Phenotypic and in vivo functional characterization of immortalized human fetal liver cells.

We report the establishment and characterization of immortalized human fetal liver progenitor cells by expression of the Simian virus 40 large T (SV40 LT) antigen. Well-characterized cells at various passages were transplanted into nude mice with acute liver injury and tested for functional capacity. The SV40LT antigen-immortalized fetal liver cells showed a morphology similar to primary cells. Cultured cells demonstrated stable phenotypic expression in various passages, of hepatic markers such as albumin, CK 8, CK18, transcription factors HNF-4α and HNF-1α and CYP3A/7. The cells did not stain for any of the tested cancer-associated markers. Albumin, HNF-4α and CYP3A7 expression was confirmed by reverse transcription polymerase chain reaction (RT-PCR). Flow cytometry showed expression of some progenitor cell markers. In vivo study showed that the cells expressed both fetal and differentiated hepatocytes markers. Our study suggests new approaches to expand hepatic progenitor cells, analyze their fate in animal models aiming at cell therapy of hepatic diseases.

Characterization and engraftment of long-term serum-free human fetal liver cell cultures.

BACKGROUND AIMS: Cultured human hepatocytes have extensive diagnostic and clinical applications. However, the setting-up of new in vitro culture techniques allowing the long-term survival and functional maintenance of adult human hepatocytes represents a formidable challenge. Fetal liver cells (FLC) are attractive candidate donor cells because of their high proliferative capacity. METHODS: Using cell culture and molecular techniques, we studied the in vitro and in vivo characteristics of FLC grown long-term in serum-free conditions. RESULTS: Serum-free FLC obtained from 6-10-week-old human fetal livers grew as multiple clusters in suspension and could be subcultured for at least six passages. These cells maintained stable hepatocyte phenotypes and gene expression patterns in culture for up to 6 months. When a cluster of these cells in various passages was placed on collagen-coated plates, they formed a monolayer and morphologically resembled hepatocytes. The cells expressed alpha -fetoprotein, cytokeratin (CK) 8, CK18 and CK19 and albumin (ALB). Hepatocyte nuclear factor 4alpha and 1beta and cytochrome P450 (CYP) 3A4 and CYP3A7 mRNA expression was demonstrated by reverse transcriptase-polymerase chain reaction (RT-PCR). Cells at different passages, when transplanted into nude mice with liver injury, engrafted successfully, as detected by in situ hybridization using a human-specific DNA probe. Colonies of human-specific CK8, CK18, c-Met nuclear antigen (Ag), mitochondrial Ag, hepatocyte-specific Ag and ALB-expressing cells were present in the livers of recipient animals. CONCLUSIONS: Primary human FLC can be kept in culture consistently over a long time period and are potential candidates for cell therapy and in vitro diagnostics.

Optimal conditions for freezing CHO-S and HEK293-EBNA cell lines: influence of Me2SO, freeze density, and PEI-mediated transfection on revitalization and growth of cells, and expression of recombinant protein.

To avoid the time consuming, labor intensive seed-train expansion and to improve production reliability and consistency, portions of bulk cryopreserved cells from the same cultivation can be utilized as inocula or alternatively may be used to undertake transient transfections for large-scale bioreactor production. In this study, the conditions for large-scale freezing in cryobags were optimized utilizing a design of experiment approach. We showed that relatively high density of 30-40 x 10(6) cells/mL and relatively low Me(2)SO concentrations of 5-6% in the freezing media are optimal to freeze HEK293-EBNA and CHO-S cells in a controlled manner in order to achieve high viable cell recovery and growth post-thawing. The immediate transfer of freshly thawed cells into culture medium resulted in better cell growth compared to cells that were centrifuged in order to remove Me(2)SO. This was the case as long as the residual Me(2)SO did not exceed 0.2-0.3%. The best time to perform transient 25 kDa polyethylenimine-mediated transfection of pCEP4-EGFP plasmid into freshly thawed, one-step inoculated cells is after 72-96 h in culture. At this time point, the numbers of EGFP-positive cells in the freshly thawed culture mimic perfectly that of cells grown continuously. Finally, our data showed that it is possible to freeze transiently polyethyleneimine-transfected HEK293-EBNA cells and maintain growth rate and expression of recombinant protein following thawing. The optimal time point for freezing cells was 4 h after transfection.

Targeted expression of GLI1 in the mammary gland disrupts pregnancy-induced maturation and causes lactation failure.

The Hedgehog signaling pathway regulates the development and function of numerous tissues and when mis-regulated causes tumorigenesis. To assess the role of a deregulated Hedgehog signaling pathway in the mammary gland we targeted the expression of the Hedgehog effector protein, GLI1, to mammary epithelial cells using a bigenic inducible system. A constitutively active Hedgehog signaling pathway resulted with 100% penetrance in an undifferentiated mammary lobuloalveolar network during pregnancy. GLI1-expressing transgenic females were unable to lactate and milk protein gene expression was essentially absent. The inability to lactate was permanent and independent of continued GLI1 transgene expression. An increased expression of the GLI1 response gene Snail coupled to reduced expression of E-cadherin and STAT5 in the transgenic mammary gland provides a likely molecular explanation, underlying the observed phenotypic changes. In addition, remodeling of the mammary gland after parturition was impaired and expression of GLI1 was associated with accumulation of cellular debris in the mammary ducts during involution, indicating a defect in the clearance of dead cells. Areas with highly proliferative epithelial cells were observed in mammary glands with induced expression of GLI1. Within such areas an increased frequency of cells expressing nuclear Cyclin D1 was observed. Taken together the data support the notion that correct regulation of Hedgehog signaling within the epithelial cell compartment is critical for pregnancy-induced mammary gland development and remodeling.

Expression of the PTCH1 tumor suppressor gene is regulated by alternative promoters and a single functional Gli-binding site.

The PTCH1 tumor suppressor gene encodes a receptor for secreted hedgehog (HH) ligands and is important for proper proliferation, differentiation and patterning in almost every tissue and organ during embryogenesis. The PTCH1 protein works as a negative regulator of the HH-signaling pathway by repressing downstream signaling by the coreceptor smoothened (SMOH). Mutations in PTCH1 lead to constitutive expression of HH target genes and a relationship between mutated PTCH1 and the most common tumor form in the Western world, Basal Cell Carcinoma (BCC) has been clearly established. We here show that PTCH1 is transcriptionally regulated by three independent promoters generating transcripts with alternative first exons. We demonstrate that only one of two putative Gli-binding sites that were identified in the promoter region of PTCH1 is functional, and that the transactivating Gli proteins, GLI1, Gli2 and GLI3, bind and enhance transcription through this site. Moreover, a strong repression of both basal and induced PTCH1 transcription was observed following expression of a truncated version of GLI3. Most interestingly, the upstream components in the HH-signaling cascade, Sonic HH (SHH) and SMOH, solely operate through the functional Gli-binding site because mutation of the Gli-binding site resulted in the disappearance of the enhanced transcription induced by the Gli proteins, as well as by SHH or SMOH. This finding suggests that transcriptional activation of the PTCH1 gene mediated via the HH-signaling pathway is dependent on the single functional Gli-binding site.