Khan Muti ur Rehman, Ali Ijaz, Jiao Wei, Wang Yun, Masood Saima, Yousaf Muhammad Zubair, Javaid Aqeel, Ahmad Shafique, Feng Meifu
Department of Pathology, University of Veterinary and Animal Sciences, Lahore 54000, Pakistan.
Institute of Biotechnology and Genetic Engineering, University of Agriculture, Peshawar 25000, Pakistan.
Biomed Res Int. 2014;2014:412075. doi: 10.1155/2014/412075. Epub 2014 Feb 25.
Kiaa1867 (human Kirre, hKirre) has a critical role in brain development and/or maintenance of the glomerular slit diaphragm in kidneys. Murine homolog of this gene, mKirre expressed in OP9 and AFT024 cells could support hematopoietic stem cells/hematopoietic progenitor cells (HSC/HPC) expansion in vitro. HKirre is also expressed in human FBMOB-hTERT cell line and fetal liver fibroblast-like cells but its function has remained unclear. In this paper, we cloned a hKirre gene from human fetal liver fibroblast-like cells and established a stably overexpressing hKirre-AFT024 cell line. Resultant cells could promote self-renewal and ex vivo expansion of HSCs/HPCs significantly higher than AFT024-control cells transformed with mock plasmid. The Expanded human umbilical cord blood (hUCB) CD34(+) cells retained the capacity of multipotent differentiation as long as 8 weeks and successfully repopulated the bone marrow of sublethally irradiated NOD/SCID mice, which demonstrated the expansion of long-term primitive transplantable HSCs/HPCs. Importantly, hkirre could upregulate the expressions of Wnt-5A, BMP4, and SDF-1 and downregulate TGF- β with other hematopoietic growth factors. By SDS-PAGE and Western Blot analysis, a ~89 kDa protein in total lysate of AFT024-hKirre was identified. Supernatants from AFT024-hkirre could also support CD34(+)CD38(-) cells expansion. These results demonstrated that the AFT024-hKirre cells have the ability to efficiently expand HSCs/HPCs.
Kiaa1867（人类Kirre，hKirre）在大脑发育和/或肾脏肾小球裂孔隔膜的维持中起关键作用。该基因的小鼠同源物mKirre在OP9和AFT024细胞中表达，可在体外支持造血干细胞/造血祖细胞（HSC/HPC）的扩增。HKirre也在人FBMOB-hTERT细胞系和胎儿肝成纤维细胞样细胞中表达，但其功能仍不清楚。在本文中，我们从人胎儿肝成纤维细胞样细胞中克隆了hKirre基因，并建立了稳定过表达hKirre的AFT024细胞系。所得细胞促进HSC/HPC自我更新和体外扩增的能力显著高于用空质粒转化的AFT024对照细胞。扩增后的人脐带血（hUCB）CD34(+)细胞在长达8周的时间内保持多能分化能力，并成功重建造血亚致死剂量照射的NOD/SCID小鼠的骨髓，这证明了长期原始可移植HSC/HPC的扩增。重要的是，hkirre可上调Wnt-5A、BMP4和SDF-1的表达，并与其他造血生长因子一起下调TGF-β。通过SDS-PAGE和Western Blot分析，在AFT024-hKirre的总裂解物中鉴定出一种约89 kDa的蛋白质。AFT024-hkirre的上清液也可支持CD34(+)CD38(-)细胞的扩增。这些结果表明，AFT024-hKirre细胞具有有效扩增HSC/HPC的能力。
