Senescence and quiescence in adipose-derived stromal cells:Effects of human platelet lysate, fetal bovine serum and hypoxia

AbstractBackground aims. Adipose-derived stromal cells (ASCs) are attractive sources for cell-based therapies. The hypoxic niche of ASCs in vivo implies that cells will benefit from hypoxia during in vitro expansion. Human platelet lysate (hPL) enhances ASC proliferation rates, compared with fetal bovine serum (FBS) at normoxia. However, the low proliferation rates of FBS-expanded ASCs could be signs of senescence or quiescence. We aimed to determine the effects of hypoxia and hPL on the expansion of ASCs and whether FBS-expanded ASCs are senescent or quiescent. Methods. ASCs expanded in FBS or hPL at normoxia or hypoxia until passage 7 (P7), or in FBS until P5 followed by culture in hPL until P7, were evaluated by proliferation rates, cell cycle analyses, gene expression and β-galactosidase activity. Results. hPL at normoxia and hypoxia enhanced proliferation rates and expression of cyclins, and decreased G0/G1 fractions and expression of p21 and p27, compared with FBS. The shift from FBS to hPL enhanced cyclin levels, decreased p21 and p27 levels and tended to decrease G0/G1 fractions. Conclusion. Hypoxia does not add to the effect of hPL during ASC expansion with regard to proliferation, cell cycle regulation and expression of cyclins, p21 and p27. hPL rejuvenates FBS-expanded ASCs with regard to cell cycle regulation and expression of cyclins, p21 and p27. This indicates a reversible arrest. Therefore, we conclude that ASCs expanded until P7 are not senescent regardless of culture conditions

Retention and Functional Effect of Adipose-Derived Stromal Cells Administered in Alginate Hydrogel in a Rat Model of Acute Myocardial Infarction

Background. Cell therapy for heart disease has been proven safe and efficacious, despite poor cell retention in the injected area. Improving cell retention is hypothesized to increase the treatment effect. In the present study, human adipose-derived stromal cells (ASCs) were delivered in an in situ forming alginate hydrogel following acute myocardial infarction (AMI) in rats. Methods. ASCs were transduced with luciferase and tested for ASC phenotype. AMI was inducted in nude rats, with subsequent injection of saline (controls), 1 × 106 ASCs in saline or 1 × 106 ASCs in 1% (w/v) alginate hydrogel. ASCs were tracked by bioluminescence and functional measurements were assessed by magnetic resonance imaging (MRI) and 82rubidium positron emission tomography (PET). Results. ASCs in both saline and alginate hydrogel significantly increased the ejection fraction (7.2% and 7.8% at 14 days and 7.2% and 8.0% at 28 days, resp.). After 28 days, there was a tendency for decreased infarct area and increased perfusion, compared to controls. No significant differences were observed between ASCs in saline or alginate hydrogel, in terms of retention and functional salvage. Conclusion. ASCs improved the myocardial function after AMI, but administration in the alginate hydrogel did not further improve retention of the cells or myocardial function

Adipose-derived stromal cells increase the formation of collagens through paracrine and juxtacrine mechanisms in a fibroblast co-culture model utilizing macromolecular crowding

BACKGROUND: Adipose-derived stromal cells (ASCs) possess a multitude of regenerative capabilities, which include immunomodulation, angiogenesis, and stimulation of extracellular matrix (ECM) remodeling. However, the underlying mechanisms leading to ECM remodeling remain largely elusive and highlight the need for functional in vitro models for mode of action studies. Therefore, the purpose of this study was to develop an in vitro co-culture model to investigate the capabilities of ASCs to modulate fibroblasts and ECM. METHODS: An ECM in vitro model with ASCs and normal human dermal fibroblasts (NHDFs) was established utilizing macromolecular crowding, ascorbic acid, and TGF-β stimulation. Paracrine and juxtacrine co-cultures were created using transwell inserts and cell cultures with direct cell–cell contacts. The cultures were screened using RT(2) PCR Profiler Arrays; the protein levels of myofibroblast differentiation marker alpha smooth muscle actin (αSMA) and ECM remodeling enzymes were analyzed using western blot on cell lysates; the formation of collagen type I, III, VI, and fibronectin was investigated using ELISA on culture supernatants; and the deposition of collagens was analyzed using immunocytochemistry. RESULTS: TGF-β stimulation of NHDF monocultures increased the expression of 18 transcripts relevant for ECM formation and remodeling, the protein levels of αSMA and matrix metalloproteinase-2 (MMP-2), the formation of collagen type I, III, VI, and fibronectin, and the deposition of collagen type I and VI and decreased the protein levels of MMP-14. Inclusion of ASCs in the ECM co-culture model increased the formation of collagen type I and III through paracrine mechanisms and the formation of collagen type VI through juxtacrine mechanisms. CONCLUSIONS: The co-culture model provides effective stimulation of NHDF monocultures by TGF-β for enhanced formation and deposition of ECM. In the model, ASCs induce changes in ECM by increasing formation of collagen type I, III and VI. The obtained results could guide further investigations of ASCs’ capabilities and underlying mechanisms related to ECM formation and remodeling. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s13287-022-02923-y