Authors: Dong Hwan Lee, Seon Ae Kim, Eun Jeong Go, Chi Young Yoon, Mi-La Cho, Asode Ananthram Shetty, Seok Jung Kim

The study investigates the characteristics and therapeutic potential of mesenchymal stem cells (MSCs) derived from hemovac blood concentrates (HVBCs) collected during total knee arthroplasty (TKA). The primary focus is on comparing MSCs obtained from osteoarthritis patients (OA-MSCs) with those where the signal transducer and activator of transcription 3 (STAT3) signaling has been suppressed (iSTAT3-MSCs). STAT3 is known to play a crucial role in inflammation and cell differentiation, making it a target for improving MSC function in therapeutic applications.

Methodology:

• Patient Sample: The study included 20 patients undergoing TKA. Hemovac blood was collected post-surgery and concentrated to isolate MSCs.
• Cell Culturing: MSCs were cultured from HVBCs, and STAT3 signaling was inhibited in a subset using the STAT3 inhibitor STA21.
• Characterization: The cells were characterized for their surface marker expression, morphology, and differentiation potential into osteogenic, adipogenic, and chondrogenic lineages. Additionally, the anti-inflammatory properties of the cells were assessed by measuring cytokine levels.

Results:

1. Cell Morphology and Marker Expression:

   • Both OA-MSCs and iSTAT3-MSCs exhibited typical fibroblast-like morphology and high expression of MSC-specific markers, including CD73, CD90, and CD105. The suppression of STAT3 did not alter these fundamental characteristics.
   • Importantly, both cell types showed low expression of hematopoietic and leukocyte markers (CD34, CD45) and antigen-presenting cell marker HLA-DR, indicating a high purity of MSCs.

2. Differentiation Potential:

   • Osteogenic Differentiation: iSTAT3-MSCs showed a significantly higher expression of osteogenic markers (RUNX2, ALP, osteocalcin, osteopontin) compared to OA-MSCs. Enhanced mineralization was also observed in iSTAT3-MSCs, indicating superior osteogenic differentiation.
   • Adipogenic Differentiation: Both OA-MSCs and iSTAT3-MSCs differentiated into adipocytes, as evidenced by the formation of lipid droplets and expression of adipogenic markers (C/EBPα, PPARγ). There was no significant difference between the two cell types in adipogenic differentiation.
   • Chondrogenic Differentiation: iSTAT3-MSCs demonstrated a greater chondrogenic potential than OA-MSCs, with higher expression of chondrogenic markers (SOX9, aggrecan, type II collagen) and stronger staining for proteoglycans and type II collagen in the extracellular matrix.

3. Anti-Inflammatory Properties:

   • iSTAT3-MSCs exhibited enhanced anti-inflammatory properties, characterized by higher levels of anti-inflammatory cytokines (IL-10, TGF-β) and lower levels of pro-inflammatory cytokines (IL-6, IL-1β, IL-8, VEGF). This suggests that STAT3 suppression improves the immunomodulatory function of MSCs, making them more suitable for therapeutic applications in inflammatory conditions like osteoarthritis.

Discussion:

• The findings suggest that MSCs isolated from HVBCs after TKA can be effectively used in stem cell therapy, particularly when STAT3 signaling is suppressed to enhance their therapeutic properties.
• The study highlights the potential of using HVBCs as a non-invasive, easily accessible source of MSCs for clinical applications, reducing the need for more invasive procedures like bone marrow aspiration.
• The enhanced osteogenic and chondrogenic differentiation capabilities of iSTAT3-MSCs, along with their superior anti-inflammatory profile, make them promising candidates for treating bone and cartilage disorders, such as fractures, non-union fractures, avascular necrosis, and cartilage defects.

Conclusion: The research demonstrates that STAT3 signaling suppression in MSCs derived from HVBCs improves their differentiation potential and anti-inflammatory properties. This suggests that iSTAT3-MSCs could be a valuable resource in stem cell therapy, particularly for conditions involving bone and cartilage damage. The ability to harvest these cells during routine TKA without additional invasive procedures further underscores their potential in clinical settings.

Implications for Future Research:

• The study paves the way for further clinical trials to assess the therapeutic efficacy of iSTAT3-MSCs in treating musculoskeletal disorders.
• Additional research is needed to explore the long-term stability and functionality of these cells in vivo, as well as their potential in other therapeutic areas.