Induced Differentiation of Macaque Adipose-Derived Stem Cells in Vitro

0
0
0

Induced Differentiation of Macaque Adipose-Derived Stem Cells in Vitro

Stem cells are a cornerstone of regenerative medicine, but one of the most studied types—bone marrow mesenchymal stem cells (BMSCs)—has significant limitations: low purification rates, limited availability, and an invasive collection process that causes pain. This has driven interest in adipose-derived stem cells (ADSCs), a more accessible alternative found in fat tissue. ADSCs share BMSCs’ ability to differentiate into multiple cell types but are easier to collect, proliferate faster, and avoid immune rejection or ethical concerns. However, most ADSC research relies on small mammals like mice or rabbits. A critical gap remains: how do ADSCs from primates—species anatomically and physiologically closer to humans—behave? A 2021 study by Chinese researchers aimed to answer this by investigating macaque ADSCs.

Led by Jun-Liang Jiang, Tao Li, and colleagues from the Department of Orthopedics and Trauma at the Second People’s Hospital of Yunnan Province, the study focused on isolating macaque ADSCs and testing their multipotent differentiation potential. The work, published in the Chinese Medical Journal, followed strict ethical protocols approved by the hospital’s animal ethics committee and used abdominal subcutaneous fat from macaques provided by the Kunming Primate Research Center of the Chinese Academy of Sciences.

To isolate ADSCs, the team used collagenase and neutral protease to digest fat tissue, then cultured the cells in standard lab conditions. They measured cell growth with a Cell Counting Kit-8 (CCK-8) and analyzed the cell cycle—whether cells were actively dividing—using flow cytometry. To test multipotency (the ability to become different cell types), they exposed ADSCs to three specialized “induction media”: one to trigger chondrogenic (cartilage) differentiation, one for osteogenic (bone) differentiation, and one for adipogenic (fat) differentiation. They used staining techniques to confirm changes: toluidine blue for cartilage proteins, Von Kossa for calcium (a bone marker), and Oil Red O for fat droplets.

The results showed that macaque ADSCs behaved similarly to ADSCs from other species but with key primate-specific stability. Primary cells began adhering to the dish within 2–3 hours, starting as round or elliptical shapes before stretching into spindle or triangular forms after 12 hours. By 24 hours, most cells were attached, and after 3–4 days of slow initial growth, they began multiplying rapidly—forming a dense layer of spindle-shaped cells that covered the dish. Even after three passages (splitting cells to keep them growing), the ADSCs retained their shape and growth rate, showing long-term stability.

Proliferation data reinforced this activity: the CCK-8 assay found growth rates nearly doubled from day 2 to day 4. Flow cytometry of passage 3 cells revealed 75.1% were in the G1 phase (a resting state before division) and 5.42% in the S phase (actively replicating DNA)—signs the cells were ready to proliferate.

When induced to form cartilage (chondrogenic), ADSCs accelerated growth by days 5–6, changing from spindle-shaped to oval or irregular. By day 8, cells increased in size, and by day 21, they secreted a matrix that blurred cell boundaries. Toluidine blue staining at 3 weeks confirmed cartilage-specific proteins, marking successful differentiation.

For bone (osteogenic) induction, ADSCs became cubic, polygonal, or irregular by day 7, with larger cell volumes. By days 13–14, calcium nodules—early signs of bone formation—appeared, and by day 21, cells were covered in matrix. Von Kossa staining at 3 weeks highlighted calcium deposits, confirming osteoblast activity.

Adipogenic induction (fat) caused ADSCs to turn round or irregular by day 10. Lipid droplets appeared in cells by day 14 and grew larger over time. Oil Red O staining at 3 weeks showed red-stained fat droplets in the cytoplasm—clear evidence of adipocyte differentiation.

These findings underscore the promise of macaque ADSCs for regenerative medicine. Compared to BMSCs, ADSCs are far more accessible: fat tissue can be collected via minimally invasive procedures like liposuction, and ADSCs proliferate faster in culture. Primates like macaques are critical because their biology closely mirrors humans, making their ADSCs more relevant for translating lab results to clinical applications. However, as the authors note, most ADSC research remains in preclinical stages. While this study shows macaque ADSCs can differentiate into cartilage, bone, and fat cells, more work is needed to understand their behavior in living organisms (in vivo) and address safety and efficacy questions for human use.

The study was supported by the Yunnan Provincial Science and Technology Department Kunming Medical University Joint Special Project (No. 2019FE001-170). The authors declare no conflicts of interest.

References

  1. Fang B, Liu Y, Zheng D, Shan S, Wang C, Gao Y, et al. The effects of mechanical stretch on the biological characteristics of human adipose-derived stem cells. J Cell Mol Med 2019;23:4244–4255. https://doi.org/10.1111/jcmm.14314
  2. Trapanese C, Meunier H, Masi S. What, where and when: spatial foraging decisions in primates. Biol Rev Camb Philos Soc 2019;94:483–502. https://doi.org/10.1111/brv.12462
  3. Gagliardi C, Bunnell BA. Isolation and culture of rhesus adipose-derived stem cells. Methods Mol Biol 2011;702:3–16. https://doi.org/10.1007/978-1-61737-960-4_1
  4. Estes JD, Wong SW, Brenchley JM. Nonhuman primate models of human viral infections. Nat Rev Immunol 2018;18:390–404. https://doi.org/10.1038/s41577-018-0005-7
  5. Izadpanah R, Trygg C, Patel B, Kriedt C, Dufour J, Gimble JM, et al. Biologic properties of mesenchymal stem cells derived from bone marrow and adipose tissue. J Cell Biochem 2010;99:1285–1297. https://doi.org/10.1002/jcb.20904

https://doi.org/10.1097/CM9.0000000000001486

Was this helpful?

0 / 0