Katherine S. Koch

Summary: Embryonic mouse STO (S, SIM; T, 6‐thioguanine resistant; O, ouabain resistant) and 3(8)21‐enhanced green fluorescent protein (EGFP) cell lines exhibit long‐term survival and hepatic progenitor cell behaviour after xenogeneic engraftment in non‐immunosuppressed inbred rats, and were previously designated major histocompatibility complex (MHC) class I‐ and class II‐negative lines. To determine the molecular basis for undetectable MHC determinants, the expression and haplotype of H‐2K, H‐2D, H‐2L and I‐A proteins were reassessed by reverse transcriptase–polymerase chain reaction (RT‐PCR), cDNA sequencing, RNA hybridization, immunoblotting, quantitative RT‐PCR (QPCR), immunocytochemistry and flow cytometry. To detect cell differentiation (CD) surface antigens characteristic of stem cells, apoptotic regulation or adaptive immunity that might facilitate progenitor cell status or immune privilege, flow cytometry was also used to screen untreated and cytokine [interferon (IFN)‐γ]‐treated cultures. Despite prior PCR genotyping analyses suggestive of H‐2^q haplotypes in STO, 3(8)21‐EGFP and parental 3(8)21 cells, all three lines expressed H‐2K cDNA sequences identical to those of d‐haplotype BALB/c mice, as well as constitutive and cytokine‐inducible H‐2K^d determinants. In contrast, apart from H‐2L^d[LOW] display in 3(8)21 cells, H‐2D^d, H‐2L^d and I‐A^d determinants were undetectable. All three lines expressed constitutive and cytokine‐inducible CD34; however, except for inducible CD117^[LOW] expression in 3(8)21 cells, no expression of CD45, CD117, CD62L, CD80, CD86, CD90·1 or CD95L/CD178 was observed. Constitutive and cytokine‐inducible CD95^[LOW] expression was detected in STO and 3(8)21 cells, but not in 3(8)21‐EGFP cells. MHC (class I^+[LOW]/class II^–) and CD (CD34⁺/CD80^–/CD86^–/CD95L^–) expression patterns in STO and STO cell‐derived progenitor cells resemble patterns reported for human embryonic stem cell lines. Whether these patterns reflect associations with mechanisms that are regulatory of immune privilege or functional tissue‐specific plasticity is unknown.

Cells derived from embryonic mouse STO cell lines differentiate into hepatocytes when transplanted into the livers of nonimmunosuppressed dipeptidylpeptidase IV (DPPIV)–negative F344 rats. Within 1 day after intrasplenic injection, donor cells moved rapidly into the liver and were found in intravascular and perivascular sites; by 1 month, they were intrasinusoidal and also integrated into hepatic plates with approximately 2% efficiency and formed conjoint bile canaliculi. Neither donor cell proliferation nor host inflammatory responses were observed during this time. Detection of intrahepatic mouse COX1 mitochondrial DNA and mouse albumin mRNA in recipient rats indicated survival and differentiation of donor cells for at least 3 months. Mouse COX1 targets were also detected intrahepatically 4–9 weeks after STO cell injection into nonimmunosuppressed wild‐type rats. In contrast to STO‐transplanted rats, mouse DNA or RNA was not detectable in untreated or mock‐transplanted rats or in rats injected with donor cell DNA. In cultured STO donor cells, DPPIV and glucose‐6‐phosphatase activities were observed in small clusters; in contrast, mouse major histocompatibility complex class I H‐2K^q, H‐2D^q, and H‐2L^q and class II I‐A^q markers were undetectable in vitro before or after interferon gamma treatment. Together with H‐2K allele typing, which confirmed the Swiss mouse origin of the donor cells, these observations indicate that mouse‐derived STO cell lines can differentiate along hepatocytic lineage and engraft into rat liver across major histocompatibility barriers.