Undifferentiated cells have been identified in the prenatal blastocyst, inner cell mass, and gonadal ridges of rodents and primates, including humans. After isolation these cells express molecular and immunological markers for embryonic cells, capabilities for extended self‐renewal, and telomerase activity. When allowed to differentiate, embryonic stem cells express phenotypic markers for tissues of ectodermal, mesodermal, and endodermal origin. When implanted in vivo, undifferentiated noninduced embryonic stem cells formed teratomas. In this report we describe a cell clone isolated from postnatal rat skeletal muscle and derived by repetitive single‐cell clonogenic analysis. In the undifferentiated state it consists of very small cells having a high ratio of nucleus to cytoplasm. The clone expresses molecular and immunological markers for embryonic stem cells. It exhibits telomerase activity, which is consistent with its extended capability for self‐renewal. When induced to differentiate, it expressed phenotypic markers for tissues of ectodermal, mesodermal, and endodermal origin. The clone was designated as a postnatal pluripotent epiblastic‐like stem cell (PPELSC). The undifferentiated clone was transfected with a genomic marker and assayed for alterations in stem cell characteristics. No alterations were noted. The labeled clone, when implanted into heart after injury, incorporated into myocardial tissues undergoing repair. The labeled clone was subjected to directed lineage induction in vitro, resulting in the formation of islet‐like structures (ILSs) that secreted insulin in response to a glucose challenge. This study suggests that embryonic‐like stem cells are retained within postnatal mammals and have the potential for use in gene therapy and tissue engineering. Anat Rec Part A 277A:178–203, 2004. © 2004 Wiley‐Liss, Inc.

The MHC Class II transactivator (CIITA) acts in the cell nucleus as the master regulator of MHC class II (MHC II) gene expression. It is important to study CIITA regulation in multiple myeloma since MHC expression is central to ability of myeloma cells to present antigen and to the ability of the immune system to recognize and destroy this malignancy. Regulation of CIITA by IFN-γ in B lymphocytes occurs through the CIITA type IV promoter (pIV), one of the four potential promoters (pI-pIV) of this gene. To investigate regulation of CIITA by IFN-γ in multiple myeloma cells, first the ability of these cells to respond to IFN-γ was examined. RTPCR analyses show that IFN-γR1, the IFN-γ-binding chain of the IFN-γ receptor, is expressed in myeloma cells and IRF-1 expression increases in response to IFN-γ treatment. Western blotting demonstrates that STAT1 is activated by phosphorylation in response to IFN-γ. RT-PCR and functional promoter analyses show that IFN-γ up regulates the activity of CIITA pIV, as does ectopic expression of IRF-1 or IRF-2. In vivo protein/DNA binding studies demonstrate protein binding at the GAS, E box and IRF-E sites. In vitro studies confirm the binding of IRF-1 and IRF-2 to CIITA pIV. Although multiple myeloma cells express PRDI-BF1/Blimp-1, a factor that represses both the CIITA type III and IV promoters, they retain the capability to up regulate CIITA pIV and MHC II expression in response to IFN-γ treatment. These findings are the first to demonstrate that although PRDI-BF1/Blimp-1 diminishes the constitutive ability of these cells to present antigen by limiting CIITA and MHC II expression, it is possible to enhance this expression through the use of cytokines, like IFN-γ.

MHC Class II transactivator (CIITA) is the master transcriptional regulator of MHC class II (MHC II) genes. MHC II expression is central to antigen presentation and the immune recognition of tumor cells. The human CIITA gene, MHC2TA, is regulated in a cell-type specific manner by multiple promoters. Regulation by IFN-γ is generally via the type IV promoter (pIV) and involves the binding of IFN regulatory factor (IRF)-1 and IRF-2. The response of pIV to IFN-γ was examined in myeloma cells. Western blot and RT-PCR show that STAT1 is activated by phosphorylation and IRF-1 RNA expression increases in response to IFN-γ. RT-PCR and functional promoter analyses show that IFN-γ up-regulates the activity of MHC2TA pIV as does ectopic expression of IRF-1 or IRF-2, and the IRF-E site is required. Binding studies confirm IRF-1 and IRF-2 binding to MHC2TA pIV. Myeloma cells express positive regulatory domain I-binding factor 1 (PRDI-BF1), also called B lymphocyte-induced maturation protein-1 (Blimp-1), which represses both MHC2TA pIII and pIV. Importantly, these cells retain the ability to up-regulate CIITA pIV and MHC II in response to IFN-γ. Although PRDI-BF1/Blimp-1 diminishes the ability of myeloma cells to present antigen by limiting CIITA and MHC II expression, these studies indicate that expression can be restored by the use of cytokines like IFN-γ. Research support: RO1CA10220, RO1CA114504, MEDCEN Foundation of Central GA