At present, criteria for determining and characterizing MSCs follow those proposed by the ISCT [ 28 ]. Accordingly, MSCs are defined as cells that will adhere to TCP under standard culture conditions, express particular cell surface antigens (> 95% positive meant for CD73, CD90, and CD105), and have trilineage differentiation possible. As reported by others [ 25 , 26 , 29 ], and demonstrated in the present study, these criteria are inadequate. First, plastic-type material adhesion of very early-stage MSCs is poor [ 29 , 30 ] and accumulating evidence indicates that tradition on TCP alters the MSC phenotype [ 10 , 31 , 32 ]. Second, cell surface marker expression, which is routinely utilized to define MSCs, does not correlate well with the differentiation condition of the cells or their potency, and does not distinguish between MSCs obtained from young versus old donors (Additional file 1 : Figure S1). These weak points are highlighted in the current study.
In the present study, we have uncovered several features that correlate well with functional properties of the cellular material. With aging, loss of stemness was accompanied by a decrease in cellular proliferation and differentiation, SSEA-4 expression, and ATP content material, and an increase in cell size and expression associated with β -Gal, intracellular ROS, and Annexin-V. However , in contrast to our previous report in mice [ 15 ], the current study showed that exposure to young ECM only reasonably restored the proliferation and differentiation of elderly MSCs (Fig. 4b ). Curiously, the percentage of SSEA-4 in elderly MSCs classy on young ECM was rescued, but without raising the total number of cells, suggesting that cell death is happening at a higher rate in the elderly cells. To confirm that will elderly MSCs produce cytokines with deleterious paracrine results, we treated young MSCs with media conditioned simply by elderly cells and found that proliferation was under control (Fig. 3a ). Consequently, we found that conditioned media from elderly MSC cultures contained significantly increased levels of cytokines associated with the SASP [ 27 ]. We hypothesize that, in vivo, small(+) cells fail to thrive because they are exposed to various SASP cytokines which negatively impact their behavior [ 23 , 33 – 35 ].
Prior work on the particular SASP has been performed on fetal cells or immortalized cell lines [ 27 , 36 – 38 ]. To the best of our understanding, the current study is the first to report that aged MSCs display the SASP. This observation may have excellent relevance for stem cell-based regenerative therapies, since the existing logic is that MSCs have potent immunomodulatory capabilities and really should be able to combat inflammation and reverse the effects of the SASP in the elderly population [ 39 , 40 ]. In contrast, we display that elderly MSCs, which contain a considerable number of senescent cells, might actually contribute to inflammation and further diminish endogenous MSC function. Therefore, autologous stem cell therapies in elderly individuals might actually compound or promote age-related degeneration. In the present study, all of us show that in contrast to the original population of elderly MSCs, the “ youthful” subpopulation expresses fewer SASP cytokines at levels similar to young MSCs (Fig. 3b ), but also displays a non-SASP cytokine profile reminiscent of the parent elderly population (Fig. 3c ). These results support our hypothesis that “ youthful” MSCs could be rescued from the deleterious environment in the elderly MSC human population. Interestingly, the cytokine array also called to our attention that will conditioned media of young MSCs contained less IL-6 and more VEGF and GDNF when cultured on ECM vs TCP, suggesting that the therapeutic efficacy of MSCs may be enhanced by maintenance on ECM.
While the results are promising, 1 drawback of the study is that cells from young and aged donors were obtained from different sources. Young MSCs had been obtained from iliac crest BM aspirates, while elderly MSCs were obtained from surgical waste (bone chips, BM, and so forth ) of patients undergoing total joint arthroplasty. Hence, there were substantial differences in age, donor site (femur versus iliac crest), and presence/absence of disease between the groupings. These issues do not significantly detract from our findings as we could obtain MSCs with characteristics consistent with other reports within the literature. However , it will be necessary in the future to address these possible issues in “ healthy” elderly donors using an suitable animal model.
Recently, others have verified that MSCs maintained on native ECM (vs TCP) are smaller in size and of higher quality [
]. In the current research, we were able to consistently isolate a subpopulation of “ youthful” cells from preparations of elderly MSCs providing a few criteria and then successfully expand them on a previously recognized culture system that promotes proliferation and retention associated with stemness [
]. The number of cells obtained at the end of 3 weeks was more than 28-fold higher than when unfractionated aged MSCs were cultured on TCP (fold-changes: 17, 120/608) (Fig.
). In Fig.
, we propose that the ratio of younger to elderly MSCs changes with aging and summarize a strategy for establishing personal stem cell banks along with sufficient numbers of high-quality cells to support multiple infusions associated with autologous “ youthful” MSCs. Based on data in the current research, 2 . 5 × 10
BM-MSCs from an 80-year-old 90-kg donor would be expected to contain about 2 × 10
small(+) cells. After 3 weeks of subculture on BM-ECM (initial seeding density of 2 × 10
), we would expect that the small(+) cell population could have increased to approximately 3 × 10
cells. To deal with this patient (donor) with an infusion of 1 × 10
cells/kg every 3 months, 3 × 10
cells would support more than 32 infusions of cell-based therapy over 8 years. To the best of our understanding, there is no currently available system able to rapidly amplify such a few cells from an elderly patient and meet the medical demand for large numbers of high-quality MSCs. This approach would be specifically valuable for patients requiring multiple infusions to treat age-related degenerative diseases that are virtually unaffected by a single infusion of stem cells [
]. Furthermore, repeated infusion of high-quality MSCs would provide a strategy for slowing down or even reversing the well-known deleterious effects of aging on the microenvironment and its effects on stem cell viability and function/activity. However , a significant amount of work remains to achieve this goal, including the marketing of cell dose, length of treatment, and route associated with administration (systemic vs local), and enable the progressive reversal of the aged MSC microenvironment.
Elderly MSCs with a “ youthful” phenotype can be expanded on young ECM for cell-based remedies. a Ratio associated with young to elderly MSCs changes with chronological age group. In the elderly, the proportion of young (“ youthful” ) MSCs has dropped to approximately 8– 10% of the total population. This can be clearly seen in the brightfield micrographs. In the young, virtually all cells display a fibroblastic spindle-shaped morphology. With advancing age, cells become bigger and spread across the surface (black arrows), but fibroblastic cells can still be found (white arrows). b Our current studies strongly recommend it is possible to rescue these “ youthful” cells by splitting up from the parent MSC population, based on cell size plus SSEA-4 expression, and then expand them on a young ECM to bank large quantities of high-quality autologous MSCs for more effective treatment of age-related diseases. ECM extracellular matrix, FSC forward scatter, MSC mesenchymal stem cell, SSEA-4 stage-specific embryonic antigen-4