Several Important In Vitro Improvements in the Amplification, Differentiation and Tracing of Fetal Liver Stem/Progenitor Cells

Abstract

ObjectiveWe previously isolated fetal liver stem/progenitor cells (FLSPCs), but there is an urgent need to properly amplify FLSPCs, effectively induce FLSPCs differentiation, and steadily trace FLSPCs for in vivo therapeutic investigation.MethodsFLSPCs were maintained in vitro as adherent culture or soft agar culture for large-scale amplification. To direct the differentiation of FLSPCs into hepatocytes, FLSPCs were randomly divided into four groups: control, 1% DMSO-treated, 20 ng/ml HGF-treated and 1% DMSO+20 ng/ml HGF-treated. To trace FLSPCs, the GFP gene was introduced into FLSPCs by liposome-mediated transfection.ResultsFor amplifying FLSPCs, the soft agar culture were more suitable than the adherent culture, because the soft agar culture obtained more homogeneous cells. These cells were with high nuclear:cytoplasmic ratio, few cell organelles, high expression of CD90.1 and CD49f, and strong alkaline phosphatase staining. For inducing FLSPCs differentiation, treatment with HGF+DMSO was most effective (P<0.05), which was strongly supported by the typical morphological change and the significant decrease of OV-6 positive cells (P<0.01). In addition, the time of indocyanine green elimination, the percentage of glycogen synthetic cells, and the expressions of ALB, G-6-P, CK-8, CK-18 and CYP450-3A1 in HGF+DMSO-treated group were higher than in any other group. For tracing FLSPCs, after the selection of stable FLSPC transfectants, GFP expression continued over successive generations.ConclusionsFLSPCs can properly self-renew in soft agar culture and effectively differentiate into hepatocyte-like cells by HGF+DMSO induction, and they can be reliably traced by GFP expression.

Unbalanced distribution of materials: the art of giving rise to hepatocytes from liver stem/progenitor cells

“…A). When FLSPCs were grown in 3D suspension culture, they were likely to self‐renew; in contrast, FLSPCs that were maintained in 2D adherent culture were prone to differentiation . These data are consistent with others’ report .…”

“…Based on our experiences, the manner of culture will also affect the cell fate of LSPCs (Fig.#x00A0; 3 A). When FLSPCs were grown in 3D suspension culture, they were likely to self-renew; in contrast, FLSPCs that were maintained in 2D adherent culture were prone to differentiation 65 . These data are consistent with others#x2019; report 66 .…”

“…A study by our group revealed that HGF can only induce the early transition of ALB-negative LSPCs to ALB-positive hepatocyte-like cells. Furthermore, a study by our group revealed that the combination of HGF with dimethyl sulphoxide (DMSO) accelerated the hepatic differentiation of LSPCs 65 . Based on these theories, many groups combine several types of GFs to efficiently induce LSPC differentiation.…”

“…Why are these LETFs so important for the hepatic differentiation of LSPCs? It is because hepatocyte-specific gene expression is regulated by the orchestrated action of sets of LETFs 52 – 65 . For example, HNF4#x03B1; is known to be a key regulator of both hepatic differentiation during embryonic development and the maintenance of a differentiated phenotype in the adult liver 118 .…”

Multidynamic Liposomes in Nanomedicine: Technology, Biology, Applications, and Disease Targeting

“…The self-renewal property of FLSPCs in soft agar culture and effectively differentiation into hepatocyte-like cells was mediated by GFP traced liposome-mediated transfection continued over successive generations [82].…”