impact on mid-old cells. Treatment with recombinant SLIT2 protein resulted in decreased expression of inflammatory genes and proteins, as well as a restoration of the proliferative capacity of mid-old cells. Downregulation of SLIT2 in young cells had the opposite effect, leading to increased expression of inflammatory genes and decreased proliferation.

Anti-Aging Effects in Aged Mice:

To validate the anti-aging effects of SLIT2, researchers conducted experiments using aged mice. Treatment with recombinant mouse SLIT2 protein resulted in increased activity, inhibition of muscle mass reduction, and a decrease in the number of mid-old cells in various tissues. These findings suggest that SLIT2 functions as an anti-aging molecule in aged mice.


The study of mid-old cells provides valuable insights into the aging process and potential strategies for reversing the aging phenotype. Through in vitro studies and analysis of aged tissues, researchers have identified markers and functional changes associated with mid-old cells. Factors secreted from young cells, such as SLIT2, have been found to reverse the aging phenotype of mid-old cells and exhibit anti-aging effects in aged mice. These findings pave the way for further research into the mechanisms of aging and the development of interventions to promote healthy aging.

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