Downregulation of Signal Transducer and Activator of Transcription 4 Contributes to Impaired Osteogenic Differentiation of Human Bone Marrow Stem Cells during in vitro Expansion.

INTRODUCTION: In vitro expansion of primary human bone marrow stem cells (hBMSCs) is necessary to obtain sufficient cells for therapeutic uses. Unfortunately, hBMSCs rapidly lose their osteogenic differentiation potential during expansion, significantly limiting their applications. Signal transducer and activator of transcription 4 (STAT4) is known to play roles in cell migration, proliferation, and differentiation. This study aimed to determine the expression and the role of STAT4 during the expansion of hBMSCs. METHODS: STAT4 expression in different passages of hBMSCs was evaluated using qRT-PCR and Western blotting. RNA interference and adeno-associated virus serotype 2-mediated gene overexpression were employed to assess the function of STAT4. RNA samples from STAT4-overexpressing hBMSCs were analyzed by RNA-seq to identify differentially expressed genes (DEGs), followed by bioinformatics analyses to determine the pathways affected by STAT4. RESULTS: STAT4 expression progressively decreases during the in vitro expansion of hBMSCs, concomitant with the loss of osteogenic differentiation potential. STAT4 knockdown in early passage hBMSCs significantly inhibits their osteogenic differentiation, evidenced by markedly reduced calcium deposition and downregulation of osteogenic markers. STAT4 knockdown also reduces hBMSCs' proliferation ability. Conversely, STAT4 overexpression notably increases calcium deposition in passage 3 to passage 7 cells, suggesting that enhanced STAT4 expression can mitigate the loss of osteogenic potential during hBMSC expansion. Transcriptomic analysis revealed DEGs in STAT4-overexpressing hBMSCs. Subsequent bioinformatics analyses indicated that some of these DEGs are involved in pathways regulating cell differentiation and senescence. CONCLUSION: The in vitro expansion of hBMSCs leads to the downregulation of STAT4, which contributes to the impairment of their osteogenic potential and may affect cell self-renewability. This study provides insight into the molecular mechanisms underlying the loss of osteogenic differentiation during hBMSC expansion and identifies STAT4 as a potential target for hBMSC-based bone regeneration therapies.