Induced Differentiation of Macaque Adipose-Derived Stem Cells In Vitro
Bone marrow mesenchymal stem cells (BMSCs) have long been the focus of adult stem cell research due to their multipotent differentiation capabilities. However, BMSCs present several limitations, including low purification rates, limited availability, and the invasive nature of their extraction, which can cause trauma to the human body. In recent years, adipose-derived stem cells (ADSCs) have emerged as a promising alternative. ADSCs, derived from adipose tissue, exhibit similar differentiation potential to BMSCs but offer additional advantages, such as a wider range of sources, easier collection, reduced patient discomfort, rapid proliferation, and the absence of immune rejection or ethical concerns. While ADSCs have been extensively studied in lower mammals like rabbits and mice, research on primates remains limited. Given the anatomical and physiological similarities between primates and humans, studying ADSCs in primates provides valuable insights for regenerative medicine. This study aimed to explore the isolation methods and multipotent differentiation potential of ADSCs derived from macaques in vitro, contributing to the growing body of knowledge on primate ADSCs.
The study was conducted under the approval and supervision of the animal ethics committee of the Second People’s Hospital of Yunnan Province, ensuring compliance with ethical standards for experimental animals. ADSCs were isolated from the abdominal subcutaneous adipose tissue of macaques (obtained from the Kunming Primate Research Center, Chinese Academy of Sciences) using collagenase and neutral protease digestion under aseptic conditions. The morphology, growth, and cell cycle status of the isolated ADSCs were observed and recorded using an inverted microscope (DS-Ri2, Nikon, Japan). Cell growth was assessed using a cell counting kit-8 (CCK-8; Beyotime Biotechnology, Shanghai, China), while fat visualization was achieved through Oil Red O staining (Solarbio, Beijing, China). The cell cycle was analyzed using flow cytometry (Accuri C6 Plus, BD, US). Additionally, the multipotent differentiation potential of ADSCs was evaluated by culturing the cells in chondrogenic, osteogenic, and adipogenic induction media. Specific staining methods, including toluidine blue for chondrogenic induction, Von Kossa for osteogenic induction, and Oil Red O for adipogenic induction, were used to confirm the differentiation outcomes.
The primary ADSCs began to adhere to the culture flask 2 to 3 hours after seeding, initially appearing as round or elliptical cells of varying sizes. Within 12 hours, some cells began to stretch into short spindle and triangular shapes, and by 24 hours, most cells had adhered to the flask. During the first 3 to 4 days, ADSCs exhibited slow growth, followed by a significant acceleration in proliferation. The number of fusiform-shaped cells increased markedly, eventually covering the entire flask surface. By the third passage, the cells maintained their morphology and continued to proliferate vigorously. The CCK-8 assay results revealed a transition from slow to rapid growth, with the growth rate on day 4 nearly doubling that of day 2. Flow cytometry analysis of passage 3 ADSCs showed that 75.1% of cells were in the G1 phase, while 5.42% were in the S phase, indicating active proliferation.
Chondrogenic induction of ADSCs resulted in accelerated growth by days 5 to 6, with cells transitioning from fusiform to oval or irregular shapes. By day 8, cell volume increased, and the secreted matrix began to cover the cell surface, blurring cell boundaries. By day 21, the cells exhibited a morphology consistent with chondrocytes, confirmed by toluidine blue staining at week 3. Osteogenic induction led to a morphological shift from fusiform to cubic, polygonal, or irregular shapes by day 7, with increased cell volume and size. Calcified nodules were observed by days 13 to 14, and Von Kossa staining at week 3 confirmed osteogenic differentiation. Adipogenic induction caused ADSCs to transition from fusiform to round or irregular shapes by day 10, with lipid droplets widely distributed by day 14. By week 3, the lipid droplets had enlarged, and Oil Red O staining confirmed adipogenic differentiation.
ADSCs are found in the capillaries and adventitia of large blood vessels within adipose tissue, sharing morphological and differentiation characteristics with BMSCs. While most ADSC research has focused on small animals, primates offer a closer physiological and anatomical resemblance to humans, making them a valuable model for regenerative medicine studies. This study successfully isolated macaque ADSCs from abdominal subcutaneous adipose tissue, demonstrating their ability to differentiate into chondrocytes, osteoblasts, and adipocytes. The cells exhibited stable and rapid growth, maintaining their characteristics through multiple passages.
The chondrogenic, osteogenic, and adipogenic induction experiments confirmed the multipotent differentiation potential of macaque ADSCs. These findings align with previous studies showing that primate ADSCs can retain stable characteristics even after extensive passaging. The study highlights the potential of ADSCs in tissue engineering and regenerative medicine, particularly in applications requiring chondrogenic, osteogenic, or adipogenic differentiation. However, while in vitro and in vivo animal studies have provided valuable insights, further research is needed to translate these findings into clinical practice.
In conclusion, this study provides a comprehensive analysis of the isolation and multipotent differentiation of macaque ADSCs in vitro. The results underscore the potential of ADSCs in regenerative medicine, particularly in primate models that closely mimic human physiology. Future research should focus on further characterizing the biological properties of ADSCs and exploring their therapeutic applications in clinical settings.
doi.org/10.1097/CM9.0000000000001486
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