Rat Bone Marrow Mononuclear Cells

PRPs Upregulated Proliferative Ability of BM MSCs

Cartilage defects often lead to osteoarthritis due to limited self-regeneration ability. Traditional bone marrow stimulation (BMS) techniques, such as multiple channeling, fail to generate hyaline cartilage, forming fibrocartilage instead. The restriction of endogenous bone marrow mesenchymal stem cells (BM MSCs) in number and strength contributes to this outcome. Research indicates that platelet-rich plasma (PRP) demonstrates potential benefits for tissue healing and increasing stem cell multiplication. Lee et al. investigated if second-generation PRP (2G PRP) with extended retention time improves cartilage repair through enhanced local proliferation and migration of BM MSCs to repair sites.

They isolated mononuclear cells from the bone marrow of femurs and tibias in 8-week-old SD rats. After subculturing adherent BM MSCs on the 10th day researchers used BM MSCs that were in passages 3 and 4 for this study. The influence of PRPs on BM MSC proliferation was evaluated by treating MSCs with 10% (v/v) PRP supernatant followed by measurements of proliferation across a 10-day period (Fig. 1A). BM MSC proliferation showed peak levels on days 3 and 5. On days 3 and 5 the 2G PRP group as well as the groups treated with PRP with Ca²⁺ + THRB and PRP with Ca²⁺ showed significantly greater growth compared to the control group. The MSC proliferation increased 5.96-fold, 5.68-fold, and 5.39-fold in the 2G PRP group and Ca²⁺ + THRB and Ca²⁺ groups respectively compared to the baseline measurement at day 0 after 5 days. The PRP group did not exhibit any growth improvements over the control group. The absence of soluble factors in washed-out PRP demonstrates its inability to stimulate cell growth. The 2G PRP treatment demonstrated the greatest proliferation enhancement at 1.72-fold over the PRP group on days 3 and 5, whereas Ca²⁺ + THRB and Ca²⁺ treatments also improved proliferation beyond PRP levels but showed no significant differences between them.

Fig. 1. Effects of 2G PRP on proliferation and migration of rat BM MSCs (Lee MJ, Jiang J, et al., 2024).

Isolation, Induction and Identification of NTCSCs

Yu's team investigated the efficacy of transplanting bone marrow neural tissue-committed stem cell-derived sensory neuron-like cells for repairing peripheral nerve sensory impairments in rats. SD rat bone marrow mononuclear cells were isolated and cultured as previously described, and cell spheres were obtained through single-cell cloning and serum-free suspension culture (Fig. 2a). After culturing to the third generation, neuronal cells were induced from neural tissue-committed stem cells (NTCSCs) (Fig. 2b). The NTCSCs exhibited neuron-like morphology, with cytoplasm retracting to form round cells with a typical perikaryon shape, enhanced stereoscopic sensation, and refraction. The cells had 2 to 5 elongated protrusions resembling neuronal axons, with dendrite-like branches at their ends. These protrusions intertwined to form a network. Immunofluorescence staining was performed on the induced and differentiated cells for neuronal markers NeuN, MAP-2, and GluR4. Results showed that NeuN, MAP-2, and GluR4 were expressed in the induced cells (Fig. 3). MAP-2 and NeuN fluorescence was red, with MAP-2 mainly in the cytoplasm and NeuN in both the nucleus and cytoplasm. GluR4 fluorescence was green, with most expression in the cytoplasm and a small amount on the cell membrane. DAPI staining of the nucleus was blue.

Fig. 2. Morphological characteristics of cell spheres and neuron-like cells (Yu Z, Xu N, et al., 2019).

Fig. 3. Characteristic proteins of NeuN, MAP-2, and GluR4 were expressed in the cells (Yu Z, Xu N, et al., 2019).