(T1216) DERIVATION OF HIGHLY PURE POPULATION OF MESENCHYMAL STEM CELLS (MSCs) DERIVED FROM INDUCED PLURIPOTENT STEM CELLS (iPSCs) USING A XENO-FREE CULTURE SYSTEM

Abstract: The application potential of mesenchymal stromal cells (MSCs) for cell therapy products is widely known in regenerative medicine. Although MSCs can be isolated from sources such as bone marrow, umbilical cord, and adipose tissue, their clinical application is often hindered by scalability challenges due to limited proliferative capacity and donor variability. To overcome these limitations, we developed an alternative source of MSCs by deriving them from induced pluripotent stem cells (iPSCs), which offers a promising solution due to its greater expandability and potentially continuous supply. However, a critical quality concern for iPSC-derived cell therapy products is the presence of residual undifferentiated cells, which pose a risk of tumorigenicity. In this study, we established a xeno-free differentiation protocol to derive pure MSCs from iPSCs. The resulting iMSCs demonstrates stable proliferation over 30 population doublings (PDs) while maintaining consistent expression of MSC-specific markers (CD105, CD73, CD90, CD29, and CD44). Genomic stability was preserved, as confirmed by short tandem repeat (STR) analysis and karyotyping, indicating no genetic alterations during differentiation. Functionally, iMSCs exhibited trilineage differentiation potential into osteocytes, adipocytes, and chondrocytes, comparable to traditional MSCs. To address the concern of impurities undifferentiated cells, we developed a sensitive quantitative real-time polymerase chain reaction (qRT-PCR) assay targeting LIN28A, a pluripotency marker highly expressed in undifferentiated iPSCs. Spike-in experiments determined the assay's limit of detection (LOD) to be < 1 iPSC per 10,000 iMSC cells. This xeno-free protocol efficiently generates a highly pure MSC population and provides an up-scalable source of MSC production. Our studies highlight the potential of iMSCs as a reliable source for cGMP-grade MSC-based therapies, ensuring safety and reliability for future clinical applications.

Funding Source: This work was fully funded and supported by PT. Kalbe Farma Tbk.

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