Croce Stefania, Cobianchi Lorenzo, Zoro Tamara, Dal Mas Francesca, Icaro Cornaglia Antonia, Lenta Elisa, Acquafredda Gloria, De Silvestri Annalisa, Avanzini Maria Antonietta, Visai Livia, Brambilla Szandra, Bruni Giovanna, Gravina Giulia Di, Pietrabissa Andrea, Ansaloni Luca, Peloso Andrea
Department of Clinical, Surgical, Diagnostic & Pediatric Sciences, University of Pavia, 27100 Pavia, Italy.
Department of General Surgery, Fondazione IRCCS Policlinico San Matteo, 27100 Pavia, Italy.
Biomedicines. 2022 Nov 4;10(11):2817. doi: 10.3390/biomedicines10112817.
In end-stage chronic liver disease, transplantation represents the only curative option. However, the shortage of donors results in the death of many patients. To overcome this gap, it is mandatory to develop new therapeutic options. In the present study, we decellularised pig livers and reseeded them with allogeneic porcine mesenchymal stromal cells (pMSCs) to understand whether extracellular matrix (ECM) can influence and/or promote differentiation into hepatocyte-like cells (HLCs).
After decellularisation with SDS, the integrity of ECM-scaffolds was examined by histological staining, immunofluorescence and scanning electron microscope. DNA quantification was used to assess decellularisation. pMSCs were plated on scaffolds by static seeding and maintained in in vitro culture for 21 days. At 3, 7, 14 and 21 days, seeded ECM scaffolds were evaluated for cellular adhesion and growth. Moreover, the expression of specific hepatic genes was performed by RT-PCR.
The applied decellularisation/recellularisation protocol was effective. The number of seeded pMSCs increased over the culture time points. Gene expression analysis of seeded pMSCs displayed a weak induction due to ECM towards HLCs.
These results suggest that ECM may address pMSCs to differentiate in hepatocyte-like cells. However, only contact with liver-ECM is not enough to induce complete differentiation.
在终末期慢性肝病中,肝移植是唯一的治愈选择。然而,供体短缺导致许多患者死亡。为了克服这一差距,开发新的治疗方法势在必行。在本研究中,我们对猪肝脏进行脱细胞处理,并用同种异体猪间充质基质细胞(pMSCs)重新接种,以了解细胞外基质(ECM)是否能够影响和/或促进其向肝细胞样细胞(HLCs)分化。
用十二烷基硫酸钠进行脱细胞处理后,通过组织学染色、免疫荧光和扫描电子显微镜检查ECM支架的完整性。采用DNA定量法评估脱细胞效果。通过静态接种将pMSCs接种到支架上,并在体外培养21天。在第3、7、14和21天,对接种后的ECM支架进行细胞黏附和生长评估。此外,通过逆转录聚合酶链反应(RT-PCR)检测特定肝脏基因的表达。
所应用的脱细胞/再细胞化方案是有效的。接种的pMSCs数量在培养时间点上有所增加。接种的pMSCs的基因表达分析显示,ECM对HLCs有微弱的诱导作用。
这些结果表明,ECM可能促使pMSCs向肝细胞样细胞分化。然而,仅与肝脏ECM接触不足以诱导完全分化。