Cyclical aggregation extends in vitro expansion potential of human mesenchymal stem cells

Abstract

Mesenchymal stem cell (MSC)-based therapy has shown great promises in various animal disease models. However, this therapeutic potency has not been well claimed when applied to human clinical trials. This is due to both the availability of MSCs at the time of administration and lack of viable expansion strategies. MSCs are very susceptible to in vitro culture environment and tend to adapt the microenvironment which could lead to cellular senescence and aging. Therefore, extended in vitro expansion induces loss of MSC functionality and its clinical relevance. To combat this effect, this work assessed a novel cyclical aggregation as a means of expanding MSCs to maintain stem cell functionality. The cyclical aggregation consists of an aggregation phase and an expansion phase by replating the dissociated MSC aggregates onto planar tissue culture surfaces. The results indicate that cyclical aggregation maintains proliferative capability, stem cell proteins, and clonogenicity, and prevents the acquisition of senescence. To determine why aggregation was responsible for this phenomenon, the integrated stress response pathway was probed with salubrial and GSK-2606414. Treatment with salubrial had no significant effect, while GSK-2606414 mitigated the effects of aggregation leading to in vitro aging. This method holds the potential to increase the clinical relevance of MSC therapeutic effects from small model systems (such as rats and mice) to humans, and may open the potential of patient-derived MSCs for treatment thereby removing the need for immunosuppression.

In Vitro Culture Expansion Shifts the Immune Phenotype of Human Adipose-Derived Mesenchymal Stem Cells

“…On the other hand, adipogenic differentiation of P12 hASCs all showed decreased mRNA levels for genes responsible for early differentiation regulation and late-stage maturation of adipocytes ( C/EBPα, FABP4, LPL , and PPARγ ) compared to the P5 group ( Figure 1F ). The representative images of hASC differentiation into adipocytes and osteoblasts were shown by Oil Red O staining and Von Kossa staining respectively ( Supplementary Figure S2 ) and in our previous publication ( 42 ). hASCs’ functionality at high passages was also tested for CFU-F, which showed colony-forming ability decreased from about 35 colonies for P5 cells to less than 20 colonies for P12 cells ( Figure 1G ) .…”

“…hASCs subjected to extended in vitro culture had moderately increased population doubling times (although there was no statistical significance), maintaining a ratio of a population doubling roughly every 3 days ( Figure 1B ). However, the accumulative cell doublings for hASCs of aged donors in our previous publication revealed the prolonged doubling time ( 42 ). Similarly, mRNA levels encoding for p53 and cyclin-dependent kinase inhibitor proteins ( p15 and p21 ) responsible for regulating cell cycle and cellular senescence show no difference between P4 and P12 cells ( Figure 1C and Supplementary Figure S1 ).…”

“…Frozen hASCs at passage 1 were acquired from the Tulane Center for Stem Cell Research and Regenerative Medicine. The hASCs were isolated from the subcutaneous abdominal adipose tissue from three de-identified healthy donors that were younger than 45 years old with a body mass index lower than 25 ( Supplementary Table S1 ) ( 31 , 42 ). The isolated cells were characterized for their MSC properties through colony-forming unit (CFU) assays as well as tri-lineage differentiation potential (osteogenic, adipogenic, and chondrogenic differentiation) in vitro .…”

Engineering extracellular vesicles by three‐dimensional dynamic culture of human mesenchymal stem cells

“…The cargo profiles of EVs from 3D cortical spheroids derived from human induced pluripotent stem cells (hiPSCs) have been reported to reflect lineage‐specific developmental stages of stem cell differentiation in previous studies (Marzano et al., 2019 ; Marzano et al., 2021 ). Moreover, recent studies provide strong evidence that 3D aggregate culture induces drastic changes in hMSC homeostasis, characterized by enhanced glycolysis, activation of autophagy, and anti‐senescence property, retention of the primitive phenotype of hMSCs in vitro (Bijonowski et al., 2020 ; Bijonowski et al., 2020 ; Liu et al., 2017 ; Sart et al., 2014 ; Yuan et al., 2019 ; Yuan et al., 2019 ). In particular, the secretome of 3D hMSC aggregates exhibited enhanced expression of cytokines associated with immunosuppression and anti‐apoptosis compared to 2D culture (Bartosh et al., 2010 ; Sart et al., 2014 ).…”

Spatiotemporal Regulation of Cell–Cell Adhesions

“…Self-assembled multicellular aggregates form by mixing multiple cell types such that microtissues with desired organization form. Generally, these structures form based on minimizing the potential internal energy resulting from cell-cell adhesions [24,25]. Self-assembled aggregates have been used to construct multicell neuro-organoids comprised of cortical neural progenitor cells, endothelial cells, and mesenchymal stem cells.…”