Journal: Aging Cell

Article Title: Aging Adipose‐Derived Mesenchymal Stem Cells, Cultured on a Native Young Extracellular Matrix, Are Protected From Senescence and Apoptosis Along With Increased Expression of HLA ‐ DR and CD74 Associated With PI3K Signaling

doi: 10.1111/acel.70165

Figure Lengend Snippet: Differentiation capacity of aging AD‐MSCs is enhanced with maintenance on ECM Plus (ECM) as compared to TCP. (A) Formation of colony‐forming units (CFUs) was enhanced when cells were pre‐cultured on ECM as compared to TCP (P1 and P3). CFU‐fibroblasts (CFU‐F) were stained with crystal violet (blue); CFU‐adipocytes (CFU‐AD) were stained with Oil Red O (red); and CFU‐osteoblasts (CFU‐OB) were stained with von Kossa. (B–D) CFU quantification was performed by counting the number of colonies/well (CFU#/well) or pixels per 10 cm 2 well. * p < 0.05 ( n = 3), versus TCP at the same passage. (E, F) Generation of cartilage was enhanced when cells were pre‐cultured on ECM as compared to TCP. Transverse sections of cartilage pellets, stained with Alcian Blue, clearly demonstrated the presence of mucins and proteoglycans. In addition, the expression of type II collagen (COL2A1), a major collagenous protein of cartilage, was also significantly increased by pre‐culture on ECM. * p < 0.05 ( n = 3), versus TCP group treated with chondrogenic media. (G–I) Neurogenesis was enhanced when the cells were pre‐cultured on ECM as compared to TCP. After overnight incubation in neurogenic induction media, a marked morphological difference between cells pre‐cultured on ECM versus TCP could be seen under phase contrast microscopy. Moreover, neuron‐specific Nissl body staining was observed and transcripts for neurogenic markers, musashi1 (Msi1) and microtubule associated protein 2 (MAP2) were increased. * p < 0.05 ( n = 3), versus TCP group treated with neurogenic media. (J) In vivo bone formation capacity of cells pre‐cultured on ECM was enhanced compared to TCP (P1 or P3). Following each passage, aging AD‐MSCs (ASCs) or Wharton Jelly (WJ) cells pre‐cultured on TCP or ECM (1 × 10 6 cells) were loaded onto HA/TCP particles and implanted subcutaneously into the dorsal surface of 10‐week‐old immunodeficient mice. After 8 weeks in vivo, three implants for each group were harvested for histological analysis. B, bone‐like regions; Ft, fat tissue; and HA, HA/TCP. Low (L) magnification: Scale bar: 200 μm. High (H) magnification: Scale bar: 100 μm. (K) Quantification of new bone formation (“% bone area”) was histomorphometrically determined using Image J analysis software. The data shown in the figure represents the mean and standard deviation that was calculated using 9 H&E sections (3 per implant, repeated 3 times to have 3 implants with cells from 3 different donors). * p < 0.05 ( n = 9), versus TCP group. (L, M) Trophic factor expression by the cells was increased with maintenance on ECM as compared to TCP. To simulate an inflammatory environment, cells pre‐cultured on ECM or TCP were treated with TNF‐α (20 ng/mL) for 48 h and the expression of prostaglandin‐endoperoxide synthase 2 (PTGS2), which encodes the cyclooxygenase 2 (COX‐2) enzyme, and TNF‐α induced protein 6 (TNFA1P6) was measured by RT‐PCR. * p < 0.05 ( n = 3), versus TCP group treated with TNF‐α.

Article Snippet: MSCs cultured on TCP or ECM Plus were re‐suspended at 10 6 cells/mL in either control media (DMEM containing 2 mM L‐glutamine and 10% FBS) or Promocell Chondrogenic Differentiation Medium (Fisher Scientific, Waltham, MA) and seeded into U‐Bottom Suspension 96‐well plates (Genesee Scientific, San Diego, CA).

Techniques: Cell Culture, Staining, Expressing, Incubation, Microscopy, In Vivo, Software, Standard Deviation, Reverse Transcription Polymerase Chain Reaction