Discussion

Campagnoli et al [20] have reported that mesenchymal stem/progenitor cells could be obtained from human first-trimester fetal liver (FL), which are similar to those derived from adult bone marrow. However, the limited life span of the primary cells is the main obstacle for their further use. So we tried to transfect the primary FL stromal cells (FLSC) with hTERT gene to overcome cell crisis. However, whether immortalization of primary human cells could be achieved by the ectopic expression of hTERT might depend on cell types or culture conditions, and the ability of ectopic hTERT to extend life-span may also be related to the site of integration and the levels of telomere- or telomerase-associated proteins in a cell type-specific manner [31, 32, 33, 34]. In this paper, we reported the establishment and characteristics of mesenchymal stem-like cell line, through transfecting the primary FL adherent stromal cells with retrovirus containing hTERT. The cell line had the potential of mesenchymal stem cells (MSCs) because it could differentiate into osteocytes and adipocytes, which have been demonstrated by histochemical evaluations and RT-PCR analysis (Figure 2), under the conditions that favored adipogenic and osteogenic differentiation of adult MSCs [23]. Consistently, the FLMSL-hTERT cells were found to be positive for CD29 (01-integrin), CD44 (the hyaluronate receptor), the intercellular adheresive molecular, CD58 and CD105 (endoglin) (Figure 3), which are similar with those reported for MSCs derived from other tissues [27, 28, 29, 30]. The expression of surface antigens on the FLMSL-hTERT cells kept unchanged (Figure 3) and the hTERT activity in these cells were stable (Figure 1) even after expanded for about two years in vitro.

Ectopic expression of hTERT in primary human cells may inhibit the replicative senescence, leading to immortalization. The immortalized cell lines such as FLMSL-hTERT do not necessarily undergo malignant transformation. Several reports have demonstrated that hTERT-transduced human cells develop into tumor at more than 100 PD [35, 36]. However, the tumorigenicity of the hTERT-transduced cell lines is inevitably conferred by the concomitant epigenetic alterations or oncogenic mutations [35, 36, 37]. Both the results of tumorigenicity assay and the fact that FLMSL-hTERT has been maintained in culture more than 150 PD without alteration of the phenotype, morphology and biological functions suggests that the cells have been immortalized, but not yet underwent significant oncogenic mutations.

FL is one of the important sites that HSCs emerge during ontogeny and can provide a conducive microenvironment to support HSCs [10, 11, 12, 13]. Here, we found that the FLMSL-hTERT cells expressed detectable levels of SCF, Wnt5A, FL, KIAA1867, TGF-p, Delta-like, VEGF, SDF-1, PLGF and Jagged-1, which are necessary in hematopoietic support, but not TPO and Shh. We further analyzed the function of the cell line in the HSC/HPC expansion. As expected, the FLMSL-hTERT can dramatically expand CB HSCs/HPCs ex vivo (Figure 5), especially maintaining HSC properties in vitro at least for 8 wk (Figure 6). The results also showed that the FLMSL-hTERT cells are superior to primary FLSCs in expanding HSCs/HPCs in vitro, especially for maintaining hematopoietic activity during long-term culture (Figure 6). The mechanisms underlying the enhanced expansion are not yet clear. However, this may be related to longer survival time of FLMSL-hTERT cells than primary FLSCs in the cultures.

In a conclusion, through transfection with retrovirus containing hTERT gene, we have developed an immortalized mesenchymal stem-like cell line derived from human fetal liver. The normal biological potential of these cells could be maintained. In this study, we confirmed and extended the findings that FL stromal cells form an important constituent of the hematopoietic niche and they are known to support self-renewal and proliferation of HSCs/HPCs. This cell line is superior to primary FLSCs in supporting HSC/HPC expansion in vitro. This newly established cell line might be of value for studying the mechanisms by which stromal cells regulate HSC/HPC expansion in FL and for developing a novel strategy for ex vivo expansion of human transplantable HSCs/HPCs.

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