Supplementary Material for: Downregulation of Signal Transducer and Activator of Transcription 4 (STAT4) contributes to impaired osteogenic differentiation of human bone marrow stem cells (hBMSCs) 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 aims 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 (AAV2)-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.

### 引言
人原代骨髓干细胞（primary human bone marrow stem cells, hBMSCs）的体外扩增是获取足量治疗用细胞的必要手段。然而，hBMSCs在扩增过程中会迅速丧失成骨分化潜能，极大限制了其应用。信号转导及转录激活因子4（signal transducer and activator of transcription 4, STAT4）已知在细胞迁移、增殖及分化中发挥作用。本研究旨在明确STAT4在hBMSCs扩增过程中的表达模式及其作用。

### 方法
采用实时定量聚合酶链反应（quantitative real-time polymerase chain reaction, qRT-PCR）和蛋白质印迹法（Western blotting）检测不同传代hBMSCs中STAT4的表达水平。通过RNA干扰（RNA interference）及腺相关病毒2型（adeno-associated virus serotype 2, AAV2）介导的基因过表达技术评估STAT4的功能。对STAT4过表达的hBMSCs样本进行转录组测序（RNA-seq）分析以筛选差异表达基因（differentially expressed genes, DEGs），随后通过生物信息学分析明确STAT4影响的信号通路。

### 结果
hBMSCs体外扩增过程中STAT4表达水平逐渐降低，且与成骨分化潜能的丧失同步发生。早期传代hBMSCs中STAT4的敲低显著抑制其成骨分化，表现为钙沉积明显减少及成骨标志物表达下调；同时还会降低hBMSCs的增殖能力。反之，STAT4过表达可显著增加第3至第7代细胞的钙沉积，提示上调STAT4表达能缓解hBMSCs扩增过程中骨分化潜能的丧失。转录组分析显示STAT4过表达的hBMSCs中存在差异表达基因；后续生物信息学分析表明，部分差异基因参与调控细胞分化及衰老的信号通路。

### 结论
hBMSCs体外扩增会导致STAT4表达下调，这不仅是其成骨潜能受损的原因之一，还可能影响细胞的自我更新能力。本研究揭示了hBMSCs扩增过程中成骨分化潜能丧失的分子机制，并将STAT4确定为基于hBMSC的骨再生治疗的潜在靶点。